Sulforaphane for treating thalassemia and sideroblastic anemia

Abstract

The present invention relates to the use of sulforaphane as an inhibitor of NLRP1 and of inflammasomes comprising NLRP1 as a sensor protein, and to the use thereof as a drug for treating and / or preventing diseases characterised by the activation of inflammasomes comprising NLRP1 as a sensor protein. Specifically, the use of sulforaphane for treating thalassemia and sideroblastic anemia is described.

Description

[0001] Sulforaphane for the treatment of thalassemia and sideroblastic anemia

[0002] DESCRIPTION

[0003] TECHNICAL FIELD

[0004] The present invention relates to the field of medicine, in particular to the treatment of thalassemia and sideroblastic anemia with an inflammasome inhibitor compound.

[0005] PRIOR ART

[0006] Hematopoiesis is the process by which all types of blood cells are produced from hematopoietic stem cells (HSCs) in the bone marrow. This complex and highly regulated process ensures the continuous replacement of blood cells, which have a limited lifespan and are essential for various physiological functions, such as oxygen transport, immune defense, and blood clotting. The bone marrow provides a specialized microenvironment, or niche, that supports hematopoiesis. This includes stromal cells, components of the extracellular matrix, and various signaling molecules that interact with HSCs and progenitor cells to regulate their function.

[0007] Erythropoiesis is the process by which red blood cells (erythrocytes) are produced. It occurs primarily in the bone marrow and is tightly regulated to maintain adequate oxygen-carrying capacity in the blood. The main function of erythrocytes is to transport oxygen from the lungs to the tissues and return carbon dioxide from the tissues to the lungs for exhalation.

[0008] The process of erythropoiesis begins with multipotent hematopoietic stem cells (HSCs), which can differentiate into two types of blood cells. HSCs give rise to common myeloid progenitors, which are precursor cells committed to the myeloid lineage. From common myeloid progenitors, the pathway narrows further to form erythroid progenitors, specifically burst-forming erythroid units (BFU-E) and colony-forming erythroid units (CFU-E). CFU-E cells differentiate into proethroblasts, which are the first recognizable erythroid precursors. Following nuclear extrusion, the cell becomes a reticulocyte, which still contains some residual RNA and can synthesize hemoglobin. Reticulocytes leave the bone marrow and enter the bloodstream. In the bloodstream, reticulocytes lose their remaining RNA and fully mature into erythrocytes.Mature erythrocytes are biconcave discs without a nucleus, optimized for gas exchange.

[0009] Erythropoiesis is regulated by erythropoietin (EPO), a key hormone produced by the kidneys in response to low oxygen levels (hypoxia) that stimulates the proliferation and differentiation of erythroid progenitors in the bone marrow. Iron also plays an essential role in hemoglobin synthesis. Iron deficiency can lead to reduced erythropoiesis and anemia. Vitamin B12 and folate are necessary for DNA synthesis in erythroid progenitors. Deficiency in these vitamins can cause megaloblastic anemia, characterized by the presence of abnormally large and immature red blood cells. Another factor to consider in erythrocyte development is the bone marrow microenvironment, which provides support and signals that regulate erythropoiesis, including growth factors, cytokines, and interactions with stromal cells.

[0010] Several diseases are linked to impaired erythropoiesis, most notably anemia. Anemia is characterized by a decrease in the number of red blood cells or hemoglobin, which reduces the body's oxygen-carrying capacity. Its causes can include nutritional deficiencies (iron, vitamin B12, folate), bone marrow disorders, chronic diseases, and genetic conditions such as sideroblastic anemia and thalassemia.

[0011] Thalassemia encompasses a group of inherited blood disorders characterized by abnormal production of hemoglobin, the red blood cell protein responsible for oxygen transport. The disorder results from mutations in the genes that produce hemoglobin, leading to reduced or absent production of one of its two components: the alpha or beta globin chains. This imbalance causes the formation of defective red blood cells, leading to anemia and other complications. There are several types of thalassemia:

[0012] - Alpha Thalassemia: caused by mutations or deletions in the genes responsible for the production of alpha globin chains. Normally, there are four genes involved in alpha globin production (two on each chromosome 16). The severity of alpha thalassemia depends on the number of genes affected.

[0013] - Silent carrier: an affected gene, usually without symptoms.

[0014] - Alpha thalassemia trait (minor): two genes affected, causing mild anemia. - Hemoglobin H disease: three genes affected.

[0015] - Alpha Thalassemia Major (Bart's Hemoglobin): all four genes are affected, which usually leads to a serious, often fatal disease in utero or shortly after birth if left untreated.

[0016] -Beta Thalassemia: caused by mutations in the genes responsible for producing beta globin chains. Humans have two genes for beta globin (one on each chromosome 11). The severity of beta thalassemia depends on whether the mutations reduce or completely eliminate beta globin production.

[0017] - Beta thalassemia minor (trait): an affected gene, which usually causes mild anemia.

[0018] - Beta Thalassemia Intermedia: two genes affected but with milder mutations, which cause moderate anemia and variable symptoms.

[0019] - Beta Thalassemia Major (Cooley's Anemia): two genes affected with serious mutations, which cause severe anemia, requiring regular blood transfusions and other treatments.

[0020] Patients with thalassemia major have more severe anemia from an early age and require regular blood transfusions along with lifelong intensive iron chelation therapy, while thalassemia minor, the less severe form, is characterized by individuals with mild, asymptomatic anemia (thalassemia trait). Intermediate forms constitute a group with a highly variable clinical spectrum, ranging from mild to moderate to moderately severe anemia, but they do not require regular blood transfusions, only occasional ones, although they do develop various complications typical of thalassemia major, such as iron overload, osteoporosis and skeletal abnormalities, extramedullary hematopoiesis, liver disease, endocrine organ involvement, lower extremity ulcers, pulmonary hypertension, etc.

[0021] In 2012, the International Thalassemia Federation adopted new terminology for the clinical classification of thalassemia to better understand the pathophysiology of the disease and improve clinical management and the management of complications. This classification distinguishes between transfusion-dependent thalassemia (TDT) and non-transfusion-dependent thalassemia (NTDT), depending on whether patients require regular transfusion therapy for life to survive (such as patients with p-thalassemia major or hemoglobin E / p-thalassemia severe) or do not require regular transfusions throughout their lives to survive (such as patients with p-thalassemia intermedia or hemoglobin E / p-thalassemia mild or moderate, hemoglobin H disease, and other combinations), although they may require occasional or even frequent transfusions in certain clinical settings and for defined periods of time.

[0022] The symptoms of thalassemia vary depending on the type and severity of the thalassemia and may include mild forms (silent carrier, minor), often asymptomatic or with mild anemia; and moderate to severe forms (intermediate, major) that may present with severe anemia, fatigue, weakness, pale or yellowish skin (jaundice), delayed growth and development in children, bone deformities, especially in the face and skull, enlarged spleen (splenomegaly), and increased risk of infections.

[0023] Differentiating a new patient with thalassemia such as DDT or NTDT is essential and requires accurate medical evaluation using indicator vapors such as hematological parameters, particularly baseline hemoglobin levels, and follow-up of at least 3 to 6 months is recommended to determine clinical severity before making the diagnosis of DDT or NTDT.

[0024] Untreated or inadequately treated thalassemia can lead to complications such as severe anemia, heart failure, liver disease, diabetes, growth retardation, and bone deformities. Regular medical care and adherence to treatment regimens are crucial for managing the disease and improving quality of life, as these rare diseases often present with complications.

[0025] Sideroblastic anemia is a group of disorders characterized by the body's inability to properly incorporate iron into hemoglobin, despite having adequate or even excessive iron stores. This results in the presence of ringed sideroblasts in the bone marrow: immature red blood cells with iron-laden mitochondria arranged in a ring around the nucleus. The disease can be congenital (inherited) or acquired, with a wide range of underlying causes and varying severity.

[0026] Congenital sideroblastic anemia can be X-linked. The most common congenital form is caused by mutations in the ALAS2 gene, which encodes the enzyme delta-aminolevulinic acid synthase 2, crucial for the first step in heme synthesis. Congenital sideroblastic anemia can also be inherited in an autosomal recessive pattern or be mitochondrial in origin, involving mutations in other genes involved in iron metabolism and heme synthesis. These are less common forms of sideroblastic anemia.

