Methods for treating sickle cell disease by administering a BTK inhibitor

Abstract

Methods for treating Sickle Cell Disease (SCD) comprising administering a BTKi, such as at least one compound chosen from (R)-2-[3-[4-amino-3-(2-fluoro-4-phenoxy-phenyl)pyrazolo[3,4-d]pyrimidin-1-yl]piperidine-1-carbonyl]-4-methyl-4-[4-(oxetan-3-yl)piperazin-1-yl]pent-2-enenitrile (rilzabrutinib) and pharmaceutically acceptable salts thereof, are disclosed.

Description

Attorney Docket No. 01183-0329-00PCT-PRNMETHODS FOR TREATING SICKLE CELL DISEASE BY ADMINISTERING A BTK INHIBITORCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 680,784, filed August 8, 2024 and U.S. Provisional Application No. 63 / 726,794, filed December 2, 2024, the entire contents of which are incorporated by reference herein in their entireties for all purposes.SUMMARY

[0002] Disclosed herein are methods for treating Sickle Cell Disease (SCD). BTK inhibitors, and pharmaceutical compositions comprising the same, are also disclosed.

[0003] Sickle Cell Disease, commonly referred to as SCD, is a debilitating red blood cell (RBC) disorder in which a pathogenic variant of P-hemoglobin (Hb), termed sickle hemoglobin (HbS), polymerizes under conditions of low oxygen tension, leading red blood cells (RBCs) to adopt a classic crescent or sickle shape. The complex pathophysiology of SCD is characterized by the inherent instability of HbS, oxidative stress, chronic inflammation, hemolysis, and vaso-occlusive crises (Hebbel 2011; Hebbel et al. 1988). In individuals with SCD, endothelial activation and upregulation of adhesion molecules (e.g., L- selectin, P-selectin, E-selectin, and integrins) leads to increased interactions between leukocytes, RBCs, platelets and the surrounding endothelium (z.e., hypercoagulability and endothelial activation). This, in turn, obstructs microvascular blood flow, resulting in vasoocclusion and tissue ischemia. Subsequent reperfusion triggers additional inflammatory signaling, resulting in exacerbated tissue injury (Fig. 1) (Ataga and Stocker 2015, Rai and Ataga 2020). Over time, repeated tissue injury may lead to multiorgan dysfunction. Thus, SCD is associated with negative effects on quality of life, is a major cause of hospitalization, and incurs an elevated risk for death.

[0004] Existing and emerging curative therapies for SCD have garnered considerable interest. Allogeneic bone marrow transplantation, the only available curative treatment for SCD, results in disease-free survival in a majority of patients. However, its utility is restricted to eligible patients with an available donor (approximately 17% of patients with SCD). Subsequently, ongoing clinical trials are also investigating the ability of gene editing to modify a patient’s own hematopoietic stem cells, thus obviating the need for compatible donors (Rai and Ataga 2020).Attorney Docket No. 01183-0329-00PCT-PRN

[0005] Although curative therapies present an exciting avenue of treatment, they remain largely out of reach to the vast majority of SCD patients. Thus, there is an ongoing need for safe, effective, and affordable treatments for managing the clinical manifestations of SCD. Current non-curative therapies for SCD include treatment with drugs such as hydroxycarbamide (hydroxyurea), which induces fetal hemoglobin production; L-glutamine, which promotes the scavenging of reactive oxygen species implicated in inflammation; and voxelotor, which stabilizes hemoglobin in its oxyhemoglobin state (Rai and Ataga 2020). Crizanlizumab, an anti-p-selectin antibody that prevents the adhesion of RBCs to the endothelium of blood vessels, is still available in the United States but was recently taken off the market in the European Union after clinical trials failed to confirm its ability to reduce the frequency and severity of vaso-occlusive crises.

[0006] The diverse therapeutic mechanisms employed by these drugs attests to the complex pathophysiology of SCD and hints at the broad potential of drugs targeting alternative disease pathways. As a non-limiting example, there is a need for drugs that reduce chronic inflammation and hemolysis, increase hemoglobin levels, prevent vaso-occlusion, and ameliorate ischemia-reperfusion injury following vaso-occlusive crises, thereby improving quality of life and mitigating SCD-associated end-organ damage.

[0007] Bruton’s agammaglobulinemia tyrosine kinase (BTK) is an essential signaling element in B cells and innate immune cells, is expressed downstream of the B-cell receptor (BCR), Fc-gamma receptor (FcyR), and Fc-epsilon receptor (FcsR), and serves as a direct regulator of the NERP3 inflammasome. BTK is a non-receptor tyrosine kinase and a member of the TEC family of kinases. BTK is essential to B cell differentiation, development, and antibody production. Illustratively, inhibition of BTK activity produces phenotypic changes consistent with blockade of the BCR, including the down-regulation of cell proliferation, differentiation, maturation, and survival, as well as the up-regulation of apoptosis.

[0008] Rather than acting in an “on / off switch” manner, BTK may be best viewed as an immune function “modulator” (Crofford et al., 2016; Pal Singh et al., 2018). Important insights into BTK function come from loss of function analyses in humans and mice. Individuals with loss of function mutations in the BTK gene develop X-linked agammaglobulinemia (XLA), characterized by a complete absence of circulating B cells and plasma cells, and very low levels of immunoglobulins of all classes (Tsukada 1993, Vetrie 1993). This indicates the potential for BTK inhibition to suppress production ofAttorney Docket No. 01183-0329-00PCT-PRN autoantibodies thought to be important in the development of autoimmune diseases, such as ITP.

[0009] While BTK is not expressed in T cells, natural killer cells, or plasma cells and has no traceable direct functions in T cells or plasma cells (Sideras and Smith 1995; Mohamed et al., 2009), the enzyme regulates the activation of other hematopoietic cells, such as B cells, monocytes, basophils, mast cells, macrophages, neutrophils, and platelets. For example, BTK plays a role in the activation of neutrophils, which are key players in the inflammatory response that contributes to wound healing but may also cause tissue damage (Volmering S et al., 2016).

[0010] Accordingly, a selective BTK inhibitor (BTKi) has the potential to target multiple pathways involved in inflammation and autoimmunity, including, but not limited to: blocking BCR signaling, B cell activation, and autoantibody production; inhibiting plasma cell differentiation and antibody production; blocking IgG-mediated FcyR activation, phagocytosis, and inflammatory mediators in monocytes or macrophages; blocking IgE- mediated FcsR activation, migration, and degranulation in mast cells or basophils; and inhibiting activation, adhesion, recruitment, and oxidative burst in neutrophils. Based on these effects, a selective BTK inhibitor may block the initiation and progression of various inflammatory diseases and mitigate tissue damage resulting from these diseases. Although individuals with loss of function mutations in the BTK gene have decreased humoral immunity and are susceptible to pyogenic bacterial and enterovirus infections, requiring treatment with intravenous immunoglobulin, inhibition of BTK in individuals with an intact immune system is not predicted to produce similar susceptibility to infection.

[0011] Several orally administered BTK inhibitors, including ibrutinib (PCI-32765) and spebrutinib (CC-292), are currently marketed or in clinical development for a range of indications (Lee A et al., 2017). For example, ibrutinib has provided further clinical validation of the BTK target and was recently approved for human use in mantle cell lymphoma, Waldenstrom’s macroglobulinemia, and chronic lymphocytic leukemia by the U.S. Food and Drug Administration (FDA). Ibrutinib has also demonstrated activity in other hematological malignancies (Wang 2013; Byrd 2013, Imbruvica Package Insert, 2015). In addition, CC-292 has been reported to be well tolerated in a healthy volunteer population at doses which provide 100% occupancy of the BTK enzyme (Evans 2013). Furthermore, evobrutinib recently demonstrated efficacy for multiple sclerosis in a Phase 2 trial (Montalban et al., 2019). Other BTKi compounds are in clinical development for variousAttorney Docket No. 01183-0329-00PCT-PRN immune-mediated disorders, such as rheumatoid arthritis (NCT03823378, NCT03682705, NCT03233230), and asthma (NCT03944707) (Montalban et al., 2019; Norman 2016; Tam et al., 2018; Crawford et al., 2018; Min et al., 2019; Gillooly 2017; Nadeem et al., 2019).

[0012] While covalent BTKi, such as ibrutinib and acalabrutinib, improved on the selectivity issues that plagued many first-generation kinase inhibitors, these inhibitors are typically irreversible causing permanent modification of both on- and off-target kinases and side effects such as thrombocytopenia, anemia, platelet aggregation, and hepatotoxicity (RITUXAN Prescribing Information, 2018; Drug Record Kinase Inhibitors, 2019; Khan Y et al., 2019; Paydas S, 2019; IMBRUVICA, 2013; Rigg RA et al., 2016; Tang CPS et al., 2018). Thus, there is a need for treatment modalities for immune-mediated diseases such as SCD, based on BTKi with reduced side effects.

[0013] The methods of modulating immune-mediated diseases such as SCD described herein include administration of a BTKi. In some embodiments, the BTKi is a reversible inhibitor of BTK. In some embodiments, the BTKi is a hybrid BTK inhibitor in which the inhibitor binds to BTK in a reversible covalent manner. In some embodiments, the BTKi is an irreversible inhibitor of BTK. In some embodiments, the BTKi is a BTKi described in Tasso et al., Molecules, 2021, 26, 7411, the disclosure of which is incorporated herein by reference.

[0014] In some embodiments, the BTKi is (R)-2-[3-[4-amino-3-(2-fluoro-4-phenoxy- phenyl)pyrazolo[3,4-d]pyrimidin-l-yl]piperidine-l-carbonyl]-4-methyl-4-[4-(oxetan-3- yl)piperazin-l-yl]pent-2-enenitrile or a pharmaceutically acceptable salt thereof (rilzabrutinib). In some embodiments, the BTKi is l-[(3R)-3-[4-amino-3-(4-phenoxyphenyl)- lH-pyrazolo[3,4-d]pyrimidin-l-yl]-l-piperidinyl]-2-propen-l-one or a pharmaceutically acceptable salt thereof (ibrutinib). In some embodiments, the BTKi is (4-amino-3-(4- phenoxyphenyl)-l-[(3R)-l-(prop-2-enoyl)piperidin-3-yl]-l,3-dihydro-2H-imidazo[4,5- c]pyridin-2-one) or a pharmaceutically acceptable salt thereof (tolebrutinib). In some embodiments, the BTKi is PRN2675.

[0015] In some embodiments, the BTKi is fenebrutinib. In some embodiments, the BTKi is evobrutinib. In some embodiments, the BTKi is orelabrutinib. In some embodiments, the BTKi is remibrutinib. In some embodiments, the BTKi is BIIB-091. In some embodiments, the BTK inhibitor tirabrutinib. In some embodiments, the BTKi is acalabrutinib. In some embodiments, the BTKi is vecabrutinib. In some embodiments, the BTKi is zanubrutinib. In some embodiments, the BTKi is poseltinib. In some embodiments, the BTKi is pirtobrutinib. In some embodiments, the BTKi is spebrutinib. In someAttorney Docket No. 01183-0329-00PCT-PRN embodiments, the BTKi is olmutinib. In some embodiments, the BTKi is branebrutinib. In some embodiments, the BTKi is TAK-020. In some embodiments, the BTKi is elsubrutinib. In some embodiments, the BTK inhibitor is tolebrutinib.

[0016] In some embodiments, the BTKi is selected from rilzabrutinib, ibrutinib, tolebrutinib, PRN2675, fenebrutinib, evobrutinib, orelabrutinib, remibrutinib, BUB-091, tirabrutinib, acalabrutinib, vecabrutinib, zanubrutinib, poseltinib, pirtobrutinib, spebrutinib, olmutinib, branebrutinib, TAK-020, elsubrutinib, and tolebrutinib.

