Use of caspase-1 inhibitors for treating ineffective erythropoiesis in patients suffering from sickle cell disease

Abstract

The present invention involves the use of Caspase-1 inhibitors for treating ineffective erythropoiesis in patients with sickle cell disease (SCA). It highlights increased levels of IL-18 positive cells and Caspase-1 activity in hematopoietic stem cells (HSCs) of SCA patients compared to healthy donors. The invention demonstrates that treatment with a Caspase-1 inhibitor reduces this activity, suggesting its potential therapeutic benefit.

Description

[0001] USE OF CASPASE-1 INHIBITORS FOR TREATING INEFFECTIVE

[0002] ERYTHROPOIESIS IN PATIENTS SUFFERING FROM SICKLE CELL DISEASE

[0003] FIELD OF THE INVENTION:

[0004] The present invention is in the field of medicine, in particular haematology.

[0005] BACKGROUND OF THE INVENTION:

[0006] Sickle cell disease (SCD) is a recessively inherited disease and one of the most common severe monogenic disorders worldwide affecting more than 8 million people. SCD is due to a single mutation in the P-globin chain (PS) of human hemoglobin resulting in HbS. This abnormal haemoglobin polymerizes in the deoxygenated state and causes the sickling of the red blood cell (RBC). Although every person with SCD has the same underlying genotype, being homozygous for the P-globin variant (HBB; c.20A>T,p.Glu7Val), it is a remarkably variable condition, with a wide range of clinical manifestations. One of the critical aspects of SCD is the ineffective erythropoiesis, the process by which new red blood cells are produced. Recent work has shown that defects in erythropoiesis in SCD could be due to increased inflammation in the bone marrow niche. Among the various inflammatory mediators, interleukin- 18 (IL-18) plays a significant role in exacerbating the condition. IL-18 is known to induce severe oxidative stress, which hampers the normal maturation and proliferation of erythroid progenitor cells, leading to ineffective erythropoiesis.

[0007] The SS Townes mouse model (Ryan TM et al. 1997), chronic hemolysis and defective erythropoiesis in the bone marrow lead to compensatory activation of stress erythropoiesis in the spleen, reflected by marked splenomegaly and expansion of erythroid progenitors. Treatment with a caspase-1 inhibitor restored erythroid maturation, resulting in increased hemoglobin levels in peripheral blood and reduction of anemia. This hematologic improvement was accompanied by decreased splenic erythropoietic activity, indicating normalization of erythropoietic dynamics and reduced inflammatory stress. These findings confirm the causal role of caspase- 1 -driven inflammation in ineffective erythropoiesis and demonstrate the therapeutic potential of targeting this pathway to restore erythroid homeostasis in SCD.

[0008] SUMMARY OF THE INVENTION: The present invention is defined by the claims. In particular, the present invention relates to the use of caspase-1 inhibitors for treating ineffective erythropoiesis in patients suffering from sickle cell disease.

[0009] DETAILED DESCRIPTION OF THE INVENTION:

[0010] The present invention relates to a method of treating ineffective erythropoiesis in a patient suffering from sickle cell disease comprising administering to the patient a therapeutically effective amount of a caspase- 1 inhibitor.

[0011] As used herein, the term “patient” is interchangeable with the term “individual” or “subject”, and may refer to a patient to be treated by the methods disclosed herein. In particular, the patient suffers from sickle cell disease. In some embodiments, the patient is a human infant. In some embodiments, the patient is a human child. In some embodiments, the patient is a human adult. In some embodiments, the patient is an elderly.

[0012] As used herein, the term “sickle cell disease” or “SCD” has its general meaning in the art and refers to a hereditary blood disorder characterized by the production of abnormal hemoglobin known as hemoglobin S (HbS). This abnormal hemoglobin causes red blood cells to become rigid, sticky, and shaped like crescent moons or sickles. These sickle-shaped cells can get stuck in small blood vessels, which can slow or block blood flow and oxygen to parts of the body, leading to severe pain, organ damage, and increased risk of infection.

[0013] As used herein, the term "ineffective erythropoiesis" refers to the defective production of red blood cells (RBCs) in the bone marrow, which is characterized by the failure of erythroid progenitor cells to mature and proliferate normally. This condition results from various factors, including oxidative stress and chronic inflammation, which inhibit the proper development of these progenitor cells, leading to a reduced number of functional RBCs in the bloodstream. Ineffective erythropoiesis is a significant concern in diseases such as sickle cell disease, where the abnormal hemoglobin and inflammatory mediators further exacerbate the inefficiency of erythropoiesis. In particular, the caspase-1 inhibitor is particularly suitable for reducing inflammation that leads to the ineffective erythropoiesis.

