WO2025032465A1 - Use of gardos channel inhibitors in the treatment of sickle cell disease - Google Patents

Abstract

Provided herein are methods of treating sickle cell disease, methods of increasing hemoglobin levels in a subject diagnosed with sickle cell disease, and methods of reducing hemolysis-associated complications in a subject diagnosed with sickle cell disease, in a subject in need thereof, comprising administering a Gardos channel inhibitor such as 2,2- bis(4-fluorophenyl)-2-phenylacetamide to the subject. Subjects eligible for treatment include subjects as having hemolysis dominant (HD) sickle cell disease on the basis of laboratory markers, clinical history, and/or sequelae.

Description

USE OF GARDOS CHANNEL INHIBITORS IN THE TREATMENT OF SICKLE CELL DISEASE CROSS-REFERENCES TO RELATED APPLICATIONS [0001] This application claims the benefit of priority to U.S. Provisional Patent Application No.63,530,887, filed August 4, 2023; and U.S. Provisional Patent Application No. 63/627,319, filed January 31, 2024, each of which are incorporated by reference herein in their entirety. TECHNICAL FIELD [0002] The following relates generally to methods of treatment for Sickle cell disease (SCD) comprising the administration of a Gardos channel inhibitor, in one embodiment 2,2-bis(4- fluorophenyl)-2-phenylacetamide, to subjects of a defined patient population or theratype of SCD. In one embodiment, subjects contained within said population or theratype are characterized by and/or identified through clinical history, laboratory markers, genotype, or a combination thereof. In one embodiment subjects of a defined hemolytic dominant (HD) SCD theratype are treated with the administration of a Gardos channel inhibitor such as 2,2- bis(4- fluorophenyl)-2-phenylacetamide. BACKGROUND [0003] Sickle cell disease (SCD) refers to a group of inherited red blood cell disorders characterized by the presence of abnormal hemoglobin S (HbS). SCD is among the most common monogenic diseases worldwide, with an estimated 300,000 infants born globally each year with severe homozygous SCA (HbSS disease), the most prevalent and severe form of SCD. More than half of SCA births occur in sub-Saharan Africa, in particular in West and Central Africa where carrier frequencies approach 25% in some regions. SCA is also common in India, the Middle East, and Southern European countries. In the United States, SCA affects an estimated 100,000 individuals. [0004] For nearly a century after its initial description in 1910, the management of SCD was largely supportive, focusing on symptomatic relief and prevention of infections. Hydroxyurea, approved by the US Food and Drug Administration in 1998, was the first disease- modifying therapy shown to reduce certain SCD complications via fetal hemoglobin (HbF) induction. Due to its efficacy, hydroxyurea is now recommended first-line for most SCD patients. However, major barriers persist in access and adherence to hydroxyurea therapy worldwide, and despite ongoing scientific advancements, most individuals with SCD worldwide still receive inadequate care. The vast majority of those affected reside in low- resource countries in sub-Saharan Africa and India where early childhood mortality with SCD remains as high as 50-90%. Even in high-income nations like the United States, significant disparities exist in life expectancy, complication rates, and access to specialized treatments compared to the general population. [0005] Though additional therapeutics have been brought to market for SCD, these therapies are very expensive, and require significant infrastructure to manufacture and distribute. By way of example only, monoclonal antibodies and gene therapies can cost anywhere from $100,000 per year, to several million for potentially curative treatment, and require highly specialized manufacturing and treatment infrastructure. Moreover, market penetration of these drugs has remained modest. Other potential therapeutics developed for SCD, including 2,2-bis(4- fluorophenyl)-2-phenylacetamide, have been unable to achieve regulatory approval due to failures in the clinical setting. There remains a very strong need for safe, scalable, and accessible treatments to manage long-term SCD. Additionally, there remains a need for therapies that are accessible to parts of the world most affected by SCD (namely sub- Saharan Africa), which reduce the prohibitive cost and requisite infrastructure requirements of recent therapies. SUMMARY [0006] In one broad aspect, there is provided a use of a Gardos channel inhibitor in the treatment of a specific subpopulation of subjects with sickle cell disease. [0007] In another aspect, provided is a use of a Gardos channel inhibitor in the treatment of sickle cell disease in a specific subpopulation of subjects wherein the specific subpopulation of subjects has hemolysis dominant (HD) sickle cell disease. In some embodiments, subjects are characterized as having HD sickle cell disease on the basis of clinical history. In some embodiments, subjects are characterized as having HD sickle cell disease on the basis of clinical history and/or laboratory markers. In some embodiments, subjects are characterized as having HD sickle cell disease on the basis of clinical history, laboratory markers, and/or sequelae. In some embodiments, subjects are characterized as having hemolysis dominant (HD) sickle cell disease on the basis of laboratory markers, clinical history, and/or sequelae, and are differentially associated with distinct genetic backgrounds and ancestries. [0008] In another aspect, provided is a method of treating sickle cell disease in a subject in need thereof, comprising administering a Gardos channel inhibitor to the subject, wherein the subject has experienced fewer than 4 vaso-occlusive pain crises (VOCs) in the preceding 24 months. [0009] In another aspect, provided is a method of increasing hemoglobin levels in a subject diagnosed with sickle cell disease, comprising administering a Gardos channel inhibitor to the subject, wherein the subject has experienced fewer than 4 vaso-occlusive pain crises (VOCs) in the preceding 24 months. [0010] In another aspect, provided is a method of reducing hemolysis-associated complications in a subject diagnosed with sickle cell disease, comprising administering a Gardos channel inhibitor to the subject, wherein the subject has experienced fewer than 4 vaso-occlusive pain crises (VOCs) in the preceding 24 months. In some embodiments, the hemolysis-associated complications are selected from elevated tricuspid regurgitant jet velocity, glomerulopathy, renal disease, hepatobiliary disease, lower extremity ulceration, priapism, infarctive stroke, or heart disease. In some embodiments, the hepatobiliary disease is cholelithiasis. In some embodiments, the hemolysis-associated complications are selected from pulmonary hypertension, glomerulopathy, renal disease, cholelithiasis, lower extremity ulceration, priapism, infarctive stroke, or heart disease. [0011] In another aspect, provided is a method of aiding in treatment of a subject with sickle cell disease, comprising administering a Gardos channel inhibitor to the subject, wherein the subject has experienced fewer than 4 vaso-occlusive pain crises (VOCs) in the preceding 24 months. [0012] In another aspect, there is provided a use of a Gardos channel inhibitor in the treatment of sickle cell disease in a specific subpopulation of subjects wherein the Gardos channel inhibitor is 2,2-bis(4-fluorophenyl)-2-phenylacetamide, which has the structure: . BRIEF D DRAWINGS [0013] The features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings. [0014] FIG.1 illustrates a Uniform Manifold Approximation and Projection (UMAP) of genome wide expression data from patients with Sickle Cell Disease who rank in the upper- and lower-quartiles for HMOX1 expression (a key driver of hemolysis). Shapes represent the results of leiden clustering performed on the gene expression data; samples of the same shape have similar gene expression profiles. [0015] FIG.2 illustrates a UMAP of genome wide expression data from patients with Sickle Cell Disease who rank in the upper- and lower-quartiles for HMOX1 expression (a key driver of hemolysis). Shape represents the membership of each sample into either the upper-quartile HMOX1 expression group or the lower-quartile HMOX1 expression group. [0016] FIG.3 illustrates Z-Scores of drug targets among approved and experimental SCD agents as a measure of statistical proximity to a HD pathway (RBC density) of SCD, wherein target 8 is the Gardos channel. DETAILED DESCRIPTION [0017] The invention described herein is directed to, inter alia, identification of a defined patient population (or theratype) of Sickle cell disease (SCD) that would benefit from treatment. In addition, the invention is directed to treating and/or aiding in the treatment of SCD for this defined patient population (or theratype) of SCD. The treatment includes administering one or more Gardos channel inhibitors, such as 2,2-bis(4- fluorophenyl)-2- phenylacetamide, to subjects of a defined patient population or theratype of SCD. In one embodiment, subjects contained within the population or theratype are characterized by and/or identified through clinical history, laboratory markers, genotype, or a combination thereof. In some embodiments, the subject has a defined hemolytic dominant (HD) SCD theratype, wherein patients with a clinical history of less than 4 vaso-occlusive crises over the preceding 24 months are deemed to belong to a predominantly hemolytic phenotype. In one embodiment, subjects of a defined hemolytic dominant (HD) SCD theratype are treated with the administration of a Gardos channel inhibitor such as 2,2-bis(4-fluorophenyl)-2- phenylacetamide. [0018] SCD is caused by a single nucleotide polymorphism (SNP) in the β-globin gene (HBB) on chromosome 11. This SNP leads to the amino acid substitution of valine for glutamic acid at position 6 of the β-globin chain, producing HbS. The hydrophobic valine residue promotes polymerization of deoxygenated HbS into rigid intracellular fibers that distort red blood cells into the characteristic sickled shape. These abnormally shaped red blood cells