Combination therapy for the treatment of sickle cell disease

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, together with kits for such treatments, in a subject in need thereof, comprising administering a Gardos channel inhibitor such as 2,2-bis(4-fluorophenyl)-2-phenylacetamide and a pyruvate kinase activator such as N-(4-((4-(Cyclopropylmethyl)-1-piperazinyl)carbonyl)phenyl)-8- quinolinesulfonamide 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

Atorney Docket No.: 063697-506001 WO COMBINATION THERAPY FOR THE TREATMENT OF SICKLE CELL DISEASE CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] This application claims benefit of priority to U.S. Provisional Patent Application No.63 / 738,423, filed December 23, 2024, which is incorporated by reference herein in its 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 or a pharmaceutically acceptable salt thereof, in combination with the administration of a pyruvate kinase activator, in one embodiment N-(4-((4-(Cyclopropylmethyl)-l -piperazinyl)carbonyl)phenyl)-8-quinolinesulfonamide or a pharmaceutically acceptable salt thereof, to a subject afflicted with SCD. In one aspect, provided is a medicament comprising a Gardos channel inhibitor (GCI) or a pharmaceutically acceptable salt or ester thereof, and a medicament comprising pyruvate kinase activator (PKA) or a pharmaceutically acceptable salt or ester thereof, and pharmaceutically acceptable carrier, excipient or stabilizer, and for use in a combination therapy for treating a sickle cell disease in a subject in need thereof. In one embodiment, provided is a kit comprising a first container, a second container, and a package insert, wherein the first container comprises at least one dose of a medicant comprising a Gardos channel inhibitor (GCI), the second container comprises at least one dose of a medicant comprising a pyruvate kinase activator (PKA), and the package insert comprises instructions for treating a subject for sickle cell disease using the medicament in the first container and the medicament in the second container. In one embodiment, the subjects afflicted with SCD are a defined patient population or theratype of SCD. In one embodiment, the subject contained within said population or theratype is characterized by and / or identified through clinical history, laboratory markers, genotype, or a combination thereof. In one embodiment, the subject of a defined hemolytic dominant (HD) SCD theratype is treated with the administration of a Gardos channel inhibitor such as 2,2-bis(4- fluorophenyl)-2-phenylacetamide.Atorney Docket No.: 063697-506001 WOBACKGROUND

[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 bom 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. Other forms of SCD are also known, and these include sickle cell disease with hemoglobin C (HbSC), sickle beta-plus thalassemia (HbSB+ Thal), sickle beta-zero thalassemia (HbSBO Thai) and sickle cell trait (HbAS).

[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 forAtorney Docket No.: 063697-506001 WOtherapies 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 aspect, provided is a method of treating sickle cell disease in a subject in need thereof, comprising administering to the subject a Gardos channel inhibitor (GCI) and a pyruvate kinase activator (PKA).

[0007] In another aspect, provided is a method of increasing hemoglobin levels in a subject diagnosed with sickle cell disease, comprising administering to the subject a Gardos channel inhibitor (GCI) and a pyruvate kinase inhibitor (PKA), wherein the subject has experienced fewer than 4 vaso-occlusive pain crises (VOCs) in the preceding 24 months.

[0008] In another aspect, provided is a method of reducing hemolysis-associated complications in a subject diagnosed with sickle cell disease, comprising administering to the subject a Gardos channel inhibitor (GCI) and a pyruvate kinase inhibitor (PKA), 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 improving hemolysis-associated laboratory markers in a subject diagnosed with sickle cell disease, comprising administering to the subject a Gardos channel inhibitor (GCI) and a pyruvate kinase inhibitor (PKA), 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 aiding in treatment of a subject with sickle cell disease, comprising administering to the subject a Gardos channel inhibitor (GCI) and a pyruvate kinase inhibitor (PKA), wherein the subject has experienced fewer than 4 vasoocclusive pain crises (VOCs) in the preceding 24 months.

[0011] In another aspect, provided is a kit comprising a first container, a second container, and a package insert, wherein the first container comprises at least one dose of a medicament comprising a Gardos channel inhibitor (GCI), the second container comprises at least one dose of a medicament comprising a pyruvate kinase activator (PKA), and the package insertAttomey Docket No.: 063697-506001 WOcomprises instructions for treating a subject for sickle cell disease using the medicament in the first container and the medicament in the second container.

[0012] In another aspect, provided is a pharmaceutical composition comprising a Gardos channel inhibitor (GCI) or a pharmaceutically acceptable salt or ester thereof, and a pyruvate kinase activator (PKA) or a pharmaceutically acceptable salt or ester thereof, and a pharmaceutically acceptable carrier, excipient or stabilizer.

[0013] In another aspect, provided is a combination product comprising a Gardos channel inhibitor (GCI) or a pharmaceutically acceptable salt or ester thereof, and a pyruvate kinase inhibitor (PKA) or a pharmaceutically acceptable salt or ester thereof. In embodiments, the GCI and the PKA are in separate dosage forms. In embodiments, the GCI and the PKA are in a single dosage form.

[0014] Also provided are pharmaceutical compositions and combination products as disclosed herein, for use in the methods disclosed herein.

[0015] Also provided are pharmaceutical compositions and combination products as disclosed herein, for use in the manufacture of a medicament for use in the methods disclosed herein.

[0016] Also provided are uses of the pharmaceutical compositions and combination products as disclosed herein in the methods disclosed herein.

[0017] In embodiments, the GCI is 2,2-bis(4-fluorophenyl)-2-phenylacetamide, which has the structure:or a pharmaceutically acceptable salt thereof.

[0018] In embodiments, the PKA is selected from N-(4-((4-(Cyclopropylmethyl)-l-piperazinyl)carbonyl)phenyl)-8-quinolinesulfonamide, (2S)-l-(5-{2H,3H-[l,4]dioxino[2,3-b]pyridine-7-sulfonyl}-lH,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrol-2-yl)-3-hydroxy-2-Attorney Docket No.: 063697-506001 WOphenylpropan-1 -one, and 6-[(6-aminopyridin-2-yl)methyl]-4-methyl-2-[(lH-pyrazol-3-yl)methyl]-4,6-dihydro-5H-[l,3]thiazolo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5-one. In embodiments, the PKAis N-(4-((4-(Cyclopropylmethyl)-l-piperazinyl)carbonyl)phenyl)-8-quinolinesulfonamide, which has the structure:

[0019] In embodiments, the sickle cell disease is selected from sickle cell anemia (SCA, HbSS or SS), sickle cell disease with hemoglobin C (HbSC), sickle beta-plus thalassemia (HbSB+ Thai), sickle beta-zero thalassemia (HbSBO Thai) and sickle cell trait (HbAS). In embodiments, the sickle cell disease is sickle cell anemia.

[0020] In embodiments, the methods provided herein are for treatment of a specific subpopulation of subjects with sickle cell disease.

[0021] In embodiments, the methods provided herein are for 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.Attorney Docket No.: 063697-506001 WOBRIEF DESCRIPTION OF THE DRAWINGS

[0022] 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.

[0023] 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 HM0X1 expression (a key driver of hemolysis). Shapes represent the results of lei den clustering performed on the gene expression data; samples of the same shape have similar gene expression profdes.

[0024] 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 HM0X1 expression (a key driver of hemolysis). Shape represents the membership of each sample into either the upper-quartile HM0X1 expression group or the lower-quartile HM0X1 expression group.

[0025] FIG. 3 illustrates Z-S cores 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

[0026] The invention described herein is directed to, inter alia, treating and / or aiding in the treatment of Sickle cell disease (SCD) in patients afflicted with SCD, and moreover, for subjects of a defined patient population or theratype 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, in combination with administering one or more pyruvate kinase activators, such as N-(4-((4-(Cyclopropylmethyl)-l-piperazinyl)carbonyl)phenyl)-8-quinolinesulfonamide, to subjects afflicted with SCD, and moreover, 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 combinationAtorney Docket No.: 063697-506001 WOthereof. 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.

[0027] SCD is caused by a single nucleotide polymorphism (SNP) in the [3-globin gene (HBB) on chromosome 11. This SNP leads to the amino acid substitution of valine for glutamic acid at position 6 of the P-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.

[0028] 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.

[0029] 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 influxAttorney Docket No.: 063697-506001 WOwhich 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.

[0030] 2,2-bis(4-fluorophenyl)-2-phenylacetamide, also known formally as 4-fluoro-a-(4-fhrorophenylj-a-phenyl-benzeneacetamide, as 1S / C2OHI5F2NO / C21-17-10-6-15(7-11-17)20(19(23)24, 14-4-2-l-3-5-14)16-8-12-18(22)13-9-16 / hl-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.