[0014] As used herein, the term "treatment" or "treat" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of patient at risk of contracting the disease or suspected to have contracted the disease as well as patients who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a patient having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a patient beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a patient during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a patient during treatment of an illness, e.g., to keep the patient in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., disease manifestation, etc.]).

[0015] As used herein, the term "caspase-1" refers to an enzyme that plays a pivotal role in the inflammatory process and apoptosis. Caspase-1 is part of the cysteine-aspartic acid protease (caspase) family and is responsible for cleaving and activating the precursors of inflammatory cytokines such as interleukin- 1|3 (IL-ip) and interleukin- 18 (IL- 18). The activation of caspase- 1 is typically regulated by multiprotein complexes known as inflammasomes, which respond to various pathogenic infections and cellular stress signals. In the context of sickle cell disease, the inventors showed that caspase- 1 activity contributes to the inflammatory milieu and oxidative stress that impair erythropoiesis and exacerbate the disease pathology. As used herein, the term “inhibitor” refers to a compound that decreases the magnitude of at least one activity, signaling or expression of a molecule compared to the magnitude of the activity, signaling or expression observed in the absence of the inhibitor. In some instances, an inhibitor will substantially decrease the magnitude of at least one activity, signaling or expression of a molecule compared to the magnitude of the activity or expression observed in the absence of the inhibitor. In some instances, an inhibitor will completely diminish the magnitude of at least one activity, signaling or expression of a molecule compared to the magnitude of the activity, signaling or expression observed in the absence of the inhibitor. As used herein, the term "caspase-1 inhibitor" refers to a compound that can inhibit the activity and / or expression of caspase-1. Without wishing to be bound by a theory, a caspase-1 inhibitor can act by a number of different pathways. The caspase-1 inhibitors can encompass numerous classes of chemical molecules, e.g., small organic or inorganic molecules, polysaccharides, biological macromolecules, e.g., peptides, proteins, peptide analogs and derivatives, peptidomimetics, antibodies, antibody fragments, nucleic acids, nucleic acid analogs and derivatives, an extract made from biological materials such as bacteria, plants, fungi, or animal cells or tissues, naturally occurring or synthetic compositions. Thus, a caspase-1 inhibitor can be a molecule of any type that interferes with the activity or expression of caspase- 1, for example, either by decreasing transcription or translation of caspase- 1 encoding nucleic acid, or by inhibiting or blocking caspase- 1 activity, or both. Examples of caspase- 1 inhibitors include, but are not limited to, antisense polynucleotides, interfering RNAs, catalytic RNAs, RNA-DNA chimeras, caspase- 1 -specific aptamers, anti-caspase- 1 antibodies, caspase- 1- binding fragments of anti-caspase- 1 antibodies, caspase- 1 -binding small molecules, caspase- 1- binding peptides, and other polypeptides that specifically bind caspase- 1 (including, but not limited to, caspase- 1 -binding fragments of one or more caspase- 1 ligands, optionally fused to one or more additional domains), such that the interaction between the caspase- 1 inhibitor and caspase- 1 results in a reduction or cessation of caspase- 1 activity or expression. It will be appreciated that caspase-1 inhibitors described herein may be strong inhibitors of caspase-1. In some embodiments, a caspase- 1 inhibitor inhibits the biological activity of the caspase- 1 receptor by at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% relative to a control. In some embodiments, a caspase- 1 inhibitor completely abrogates the biological activity of the caspase- 1 receptor relative to a control. A control can comprise a sample that is not treated with a caspase-1 inhibitor. In some embodiments, the caspase-1 inhibitor is a small molecule. As used herein, the term “small molecule” refers to a low molecular weight organic compound, typically less than 900 daltons, which can regulate a biological process, with a size small enough to rapidly diffuse across cell membranes and reach intracellular sites of action. Small molecules can be designed to interact with specific proteins, nucleic acids, or other biomolecules to modulate their activity. Small molecules that act as caspase- 1 inhibitors are well known in the art and include those described in W093 / 05071, WO93 / 09135, WO93 / 14777, WO95 / 26958, WO95 / 29672, WO95 / 33751, WO95 / 35308, WO96 / 03982, WO96 / 30395, W097 / 07805, WO97 / 08174, WO97 / 22618, WO97 / 22619, WO97 / 27220, WO98 / 11109, WO98 / 11129, WO98 / 16502, WO98 / 16504, WO98 / 16505, WO98 / 24804, WO98 / 24805, WO99 / 47545, W001 / 90063, EP 519748 (U.S. equivalents are U.S. Pat. Nos. 5,430,128 and 5,434,248), EP 547699, EP 618223, EP 623592 (U.S. equivalents are U.S. Pat. Nos. 5,985,838, and 6,576,614), EP 623606 (U.S. equivalents are U.S. Pat. Nos. 5,462,939 and 5,585,486), EP 628550 (U.S. equivalents are U.S. Pat. Nos. 5,585,357 and 5,677,283), EP 644198, U.S. Pat. No. 5,430,128, U.S. Pat. No. 5,434,248, U.S. Pat. No. 5,462,939, U.S. Pat. No. 5,552,400, U.S. Pat. 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No. 7,417,029, US 2006 / 0128696, Mjalli et al., 1993, Bioorg Med Chem Lett 3:2689-2693, Mjalli et al., 1994, Bioorg Med Chem Lett 4: 1965-1968, Mjalli et al., 1995 Bioorg Med Chem Lett 5: 1405-1408, Mjalli et al., 1995, Bioorg Med Chem Lett 5: 1409-1414, Thomberry et al., 1994, Biochem 33:3934-3940, Dolle et al., 1994, J Med Chem 37:563-564, Dolle et al., 1994, J Med Chem 37: 3863-3866, Dolle et al., 1995, J Med Chem 38: 220-222, Graybill et al., 1997 Bioorg Med Chem Lett 7:41-46, Semple et al., 1998, Bioorg Med Chem Lett 8:959-964, and Okamoto et al., 1999, Chem Pharm Bull 47: 11-21, herewith incorporated by reference in their entireties for all purposes.