[0017] Compound (I) is a BTK inhibitor of the following structure:wherein *C is a stereochemical center. See PCT Publication No. WO 2014 / 039899, which is incorporated herein by reference, e.g., Example 31.(R)-2-[3-[4-amino-3-(2-fluoro-4-phenoxy-phenyl)pyrazolo[3,4-d]pyrimidin-l- yl]piperidine- 1 -carbonyl]-4-methyl-4-[4-(oxetan-3 -yl)piperazin- 1 -yl]pent-2-enenitrile, having the following structure:is also known as PRN1008 and rilzabrutinib. This compound has been disclosed in several patent publications, such as, e.g., PCT Publication Nos. WO 2014 / 039899, WO 2015 / 127310, WO 2016 / 100914, WO 2016 / 105531, WO 2018 / 005849, and WO 2021 / 150723 the contents of each of which are incorporated by reference herein.

[0018] Rilzabrutinib is a novel, highly selective, and potent small molecule inhibitor of non-T cell white blood cell signaling via B-cell receptor, FcyR, and / or FcsR signaling ofAttorney Docket No. 01183-0329-00PCT-PRN the BTK pathway. Rilzabrutinib functions as a reversible covalent BTK inhibitor and forms both a non-covalent and a covalent bond with its target; in particular, its reversible cysteine binding enables high selectivity and precise BTK inhibition without a permanent modification of proteins and peptides (Langrish et al. 2021, Owens et al. 2022, Smith PF et al. 2017). Taken together, these properties allow for enhanced selectivity and extended inhibition with low systemic exposure. In comparison to first and second generation BTKi, rilzabrutinib has shown minimal cross-reactivity with other molecules and is low risk for off- target effects (Smith et al. 2017). Importantly, rilzabrutinib ’s reversible binding minimizes the likelihood of permanently modified peptides (Serafimova 2012). In addition, rilzabrutinib shows improved kinase selectivity relative to the covalent BTK inhibitor ibrutinib. Preclinical studies in a broad kinase enzyme inhibition panel showed that 1 pM rilzabrutinib achieved >90% inhibition of just 6 of 251 kinases sharing a common cysteine in their active site. By contrast, 1 pM ibrutinib inhibited 21 kinases. Rilzabrutinib ’s IC50 values were 1.3 nM for BTK, 0.8 nM for tyrosine protein kinase TEC, 1.0 nM for bone marrow tyrosine kinase on chromosome X (BMX), 1.2 nM for receptor-like kinase (RLK), 6.3 nM for B cell lymphocyte kinase (BLK), and 11 nM for ERBB4. Further preclinical assays with rilzabrutinib showed that binding to BTK persisted while that for other TEC family members decayed rapidly over time.

[0019] Rilzabrutinib has shown encouraging results for the treatment of immune- mediated diseases. In humans, rilzabrutinib is rapidly absorbed following oral administration, with a fast half-life (3-4 h) and variable pharmacokinetics (Smith et al., 2017).

[0020] In Phase 1 studies of rilzabrutinib with 114 healthy volunteers, target BTK occupancy levels were safely and consistently exceeded, suggesting rilzabrutinib may be highly effective in treating autoimmune diseases. Moreover, preclinical and clinical pharmacokinetic and pharmacodynamic data showed that treatment effects endured even after the compound was cleared from circulation, consistent with an extended target residence time (Hill et al., 2015) and high target occupancy rate (> 90% within four hours and high sustained occupancy over 24 h) (Smith et al., 2015).

[0021] Rilzabrutinib has also demonstrated a favorable safety profile in clinical studies. In contrast with non-selective, irreversible BTK inhibitors, rilzabrutinib does not alter platelet aggregation in healthy volunteers or patients with ITP and thus does not lead to bleeding problems (Langrish et al. 2021, von Hundelshausen and Seiss 2021). As an additional point of contrast with irreversible BTK inhibitors, rilzabrutinib treatment does not exert clinically relevant effects on cardiac repolarization (electrocardiogram parametersAttorney Docket No. 01183-0329-00PCT-PRN including corrected QT interval) in healthy volunteers (N=51), even when administered at supratherapeutic doses (Lipsky and Lamanna 2020). Indeed, the most commonly reported adverse events in healthy volunteers were gastrointestinal adverse events, including nausea / vomiting and diarrhea. No serious adverse events or deaths were reported, and no participants discontinued treatment due to an adverse event (Smith PF 2017).

[0022] There is preliminary evidence to support the role of BTK inhibition in patients with autoimmune cytopenias (Rogers 2016, Montillo 2017), where sequential episodes of severe autoimmune hemolytic anemia and ITP ceased after initiation of treatment with ibrutinib, a BTK / EGFR / ITK inhibitor, in patients with chronic lymphatic leukemia.Additionally, rilzabrutinib treatment in vitro profoundly inhibits human B cell activation and blocks antibody (IgG, IgE) mediated activation of immune cells via Fc receptor signaling.

[0023] In nonclinical studies, rilzabrutinib demonstrates a significant dose dependent reduction of platelet-loss (consumption) in a mouse model of ITP. Rilzabrutinib also shows rapid and significant anti-inflammatory effects in a rat collagen-induced arthritis model, a rat antibody-mediated Arthus model, spontaneous canine pemphigus foliaceus, and human pemphigus vulgaris.

[0024] In addition, a growing body of evidence also suggests multiple roles for BTK in innate immunity, especially as a regulator of Toll-like receptor 4 (TLR4) and the NLR family pyrin domain containing 3 (NLRP3) inflammasome (Liu et al. 2017, Weber et al.2017, Weber et al. 2021). Both TLR4 and NLRP3 play well-established roles in the inflammatory response. Moreover, inflammation instigated by NLRP3 has been shown to be fundamental to pathophysiological changes in diseases such as stroke (Ito et al. 2015), liver inflammation (Were et al. 2014), Type 2 diabetes (Masters et al. 2010), Alzheimer’s disease (Heneka et al. 2013), and Parkinson’s disease (Gordon et al. 2018).

[0025] Consistent with its role in innate immunity, BTK has been shown to mediate the inflammatory response in a number of different cell types. For example, BTK activation led to increased oxidative stress in dendritic cells, neutrophils, and B cells in a mouse model of acute kidney injury (Nadeem et al. 2021). Meanwhile, in platelets, BTK regulates thrombo-inflammation, and BTK inhibition impairs platelet aggregation (Nicolson et al.2018, Nicolson et al. 2021).

[0026] In neutrophils BTK activation appears to promote recruitment and activation, as well as the subsequent formation of neutrophil extracellular traps (NETs). As a first point of evidence, BTK-deficient neutrophils exhibit a significant reduction in crawling velocity and post-adhesion strengthening relative to wild-type neutrophils (Volmering et al. 2016).Attorney Docket No. 01183-0329-00PCT-PRNMoreover, inhibition of BTK with ibrutinib markedly diminished the number of NETs in the alveoli of mice infected with influenza (Florence et al. 2018).

[0027] A wide body of literature supports a role for chronic inflammation in the pathophysiology of SCD. As a first point of evidence, hallmarks of hemolysis, including free heme and cell-free DNA, have been shown to activate the SCD inflammasome under steadystate conditions (Beckman et al. 2021, Belcher et al. 2014). In particular, free heme has been shown to interact with TLR4 to promote inflammation (Beckman et al. 2021; Liu et al. 2017).

[0028] Additionally, steady-state levels of inflammasome markers such as caspase-1, IL-ip, and IL-18 are elevated in SCD (Cerqueira et al. 2011, Vogel et al. 2018). In keeping with this, clinical studies show an increase in baseline activation of the NLRP3 inflammasome, with additional activation during ischemic vaso-occlusive crises, in patients with SCD (Vogel et al. 2018). These observations on NLRP3 are further supported by studies in a murine SCD model. Townes mice, which express human wild-type (HbAA) or sickle (HbSS) hemoglobin in place of murine hemoglobin, exhibit signs of chronic inflammation in the absence of an experimental challenge and respond to ischemia-reperfusion by further activating several inflammatory pathways, including the NLRP3 inflammasome pathway.

[0029] Studies were initiated to evaluate the effect of an exemplary BTKi, rilzabrutinib, on SCD. A first study, which investigated the baseline status of inflammatory pathways in Townes HbSS mice as compared to HbAA controls, was used to validate analytical methods for monitoring disease severity. To this end, biomarkers for hemolysis (cell-free heme and cell-free hemoglobin), reactive oxidative stress (ROS) malondialdehyde (MDA), RBC oxidative stress, coagulation (fibrin, D-dimer), and neutrophil activation (neutrophil elastase, myeloperoxidase (MPO), citrullinated histone H3, and cell-free DNA) were monitored. Additionally, a multiplex quantitative real time PCR (qRT-PCR) assay was used to assess expression of 16 target genes in the liver, kidney, lung, and spleen.

[0030] The study was performed on N=12 Townes mice as described in Table 1. The mice, which were 8 weeks old at the beginning of the study, were aged to 14 weeks and euthanized by CO2 narcosis.Attorney Docket No. 01183-0329-00PCT-PRNTable 1. Experimental Design.

[0031] Following euthanasia, whole blood was collected by cardiac puncture and aliquoted into one of two tubes: 500-800 pL was placed in a tube with EDTA as an anticoagulant and stored on ice, and 200-300 pL was placed in a tube with 3.8% sodium citrate. Blood was processed to plasma by centrifuging for 15 min at a minimum of 1000 x g at 4°C. Aliquots of 10-50 or 100 pL were flash frozen and stored at -80°C prior to use for the piloting and validation of the following assays at JAX as described below. a. QuantiChrom™ Heme Assay Kit (MAK316-1KT) for free heme: blood was collected in EDTA tubes and processed to plasma by centrifuging for 10 min at 2000 x g at room temperature. Five (5) to fifty (50) pL of plasma or diluted plasma was used per well. b. Abeam MDA Competitive ELISA Assay Kit (ab238537) for Malondialdehyde (MDA) or MyBioSource Mouse Malondialdehyde (MDA) ELISA Kit (MBS269473): blood was collected in EDTA tubes and processed to plasma by centrifuging for 10 min at 2000 x g at room temperature. Fifty (50) pL of plasma was required per well. c. Abbexa® mouse D-Dimer ELISA kit (abx258705) for fibrinogen D- dimer: blood was collected in tubes with 3.8% sodium citrate as an anti-coagulant and processed to plasma by centrifuging for 10 min at 2000 x g at room temperature. Plasma was diluted 100-fold, and 50 pL of diluted plasma was added to each well. d. Novus™ mouse Free Hemoglobin ELISA kit (NBP2-59999-1) for cell- free hemoglobin: blood was collected in EDTA tubes and processed to plasma by centrifuging for 15 min at 1000 x g at 4°C. Plasma was diluted 10 pL to 40 pL sample diluent. 15 pL of this solution was subsequently diluted with 285 pL sample diluent.Attorney Docket No. 01183-0329-00PCT-PRN e. Abeam™ mouse Neutrophil Elastase ELISA kit (ab252356) for neutrophil elastase: blood was collected in EDTA tubes and processed to plasma by centrifuging for 10 min at 2000 x g at room temperature. Plasma was diluted 1 : 10 in sample diluent NS. f. Abeam™ mouse Myeloperoxidase ELISA kit (ab275109) for myeloperoxidase (MPO): blood was collected in EDTA tubes and processed to plasma by centrifuging for 10 min at 2000 x g at room temperature. Plasma was diluted 1 : 10 in Sample diluent NS. g. Cayman Citrullinated Histone H3 ELISA kit (501620) for citrullinated H3: blood was collected in tubes with 3.8% sodium citrate as an anticoagulant and processed to plasma by centrifuging for 10 min at 2000 x g at room temperature. Plasma was diluted a minimum of 1 :2 in assay buffer. h. Active Motif Cell-Free DNA (cfDNA) purification kit (cat# 25503).