[0027] Within acquired sideroblastic anemia, we find the so-called "primary" or "clonal" type, which includes myelodysplastic syndromes (MDS) with ring sideroblasts. This subtype of acquired sideroblastic anemia is considered a neoplastic disorder of the bone marrow. The so-called "secondary" or "reversible" type, on the other hand, is due to external factors such as chronic alcoholism (since alcohol can interfere with heme synthesis), the use of certain medications (for example, isoniazid, chloramphenicol), some toxins and heavy metals (for example, lead, zinc) that can alter iron metabolism and heme synthesis, and nutritional deficiencies such as vitamin B6 (pyridoxine) deficiency, which can cause sideroblastic anemia because it is a cofactor of the ALAS2 enzyme.

[0028] The symptoms of sideroblastic anemia can vary, but they usually include fatigue and weakness, pale or yellowish skin, shortness of breath, palpitations or irregular heartbeat, signs of iron overload such as enlarged liver or heart problems, especially in chronic cases.

[0029] Current treatments for anemia include regular red blood cell transfusions to maintain hemoglobin levels; chelation therapies to remove excess iron from the body (which can accumulate due to frequent transfusions); and bone marrow or stem cell transplantation, which is the only potential cure for the disease, although it is not suitable for all patients.

[0030] The possibility of using gene therapy in the treatment of anemia is an emerging area of ​​treatment, but still largely in the experimental phase.

[0031] Sulforaphane is a naturally occurring compound found primarily in cruciferous vegetables, and in high concentrations in broccoli, Brussels sprouts, cabbage, cauliflower, kale, and bok choy. It belongs to a group of plant compounds known as isothiocyanates, which are derived from glucosinolates. Specifically, it is 1-isothiocyanato-4-(methylsulfinyl)butane, or compound with formula (I):

[0032] Formula (I)

[0033] Sulforaphane is well known for its potential health benefits, especially in relation to its antioxidant, anti-inflammatory, and anti-cancer properties.

[0034] Sulforaphane supplements, usually derived from broccoli seed extract, are currently available on the market. While these supplements can be a convenient way to increase intake, it is generally recommended to obtain nutrients from whole foods whenever possible. Overall, sulforaphane is considered safe for consumption through diet.

[0035] To date, sulforaphane has been associated with antioxidant properties, as it has been shown to help induce the production of phase II detoxification enzymes, such as glutathione S-transferase. These enzymes improve the body's ability to neutralize reactive oxygen species and detoxify harmful substances.

[0036] Several studies have shown that sulforaphane induces apoptosis (programmed cell death) in cancer cells and inhibits tumor growth, leading to its proposal as a useful compound in cancer prevention. It can also inhibit histone deacetylase, an enzyme involved in regulating gene expression, which could affect the growth of cancer cells.

[0037] Other research suggests that sulforaphane may have neuroprotective effects and protect against neurodegenerative diseases by activating the Nrf2 pathway, which increases the production of antioxidant proteins that protect against oxidative damage triggered by inflammation.

[0038] It has also been attributed with anti-inflammatory effects, mainly due to its ability to modulate several key pathways and molecules involved in inflammation, including:

[0039] - Activation of Nrf2; sulforaphane is known to activate the nuclear erythroid factor-related factor 2 (Nrf2) pathway. Nrf2 is a transcription factor that regulates the expression of antioxidant proteins that protect against oxidative damage triggered by injury and inflammation. By promoting the expression of these antioxidant enzymes, sulforaphane helps reduce oxidative stress, which is closely linked to inflammatory processes.

[0040] - Inhibition of NF-κB; nuclear factor kappa B (NF-κB) is a protein complex that plays a central role in regulating the immune response to infection and inflammation. Activation of NF-κB leads to the expression of various pro-inflammatory cytokines, chemokines, and adhesion molecules. Sulforaphane has been shown to inhibit NF-κB activation, thereby reducing the production of these inflammatory mediators.

[0041] - Suppression of pro-inflammatory cytokines; sulforaphane can reduce the production of pro-inflammatory cytokines such as interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), and others. These cytokines are involved in the spread of the inflammatory response and are often elevated in chronic inflammatory diseases.

[0042] - Modulation of enzymes involved in inflammation; sulforaphane can inhibit the activity of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (NOS), enzymes that contribute to inflammatory processes by producing pro-inflammatory mediators such as prostaglandins and nitric oxide.

[0043] - Epigenetic modulation; sulforaphane can influence inflammation by altering gene expression through epigenetic mechanisms, such as the inhibition of histone deacetylase (HDAC). This can lead to changes in the expression of genes involved in inflammation.

[0044] In addition, some clinical trials suggest that sulforaphane may reduce markers of inflammation in conditions such as obesity, type 2 diabetes, and other chronic inflammatory conditions.

[0045] Although promising, most research on its anti-inflammatory effect is still in its early stages, and further clinical studies are needed to determine the optimal dose, safety, and efficacy of sulforaphane for treating inflammation-related conditions in humans.

[0046] Some studies have indicated the possibility of using sulforaphane to promote the production of white blood cells and thus treat anemia caused by oxidative stress resulting from exposure to gamma X-rays or proton radiation [WO2007131973A2]. Sulforaphane has also been described as a component of complex compositions that include other natural compounds such as medium-chain triglycerides, green tea extract, and curcumin to treat anemia developed as an unwanted side effect of radiotherapy used in cancer treatments [US11020372B2], without establishing its relevance within the composition, nor describing a clear pathway of action that can specifically attribute these properties to sulforaphane.

[0047] DESCRIPTION OF THE INVENTION

[0048] The present invention describes for the first time the use of sulforaphane to treat thalassemia and sideroblastic anemia due to its properties as a potent inhibitor of the NLRP1 inflammasome. Sulforaphane has been identified as a potent inhibitor of NLRP1. NLRP1 is a sensor protein that is part of inflammasomes.

[0049] Inflammasomes are multiprotein complexes that play a crucial role in the innate immune system, particularly in the response to infections and cell damage. They are responsible for activating inflammatory processes, promoting the maturation of pro-inflammatory cytokines such as interleukin-1fJ and interleukin-18. They have been shown to induce pyroptosis, a programmed cell death process distinct from apoptosis.

[0050] They consist of a sensor protein, an adaptor protein, and an effector protein.

[0051] The sensor protein is typically a pattern recognition receptor (PRR) that detects pathogenic microorganisms or stress signals. Examples of these receptors include NOD-like receptors (nucleotide-binding oligomerization domains), NLRs (receptors containing leucine-rich repeats) such as NLRP3 (NLR family receptor containing a pyrin or PYD 3 domain) and NLRP1 (NLR family receptor containing a pyrin 1 domain), and AIM2 (absent in melanoma 2).

[0052] The adaptor protein is typically a speckled-like protein containing a CARD (caspase recruitment and activation domain). It is an apoptosis-associated protein that facilitates inflammasome assembly by linking the sensor protein to the effector protein. These adaptor proteins are also known as ASCs or PYCARDs. The effector protein is typically Caspase-1, an enzyme that, once activated, processes pro-inflammatory cytokines.

[0053] Inflammasome activation can be measured by ASC oligomerization and the formation of specks or spots.

[0054] The role of NLRP1 in hematopoiesis has been linked to anemia associated with chronic inflammation. Recent studies indicate that NLRP1 critically regulates hematopoiesis [1].

[0055] NLRP1 corresponds to the protein of any of the sequences SEQ. ID. No. 1 to SEQ. ID. No. 5 shown below:

[0056] Isoforma 5 of NLRP1, with access number NP_001028225.1, which corresponds to SEQ.