obstruct blood flow causing tissue ischemia and infarction in multiple organs. Recurrent sickling and hemolysis coupled with chronic inflammation and dysregulated cellular interactions underlie the pathophysiology of SCD. [0019] The diverse clinical spectrum of SCD correlates with the degree of anemia and hemolysis. Some individuals exhibit a relatively milder disease course, while others suffer severe, recurrent complications requiring frequent hospitalizations. Common complications include episodic vaso-occlusive pain crises, acute chest syndrome, stroke, avascular necrosis, nephropathy, retinopathy, pulmonary hypertension, and life-threatening infections due to functional asplenia. Despite recent therapeutic advancements, the average life expectancy of individuals with SCD remains approximately 20 years lower than the general population. Critically, though the clinical manifestations of SCD are highly variable, therapeutic programs have historically focused on unstratified SCD populations. Given the lifelong nature of SCD, precision medicine approaches such as genetic testing to identify theratypes, or disease subgroups with differential therapeutic responses, are a worthwhile investment. The upfront costs of diagnostic screening will likely be offset by improved clinical outcomes and reduced treatment expenses over a patient's lifetime. [0020] The Gardos channel, encoded by the KCNN4 gene, is a calcium-activated potassium channel highly expressed in human erythrocytes. When intracellular calcium levels rise, the Gardos channel opens allowing efflux of potassium and water loss, leading to cellular dehydration. In SCD, the Gardos channel is pathologically overactivated. Intracellular hemoglobin S polymerization causes recurrent membrane damage, allowing calcium influx which triggers the channel to open. This leads to a cycle of cellular dehydration, increasing hemoglobin concentration and further sickling. The dehydrated, dense sickle erythrocytes have exaggerated adhesive properties and are prone to get trapped in small vessels. These poorly deformable cells play a central role in initiating vaso-occlusion and downstream ischemia-reperfusion injury. Inhibitors of the Gardos channel can block potassium and water loss from erythrocytes, reducing the number of dehydrated dense cells and decreases intracellular hemoglobin S concentration. Lowering hemoglobin S levels delays the kinetics of polymerization, reducing RBC sickling. The improved hydration and rheology of Gardos channel inhibitor-effected erythrocytes could therefore decrease intravascular hemolysis. [0021] 2,2-bis(4-fluorophenyl)-2-phenylacetamide, also known formally as 4-fluoro-α-(4- fluorophenyl)-α-phenyl-benzeneacetamide, as 1S/C20H15F2NO/c21-17-10-6-15(7-11- 17)20(19(23)24,14-4-2-1-3-5-14)16-8-12-18(22)13-9-16/h1-13H,(H2,23,24), by its molecular formula C20H15F2NO, also known as senicapoc, is a potent Gardos channel inhibitor that has previously been studied for treatment of SCD. As discussed in greater detail herein, the compound was studied in Phase 2 and Phase 3 clinical trials for the treatment of SCD but was ultimately not approved for use in humans by the FDA or any other regulatory body due to its failure to meet clinical endpoints in a Phase 3 trial. Given that in the pharmaceutical industry, any commercial success must first be predicated on regulatory approval, the compound has hitherto not displayed any commercial success. [0022] The present disclosure pertains to identification of a defined patient population (or theratype) of SCD that would benefit from treatment as well as treatment and aiding in the treatment of defined subgroups of patients who may be effectively treated for sickle-cell disease by administering a therapeutically effective amount of one or more Gardos channel inhibitors, such as 2,2-bis(4-fluorophenyl)-2-phenylacetamide, in spite of previous failures of this drug in the clinic. Embodiments herein disclosed may be shown to be efficacious in the selected patient population, and would therefore be approved by regulatory bodies, and thus be able to demonstrate commercial success where none has existed prior. In particular, the present disclosure pertains to the treatment of a theratype (hemolysis dominant sickle cell disease) characterized by increased intravascular hemolysis, endothelial dysfunction, and inflammation, and decreased vaso-occlusion, with a Gardos channel inhibitor, e.g., 2,2-bis(4- fluorophenyl)-2-phenylacetamide. In this theratype, 2,2- bis(4-fluorophenyl)-2- phenylacetamide may show improved treatment response by ameliorating hemolysis- associated complications. Moreover, this compound, being a scalable small molecule, can immediately ameliorate the standard of care in parts of the world most impacted by SCD in which other therapies are poorly accessible. Definitions [0023] Unless otherwise defined, all terms of art, notations, and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this application pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. Many of the techniques and procedures described or referenced herein are well understood and commonly employed using conventional methodology by those skilled in the art. [0024] The singular form “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. “A and/or B” is used herein to include all of the following alternatives: “A”, “B”, “A or B”, and “A and B”. [0025] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure. [0026] Certain ranges are presented herein with numerical values being preceded by the term “about.” The term “about” is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number. If the degree of approximation is not otherwise clear from the context, “about” means either within plus or minus 10% of the provided value, or rounded to the nearest significant figure, in all cases inclusive of the provided value. In some embodiments, the term “about” indicates the designated value ± up to 10%, up to ± 5%, or up to ± 1%. [0027] As used herein, a “subject” or an “individual” includes animals, such as human (e.g., human individuals) and non-human animals. In some embodiments, a “subject” or “individual” is a patient under the care of a physician. Thus, the subject can be a human patient or an individual who has, is at risk of having, or is suspected of having a health condition of interest and/or one or more symptoms of the health condition. The subject can also be an individual who is diagnosed with a risk of the health condition of interest at the time of diagnosis or later. The term “non-human animals” includes all vertebrates, e.g., mammals, e.g., rodents, e.g., mice, non-human primates, and other mammals, such as e.g., sheep, dogs, cows, chickens, and non-mammals, such as amphibians, reptiles, etc. [0028] As used herein, the terms “administration” and “administering” refer to the delivery of a compound or composition by an administration route including, but not limited to, oral, intravenous, intra-arterial, intramuscular, intraperitoneal, subcutaneous, intramuscular, and topical administration, or combinations thereof. The term includes, but is not limited to, administering by a medical professional and self-administering. [0029] As used herein, and unless otherwise specified, different “theratypes” refer to defined sub-populations of subjects, with variation in disease sequelae and therapy response. [0030] As used herein, and unless otherwise specified, a “therapeutically effective” or “pharmaceutically effective” amount of a compound or composition of the disclosure generally refer to an amount or number sufficient for a compound or composition to accomplish a stated purpose relative to the absence of the composition, e.g., to provide a therapeutic benefit in the treatment or management of the SCD, or to delay or minimize one or more symptoms associated with the SCD. A therapeutically effective amount of a compound means an amount of therapeutic agent, alone or in combination with other therapeutic agents, which provides a therapeutic benefit in the treatment or management of the SCD. The term “therapeutically effective amount” can encompass an amount that improves overall therapy, reduces or avoids symptoms or causes of the SCD, or enhances the therapeutic efficacy of another therapeutic agent. An example of an “effective amount” is an amount sufficient to contribute to the treatment, prevention, or reduction of a symptom or symptoms of a disease, which could also be referred to as a “therapeutically effective amount.” A “reduction” of a symptom means decreasing of the severity or frequency of the symptom(s), or elimination of the symptom(s). The exact amount of a composition including a “therapeutically effective amount” will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols.1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins). 