[0031] The present disclosure pertains to the treatment 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, in combination with administering one or more pyruvate kinase activators, such as N-(4-((4-(Cyclopropylmethyl)-l-piperazinyl)carbonyl)phenyl)-8-quinolinesulfonamide, to subjects afflicted with SCD. 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 GardosAttorney Docket No.: 063697-506001 WOchannel inhibitor, e.g., 2,2-bis(4-fluorophenyl)-2-phenylacetamide in combination with a pyruvate kinase activators, N-(4-((4-(Cyclopropylmethyl)-l-piperazinyl)carbonyl)phenyl)-8-quinolinesulfonamide. 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

[0032] 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.

[0033] 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”.

[0034] 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.

[0035] 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 termAttorney Docket No.: 063697-506001 WOprecedes. 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%.

[0036] 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.

[0037] 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.

[0038] As used herein, and unless otherwise specified, different “theratypes” refer to defined sub-populations of subjects, with variation in disease sequelae and therapy response.

[0039] 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 ofAtorney Docket No.: 063697-506001 WOthe 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).

[0040] As used herein, and unless otherwise specified, the term “NOAEL” means No Oberved Adverse Event Level, as a term commonly used in toxicology and pharmacology to denote the highest dose or exposure level of a substance at which there are no significant adverse effects observed in the subjects, for example the animals used during a study.NOAEL may be determined through systematic studies, such as acute, sub-chronic, or chronic toxicity studies, whereby. Researchers evaluate various endpoints such as physiological changes, behavioral effects, and histopathological changes are evaluated to identify the highest dose without adverse outcomes.2,2-Bis(4-fluorophenyl)-2-phenylacetamide

[0041] 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.Attorney Docket No.: 063697-506001 WO

[0042] 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.

[0043] However, the phase III trial in 297 SCD patients failed to show a reduction in vasoocclusive 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 hydroxy carbamide. 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

[0044] 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 Nonhemolysis 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.

[0045] 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 distinctAtorney Docket No.: 063697-506001 WOtranscriptional signatures. Gene-target based ranking of drug candidates was then performed for each respective theratype.Sequelae

[0046] 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.

[0047] 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

[0048] 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.

[0049] 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 [3-globin chain causesAtorney Docket No.: 063697-506001 WOpolymerization 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.

[0050] 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.

[0051] 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 pmol / L or greater prior to treatment with a Gardos channel inhibitor.

[0052] 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, sickleshaped erythrocytes elicits a compensatory response wherein the bone marrow increasesAtorney Docket No.: 063697-506001 WOerythropoietic activity. This manifests as reticulocytosis, defined as elevation of the reticulocyte count above the standard reference range.

[0053] 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.

[0054] 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.

[0055] 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.

[0056] 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 pmol / mL / hr or greater.

[0057] 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 sickleAtorney Docket No.: 063697-506001 WOerythrocyte 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.

[0058] 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

[0059] Various genotypes may be associated with the two disclosed theratypes. This is summarized in Table 2 below.

[0060] Genetic mutations associated with SCD may be further associated with various sequelae, and thus disease theratype.

[0061] Pulmonary hypertension is characteristic of the HD theratype. Pulmonary hypertension is impacted by the TGF- / BMP pathway genes, with individual gene Single Nucleotide Polymorphisms (SNPs) being associated with greater preponderance for pulmonary hypertension.Atorney Docket No.: 063697-506001 WO

[0062] The ACVRL1 gene encodes for a receptor in the TGF- 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- signaling, leading to abnormal vascular inflammation and endothelial cell dysfunction. Polymorphisms rs3759178, rs3847859, and rs706814 are associated with pulmonary hypertension risk.

[0063] The BMPR2 gene encodes for a TGF- receptor, and has mutations linked to familial pulmonary arterial hypertension, and is associated with pulmonary hypertension risk in sickle cell disease. Altered TGF- signaling affects vascular inflammation, with polymorphisms rsl7199249 and rs35711585 nominally associated with pulmonary hypertension risk.

[0064] The BMP6 gene is a member of the TGF- [3 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.

[0065] In some embodiments, subjects having an rs3759178, rs3847859, or rs706814 polymorphism in the ACVRL1 gene; or polymorphism rs 17199249 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); rs!7199249, rs35711585 (in the BMPR2 gene); rs267192, rs267196, and rs267201 (in the BMP6 gene).

[0066] 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 genderspecific genetic modifier for ACS susceptibility in females with SCD. The NOS3 T-786CAttorney Docket No.: 063697-506001 WOpolymorphism is thus associated with reduced NO levels and increased ACS risk specifically in female SCD patients.

[0067] 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.

[0068] 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.

[0069] Embodiments disclosed herein pertain to the administration of a Gardos channel inhibitor to hemolysis dominant (HD) SCD subjects.Combination Treatments for HD Theratype

[0070] 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

[0071] 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. WhenAtorney Docket No.: 063697-506001 WOhemoglobin 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.

[0072] 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.

[0073] 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.

[0074] 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.Pyruvate Kinase Activators

[0075] Pyruvate kinase activators (PKAs) are compounds that enhance the activity of pyruvate kinase (PK); PK is responsible for catalyzing the final step in the glycolytic pathway (the series of biochemical reactions that convert glucose into pyruvate and generates energy in the form of ATP) whereby, in the last step, phosphoenolpyruvate (PEP) and ADP are converted into pyruvate and ATP. As such, PK activity plays a crucial role for energy production in cells.

[0076] An increased binding of oxygen reduces sickle hemoglobin polymerization and sickling. In addition, PKR activation increases adenosine triphosphate (ATP) produced via glycolytic flux, which helps preserve membrane integrity and RBC deformability.Attorney Docket No.: 063697-506001 WO

[0077] In ex-vivo studies wherein RBCs from patients with SCD have been incubated with the PK activator N-(4-((4-(Cyclopropylmethyl)-l-piperazinyl)carbonyl)phenyl)-8-quinolinesulfonamide (mitapivat), the RBCs have had their erythrocyte PK (PKR) activity restored and a reduction in their 2,3-DPG content, which increased haemoglobin-oxygen affinity and RBC deformability and reduced RBC sickling. The increase in cellular ATP levels improved RBC integrity and cellular hydration. Similar affects have been shown for another PK activator, 6-[(6-aminopyridin-2-yl)methyl]-4-methyl-2-[(lH-pyrazol-3-yl)methyl]-4,6-dihydro-5H-[l,3]thiazolo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5-one (AG- 946), and the investigational PK activator (2S)-l-(5-{2H,3H-[l,4]dioxino[2,3-b]pyridine-7-sulfonyl}-lH,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrol-2-yl)-3-hydroxy-2-phenylpropan-l-one also has demonstrated such activity.Combination Therapy using a Gardos Channel Inhibitor and a Positive Allosteric Modifier of Hemoglobin or a Pyruvate Kinase Activator

[0078] 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.

[0079] 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.

[0080] 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 anAtorney Docket No.: 063697-506001 WOintracellular 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.

[0081] A Gardos channel inhibitor and a pyruvate kinase activator may be used in combination to reduce the rate of HbS polymerization and subsequent sickling. Gardos channel inhibition reduces erythrocyte dehydration and therefore results in lower intracellular HbS concentration; pyruvate kinase activation may directly reduce sickling through its effects of reducing the 2,3 -DPG content of the afflicted RBCs leasing to an increased oxygen affinity and increased ATP production. 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

[0082] Provided herein are methods of treating of sickle cell disease in a subject in need thereof, by administering to the subject a Gardos channel inhibitor in combination with administering a pyruvate kinase activator. 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 combination with the pyruvate kinase activator. 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.

[0083] The invention contemplates, inter alia, methods of increasing hemoglobin levels in a subject diagnosed with SCD or suspected to have SCD, by administering to the subject a Gardos channel inhibitor in combination with administering a pyruvate kinase activator,Atorney Docket No.: 063697-506001 WOwherein 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.

[0084] 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 to the subject a Gardos channel inhibitor in combination with administering a pyruvate kinase activator, 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.

[0085] In an embodiment, there is provided is a method of aiding in treatment of a subject with sickle cell disease, by administering to the subject a Gardos channel inhibitor in combination with administering a pyruvate kinase activator, 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 whoAtorney Docket No.: 063697-506001 WOwould 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.