[0057] ID. No. 1:

[0058] MAGGAWGRLACYLEFLKKEELKEFQLLLANKAHSRSSSGETPAQPEKTSGMEVASYLVA

[0059] QYGEQRAWDLALHTWEQMGLRSLCAQAQEGAGHSPSFPYSPSEPHLGSPSQPTSTAV

[0060] LMPWIHELPAGCTQGSERRVLRQLPDTSGRRWREISASLLYQALPSSPDHESPSQESPN

[0061] APTSTAVLGSWGSPPQPSLAPREQEAPGTQWPLDETSGIYYTEIREREREKSEKGRPP

[0062] WAAWGTPPQAHTSLQPHHHPWEPSVRESLCSTWPWKNEDFNQKFTQLLLLQRPHPR

[0063] SQDPLVKRSWPDYVEENRGHLIEIRDLFGPGLDTQEPRIVILQGAAGIGKSTLARQVKEA

[0064] WGRGQLYGDRFQHVFYFSCRELAQSKVVSLAELIGKDGTATPAPIRQILSRPERLLFILDG

[0065] VDEPGWVLQEPSSELCLH WSQPQPADALLGSLLGKTI LPEASFLITARTTALQN LI PSLEQ

[0066] ARWVEVLGFSESSRKEYFYRYFTDERQAIRAFRLVKSNKELWALCLVPWVSWLACTCL

[0067] MQQMKRKEKLTLTSKTTTTLCLHYLAQALQAQPLGPQLRDLCSLAEGIWQKKTLFSPD

[0068] DLRKHGLDGAIISTFLKMGILQEHPIPLSYSFIHLCFQEFFAAMSYVLEDEKGRGKHSNCII

[0069] DLEKTLEAYGIHGLFGASTTRFLLGLLSDEGEREMENIFHCRLSQGRNLMQWVPSLQLLL

[0070] QPHSLESLHCLYETRNKTFLTQVMAHFEEMGMCVETDMELLVCTFCIKFSRHVKKLQLIE

[0071] GRQHRSTWSPTMVVLFRWVPVTDAYWQILFSVLKVTRNLKELDLSGNSLSHSAVKSLCK

[0072] TLRRPRCLLETLRLAGCGLTAEDCKDLAFGLRANQTLTELDLSFNVLTDAGAKHLCQRLR

[0073] QPSCKLQRLQLVSCGLTSDCCQDLASVLSASPSLKELDLQQNNLDDVGVRLLCEGLRHP

[0074] ACKLIRLGLDQTTLSDEMRQELRALEQEKPQLLIFSRRKPSVMTPTEGLDTGEMSNSTSS

[0075] LKRQRLGSERAASHVAQANLKLLDVSKIFPIAEIAGKSHEESSPEWPVELLCVPSPASQG

[0076] DLHTKPLGTDDDFWGPTGPVATEVVDKEKNLYRVHFPVAGSYRWPNTGLCFVMREAVT VEI EFCVWDQFLGEI N PQHSWMVAGPLLDI KAEPGAVEAVH LPH FVALQGGHVDTSLFQ

[0077] MAHFKEEGMLLEKPARVELHHIVLENPSFSPLGVLLKMIHNALRFIPVTSVVLLYHRVHPE EVTFHLYLIPSDCSIRKAIDDLEMKFQFVRIHKPPPLTPLYMGCRYTVSGSGSGMLEILPKE LELCYRSPGEDQLFSEFYVGHLGSGIRLQVKDKKDETLVWEALVKPGRNTSQPWNLRC

[0078] NRDARRY

[0079] Isoforma 2 of NLRP1, with access number NP_055737.1, which corresponds to SEQ. ID.

[0080] No. 2:

[0081] MAGGAWGRLACYLEFLKKEELKEFQLLLANKAHSRSSSGETPAQPEKTSGMEVASYLVA

[0082] QYGEQRAWDLALHTWEQMGLRSLCAQAQEGAGHSPSFPYSPSEPHLGSPSQPTSTAV LMPWIHELPAGCTQGSERRVLRQLPDTSGRRWREISASLLYQALPSSPDHESPSQESPN APTSTAVLGSWGSPPQPSLAPREQEAPGTQWPLDETSGIYYTEIREREREKSEKGRPP

[0083] WAAWGTPPQAHTSLQPHHHPWEPSVRESLCSTWPWKNEDFNQKFTQLLLLQRPHPR SQDPLVKRSWPDYVEENRGHLIEIRDLFGPGLDTQEPRIVILQGAAGIGKSTLARQVKEA WGRGQLYGDRFQHVFYFSCRELAQSKVVSLAELIGKDGTATPAPIRQILSRPERLLFILDG

[0084] VDEPGWVLQEPSSELCLH WSQPQPADALLGSLLGKTI LPEASFLITARTTALQN LI PSLEQ ARWVEVLGFSESSRKEYFYRYFTDERQAIRAFRLVKSNKELWALCLVPWVSWLACTCL MQQMKRKEKLTLTSKTTTTLCLHYLAQALQAQPLGPQLRDLCSLAAEGIWQKKTLFSPD

[0085] DLRKHGLDGAIISTFLKMGILQEHPIPLSYSFIHLCFQEFFAAMSYVLEDEKGRGKHSNCII

[0086] DLEKTLEAYGIHGLFGASTTRFLLGLLSDEGEREMENIFHCRLSQGRNLMQWVPSLQLLL

[0087] QPHSLESLHCLYETRNKTFLTQVMAHFEEMGMCVETDMELLVCTFCIKFSRHVKKLQLIE

[0088] GRQHRSTWSPTMWLFRWVPVTDAYWQILFSVLKVTRNLKELDLSGNSLSHSAVKSLCK

[0089] TLRRPRCLLETLRLAGCGLTAEDCKDLAFGLRANQTLTELDLSFNVLTDAGAKHLCQRLR

[0090] QPSCKLQRLQLVSCGLTSDCCQDLASVLSASPSLKELDLQQNNLDDVGVRLLCEGLRHP

[0091] ACKLIRLGLDQTTLSDEMRQELRALEQEKPQLLIFSRRKPSVMTPTEGLDTGEMSNSTSS LKRQRLGSERAASHVAQANLKLLDVSKIFPIAEIAEESSPEVVPVELLCVPSPASQGDLHT KPLGTDDDFWGPTGPVATEVVDKEKNLYRVHFPVAGSYRWPNTGLCFVMREAVTVEIE

[0092] FCVWDQFLGEINPQHSWMVAGPLLDIKAEPGAVEAVHLPHFVALQGGHVDTSLFQMAH FKEEGMLLEKPARVELHHIVLENPSFSPLGVLLKMIHNALRFIPVTSVVLLYHRVHPEEVTF HLYLIPSDCSIRKELELCYRSPGEDQLFSEFYVGHLGSGIRLQVKDKKDETLVWEALVKP

[0093] GDLMPATTLIPPARIAVPSPLDAPQLLHFVDQYREQLIARVTSVEVVLDKLHGQVLSQEQY ERVLAENTRPSQMRKLFSLSQSWDRCKDGLYQALKETHPHLIMELWEKGSKKGLLPLS S

[0094] Isoform 1 of NLRP1, with access number NP_127497.1, which corresponds to SEQ. ID.

[0095] No. 3:

[0096] MAGGAWGRLACYLEFLKKEELKEFQLLLANKAHSRSSSGETPAQPEKTSGMEVASYLVA QYGEQRAWDLALHTWEQMGLRSLCAQAQEGAGHSPSFPYSPSEPHLGSPSQPTSTAV LMPWIHELPAGCTQGSERRVLRQLPDTSGRRWREISASLLYQALPSSPDHESPSQESPN