2,2-Bis(4-fluorophenyl)-2-phenylacetamide [0031] Preclinical studies demonstrated 2,2-bis(4-fluorophenyl)-2-phenylacetamide acts as a potent Gardos channel inhibitor, preventing pathological erythrocyte dehydration and sickling in vitro and in SAD transgenic mice. In cultured human sickle red blood cells, 2,2-bis(4- fluorophenyl)-2-phenylacetamide inhibited Gardos channel activity with an IC50 of 12 nM and reduced formation of dense, dehydrated cells. Oral 2,2-bis(4-fluorophenyl)-2- phenylacetamide treatment of SAD mice for 21 days significantly improved hematological parameters including decreased mean corpuscular hemoglobin concentration and increased hematocrit, indicating reduced red cell dehydration. However, 2,2-bis(4-fluorophenyl)-2- phenylacetamide failed to meet its endpoints in clinical trials and was never approved for use. [0032] A phase II randomized trial evaluated 2,2-bis(4-fluorophenyl)-2- phenylacetamide in 169 SCD patients over 12 weeks. Those treated with 10 mg/day 2,2-bis(4- fluorophenyl)-2- phenylacetamide exhibited a significant 0.68 g/dL increase in hemoglobin and reductions in hemolysis markers like reticulocyte count, bilirubin, and LDH compared to placebo. This indicated 2,2-bis(4-fluorophenyl)-2-phenylacetamide decreased sickling and improved erythrocyte survival in SCD patients as hypothesized based on preclinical data. [0033] However, the phase III trial in 297 SCD patients failed to show a reduction in vaso- occlusive crises over 48 weeks with 2,2-bis(4-fluorophenyl)-2-phenylacetamide 10 mg/day compared to placebo. In fact, a statistically significant increase in the crisis rate was reported in patients receiving senicapoc and not concomitantly taking hydroxycarbamide. Post-hoc analysis identified a subset of “hemoglobin responders” with increased hemoglobin on 2,2- bis(4-fluorophenyl)-2-phenylacetamide, but this did not correlate with fewer crises. As a result, further clinical development of 2,2-bis(4-fluorophenyl)-2- phenylacetamide for SCD was discontinued after the phase III trial. SCD Theratypes [0034] Although sickle-cell disease has been described as including two sub-phenotypes including a hemolytic phenotype and a vaso-occlusive phenotype, these sub-phenotypes of clinical manifestation are known to be overlapping, and as yet there is no clear methodology to disaggregate the two sub-phenotypes, or to identify distinct patient populations for differential treatment. In this disclosure, we characterize two theratypes of SCD: the Non- hemolysis dominant (NHD) theratype, and the hemolysis dominant theratype (HD). These theratypes may be simultaneously manifested with varying dominance across patient populations and individual patients. These theratypes may be differentiated on the basis of laboratory markers, clinical history, and/or sequelae, and are differentially associated with distinct genetic backgrounds and ancestries. [0035] Herein, gene-expression data was obtained from SCD patients and was subjected to principal component analysis and unsupervised clustering to assess i) if the disease population stratified into distinct transcriptional profiles, and ii) if these profiles separated by transcriptional signature for hemolysis. The results of these experiments (shown in FIG.1 and FIG.2), indicated that the proposed theratypes were emergent from distinct transcriptional signatures. Gene-target based ranking of drug candidates was then performed for each respective theratype. Sequelae [0036] The NHD theratype is characterized by sequelae that includes vaso-occlusive pain crises, acute chest syndrome, splenic infarct, renal failure, avascular necrosis, acute renal injury, and hand foot syndrome. Emphasis however should be placed on vaso-occlusive pain crises, acute chest syndrome, avascular necrosis, and in particular vaso-occlusive pain crises, the reduction of which constituted the primary end point in historic 2,2-bis(4-fluorophenyl)- 2-phenylacetamide trials. [0037] The HD theratype by contrast is characterized by lower extremity ulceration, elevated tricuspid regurgitant jet velocity, glomerulopathy, renal disease, priapism, hepatobiliary disease, infarctive stroke, heart failure or heart disease, and/or acute anemia. In embodiments, the HD theratype is characterized by lower extremity ulceration such as leg ulcers, pulmonary hypertension, glomerulopathy, renal disease, priapism, cholelithiasis, infarctive stroke, heart failure or heart disease, and acute anemia. Laboratory Markers [0038] Lactate dehydrogenase (LDH) is an intracellular enzyme involved in anaerobic glycolysis that catalyzes the interconversion of pyruvate and lactate. In sickle cell disease (SCD), intravascular hemolysis releases LDH into the plasma. Markedly elevated plasma LDH activity serves as a biomarker of accelerated erythrocyte destruction and is associated with phenotypes of SCD at high risk for early mortality and portends greater disease severity. Hyper- elevated LDH levels are indicative of the HD theratype. In some embodiments, the HD theratype is characterized by a plasma lactate dehydrogenase (LDH) level of 408 U/L or greater prior to treatment with a Gardos channel inhibitor. [0039] Hemoglobin is a heterotetrametric hemeprotein present within erythrocytes that serves as the major oxygen transporter in the body. Each hemoglobin molecule contains four globin chains, each associated with an iron-containing heme group. Binding of oxygen to ferrous iron in the heme moiety enables hemoglobin to carry oxygen from the lungs to peripheral tissues. In SCD, a single amino acid substitution in the β-globin chain causes polymerization of deoxygenated hemoglobin S, promoting erythrocyte rigidification and fragility. Intravascular hemolysis coupled with shortened erythrocyte lifespan leads to anemia. Quantifying blood hemoglobin concentration provides an index of the oxygen carrying capacity of the blood. [0040] Typically, hemoglobin levels in people with SCD range between 6 and 8 grams per deciliter (g/dL), which is significantly lower than the normal range (approximately 13.5-17.5 g/dL for men and 12.0-15.5 g/dL for women). This reduction in hemoglobin translates to decreased arterial oxygen content, which can impair oxygen delivery to vital organs. Low hemoglobin levels are associated with fatigue, dyspnea, and other sequelae resulting from tissue hypoxia. Furthermore, severe anemia exacerbates the complications of SCD by heightening circulatory demands leading to high-output and left ventricular heart failure. Significantly reduced hemoglobin levels are indicative of the HD theratype in SCD. In some embodiments, the HD theratype is characterized by a hemoglobin level of less than about 9.2 g/dL prior to treatment with a Gardos channel inhibitor. [0041] Bilirubin is a tetrapyrrole pigment produced from heme catabolism. During physiological erythrocyte turnover and intravascular hemolysis in sickle cell disease, heme oxygenase cleaves the heme ring, generating equimolar quantities of biliverdin, carbon monoxide, and iron. Biliverdin reductase subsequently converts biliverdin into bilirubin. Indirect bilirubin binds to albumin and is transported to the liver, where it is conjugated to glucuronic acid for biliary excretion. Hyperbilirubinemia is common in SCD due to accelerated erythrocyte hemolysis coupled with hepatic uridine diphosphate- glucuronosyltransferase deficiency, which impairs bilirubin conjugation. Serum levels of unconjugated and indirect- reacting bilirubin are thereby elevated. High total serum indirect bilirubin levels are indicative of clinically significant hemolysis. Elevated total indirect bilirubin is a laboratory marker of the HD theratype and is linked to greater disease severity. In some embodiments, the HD theratype is characterized by a total serum indirect bilirubin level of 45 μmol/L or greater prior to treatment with a Gardos channel inhibitor. [0042] Reticulocytes are immature erythrocytes containing residual RNA that are released from the bone marrow into circulation. In healthy individuals, reticulocytes comprise a small percentage of the total red blood cell mass. In SCD chronic hemolysis of rigidified, sickle- shaped erythrocytes elicits a compensatory response wherein the bone marrow increases erythropoietic activity. This manifests as reticulocytosis, defined as elevation of the reticulocyte count above the standard reference range. [0043] Quantification of circulating reticulocytes provides an indirect measure of the erythropoietic rate and allows assessment of the bone marrow’s compensatory capacity in response to chronic hemolysis. Reticulocytosis is nearly universal in SCD. The degree of reticulocytosis positively correlates with the hemolytic rate. Significant reticulocytosis signifies hyper-hemolysis and correlates to the HD theratype. In some embodiments, the HD theratype is characterized by a reticulocyte count of 11% or greater prior to treatment with a Gardos channel inhibitor. [0044] Hemolytic indices are composite scores derived from multiple biomarkers including bilirubin, LDH, and AST that provide an integrated measure of hemolysis and are positively correlated with hemolytic complications of SCD including vasculopathies among others. [0045] Although LDH, bilirubin and AST counts can indicate increased hemolysis, they can lack specificity when used alone. The hemolytic index combines these variables to better quantify ongoing hemolysis. An elevated hemolytic index can more accurately identify SCD patients at high risk for complications and early mortality compared to individual hemolysis markers. In one study, a high hemolytic index predicted a significantly increased risk of death at 2 years. The index circumvents the poor specificity of LDH, bilirubin or AST alone, providing a more discerning biomarker of clinically significant hemolysis, and correlates to the HD sub- theratype of SCD. [0046] Arginase is an erythrocyte-associated manganese metalloenzyme that catalyzes the hydrolysis of L-arginine to ornithine and urea. In SCD, intravascular hemolysis releases arginase into plasma. Elevated plasma arginase depletes circulating L-arginine, the substrate for endothelial nitric oxide synthase. This impairs nitric oxide generation, promoting endothelial dysfunction. Thus, increased plasma arginase reflects hemolysis and serves as an indirect biomarker of the HD theratype. In some embodiments, the HD theratype is characterized by a plasma arginase activity of 2.6 μmol/mL/hr or greater. [0047] Increased systolic blood pressure is another laboratory indicator that can identify SCD patients at greater risk for stroke and other vascular complications. Higher systolic blood pressure elevates shear stress on the vascular endothelium, amplifying sickle erythrocyte adhesion and vascular occlusion. Through this mechanism, elevated systolic blood pressure may exacerbate the vasculopathy associated with the HD theratype. Subjects meeting HD laboratory criteria with increased systolic blood pressure may derive particular benefit from therapies that improve nitric oxide bioavailability and inhibit aberrant sickle red blood cell adhesion, in one embodiment, 2,2-bis(4-fluorophenyl)-2-phenylacetamide. In some embodiments, the HD theratype is characterized by a systolic blood pressure of 120 or greater prior to treatment with a Gardos channel inhibitor. [0048] The foregoing list of laboratory markers is not intended to be comprehensive, nor intended to be analyzed in strict terms of binary or temporal thresholds. For instance, a history of acute anemic events in patients may also be an indicator of HD. Despite having high levels