[0086] In some embodiments, the Gardos channel inhibitor and the pyruvate kinase activator are administered orally. In some embodiments, the Gardos channel inhibitor and the pyruvate kinase activator are administered daily. In some embodiments, the Gardos channel inhibitor and the pyruvate kinase activator are administered once or twice daily. In some embodiments, the Gardos channel inhibitor and the pyruvate kinase activator are administered once daily. In some embodiments, the Gardos channel inhibitor is administered twice daily.

[0087] In some embodiments, the Gardos channel inhibitor (e.g., 2,2-bis(4-fluorophenyl)-2-phenylacetamide) and the pyruvate kinase activator are administered at a therapeutically effective dose.

[0088] 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 pmol / 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 pmol / 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 pmol / L or greater;Atorney Docket No.: 063697-506001 WO(d) a reticulocyte count of about 11% or greater;(e) a plasma arginase activity of about 2.6 pmol / mL / hr or greater; and / or(f) a systolic blood pressure of about 120 or greater.

[0089] In some embodiments of the methods disclosed herein, prior to administering the Gardos channel inhibitor of the combination therapy treatment, 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 pmol / L or greater;(d) a reticulocyte count of about 11% or greater;(e) a plasma arginase activity of about 2.6 pmol / mL / hr or greater; and / or(f) a systolic blood pressure of about 120 or greater.

[0090] In some embodiments of the methods disclosed herein, prior to administering the Gardos channel inhibitor of the combination therapy treatment, 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 pmol / 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 pmol / mL / hr or greater.

[0091] In some embodiments of the methods disclosed herein, prior to administering the Gardos channel inhibitor of the combination therapy treatment, the subject has a hemoglobin level of less than about 9.2 g / dL and a systolic blood pressure of about 120 or greater.Atorney Docket No.: 063697-506001 WO

[0092] 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.

[0093] 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.

[0094] In some embodiments, the total serum indirect bilirubin level of about 45 pmol / L or greater is an indirect bilirubin level of between about 45 pmol / L and about 135 pmol / L.

[0095] In some embodiments, the reticulocyte count of about 11% or greater is a reticulocyte count of between about 11% and about 19%.

[0096] In some embodiments, the plasma arginase activity of about 2.6 pmol / mL / hr or greater is a plasma arginase activity of between about 2.6 pmol / mL / hr and about 9.8 pmol / mL / hr.

[0097] In some embodiments, the systolic blood pressure of 120 or greater is a systolic blood pressure of between about 120 and about 155.

[0098] In some embodiments, eligibility for 2,2-bis(4-fluorophenyl)-2-phenylacetamide is determined by clinical history.

[0099] 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 j et velocity, glomerulopathy, renal disease, hepatobiliary disease, lower extremity ulceration, priapism, infarctive stroke, or heart disease. In some embodiments, subjects with ahistory 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 channelAtorney Docket No.: 063697-506001 WOinhibitor of the combination therapy treatment, the subject has a history of pulmonary hypertension, glomerulopathy, renal disease, cholelithiasis, lower extremity ulceration, priapism, infarctive stroke, or high output heart failure.

[0100] In some embodiments, subjects with ahistory 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.

[0101] In some embodiments, subjects with ahistory of NHD-associated complications are excluded from treatment with 2, 2-bis(4-fluorophenyl)-2 -phenylacetamide.

[0102] In some embodiments, subjects with ahistory of HD-associated complications are included for treatment with 2,2-bis(4-fluorophenyl)-2-phenylacetamide.

[0103] In some embodiments, subjects receiving 2,2-bis(4-fluorophenyl)-2-phenylacetamide are subject to ongoing monitoring.

[0104] In some embodiments, subjects who, at a set time proceeding first treatment with 2,2-bis(4-fluorophenyl)-2 -phenylacetamide, display insufficient hemoglobin increases posttreatment, or insufficient total hemoglobin levels, will have their therapy terminated.

[0105] In some embodiments, subjects who, after 24 weeks, demonstrate hemoglobin increases of less than 10g per litre will have their therapy terminated.

[0106] In some embodiments, subject eligibility for treatment with 2,2-bis(4-fluorophenyl)-2- phenylacetamide is determined by one or more genes.

[0107] 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.

[0108] In some embodiments, subjects having one or more of polymorphisms: rs2208139 on the TGFBR3 gene, rs6586039, hCV1663921 on the BMPR1 A gene, rs5014202 on theAttomey Docket No.: 063697-506001 WOSMAD6 gene, rs 10518707 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, rs!7199249, rs35711585 on the BMPR2 gene, rs267192, rs267196, rs267201 on the BMP6 gene, rs!0874940 on the TGFBR3 gene, 4a allele on the NOS3 gene, rs 10857560 on the MAPK8 gene, are included for treatment with 2,2-bis(4-fluorophenyl)-2-phenylacetamide.

[0109] 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.

[0110] In some embodiments, subjects having polymorphisms rs267192, rs267196, rs267201, rs408505, rs449853, rs!225934, or rs3812163 on the BMP6 gene, are excluded from treatment with 2,2-bis(4-fluorophenyl)-2-phenylacetamide.

[0111] In some embodiments, subjects having polymorphisms rs73885319 (Gl) or G1 / G2 on the APOL1 gene, are excluded from treatment with 2,2-bis(4-fluorophenyl)-2-phenylacetamide.

[0112] In some embodiments a composite clinical score is calculated from genetic background, clinical history, and laboratory parameters to assess HD-theratype.

[0113] In some embodiments, a point-of-care genetic test is used to identify subjects with HD-theratype.

[0114] In some embodiments, pediatric HD-theratypes are identified prior to onset of HD complications by family history of HD complications or genotype.

[0115] In some embodiments, the methods disclosed herein do not increase the rate of vasoocclusive crises in the subject.

[0116] Also provided herein are a Gardos channel inhibitor and a pyruvate kinase activator as a combination therapy for use in 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-Attomey Docket No.: 063697-506001 WOfluorophenyl)-2- phenylacetamide, having chemical structure C20H15F2NO and commercial name senicapoc. In some embodiments, the sickle cell disease is sickle cell anemia.

[0117] Also provided herein are a Gardos channel inhibitor and a pyruvate kinase activator as a combination therapy for use in 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.

[0118] Also provided herein are a Gardos channel inhibitor and a pyruvate kinase activator as a combination therapy for use in reducing hemolysis-associated complications in a subject diagnosed with sickle cell disease, wherein the subject has experienced fewer than 4 vasoocclusive 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.

[0119] Also provided herein are a Gardos channel inhibitor and a pyruvate kinase activator as a combination therapy for use in aiding in treatment of a subject 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 sickle cell disease is sickle cell anemia.

[0120] Also provided herein are uses of a Gardos channel inhibitor and a pyruvate kinase activator as a combination therapy 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-Atorney Docket No.: 063697-506001 WOfluorophenyl)-2- phenylacetamide, having chemical structure C20H15F2NO and commercial name senicapoc. In some embodiments, the sickle cell disease is sickle cell anemia.

[0121] Also provided herein are uses of a Gardos channel inhibitor and a pyruvate kinase activator as a combination therapy 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.

[0122] Also provided herein are uses of a Gardos channel inhibitor and a pyruvate kinase activator as a combination therapy 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.

[0123] Also provided herein are uses of a Gardos channel inhibitor and a pyruvate kinase activator as a combination therapy for aiding in treatment of a subject 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 sickle cell disease is sickle cell anemia.

[0124] Also provided herein are uses of a Gardos channel inhibitor and a pyruvate kinase activator for the manufacture of a medicament or medicaments 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 structureAtorney Docket No.: 063697-506001 WOC20H15F2NO and commercial name senicapoc. In some embodiments, the pyruvate kinase activator is N-(4-((4-(Cyclopropylmethyl)-l-piperazinyl)carbonyl)phenyl)-8-quinolinesulfonamide having the chemical formula C24H26N4O3S and the commercial name mitapivat. In some embodiments, the sickle cell disease is sickle cell anemia. In some embodiments, the medicaments comprise a kit, and the kit also comprises a package insert comprising instructions for using the Gardos channel inhibitor in combination with a pyruvate kinase activator to treat a sickle cell disease in a subject. In some embodiments of the kit, the Gardos channel inhibitor is 2,2-bis(4-fluorophenyl)-2- phenylacetamide and the pyruvate kinase activator is N-(4-((4-(Cyclopropylmethyl)-l-piperazinyl)carbonyl)phenyl)-8-quinolinesulfonamide and the instructions state that the medicaments are intended for use in treating a subject having a sickle cell disease that is sickle cell anemia. In some embodiments of the kit, the instructions state the order in which the medicaments are to be administered to the subjects, and in some further embodiments of the kit, the instructions state the period of administration of the Gardos channel inhibitor and the pyruvate kinase activator to the subject relative to each the administration of each medicament to the other.