[0097] APTSTAVLGSWGSPPQPSLAPREQEAPGTQWPLDETSGIYYTEIREREREKSEKGRPP

[0098] WAAWGTPPQAHTSLQPHHHPWEPSVRESLCSTWPWKNEDFNQKFTQLLLLQRPHPR

[0099] SQDPLVKRSWPDYVEENRGHLIEIRDLFGPGLDTQEPRIVILQGAAGIGKSTLARQVKEA

[0100] WGRGQLYGDRFQHVFYFSCRELAQSKVVSLAELIGKDGTATPAPIRQILSRPERLLFILDG

[0101] VDEPGWVLQEPSSELCLH WSQPQPADALLGSLLGKTI LPEASFLITARTTALQN LI PSLEQ

[0102] ARWVEVLGFSESSRKEYFYRYFTDERQAIRAFRLVKSNKELWALCLVPWVSWLACTCL

[0103] MQQMKRKEKLTLTSKTTTTLCLHYLAQALQAQPLGPQLRDLCSLAAEGIWQKKTLFSPD

[0104] DLRKHGLDGAIISTFLKMGILQEHPIPLSYSFIHLCFQEFFAAMSYVLEDEKGRGKHSNCII

[0105] DLEKTLEAYGIHGLFGASTTRFLLGLLSDEGEREMENIFHCRLSQGRNLMQWVPSLQLLL

[0106] QPHSLESLHCLYETRNKTFLTQVMAHFEEMGMCVETDMELLVCTFCIKFSRHVKKLQLIE

[0107] GRQHRSTWSPTMVVLFRWVPVTDAYWQILFSVLKVTRNLKELDLSGNSLSHSAVKSLCK

[0108] TLRRPRCLLETLRLAGCGLTAEDCKDLAFGLRANQTLTELDLSFNVLTDAGAKHLCQRLR

[0109] QPSCKLQRLQLVSCGLTSDCCQDLASVLSASPSLKELDLQQNNLDDVGVRLLCEGLRHP

[0110] ACKLIRLGLDQTTLSDEMRQELRALEQEKPQLLIFSRRKPSVMTPTEGLDTGEMSNSTSS

[0111] LKRQRLGSERAASHVAQANLKLLDVSKIFPIAEIAEESSPEWPVELLCVPSPASQGDLHT

[0112] KPLGTDDDFWGPTGPVATEVVDKEKNLYRVHFPVAGSYRWPNTGLCFVMREAVTVEIE

[0113] FCVWDQFLGEINPQHSWMVAGPLLDIKAEPGAVEAVHLPHFVALQGGHVDTSLFQMAH

[0114] FKEEGMLLEKPARVELHHIVLENPSFSPLGVLLKMIHNALRFIPVTSVVLLYHRVHPEEVTF

[0115] HLYLIPSDCSIRKAIDDLEMKFQFVRIHKPPPLTPLYMGCRYTVSGSGSGMLEILPKELELC

[0116] YRSPGEDQLFSEFYVGHLGSGIRLQVKDKKDETLVWEALVKPGDLMPATTLIPPARIAVP

[0117] SPLDAPQLLHFVDQYREQLIARVTSVEVVLDKLHGQVLSQEQYERVLAENTRPSQMRKL

[0118] FSLSQSWDRKCKDGLYQALKETHPHLIMELWEKGSKKGLLPLLSS

[0119] Isoforma 3 of NLRP1, with access number NP_127499.1, which corresponds to SEQ. ID.

[0120] No. 4:

[0121] MAGGAWGRLACYLEFLKKEELKEFQLLLANKAHSRSSSGETPAQPEKTSGMEVASYLVA

[0122] QYGEQRAWDLALHTWEQMGLRSLCAQAQEGAGHSPSFPYSPSEPHLGSPSQPTSTAV

[0123] LMPWIHELPAGCTQGSERRVLRQLPDTSGRRWREISASLLYQALPSSPDHESPSQESPN

[0124] APTSTAVLGSWGSPPQPSLAPREQEAPGTQWPLDETSGIYYTEIREREREKSEKGRPP

[0125] WAAVVGTPPQAHTSLQPHHHPWEPSVRESLCSTWPWKNEDFNQKFTQLLLLQRPHPR

[0126] SQDPLVKRSWPDYVEENRGHLIEIRDLFGPGLDTQEPRIVILQGAAGIGKSTLARQVKEA

[0127] WGRGQLYGDRFQHVFYFSCRELAQSKVVSLAELIGKDGTATPAPIRQILSRPERLLFILDG

[0128] VDEPGWVLQEPSSELCLH WSQPQPADALLGSLLGKTI LPEASFLITARTTALQN LI PSLEQ

[0129] ARWVEVLGFSESSRKEYFYRYFTDERQAIRAFRLVKSNKELWALCLVPWVSWLACTCL

[0130] MQQMKRKEKLTLTSKTTTTLCLHYLAQALQAQPLGPQLRDLCSLAAEGIWQKKTLFSPD DLRKHGLDGAIISTFLKMGILQEHPIPLSYSFIHLCFQEFFAAMSYVLEDEKGRGKHSNCII

[0131] DLEKTLEAYGIHGLFGASTTRFLLGLLSDEGEREMENIFHCRLSQGRNLMQWVPSLQLLL

[0132] QPHSLESLHCLYETRNKTFLTQVMAHFEEMGMCVETDMELLVCTFCIKFSRHVKKLQLIE

[0133] GRQHRSTWSPTMWLFRWVPVTDAYWQILFSVLKVTRNLKELDLSGNSLSHSAVKSLCK

[0134] TLRRPRCLLETLRLAGCGLTAEDCKDLAFGLRANQTLTELDLSFNVLTDAGAKHLCQRLR

[0135] QPSCKLQRLQLVSCGLTSDCCQDLASVLSASSPSLKELDLQQNNLDDVGVRLLCEGLRHP

[0136] ACKLIRLGKPSVMTPTEGLDTGEMSNSTSSLKRQRLGSERAASHVAQANLKLLDVSHIPPI

[0137] AEIAESSPEWPVELLCVPSPASQGDLHTKPLGTDDDFWGPTGPVATEVVDKEKNLYR

[0138] VHFPVAGSYRWPNTGLCFVMREAVTVEIEFCVWDQFLGEINPQHSWMVAGPLLDIKAEP

[0139] GAVEAVHLPHFVALQGGHVDTSLFQMAHFKEEGMLLEKPARVELHHIVLENPSFSPLGV

[0140] LLKMIHNALRFIPVTSVVLLYHRVHPEEVTFHLYLIPSDCSIRKAIDDLEMKFQFVRIHKPPP

[0141] LTPLYMGCRYTVSGSGSGGMLEILPKELELCYRSPGEDQLFSEFYVGHLGSGIRLQVKDKK

[0142] DETLVWEALVKPGDLMPATTLIPPARIAVPSPLDAPQLLHFVDQYREQLIARVTSVEVVLD

[0143] KLHGQVLSQEQYERVLAENTRPSQMRKLFSLSQSWDRKCKDGLYQALKETHPHLIMEL

[0144] WEKGSKKGLLPLSS

[0145] NLRP1 isoform 4 , with accession number NP_127500.1 , corresponding to SEQ. ID.

[0146] No. 5:

[0147] MAGGAWGRLACYLEFLKKEELKEFQLLLANKAHSRSSSGETPAQPEKTSGMEVASYLVA

[0148] QYGEQRAWDLALHTWEQMGLRSLCAQAQEGAGHSPSFPYSPSEPHLGSPSQPTSTAV

[0149] LMPWIHELPAGCTQGSERRVLRQLPDTSGRRWREISASLLYQALPSSPDHESPSQESPN

[0150] APTSTAVLGSWGSPPQPSLAPREQEAPGTQWPLDETSGIYYTEIREREREKSEKGRPP

[0151] WAAWGTPPQAHTSLQPHHHPWEPSVRESLCSTWPWKNEDFNQKFTQLLLLQRPHPR

[0152] SQDPLVKRSWPDYVEENRGHLIEIRDLFGPGLDTQEPRIVILQGAAGIGKSTLARQVKEA

[0153] WGRGQLYGDRFQHVFYFSCRELAQSKVVSLAELIGKDGTATPAPIRQILSRPERLLFILDG

[0154] VDEPGWVLQEPSSELCLH WSQPQPADALLGSLLGKTI LPEASFLITARTTALQN LI PSLEQ

[0155] ARWVEVLGFSESSRKEYFYRYFTDERQAIRAFRLVKSNKELWALCLVPWVSWLACTCL

[0156] MQQMKRKEKLTLTSKTTTTLCLHYLAQALQAQPLGPQLRDLCSLAEGIWQKKTLFSPD

[0157] DLRKHGLDGAIISTFLKMGILQEHPIPLSYSFIHLCFQEFFAAMSYVLEDEKGRGKHSNCII

[0158] DLEKTLEAYGIHGLFGASTTRFLLGLLSDEGEREMENIFHCRLSQGRNLMQWVPSLQLLL

[0159] QPHSLESLHCLYETRNKTFLTQVMAHFEEMGMCVETDMELLVCTFCIKFSRHVKKLQLIE

[0160] GRQHRSTWSPTMWLFRWVPVTDAYWQILFSVLKVTRNLKELDLSGNSLSHSAVKSLCK

[0161] TLRRPRCLLETLRLAGCGLTAEDCKDLAFGLRANQTLTELDLSFNVLTDAGAKHLCQRLR

[0162] QPSCKLQRLQLVSCGLTSDCCQDLASVLSASPSLKELDLQQNNLDDVGVRLLCEGLRHP

[0163] ACKLIRLGKPSVMTPTEGLDTGEMSNSTSSLKRQRLGSERAASHVAQANLKLLDVSKIFPI

[0164] AEIAEESSPEVVPVELLCVPSPASQGDLHTKPLGTDDDFWGPTGPVATEVVDKEKNLYR VHFPVAGSYRWPNTGLCFVMREAVTVEIEFCVWDQFLGEINPQHSWMVAGPLLDIKAEP GAVEAVHLPHFVALQGGHVDTSLFQMAHFKEEGMLLEKPARVELHHIVLENPSFSPLGV LLKMIHNALRFIPVTSWLLYHRVHPEEVTFHLYLIPSDCSIRKELELCYRSPGEDQLFSEF YVGHLGSGIRLQVKDKKDETLVWEALVKPGDLMPATTLIPPARIAVPSPLDAPQLLHFVD QYREQLIARVTSVEWLDKLHGQVLSQEQYERVLAENTRPSQMRKLFSLSQSWDRKCK DGLYQALKETHPHLIMELWEKGSKKGLLPLSS

[0165] The present invention demonstrates the role of sulforaphane as an NLRP1 inhibitor through screening with a library of bioactive compounds, where compound 6 (corresponding to sulforaphane) was found to have a potent capacity to inhibit NLRP1 activation observed through ASC oligomerization. For these assays, cells stably expressing the ASC-GFP fusion protein were used to express NLRP1, and the inhibitory effect of NLRP1-dependent ASC oligomerization was observed by fluorescence microscopy (Figure 1).

[0166] Next, the response of these cells to different doses of sulforaphane was tested to verify the efficacy of this compound in inhibiting NLRP1 activity. Potent inhibition of NLRP1 activity by sulforaphane at 5 pM was observed (Figure 2), even capable of reversing the activation of NLRP1 activity mediated by anisomycin (Figures 3 and 4).

[0167] To verify that the inhibition is specific to NLRP1, a developed knockout cell line lacking NLRP1 in the erythrocyte context was used. The knockout cell line used is K562, which can be differentiated into erythrocytes using hemin (Figure 5). Greater toxicity was detected in cells with NLRP1 than in cells lacking NLRP1, indicative of sulforaphane's specificity for NLRP1.

[0168] After observing the effect of sulforaphane on NLRP1 inhibition, the potential of this inhibition to restore erythropoiesis was tested. To this end, several erythroid colony-forming assays were performed using ex vivo samples from patients with thalassemia and sideroblastic anemia. It was found that NLRP1 inhibition with sulforaphane had a positive effect on erythrocyte production (Figure 6). These results support a novel function of sulforaphane: on the one hand, inhibiting NLRP1 activation, and on the other hand, restoring erythropoiesis in patients with thalassemia and sideroblastic anemia. Therefore, in a first aspect of the present invention, sulforaphane is described for use as an NLRP1 inhibitor.

[0169] In a second aspect of the present invention, sulforaphane is described for use as an inhibitor of inflammasomes characterized by comprising NLRP1 as a sensor protein.

[0170] The term “compound,” “active principle,” “active substance,” “pharmaceutically active substance,” “drug,” “active ingredient,” or “pharmaceutically active ingredient” means any component that potentially provides pharmacological activity or another distinct effect in the diagnosis, cure, mitigation, treatment, or prevention of a disease, or that affects the structure or function of the human or other animal body. The term includes those components that promote a chemical change in the preparation of the drug and are present in the drug in an intended modified form that provides the specific activity or effect.

[0171] The terms "inhibit" and "inhibition" refer to slowing down, stopping, or reversing the activity, activation pathway, or mechanism of action of a molecule. Inhibition can be greater than approximately 10%, 20%, 40%, 60%, 80%, 90%, 95%, or 99%, for example, compared to the activity that occurs in the absence of treatment.

[0172] In a third aspect of the invention, sulforaphane is described for use as a drug in the treatment and / or prevention of diseases involving the activation of inflammasomes characterized by comprising NLRP1 as a sensor protein.

[0173] A fourth aspect of the present invention describes sulforaphane for use in the treatment and / or prevention of thalassemia and sideroblastic anemia.

[0174] A fifth aspect of the invention relates to a composition comprising sulforaphane for use in the treatment and / or prevention of thalassemia and sideroblastic anemia.

[0175] Preferably, said composition is a pharmaceutical composition and more preferably, it further comprises a pharmaceutically acceptable vehicle and / or pharmaceutically acceptable excipients.

[0176] As used herein, the term "pharmaceutical composition" or "medicinal product" refers to any substance used for the diagnosis, prevention, relief, treatment, or cure of a disease in humans or animals. The pharmaceutical composition of the invention may be used alone or in combination with other pharmaceutical compositions.

[0177] The term "pharmaceutically acceptable excipient" refers to a substance that aids in the absorption of the pharmaceutical composition comprising the composition of the invention, stabilizes said pharmaceutical composition, or assists in its manufacture by providing consistency, shape, flavor, or any other specific functional characteristic. Thus, excipients may have the function of binding the ingredients together, such as starches, sugars, or cellulose; a sweetening function; a coloring function; a protective function, such as isolating it from air and / or moisture; a filler function for a tablet, capsule, or any other form of formulation; a disintegrating function to facilitate the dissolution of the components and their absorption; and include other types of excipients not mentioned in this paragraph.

[0178] A "pharmaceutically acceptable carrier" (or "pharmacologically acceptable") refers to any substance, or combination of substances, known in the pharmaceutical industry, used in the manufacture of dosage forms and includes, among others, solids, liquids, solvents, or surfactants. The carrier may be an inert substance or have an action similar to any of the compounds of the present invention, serving to facilitate the incorporation of the drug, as well as other compounds, allowing for improved dosage and administration, or providing consistency and form to the pharmaceutical composition. When the dosage form is liquid, the carrier is the diluent. The term "pharmacologically acceptable" refers to the fact that the compound in question is permitted and evaluated to be harmless to the organisms to which it is administered.

[0179] The pharmaceutical composition of the invention can be administered via any route of administration, and as such, said composition will be formulated in the pharmaceutical form appropriate to the chosen route of administration. Thus, the pharmaceutical composition of the invention can be administered orally, nasally, ocularly, topically, intradermally, intracranially, intramuscularly, intravenously, or intraperitoneally.

[0180] The pharmaceutical composition may further comprise another active ingredient or compound useful in the treatment of anemia in general, and anemia caused by thalassemia and sideroblastic anemia in particular. Preferably, the composition comprises at least one of the compounds selected from the list consisting of: chelating agents, folic acid, vitamin B12, and vitamin C.

[0181] The pharmaceutical composition may include a single composition or separate compositions.

[0182] Another aspect of the invention relates to a combination preparation or kit comprising: (a) a compound according to claim 1 or a composition according to any of claims 2 to 4, and (b) an active ingredient selected from the list consisting of chelating agents, folic acid, vitamin B12, vitamin C, or any combination thereof, for simultaneous, combined, or sequential use in the treatment and / or prevention of human thalassemia and human sideroblastic anemia. The term "combination preparation" or "juxtaposition" in this specification means that the components of the combination preparation need not be present as a single unit, for example, in an actual composition, to be available for combined, separate, or sequential use.Thus, the expression "juxtaposed" implies that it does not necessarily result in a true combination, given the physical separation of the components.

[0183] The pharmaceutical composition may include an effective amount of sulforaphane.

[0184] The term "effective amount" used herein refers to an amount sufficient to block or inhibit NLRP1 and thereby prevent or treat thalassemia and sideroblastic anemia in an individual in need of such prevention or treatment.