of hemoglobin at a point in time, a patient with history of acute anemic events could still be eligible for 2,2-bis(4- fluorophenyl)-2-phenylacetamide. In other words, foregoing list of laboratory markers is not intended to exclude patients whose ranges do not contemporaneously reflect high hemolysis, if they have a history of acute anemic events punctuating otherwise stable hemoglobin levels. For instance, a history of hospitalization or transfusion or intermittently severe anemia can contribute to theratype inclusion, regardless of hemoglobin levels at any single point in time. Stated otherwise, the totality of clinical history including historical laboratory markers informs theratype inclusion rather than reliance solely on most recent values. Genotype [0049] Various genotypes may be associated with the two disclosed theratypes. This is summarized in Table 2 below. [0050] Genetic mutations associated with SCD may be further associated with various sequelae, and thus disease theratype. [0051] Pulmonary hypertension is characteristic of the HD theratype. Pulmonary hypertension is impacted by the TGF-β/BMP pathway genes, with individual gene Single Nucleotide Polymorphisms (SNPs) being associated with greater preponderance for pulmonary hypertension. [0052] The ACVRL1 gene encodes for a receptor in the TGF-β signaling pathway and is implicated with a higher risk for primary and sickle cell disease-associated pulmonary hypertension. The effect of the mutation is to disrupt TGF-β signaling, leading to abnormal vascular inflammation and endothelial cell dysfunction. Polymorphisms rs3759178, rs3847859, and rs706814 are associated with pulmonary hypertension risk. [0053] The BMPR2 gene encodes for a TGF-β receptor, and has mutations linked to familial pulmonary arterial hypertension, and is associated with pulmonary hypertension risk in sickle cell disease. Altered TGF-β signaling affects vascular inflammation, with polymorphisms rs17199249 and rs35711585 nominally associated with pulmonary hypertension risk. [0054] The BMP6 gene is a member of the TGF- β superfamily and regulates inflammation. BMP6 is strongly implicated in pulmonary hypertension susceptibility by disrupting leukocyte differentiation and function. Polymorphisms rs267192, rs267196, or rs267201 are significantly associated with pulmonary hypertension risk. As pulmonary hypertension correlates to the HD theratype in SCD, inferences respecting these mutations are made to inform treatment. [0055] In some embodiments, subjects having an rs3759178, rs3847859, or rs706814 polymorphism in the ACVRL1 gene; or polymorphism rs17199249 or rs35711585 on the BMPR2 gene; or polymorphism rs267192, rs267196, or rs267201 on the BMP6 gene, or a combination thereof, meet the inclusion criteria for treatment with 2,2-bis(4-fluorophenyl)-2- phenylacetamide. In some embodiments of the methods disclosed herein, the subject has one, two, three, or more polymorphisms selected from the group consisting of: rs3759178, rs3847859, rs706814 (in the ACVRL1 gene); rs17199249, rs35711585 (in the BMPR2 gene); rs267192, rs267196, and rs267201 (in the BMP6 gene). [0056] Conversely Acute Chest Syndrome (ACS) has been associated with the NHD theratype in SCD. The NOS3 gene encodes endothelial nitric oxide synthase (eNOS), which regulates nitric oxide (NO) levels. The NOS3 T-786C polymorphism in the promoter region reduces eNOS expression. Female SCD patients with the TC/CC genotype had significantly increased risk of developing ACS compared to TT homozygotes. In contrast, the T- 786C variant did not affect ACS risk in male SCD patients. This suggests T-786C is a gender- specific genetic modifier for ACS susceptibility in females with SCD. The NOS3 T-786C polymorphism is thus associated with reduced NO levels and increased ACS risk specifically in female SCD patients. [0057] In some embodiments, female subjects having the NOS3 T-786C polymorphism are excluded from treatment with 2,2-bis(4-fluorophenyl)-2-phenylacetamide, due to the genotype’s correlation with ACS, a sequela of the NHD theratype. [0058] Embodiments of the invention disclosed herein describe a method of identify SCD subjects with the HD theratype for targeted treatment with a Gardos channel inhibitor to reduce laboratory markers of hemolysis and hemolytic-associated complications, i.e., leg ulcers, priapism, stroke, biliary pathology, etc. In some embodiments, HD subjects are identified by clinical history, laboratory markers, and/or genotypes. In some embodiments these features are used autonomously to differentiate HD from non-HD theratypes, and in others are aggregated into a composite clinical score. [0059] Embodiments disclosed herein pertain to the administration of a Gardos channel inhibitor to hemolysis dominant (HD) SCD subjects. Combination Treatments for HD Theratype [0060] In some embodiments, the combination of a Gardos channel inhibitor, together with a secondary therapy acting on hemolysis such as a positive allosteric modifier of hemoglobin, is clinically additive in the treatment of patients having the HD theratype. In some embodiments, the combination of a Gardos channel inhibitor, together with a secondary therapy acting on hemolysis such as a positive allosteric modifier of hemoglobin, is clinically synergistic in the treatment of patients having the HD theratype. Positive Allosteric Modifiers [0061] Allosteric modifiers of hemoglobin are molecules that bind to a site on the hemoglobin protein distinct from the oxygen-binding site, leading to a change in the protein's conformation and thereby affecting its affinity for oxygen. Positive allosteric modifiers such as 2-hydroxy-6-[[2-(2-propan-2-ylpyrazol-3-yl)pyridin-3-yl]methoxy]benzaldehyde (also known as voxelotor or Oxbryta) increase hemoglobin’s affinity for oxygen. When hemoglobin has a higher affinity for oxygen, it is more likely to pick up oxygen in the lungs. This can be beneficial in conditions where oxygen uptake is impaired. [0062] The binding of an allosteric modifier can induce a conformational change in hemoglobin that is propagated to the oxygen-binding sites. Hemoglobin is a tetramer consisting of two alpha and two beta subunits, and it has an R-state (relaxed, high affinity for oxygen) and a T-state (tense, low affinity for oxygen). Allosteric modifiers can stabilize either of these states, thereby influencing the overall affinity for oxygen. [0063] In the case of SCD, positive allosteric modifiers of hemoglobin can increase hemoglobin’s affinity for oxygen, thus stabilizing the R-state and reducing the tendency of the deoxygenated sickle hemoglobin (HbS) to polymerize, which is a key event in the pathogenesis of the disease. By reducing this polymerization, these compounds ameliorate some of the symptoms and complications of SCD. [0064] By reducing the rate of hemolysis, positive allosteric modifiers can mitigate some of the complications associated with SCD. Hemolysis leads to anemia, which in turn can cause a host of problems, including fatigue, weakness, and an increased risk for infections and other complications. Combination Therapy using a Gardos Channel Inhibitor and a Positive Allosteric Modifier of Hemoglobin [0065] As Gardos channel inhibitors are not currently approved for therapeutic use in SCD, a beneficial therapeutic effect in combination with a secondary therapeutic agent such as a positive allosteric modifier of hemoglobin, either additive or synergistic, would be significant. [0066] Synergism refers to the dynamic interaction among two or more drugs, resulting in a combined effect that is greater than the sum of their individual effects. Gardos channel inhibition together with a positive allosteric modifier of hemoglobin may act additively or synergistically to address different aspects of the pathophysiology of the HD theratype. Each drug targets a different mechanism that contributes to the severity and complications of the HD theratype, thereby providing a more comprehensive approach to management. [0067] Both Gardos channel inhibitors and the positive allosteric modifiers of hemoglobin act to reduce the rate of HbS polymerization and subsequent sickling. Gardos channel inhibition reduces erythrocyte dehydration and therefore results in lower intracellular HbS concentration; and hemoglobin allosteric modification directly reduces sickling by stabilizing the relaxed oxygenated state. Without being bound by theory, it is believed that lowering the intrinsic polymerization rate of deoxy-HbS through allosteric modification is more effective when the red blood cells are better hydrated (e.g., via Gardos channel inhibition), creating an intracellular environment less conducive to HbS aggregation. Together, these mechanisms may synergistically or additively reduce the rate of HbS polymerization through complementary pathways. By acting synergistically on separate but complementary pathways, these two pharmacological agents could offer a more comprehensive approach to SCD disease management, addressing both the triggers of erythrocyte sickling and the sickling process itself. Methods of Treatment and Aiding in the Treatment [0068] Provided herein are methods of treating of sickle cell disease in a subject in need thereof, by administering a Gardos channel inhibitor to the subject. In some embodiments, the subject has experienced fewer than 4 vaso-occlusive pain crises (VOCs) in the preceding 24 months. In some embodiments, the Gardos channel inhibitor is 2,2-bis(4-fluorophenyl)-2- phenylacetamide, having chemical structure C20H15F2NO and commercial name senicapoc. A therapeutically effective amount of 2,2-bis(4-fluorophenyl)-2- phenylacetamide can used. In some embodiments, the sickle cell disease is sickle cell anemia. In some embodiments, subjects who have experienced 4 or more VOCs in the preceding 24 months are excluded from treatment. In some embodiments, subjects who have experienced 2 or more VOCs in the preceding 12 months are excluded from treatment. [0069] The invention contemplates methods of increasing hemoglobin levels in a subject diagnosed with SCD or suspected to have SCD, by administering a Gardos channel inhibitor to the subject, wherein the subject has experienced fewer than 4 vaso-occlusive