Compounds for treatment of hemolysis- and inflammasome-associated diseases

Abstract

Disclosed herein are methods of treating a complication of a hemolysis and / or an inflammasome activation-associated disease comprising administering to a patient in need thereof an inflammasome inhibitor and a hemolysis inhibitor.

Description

COMPOUNDS FOR TREATMENT OF HEMOLYSIS- AND INFLAMMASOME-ASSOCIATED DISEASESCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application Serial No. 63 / 674,717, filed on July 23, 2024, and U.S. Provisional Patent Application Serial No. 63 / 704,959, filed on October 8, 2024. The entire contents of the foregoing are hereby incorporated by reference in their entireties.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under Grant Nos. P01 HL149626 and R35 HL161239 awarded by the National Institutes of Health. The government has certain rights in the invention.BACKGROUND

[0003] Hemolysis is a hallmark of sickle cell disease (SOD). When deoxygenated, sickle hemoglobin polymerizes, causing changes in RBC membrane shape and function that increase its fragility and ultimately leads to RBC destruction and hemoglobin release. Increasing evidence suggest that hemoglobin and its oxidized form of free heme, a lipophilic bioactive molecule, play a key role in the initiation and progression of hemolytic complications due to their ability to trigger oxidative stress, sterile inflammation, cell death and tissue injury.

[0004] The inflammasome signaling pathway is a key host inflammatory response that promotes IL-ip production by processing pro-IL-1 p into cleaved mature IL-i p. The inflammasome signaling pathway is activated in numerous inflammatory diseases and upregulate in SCD. inflammasome activation are critical for the initiation, development, treatment, and prognosis of multiple disease which include infectious diseases, autoimmune diseases, cancer, and metabolic disorder and other disease including SCD, and pharmacological inhibition of inflammasome pathway are considered as a promising therapeutic strategy in several inflammatory disease models (Guo H et al. Nat Med. 21 :677- 87, 2015; Mangan MSJ, et al. Nat Rev Drug Discov. 17:588-606, 2018).

[0005] Quinine can bind with cell-free heme / hemin released by hemolysis and inhibit multiple immune effector cell function including secretion of antibodies by B cells and release of inflammatory cytokines by innate immune cells.SUMMARY

[0006] In some embodiments provided herein is a method of treating a hemolysis- associated disease, comprising administering to a patient in need thereof a therapeutically effective dose of a hemolysis inhibitor and a therapeutically effective dose of an inflammasome inhibitor.

[0007] In some embodiments, the hemolysis-associated disease is sickle cell disease

[0008] In some embodiments, the inflammasome-associated disease is an infectious disease, autoimmune disease, cancer, metabolic disorder, or sickle cell disease.

[0009] In some embodiments, the patient exhibits hemolysis.

[0010] In some embodiments, the hemolysis inhibitor is selected from inhibitors of HbS polymerization, potentiators of HbS oxygen affinity, and potentiators of RBC ATP levels.

[0011] In some embodiments, the inhibitor of HbS polymerization is one or more of hydroxyurea, tetrahydrouridine, decitabine, and pociredir.

[0012] In some embodiments, the potentiator of HbS oxygen affinity is voxelotor.

[0013] In some embodiments, the potentiator of RBC ATP levels is one or more of mitapivat or etavopivat.

[0014] In some embodiments, the inflammasome inhibitor is one or more of interleukin-1 receptor agonist (IL-1 RA), a spleen tyrosine kinase (SYK) inhibitor, epeleuton, flurbiprofen, MCC950, anti-IL-1 p antibody, and the combination of hemin and quinine.

[0015] In some embodiments, the SYK inhibitor is one or more of ostamatinib, entospletinib, lanraplenib, and sovleplenib.

[0016] In some embodiments, the inflammasome inhibitor is the combination of quinine and hemin.

[0017] In some embodiments, the inflammasome inhibitor is quinine alone.

[0018] In some embodiments, the quinine is in the form of a quinine salt.

[0019] In some embodiments, the quinine is in the form of quinine free base.

[0020] In some embodiments, provided herein is a method of treating a hemolysis- associated disease, comprising administering to a patient exhibiting hemolysis a therapeutically effective dose of an inflammasome inhibitor.