[0125] Also provided herein are uses of a Gardos channel inhibitor and a pyruvate kinase activator for the manufacture of a medicament or medicaments 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 pyruvate kinase activator is N-(4-((4-(Cyclopropylmethyl)-l-piperazinyl)carbonyl)phenyl)-8-quinolinesulfonamide having the chemical formula C24H26N4O3S and the commercial name mitapivat. In some embodiments, the sickle cell disease is sickle cell anemia. In some embodiments, the medicaments comprise a kit, and the kit also comprises a package insert comprising instructions for using the Gardos channel inhibitor in combination with a pyruvate kinase activator to treat a sickle cell disease in a subject. In some embodiments of the kit, the Gardos channel inhibitor is 2,2-bis(4-fluorophenyl)-2- phenylacetamide and the pyruvate kinase activator is N-(4-((4-(Cyclopropylmethyl)-l-piperazinyl)carbonyl)phenyl)-8-quinolinesulfonamide and the instructions state that the medicaments are intended for use in treating a subject having a sickle cell disease that is sickle cell anemia. In some embodiments of the kit, the instructions state the order in which the medicaments are to be administered to the subjects, and in someAtorney Docket No.: 063697-506001 WOfurther embodiments of the kit, the instructions state the period of administration of the Gardos channel inhibitor and the pyruvate kinase activator to the subject relative to each the administration of each medicament to the other.

[0126] Also provided herein are uses of a Gardos channel inhibitor and a pyruvate kinase activator for the manufacture of a medicament or medicaments 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 j et 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 pyruvate kinase activator is N-(4-((4-(Cyclopropylmethyl)-l-piperazinyl)carbonyl)phenyl)-8-quinolinesulfonamide having the chemical formula C24H26N4O3S and the commercial name mitapivat. In some embodiments, the sickle cell disease is sickle cell anemia. In some embodiments, the medicaments comprise a kit, and the kit also comprises a package insert comprising instructions for using the Gardos channel inhibitor in combination with a pyruvate kinase activator to treat a sickle cell disease in a subject. In some embodiments of the kit, the Gardos channel inhibitor is 2,2-bis(4-fluorophenyl)-2- phenylacetamide and the pyruvate kinase activator is N-(4-((4-(Cyclopropylmethyl)-l-piperazinyl)carbonyl)phenyl)-8-quinolinesulfonamide and the instructions state that the medicaments are intended for use in treating a subject having a sickle cell disease that is sickle cell anemia. In some embodiments of the kit, the instructions state the order in which the medicaments are to be administered to the subjects, and in some further embodiments of the kit, the instructions state the period of administration of the Gardos channel inhibitor and the pyruvate kinase activator to the subject relative to each the administration of each medicament to the other.Attomey Docket No.: 063697-506001 WO

[0127] Additional non-limiting embodiments are enumerated in the lists below.Enumerated Embodiments A:

[0128] Embodiment 1. A method of treating sickle cell disease in a subject in need thereof, comprising administering to the subject a Gardos channel inhibitor (GCI) and a pyruvate kinase activator (PKA).

[0129] Embodiment 2. A method of increasing hemoglobin levels in a subject diagnosed with sickle cell disease, comprising administering to the subject a Gardos channel inhibitor (GCI) and a pyruvate kinase inhibitor (PKA), wherein the subject has experienced fewer than 4 vaso-occlusive pain crises (VOCs) in the preceding 24 months.

[0130] Embodiment 3. A method of reducing hemolysis-associated complications in a subject diagnosed with sickle cell disease, comprising administering to the subject a Gardos channel inhibitor (GCI) and a pyruvate kinase inhibitor (PKA), wherein the subject has experienced fewer than 4 vaso-occlusive pain crises (VOCs) in the preceding 24 months.

[0131] Embodiment 4. A method of improving hemolysis-associated laboratory markers in a subject diagnosed with sickle cell disease, comprising administering to the subject a Gardos channel inhibitor (GCI) and a pyruvate kinase inhibitor (PKA), wherein the subject has experienced fewer than 4 vaso-occlusive pain crises (VOCs) in the preceding 24 months.

[0132] Embodiment 5. A method of aiding in treatment of a subject with sickle cell disease, comprising administering to the subject a Gardos channel inhibitor (GCI) and a pyruvate kinase inhibitor (PKA), wherein the subject has experienced fewer than 4 vaso-occlusive pain crises (VOCs) in the preceding 24 months.

[0133] Embodiment 6. The method of any one of embodiments 1-5, wherein the GCI is 2,2-bis(4-fluorophenyl)-2 -phenylacetamide or a pharmaceutically acceptable salt thereof.

[0134] Embodiment 7. The method of any one of embodiments 1-6, wherein the PKA is selected from N-(4-((4-(Cyclopropylmethyl)-l-piperazinyl)carbonyl)phenyl)-8-quinolinesulfonamide, (2 S ) - 1 -(5 - {2H,3H-[ 1 ,4] dioxino[2,3 -b] py ridine-7 -sulfonyl } -lH,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrol-2-yl)-3-hydroxy-2-phenylpropan-l-one, and 6-[(6-Atorney Docket No.: 063697-506001 WOaminopyridin-2-yl)methyl]-4-methyl-2-[(lH-pyrazol-3-yl)methyl]-4,6-dihydro-5H-[l,3]thiazolo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5-one.

[0135] Embodiment 8. The method of embodiment 7, wherein the PKA is N-(4-((4-(Cyclopropylmethyl)-l-piperazinyl)carbonyl)phenyl)-8-quinolinesulfonamide.

[0136] Embodiment 9. The method of any one of the preceding embodiments, wherein the sickle cell disease is selected from sickle cell anemia (SCA, HbSS or SS), sickle cell disease with hemoglobin C (HbSC), sickle beta-plus thalassemia (HbSB+ Thai), sickle beta-zero thalassemia (HbSBO Thai) and sickle cell trait (HbAS).

[0137] Embodiment 10. The method of any one of the preceding embodiments, wherein the sickle cell disease is sickle cell anemia.

[0138] Embodiment 11. The method of any one of embodiments 1 to 10, wherein the subject has a hemoglobin level of less than about 9.2 g / dL prior to administering the combination therapy.

[0139] Embodiment 12. The method of any one of embodiments 1 to 10, wherein the subject has a plasma lactate dehydrogenase (LDH) level of about 408 U / L or greater prior to administering the combination therapy.

[0140] Embodiment 13. The method of any one of embodiments 1 to 10, wherein the subject has a total serum indirect bilirubin level of about 45 pmol / L or greater prior to administering the combination therapy.

[0141] Embodiment 14. The method of any one of embodiments 1 to 10, wherein the subject has a reticulocyte count of about 11% or greater prior to administering the combination therapy.

[0142] Embodiment 15. The method of any one of embodiments 1 to 10, wherein the subject has a plasma arginase activity of about 2.6 pmol / mL / hr or greater.

[0143] Embodiment 16. The method of any one of embodiments 1 to 10, wherein the subject has a systolic blood pressure of about 120 or greater prior to administering the combination therapy.Atorney Docket No.: 063697-506001 WO

[0144] Embodiment 17. The method of any one of embodiments 1 to 16, wherein the subject has a history of pulmonary hypertension, glomerulopathy, renal disease, cholelithiasis, lower extremity ulceration, priapism, infarctive stroke, or high output heart failure.

[0145] Embodiment 18. The method of any one of embodiments 1 to 17, wherein the subject does not have ahistory of avascular necrosis or osteonecrosis.

[0146] Embodiment 19. The method of any one of the preceding embodiments, wherein the combination therapy comprises a co-administration of the GCI and the PKA.

[0147] Embodiment 20. The method of any one of the preceding embodiments, wherein the combination therapy comprises a sequential administration of the GCI and the PKA.

[0148] Embodiment 21. The method of any one of the preceding embodiments, wherein the combination therapy is administered at a therapeutically effective dose for each of the GCI and the PKA.

[0149] Embodiment 22. The method of any one of the preceding embodiments, wherein the PKA in the composition has a concentration that does not exceed a concentration within a range of concentrations for a no observed adverse effect level (NOAEL).