[0016] In some embodiments of the present invention, a caspase-1 inhibitor is a peptide caspase-1 inhibitor or single stereoisomers, mixtures of stereoisomers, pharmaceutically acceptable salts or prodrugs thereof. In some embodiments of the present invention, a peptide caspase-1 inhibitor is selected from the group consisting of BACMK (Boc-Asp(Obzl)-CMK, z-VAD (Z-Val-Ala-Asp), BocD, LY333531, casputin, Ac-DQMD-CHO, CV-1013, VX-740, VX-765, VX-799, Ac-YVAD- CMK, IDN-5370, IDN-6556, IDN-6734, IDN-1965, IDN-1529, z-VAD-fmk (Z-Val-Ala- Asp(OMe)-Fluoro methyl ester), z-DEVD-cmk, Z-DEVD, Ac-YVAD-fmk, z-Asp-Ch2-DCB, Ac-IETD, Ac-VDVAD, Ac-DQMD, Ac-LEHD, Z-WEHD, Z-WEHD-fmk, Z- WE(OMe)HD(OMe)-fmk, Z-YVAD, Z-YVAD-fmk, Ac-YVAD-cmk, Ac-VEID and single stereoisomers, mixtures of stereoisomers, pharmaceutically acceptable salts or prodrugs thereof.

[0017] In some embodiments, a caspase-1 inhibitor for use in a method of the present invention is VX- 765 or single stereoisomers, mixtures of stereoisomers, pharmaceutically acceptable salts or prodrugs thereof. VX-765 is also known as is (S)-l-((S)-2-{[l-(4-Amino-3-chloro-phenyl)- methanoyl]-amino}-3,3-dimethyl-butanoyl)-pyrrolidine-2-carboxylic acid ((2R,3 S)-2-ethoxy- 5-oxo-tetrahydro-furan-3-yl)-amide.

[0018] In some embodiments, the caspase-1 inhibitor is selected from the group consisting of VX-765, ML132, VX-740, VRT-018858, YVAD, and WEHD.