[0185] The effective dose can be appropriately selected for each individual by a qualified healthcare professional, taking into account the severity of the disease, the patient's age, body weight, health conditions, sex, drug sensitivity, duration of administration, route of administration, excretion rate, treatment duration, and other factors, including whether the drug is used in combination with or concurrently with other pharmaceutical formulations, and other factors known in the field of medicine. Therapy is considered "personalized" when the compound administered to an individual to treat a disease is specifically tailored to the genotypic and phenotypic characteristics of the individual being treated, thus avoiding the waste of time with ineffective therapies.The term "prevention", as used herein, refers to the ability of the compound of the invention or the pharmaceutical composition of the invention or the combined preparation of the invention, to prevent, minimize or impede the progression of thalassemia and sideroblastic anemia.

[0186] The term "treatment", as used herein, refers to the ability of the compound of the invention or the pharmaceutical composition of the invention or the combined preparation of the invention to minimize, reduce or totally or partially reverse the effects of thalassemia and sideroblastic anemia.

[0187] The term "patient," "individual," or "subject," as used herein, refers to any animal, preferably a mammal, preferably primates and human beings. In a preferred embodiment, the subject is a human being.

[0188] A final aspect of the invention relates to a method for selecting therapeutic agents useful in the prevention, improvement, relief, and / or treatment of thalassemia and sideroblastic anemia, comprising: a) determining the activity of NLRP1 at a specified concentration of the compound to be analyzed or in the absence of said compound, b) determining the activity of NLRP1 at a concentration of the compound to be analyzed different from that of a).

[0189] Compounds that inhibit NLRP1 activity could be identified as potential therapeutic agents against thalassemia and sideroblastic anemia.

[0190] Determining a compound's ability to inhibit NLRP1 activity can be done, for example, by determining NLRP1's capacity to bind or interact with a target molecule of that compound, either directly or indirectly. This can also involve activity assays, directly or indirectly measuring NLRP1 activity. Alternatively, it can be an expression assay, directly or indirectly determining the expression of NLRP1 mRNA or NLRP1 protein. These assays can also be combined with an in vivo assay measuring the effect of a compound on the symptoms of NLRP1-related diseases, specifically anemia, and more specifically thalassemia and sideroblastic anemia (for example, but not limited to, in animal models or other known model systems).The compounds to be tested used in the therapeutic agent selection method are not limited to low molecular weight organic molecules, proteins (including antibodies), peptides, oligonucleotides, etc. They can be natural and / or synthetic compounds.

[0191] For example, antibodies capable of binding to NLRP1, which can be used therapeutically as previously discussed, can also be employed in immunohistochemical assays, such as Western blots, ELISAs, radioimmunoassays, immunoprecipitation assays, or other immunohistochemical assays known in the prior art. NLRP1 polypeptides can be used to immunize an animal to obtain polyclonal antibodies. Monoclonal antibodies can also be prepared using techniques that allow antibody production by cultured cell lines, including, but not limited to, hybridomas and human B-cell hybridomas. Techniques for producing chimeric, humanized, or synthetic antibodies are known.

[0192] Finally, a last aspect of the invention describes the non-therapeutic use of sulforaphane in a food or nutritive composition to promote erythropoiesis.

[0193] Unless otherwise defined, all technical and scientific terms used herein have the same meaning commonly understood by a person skilled in the art to which this invention pertains. Similar or equivalent methods and materials to those described herein may be used in the practice of the present invention. Throughout the description and claims, the word "comprises" and its variations are not intended to exclude other technical features, additives, components, or steps. Other objects, advantages, and features of the invention will be apparent to those skilled in the art upon examination of the description or may be discovered through the practice of the invention. The following examples and drawings are provided for illustrative purposes and are not intended to limit the scope of the present invention.

[0194] DESCRIPTION OF THE FIGURES

[0195] Figure 1. Inflammasome inhibition assay with a panel of compounds. (A) Representative fluorescence images of the compounds tested as NLRP1 inhibitors in the HEK293 cell line. (B) Quantification of cells in which ASC is oligomerized (speck formation), corresponding to NLRP1 inflammasome activation. The positive control (+) refers to cells transfected with untreated NLRP1 and ASC-GFP, the negative control (-) to cells transfected only with ASC-GFP, and the DMSO control is similar to the positive control with the addition of the compound vehicle. Cells treated with compound 6 show fewer specks. Three independent experiments were performed (n=3). Statistical analysis was performed using one-way ANOVA, comparing the compounds under the DMSO condition. ** p-value less than 0.01.

[0196] Figure 2. Dose-response assay of sulforaphane on NLRP1 inflammasome activation in HEK293 ASC-GFP cells. HEK293 ASC-GFP cells were transfected with NLRP1 and treated with sulforaphane (SFN) for 24 hours at different concentrations: 2.5 pM, 5 pM, 10 pM, 20 pM, 40 pM, and no sulforaphane as a control (C-). (A) GFP fluorescence microscopy images, Hoechst staining, and superimposition of both. (B) Quantification of ASC-GFP speck formation, indicative of NLRP1 activation. Three independent experiments were performed (n=3). Statistical analysis was performed using one-way ANOVA, comparing the compounds to the control condition. ** p-value less than 0.01 and **** p-value less than 0.0001.

[0197] Figure 3. Dose-response assay of sulforaphane on NLRP1 inflammasome activation in anisomycin-activated HEK293 cells. HEK293 ASC-GFP NLRP1 cells were treated with sulforaphane (SNF) at concentrations of 5 pM or 10 pM, or without sulforaphane as a control (C-), for 24 hours. Three hours prior to visualization, they were treated with anisomycin (Aniso), a previously described activator for NLRP1, at 10 pg / ml. (A) GFP fluorescence microscopy images, Hoechst staining, and superimposition of both. (B) Quantification of ASC-GFP speck formation. Three independent experiments were performed (n=3). Statistical analysis was performed using one-way ANOVA, comparing the compounds with the anisomycin (Aniso) condition. **** p-value less than 0.0001.

[0198] Figure 4. Dose-response assay of sulforaphane on NLRP1 inflammasome activation in anisomycin-activated A549 cells. A549 ASC-GFP NLRP1 cells were treated with sulforaphane (SNF) concentrations of 2.5 pM, 25 pM, 40 pM, and 50 pM, or without sulforaphane as a control (C-), for 24 hours. Three hours prior to visualization, they were treated with anisomycin (Aniso), a previously described activator for NLRP1, at 10 pg / ml. (A) GFP fluorescence microscopy images, Hoechst staining, and superimposition of both. (B) Quantification of ASC-GFP speck formation. Three independent experiments were performed (n=3). Statistical analysis was performed using one-way ANOVA, comparing the compounds with the anisomycin (Aniso) condition. **** p-value less than 0.0001.

[0199] Figure 5. MTT assay to test sulforaphane toxicity in wild-type (WT) and NLRP1-deficient (NLRPI) K562 cells 7(A) Western blot. NLRP1 protein expression in two different clones of NLRP1-deficient K562 cells, generated using CRISPR-Cas9 technology, compared with two clones of the wild-type (WT) strain, with actin used as a positive control. (B) Representative image of wild-type (WT) and NLRP1-deficient (NLRPT) K562 cells / _ (C) Western blot. NLRP1, GATA1, NLRP3, and actin (as a control) protein levels at different times (0, 24, 48, 96 h) after hemin differentiation in wild-type (WT) and NLRP1-deficient (NLRPT) K562 cells. NLRP1-deficient cells show a greater accumulation of hemoglobin, corresponding to a more advanced state of erythroid differentiation. / _(D) MTT assay (viability study) of K562 cells treated for 48 hours prior to the assay with different concentrations of sulforaphane (SNF): 1 pM, 2.5 pM, 5 pM, 10 pM, 25 pM, 50 pM, and 0 pM (control). Measurements are shown as arbitrary fluorescence units (AU). (E) Western blot. NLRP1, GATA1, and actin protein levels (positive control) in K562 cells at 0, 24, and 48 h post-differentiation with hemin. Results are shown for cells treated with 25 pM sulforaphane or DMSO (control). Two (DE) or three (AC) independent experiments were performed. Statistical analysis was performed using two-way ANOVA, comparing WT cells with NLRPT cells. / _ *** p-value less than 0.001 and **** less than 0.0001.