[0150] Embodiment 23. A kit comprising a first container, a second container, and a package insert, wherein the first container comprises at least one dose of a medicament comprising a Gardos channel inhibitor (GCI), the second container comprises at least one dose of a medicament comprising a pyruvate kinase activator (PKA), and the package insert comprises instructions for treating a subject for sickle cell disease using the medicament in the first container and the medicament in the second container.

[0151] Embodiment 24. The kit of embodiment 23, wherein the medicament in the first container comprises a pharmaceutical composition comprising 2,2-bis(4-fluorophenyl)-2-phenylacetamide or a pharmaceutically acceptable salt or ester thereof, and a pharmaceutically acceptable carrier, excipient or stabilizer.

[0152] Embodiment 25. The kit of embodiment 23, wherein the medicament in the second container comprises a pharmaceutical composition comprising a pyruvate kinase activatorAttomey Docket No.: 063697-506001 WO(PKA) or a pharmaceutically acceptable salt or ester thereof, and a pharmaceutically acceptable carrier, excipient or stabilizer, wherein the PKA is selected fromN-(4-((4-(Cyclopropylmethyl)-l-piperazinyl)carbonyl)phenyl)-8-quinolinesulfonamide, (2S)-l-(5-{2H,3H-[l,4]dioxino[2,3-b]pyridine-7-sulfonyl}-lH,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrol-2-yl)-3-hydroxy-2-phenylpropan-l-one and 6-[(6-aminopyridin-2-yl)methyl]-4-methyl-2-[(lH-pyrazol-3-yl)methyl]-4,6-dihydro-5H-[l,3]thiazolo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5-one.

[0153] Embodiment 26. The kit of any of embodiments 23-25, wherein the instructions state that the medicament in the first container and the medicament in the second container are to be administered concurrently to the subject.

[0154] Embodiment 27. The kit of any of embodiments 23-25, wherein the instructions state that the medicament in the first container and the medicament in the second container are to be administered to the subject sequentially relative to each other.

[0155] Embodiment 28. The kit of any of embodiments 23-27, wherein the instructions recite an administration of the PKA at a dose concentration level of at least 5 mg / ml.

[0156] Embodiment 29. The kit of any of embodiments 23-27, wherein the instructions recite an administration of the PKA at a dose concentration level of no greater than a concentration within a range of concentrations for a no observed adverse effect level (NOAEL).

[0157] Embodiment 30. A pharmaceutical composition comprising a Gardos channel inhibitor (GCI) or a pharmaceutically acceptable salt or ester thereof, and a pyruvate kinase activator (PKA) or a pharmaceutically acceptable salt or ester thereof, and a pharmaceutically acceptable carrier, excipient or stabilizer.

[0158] Embodiment 31. The pharmaceutical composition of embodiment 30, wherein the GCI is 2,2-bis(4-fluorophenyl)-2-phenylacetamide.

[0159] Embodiment 32. The pharmaceutical composition of embodiment 31, wherein PKA is selected from N-(4-((4-(Cyclopropylmethyl)-l-piperazinyl)carbonyl)phenyl)-8-quinolinesulfonamide, (2 S ) - 1 -(5 - {2H,3H-[ 1 ,4] dioxino[2,3 -b] py ridine-7 -sulfonyl } -lH,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrol-2-yl)-3-hydroxy-2-phenylpropan-l-one, and 6-[(6-Atorney Docket No.: 063697-506001 WOaminopyridin-2-yl)methyl]-4-methyl-2-[(lH-pyrazol-3-yl)methyl]-4,6-dihydro-5H-[l,3]thiazolo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5-one.

[0160] Embodiment 33. The pharmaceutical composition of embodiment 32, wherein PKA is N-(4-((4-(Cyclopropylmethyl)-l-piperazinyl)carbonyl)phenyl)-8-quinolinesulfonamide.

[0161] Embodiment 34. The pharmaceutical composition of any one of embodiments 30-33, wherein the GCI in the composition has a concentration of at least 5mg / ml.Enumerated Embodiments B:

[0162] Embodiment 1. A pharmaceutical composition comprising a Gardos channel inhibitor (GCI) or a pharmaceutically acceptable salt or ester thereof, a pyruvate kinase activator (PKA) or a pharmaceutically acceptable salt or ester thereof, and a pharmaceutically acceptable carrier, excipient or stabilizer.

[0163] Embodiment 2. A combination product comprising a Gardos channel inhibitor (GCI) or a pharmaceutically acceptable salt or ester thereof, and a pyruvate kinase activator (PKA) or a pharmaceutically acceptable salt or ester thereof.

[0164] Embodiment 3. The combination product of embodiment 2, wherein the GCI and the PKA are in separate dosage forms.

[0165] Embodiment 4. The combination product of embodiment 2, wherein the GCI and the PKA are in a single dosage form.

[0166] Embodiment 5. The pharmaceutical composition or combination product of any one of embodiments 1 to 4, wherein the GCI is 2,2-bis(4-fluorophenyl)-2-phenylacetamide or a pharmaceutically acceptable salt thereof.

[0167] Embodiment 6. The pharmaceutical composition or combination product of any one of embodiments 1 to 5, wherein the PKA is selected fromN-(4-((4-(Cyclopropylmethyl)-l-piperazinyl)carbonyl)phenyl)-8-quinolinesulfonamide, (2S)-l-(5-{2H,3H-[l,4]dioxino[2,3-b]pyridine-7-sulfonyl}-lH,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrol-2-yl)-3-hydroxy-2-phenylpropan-1 -one, and 6-[(6-aminopyridin-2-yl)methyl]-4-methyl-2-[(lH-pyrazol-3-yl)methyl]-4,6-dihydro-5H-[l,3]thiazolo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5-one.Atorney Docket No.: 063697-506001 WO

[0168] Embodiment 7. The pharmaceutical composition or combination product of any one of embodiments 1 to 6, wherein the PKAis N-(4-((4-(Cyclopropylmethyl)-l-piperazinyl)carbonyl)phenyl)-8-quinolinesulfonamide.

[0169] Embodiment 8. The pharmaceutical composition or combination product of any one of embodiments 1 to 7, wherein the GCI in the composition or combination product has a concentration of at least 5mg / ml.

[0170] Embodiment 9. The pharmaceutical composition or combination product of any one of embodiments 1 to 8, for use in treating sickle cell disease.

[0171] Embodiment 10. The pharmaceutical composition or combination product of any one of embodiments 1 to 8, for use in a method of increasing hemoglobin levels in a subject diagnosed with sickle cell disease, wherein the subject has experienced fewer than 4 vasoocclusive pain crises (VOCs) in the preceding 24 months.

[0172] Embodiment 11. The pharmaceutical composition or combination product of any one of embodiments 1 to 8, for use in a method of 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.

[0173] Embodiment 12. The pharmaceutical composition or combination product of any one of embodiments 1 to 8, for use in a method of improving hemolysis-associated laboratory markers 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.

[0174] Embodiment 13. The pharmaceutical composition or combination product of any one of embodiments 1 to 8, for use in a method of aiding in treatment of a subject with sickle cell disease, wherein the subject has experienced fewer than 4 vaso-occlusive pain crises (VOCs) in the preceding 24 months.

[0175] Embodiment 14. The pharmaceutical composition or combination product for use of any one of embodiments 9 to 13, wherein the sickle cell disease is selected from sickle cell anemia (SCA, HbSS or SS), sickle cell disease with hemoglobin C (HbSC), sickle beta-plus thalassemia (HbSB+ Thai), sickle beta-zero thalassemia (HbSBO Thai) and sickle cell trait (HbAS).Atorney Docket No.: 063697-506001 WO

[0176] Embodiment 15. The pharmaceutical composition or combination product for use of any one of embodiments 9 to 14, wherein the sickle cell disease is sickle cell anemia.

[0177] Embodiment 16. The pharmaceutical composition or combination product for use of any one of embodiments 9 to 15, wherein the subject has a hemoglobin level of less than about 9.2 g / dL.

[0178] Embodiment 17. The pharmaceutical composition or combination product for use of any one of embodiments 9 to 16, wherein the subject has a plasma lactate dehydrogenase (LDH) level of about 408 U / L or greater.

[0179] Embodiment 18. The pharmaceutical composition or combination product for use of any one of embodiments 9 to 17, wherein the subject has a total serum indirect bilirubin level of about 45 pmol / L or greater.

[0180] Embodiment 19. The pharmaceutical composition or combination product for use of any one of embodiments 9 to 18, wherein the subject has a reticulocyte count of about 11% or greater.