pain crises (VOCs) in the preceding 24 months. In some embodiments, the Gardos channel inhibitor is 2,2-bis(4-fluorophenyl)-2- phenylacetamide, having chemical structure C₂₀H₁₅F₂NO and commercial name senicapoc. In some embodiments, the sickle cell disease is sickle cell anemia. In some embodiments, the sickle cell disease is sickle cell anemia. In some embodiments, subjects who have experienced 4 or more VOCs in the preceding 24 months are excluded from treatment. In some embodiments, subjects who have experienced 2 or more VOCs in the preceding 12 months are excluded from treatment. [0070] In some embodiments, provided is a method of reducing hemolysis-associated complications in a subject diagnosed with SCD or suspected to have SCD, by administering a Gardos channel inhibitor to the subject, wherein the subject has experienced fewer than 4 vaso-occlusive pain crises (VOCs) in the preceding 24 months. In some embodiments, the hemolysis-associated complications are selected from elevated tricuspid regurgitant jet velocity, glomerulopathy, renal disease, hepatobiliary disease, lower extremity ulceration, priapism, infarctive stroke, or heart disease. In some embodiments, the hepatobiliary disease is cholelithiasis. In some embodiments, the hemolysis-associated complications are selected from pulmonary hypertension, glomerulopathy, renal disease, cholelithiasis, lower extremity ulceration, priapism, infarctive stroke, or heart disease. In some embodiments, the Gardos channel inhibitor is 2,2-bis(4-fluorophenyl)-2- phenylacetamide, having chemical structure C₂₀H₁₅F₂NO and commercial name senicapoc. In some embodiments, the sickle cell disease is sickle cell anemia. In some embodiments, the sickle cell disease is sickle cell anemia. In some embodiments, subjects who have experienced 4 or more VOCs in the preceding 24 months are excluded from treatment. In some embodiments, subjects who have experienced 2 or more VOCs in the preceding 12 months are excluded from treatment. [0071] In an embodiment, provided is a method of aiding in treatment of a subject with sickle cell disease, by administering a Gardos channel inhibitor to the subject, wherein the subject has experienced fewer than 4 vaso-occlusive pain crises (VOCs) in the preceding 24 months. Aiding in the treatment may encompass activities that one of skill in the art could take to identify a defined patient population for receiving the 2,2-bis(4-fluorophenyl)-2- phenylacetamide. Such activities include determination of the theratype of subjects who would be receiving the 2,2-bis(4-fluorophenyl)-2- phenylacetamide as described herein. Sharing of theratype information for treatment is encompassed as aiding in the treatment. [0072] In some embodiments, the Gardos channel inhibitor is administered orally. In some embodiments, the Gardos channel inhibitor is administered daily. In some embodiments, the Gardos channel inhibitor is administered once or twice daily. In some embodiments, the Gardos channel inhibitor is administered once daily. In some embodiments, the Gardos channel inhibitor is administered twice daily. [0073] In some embodiments, the Gardos channel inhibitor (e.g., 2,2-bis(4-fluorophenyl)-2- phenylacetamide) is administered at a therapeutically effective dose. [0074] Eligibility for the Gardos channel inhibitor, e.g., 2,2-bis(4-fluorophenyl)-2- phenylacetamide, can be determined by one or more laboratory indicators, including but not limited to, hemoglobin levels (reduced), LDH levels (elevated), indirect bilirubin levels (elevated), reticulocyte levels (elevated), plasma arginase (elevated), and systolic blood pressure (elevated) (see Table 1). In some embodiments, the subject has a hemoglobin level of less than about 9.2 g/dL prior to administering the Gardos channel inhibitor. In some embodiments, the subject has a total serum indirect bilirubin level of about 45 μmol/L or greater prior to administering the Gardos channel inhibitor. In some embodiments, the subject has a reticulocyte count of about 11% or greater prior to administering the Gardos channel inhibitor. In some embodiments, the subject has a plasma arginase activity of about 2.6 μmol/mL/hr or greater prior to administering the Gardos channel inhibitor. In some embodiments, the subject has a systolic blood pressure of about 120 or greater prior to administering the Gardos channel inhibitor. In some embodiments of the methods disclosed herein, prior to administering the Gardos channel inhibitor, the subject has one, two, three, four, five, or six of: (a) a hemoglobin level of less than about 9.2 g/dL; (b) a plasma lactate dehydrogenase (LDH) level of about 408 U/L or greater; (c) a total serum indirect bilirubin level of about 45 μmol/L or greater; (d) a reticulocyte count of about 11% or greater; (e) a plasma arginase activity of about 2.6 μmol/mL/hr or greater; and/or (f) a systolic blood pressure of about 120 or greater. [0075] In some embodiments of the methods disclosed herein, prior to administering the Gardos channel inhibitor, the subject has a hemoglobin level of less than about 9.2 g/dL and at least one of: (b) a plasma lactate dehydrogenase (LDH) level of about 408 U/L or greater; (c) a total serum indirect bilirubin level of about 45 μmol/L or greater; (d) a reticulocyte count of about 11% or greater; (e) a plasma arginase activity of about 2.6 μmol/mL/hr or greater; and/or (f) a systolic blood pressure of about 120 or greater. [0076] In some embodiments of the methods disclosed herein, prior to administering the Gardos channel inhibitor, the subject has a systolic blood pressure of about 120 or greater, and at least one of (a) a hemoglobin level of less than about 9.2 g/dL; (b) a plasma lactate dehydrogenase (LDH) level of about 408 U/L or greater; (c) a total serum indirect bilirubin level of about 45 μmol/L or greater; and/or (d) a reticulocyte count of about 11% or greater; and/or (e) a plasma arginase activity of about 2.6 μmol/mL/hr or greater. [0077] In some embodiments of the methods disclosed herein, prior to administering the Gardos channel inhibitor, the subject has a hemoglobin level of less than about 9.2 g/dL and a systolic blood pressure of about 120 or greater. [0078] In some embodiments, the hemoglobin level of less than about 9.2 g/dL is a hemoglobin level of between about 4.5 g/dL and about 9.2 g/dL, between about 4.5 g/dL and about 9 g/dL, between about 6 g/dL and about 9.2 g/dL, between about 6 g/dL and about 9 g/dL, or between about 6 g/dL and about 8 g/dL. [0079] In some embodiments, the LDH level of about 408 U/L or greater is a LDH level of between about 408 U/L and about 1,200 U/L. [0080] In some embodiments, the total serum indirect bilirubin level of about 45 μmol/L or greater is an indirect bilirubin level of between about 45 μmol/L and about 135 μmol/L. [0081] In some embodiments, the reticulocyte count of about 11% or greater is a reticulocyte count of between about 11% and about 19%. [0082] In some embodiments, the plasma arginase activity of about 2.6 μmol/mL/hr or greater is a plasma arginase activity of between about 2.6 μmol/mL/hr and about 9.8 μmol/mL/hr. [0083] In some embodiments, the systolic blood pressure of 120 or greater is a systolic blood pressure of between about 120 and about 155. [0084] In some embodiments, eligibility for 2,2-bis(4-fluorophenyl)-2-phenylacetamide is determined by clinical history. [0085] In some embodiments, subjects with a history of one or more of elevated tricuspid regurgitant jet velocity, glomerulopathy, renal disease, hepatobiliary disease, lower extremity ulceration, priapism, infarctive stroke, or heart disease, or a combination thereof, are included for treatment with 2,2-bis(4-fluorophenyl)- 2-phenylacetamide. In some embodiments, the hepatobiliary disease is cholelithiasis. In some embodiments of the methods disclosed herein, prior to administering the Gardos channel inhibitor, the subject has a history of elevated tricuspid regurgitant jet velocity, glomerulopathy, renal disease, hepatobiliary disease, lower extremity ulceration, priapism, infarctive stroke, or heart disease. In some embodiments, subjects with a history of one or more of skin ulcers, pulmonary hypertension, priapism, cholelithiasis, left ventricular heart failure, acute anemic events, stroke, or a combination thereof, are included for treatment with 2,2-bis(4-fluorophenyl)- 2-phenylacetamide. In some embodiments of the methods disclosed herein, prior to administering the Gardos channel inhibitor, the subject has a history of pulmonary hypertension, glomerulopathy, renal disease, cholelithiasis, lower extremity ulceration, priapism, infarctive stroke, or high output heart failure. [0086] In some embodiments, subjects with a history of multiple pain crises, acute chest syndrome, splenic infarct, renal failure, avascular necrosis, acute kidney injury, and hand foot syndrome, or a combination thereof, are excluded from treatment with a Gardos channel inhibitor, such as 2,2-bis(4-fluorophenyl)-2-phenylacetamide. In some embodiments of the methods disclosed herein, prior to administering the Gardos channel inhibitor, the subject does not have a history of avascular necrosis or osteonecrosis. [0087] In some embodiments, subjects with a history of NHD-associated complications are excluded from treatment with 2,2-bis(4-fluorophenyl)-2-phenylacetamide. [0088] In some embodiments, subjects with a history of HD-associated complications are included for treatment with 2,2-bis(4-fluorophenyl)-2-phenylacetamide. [0089] In some embodiments, subjects receiving 2,2-bis(4-fluorophenyl)-2-phenylacetamide are subject to ongoing monitoring. [0090] In some embodiments, subjects who, at a set time proceeding first treatment with 2,2- bis(4-fluorophenyl)-2-phenylacetamide, display insufficient hemoglobin increases post- treatment, or insufficient total hemoglobin levels, will have their therapy terminated. [0091] In some embodiments, subjects who, after 24 weeks, demonstrate hemoglobin increases of less than 10g per litre will have their therapy terminated. [0092] In some embodiments, subject eligibility for treatment with 2,2-bis(4-fluorophenyl)- 2- phenylacetamide is determined by one or more genes. [0093] In some embodiments, subject