[0032] Spleen, liver, kidney, and lung samples were harvested and analyzed as described below. a. The spleen was collected, weighed, dissected into five equal pieces and individually flash frozen in separate tubes. Four tubes contained RNAlater® RNA stabilization solution for qRT-PCR. b. The liver was collected and weighed, and four pieces of -100 mg sections removed from the left lobe. Each piece was individually flash frozen in separate tubes containing RNAlater® RNA stabilization solution for qRT-PCR. c. Each kidney was dissected out, weighed, and cut in half. Each piece was individually flash frozen in separate tubes containing RNAlater® RNA stabilization solution for qRT-PCR. d. The lungs were dissected out and weighed, and four pieces of -100 mg sections were removed from the lobes. Each piece was individually flash frozen in separate tubes containing RNAlater® RNA stabilization solution for qRT-PCR.

[0033] For each tissue, one aliquot was used for validation of biomarkers (see Table 2) using multiplex TaqMan™ qRT-PCR assays by the JAX Molecular Biology and Biomarker Core. The additional aliquots were stored at -80C for potential future analyses.Attorney Docket No. 01183-0329-00PCT-PRN

[0034] qRT-PCR was performed as described below. a. RNA extraction: starting with -100 mg tissue, each sample was homogenized in QIAzol® Lysis Reagent, following manufacturer’s instructions to obtain -50 pL RNA that was subsequently stored at - 80°C. b. cDNA synthesis: starting with 2000 ng total RNA per reaction, samples were processed following manufacturer’s instructions (Applied Biosystems™ High-Capacity cDNA Reverse Transcription Kit Cat No. 4368814). Samples were processed in a thermocycler, then stored at -20°C. c. qPCR: the cDNA samples and TaqMan™ Fast Advanced Master Mix (cat# 4444557) were thawed on ice and protected from excessive exposure to light. The cDNA +RT samples are run next to cDNA no RT sample controls and a no-template control sample. There were four technical replicates of 100 ng per well. Following manufacturer’s instructions, sample were combined with kit components and run on a ViiA™ 7 Applied Biosystems™ Real-Time PCR machine with QUANTSTUDIO™ software. All samples for each probe were run on the same 384 well plate. d. qPCR probes: all probe sets were purchased from IDT. Probes were conjugated to FAM and double quenchers. e. qPCR validation: the assay for each gene was validated for amplification efficiency such that the results for the target gene assay were similar to the reference assay.Table 2. Biomarkers for qRT-PCR Analysis.Attorney Docket No. 01183-0329-00PCT-PRN

[0035] Average levels of cell-free DNA were significantly higher in SS mice (2.5 ng / pL) than in AA mice (0.34 ng / pL) (p=0.0018; unpaired t-test) (Fig. 2).

[0036] Target gene expression varied in samples taken from the liver, kidney, lung, and spleen of SS and AA mice (Figs. 3-8).

[0037] Numerous target genes were upregulated in liver samples from SS mice relative to liver samples from control mice (Fig. 3). Itga4, Nlrp3, Sele, Selp, Caspl, Hmoxl, Pycard, 1110, Vcaml, Tnfl, 116, Cxcll, and Ednl showed at least ~1.5-fold higher average expression levels in AA mice. Notably, Itga4, Sele, Selp, Hmoxl, Pycard, 1110, Vcaml, Tnfl, 116, and Cxcll were upregulated by more than 4-fold, with Itga4 and Sele showing increases of more than 10-fold and 30-fold, respectively.

[0038] Kidney samples from SS mice also exhibited a notable increase in target gene expression (Fig. 4). Itga4, Nlrp3, Sele, Selp, Caspl, Hmoxl, 1110, Illb, Tnfl, 116, Cxcll, and Ednl were upregulated by at least ~1.5-fold in SS samples compared to AA samples. Moreover, Selp, 1110, and 116 expression showed a more than 4-fold increase in SS tissue samples relative to control tissue samples.

[0039] Significantly, both Nlrp3 and Caspl were upregulated in liver and kidney samples from SS mice. Nlrp3 and Caspl are components of the Nlrp3 inflammasome, which acts downstream to BTK; thus, these data suggest that Nlrp3 and Caspl mRNA expression may serve as a readout of Nlrp3 inflammasome activation in both untreated and rilzabrutinib- treated SS mice.

[0040] Comparably fewer target genes were overexpressed in lung samples from SS mice relative to samples from AA mice. Selp, Hmoxl, 1110, and Illb exhibited modest upregulation in SS mice (Figure 5). Additionally, and consistent with data from liver and kidney samples, Caspl and Nlrp3 were slightly upregulated in SS mice. Of note, the present study employed a small sample size (N=6 HbSS and N=6 HbAA mice); subsequent studies may therefore reveal more significant increases in Casp and Nlrp3 expression in SS mice relative to their control counterparts.

[0041] Several genes were downregulated by more than 50% in spleen samples from SS mice relative to samples from AA mice (Figure 6). RelA, Nlrp3, Caspl, 1110, ILlb, Tnfl, 116, and Cxcll exhibited reduced expression. However, these data may reflect the small sample size associated with the present study and the known methodological challenges associated with isolating RNA from the spleen.

[0042] Blood analyte levels also varied between HbSS and HbAA mice. Average levels of neutrophil elastase and myeloperoxidase were increased in SS mice as compared toAttorney Docket No. 01183-0329-00PCT-PRNAA control mice (Figures 9 and 10). SS mice exhibited an average neutrophil elastase concentration of 33.3 ng / mL, while AA mice had an average concentration of 23.8 ng / mL (p=0.0142; unpaired t-test). For myeloperoxidase, SS mice and AA mice had average concentrations of 54.6 ng / mL and 31.6 ng / mL, respectively (p=0.0036; unpaired t-test).

[0043] Both SS and AA mice exhibited high levels of free hemoglobin (Figures 11 and 12). Samples from SS mice had an average free hemoglobin concentration of 2.84 pg / mL, while samples from AA mice had an average concentration of 2.42 pg / mL (p=0.4113; unpaired t-test); this modest difference likely stems from the small study size and may additionally reflect in vitro hemolysis during sample processing. In keeping with this, SS mice exhibited significantly higher levels of free heme (51.3 pg / mL) than AA mice (25.8 pg / mL) (p<0.0001; unpaired t-test).

[0044] Following completion of the first study, a second study was performed to examine whether rilzabrutinib has a therapeutic effect on vasculopathy due to chronic sterile inflammation, specifically hemolysis-driven vaso-occlusive crises, in HbSS mice. Specifically, the study examined the effects of rilzabrutinib treatment on chronic inflammatory state, blood cell adhesion, oxidative stress, thromboinflammation, vascular stasis, and, ultimately, vaso-occlusive crises and hemolysis. To this end, Townes mice were pretreated with rilzabrutinib, an irreversible covalent BTKi, or a P-selectin-blocking antibody for two weeks prior to challenge with hypoxia / reperfusion (HR) or hemoglobin (Hb) injury.

[0045] Disclosed herein are methods of treating sickle cell disease (SCD) in a mammal in need thereof, comprising administering to the mammal in need thereof a therapeutically effective amount of a BTK inhibitor (BTKi).

[0046] Also disclosed herein are methods of treating sickle cell disease (SCD) in a patient in need thereof, comprising administering to the patient in need thereof a therapeutically effective amount of a BTK inhibitor (BTKi).BRIEF DESCRIPTION OF DRAWINGS

[0047] Fig. 1 shows a schematic of the pathophysiology of sickle cell disease (SCD).

[0048] Fig. 2 shows the concentration of cell free DNA (cfDNA) in HbAA and HbSS mice.

[0049] Fig. 3 shows the relative expression of 16 target mRNAs in liver samples from HbAA and HbSS mice.Attorney Docket No. 01183-0329-00PCT-PRN

[0050] Fig. 4 shows the relative expression of 16 target mRNAs in kidney samples from HbAA and HbSS mice.

[0051] Fig. 5 shows the relative expression of 16 target mRNAs in lung samples from HbAA and HbSS mice.

[0052] Fig. 6 shows the relative expression of 16 target mRNAs in spleen samples from HbAA and HbSS mice.

[0053] Fig. 7 shows the relative expression of 16 target mRNAs in spleen, liver, kidney, and lung samples from HbAA and HbSS mice.

[0054] Fig. 8 shows the relative expression of 16 target mRNAs in spleen, liver, kidney, and lung samples from HbAA and HbSS mice.

[0055] Fig. 9 shows the concentration of neutrophil elastase in HbAA and HbSS mice.

[0056] Fig. 10 shows the concentration of myeloperoxidase in HbAA and HbSS mice.

[0057] Fig. 11 shows the concentration of heme in HbAA and HbSS mice.

[0058] Fig. 12 shows the concentration of free hemoglobin in HbAA and HbSS mice.

[0059] Figs. 13A and B show the inhibition of microvascular stasis by rilzabrutinib and the irreversible BTKi. Townes HbAA and HbSS mice were pretreated with the vehicle control, rilzabrutinib, the irreversible BTKi, an anti-P-selectin mAb, or an isotype control mAb. On day 14, Townes HbSS and HbAA mice (N=12 per group) were implanted with a dorsal skinfold chamber (DSFC). At baseline, flowing subcutaneous venules (20-23 venules per mouse) were selected and mapped. Mice were then challenged with (A) hypoxia / reoxygenation (HR; N=6 per group) or (B) human hemoglobin (Hb; N=6 per group). Microvascular stasis (vaso-occlusion) was measured at the indicated times after challenge. Values are means ± SEM. **P< 01, ***p<.001, ****p<0.0001, HbSS + vehicle compared to HbAA + vehicle, HbSS + rilzabrutinib, HbSS + irreversible BTKi, or HbSS + anti-P-selectin mAb, 2-way ANOVA, with the Dunnett multiple comparisons test.

[0060] Fig. 14 shows spleen weight as a percentage of body weight. Body weights were measured in HbAA and HbSS mice before DSFC implantation. Spleens from mice with DSFCs were collected and weighed 4 hours after an intravenous challenge with HR (N=6 per group) or Hb (N=6 per group). Data from mice in each treatment group challenged with HR or Hb were combined, as there were no significant differences between the means of each challenge group. Data are means ± SEM, **p<0.01 and ***p<0.001, one-way ANOVA with the Dunnett multiple comparison test. Treatments were compared to HbSS + Vehicle.Attorney Docket No. 01183-0329-00PCT-PRN

[0061] Figs. 15A-D show leukocyte counts. (A) white blood cell (WBC), (B) lymphocyte, (C) neutrophil, and (D) monocyte counts were measured in Townes HbAA and HbSS mice with an implanted DSFC using blood collected 4 hours after a challenge with HR (N=6 per group) or intravenous Hb (N=6 per group). Data from mice in each treatment group challenged with HR or Hb were combined (N=12 per group), as there were no significant differences between the means of each challenge group. Data are means ± SEM, **p<0.01 and ***p<0.001, one-way ANOVA with the Dunnett multiple comparison test. Treatments were compared to HbSS + Vehicle.

[0062] Figs. 16A-G show that red blood cells, hematocrits, corpuscular hemoglobin and reticulocytes were unchanged in any of the HbSS treatment groups. (A) RBC counts, (B) hematocrit, (C) Hb, (D) mean corpuscular hemoglobin concentration (MCHC), (E) reticulocyte (Retie) counts, (F) mean corpuscular volume (MCV), and (G) mean corpuscular hemoglobin (MCH) were measured in Townes HbSS mice with an implanted DSFC using blood collected 4 hours after a challenge with HR (N=6 per group) or intravenous Hb (N=6 per group). Data from mice in each treatment group challenged with HR or Hb were combined (N=12 per group), as there were no significant differences between the means of each challenge group. Data are means ± SEM, ***p<0.001, one-way ANOVA with the Dunnett multiple comparison test. Treatments were compared to HbSS + Vehicle.