[0181] Embodiment 20. The pharmaceutical composition or combination product for use of any one of embodiments 9 to 19, wherein the subject has a plasma arginase activity of about 2.6 pmol / mL / hr or greater.

[0182] Embodiment 21. The pharmaceutical composition or combination product for use of any one of embodiments 9 to 20, wherein the subject has a systolic blood pressure of about 120 or greater.

[0183] Embodiment 22. The pharmaceutical composition or combination product for use of any one of embodiments 9 to 21, wherein the subject has a history of pulmonary hypertension, glomerulopathy, renal disease, cholelithiasis, lower extremity ulceration, priapism, infarctive stroke, or high output heart failure.

[0184] Embodiment 23. The pharmaceutical composition or combination product for use of any one of embodiments 9 to 22, wherein the subject does not have a history of avascular necrosis or osteonecrosis.Atorney Docket No.: 063697-506001 WO

[0185] Embodiment 24. The pharmaceutical composition or combination product of any one of embodiments 1 to 8, for use in the manufacture of a medicament for treating sickle cell disease.

[0186] Embodiment 25. The pharmaceutical composition or combination product of any one of embodiments 1 to 8, for use in 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.

[0187] Embodiment 26. The pharmaceutical composition or combination product of any one of embodiments 1 to 8, for use 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.

[0188] Embodiment 27. The pharmaceutical composition or combination product of any one of embodiments 1 to 8, for use in the manufacture of a medicament for improving hemolysis-associated laboratory markers 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.

[0189] Embodiment 28. The pharmaceutical composition or combination product of any one of embodiments 1 to 8, for use in the manufacture of a medicament for aiding in treatment of a subject with sickle cell disease, wherein the subject has experienced fewer than 4 vaso-occlusive pain crises (VOCs) in the preceding 24 months.

[0190] Embodiment 29. The pharmaceutical composition or combination product for use of any one of embodiments 24 to 28, wherein the sickle cell disease is selected from sickle cell anemia (SCA, HbSS or SS), sickle cell disease with hemoglobin C (HbSC), sickle beta-plus thalassemia (HbSB+ Thai), sickle beta-zero thalassemia (HbSBO Thai) and sickle cell trait (HbAS).

[0191] Embodiment 30. The pharmaceutical composition or combination product for use of any one of embodiments 24 to 29, wherein the sickle cell disease is sickle cell anemia.Atorney Docket No.: 063697-506001 WO

[0192] Embodiment 31. The pharmaceutical composition or combination product for use of any one of embodiments 24 to 30, wherein the subject has a hemoglobin level of less than about 9.2 g / dL.

[0193] Embodiment 32. The pharmaceutical composition or combination product for use of any one of embodiments 24 to 31, wherein the subject has a plasma lactate dehydrogenase (LDH) level of about 408 U / L or greater.

[0194] Embodiment 33. The pharmaceutical composition or combination product for use of any one of embodiments 24 to 32, wherein the subject has a total serum indirect bilirubin level of about 45 pmol / L or greater.

[0195] Embodiment 34. The pharmaceutical composition or combination product for use of any one of embodiments 24 to 33, wherein the subject has a reticulocyte count of about 11% or greater.

[0196] Embodiment 35. The pharmaceutical composition or combination product for use of any one of embodiments 24 to 34, wherein the subject has a plasma arginase activity of about 2.6 pmol / mL / hr or greater.

[0197] Embodiment 36. The pharmaceutical composition or combination product for use of any one of embodiments 24 to 35, wherein the subject has a systolic blood pressure of about 120 or greater.

[0198] Embodiment 37. The pharmaceutical composition or combination product for use of any one of embodiments 24 to 36, wherein the subject has a history of pulmonary hypertension, glomerulopathy, renal disease, cholelithiasis, lower extremity ulceration, priapism, infarctive stroke, or high output heart failure.

[0199] Embodiment 38. The pharmaceutical composition or combination product for use of any one of embodiments 24 to 37, wherein the subject does not have a history of avascular necrosis or osteonecrosis.

[0200] Embodiment 39. Use of the pharmaceutical composition or combination product of any one of embodiments 1 to 8, for treating sickle cell disease.Atorney Docket No.: 063697-506001 WO

[0201] Embodiment 40. Use of the pharmaceutical composition or combination product of any one of embodiments 1 to 8, 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.

[0202] Embodiment 41. Use of the pharmaceutical composition or combination product of any one of embodiments 1 to 8, for reducing hemolysis-associated complications in a subject diagnosed with sickle cell disease, wherein the subject has experienced fewer than 4 vasoocclusive pain crises (VOCs) in the preceding 24 months.

[0203] Embodiment 42. Use of the pharmaceutical composition or combination product of any one of embodiments 1 to 8, for improving hemolysis-associated laboratory markers 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.

[0204] Embodiment 43. Use of the pharmaceutical composition or combination product of any one of embodiments 1 to 8, for aiding in treatment of a subject with sickle cell disease, wherein the subject has experienced fewer than 4 vaso-occlusive pain crises (VOCs) in the preceding 24 months.

[0205] Embodiment 44. The use of any one of embodiments 39 to 43, wherein the sickle cell disease is selected from sickle cell anemia (SCA, HbSS or SS), sickle cell disease with hemoglobin C (HbSC), sickle beta-plus thalassemia (HbSB+ Thai), sickle beta-zero thalassemia (HbSBO Thai) and sickle cell trait (HbAS).

[0206] Embodiment 45. The use of any one of embodiments 39 to 44, wherein the sickle cell disease is sickle cell anemia.

[0207] Embodiment 46. The use of any one of embodiments 39 to 45, wherein the subject has a hemoglobin level of less than about 9.2 g / dL.

[0208] Embodiment 47. The use of any one of embodiments 39 to 46, wherein the subject has a plasma lactate dehydrogenase (LDH) level of about 408 U / L or greater.

[0209] Embodiment 48. The use of any one of embodiments 39 to 47, wherein the subject has a total serum indirect bilirubin level of about 45 pmol / L or greater.Atorney Docket No.: 063697-506001 WO

[0210] Embodiment 49. The use of any one of embodiments 39 to 48, wherein the subject has a reticulocyte count of about 11% or greater.

[0211] Embodiment 50. The use of any one of embodiments 39 to 49, wherein the subject has a plasma arginase activity of about 2.6 pmol / mL / hr or greater.

[0212] Embodiment 51. The use of any one of embodiments 39 to 50, wherein the subject has a systolic blood pressure of about 120 or greater.

[0213] Embodiment 52. The use of any one of embodiments 39 to 51, wherein the subject has a history of pulmonary hypertension, glomerulopathy, renal disease, cholelithiasis, lower extremity ulceration, priapism, infarctive stroke, or high output heart failure.

[0214] Embodiment 53. The use of any one of embodiments 39 to 52, wherein the subject does not have a history of avascular necrosis or osteonecrosis.

[0215] Embodiment 54. A Gardos channel inhibitor (GCI) and a pyruvate kinase activator (PKA) as a combination therapy for use in treating sickle cell disease.

[0216] Embodiment 55. A Gardos channel inhibitor (GCI) and a pyruvate kinase activator (PKA) as a combination therapy for use in a method of 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.

[0217] Embodiment 56. A Gardos channel inhibitor (GCI) and a pyruvate kinase activator (PKA) as a combination therapy for use in a method of 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.

[0218] Embodiment 57. A Gardos channel inhibitor (GCI) and a pyruvate kinase activator (PKA) as a combination therapy for use in a method of improving hemolysis-associated laboratory markers 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.

[0219] Embodiment 58. A Gardos channel inhibitor (GCI) and a pyruvate kinase activator (PKA) as a combination therapy for use in a method of aiding in treatment of a subject withAtorney Docket No.: 063697-506001 WOsickle cell disease, wherein the subject has experienced fewer than 4 vaso-occlusive pain crises (VOCs) in the preceding 24 months.

[0220] Embodiment 59. The GCI and PKA as a combination therapy for use of any one of embodiments 54 to 58, wherein the GCI is 2, 2-bis(4-fluorophenyl)-2 -phenylacetamide or a pharmaceutically acceptable salt thereof.

[0221] Embodiment 60. The GCI and PKA as a combination therapy for use of any one of embodiments 54 to 59, wherein the PKA is selected from N-(4-((4-(Cyclopropylmethyl)-l-piperazinyl)carbonyl)phenyl)-8-quinolinesulfonamide, (2S)-l-(5-{2H,3H-[l,4]dioxino[2,3-b]pyridine-7-sulfonyl}-lH,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrol-2-yl)-3-hydroxy-2-phenylpropan-1 -one, and 6-[(6-aminopyridin-2-yl)methyl]-4-methyl-2-[(lH-pyrazol-3-yl)methyl]-4,6-dihydro-5H-[l,3]thiazolo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5-one.