eligibility is determined by an aggregated genetic score. Said genetic score may be consisting of, but not limited to, all or some of the non- comprehensive genes and polymorphisms listed in Table 2. [0094] In some embodiments, subjects having one or more of polymorphisms: rs2208139 on the TGFBR3 gene, rs6586039, hCV1663921 on the BMPR1A gene, rs5014202 on the SMAD6 gene, rs10518707 on the SMAD 3 gene, (TA)7, (TA)8 repeats on the UGT1A gene, rs685417, rs516306, rs2149860, hCV3118898 on the KL gene, -597G>A, -174G>C on the IL-6 gene, rs2249358, rs211239 on the KL gene, rs3759178, rs3847859, rs706814 on the ACVRL1 gene, rs17199249, rs35711585 on the BMPR2 gene, rs267192, rs267196, rs267201 on the BMP6 gene, rs10874940 on the TGFBR3 gene, 4a allele on the NOS3 gene, rs10857560 on the MAPK8 gene, are included for treatment with 2,2-bis(4-fluorophenyl)-2- phenylacetamide. [0095] In some embodiments, female subjects having polymorphism T-786C on the NOS3 gene, are excluded from treatment with 2,2-bis(4-fluorophenyl)-2-phenylacetamide. [0096] In some embodiments, subjects having polymorphisms rs267192, rs267196, rs267201, rs408505, rs449853, rs1225934, or rs3812163 on the BMP6 gene, are excluded from treatment with 2,2-bis(4-fluorophenyl)-2-phenylacetamide. [0097] In some embodiments, subjects having polymorphisms rs73885319 (G1) or G1/G2 on the APOL1 gene, are excluded from treatment with 2,2-bis(4-fluorophenyl)-2- phenylacetamide. [0098] In some embodiments of the methods disclosed herein, the method further comprises administering a secondary therapeutic agent. In some embodiments, the secondary therapeutic agent is administered at a therapeutically effective amount. [0099] In some embodiments, the secondary therapeutic agent is hydroxycarbamide. [0100] In some embodiments, the secondary therapeutic agent is a positive allosteric modifier of hemoglobin. In some embodiments, the positive allosteric modifier of hemoglobin is 2-hydroxy-6-[[2-(2-propan-2-ylpyrazol-3-yl)pyridin-3- yl]methoxy]benzaldehyde (voxelotor). In some embodiments, the positive allosteric modifier of hemoglobin is 2-hydroxy-6-{[(3S)-4-{[2-(2-hydroxyethyl)pyridin-3- yl]carbonyl}morpholin-3-yl]methoxy}benzaldehyde (osivelotor). [0101] In some embodiments, subjects are eligible for treatment with a Gardos channel inhibitor, such as 2,2-bis(4-fluorophenyl)-2-phenylacetamide, only in the setting of combination treatment with hydroxyurea. [0102] In some embodiments, subjects are eligible for treatment with a Gardos channel inhibitor, such as 2,2-bis(4-fluorophenyl)-2-phenylacetamide, only in the setting of combination treatment with 6-((3S,4S)-4-methyl-1-(pyrimidin-2-ylmethyl)pyrrolidin-3-yl)-1- (tetrahydro-2H- pyran-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one. [0103] In some embodiments a composite clinical score is calculated from genetic background, clinical history, and laboratory parameters to assess HD-theratype. [0104] In some embodiments, a point-of-care genetic test is used to identify subjects with HD-theratype. [0105] In some embodiments, pediatric HD-theratypes are identified prior to onset of HD complications by family history of HD complications or genotype. [0106] In some embodiments, the methods disclosed herein do not increase the rate of vaso- occlusive crises in the subject. [0107] Also provided herein are uses of a Gardos channel inhibitor for treating sickle cell disease in a subject in need thereof, wherein the subject has experienced fewer than 4 vaso- occlusive pain crises (VOCs) in the preceding 24 months. In some embodiments, the Gardos channel inhibitor is 2,2-bis(4-fluorophenyl)-2- phenylacetamide, having chemical structure C20H15F2NO and commercial name senicapoc. In some embodiments, the sickle cell disease is sickle cell anemia. [0108] Also provided herein are uses of a Gardos channel inhibitor for increasing hemoglobin levels in a subject diagnosed with sickle cell disease, wherein the subject has experienced fewer than 4 vaso-occlusive pain crises (VOCs) in the preceding 24 months. In some embodiments, the Gardos channel inhibitor is 2,2-bis(4-fluorophenyl)-2- phenylacetamide, having chemical structure C20H15F2NO and commercial name senicapoc. In some embodiments, the sickle cell disease is sickle cell anemia. [0109] Also provided herein are uses of a Gardos channel inhibitor for reducing hemolysis- associated complications in a subject diagnosed with sickle cell disease, wherein the subject has experienced fewer than 4 vaso-occlusive pain crises (VOCs) in the preceding 24 months. In some embodiments, the hemolysis-associated complications are selected from elevated tricuspid regurgitant jet velocity, glomerulopathy, renal disease, hepatobiliary disease, lower extremity ulceration, priapism, infarctive stroke, or heart disease. In some embodiments, the hepatobiliary disease is cholelithiasis. In some embodiments, the hemolysis-associated complications are selected from pulmonary hypertension, glomerulopathy, renal disease, cholelithiasis, lower extremity ulceration, priapism, infarctive stroke, or heart disease. In some embodiments, the Gardos channel inhibitor is 2,2-bis(4-fluorophenyl)-2- phenylacetamide, having chemical structure C₂₀H₁₅F₂NO and commercial name senicapoc. In some embodiments, the sickle cell disease is sickle cell anemia. [0110] Also provided herein are uses of a Gardos channel inhibitor for aiding in treatment of a subject with sickle cell disease, comprising administering a Gardos channel inhibitor to the subject, wherein the subject has experienced fewer than 4 vaso-occlusive pain crises (VOCs) in the preceding 24 months. In some embodiments, the sickle cell disease is sickle cell anemia. [0111] Also provided herein are uses of a Gardos channel inhibitor for the manufacture of a medicament for treating sickle cell disease in a subject in need thereof, wherein the subject has experienced fewer than 4 vaso-occlusive pain crises (VOCs) in the preceding 24 months. In some embodiments, the Gardos channel inhibitor is 2,2-bis(4-fluorophenyl)-2- phenylacetamide, having chemical structure C20H15F2NO and commercial name senicapoc. In some embodiments, the sickle cell disease is sickle cell anemia. [0112] Also provided herein are uses of a Gardos channel inhibitor for the manufacture of a medicament for increasing hemoglobin levels in a subject diagnosed with sickle cell disease, wherein the subject has experienced fewer than 4 vaso-occlusive pain crises (VOCs) in the preceding 24 months. In some embodiments, the Gardos channel inhibitor is 2,2-bis(4- fluorophenyl)-2- phenylacetamide, having chemical structure C₂₀H₁₅F₂NO and commercial name senicapoc. In some embodiments, the sickle cell disease is sickle cell anemia. [0113] Also provided herein are uses of a Gardos channel inhibitor for the manufacture of a medicament for reducing hemolysis-associated complications in a subject diagnosed with sickle cell disease, wherein the subject has experienced fewer than 4 vaso-occlusive pain crises (VOCs) in the preceding 24 months. In some embodiments, the hemolysis-associated complications are selected from elevated tricuspid regurgitant jet velocity, glomerulopathy, renal disease, hepatobiliary disease, lower extremity ulceration, priapism, infarctive stroke, or heart disease. In some embodiments, the hepatobiliary disease is cholelithiasis. In some embodiments, the hemolysis-associated complications are selected from pulmonary hypertension, glomerulopathy, renal disease, cholelithiasis, lower extremity ulceration, priapism, infarctive stroke, or heart disease. In some embodiments, the Gardos channel inhibitor is 2,2-bis(4-fluorophenyl)-2- phenylacetamide, having chemical structure C₂₀H₁₅F₂NO and commercial name senicapoc. In some embodiments, the sickle cell disease is sickle cell anemia. [0114] Numerous specific details are set forth in order to provide a thorough understanding of the examples described herein. However, it will be understood by those of ordinary skill in the art that the examples described herein may be practiced without these specific details. In other instances, well-known methods, procedures and components have not been described in detail so as not to obscure the examples described herein. Also, the description is not to be considered as limiting the scope of the examples described herein. Table 1 Laboratory Marker Theratype Hemoglobin - Baseline, Elevated non-HD, NHD Hemoglobin - Low HD Lactate Dehydrogenase - Elevated HD Lactate Dehydrogenase – Within normal range non-HD, NHD Indirect Bilirubin - Elevated HD Indirect Bilirubin - Within normal range non-HD, NHD Reticulocytes - Elevated HD Reticulocytes - Within normal range non-HD, NHD Plasma Arginase - Elevated HD Plasma Arginase – Within normal range non-HD, NHD Systolic Blood Pressure - Elevated HD Systolic Blood Pressure - Within normal range non-HD, NHD Table 2 Gene Polymorphism Sequelae Influence Associated Theratype TGFBR3 rs2208139 Bacteremia Increases risk HD BMPR1 rs6586039, hCV1663921 Bacteremia Increases risk HD A SMAD6 rs5014202 Bacteremia Increases risk HD SMAD3 rs10518707 Bacteremia Increases risk HD NOS3 T-786C Acute chest Increases risk NHD syndrome (females) UGT1A (TA)7 repeat Bilirubin/ Increases risk HD gallstones UGT1A (TA)7, (TA)8 repeats Bilirubin/ Increases risk HD gallstones KL rs685417, rs516306, Leg ulcers Increases risk HD rs2149860, hCV3118898 IL-6 -597G>A, -174G>C Leg ulcers Increases risk HD BMP6 rs267192, rs267196, Osteonecrosi Increases risk HD rs267201, rs408505, s rs449853, rs1225934 BMP6 rs3812163 Osteonecrosi Increases risk HD s IL-1β -511C>T, +3954C>T Osteonecrosi Increases risk HD s KL rs2249358, rs211239 Priapism Increases risk HD ACVRL rs3759178, rs3847859, Pulmonary Increases risk HD 1 rs706814 hypertension BMPR2 rs17199249, rs35711585 Pulmonary Increases risk HD hypertension BMP6 rs267192, rs267196, Pulmonary Increases risk HD rs267201 hypertension TGFBR3 rs10874940 Pulmonary Increases risk HD hypertension NOS3 4a allele Pulmonary Increases risk HD hypertension MAPK8 rs10857560 Pulmonary Increases risk HD hypertension APOL1 rs73885319 (G1) Renal Increases risk NHD dysfunction APOL1 G1/G2 Renal Increases risk NHD dysfunction TNF-α -308G>A Stroke Increases risk NHD ENPPI K173Q