[0200] Figure 6. Effect of sulforaphane on restoring erythrocyte production in primary cells from healthy donors and patients with sideroblastic anemia. Erythroid colony counts in methylcellulose assays using PBMCs from healthy donors (A), one patient with congenital sideroblastic anemia (B), and four different thalassemia patients (C). Two independent experiments were performed with each patient (n=2). Statistical analysis was performed using one-way ANOVA, comparing sulforaphane treatment at different concentrations with the DMSO control. ** p-value less than 0.01 and **** less than 0.0001.

[0201] DETAILED DESCRIPTION OF THE INVENTION

[0202] Cell cultures For the different inflammasome inhibition assays, HEK293 cells (human embryonic kidney cells) stably expressing ASC-GFP, and A549 cells (human basal alveolar epithelial adenocarcinoma cells) stably expressing ASC-GFP and NLRP1 were used.

[0203] For sulforaphane toxicity assays, K562 cells (immortalized human chronic myeloid leukemia cells) were used and a NLRP1-deficient K562 cell line was generated.

[0204] The different cell lines were verified to be free of mycoplasma contamination and were authenticated by STR profiling. The cells were maintained and subcultured before reaching confluence every 72 hours.

[0205] Inflammasome inhibition assay with a battery of compounds.

[0206] HEK293 cells were maintained in DMEN culture medium supplemented with 10% fetal bovine serum (FCS), 2 mM glutamine, and 1% penicillin-streptomycin (Life Technologies).

[0207] HEK293 cells at 70-80% confluence in 24-well plates were transfected with NLRP1 and ASC-GFP using Lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturer's protocol. The negative control was transfected with ASC-GFP only.

[0208] At 4 hours after transfection, the culture medium was changed and the following battery of compounds was added: compound 1: p-coumaric acid; compound 2: ferulic acid; compound 3: caffeic acid; compound 4: rosmarinic acid; compound 5: vanillic acid; compound 6: sulforaphane; compound 7: syringic acid; compound 8: protocatechuic acid; compound 9: chlorogenic acid; compound 10: sinapinic acid; compound 11: homovanillic acid; compound 12: 2-picolinic acid; compound 13: nicotinic acid; compound 14: tyrosol and compound 15: dehydroacetic acid.

[0209] To evaluate the inhibitory capacity of the compounds, ASC oligomerization was observed using fluorescence microscopy. As shown in Figure 1A, images of the specks (green fluorescence) were captured using a Leica DM18 fluorescence microscope. Speck formation corresponds to activation of the NLRP1 inflammasome; therefore, a lower number of specks indicates a greater inhibitory capacity of the tested compounds. Cells treated with compound 6 showed a lower number of specks. Quantification was performed using Fiji software. A threshold was applied, and a particle analyzer was used to count the total number of cells. A multipoint instrument was used to count the number of fluorescent spots (specks). A graph was created calculating the percentage of spots in relation to the total number of cells and spots (Figure 1B).Three independent experiments were performed (n=3). It was clearly observed that cells treated with compound 6 showed a lower number of specks compared to the positive control (cells transfected with NLRP1 and ASC-GFP and untreated cells) and compared to the other compounds tested. The result for compound 6 was similar to that of the negative control (cells transfected only with ASC-GFP, not transfected with NLRP1, and therefore not expressing NLRP1).

[0210] Effect of sulforaphane on activation of the NLRP1 inflammasome.

[0211] The HEK293 cell line, which stably expresses ASC-GFP, and the A549 cell line, which stably expresses ASC-GFP and NLRP1 (donated by Dr. E. Meunier), were used. They were maintained and cultured under the same conditions as the HEK293 cells.

[0212] Trials were conducted on the effect of different concentrations of sulforaphane with the HEK293 cell line transfected with NLRP1 (Figure 2), the HEK293 cell line transfected with NLRP1 and activated with anisomycin (an activator described for human NLRP1) (Figure 3) and with the A549 cell line, which stably expresses ASC-GFP and NLRP1, activated with anisomycin (Figure 4).

[0213] Evaluation of ASC oligomerization by fluorescence microscopy

[0214] At 24 hours after transfection, Hoechst staining (H3570, Invitrogen) was performed and images were captured at 4X and 20X of the nuclei (blue fluorescence) and the dots (green fluorescence) using a Leica DM18 fluorescence microscope (Figure 2A, 3A and 4A).

[0215] Quantification was performed using Fiji software. A threshold was applied, and a particle analyzer was used to count the total number of cells. A multipoint instrument was used to count the number of fluorescent specks. The formation of ASC-GFP specks is indicative of NLRP1 activation.

[0216] A graph was created calculating the percentage of points in relation to the total number of cells and points (Figure 2B, 3B and 4B). Three independent experiments were performed (n=3). sulforaphane

[0217] HEK293 cells with stable ASC-GFP expression transfected with NLRP1 were treated with sulforaphane for 24 hours at different concentrations: 2.5pM, 5 pM, 10 pM, 20 pM and 40 pM, including a sulforaphane-free condition as a control.

[0218] Figure 2A shows the fluorescence microscopy images whose results can be verified in the graph in Figure 2B, where a clear inhibition of NLRP1 is observed compared to the control starting from a sulforaphane concentration of 5 pM.

[0219] Sulforaphane dose-response assay in anisomycin-activated cells

[0220] HEK293 cells with stable ASC-GFP expression were transfected with NLRP1 and treated with sulforaphane at concentrations of 5 pM and 10 pM for 24 hours, including a sulforaphane-free condition as a control.

[0221] Three hours before visualization, they were treated with an activator described for NLRP1, anisomycin, at 10 pg / ml.

[0222] Figure 3A shows the fluorescence microscopy images whose results can be seen in the graph in Figure 3B. This graph illustrates the activating effect of anisomycin compared to the control and how treatment with sulforaphane at 5 pM reverses this activation, returning to the control values. Treatment with 10 pM has such an inhibitory effect that, in addition to completely reversing the activation caused by anisomycin, it reduces the number of cells with ASCs even below the control values.

[0223] The A549 cell line, which stably expresses ASC-GFP and NLRP1, was treated with sulforaphane at 2.5 pM, 25 pM, 40 pM, and 50 pM for 24 hours, including a sulforaphane-free condition as a control, and 3 hours before visualization, it was treated with anisomycin at 10 pg / ml. The doses used were higher than those used in HEK293 cells because these cells are more resistant to treatment.

[0224] Figure 4A shows the fluorescence microscopy images whose results can be seen in the graph in Figure 4B, where it can be observed that sulforaphane is able to cancel the activating effect of anisomycin at a concentration of 2.5 pM, and to induce inflammasome inhibition, despite the use of anisomycin, at concentrations of 25 pM and above. MTT assay to test the toxicity of sulforaphane in wild-type (WT) and NLRP1-deficient (NLRPI) K562 cells 7 ) and acceleration of erythroid differentiation in vitro with sulforaphane.

[0225] K562 cell line generation deficient in NLRP1

[0226] K562 cells were maintained in RPMI culture medium supplemented with 10% fetal bovine serum (FCS), 2 mM glutamine and 1% penicillin-streptomycin (Life Technologies).

[0227] To perform NLRP1 knockout in K562 cells, the NEPA21 electroporator was used to introduce Cas9 protein and guides into the cells. The cells were washed with Optimem to remove excess serum and antibiotic residues before electroporation, and no antibiotics were added during or immediately after the electroporation process. Electroporation was performed at 200 V and 3.0 ms, achieving 80% positive cells. A concentration of 1 x 10⁻⁵ was used for the K562 cells. 6 cells in 100 pl of Optimem. The Cas9 D10A and guides were acquired from IDT. Two different guide combinations were selected: 5'-ccaccgcagtgctaatgccc-3' (SEQ. ID. No. 6), 5'-accccaggtggggttcactt-3' (SEQ. ID. No. 7), and 5'-ctggatccatgaattgccgg-3' (SEQ. ID. No. 8), 5'-attagcactgcggtggaggt-3' (SEQ. ID. No. 9).

[0228] Protein expression analysis by immunoblotting

[0229] The expression level of the NLRP1 protein (using actin as a positive control) was determined in different clones of NLRP1-deficient K562 cells (NLRPT). / _ ) compared with clones of the wild-type strain (WT) (Figure 5A).