[0222] Embodiment 61. The GCI and PKA as a combination therapy for use of any one of embodiments 54 to 60, wherein the PKA is N-(4-((4-(Cyclopropylmethyl)-l-piperazinyl)carbonyl)phenyl)-8-quinolinesulfonamide.

[0223] Embodiment 62. The GCI and PKA for use of any one of embodiments 54 to 61, wherein the sickle cell disease is selected from sickle cell anemia (SCA, HbSS or SS), sickle cell disease with hemoglobin C (HbSC), sickle beta-plus thalassemia (HbSB+ Thai), sickle beta-zero thalassemia (HbSBO Thai) and sickle cell trait (HbAS).

[0224] Embodiment 63. The GCI and PKA for use of any one of embodiments 54 to 62, wherein the sickle cell disease is sickle cell anemia.

[0225] Embodiment 64. The GCI and PKA for use of any one of embodiments 54 to 63, wherein the subject has a hemoglobin level of less than about 9.2 g / dL.

[0226] Embodiment 65. The GCI and PKA for use of any one of embodiments 54 to 64, wherein the subject has a plasma lactate dehydrogenase (LDH) level of about 408 U / L or greater.

[0227] Embodiment 66. The GCI and PKA for use of any one of embodiments 54 to 65, wherein the subject has a total serum indirect bilirubin level of about 45 pmol / L or greater.Attorney Docket No.: 063697-506001 WO

[0228] Embodiment 67. The GCI and PKA for use of any one of embodiments 54 to 66, wherein the subject has a reticulocyte count of about 11% or greater.

[0229] Embodiment 68. The GCI and PKA for use of any one of embodiments 54 to 67, wherein the subject has a plasma arginase activity of about 2.6 pmol / mL / hr or greater.

[0230] Embodiment 69. The GCI and PKA for use of any one of embodiments 54 to 68, wherein the subject has a systolic blood pressure of about 120 or greater.

[0231] Embodiment 70. The GCI and PKA for use of any one of embodiments 54 to 69, wherein the subject has a history of pulmonary hypertension, glomerulopathy, renal disease, cholelithiasis, lower extremity ulceration, priapism, infarctive stroke, or high output heart failure.

[0232] Embodiment 71. The GCI and PKA for use of any one of embodiments 54 to 70, wherein the subject does not have a history of avascular necrosis or osteonecrosis.

[0233] 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 1Attorney Docket No.: 063697-506001 WOTable 2> >Attomey Docket No.: 063697-506001 WO>>EXAMPLESExample 1: Identification of a hemolytic endotype of SCD from SCD patient blood samples

[0234] A transcriptomic analysis was employed to detect the existence of a distinct hemolytic endotype in SCD. Principal component analysis was performed on transcriptome-Atorney Docket No.: 063697-506001 WOwide 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).

[0235] Stratification of patients based on a primary regulator of hemolysis (HM0X1) 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).

[0236] 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.

[0237] 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.

[0238] 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.Attorney Docket No.: 063697-506001 WOExample 2: Multi-Omics Analysis of Combination Therapies for SCD based on SCD patient samples

[0239] 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.

[0240] 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.

[0241] Compiling a comprehensive representation of the SCD disease module requires the integration of multiple gene sets from curated disease databases and differential expression studies.

[0242] 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.

[0243] 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.Atorney Docket No.: 063697-506001 WOExample 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

[0244] 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.

[0245] 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.

[0246] 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.

[0247] 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.

[0248] 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).

[0249] Patients with more than 4 vaso-occlusive pain crises over the preceding 24 months are excluded from treatment.Atorney Docket No.: 063697-506001 WOExample 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

[0250] 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.

[0251] 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.

[0252] Analysis of the primary endpoint of reticulocyte response, defined as the proportion of patients achieving decrease in reticulocyte count from baseline, is assessed.

[0253] 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.

[0254] 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).

[0255] Patients with more than 4 vaso-occlusive pain crises over the preceding 24 months are excluded from treatment.

[0256] Patients with baseline hemoglobin levels of more than 9.2 g / dL are excluded from treatment.Atorney Docket No.: 063697-506001 WO

[0257] Patients with reticulocyte counts of less than 11% are excluded from treatment.

[0258] 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.Example 5: Ex vivo Analysis of Combination Therapies for SCD Using a Sickle Cell Mouse Model System

[0259] To assess the combination of Gardos channel inhibition and pyruvate kinase activation in erythrocytes (RBCs) as a potent drug synergy for the treatment oof sickle cell disease, a sickle cell mouse model system will be utilized wherein RBCs from sickle cell mice lines (as detailed below) will be treated with concentrations of the Gardos channel inhibitor 2, 2-bis(4-fluorophenyl)-2 -phenylacetamide (Senicapoc) and the pyruvate kinase activator N-(4-((4-(Cyclopropylmethyl)-l-piperazinyl)carbonyl)phenyl)-8-quinolinesulfonamide (Mitapivat), and also for comparative purposes with the positive allosteric modifier of hemoglobin namely 2-hydroxy-6-[[2-(2-propan-2-ylpyrazol-3-yl)pyridin-3-yl]methoxy]benzaldehyde (Voxelotor), as provided in the experimental protocol detailed below:A) Samples

[0260] Townes mice, available from The Jackson Laboratory (Bar Harbor, ME), expressing either the sickle cell trait (HbAS), or the sickle cell disease (SCD) phenotype (HbS), will be utilized in the study. Details on the genotype and phenotype of these mouse strains can be found on The Jackson Laboratory website pages:https : / / www.j ax. org / strain / 013071#, andhttps: / / www.jax.org / jax-mice-and-services / strain-data-sheet-pages / phenotype-information-for-013071-and-003342,and at the original peer-reviewed publication: Wu et al. “Correction of sickle cell disease by homologous recombination in embryonic stem cells”, Blood, 2006 Aug 15; 108(4): 1183-8, which is herein incorporated by reference in its entirety

[0261] For the experiments listed below, 3 mL Townes (HbSS) whole blood derived from 3 exsanguinated mice (Iml / mouse) will be utilized, and .3 mL Townes (AS) whole blood derived from 3 exsanguinated mice (Iml / mouse) will be utilizedAtorney Docket No.: 063697-506001 WO

[0262] The blood samples will undergo Ficoll separation upon the day of their reception at the research center where the experimental work will be performed.B) Treatments (each treatment combination and oxygen condition will be analyzed at two timepoints (H4 and H24; 30 conditions) x duplicate per condition = 60 wells)(i) Medicaments & Combinations

[0263] RBC thawed for one HbSS mouse will be split for use in the conditions as detailed in Table 3 below. For the HbAS mouse, only combinations A, B, and C will be performed.ii) Oxygen

[0264] Each condition will be split in two partial pressure conditions: 40% and 100% Table 3*Duplicate: Each replicate contains un-pooled RBC from an individual mouse.Atorney Docket No.: 063697-506001 WOC) Tests re Morphology & Cellular Integrity(i) RBC Morphology

[0265] To evaluate RBC morphology, starting with a 0.05 mL sample of RBC suspended in phosphate-buffered saline (PBS) to a hematocrit of 20%. RBCs will be chemically fixated (2.5% glutaraldehyde in PBS), stained (Sure-Stain Wright- Giemsa (CS434D; Fisher Scientific), and examined via light microscopy (high magnification, e.g., lOOx objective) to assess their shape, size, and structural characteristics. The number of sickled erythrocytes per high power field are counted for 500 cells in triplicate per treatment condition.(ii) Hemolysis

[0266] To evaluate RBC hemolysis. The supernatant of each well for each condition are sampled (10 pL) to analyze bilirubin, aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) as markers of hemolysis.(iii) RBC Density via percoll density gradient