[0063] Figs. 17A-D show that NF-KB activation is inhibited with BTK inhibitors. Townes HbAA and HbSS mice were pretreated with the vehicle control, rilzabrutinib, the irreversible BTKi, an anti-P-selectin mAb, or an isotype control mAb. On day 14, Townes HbSS and HbAA mice (N=3 per group) were challenged with (A and B) HR or (C and D) intravenous human Hb. Four hours after challenge, mice were euthanized, and livers were collected and flash frozen. (A and C) Immunoblots of liver nuclear extracts were stained with antibodies to NF-KB phospho-p65 and total p65 to assess NF-KB activation. (B and D) Phospho-p65 and total p65 bands were quantitated using densitometry. Phospho-p65 levels are mean densities relative to total p65 control. Values are means ± SEM. ***P< 001, HbSS + vehicle compared to HbAA + vehicle, HbSS + rilzabrutinib, HbSS + irreversible BTKi, or HbSS + anti-P-selectin mAb, one-way ANOVA, with the Dunnett multiple comparisons test.

[0064] Figs. 18A-F show lung inflammation in response to proinflammatory challenge. Townes HbAA and HbSS mice were pretreated with the vehicle control, rilzabrutinib, the irreversible BTKi, an anti-P-selectin mAb, or an isotype control mAb. On day 14, Townes HbSS and HbAA mice (N=3 per group) were challenged with (A, B, and C) HR or (D, E and F) intravenous human Hb. Four hours after challenge, mice wereAttorney Docket No. 01183-0329-00PCT-PRN euthanized, and lungs were inflated in OCT media and flash frozen. As a marker of inflammation, lung P-selectin and von Willebrand factor (VWF) expression on blood vessel endothelial cells (CD31+) in the lungs were measured by immunofluorescence staining. (A) In mice challenged with HR, representative lung P-selectin and VWF images are presented. P-selectin (B) and VWF (C) were quantified by dividing the number of P-selectin- or VWF- positive pixels by the number of CD31 -positive pixels in the images to generate P- selectin / CD31 and VWF / CD31 ratios. Results were counted from 3 independent image fields collected from each lung (N=3 per group) for a total of 9 fields used for each condition. (D) In mice challenged with Hb, representative lung P-selectin and VWF images are presented. P-selectin (E) and VWF (F) were quantified to generate P-selectin / CD31 and VWF / CD31 ratios. Values are means ± SEM, one-way ANOVA with pairwise comparisons and Sidak's multiple comparisons test.

[0065] Figs. 19A-E show the effect of rilzabrutinib pretreatment on gene expression in kidney and liver samples from HbSS mice challenged with HR or Hb. Twist capture exome sequencing was used to evaluate the expression of genes associated with BTK and inflammasome components and regulation (A), pro-inflammatory cytokines and chemokines(B), adhesion molecules, toll-like receptors, and transcription factors and signaling pathways(C), complement system components (D), and platelet activation / hemostasis and oxidative stress / enzymatic activity (E).Definitions:

[0066] Unless otherwise stated, the following terms used in the specification and claims are defined for the purposes of this Application and have the following meanings. All undefined technical and scientific terms used in this Application have the meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0067] As used herein, “a” or “an” entity refers to one or more of that entity; for example, a compound refers to one or more compounds or at least one compound unless stated otherwise. As such, the terms “a” (or “an”), “one or more,” and “at least one” can be used interchangeably herein.

[0068] As used herein, the term “about” is used herein to mean approximately, in the region of, roughly, or around. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term “about” is used herein to modify a numerical value above and below the stated value by a variance of 5%. With regard to specific values, itAttorney Docket No. 01183-0329-00PCT-PRN should be understood that specific values described herein for subject populations (e.g., the subject of the described clinical trial) represent median, mean, or statistical numbers, unless otherwise provided. Accordingly, aspects of the present disclosure requiring a particular value in a subject are supported herein by population data in which the relevant value is assessed to be a meaningful delimitation on the subject population.

[0069] As used herein, the term “active pharmaceutical ingredient” or “therapeutic agent” (“API”) refers to a biologically active compound.

[0070] As used herein, the terms “administer,” “administering,” or “administration” herein refer to providing, giving, dosing, and / or prescribing by either a health practitioner or an authorized agent and / or putting into, taking, or consuming by the patient or person himself or herself. For example, “administration” of an API to a patient refers to any route (e.g., oral delivery) of introducing or delivering the API to the patient. Administration includes selfadministration and administration by another.

[0071] As used herein, “BID” and “bid” are used interchangeably to refer to twice a day.

[0072] As used herein, the term “cell adhesion” or “blood cell adhesion” refers to adhesive interactions between two or more cells selected from RBCs, white blood cells, platelets, and endothelial cells. For example, in some embodiments, “cell adhesion” may refer to an interaction between two RBCs or an interaction between an RBC and an endothelial cell.

[0073] As used herein, the term “hemoglobin” encompasses both hemoglobin within red blood cells, i.e., corpuscular, cellular, or intracellular hemoglobin, and cell-free hemoglobin, i.e., extracellular hemoglobin. In some embodiments, wherein hemoglobin is administered intravenously in order to induce hemoglobin injury, i.e., hemoglobin challenge, it is to be understood that the intravenous hemoglobin is cell-free.

[0074] As used herein, the term “in combination with,” when referring to two or more compounds, agents, or additional active pharmaceutical ingredients, means the administration of two or more compounds, agents, or active pharmaceutical ingredients to the patient prior to, concurrent with, or subsequent to each other during a treatment period. Unless specified otherwise, the two or more compounds, agents, or active pharmaceutical ingredients may be administered on different schedules during the treatment period, such as, e.g., with one or more compounds, agents, or active pharmaceutical ingredients being administered once a day and one or more other compounds, agents, or active pharmaceutical ingredients being administered twice a day.Attorney Docket No. 01183-0329-00PCT-PRN

[0075] As used herein, an amount expressed in terms of “mg of [X]” refers to the total amount in milligrams of [X], i.e., the free base. In some embodiments, rilzabrutinib may be administered as a pharmaceutically acceptable salt of rilzabrutinib, in which case an amount expressed in terms of “mg of rilzabrutinib” refers to the total amount in milligrams of rilzabrutinib, i.e., the free base, plus the equivalent amount of one or more pharmaceutically acceptable salts of rilzabrutinib based on the weight of free base therein. For example, “400 mg of at least one compound chosen from rilzabrutinib and pharmaceutically acceptable salts thereof’ includes 400 mg of rilzabrutinib and a concentration of one or more pharmaceutically acceptable salts of rilzabrutinib equivalent to 400 mg of rilzabrutinib.

[0076] As used herein, the term “oxidative stress” may encompass both total oxidative stress and RBC oxidative stress. Total oxidative stress refers to systemic oxidative stress as measured using a biomarker such as MDA, while RBC oxidative stress refers to oxidative stress experienced by red blood cells.

[0077] As used herein, a “pharmaceutically acceptable carrier or excipient” means a carrier or an excipient that is useful in preparing a pharmaceutical composition that is generally safe, and neither biologically nor otherwise undesirable, such as, e.g., a carrier or an excipient that is acceptable for mammalian pharmaceutical use.

[0078] As used herein, the term “pharmaceutically acceptable salt” refers to a salt form, e.g., an acid addition salt, of an active pharmaceutical agent that is pharmaceutically acceptable and that possesses the desired pharmacological activity of the API of which the salt is made. Pharmaceutically acceptable salts are well known in the art and include those derived from suitable inorganic and organic acids. Such salts include, but are not limited to, salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, and the like; or formed with organic acids such as formic acid, acetic acid, propionic acid, hexanoic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid, benzenesulfonic acid, 4-toluenesulfonic acid, and the like. S. M. Berge et al. describes pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19.

[0079] As used herein, the terms “PRN1008;” “rilzabrutinib;” “(R)-2-[3-[4-amino-3- (2-fluoro-4-phenoxy-phenyl)pyrazolo[3,4-d]pyrimidin-l-yl]piperidine-l-carbonyl]-4-methyl- 4-[4-(oxetan-3-yl)piperazin-l-yl]pent-2-enenitrile;” “the compound of Formula (I);” and “2- [(3R)-3-[4-amino-3-(2-fluoro-4-phenoxy-phenyl)pyrazolo[3,4-d]-pyrimidin-l-yl]piperidine-Attorney Docket No. 01183-0329-00PCT-PRN l-carbonyl]-4-methyl-4-[4-(oxetan-3-yl)piperazin-l-yl]pent-2-enenitrile” are used interchangeably to refer to a compound having the structure:which is also referred to as 2-[(3R)-2-[4-amino-3-(2-fluoro-4-phenoxy-phenyl)pyrazolo[3,4- d]pyrimidin- 1 -yl]piperdine- 1 -carbonyl]-4-methyl-4[4-(oxetan-3 -yl)piperazin- 1 -yl]-(E and Z)- pent-2-enenitrile; (R)-2-[3-[4-amino-3-(2-fluoro-4-phenoxy-phenyl)pyrazolo[3,4- d]pyrimidin- 1 -yl]piperidine- 1 -carbonyl]-4-methyl-4-[4-(oxetan-3 -yl)piperazin- 1 -yl]pent-2- enenitrile; 1 -piperidinepropanenitrile, 3-[4-amino-3-(2-fluoro-4-phenoxyphenyl)-lH- pyrazolo[3,4-d]pyrimidin-l-yl]-a-[2-methyl-2-[4-(3-oxetanyl)-l-piperazinyl]propylidene]-P- oxo-, (3R)-; (EZ)-2-[(3R)-3-[4-amino-3-(2-fluoro-4-phenoxyphenyl)pyrazolo[3,4- d]pyrimidin-l-yl]piperidine-l-carbonyl]-4-methyl-4[4-(oxetan-3-yl)piperazin-l-yl]pent-2- enenitrile; and also by the International Nonproprietary Names for Pharmaceutical Substances (INN) as published by the World Health Organization ( / / cdn.who.int / media / docs / default-source / intemational-nonproprietary-names- (inn) / pll21.pdf?sfvrsn=69617906_15&download=true) having the following structure:. The compound of Formula (I) includesE and Z isomers, as indicated by the wavy bond in the structure shown above. The compound of Formula (I) may be present as a salt form.

[0080] A dose of the E isomer of rilzabrutinib may contain the corresponding (Z) isomer as an impurity in less than about 1% by weight; a dose of the (Z) isomer of rilzabrutinib may contain the corresponding (E) isomer as an impurity in less than about 1%Attorney Docket No. 01183-0329-00PCT-PRN by weight. When rilzabrutinib is denoted as a mixture of (E) and (Z) isomers of (R)-2-[3-[4- amino-3-(2-fluoro-4-phenoxy-phenyl)pyrazolo[3,4-d]pyrimidin-l-yl]piperi dine- 1 -carbonyl]- 4-methyl-4-[4-(oxetan-3-yl)piperazin-l-yl]pent-2-enenitrile, it means that the amount of (E) or (Z) isomer in the mixture is greater than about 1% by weight. In some embodiments, the molar ratio of (E) to (Z) isomer is 9: 1 rilzabrutinib or a pharmaceutically acceptable salt thereof may also be referred to herein as a “drug,” “active agent,” “a therapeutically active agent,” or “API.”

[0081] As used herein, “QD” and “qd” are used interchangeably to refer to once a day.

[0082] As used herein, “QWK” and “qwk” are used interchangeably to refer to once a week.