[0021] In some embodiments, provided herein is a method of treating a hemolysis- associated disease, comprising(a) identifying a patient exhibiting hemolysis; and(b) administering to the patient a therapeutically effective dose of an inflammasome inhibitor.

[0022] In some embodiments, the inflammasome inhibitor consists essentially of quinine.BRIEF DESCRIPTION OF THE DRAWINGS

[0023] FIG. 1 depicts a diagram of inflammasome signaling pathway.

[0024] FIG. 2A-D depicts human monocytes isolated from peripheral blood were cultured with LPS for 3 hours to prime pro-IL-1 p production followed by addition of various NLRP3 pathway agonists to trigger IL-1 secretion without or with therapeutic reagents including hemin, quinine or hemin plus quinine (all at 2.5pM concentration for all experiments except the dose response studies in FIGs. 2B and C). Levels of IL-1 p, IL-6 and TNF-a in the culture supernatants was analyzed 30min (for ATP treatment) or 2 hours (for nigericin and imiquimod) after addition of NLRP3 agonists. FIG. 2A: The effect of hemin, quinine and hemin plus quinine on IL-1 p secretion triggered by various NLRP3 agonists. FIG. 2B: The dose dependent effect of hemin, quinine and hemin plus quinine. FIG. 2C: The effect of QA, CQ, AQ, and DHA in the presence / absence of hemin. FIG. 2D: The effect of hemin, quinine, or hemin plus quinine on IL-6 and TNF-a.

[0025] FIG. 3 depicts human monocytes isolated from peripheral blood was cultured with LPS for 3 hours to prime pro-IL-1 p production followed by addition of ATP to trigger IL-1 p cleavage without or with 2.5|JM hemin plus quinine. After 30 min, cells were harvested and levels of pro-IL-1 p and cleaved IL-1 p analyzed by western blot.

[0026] FIG. 4 depicts human monocytes isolated from peripheral blood cultured with LPS for 3 hours to prime pro-IL-1 p production followed by addition of NLRC4, NLRP1 , AIM2 and non-canonical inflammasome pathway agonists to induce IL-1 p secretion together with hemin, quinine or hemin plus quinine. Levels of IL-1 p level in the culture medium at 3 hours post addition of agonists are shown.

[0027] FIG. 5A-B: Mice were I.P. injected with alum (700pg / mice) along with D-PBS as control or quinine + heme (Q+H) as treatment. Levels of IL-1 p, IL-6 (FIG. 5A) as well as numbers of white blood cells, including neutrophil, monocyte T and B cells (FIG. 5B) in the peritoneal cavity at 16 hours post injection are shown.

[0028] FIG. 6A-B. FIG. 6A: SOD mice were I.P. injected with alum (700pg / mice) along with D-PBS as control, hemin, quinine, or Q+H. Mice survival were monitored for 24 hours.FIG. 6B: The effects of hemin, quinine, Q+H on IL-1 p were tested in monocytes from SCD patients treated with NLRP3 inflammasome agonists nigericin and imiquimod.

[0029] Figure 7 shows the suppression of hemin induced inflammatory cytokine productions by quinine. (A) Human primary monocytes were cultured with quinine, hemin, and quinine plus hemin for 3 hours in the presence of LPS. The levels of inflammatory cytokines in culture medium were assessed using CBA kit (B) Human primary monocytes were cultured with quinine, hemin, and quinine plus hemin for 16 hours. The MCP-1 levels in culture medium were assessed. (C) Human primary monocytes were cultured with quinine, RBC lysate, and quinine plus RBC lysate for 3 hours in the presence of LPS. The levels of inflammatory cytokines in culture medium were assessed using CBA kit.