[0019] As used herein, the expression "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result. A therapeutically effective amount of drug may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of drug to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody or antibody portion are outweighed by the therapeutically beneficial effects. The efficient dosages and dosage regimens for drug depend on the disease or condition to be treated and may be determined by the persons skilled in the art. A physician having ordinary skill in the art may readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician could start doses of drug employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. In general, a suitable dose of a composition of the present invention will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect according to a particular dosage regimen. Such an effective dose will generally depend upon the factors described above. For example, a therapeutically effective amount for therapeutic use may be measured by its ability to stabilize the progression of disease. A therapeutically effective amount of a therapeutic compound may decrease tumor size, or otherwise ameliorate symptoms in a subject. One of ordinary skill in the art would be able to determine such amounts based on such factors as the subject's size, the severity of the subject's symptoms, and the particular composition or route of administration selected. An exemplary, non-limiting range for a therapeutically effective amount of drug is about 0.1-100 mg / kg, such as about 0.1-50 mg / kg, for example about 0.1-20 mg / kg, such as about 0.1-10 mg / kg, for instance about 0.5, about such as 0.3, about 1, about 3 mg / kg, about 5 mg / kg or about 8 mg / kg. An exemplary, non-limiting range for a therapeutically effective amount of an antibody of the present invention is 0.02-100 mg / kg, such as about 0.02-30 mg / kg, such as about 0.05-10 mg / kg or 0.1-3 mg / kg, for example about 0.5-2 mg / kg. Administration may e.g. be intravenous, intramuscular, intraperitoneal, or subcutaneous, and for instance administered proximal to the site of the target. Dosage regimens in the above methods of treatment and uses are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. In some embodiments, the efficacy of the treatment is monitored during the therapy, e.g. at predefined points in time. As non-limiting examples, treatment according to the present invention may be provided as a daily dosage of the inhibitor of the present invention in an amount of about 0.1-100 mg / kg, such as 0.2, 0.5, 0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100 mg / kg, per day, on at least one of days 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40, or alternatively, at least one of weeks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 after initiation of treatment, or any combination thereof, using single or divided doses every 24, 12, 8, 6, 4, or 2 hours, or any combination thereof.

[0020] Typically, the inhibitor of the present invention is administered to the subject in the form of a pharmaceutical composition which comprises a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers that may be used in these compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, di sodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene- block polymers, polyethylene glycol and wool fat. For use in administration to a subject, the composition will be formulated for administration to the subject. The compositions of the present invention may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. The used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques. Sterile injectable forms of the compositions of this invention may be aqueous or an oleaginous suspension. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example as a solution in 1,3 -butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono-or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, such as carboxymethyl cellulose or similar dispersing agents that are commonly used in the formulation of pharmaceutically acceptable dosage forms including emulsions and suspensions. Other commonly used surfactants, such as Tweens, Spans and other emulsifying agents or bioavailability enhancers which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms may also be used for the purposes of formulation. The compositions of this invention may be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, aqueous suspensions or solutions. In the case of tablets for oral use, carriers commonly used include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include, e.g., lactose. When aqueous suspensions are required for oral use, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening, flavoring or coloring agents may also be added. Alternatively, the compositions of this invention may be administered in the form of suppositories for rectal administration. These can be prepared by mixing the agent with a suitable non-irritating excipient that is solid at room temperature but liquid at rectal temperature and therefore will melt in the rectum to release the drug. Such materials include cocoa butter, beeswax and polyethylene glycols. The compositions of this invention may also be administered topically, especially when the target of treatment includes areas or organs readily accessible by topical application, including diseases of the eye, the skin, or the lower intestinal tract. Suitable topical formulations are readily prepared for each of these areas or organs. For topical applications, the compositions may be formulated in a suitable ointment containing the active component suspended or dissolved in one or more carriers. Carriers for topical administration of the compounds of this invention include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water. Alternatively, the compositions can be formulated in a suitable lotion or cream containing the active components suspended or dissolved in one or more pharmaceutically acceptable carriers. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2- octyl dodecanol, benzyl alcohol and water. Topical application for the lower intestinal tract can be effected in a rectal suppository formulation (see above) or in a suitable enema formulation. Patches may also be used. The compositions of this invention may also be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well- known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and / or other conventional solubilizing or dispersing agents. For example, an antibody present in a pharmaceutical composition of this invention can be supplied at a concentration of 10 mg / mL in either 100 mg (10 mL) or 500 mg (50 mL) single-use vials. The product is formulated for IV administration in 9.0 mg / mL sodium chloride, 7.35 mg / mL sodium citrate dihydrate, 0.7 mg / mL polysorbate 80, and Sterile Water for Injection. The pH is adjusted to 6.5. An exemplary suitable dosage range for an antibody in a pharmaceutical composition of this invention may between about 1 mg / m2and 500 mg / m2. However, it will be appreciated that these schedules are exemplary and that an optimal schedule and regimen can be adapted taking into account the affinity and tolerability of the particular antibody in the pharmaceutical composition that must be determined in clinical trials. A pharmaceutical composition of the invention for injection (e.g., intramuscular, i.v.) could be prepared to contain sterile buffered water (e.g. 1 ml for intramuscular), and between about 1 ng to about 100 mg, e.g. about 50 ng to about 30 mg or more preferably, about 5 mg to about 25 mg, of the inhibitor of the invention.