[0230] Subsequently, wild-type (WT) and NLRP1-deficient (NLRPT) K562 cells / _ ) were differentiated into erythrocytes with 50 pM hemin (Figure 5B) to observe the accumulation of hemoglobin after 24h, which corresponds to an advanced state of erythroid differentiation.

[0231] The expression levels of the NLRP1, GATA1, and NLRP3 proteins (using actin as a positive control) were also determined in wild-type (WT) and NLRP1-deficient (NLRPT) K562 cells. / _ ) differentiated with hemin 50 pM, after 24, 48 and 96 h (Figure 5C).

[0232] Finally, the expression level of the NLRP1 and GATA1 proteins (using actin as a positive control) was determined in wild-type (WT) and NLRP1-deficient (NLRPT) K562 cells. / _ ) differentiated with hemin 50 pM, after 24 and 48 h, treated with sulforaphane at 25 pM or with DMSO (control) (Figure 5E).

[0233] To quantify protein expression levels after the various treatments, cells were lysed in a solution of 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% (w / v) NP-40, and a fresh protease inhibitor cocktail (1 / 20, #P8340, Sigma-Aldrich). Protein quantification was performed using the BCA kit, with BSA as the standard. Cell lysates (40 pg) in SDS-based sample buffer were electrophoresis-treated on a polyacrylamide gel and transferred to PVDF membranes. The membranes were incubated for 1 hour with TTBS containing either 5% (w / v) skimmed milk powder or 2% (w / v) BSA. Subsequently, they were immunodetected in the same buffer for 16 hours at 4°C with the various primary antibodies diluted 1 / 1000. Next, the membranes were washed with TTBS and incubated for 1 hour at room temperature with HRP-conjugated secondary antibodies, diluted 1 / 2500 in 5% (w / v) skimmed milk in TTBS.The signal was detected using an enhanced chemiluminescence reagent and the ChemiDoc XRS system (Bio-Rad). The primary antibodies used were GATA1 (#3535, Cell Signaling), NLRP1 (#AF6788, R&D Systems), NLRP3 (#AG-20B-0014, Adipogen), and ACTIN (ACTB-HRP) (#sc-47778, Santa Cruz Biotechnology). The secondary antibodies used were anti-sheep (#31480, Thermo Fisher), anti-rabbit (#A6154, Sigma-Aldrich), and anti-mouse (#A4416, Sigma-Aldrich).

[0234] As can be seen in Figure 5A, the NLRP1-deficient K562 cells (NLRPT) / _ ) did not express the NLRP1 protein, as expected.

[0235] Differentiation with hemin resulted in a progression of the erythroid differentiation state (Figure 5B) that was greater in the case of NLRP1-deficient K562 cells, supporting the inhibition of human NLRP1 as a strategy to promote erythroid differentiation.

[0236] In cells treated with hemin (Figure 5C), inhibition of GATA1 expression was observed from 24 h onwards, both in wild-type and NLRP1-deficient cells. GATA1 is an essential transcription factor in erythroid differentiation, and its accelerated degradation indicates greater erythroid differentiation, as also observed in the accumulation of hemoglobin in pellets treated 24 h with hemin (Figure 5B).

[0237] NLRP1 was only expressed in wild-type cells, as expected, showing a peak expression 48 hours after hemin administration. NLRP3, the most studied inflammasome, was not expressed in either cell type (neither wild-type nor NLRP1-deficient), which would indicate that it plays no role in this process.

[0238] Sulforaphane administration (Figure 5E) inhibited NLRP1 expression at 24h and 48h, almost completely inhibiting it at 24h. This again led to a significant reduction in GATA1 levels in sulforaphane-treated cells, indicative of increased erythroid differentiation.

[0239] MTT Trial

[0240] The viability of differentiated cells in the presence of different concentrations of sulforaphane, measured as cellular metabolic capacity, was assessed using MTT assays (reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a yellow tetrazolium, to purple formazan by mitochondrial enzymes). In summary, 100,000 K562 cells were seeded in 96-well plates and treated with sulforaphane at concentrations of 1 pM, 2.5 pM, 5 pM, 10 pM, 25 pM, 50 pM, and 0 pM (control) for 48 hours.

[0241] MTT (Alfa Aesar, Thermo Fisher, Karlsruhe, Germany) was added to a final concentration of 483 pM (0.2 mg / ml) and the cells were incubated for 1 h at 37°C and 5% CO2. Subsequently, an acidified isopropanol solubilization solution containing 0.04 M hydrochloric acid and 0.1% NP-40 detergent was added to each well to lyse the cells and dissolve the insoluble purple formazan product formed within the cells, obtaining a colored solution.

[0242] Finally, absorbance was measured at 550 nm using a plate reader spectrophotometer (Spectrostar Nano; BMG Labtech, Ortenberg, Germany). Measurements are shown as arbitrary fluorescence units (AU). The viability of the treated cells was determined by comparing it to control conditions (100% viability, 0% cytotoxicity), in which the cells were exposed only to medium with the equivalent dose of vehicle (DMSO).

[0243] As can be seen in Figure 5D, both wild-type and NLRP1-deficient cells begin to decline at sulforaphane concentrations of 5 pM, with the loss of viability becoming more pronounced in wild-type cells at concentrations of 10 pM. However, concentrations of 25 pM and above are clearly toxic, regardless of cell type. The greater toxicity of sulforaphane in cells with NLRP1 compared to cells lacking NLRP1, which can be observed at concentrations of 10 pM, is indicative of sulforaphane's specificity for NLRP1.

[0244] In vitro effect of sulforaphane restoring erythrocyte production in primary cells from healthy donors and patients with sideroblastic anemia and thalassemia.

[0245] Blood samples were obtained from healthy donors or from patients with thalassemia and sideroblastic anemia provided by the Hematology Service of the Virgen de la Arrixaca University Clinical Hospital (HCUVA) under the ethical approval numbers (2021-11-9-HCUVA; 2023-3-5-HCUVA).

[0246] Peripheral blood mononuclear cells (PBMCs) were purified by standard gradient centrifugation with Ficoll-Paque (Cytiva).

[0247] Erythroid colony-forming unit (CFU-E) assay

[0248] PBMCs were cultured from healthy donors or from patients with thalassemia and sideroblastic anemia (1x10 5cells) in 1 ml of MethoCult™ methylcellulose-based medium (Stemcell) in the presence of sulforaphane at a concentration of 2.5 pM, and using DMSO as a vehicle control. The cells were incubated at 37°C in a humid atmosphere with 5% CO2. Erythroid colony-forming units (CFU-E) were counted after 14 days of culture.

[0249] As can be seen in Figure 6A, in cells from healthy patients, the administration of sulforaphane at concentrations of 2.5 pM has an enhancing effect on the generation of erythroid colony-forming units.

[0250] This effect is also seen in cells from patients with sideroblastic anemia (Figure 6B) and with thalassemia (Figure 6C) treated with sulforaphane at concentrations of 2.5 pM where erythropoiesis is restored.

[0251] References

[0252] 1. Rodríguez-Ruiz, L. et al. ZAKα / P38 kinase signaling pathway regulates hematopoiesis by activating the NLRP1 inflammasome. EMBO Molecular Medicine. 2023 / 10 / 11. https: / / doi.Org / 10.15252 / emmm.202318142.

Claims

CLAIMS 1. Sulforaphane for use in the treatment and / or prevention of human thalassemia and human sideroblastic anemia.

2. Composition comprising sulforaphane for use in the treatment and / or prevention of human thalassemia and human sideroblastic anemia.

3. Composition comprising sulforaphane for use according to the preceding claim, further comprising pharmaceutically acceptable excipients or carriers.

4. Composition comprising sulforaphane for use according to the preceding claim, further comprising at least one of the compounds selected from the list consisting of: chelating agents, folic acid, vitamin B12 and vitamin C, or any combination thereof.

5. A combination preparation or kit comprising: a) a compound according to claim 1 or a composition according to any of claims 2 to 4, and b) an active ingredient selected from the list consisting of chelating agents, folic acid, vitamin B12, vitamin C or any combination thereof for simultaneous, combined or sequential use in the treatment and / or prevention of human thalassemia and human sideroblastic anemia.

6. Non-therapeutic use of sulforaphane in a food or nutrient composition to promote erythropoiesis.

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