[0030] Figure 8 shows that low-dose quinine inhibits inflammatory cytokine production in SCD mice at baseline and during VOE. : (A) Control and SCD mice were feed with normal or quinine supplemented water for one week. The plasma inflammatory cytokine levels were assessed. (B) In (A), the volume of water intake of mice was examined. (C) Control and SCD mice were feed with normal or quinine supplemented water for one week before inducing VOE with TNF-a. (D) The plasma inflammatory cytokine levels were assessed. (E) The plasma inflammatory ALT / AST levels were assessed. (F) vascular occlusions (pointed out by red arrow) in the liver were examined by H&E staining.DETAILED DESCRIPTION

[0031] This disclosure is focused on hemolysis and inflammasome associated complications in sickle cell disease (SCD) and other diseases with inflammasome activation. Disclosed herein is that cell-free heme / hemin released by hemolysis may modulate the risk, initiation and development of these complications including humoral immune responses to transfused red blood cell and pain crisis. By screening multiple heme-binding small molecules, it was determined that quinine showed strong biologic activities including inhibiting B cells mat into antibody secreting cell (plasma cells) and innate immune cells secreting inflammatory cytokines in the presence of free heme or hemolysis but not in the absence of heme or hemolysis. These data indicate that through its ability to bind to heme, during hemolytic crises when there are high in vivo free heme levels, quinine alone could be used to inhibit detrimental antibody production, such as delayed hemolytic reactions after red cell transfusions, and prevent inflammatory cytokine secretion, such as during pain crisis. Thus, in some embodiments, disclosed herein is that quinine alone may modulate the risk, initiation and development of hemolysis and inflammasome associated complications in sickle cell disease (SCD) and other diseases with hemolysis and inflammasome activation complications,including humoral immune responses to transfused red blood cell and pain crisis. Quinine may also inhibit complications in other hemolytic diseases with overt intravascular hemolysis. Furthermore, a dose of an in vitro mixture of quinine plus heme is useful to inhibit detrimental antibodies and inflammatory cytokines production in the case of low or no in vivo hemolysis.

[0032] Hemolysis-associated diseases include any condition which causes lysis of red blood cells. Hemolysis inside the body can be caused by a large number of medical conditions, including infection by many Gram-positive bacteria (e.g., Streptococcus, Enterococcus, and Staphylococcus'), infection by some parasites (e.g., Plasmodium), some autoimmune disorders (e.g. , drug-induced hemolytic anemia, atypical hemolytic uremic syndrome (aHUS)), and some genetic disorders (e.g., sickle-cell disease or G6PD deficiency).

[0033] Inflammasome-associated diseases are inflammatory diseases in which inflammasome activation is critical for disease initiation, development, treatment, and / or prognosis. These diseases include infectious diseases, autoimmune diseases, cancer, metabolic disorder, and other diseases including SCD.

[0034] Sickle cell disease is a genetic blood disorder derived from a hemoglobin gene mutation, which causes red blood cell (RBCs) to stiffen, become fragile and change shape leading to premature RBC destruction. Hemolytic anemia caused by premature destruction of RBCs is a hallmark of SCD with an estimated two-thirds of hemolysis occurring extra vascularly where the reticuloendothelial cells engulf and remove RBCs from the circulation; one-third occurs intravascularly with the direct release of hemoglobin (Hb) and its breakdown product, free heme, into the circulation. The free heme released during hemolysis induces multiple inflammatory responses including inflammasome activation. Indeed, free heme has been shown to induce IL-i p and IL-18 production through activating NLRP3 pathway in monocyte, macrophage, platelet, and neutrophils. The release of IL-1 p and IL-18 will induce local and systemic inflammation leading to tissue damage and pain crisis.

[0035] Several strategies have been developed to inhibit the root cause of SCD, namely inhibition of sickle hemoglobin (HbS) polymerization, including induction of fetal hemoglobin or increased hemoglobin oxygen affinity. Other strategies involve increasing sickle RBC health using metabolic agents that increase RBC ATP levels. All these strategies inhibit hemolysis and to some extent, alleviate systemic inflammation and / or pain crisis in SCD patients, but require further optimization. Inflammasome pathway inhibitors have also been tested in sickle mouse models with some beneficial effects in improving SCD pathophysiology, but also require further improvement.

[0036] Disclosed herein are methods of lowering hemolysis through the use of a combination of therapeutic strategies that have the potential to decrease systemicinflammation, including inflammasome activation, thereby making downstream anti- inflammasome treatments more efficacious. Given that inflammasome activation can occur independent of free heme (eg anemia), it may also be that the combined use of anti- inflammasome treatment with anti-hemolytic drugs may lead to synergistic therapeutic effect on SOD complications and potentially allow for lowering the therapeutic dose of each drug class and thus minimizing their toxicity.