Stroke Increases risk NHD ANXA2 rs11853426 Stroke Increases risk NHD TEK rs489347 Stroke Increases risk NHD TGFBR3 rs284875 Stroke Increases risk NHD VCAM1 -1594C Stroke Increases risk NHD IL4R S503P Stroke Increases risk NHD TNF-α -308G>A Stroke Unclear effect NHD AGT A3, A4 alleles Stroke Increases risk NHD EXAMPLES Example 1: Identification of a hemolytic endotype of SCD from SCD patient blood samples [0115] A transcriptomic analysis was employed to detect the existence of a distinct hemolytic endotype in SCD. Principal component analysis was performed on transcriptome- wide gene expression data, which was derived from whole blood samples of patients with SCD. Genes associated with the hemolytic process showed stratified expression across the second largest axis of variation (principal component 2 from the PCA analysis of gene expression data). [0116] Stratification of patients based on a primary regulator of hemolysis (HMOX1) and repeating the analysis recapitulated the same trend across the second principal component. Performing unsupervised clustering on the stratified samples showed that one cluster had increased expression of hemolytic related genes (FIG.1 and FIG.2). [0117] Differential gene expression between samples with a high hemolytic gene signature and those with a low hemolytic gene signature showed that the transcriptomic profiles of these two groups are significantly different. Taken together, there is a subgroup of SCD patients that show increased hemolytic activity and a significantly altered transcriptomic profile as compared to SCD patients with low expression of hemolytic related genes. [0118] To corroborate this analysis, an SCD module was constructed from the total-protein coding interactome (>160,000 edges). The module contained genes identified via both GWAS and DEG studies (314) and collectively represents the molecular perturbations underlying SCD and its sequelae. Distinct biological pathways were annotated within the aggregate SCD module, and distance between each sub-module and drug-targets was calculated and compared with the distribution of closest distances of 1000 random target modules of the same degree with 1000 random disease modules of the same degree. The resulting drug-target submodule distances and Z-scores indicated that KCNN4 (Gardos channel) was the nearest target to the hemolytic submodule. FIG.3 illustrates this proximity, with KCNN4 corresponding to target 8. [0119] Clinical features, e.g., history or frequency of vaso-occlusive crises or other features, may be relied upon to circumvent transcriptional profiling to identify patients belonging to the HD theratype, and therefore identify patients eligible for treatment with a Gardos channel inhibitor in spite of previous clinical failures of such treatments in SCD patients. Example 2: Multi-Omics Analysis of Combination Therapies for SCD based on SCD patient samples [0120] To assess the combination of Gardos channel inhibition and allosteric modification of hemoglobin as a potent drug synergy, the identification of gene sets composing the disease module specific to SCD was performed. [0121] In this context, the disease module is a group of relatively interconnected nodes in the biological network that is associated with SCD. These nodes can represent various biological components, such as genes, proteins, metabolites, or other cellular components. The identification of the disease module underpinning SCD provides insight into the mechanisms underlying the disease and is an important input in the identification of optimal combination therapies. In this context, “network proximity” is an important concept, providing a measure of separation or closeness between two or more drugs within a biological or drug interaction network. The network distance can inform the design of multi-drug treatments. Drugs whose targets are more distant from each other are more likely to produce safe and synergistic effects, whereas those whose targets overlap or are in close network proximity might lead to negligible additive effects and an increase in adverse effects. [0122] Compiling a comprehensive representation of the SCD disease module requires the integration of multiple gene sets from curated disease databases and differential expression studies. [0123] Seven variations of the SCD disease module were generated, covering 396 to 631 genes associated with SCD and related sequelae. Twelve potential known compounds targeting SCD were identified, yielding 19 gene targets. A separation metric was calculated between all drug target pairs based on their network distances, quantifying distinctiveness of interaction within the disease network to identify promising candidates. [0124] Analysis of target overlaps revealed that Gardos channel inhibition, together with a positive allosteric modifier of hemoglobin, held potential for safe and synergistic or additive effect. The network analysis revealed these drugs act on separate and “distant” SCD pathways - red blood cell hydration and hemoglobin oxygen affinity, respectively. Example 3: Clinical Study of a Multicenter, Randomized, Double-blind, Placebo- controlled Study to determine Efficacy and Safety of a Gardos Channel Inhibitor in SCD Patients with a Predominantly Hemolytic Phenotype [0125] This is a multicenter, randomized, double-blind, placebo-controlled study to evaluate the clinical efficacy and safety of a Gardos Channel Inhibitor in patients with Sickle Cell Disease (SCD) with a predominantly hemolytic phenotype, wherein patients with a clinical history of less than 4 vaso-occlusive crises over the preceding 24 months are deemed to belong to a predominantly hemolytic phenotype. [0126] Following a screening period, eligible patients are randomized in a 1:1 allocation ratio to receive a Gardos Channel Inhibitor, in one embodiment senicapoc or matching placebo. Treatment is discontinued if a participant experiences any Grade 3 or higher AE or hemolytic complication as assessed by the Investigator. [0127] Analysis of the primary endpoint of RBC density response, defined as the proportion of patients achieving decrease in RBC density (% erythrocytes with hemoglobin [Hb] concentrations > 410 g/L) from baseline, is assessed. [0128] A secondary endpoint is the proportion of patients experiencing at least one hemolytic complication, defined as any of the following: skin ulcers, priapism, albuminuria, elevated tricuspid regurgitant jet velocity (TRJV), stroke, acute anemia, proteinuria, hyperbilirubinemia, cholelithiasis, echocardiographic markers, and FACIT-fatigue. [0129] Effects of treatment on certain biomarkers is also evaluated, e.g., biomarkers of red blood cell function, renal complications, or of cardiovascular health. Effect on additional clinical outcomes is also evaluated, e.g., anemia, cardiovascular complications or cardiopulmonary complications, thromboembolism, renal complications, pain, patient function, well-being, genitourinary outcomes (e.g., priapism), skin outcomes (e.g., leg ulcers), or hepatobiliary outcomes (e.g., cholelithiasis). [0130] Patients with more than 4 vaso-occlusive pain crises over the preceding 24 months are excluded from treatment. Example 4: Clinical Study of a Multicenter, Randomized, Double-blind, Placebo- controlled Study to determine Efficacy and Safety of a Gardos Channel Inhibitor in SCD Patients with a Predominantly Hemolytic Phenotype [0131] This is a multicenter, randomized, double-blind, placebo-controlled study to evaluate the clinical efficacy and safety of a Gardos Channel Inhibitor in patients with Sickle Cell Disease (SCD) with a predominantly hemolytic phenotype, wherein patients with (1) a clinical history of less than 4 vaso-occlusive crises over the preceding 24 months, (2) hemoglobin levels of less than 9.2 g/dL, and (3) reticulocyte counts of greater than 11% are deemed to belong to a predominantly hemolytic phenotype. [0132] Following a screening period, eligible patients are randomized in a 1:1 allocation ratio to receive a Gardos Channel Inhibitor, in one embodiment senicapoc or matching placebo. Treatment is discontinued if a participant experiences any Grade 3 or higher AE or hemolytic complication as assessed by the Investigator. [0133] Analysis of the primary endpoint of reticulocyte response, defined as the proportion of patients achieving decrease in reticulocyte count from baseline, is assessed. [0134] A secondary endpoint is the proportion of patients experiencing at least one hemolytic complication, defined as any of the following: skin ulcers, priapism, albuminuria, elevated tricuspid regurgitant jet velocity (TRJV), stroke, acute anemia, proteinuria, hyperbilirubinemia, cholelithiasis, echocardiographic markers, and FACIT-fatigue. [0135] Effects of treatment on certain biomarkers is also evaluated, e.g., biomarkers of red blood cell function, renal complications, or of cardiovascular health. Effect on additional clinical outcomes is also evaluated, e.g., anemia, cardiovascular complications or cardiopulmonary complications, thromboembolism, renal complications, pain, patient function, well-being, genitourinary outcomes (e.g., priapism), skin outcomes (e.g., leg ulcers), or hepatobiliary outcomes (e.g., cholelithiasis). [0136] Patients with more than 4 vaso-occlusive pain crises over the preceding 24 months are excluded from treatment. [0137] Patients with baseline hemoglobin levels of more than 9.2 g/dL are excluded from treatment. [0138] Patients with reticulocyte counts of less than 11% are excluded from treatment. [0139] It will be appreciated that the examples used herein are for illustrative purposes only. Different terminology can be used without departing from the principles expressed herein. [0140] Although the above principles have been described with reference to certain specific examples, various modifications thereof will be apparent to those skilled in the art as outlined in the appended claims. In addition, each reference provided herein is incorporated by reference in its entirety to the same extent as if each reference was individual incorporated by reference. Where a conflict exists between the instant application and a reference provided herein, the instant application shall dominate.