[0021] The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

[0022] FIGURES:

[0023] Figure 1: A histogram representing the level of IL-18 in the bone marrow serum of healthy donors (HD) (n=6) and Sickle Cell Anemia (SCA) patients (n=3).

[0024] Figure 2: A histogram representing the percentage of IL-18 positive cells in different subpopulations of PBMCs isolated from the peripheral blood of healthy donors or SCA patients.

[0025] Figure 3: A histogram representing caspase 1 activity in CD34+ (HSCs) of HD and SCA patients (with or without incubation with the inhibitor for 24 hours, at a concentration of 50uM).

[0026] Figure 4: Caspase 1 activity measured in (A) bone marrow and (B) Spleen of SS untreated and SS caspase 1 inhibitor treated mice (n=4 per group). Mice were treated for a period of 4 weeks (3 times a week) with the VX-765 (capase 1 inhibitor), administered intraperitoneally at a dose of 25 mg / kg.

[0027] Figure 5: Graph representing hemoglobin (g / dL) levels in the peripheral blood of SS untreated and SS caspase 1 inhibitor treated mice (n=4 per group). Mice were treated for a period of 4 weeks (3 times a week) with the VX-765 (capase 1 inhibitor), administered intraperitoneally at a dose of 25 mg / kg.

[0028] Figure 6: Graph representing the percentage of Ter-119 positive (Erythroblasts) in the spleen of SS untreated and SS caspase 1 inhibitor treated mice (n=4 per group). Mice were treated for a period of 4 weeks (3 times a week) with the VX-765 (capase 1 inhibitor), administered intraperitoneally at a dose of 25 mg / kg.

[0029] EXAMPLES:

[0030] EXAMPLE 1: Measurement of the level of IL-18 in the serum of the bone marrow niche in Sickle Cell Anemia and healthy individuals.

[0031] Method: Bone marrow aspirates of SCA and healthy individuals were centrifuged to isolate the serum. Using LegendPlex (BioLegend) technology cytokine levels were quantified. Results: Increased level of IL-18 was detected in SCA bone marrow compared to healthy donors (HD) (Figure 1).

[0032] EXAMPLE 2: Measurement of the intracellular level of IL-18 in PBMCs of Healthy Donors or Sickle Cell Anemia Patients.

[0033] Method: Using flow cytometry, IL-18 levels were measured in different subpopulation of cells isolated from the peripheral blood of HD and SCA individuals.

[0034] Results: Increased level of IL-18 positive cells in both the CD14+ (monocytes) and the CD34+ (Hematopoietic Stem Cells) of SCA patients when compared to healthy donors (Figure 2).

[0035] EXAMPLE 3: Quantification of Caspase 1 activity in hematopoietic stem cells (HSCs) of healthy donors and SCA patients. Incubation with Caspase 1 inhibitor to verify that the activity detected by the assay is that of caspase 1.

[0036] Method: HSCs (CD34+) cells of HD and SCA patients were isolated from the peripheral blood. Cells were cultured for 24 hours with or without the caspase 1 inhibitor. Caspase 1 activity was measured using the Caspase Gio 1 Inflammasome Assay (Promega).

[0037] Results: Caspase 1 activity is increased in HSCs of SCA patients compared to HD. Upon treatment with Caspase 1 inhibitor, the activity is decreased (Figure 3).

[0038] EXAMPLE 4: Caspase 1 activity is decreased by caspase 1 inhibitor (VX-765) in bone marrow and spleen of the SS Townes mouse model (Figure 4 A and 4B). Hemoglobin (g / dL) levels in the peripheral blood of the SS Townes mouse model is increased by caspase 1 inhibitor (VX- 765) (Figure 5). Finally, the percentage of erythroblasts in the spleen of the SS Townes mouse model is decreased by caspase 1 inhibitor (VX-765) (Figure 6).

[0039] REFERENCES:

[0040] Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

Claims

CLAIMS:

1. A method of treating ineffective erythropoiesis in a patient suffering from sickle cell disease comprising administering to the patient a therapeutically effective amount of a caspase- 1 inhibitor.

2. The method according to claim 1 wherein the caspase-1 inhibitor is selected from the group consisting of VX-765, ML132, VX-740, VRT-018858, YVAD, and WEHD.

3. The method according to claim 1 wherein the caspase-1 inhibitor is VX-765.

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