[0037] The loss of red blood cell elasticity is central to the pathophysiology of sickle cell disease. Normal red blood cells are quite elastic and have a biconcave disc shape, which allows the cells to deform to pass through capillaries. In sickle cell disease, low oxygen tension promotes red blood cell sickling and repeated episodes of sickling damage the cell membrane and decrease the cell's elasticity. These cells fail to return to normal shape when normal oxygen tension is restored. As a consequence, these rigid blood cells are unable to deform as they pass through narrow capillaries, leading to vessel occlusion and ischemia. The actual anemia of the illness is caused by hemolysis, the destruction of the red cells, because of their shape. Although the bone marrow attempts to compensate by creating new red cells, it does not match the rate of destruction. Healthy red blood cells typically function for 90-120 days, but sickled cells only last 10-20 days. A hemolytic crisis occurs when there is an acute accelerated drop in the hemoglobin level due to the breakdown of red blood cells.

[0038] In subjects with sickle cell disease, or a related disorder, physiological changes in RBCs can result in a disease with the following signs: (1) hemolytic anemia; (2) vaso-occlusive crisis; and (3) multiple organ damage from microinfarcts, including heart, skeleton, spleen, and central nervous system.

[0039] SCD is a form of hemolytic anemia, with red cell survival of around 10-20 days. Approximately one third of the hemolysis occurs intravascularly, releasing free hemoglobin (plasma free hemoglobin [PFH]) and arginase into plasma. PFH has been associated with endothelial injury including scavenging nitric oxide (NO), proinflammatory stress, and coagulopathy, resulting in vasomotor instability and proliferative vasculopathy. A hallmark of this proliferative vasculopathy is the development of pulmonary hypertension in adulthood.

[0040] Vaso-occlusive crisis occurs when the circulation of blood vessels is obstructed by sickled red blood cells, causing ischemic injuries. The most common complaint is of pain, and recurrent episodes may cause irreversible organ damage. One of the most severe forms is the acute chest syndrome which occurs as a result of infarction of the lung parenchyma. Vasoocclusive crisis can be accompanied by a pain crisis which can occur suddenly and last several hours to several days.

[0041] Current strategies that target sickle RBCs to prevent their lysis, either FDA approved or in clinical trials, include inhibition of HbS polymerization by inducing fetal hemoglobin (hydroxyurea, tetrahydrouridine and decitabine, and pociredir), or increasing HbS oxygen affinity (voxelotor) or by increasing RBC ATP levels (mitapivat, etavopivat). Several others, including WIZ inhibitors, are additional promising drugs for induction of fetal hemoglobin, but have only been tested in mice and non-human primates.

[0042] Several inhibitors that can inhibit the inflammasome pathway have been tested in sickle mice (IL-1 RA, SYK inhibitors, epeleuton, flurbiprofen, MCC950) and in patients (canakinumab, an anti-IL-1 p antibody). Additionally, a combination of hemin+quinine is a potent inflammasome inhibitor (PCT / US2021 / 054827). Additionally, quinine alone is a potent inflammasome inhibitor in the presence of hemolysis. The data demonstrate potential beneficial effects of inhibiting inflammasome pathway in SCD with further optimization.

[0043] Therefore, disclosed herein are methods to treat SCD with drugs that reduce hemolysis that will significantly decrease the systemic inflammation and inflammasome activation, rendering anti-inflammasome treatments more efficacious. Non-heme mediated mechanisms may contribute to inflammasome activation in SCD and the anti-inflammasome therapy, if combined with anti-hemolytic drugs, will have synergistic beneficial effects to alleviate SCD complications and potentially allow for lowering the therapeutic dose of each drug class and thus minimizing their toxicity. Thus, combination therapy with hemolysis inhibitors and inflammasome pathway inhibitors in SCD will be more efficacious than monotherapy with either drug class alone. It may also be used in other hemolytic anemias and disorders with increased red cell lysis.