[0267] Thalate oil mixtures of precise density are prepared by mixing 2 phthalate esters, n-butyl phthalate and dimethyl-phthalate, to achieve the following densities: 1.060, 1.064, 1.068, 1.076, 1.080, 1.084, 1.088, 1.092, 1.096, 1.100, 1.104, 1.108, 1.112, 1.116, 1.120, 1.124, 1.128, 1.132 and 1.136 g / mL at 20°C. Each each tube is filled with the RBC suspension (approximately 2 cm), the hematocrit tube is sealed at one end and spun in a temperature-controlled centrifuge (20°C) at 10000g for 10 minutes and the percentage of DRBC (below the phthalate oil layer) is calculated as follows: cells below / (cells below + cell above), with the results to be are plotted against density to generate a RBC density distribution curve for each sample For each of these curves, the DRBC percentage is determined as the percentage of RBCs with density > 1.120. The D50 is defined as the density for which cells below / (cells below + cell above) is equal to 0.5(D) RBC Senescence - evaluation via flow cytometry & ROS assay

[0268] To evaluate senescence, treated RBCs will be evaluated by viability markers in a fluorescence activated cell sorting (FACS) methodology for Ca2+; Fluo3 / AM and Annexin V and CD47. and for ROS through the use of a fluorometric assay kit to detect DCFH-DA staining.Attorney Docket No.: 063697-506001 WO(E) Data Analysis

[0269] Comparison will be made for the medicament / mice RBCs (HbSS and HbAS) condition, and also the medicament combination vs. single agent conditions. A nonparametric Fisher test will be employed to compare categorial variables (number of sickle cells, number of DRBC, number of senescent cells), and the nonparametric Wilcoxon-Mann- Whitney test will be used to compare continuous variable (hemolysis marker, D50).

[0270] 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

Atorney Docket No.: 063697-506001 WOWhat is claimed is:

1. A method of treating sickle cell disease in a subject in need thereof, comprising administering to the subject a Gardos channel inhibitor (GCI) and a pyruvate kinase activator (PKA).

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

4. A method of improving hemolysis-associated laboratory markers in a subject diagnosed with sickle cell disease, comprising administering to the subject a Gardos channel inhibitor (GCI) and a pyruvate kinase inhibitor (PKA), wherein the subject has experienced fewer than 4 vaso-occlusive pain crises (VOCs) in the preceding 24 months.

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

6. The method of any one of claims 1-5, wherein the GCI is 2,2-bis(4-fluorophenyl)-2-phenylacetamide or a pharmaceutically acceptable salt thereof.

7. The method of any one of claims 1-6, wherein the PKA is selected from N-(4-((4-(Cyclopropylmethyl)-l-piperazinyl)carbonyl)phenyl)-8-quinolinesulfonamide, (2S)-l-(5-{2H,3H-[l,4]dioxino[2,3-b]pyridine-7-sulfonyl}-lH,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrol-2-yl)-3-hydroxy-2-phenylpropan-l-one, and 6-[(6-aminopyridin-2-yl)methyl]-4-methyl-2-[(lH-pyrazol-3-yl)methyl]-4,6-dihydro-5H-[l,3]thiazolo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5-one.Atorney Docket No.: 063697-506001 WO8. The method of claim 7, wherein the PKA is N-(4-((4-(Cyclopropylmethyl)-l-piperazinyl)carbonyl)phenyl)-8-quinolinesulfonamide.

9. The method of any one of the preceding claims, wherein the sickle cell disease is selected from sickle cell anemia (SCA, HbSS or SS), sickle cell disease with hemoglobin C (HbSC), sickle beta-plus thalassemia (HbSB+ Thai), sickle beta-zero thalassemia (HbSBO Thai) and sickle cell trait (HbAS).

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

11. The method of any one of claims 1 to 10, wherein the subject has a hemoglobin level of less than about 9.2 g / dL prior to administering the combination therapy.

12. The method of any one of claims 1 to 10, wherein the subject has a plasma lactate dehydrogenase (LDH) level of about 408 U / L or greater prior to administering the combination therapy.

13. The method of any one of claims 1 to 10, wherein the subject has a total serum indirect bilirubin level of about 45 pmol / L or greater prior to administering the combination therapy.

14. The method of any one of claims 1 to 10, wherein the subject has a reticulocyte count of about 11% or greater prior to administering the combination therapy.

15. The method of any one of claims 1 to 10, wherein the subject has a plasma arginase activity of about 2.6 pmol / mL / hr or greater.

16. The method of any one of claims 1 to 10, wherein the subject has a systolic blood pressure of about 120 or greater prior to administering the combination therapy.

17. The method of any one of claims 1 to 16, wherein the subject has a history of pulmonary hypertension, glomerulopathy, renal disease, cholelithiasis, lower extremity ulceration, priapism, infarctive stroke, or high output heart failure.

18. The method of any one of claims 1 to 17, wherein the subject does not have a history of avascular necrosis or osteonecrosis.Attorney Docket No.: 063697-506001 WO19. The method of any one of the preceding claims, wherein the combination therapy comprises a co-administration of the GCI and the PKA.

20. The method of any one of the preceding claims, wherein the combination therapy comprises a sequential administration of the GCI and the PKA.

21. The method of any one of the preceding claims, wherein the combination therapy is administered at a therapeutically effective dose for each of the GCI and the PKA.

22. The method of any one of the preceding claims, wherein the PKA in the composition has a concentration that does not exceed a concentration within a range of concentrations for a no observed adverse effect level (NOAEL).

23. A kit comprising a first container, a second container, and a package insert, wherein the first container comprises at least one dose of a medicament comprising a Gardos channel inhibitor (GCI), the second container comprises at least one dose of a medicament comprising a pyruvate kinase activator (PKA), and the package insert comprises instructions for treating a subject for sickle cell disease using the medicament in the first container and the medicament in the second container.

24. The kit of claim 23, wherein the medicament in the first container comprises a pharmaceutical composition comprising 2,2-bis(4-fluorophenyl)-2-phenylacetamide or a pharmaceutically acceptable salt or ester thereof, and a pharmaceutically acceptable carrier, excipient or stabilizer.

25. The kit of claim 23, wherein the medicament in the second container comprises a pharmaceutical composition comprising a pyruvate kinase activator (PKA) or a pharmaceutically acceptable salt or ester thereof, and a pharmaceutically acceptable carrier, excipient or stabilizer, wherein the PKA is selected from N-(4-((4-(Cyclopropylmethyl)-l-piperazinyl)carbonyl)phenyl)-8-quinolinesulfonamide, (2S)-l-(5-{2H,3H-[l,4]dioxino[2,3-b]pyridine-7-sulfonyl}-lH,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrol-2-yl)-3-hydroxy-2-phenylpropan-l-one and 6-[(6-aminopyridin-2-yl)methyl]-4-methyl-2-[(lH-pyrazol-3-yl)methyl]-4,6-dihydro-5H-[l,3]thiazolo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5-one.Attorney Docket No.: 063697-506001 WO26. The kit of any of claims 23-25, wherein the instructions state that the medicament in the first container and the medicament in the second container are to be administered concurrently to the subject.

27. The kit of any of claims 23-25, wherein the instructions state that the medicament in the first container and the medicament in the second container are to be administered to the subject sequentially relative to each other.

28. The kit of any of claims 23-27, wherein the instructions recite an administration of the PKA at a dose concentration level of at least 5 mg / ml.

29. The kit of any of claims 23-27, wherein the instructions recite an administration of the PKA at a dose concentration level of no greater than a concentration within a range of concentrations for a no observed adverse effect level (NOAEL).

30. A pharmaceutical composition comprising a Gardos channel inhibitor (GCI) or a pharmaceutically acceptable salt or ester thereof, and a pyruvate kinase activator (PKA) or a pharmaceutically acceptable salt or ester thereof, and a pharmaceutically acceptable carrier, excipient or stabilizer.

31. The pharmaceutical composition of claim 30, wherein the GCI is 2,2-bis(4-fluorophenyl)-2-phenylacetamide.

32. The pharmaceutical composition of claim 31, wherein PKA is selected from N-(4-((4-(Cyclopropylmethyl)-l-piperazinyl)carbonyl)phenyl)-8-quinolinesulfonamide, (2S)-l-(5-{2H,3H-[l,4]dioxino[2,3-b]pyridine-7-sulfonyl}-lH,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrol-2-yl)-3-hydroxy-2-phenylpropan-l-one, and 6-[(6-aminopyridin-2-yl)methyl]-4-methyl-2-[(lH-pyrazol-3-yl)methyl]-4,6-dihydro-5H-[l,3]thiazolo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5-one.

33. The pharmaceutical composition of claim 32, wherein PKA is N-(4-((4-(Cyclopropylmethyl)-l-piperazinyl)carbonyl)phenyl)-8-quinolinesulfonamide.

34. The pharmaceutical composition of any one of claims 30-33, wherein the GCI in the composition has a concentration of at least 5mg / ml.

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