[0083] As used herein, the terms “sickle cell disease” and “SCD” refer to an RBC disorder in which pathogenic P-hemoglobin (sickle hemoglobin; HbS) polymerizes following deoxygenation, leading to the formation of crescent- or sickle-shaped RBCs. SCD is characterized by vaso-occlusive crises, hemolysis, and progressive multi-organ damage. Exemplary SCD variants include sickle cell anemia (HbSS), sickle cell hemoglobinopathy (HbSC), sickle P+ thalassemia (HbSp+), sickle po thalassemia (HbSpO), and the like.

[0084] As used herein, the term “therapeutically effective amount” refers to the amount of a compound that produces the desired effect for which it is administered (e.g., preventing or treating a symptom of SCD). The exact amount of an effective dose will depend on the purpose of the treatment and will be ascertainable by one skilled in the art using known techniques (see, e.g., Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding). As used herein, the term “treat,” “treating,” or “treatment,” when used in connection with a disorder or condition, includes any effect, e.g., lessening, reducing, modulating, ameliorating, or eliminating, that results in the improvement of the disorder or condition. Improvements in or lessening the severity of any symptom of the disorder or condition can be readily assessed according to standard methods and techniques known in the art.OVERVIEW

[0085] Some embodiments of the present disclosure relate to a method of treating sickle cell disease (SCD) in a mammal in need thereof, comprising administering to the mammal in need thereof a therapeutically effective amount of a BTK inhibitor (BTKi). In some embodiments the mammal is a human.Attorney Docket No. 01183-0329-00PCT-PRN

[0086] Some embodiments of the present disclosure relate to a method of treating sickle cell disease (SCD) in a patient in need thereof, comprising administering to the patient in need thereof a therapeutically effective amount of a BTK inhibitor (BTKi). In some embodiments, the patient is a human patient.

[0087] Some embodiments of the present disclosure relate to a BTKi for use in a method of treating SCD in a mammal in need thereof. In some embodiments, the method comprises administering to the mammal a therapeutically effective amount of the BTKi. In some embodiments, the mammal is a human.

[0088] Some embodiments of the present disclosure relate to a BTKi for use in a method of treating SCD in a patient in need thereof. In some embodiments, the method comprises administering to the patient a therapeutically effective amount of the BTKi. In some embodiments the patient is a human patient.

[0089] Some embodiments of the present disclosure relate to use of a BTKi for the manufacture of a medicament for treating SCD in a mammal in need thereof. In some embodiments, treating SCD in the mammal comprises administering to the mammal a therapeutically effective amount of the BTKi. In some embodiments, the mammal is a human. In some embodiments, the medicament comprises a therapeutically effective amount of the BTKi.

[0090] Some embodiments of the present disclosure relate to use of a BTKi for the manufacture of a medicament for treating SCD in a patient in need thereof. In some embodiments, treating SCD in the patient comprises administering to the patient a therapeutically effective amount of the BTKi. In some embodiments, the patient is a human. In some embodiments, the medicament comprises a therapeutically effective amount of the BTKi.

[0091] In some embodiments, treating SCD comprises reducing or preventing sickle cell crises. In some embodiments, treating SCD comprises reducing sickle cell crises. In some embodiments, treating SCD comprises preventing sickle cell crises.

[0092] In some embodiments, treating SCD comprises reducing or preventing SCD- related inflammation. In some embodiments, treating SCD comprises reducing SCD-related inflammation. In some embodiments, treating SCD comprises preventing SCD-related inflammation.

[0093] In some embodiments, treating SCD comprises reducing or preventing hemoglobin S polymerization. In some embodiments, treating SCD comprises reducingAttorney Docket No. 01183-0329-00PCT-PRN hemoglobin S polymerization. In some embodiments, treating SCD comprises preventing hemoglobin S polymerization.

[0094] In some embodiments, treating SCD comprises reducing or preventing red blood cell (RBC) sickling. In some embodiments, treating SCD comprises reducing RBC sickling. In some embodiments, treating SCD comprises preventing RBC sickling.

[0095] In some embodiments, treating SCD comprises reducing or preventing SCD- related hemolysis. In some embodiments, treating SCD comprises reducing SCD-related hemolysis. In some embodiments, treating SCD comprises preventing SCD-related hemolysis.

[0096] In some embodiments, treating SCD comprises increasing hemoglobin levels.

[0097] In some embodiments, treating SCD comprises reducing cell-free hemoglobin levels.

[0098] In some embodiments, treating SCD comprises reducing or preventing SCD- related anemia. In some embodiments, treating SCD comprises reducing SCD-related anemia. In some embodiments, treating SCD comprises preventing SCD-related anemia.

[0099] In some embodiments, treating SCD comprises reducing or preventing SCD- related hemostasis. In some embodiments, treating SCD comprises reducing SCD-related hemostasis. In some embodiments, treating SCD comprises preventing SCD-related hemostasis.

[0100] In some embodiments, treating SCD comprises reducing or preventing SCD- related vaso-occlusion or vaso-occlusive crisis. In some embodiments, treating SCD comprises reducing SCD-related vaso-occlusion or vaso-occlusive crisis. In some embodiments, treating SCD comprises preventing SCD-related vaso-occlusion or vasoocclusive crisis.

[0101] In some embodiments, treating SCD comprises reducing or preventing SCD- related ischemia / reperfusion. In some embodiments, treating SCD comprises reducing SCD- related ischemia / reperfusion. In some embodiments, treating SCD comprises preventing S CD-rel ated i schemi a / reperfusi on .

[0102] In some embodiments, treating SCD comprises reducing or preventing SCD- related thromboinflammation. In some embodiments, treating SCD comprises reducing SCD- related thromboinflammation. In some embodiments, treating SCD comprises preventing SCD-related thromboinflammation.

[0103] In some embodiments, treating SCD comprises reducing or preventing SCD- related vasculopathy. In some embodiments, treating SCD comprises reducing SCD-relatedAttorney Docket No. 01183-0329-00PCT-PRN vasculopathy. In some embodiments, treating SCD comprises preventing SCD-related vasculopathy.

[0104] In some embodiments, treating SCD comprises reducing or preventing SCD- related cell adhesion. In some embodiments, treating SCD comprises reducing SCD-related cell adhesion. In some embodiments, treating SCD comprises preventing SCD-related cell adhesion.

[0105] In some embodiments, treating SCD comprises reducing or preventing SCD- related organ damage. In some embodiments, treating SCD comprises reducing SCD-related organ damage. In some embodiments, treating SCD comprises preventing SCD-related organ damage. In some embodiments, the organ is the spleen, lung, liver, or kidney. In some embodiments, the organ is the spleen. In some embodiments, the organ is the lung. In some embodiments, the organ is the liver. In some embodiments, the organ is the kidney.

[0106] In some embodiments, the therapeutically effective amount of the BTKi is sufficient to reduce the volume or weight of the spleen relative to a baseline volume or weight. In some embodiments, the therapeutically effective amount of the BTKi is sufficient to reduce the volume of the spleen relative to a baseline volume. In some embodiments, the therapeutically effective amount of the BTKi is sufficient to reduce the weight of the spleen relative to a baseline weight.

[0107] In some embodiments, the therapeutically effective amount of the BTKi is sufficient to reduce the white blood cell count relative to a baseline count. In some embodiments, the therapeutically effective amount of the BTKi is sufficient to reduce the lymphocyte count relative to a baseline count. In some embodiments, the therapeutically effective amount of the BTKi is sufficient to reduce the neutrophil count relative to a baseline count. In some embodiments, the therapeutically effective amount of the BTKi is sufficient to reduce the monocyte count relative to a baseline count.

[0108] In some embodiments, the therapeutically effective amount of the BTKi is sufficient to increase the red blood cell (RBC) count relative to a baseline count.

[0109] In some embodiments, the therapeutically effective amount of the BTKi is sufficient to increase the hematocrit relative to a baseline hematocrit.

[0110] In some embodiments, the therapeutically effective amount of the BTKi is sufficient to increase hemoglobin levels relative to a baseline hemoglobin level.[OHl] In some embodiments, the therapeutically effective amount of the BTKi is sufficient to increase mean corpuscular hemoglobin concentration (MCHC) relative to a baseline MCHC.Attorney Docket No. 01183-0329-00PCT-PRN

[0112] In some embodiments, the therapeutically effective amount of the BTKi is sufficient to increase the reticulocyte count relative to a baseline reticulocyte count.

[0113] In some embodiments, the therapeutically effective amount of the BTKi is sufficient to increase the mean corpuscular volume (MCV) relative to a baseline MCV.

[0114] In some embodiments, the therapeutically effective amount of the BTKi is sufficient to increase the mean corpuscular hemoglobin (MCH) relative to a baseline MCH.

[0115] In some embodiments, the therapeutically effective amount of the BTKi is sufficient to reduce or prevent NF-KB activation. In some embodiments, the therapeutically effective amount of the BTKi is sufficient to reduce NF-KB activation. In some embodiments, the therapeutically effective amount of the BTKi is sufficient to prevent NF-KB activation.

[0116] In some embodiments, the therapeutically effective amount of the BTKi is sufficient to reduce or prevent p65 ser536 phosphorylation. In some embodiments, the therapeutically effective amount of the BTKi is sufficient to reduce p65 ser536 phosphorylation. In some embodiments, the therapeutically effective amount of the BTKi is sufficient to prevent p65 ser536 phosphorylation.

[0117] In some embodiments, the therapeutically effective amount of the BTKi inhibitor is sufficient to reduce or prevent the release and / or expression of at least one of P- selectin and von Willebrand factor (VWF) from Weibel-Palade bodies in vascular endothelial cells. In some embodiments, the therapeutically effective amount of the BTKi inhibitor is sufficient to reduce the release and / or expression of at least one of P-selectin and von Willebrand factor (VWF) from Weibel-Palade bodies in vascular endothelial cells. In some embodiments, the therapeutically effective amount of the BTKi inhibitor is sufficient to prevent the release and / or expression of at least one of P-selectin and von Willebrand factor (VWF) from Weibel-Palade bodies in vascular endothelial cells.

[0118] In some embodiments, the therapeutically effective amount of the BTKi inhibitor is sufficient to reduce or prevent the release of at least one of P-selectin and von Willebrand factor (VWF) from Weibel-Palade bodies in vascular endothelial cells. In some embodiments, the therapeutically effective amount of the BTKi inhibitor is sufficient to reduce or prevent the expression of at least one of P-selectin and von Willebrand factor (VWF).

[0119] In some embodiments, the therapeutically effective amount of the BTKi inhibitor is sufficient to reduce or prevent the release and / or expression of P-selectin. In some embodiments, the therapeutically effective amount of the BTKi inhibitor is sufficient to reduce or prevent the release and / or expression of VWF.Attorney Docket No. 01183-0329-00PCT-PRN

[0120] In some embodiments, the therapeutically effective amount of BTKi is sufficient to reduce the level of at least one of BTK, P-selectin, E-selectin / C62L, complement Clqb / C3 / C5ar2, MPO, CeaCaml, thrombin, fibronectin, caspase-1, interleukin ip, interleukin 18, and NACHT / LRR / PYD protein 3 in at least one organ chosen from the spleen, lung, liver, and kidney. In some embodiments, the therapeutically effective amount of BTKi is sufficient to reduce the level of at least one of BTK, caspase-1, NACHT / LRR / PYD protein 3, interleukin ip, and interleukin 18. In some embodiments, the therapeutically effective amount of BTKi is sufficient to reduce the level of at least one of P-selectin and E- selectin / C62L. In some embodiments, the therapeutically effective amount of BTKi is sufficient to reduce the level of complement Clqb / C3 / C5ar2. In some embodiments, the therapeutically effective amount of BTKi is sufficient to reduce the level of at least one of MPO and CeaCaml. In some embodiments, the therapeutically effective amount of BTKi is sufficient to reduce the level of at least one of thrombin and fibronectin.