[0044] Inflammasome inhibitors include, but are not limited to, interleukin-1 receptor agonist (IL-1RA), spleen tyosine kinase (SYK) inhibitors (e.g. fostamatinib, entospletinib, lanraplenib, sovleplenib), epeleuton, flurbiprofen, MCC950, canakinumab, and the combination of hemin+quinine. Inflammasome inhibitors also include quinine alone in the presence of hemolysis, In some embodiments, the inflammasome inhibitor is a combination of hemin+quinine.

[0045] Hemolysis inhibitors include, but are not limited to, inhibitors of HbS polymerization (e.g., hydroxyurea, tetrahydrouridine, decitabine, pociredir), potentiators of HbS oxygen affinity (e.g., voxelotor), and potentiators of RBC ATP levels (e.g., mitapivat, etavopivat).

[0046] Quinine ((R)-(6-Methoxyquinolin-4-yl)[(1 S,2S,4S,5R)-5-vinylquinuclidin-2-yl] methanol) is an antiprotozoal and an antimyotonic, and is known for the treatment of malaria caused by Plasmodium species, the treatment and prophylaxis of nocturnal recumbency leg muscle cramps, and the treatment of babesiosis caused by Babesia microti. Quinine isstructurally similar to quinidine, which is also an antiprotozoal, but can function as an antiarrhythmic. Quinidine has been associated with the prolongation of the QT interval in a dose-related fashion. Excessive QT prolongation has been associated with an increased risk of ventricular arrhythmia. Although quinine is a diastereomer of quinidine, it does not cause QT prolongation to the same degree although it has been suggested that patients with a history of cardiac arrhythmias and / or QT prolongation should carefully consider taking quinine as they may be at risk for arrhythmias.

[0047] “Pharmaceutically acceptable salts” include derivatives of the active agent (e.g. quinine), wherein the parent compound is modified by making acid or base addition salts thereof. Also included are all crystalline, amorphous, and polymorph forms. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid addition salts; and the like. The pharmaceutically acceptable salts include salts, for example, from inorganic or organic acids. For example, acid salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like. Pharmaceutically acceptable organic salts includes salts prepared from organic acids such as acetic, trifluoroacetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, mesylic, esylic, besylic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic, HOOC — (CH2)n-COOH where n is 0-4, and the like. Specific quinine salts include quinine sulfate, quinine hydrochloride, quinine dihydrochloride, and hydrates or solvates thereof. As used herein, the term “quinine” includes quinine salts. The term “quinine derivative” includes any chemical derivative of quinine and includes, but is not limited to, quinacrine, biquinolone, chloroquine, hydroxychloroquine, amodiaquine, quinine, quinidine, mefloquine, primaquine, lumefantrine, and halofantrine.

[0048] In some embodiments, quinine includes a pharmaceutically acceptable solvate, including hydrates of such compounds and salts thereof.

[0049] Hemin is an iron-containing porphyrin (iron(lll) complex of protoporphyrin IX) which is administered intravenously for treatment of certain blood disorders. Hemin is also referred to as hematin.

[0050] The term “effective amount” or “therapeutically effective amount” means an amount effective, when administered to a patient, to provide any therapeutic benefit. A therapeutic benefit may be an amelioration of symptoms, e.g., an amount effective to decrease the severity, duration, response to treatment, or incidence of one or more of the symptoms disclosed herein.

[0051] The amount that is “effective” will vary from subject to subject, depending on the age and general condition of the individual, weight of the individual, including mass or surface area, the particular active agent, and the like. Thus, it is not always possible to specify an exact “effective amount.” However, an appropriate “effective” amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation. In certain circumstances a patient may not present symptoms of a condition for which the patient is being treated. A therapeutically effective amount of an active agent may also be an amount sufficient to provide a significant positive effect on any indicium of a disease, disorder, or condition, e.g. an amount sufficient to significantly reduce the severity of a SCD, or the risk or frequency of a pain or vaso-occlusive crisis. A significant effect on an indicium of a disease, disorder, or condition is statistically significant in a standard parametric test of statistical significance. In some embodiments, a therapeutically effective amount of quinine is a dose of 2-10 mg / kg twice daily. In some embodiments, the dose of quinine is 5 mg / kg. In some embodiments, the dose is 324 mg twice a day. In some embodiments, the patient does not have heart failure, myasthenia gravis, optic neuritis, or a known hypersensitivity to quinine, mefloquine, or quinidine.