Claims

Claims: 1. A method of treating sickle cell disease in a subject in need thereof, comprising administering a Gardos channel inhibitor to the subject, wherein the subject has experienced fewer than 4 vaso-occlusive pain crises (VOCs) in the preceding 24 months.

2. A method of increasing hemoglobin levels in a subject diagnosed with sickle cell disease, comprising administering a Gardos channel inhibitor to the subject, wherein the subject has experienced fewer than 4 vaso-occlusive pain crises (VOCs) in the preceding 24 months.

3. A method of reducing hemolysis-associated complications in a subject diagnosed with sickle cell disease, comprising administering a Gardos channel inhibitor to the subject, wherein the subject has experienced fewer than 4 vaso-occlusive pain crises (VOCs) in the preceding 24 months.

4. The method of claim 3, wherein the hemolysis-associated complications are selected from elevated tricuspid regurgitant jet velocity, glomerulopathy, renal disease, cholelithiasis, lower extremity ulceration, priapism, infarctive stroke, or heart disease.

5. The method of any one of the preceding claims, wherein the Gardos channel inhibitor is 2,2-bis(4-fluorophenyl)-2-phenylacetamide (senicapoc).

6. A method of aiding in treatment of a subject with sickle cell disease, comprising administering a Gardos channel inhibitor to the subject, wherein the subject has experienced fewer than 4 vaso-occlusive pain crises (VOCs) in the preceding 24 months.

7. The method of any one of the preceding claims, wherein the sickle cell disease is sickle cell anemia.

8. The method of any one of the preceding claims, wherein the subject has a hemoglobin level of less than about 9.2 g/dL prior to administering the Gardos channel inhibitor.

9. The method of any one of the preceding claims, wherein the subject has a plasma lactate dehydrogenase (LDH) level of about 408 U/L or greater prior to administering the Gardos channel inhibitor.

10. The method of any one of the preceding claims, wherein the subject has a total serum indirect bilirubin level of about 45 μmol/L or greater prior to administering the Gardos channel inhibitor.

11. The method of any one of the preceding claims, wherein the subject has a reticulocyte count of about 11% or greater prior to administering the Gardos channel inhibitor.

12. The method of any one of the preceding claims, wherein the subject has a plasma arginase activity of about 2.6 μmol/mL/hr or greater.

13. The method of any one of the preceding claims, wherein the subject has a systolic blood pressure of about 120 or greater prior to administering the Gardos channel inhibitor.

14. The method of any one of the preceding claims, wherein the subject has a history of pulmonary hypertension, glomerulopathy, renal disease, cholelithiasis, lower extremity ulceration, priapism, infarctive stroke, or high output heart failure.

15. The method of any one of the preceding claims, wherein the subject does not have a history of avascular necrosis or osteonecrosis.

16. The method of any one of the preceding claims, wherein the Gardos channel inhibitor is administered orally.

17. The method of any one of the preceding claims, wherein the Gardos channel inhibitor is administered once or twice daily.

18. The method of any one of the preceding claims, wherein the Gardos channel inhibitor is administered at a therapeutically effective dose.

19. The method of any one of the preceding claims, wherein the method further comprises administering a secondary therapeutic agent.

20. The method of claim 19, wherein the secondary therapeutic agent is hydroxycarbamide.

21. The method of claim 19, wherein the secondary therapeutic agent is a positive allosteric modifier of hemoglobin.

22. The method of claim 21, wherein the positive allosteric modifier of hemoglobin is 2- hydroxy-6-[[2-(2-propan-2-ylpyrazol-3-yl)pyridin-3-yl]methoxy]benzaldehyde (voxelotor).

23. The method of claim 21, wherein the positive allosteric modified of hemoglobin is 2- hydroxy-6-[[(3S)-4-[2-(2-hydroxyethyl)pyridine-3-carbonyl]morpholin-3- yl]methoxy]benzaldehyde (osivelotor).