[0121] In some embodiments, the organ is the spleen. In some embodiments, the organ is the lung. In some embodiments, the organ is the liver. In some embodiments, the organ is the kidney.

[0122] In some embodiments, the therapeutically effective amount of the BTKi is sufficient to reduce the total oxidative stress.

[0123] In some embodiments, the therapeutically effective amount of the BTKi is sufficient to reduce red blood cell (RBC) oxidative stress.

[0124] In some embodiments, the therapeutically effective amount of the BTKi is sufficient to reduce the level of malondialdehyde (MDA).

[0125] In some embodiments, the therapeutically effective amount of the BTKi is sufficient to reduce the level of fibrin.

[0126] In some embodiments, the therapeutically effective amount of the BTKi is sufficient to reduce the level of D-dimer.

[0127] In some embodiments, the therapeutically effective amount of the BTKi is sufficient to reduce the level of neutrophil elastase.

[0128] In some embodiments, the therapeutically effective amount of the BTKi is sufficient to reduce the level of myeloperoxidase (MPO).

[0129] In some embodiments, the therapeutically effective amount of the BTKi is sufficient to reduce the level of citrullinated histone H3.

[0130] In some embodiments, the therapeutically effective amount of the BTKi is sufficient to reduce the level of cell-free DNA.Attorney Docket No. 01183-0329-00PCT-PRN

[0131] In some embodiments, the therapeutically effective amount of the BTKi treats or prevents one or more of: musculoskeletal pain, acute chest syndrome, hepatic crisis, sequestration crisis, priapism, hand foot syndrome, dactylitis, fatigue, SCD-associated cognitive impairment, or organ damage, including stroke, transient ischemic attack, small multiple brain infarctions, pulmonary hypertension, SCD-associated renal impairment, SCD- associated renal failure, hepatic crisis, splenic sequestration crisis, leg ulcers, SCD-associated avascular necrosis.

[0132] In some embodiments, the therapeutically effective amount of the BTKi increases the velocity of blood flow in the brain, as measured by transcranial Doppler ultrasound.

[0133] In some embodiments, the BTKi is selected from rilzabrutinib, ibrutinib, tolebrutinib, PRN2675, fenebrutinib, evobrutinib, orelabrutinib, remibrutinib, BUB-091, tirabrutinib, acalabrutinib, vecabrutinib, zanubrutinib, poseltinib, pirtobrutinib, spebrutinib, olmutinib, branebrutinib, TAK-020, elsubrutinib, and tolebrutinib.

[0134] In some embodiments, the BTKi is rilzabrutinib.EXAMPLES

[0135] The following example is intended to be illustrative and is not meant in any way to limit the scope of the disclosure.Abbreviations:Abbreviation DefinitionBID Twice a dayBW Body weightBTK Bruton’s tyrosine kinaseBTKi BTK inhibitor cDNA Complementary DNA cfDNA Cell-free DNACO2Carbon dioxideEDTA Ethylenediaminetetraacetic acidELISA Enzyme-linked immunosorbent assayEtOH EthanolF FemaleAttorney Docket No. 01183-0329-00PCT-PRNHb HemoglobinHbAA Non-sickle hemoglobinHbS / HbSS Sickle hemoglobinHEPA High efficiency particulate absorbingIACUC Institutional animal care and use committeeIP Intraperitoneal injectionJAX Jackson LaboratoryM Male mAb Monoclonal antibodyMCH Mean corpuscular hemoglobinMCHC Mean corpuscular hemoglobin concentrationMCV Mean corpuscular volumeMDA MalondialdehydeMPO MyeloperoxidaseN NumberNBF Neutral buffered formalinNational Center for BiotechnologyNCBIInformationPO Oral administrationPBMC Peripheral blood mononuclear cellQD Once a dayQWK Once a weekQuantitative real time polymerase chain qRT-PCR reactionRBC Red blood cellSCD Sickle Cell DiseaseTAT Thrombin antithrombinVWF Von Willebrand factorWBC White blood cellAttorney Docket No. 01183-0329-00PCT-PRNExample 1: The Effect of BTKi Pretreatment on the Response to Hypoxia / Reoxygenation or Hemoglobin Challenge in the Townes Sickle Cell Disease Mouse Model.A study was initiated to investigate the effect of rilzabrutinib on ischemia / reperfusion (IR) and hemoglobin (Hb) injury in Townes sickle cell disease (SCD) mice. To this end, Townes mice expressing the human wild-type (HbAA) or sickle (HbSS) hemoglobin gene in place of the mouse hemoglobin gene were pretreated with 40 mg / kg BID rilzabrutinib, 15 mg / kg QD irreversible BTKi (PRN2675), or 200 pg QWK anti-P-selectin antibody (RB40.34) for two weeks prior to hypoxia / reoxygenation (HR; to induce IR) or Hb challenge.Animal Model (Test System)

[0136] Townes mice express the HbAA or HbSS hemoglobin gene in place of the mouse hemoglobin gene. Subsequently, mice homozygous for the HbSS allele exhibit RBC sickling under hypoxia, elevated hemolysis, anemia, elevated leukocyte levels, and organ damage similar to clinical disease. Further details are provided in Table 3.

[0137] Table 3. Animal Model (Test System).Experimental Design and Timeline

[0138] N=72 Townes mice aged 10-14 weeks were assigned to the six groups shown in Table 4 (N=12 per group). Animals in Groups 1 and 2 were further divided into twoAttorney Docket No. 01183-0329-00PCT-PRN cohorts (N=6 per cohort), corresponding to once- (QD) or twice-daily (BID) dosing with the vehicle control.

[0139] All groups received pretreatment with either the vehicle control or drug for two weeks prior to the time of challenge and evaluation of stasis. Mice in Group 3 and the BID cohorts of Groups 1 and 2 received a total of 27 doses, while mice in Group 4 and the QD cohorts of Groups 1 and 2 received a total of 14 doses. For animals in each of Groups 1- 4, the final dose was administered two hours prior to the time of challenge and stasis. Mice in Groups 5 and 6 received a total of three doses, which were administered on Days 1, 8, and (24 hours prior to challenge and evaluation of stasis), respectively.

[0140] Mice were aged 12-16 weeks at the time of challenge and evaluation of stasis.

[0141] Table 4. Experimental Design.Attorney Docket No. 01183-0329-00PCT-PRNPretreatment

[0142] Mice in each of Groups 1-6 were treated as described in Table 4. Animals in Groups 1-4 were administered the vehicle control or drug via oral gavage (PO), while animals in Groups 5 and 6 were administered the antibody treatment via intraperitoneal injection (IP).

[0143] Rilzabrutinib was suspended in 0.5% methyl cellulose in water (between 1- 100 mg / mL). This formulation was stable for 15 days when refrigerated at 2-8°C. The formulation was mixed or vortexed thoroughly to provide a homogeneous suspension prior to dosing.

[0144] The irreversible BTKi was suspended at 1.5 mg / mL in 0.5% methyl cellulose / 0.2% Tween® 80 in water. The formulation was mixed or vortexed thoroughly to provide a homogeneous suspension prior to dosing.

[0145] The anti-P-selectin antibody (rat anti-mouse CD62P, RB40.34, #553741, BD Pharmingen™) is a monoclonal P-selectin-blocking IgGIX antibody, while the control antibody (rat anti-keyhole limpet hemocyanin (KLH), #559157, BD Pharmingen™) is a monoclonal isotype control IgG I Z. antibody targeting the immunostimulatory KLH antigen. Both antibodies are purified NA / LE (azide-free / low endotoxin). The antibodies were prepared in saline and administered as previously described (Novoyatleva et al. 2019).Evaluation of Microvascular Stasis after HR Challenge or Hb Injury

[0146] After pretreatment with the vehicle control or drug, mice were anesthetized with ketamine and xylazine, weighed, and implanted with a dorsal skinfold chamber (DSFC). Following surgery, the mice were placed on an intravital microscopy stage, and at least 20 flowing subcutaneous venules were selected and mapped.

[0147] Next, mice from each of the experimental groups were challenged with either HR or Hb injury. Mice in the HR challenge cohort were subjected to hypoxia (7% O2, 93% N2) for one hour prior to return to normoxia. Mice in the Hb injury cohort were infused with 1 pmol / kg cell-free human hemoglobin A via their tail vein.Attorney Docket No. 01183-0329-00PCT-PRN

[0148] At 1, 2, 3, and 4 hours after HR challenge or Hb injury, the flowing venules selected at baseline were examined for signs of stasis (no blood flow). The percentage of static venules was determined at each time point for each treatment group.Lung LavageIf possible, lung lavage was performed following evaluation of stasis. Lung lavage samples were flash frozen in liquid nitrogen and stored at -80°C.Terminal Procedures / Necropsy

[0149] Mice were euthanized via CO2 narcosis.

[0150] Terminal Blood Collection: -350 pL of whole blood was collected by cardiac puncture, aliquoted into a tube with EDTA as an anticoagulant and placed on ice. An aliquot of whole blood was collected for a manual complete blood count (CBC). The remaining whole blood was processed, and -100 pL platelet-poor plasma was aliquoted, flash froze in liquid nitrogen, and stored at -80°C for plasma analysis.

[0151] Tissue Collection: Organs, including the lungs, liver, kidney, and spleen, were harvested for further analysis.

[0152] The left lung lobe was tied off, and the right lobe was inflated in 50% optimal cutting temperature (OCT) compound in PBS. The inflated lung was excised and placed in Zamboni fixative for P-selectin, von Willebrand factor (VWF), and CD31 immunofluorescence (IF) before being flash frozen in liquid nitrogen and stored at -80°C. The non-inflated lobe was wrapped in aluminum foil, flash frozen in liquid nitrogen, and stored at -80°C prior to RNA analysis.

[0153] For the liver, two small lobes were excised for RNA analysis. Liver samples were wrapped separately in aluminum foil, flash frozen in liquid nitrogen, and stored at - 80°C.

[0154] The spleen was weighed, flash frozen in liquid nitrogen, and stored at -80°C prior to RNA analysis.

[0155] Both kidneys were excised, wrapped separately in aluminum foil, flash frozen in liquid nitrogen, and stored at -80°C prior to RNA analysis.

[0156] Frozen liver and kidney samples were shipped for RNAseq analysis.Attorney Docket No. 01183-0329-00PCT-PRNIF

[0157] The release of P-selectin and VWF from Weibel-Palade bodies of the endothelium is associated with inflammation and hemostasis. Thus, IF was used to examine P-selectin and VWF expression in N=3 OCT lung samples per treatment and challenge (a total of N=36 lung samples). The expression of P-selectin and VWF in lung blood vessels was compared to expression of the endothelial cell marker CD31. Images were visualized with FluoView and processed with Napari (Selzer et al. 2023), a multi-dimensional image viewer for Python. The number of P-selectin- and VWF-positive pixels was quantified and divided by the number of CD31 pixels in the image to generate the percent of positive pixels for each protein of interest. Results were counted from 3 independent image fields collected from each lung (N=3) for a total of 9 fields used for each condition. Results were expressed as mean percent ± SEM.Manual CBC

[0158] CBCs were measured manually for all mice using fresh blood samples. CBC included counts for white blood cells (WBCs), red blood cells (RBCs), hemoglobin (Hb), hematocrit (Het, packed cell volume), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), absolute and percent reticulocytes, absolute and percent neutrophils, absolute and percent lymphocytes, absolute and percent monocytes, absolute and percent basophils, and absolute and percent eosinophils. There were no apparent differences among HbSS treatment groups for any RBC indices.Liver Inflammation

[0159] Liver inflammation was evaluated via Western blotting (WB). WB was performed on nuclear extracts from N=3 livers per treatment and challenge, resulting in a total of N=36 samples. Nuclear extracts were isolated as previously described (Belcher et al. 2010). Nuclear subfractions (30 pg protein) were resolved on SDS-PAGE 4-20% gels (BioRad™) and transferred to PVDF membranes. Blots were immunostained with primary antibodies to NF-KB phospho-p65 (Ser536, Cell Signaling™ #3303, 1 : 1000) and total p65 (Cell Signaling™ #8242, 1 : 1000). Primary antibodies were detected with goat anti -rabbit (Abeam™ #ab97048, 1 :5000) secondary antibodies conjugated to alkaline phosphatase and visualized with ECF™ substrate (GE Healthcare™) and an Azure Biosystems C600 imager. Relative phospho- and total p65 band intensities on immunoblot images were measured usingAttorney Docket No. 01183-0329-00PCT-PRNFIJI software (NIH). Integrated phospho(ser536)-p65 band intensities were expressed relative to the total p65 band intensities as a measure of NF-KB activation and inflammatory responses to HR and Hb proinflammatory challenges (Sakurai et al. 2003).RNA sequencing

[0160] RNA from kidney and the left lobe of the liver was extracted using the RNeasy® Mini Kit (Qiagen). RNA integrity number and concentration were determined by Tapestation (Agilent) and Qubit™ Fluorometer (Invitrogen), respectively.