[0052] Quinine dosage forms existing as liquids, solutions, emulsions, or suspensions can be packaged in a container for convenient dosing of pediatric or geriatric patients. For example, prefilled droppers (such as eye droppers or the like), prefilled syringes, and similar containers housing the liquid, solution, emulsion, or suspension form are contemplated.

[0053] In some embodiments, the methods disclosed herein comprises administration of a combination or quinine, or a salt or derivative thereof, and hemin. As used herein, the term “combination” refers to administration of both compounds and does not limit the administration to a single formulation that contains both quinine, or a salt or derivative thereof. In some embodiments, quinine is administered orally and hemin is administered intravenously. The two components of the combination may be, but are not necessarily, administered at the same time or on the same schedule. However, they should be administered in sufficient proximity in time that both components are present together in the body.

[0054] In some embodiments, the methods disclosed herein comprises administration of quinine alone, or a salt or derivative thereof.

[0055] Incorporated by reference herein for all it discloses regarding compounds for treatment of hemolysis- and inflammasome-associated diseases is WQ2022 / 081742.EXAMPLESExample 1. Hemolysis Inhibits Inflammasome Activation in Sickle Cell Disease

[0056] The inflammasome signaling pathway is a key host inflammatory response that promotes IL-1 p production by processing pro-IL-1 into cleaved mature IL-1 p. It is activated in numerous inflammatory diseases with pharmacological inhibition of inflammasome pathway considered as a promising therapeutic strategy in several inflammatory disease models (Guo H et al. Nat Med. 21 :677-87, 2015; Mangan MSJ, et al. Nat Rev Drug Discov. 17:588-606, 2018. The inflammasome pathways include both canonical and non-canonical pathways (FIG. 1) with NLRP3 activation pathway considered the most important since it can sense various DAMP and PAMP stimuli.

[0057] Our findings presented here demonstrate that quinine, administered in the presence of hemolysis, inhibits NLRP3 inflammasome pathway in human monocytes activated through multiple NLRP3 agonists including ATP, nigericin, and imiquimod (FIG. 2A). An X- ray crystal structure of quinine binding with hemin has been obtained. See ACS Chem Biol. 2012 Apr 20;7(4):666-71 , doi: 10.1021 / cb200528z The complex may be formed in vitro by mixing hemin with quinine or in vivo through the binding of quinine with free hemin released during hemolysis, and it inhibits the NLRP3 inflammasome pathway in human monocytes activated through multiple NLRP3 agonists including ATP, nigericin, and imiquimod (FIG. 2A). The effect of Q+H was dose-dependent with almost complete inhibition of IL-1 p production at the highest tested concentration (2.5 M) (FIG. 2B). Inhibition of inflammasome by Q+H was specific to the heme binding ability of quinine since other hemin binding small molecules such as chloroquine (CQ), amodiaquine (AQ), and dihydroartemisinic (DHA) (FIG. 2C) had no inhibitory effect on the inflammasome activation pathway. In addition, Q+H did not alter the expression of IL-6 and TNF-a (FIG. 2D), indicating that the effect is specific to the inflammasome activation pathway rather than overall cell activation. Further support for inhibition of inflammasome activation pathway was the demonstration that Q+H prevented cleavage of pro-IL-1 p into active IL-1 p as shown by Western blotting (FIG. 3).

[0058] In addition to NLRP3 inflammasome pathway, Q+H was able to inhibit all inflammasome signaling pathways (FIG. 4).

[0059] Alum-induced peritonitis is classically used as an inflammasome-dependent inflammation animal model. Alum I.P. injection increases IL-i p and other inflammatory cytokine production as well as induces the migration of neutrophils and monocytes into the peritoneum. We found that Q+H inhibited alum-mediated IL-1 p secretion (FIG. 5A) and inflammatory cell migration in vivo (FIG. 5B).

[0060] SCD mice are more sensitive to inflammatory stimuli than control mice. 80% of sickle mice (but none of the control mice) died following I.P. injection with alum whereas all survived if they had been treated with Q+H ((40pg quinine + 80|jg hemin) / mouse, I.P. injection)(FIG. 6A). Using monocytes from SOD patients, we also found inhibition of inflammasome activation by Q+H (FIG. 6B).