[0161] Transcriptomic analysis was performed using Twist capture exome sequencing on kidney extracts and liver extracts from the left lobe. To evaluate the mouse exome library preparation kit from Twist for high quality RNA, three liver and four kidney samples were selected to perform both mRNA library preparation with TruSeq™ Stranded mRNA kit (Illumina) and mouse exome capture library (Twist). For low quality RNA, four liver and four kidney samples were used and only mouse exome capture test was performed.

[0162] ELISA Assays

[0163] ELISA Sensitivity Piloting: Frozen plasma samples were shipped for measurement of netosis markers, including cell free DNA (cfDNA), neutrophil elastase (NE), myeloperoxidase (MPO), and citrullinated histone 3 (H3cit). a. Abeam™ mouse Neutrophil Elastase ELISA kit (ab252356) for neutrophil elastaseBlood was collected in EDTA tubes and processed to plasma by centrifuging for 10 min at 2000 x g at room temperature. Plasma was diluted 1 : 10 in sample diluent NS. b. Abeam™ mouse Myeloperoxidase ELISA kit (ab275109) for myeloperoxidase (MPO)Blood was collected in EDTA tubes and processed to plasma by centrifuging for 10 min at 2000 x g at room temperature. Plasma was diluted 1 : 10 in Sample diluent NS. c. Cayman™ Citrullinated Histone H3 ELISA kit (501620) for citrullinated H3Blood was collected in tubes with 3.8% sodium citrate as an anti-coagulant and processed to plasma by centrifuging for 10 min at 2000 x g at room temperature. Plasma was diluted a minimum of 1 :2 in assay buffer.Attorney Docket No. 01183-0329-00PCT-PRN d. qPCR for cell free DNA2 kits found: Active Motif Cell-Free DNA (cfDNA) purification kit (cat# 25503).Results

[0164] Microvascular Stasis in Response to a Proinflammatory Challenge: HbAA control mice and HbSS mice treated with rilzabrutinib, the irreversible BTKi, or an anti-P- selectin mAb prior to challenge with HR (Fig. 13A) or intravenous Hb (Fig. 13B) exhibited a significant reduction microvascular stasis (vaso-occlusion) at 1, 2, 3, and 4 hours, as compared to HbSS mice treated with the vehicle control or the isotype control mAb.

[0165] Spleen Weight as a Percentage of Body Weight: Body weights were measured prior to DSFC implantation. Organs were collected after the final stasis measurement, 4 hours after challenge. Spleen weight as a percentage of body weight was significantly greater in HbSS mice compared to HbAA mice (p<0.001). Spleen weight was significantly lower in HbSS mice treated with either rilzabrutinib (p<0.001) or the irreversible BTKi (p<0.01; Fig. 14) relative to HbSS mice treated with the vehicle control.

[0166] Leukocyte Counts: Complete blood counts were measured in terminal blood samples supplemented with EDTA (Figs. 15A-D). White blood cell (WBC; Fig. 15A), lymphocyte (Fig. 15B), neutrophil (Fig. 15C), and monocyte (Fig. 15D) counts were significantly higher in HbSS sickle mice treated with vehicle as compared to HbAA control mice treated with vehicle (p<0.001 for all cell types). WBC counts were significantly lower in HbSS mice treated with rilzabrutinib (p<0.01), the irreversible BTKi (p<0.001), or anti-P- selectin (p<0.01). Similarly, lymphocyte counts were significantly lower in HbSS mice treated with either of the BTK inhibitors or anti-P-selectin (p<0.001 for all treatments). Neutrophil counts in HbSS treatment groups were not significantly different from vehicle treated HbSS mice. However, monocytes were significantly lower in HbSS mice treated with the irreversible BTKi as compared to HbSS mice treated with the vehicle control (p<0.01).

[0167] RBC Counts and RBC Indices: RBC counts and RBC indices were also measured in EDTA-treated terminal blood samples (Figs. 16A-G). Red blood cell (RBC) levels including RBC counts (Fig. 16A), hematocrits (Het; Fig. 16B), hemoglobin (Hb; Fig. 16C), and MCHC (Fig. 16D) levels, were lower, and reticulocyte counts (Retie; Fig. 16E) and MCV (Fig. 16F) were higher in vehicle-treated HbSS mice compared to vehicle treated control HbAA mice (p<0.001 for all counts and indices). MCH was not different between vehicle-treated HbAA and HbSS mice (Fig. 16G). Additionally, there were no significant differences between any treatment groups for any of the RBC measurements.Attorney Docket No. 01183-0329-00PCT-PRN

[0168] NF-KB Activation in the Liver in Response to ProinflammatoryChallenge: HbAA and HbSS mice treated with rilzabrutinib, the irreversible BTKi, or the anti-P-selectin mAb prior to challenge with HR (Figs. 17A-B) or hemoglobin A (Hb, Figs. 17C-D) had significantly reduced phospho-p65 expression (indicating decreased NF-KB activation) as compared to HbSS mice treated with vehicle or isotype control mAb (p<0.001 for Groups 3-5).

[0169] Lung Inflammation in Response to Proinflammatory Challenge: As a marker of inflammation, lung P-selectin and VWF expression on blood vessel endothelial cells (CD31+) in the lungs was measured by IF staining. HbAA control mice and HbSS sickle mice were treated with rilzabrutinib, the irreversible BTKi, or an anti-P-selectin mAb prior to challenge with HR (Figs. 18A-C) or hemoglobin (Figs. 18D-F). In mice challenged with HR, representative lung P-selectin and VWF images are presented (Fig. 18A) and P- selectin (Fig. 18B) and VWF (Fig. 18C) were quantified by the number of positive pixels and dividing that by the number of CD31 pixels in the image to generate P-selectin / CD31 and VWF / CD31 ratios. Results were counted from 3 independent image fields collected from each lung (N=3 lungs per treatment and challenge) for a total of 9 fields used for each condition. In mice challenged with HR, vehicle-treated HbSS mice had significantly increased lung P-selectin and VWF expression as compared to HbAA mice (p=0.002 for both). Importantly, HbSS mice treated with rilzabrutinib or the irreversible BTKi had significantly reduced P-selectin expression (p=0.023 and p=0.002, respectively) and VWF expression (p=0.029 and p=0.002, respectively) in the lungs as compared to the lungs of HbSS mice treated with vehicle after an HR challenge.

[0170] In mice challenged with Hb, representative lung P-selectin and VWF images are presented (Fig. 18D) and P-selectin (Fig. 18E) and VWF (Fig. 18F) were quantified. In mice challenged with Hb, vehicle treated HbSS mice had increased lung P-selectin expression (p=0.004) and VWF expression (p=0.019) as compared to HbAA mice. Importantly, HbSS mice treated with rilzabrutinib or the irreversible BTKi had reduced P- selectin expression in the lungs as compared to the lungs of HbSS mice treated with vehicle after an Hb challenge (p=0.016 and p=0.039, respectively). HbSS mice treated with the irreversible BTKi had significantly reduced VWF expression in the lungs as compared to the lungs of HbSS mice treated with vehicle after an Hb challenge (0=0.006). Quantification of P-selectin and VWF in mice treated with anti-P-selectin or control antibody is not shown due to high variability between mouse lungs in these mice.Attorney Docket No. 01183-0329-00PCT-PRN

[0171] Gene Expression in Response to Proinflammatory Challenge:Rilzabrutinib pretreatment altered the expression of genes associated with the pathophysiology of SCD in both the kidney and liver. In particular, rilzabrutinib pretreatment led to a decrease in expression of genes associated with the inflammasome, cell adhesion, the complement system, and thrombosis in the kidney and liver of HbSS mice challenged with HR or Hb injury (Table 5 and Figs. 19A-E).

[0172] In kidney samples from SCD mice challenged with Hb, treatment with rilzabrutinib resulted in a statistically significant downregulation of genes associated with adhesion, TLR5, Endothelilin-1, Myeloperoxidase, and Neutrophil elastase relative to treatment with the vehicle control. Meanwhile, in kidney samples from animals subjected to HR, rilzabrutinib pretreatment was associated with a statistically significant reduction in CCL4 relative to pretreatment with the vehicle control. For mice treated with rilzabrutinib, a statistically significant downregulation of TNF-a, CCL3, and CXCL10 was observed in both HR and lib models.

[0173] In liver samples from SCD mice challenged with Hb, treatment with rilzabrutinib resulted in downregulation of inflammasome-related genes (e.g., BTK, caspase- 1, NLRP3, IL1[3, and IL- 18), pro-inflammatory cytokines and chemokines, and transcription factors. Notably, rilzabrutinib pretreatment was associated with a statistically significant reduction in liver BTK, ASC / Pycard, NLRP3, Caspase- 1, TNF-a, CXCL10, and NF-KB expression following Hb injury. Rilzabrutinib pretreatment was also associated with a statistically significant decrease in liver IL- 10 expression following HR challenge. In both HR and Hb models, rilzabrutinib treatment led to a downregulation of gene expression of markers of inflammation, markers of adhesion (e.g., P-selectin), and complement (e.g., complement Clqb / C3 / C5ar2), NETosis (e.g., MPO).