[0061] In some embodiments, our findings demonstrate that quinine alone in the presence of hemolysis inhibits NLRP3 inflammasome pathway in human monocytes activated through multiple NLRP3 agonists including ATP, nigericin, and imiquimod.Suppression of hemin induced inflammatory cytokine productions by quinine.

[0062] Quinine is known to bind hemin, a pro-inflammatory molecule released during hemolysis. To investigate whether quinine can suppress the pro-inflammatory effect of hemolysis, we examined its impact on inflammatory cytokine production induced by hemin in human primary monocytes. Because hemin alone (25pM, 4 hours) induce extremely low IL- 10, IL-6, and TNF-a production in human primary monocytes, but its effect on these cytokines was tested in the presence of LPS. Human monocytes were incubated with quinine alone (25pM), hemin alone (25pM), and hemin (25piM) pre-mixed with different concentrations of quinine (3-25|JM) for 3 hours in the presence of LPS. The levels of IL-1 , TNF-a, and IL-6 in culture medium were assessed (Fig 7A). To induce MCP-1 production, human monocytes were incubated with quinine alone (5pM), hemin alone (5pM), and hemin (5pM) pre-mixed with different concentrations of quinine (1 .25-5|JM) for 16 hours in the absence of LPS (Fig 7B). The data show that pre-mixing hemin with quinine leads to a dose-dependent inhibition of IL-10, TNF-a, IL-6, and MCP-1 production. Similarly, we treated human monocyte with RBC lysate (final free hemoglobin concentration: 2g / L) with different dose of quinine. The result shows that quinine suppressed IL-10, TNF-a and IL-6 production induced by RBC lysate. MCP-1 is excluded because RBC lysate can induce MCP-1 production when using the same protocol as in free hemin. Notably, quinine alone have not effect in the absence of free hemin or RBC lysate, and several cytokine levels were reduced even below the basal level (dashed line), suggesting that quinine-hemin complex not only neutralizes the proinflammatory effects of free hemin and RBC lysate, but also exhibits intrinsic anti-inflammatory effects. These findings indicate that quinine exerts potent anti-inflammatory activity in the presence of free hemin and RBC lysate.Low-dose of quinine inhibits inflammatory cytokine production in SCD mice at baseline and during VOE

[0063] To examine in vivo efficacy of quinine, we administered normal or quinine- supplemented drinking water to control and SCD mice with for one week and measured plasma inflammatory cytokine levels. SCD mice exhibited elevated basal plasma levels of IL- 10, TNF-a, and IL-6 compared to control mice. Quinine treatment significantly reduced these cytokines in SCD mice (Fig8.A). The administered quinine dose (~2.5mg / kg) is substantiallylower than 20mg / kg dose used for malaria treatment [ref], having no effect on water intake of mice (Fig8.B). Given the relatively low baseline cytokine levels in SCD mice, we further tested quinine’s effect in VOE model induced by TNF-a injection [ref] . Control and SCD mice received normal or quinine-supplemented drinking water for one week prior to inducing VOE using TNF- a (I.P. injection, 500ng / mice) (FigS.C). While TNF-a triggered robust cytokine release in both control and SCD mice, quinine selectively inhibited MCP-1 , TNF-a, and IL-1 p production only in SCD mice (Fig.8D), suggesting a disease-specific anti-inflammatory effect. In SCD mice, but not in controls, TNF-a administration induced vascular occlusion in liver (Fig8.F, pointed out by red arrows), resulting in liver damage and elevated plasma ALT / AST levels (Fig8. E). Quinine treatment significantly attenuated ALT / AST level increase in SCD mice, highlighting its protective effect against VOC-associated tissue damage. In summary, our data support that quinine disease-specifically inhibit chronic inflammation at baseline and acute inflammation during VOE in SCD mice.Therefore, Figure 7 shows how quinine alone inhibits chronic inflammation in SCD mice at baseline, while it has no effect in control / healthy mice.Therefore, Figure 8 shows how quinine alone suppresses acute inflammation during vascular occlusion episode (VOE) in SCD, while it has no effect in control / healthy mice.

[0064] These results indicate that quinine alone can also have a therapeutic effect in other hemolysis-associated diseases.

[0065] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” As used herein the terms "about" and “approximately” means within 10 to 15%, preferably within 5 to 10%. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0066] The terms “a,” “an,” “the” and similar referents used in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0067] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[0068] Certain embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations on these described embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventor expects skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0069] Specific embodiments disclosed herein may be further limited in the claims using consisting of or consisting essentially of language. When used in the claims, whether as filed or added per amendment, the transition term “consisting of’ excludes any element, step, or ingredient not specified in the claims. The transition term “consisting essentially of’ limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s). Embodiments of the invention so claimed are inherently or expressly described and enabled herein.

[0070] Furthermore, numerous references have been made to patents and printed publications throughout this specification. Each of the above-cited references and printed publications are individually incorporated herein by reference in their entirety.

[0071] In closing, it is to be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the present invention. Other modifications that may be employed are within the scope of the invention. Thus, by way of example, but not of limitation, alternative configurations of the present invention may be utilized in accordance with the teachings herein. Accordingly, the present invention is not limited to that precisely as shown and described.

Claims

We claim:

1. A method of treating a hemolysis-associated disease or inflammasome- associated disease, comprising administering to a patient in need thereof a therapeutically effective dose of a hemolysis inhibitor and a therapeutically effective dose of an inflammasome inhibitor.

2. The method of claim 1 , wherein the hemolysis-associated disease is sickle cell disease.

3. The method of claim 1 , wherein the inflammasome-associated disease is an infectious disease, autoimmune disease, cancer, metabolic disorder, or sickle cell disease.

4. The method of any one of claims 1-3, wherein the patient exhibits hemolysis.

5. The method of any one of claims 1-4 wherein the hemolysis inhibitor is selected from inhibitors of HbS polymerization, potentiators of HbS oxygen affinity, and potentiators of RBC ATP levels.

6. The method of claim 5, wherein the inhibitor of HbS polymerization is one or more of hydroxyurea, tetrahydrouridine, decitabine, and pociredir.

7. The method of claim 5, wherein the potentiator of HbS oxygen affinity is voxelotor.

8. The method of claim 5, wherein the potentiator of RBC ATP levels is one or more of mitapivat or etavopivat.

9. The method of any one of claims 1-4, wherein the inflammasome inhibitor is one or more of interleukin-1 receptor agonist (IL-1 RA), a spleen tyrosine kinase (SYK) inhibitor, epeleuton, flurbiprofen, MCC950, anti-IL-1 p antibody, and the combination of hemin and quinine.

10. The method of claim 9, wherein the SYK inhibitor is one or more of ostamatinib, entospletinib, lanraplenib, and sovleplenib.11 . The method of claim 9, wherein the inflammasome inhibitor is the combination of quinine and hemin.

12. The method of claim 11 , wherein the quinine is a quinine salt.

13. The method of claim 11 , wherein the quinine is quinine free base14. The method of claim 11 , wherein the quinine is a quinine derivative selected from quinacrine, biquinolone, chloroquine, hydroxychloroquine, amodiaquine, quinine, quinidine, mefloquine, primaquine, lumefantrine, and halofantrine.

15. The method of any one of claims 1-4, wherein the inflammasome inhibitor is quinine alone.

16. The method of claim 15, wherein the quinine is a quinine salt.

17. The method of claim 15, wherein the quinine is quinine free base.

18. The method of claim 9, wherein the inflammasome inhibitor is a quinine derivative alone, selected from quinacrine, biquinolone, chloroquine, hydroxychloroquine, amodiaquine, quinine, quinidine, mefloquine, primaquine, lumefantrine, and halofantrine.

19. A method of treating a hemolysis-associated disease, comprising administering to a patient exhibiting hemolysis a therapeutically effective dose of an inflammasome inhibitor.

20. A method of treating a hemolysis-associated disease, comprising(a) identifying a patient exhibiting hemolysis; and(b) administering to the patient a therapeutically effective dose of an inflammasome inhibitor.21 . The method of claim 1 , wherein the method is a method of treating a hemolysis-associated disease..

22. The method of claim 1 , wherein the method is a method of treating an inflammasome-associated disease

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