[0174] Table 5. Summary of Gene Expression in Response to Proinflammatory Challenge in Mice Treated with Rilzabrutinib or the Vehicle ControlAtorney Docket No. 01183-0329-00PCT-PRNAttorney Docket No. 01183-0329-00PCT-PRNArrows indicate a statistically significant (<0.05) increase ) or decrease (J,) in expression of the indicated gene in animals treated with rilzabrutinib relative to animals treated with the vehicle control.Embodiments:

[0175] Non-limiting embodiments of the disclosure include:1. A method of treating sickle cell disease (SCD) in a mammal in need thereof, comprising administering to the mammal in need thereof a therapeutically effective amount of a BTK inhibitor (BTKi).2. A method of treating sickle cell disease (SCD) in a patient in need thereof, comprising administering to the patient in need thereof a therapeutically effective amount of a BTK inhibitor (BTKi).3. The method of Embodiment 1 or 2, wherein treating SCD comprises reducing or preventing sickle cell crises.4. The method of any one of the preceding Embodiments, wherein treating SCD comprises reducing or preventing SCD-related inflammation.Attorney Docket No. 01183-0329-00PCT-PRN5. The method of any one of the preceding Embodiments, wherein treating SCD comprises reducing or preventing hemoglobin S polymerization.6. The method of any one of the preceding Embodiments, wherein treating SCD comprises reducing or preventing red blood cell (RBC) sickling.7. The method of any one of the preceding Embodiments, wherein treating SCD comprises reducing or preventing SCD-related hemolysis.8. The method of any one of the preceding Embodiments, wherein treating SCD comprises increasing hemoglobin levels.9. The method of any one of the preceding Embodiments, wherein treating SCD comprises reducing cell-free hemoglobin levels.10. The method of any one of the preceding Embodiments, wherein treating SCD comprises reducing or preventing SCD-related anemia.11. The method of any one of the preceding Embodiments, wherein treating SCD comprises reducing or preventing SCD-related hemostasis.12. The method of any one of the preceding Embodiments, wherein treating SCD comprises reducing or preventing SCD-related vaso-occlusion.13. The method of any one of the preceding Embodiments, wherein treating SCD comprises reducing or preventing SCD-related ischemia / reperfusion.14. The method of any one of the preceding Embodiments, wherein treating SCD comprises reducing or preventing SCD-related thromboinflammation.15. The method of any one of the preceding Embodiments, wherein treating SCD comprises reducing or preventing SCD-related vasculopathy.Attorney Docket No. 01183-0329-00PCT-PRN16. The method of any one of the preceding Embodiments, wherein treating SCD comprises reducing or preventing SCD-related cell adhesion.17. The method of any one of the preceding Embodiments, wherein treating SCD comprises reducing or preventing SCD-related organ damage.18. The method of any one of the preceding Embodiments, wherein the therapeutically effective amount of the BTKi is sufficient to reduce the volume or weight of the spleen relative to a baseline volume or weight.19. The method of any one of the preceding Embodiments, wherein the therapeutically effective amount of the BTKi is sufficient to reduce the white blood cell count relative to a baseline count.20. The method of any one of the preceding Embodiments, wherein the therapeutically effective amount of the BTKi is sufficient to reduce the lymphocyte count relative to a baseline count.21. The method of any one of the preceding Embodiments, wherein the therapeutically effective amount of the BTKi is sufficient to reduce the neutrophil count relative to a baseline count.22. The method of any one of the preceding Embodiments, wherein the therapeutically effective amount of the BTKi is sufficient to reduce the monocyte count relative to a baseline count.23. The method of any one of the preceding Embodiments, wherein the therapeutically effective amount of the BTKi is sufficient to increase the red blood cell (RBC) count relative to a baseline count.24. The method of any one of the preceding Embodiments, wherein the therapeutically effective amount of the BTKi is sufficient to increase the hematocrit relative to a baseline hematocrit.Attorney Docket No. 01183-0329-00PCT-PRN25. The method of any one of the preceding Embodiments, wherein the therapeutically effective amount of the BTKi is sufficient to increase hemoglobin levels relative to a baseline hemoglobin level.26. The method of any one of the preceding Embodiments, wherein the therapeutically effective amount of the BTKi is sufficient to increase mean corpuscular hemoglobin concentration (MCHC) relative to a baseline MCHC.27. The method of any one of the preceding Embodiments, wherein the therapeutically effective amount of the BTKi is sufficient to increase the reticulocyte count relative to a baseline reticulocyte count.28. The method of any one of the preceding Embodiments, wherein the therapeutically effective amount of the BTKi is sufficient to increase the mean corpuscular volume (MCV) relative to a baseline MCV.29. The method of any one of the preceding Embodiments, wherein the therapeutically effective amount of the BTKi is sufficient to increase the mean corpuscular hemoglobin (MCH) relative to a baseline MCH.30. The method of any one of the preceding Embodiments, wherein the therapeutically effective amount of the BTKi is sufficient to reduce or prevent NF-KB activation.31. The method of any one of the preceding Embodiments, wherein the therapeutically effective amount of the BTKi is sufficient to reduce or prevent p65 ser536 phosphorylation.32. The method of any one of the preceding Embodiments, wherein the therapeutically effective amount of the BTKi inhibitor is sufficient to reduce or prevent the release and / or expression of at least one of P-selectin and von Willebrand factor (VWF) from Weibel- Palade bodies in vascular endothelial cells.33. The method of any one of the preceding Embodiments, wherein the therapeutically effective amount of the BTKi is sufficient to reduce the level of at least one of BTK, ASC / Pycard, NLRP3, Caspase- 1, IL-ip, TNF-a, CCL3, CCL4, CXCL10, IL- 10, VCAM-1,Attorney Docket No. 01183-0329-00PCT-PRNICAM-1, TLR5, NF-KB1, C5arl, C5ar2, ClqB, Endothelin-1, Myeloperoxidase, or Neutrophil elastase in at least one organ chosen from the spleen, lung, liver, and kidney.34. The method of any one of the preceding Embodiments, wherein the therapeutically effective amount of the BTKi is sufficient to reduce the total oxidative stress.35. The method of any one of the preceding Embodiments, wherein the therapeutically effective amount of the BTKi is sufficient to reduce red blood cell (RBC) oxidative stress.36. The method of any one of the preceding Embodiments, wherein the therapeutically effective amount of the BTKi is sufficient to reduce the level of malondialdehyde (MDA).37. The method of any one of the preceding Embodiments, wherein the therapeutically effective amount of the BTKi is sufficient to reduce the level of fibrin.38. The method of any one of the preceding Embodiments, wherein the therapeutically effective amount of the BTKi is sufficient to reduce the level of D-dimer.39. The method of any one of the preceding Embodiments, wherein the therapeutically effective amount of the BTKi is sufficient to reduce the level of neutrophil elastase.40. The method of any one of the preceding Embodiments, wherein the therapeutically effective amount of the BTKi is sufficient to reduce the level of myeloperoxidase (MPO).41. The method of any one of the preceding Embodiments, wherein the therapeutically effective amount of the BTKi is sufficient to reduce the level of citrullinated histone H3.42. The method of any one of the preceding Embodiments, wherein the therapeutically effective amount of the BTKi is sufficient to reduce the level of cell-free DNA.43. The method of any one of the preceding Embodiments, wherein the therapeutically effective amount of the BTKi treats or prevents one or more of: musculoskeletal pain, acute chest syndrome, hepatic crisis, sequestration crisis, priapism, hand foot syndrome, dactylitis, fatigue, SCD-associated cognitive impairment, or organ damage, including stroke, transientAttorney Docket No. 01183-0329-00PCT-PRN ischemic attack, small multiple brain infarctions, pulmonary hypertension, SCD-associated renal impairment, SCD-associated renal failure, hepatic crisis, splenic sequestration crisis, leg ulcers, SCD-associated avascular necrosis.44. The method of any one of the preceding Embodiments, wherein the therapeutically effective amount of the BTKi increases the velocity of blood flow in the brain, as measured by transcranial Doppler ultrasound.45. The method of any one of the preceding Embodiments, wherein the BTKi is selected from rilzabrutinib, ibrutinib, tolebrutinib, PRN2675, fenebrutinib, evobrutinib, orelabrutinib, remibrutinib, BIIB-091, tirabrutinib, acalabrutinib, vecabrutinib, zanubrutinib, poseltinib, pirtobrutinib, spebrutinib, olmutinib, branebrutinib, TAK-020, elsubrutinib, and tolebrutinib.46. The method of Embodiment 45, wherein the BTKi is rilzabrutinib.47. A BTKi for use in the method of any one of the preceding Embodiments.48. Use of a BTKi in the manufacture of a medicament of a medicament for treating SCD according to the method of any one of Embodiments 1-46.

[0176] Claims or descriptions that include “or” or “and / or” between at least one members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The disclosure includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The disclosure includes embodiments in which more than one, or all the group members are present in, employed in, or otherwise relevant to a given product or process.

[0177] Where ranges are given, endpoints are included. Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value orAttorney Docket No. 01183-0329-00PCT-PRN sub-range within the stated ranges in different embodiments of the disclosure, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0178] The foregoing disclosure has been described in some detail by way of illustration and example, for purposes of clarity and understanding. Therefore, it is to be understood that the above description is intended to be illustrative and not restrictive. The scope of the disclosure should, therefore, be determined not with reference to the above description, but should instead be determined with reference to the following appended claims, along with the full scope of equivalents to which such claims are entitled.

Claims

Attorney Docket No. 01183-0329-00PCT-PRNWhat is claimed is:

1. A method of treating sickle cell disease (SCD) in a patient in need thereof, comprising administering to the patient in need thereof a therapeutically effective amount of a BTK inhibitor (BTKi).

2. The method of claim 1, wherein treating SCD comprises reducing or preventing sickle cell crises.

3. The method of claim 1 or claim 2, wherein treating SCD comprises reducing or preventing SCD-related inflammation.

4. The method of any one of the preceding claims, wherein treating SCD comprises reducing or preventing hemoglobin S polymerization.

5. The method of any one of the preceding claims, wherein treating SCD comprises reducing or preventing red blood cell (RBC) sickling.

6. The method of any one of the preceding claims, wherein treating SCD comprises reducing or preventing SCD-related hemolysis.

7. The method of any one of the preceding claims, wherein treating SCD comprises increasing hemoglobin levels.

8. The method of any one of the preceding claims, wherein treating SCD comprises reducing cell-free hemoglobin levels.

9. The method of any one of the preceding claims, wherein treating SCD comprises reducing or preventing SCD-related anemia.

10. The method of any one of the preceding claims, wherein treating SCD comprises reducing or preventing SCD-related hemostasis.Attorney Docket No. 01183-0329-00PCT-PRN11. The method of any one of the preceding claims, wherein treating SCD comprises reducing or preventing SCD-related vaso-occlusion.

12. The method of any one of the preceding claims, wherein treating SCD comprises reducing or preventing SCD-related ischemia / reperfusion.

13. The method of any one of the preceding claims, wherein treating SCD comprises reducing or preventing SCD-related thromboinflammation.

14. The method of any one of the preceding claims, wherein treating SCD comprises reducing or preventing SCD-related vasculopathy.

15. The method of any one of the preceding claims, wherein treating SCD comprises reducing or preventing SCD-related cell adhesion.

16. The method of any one of the preceding claims, wherein treating SCD comprises reducing or preventing SCD-related organ damage.

17. The method of any one of the preceding claims, wherein the therapeutically effective amount of the BTKi is sufficient to reduce the volume or weight of the spleen relative to a baseline volume or weight.

18. The method of any one of the preceding claims, wherein the therapeutically effective amount of the BTKi is sufficient to reduce or prevent NF-KB activation.

19. The method of any one of the preceding claims, wherein the therapeutically effective amount of the BTKi inhibitor is sufficient to reduce or prevent the release and / or expression of at least one of P-selectin and von Willebrand factor (VWF) from Weibel-Palade bodies in vascular endothelial cells.

20. The method of any one of the preceding claims, wherein the therapeutically effective amount of the BTKi is sufficient to reduce the level of at least one of BTK, ASC / Pycard, NLRP3, Caspase- 1, IL-ip, TNF-a, CCL3, CCL4, CXCL10, IL- 10, VCAM-1, ICAM-1,Attorney Docket No. 01183-0329-00PCT-PRNTLR5, NF-KB1, C5arl, C5ar2, ClqB, Endothelin-1, Myeloperoxidase, or Neutrophil elastase in at least one organ chosen from the spleen, lung, liver, and kidney.

21. The method of any one of the preceding claims, wherein the BTKi is selected from rilzabrutinib, ibrutinib, tolebrutinib, PRN2675, fenebrutinib, evobrutinib, orelabrutinib, remibrutinib, BIIB-091, tirabrutinib, acalabrutinib, vecabrutinib, zanubrutinib, poseltinib, pirtobrutinib, spebrutinib, olmutinib, branebrutinib, TAK-020, elsubrutinib, and tolebrutinib.

22. The method of any one of the preceding claims, wherein the BTKi is rilzabrutinib.

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