Use of a combination of an agent that increases hdl activity and a lip-soluble antioxidant, e, pharmaceutical composition

Abstract

The present invention relates to a useful combination in the prevention and / or treatment of red blood cell disorders, in particular, acute and chronic complications associated with red blood cell dysfunction, increased cholesterol in red blood cells and decreased plasma levels of lipophilic antioxidants (sickle cell disease, thalassemia, diabetes). The invention in particular relates to pharmaceutical formulations, pharmaceutical regimens, pharmaceutical methods of treatment and uses thereof.

Description

/ 32 USE OF A COMBINATION OF AN AGENT THAT INCREASES HDL ACTIVITY AND A LIPID-SOLUBLE ANTIOXIDANT, AND PHARMACEUTICAL COMPOSITION FIELD OF THE INVENTION

[001] The present invention relates to a combined formulation or to the use of the combined formulation in the prevention and / or treatment of red blood cell disorders, in particular, acute and chronic complications associated with red blood cell dysfunction, increased cholesterol in red blood cells and decreased plasma levels of lipophilic antioxidant (sickle cell disease, thalassemia, diabetes). FUNDAMENTALS OF THE INVENTION

[002] Sickle Cell Anemia (SCA) is the common manifestation of Sickle Cell Disease (SCD) and the most frequent hemoglobinopathy in the world, affecting more than 50 million people worldwide. SCA significantly impairs quality of life and shortens lifespan. Although hemolytic anemia and frequent vaso-occlusive crises are the most common complications in patients suffering from SCA, these patients are also at high risk for developing stroke, osteonecrosis, retinopathy, priapism, leg ulcers, acute chest syndrome, and glomerulopathy (Kato et al., 2007, Blood Rev., 21(1), 37-47). Sickle-shaped Red Blood Cells (RBCs) are very rigid and fragile.The loss of deformability of red blood cells (RBCs) is considered to be the main factor responsible for vaso-occlusive events, severe hemolytic anemia, and progressive organ damage in patients suffering from the aforementioned diseases. It should also be noted that a high concentration of cholesterol in RBCs decreases their capacity for oxygen transport (Buchwald et al., 2000, J. Am. Coll. Surg., 91(5), 490-7; Buchwald). Petition 870260057648, dated 12 / 06 / 2026, p. 17 / 56 / 32 et al., 2000, Clin. Exp. Pharmacol. Fisiol., 27(12): p. 951-5), therefore being detrimental and activating the sickle cell process. It is estimated that 5% of the world's population is affected by these hemoglobinopathies for which very few treatments are available. In addition to SCD and Thalassemia, low HDL levels, high red blood cell membrane cholesterol, high blood viscosity (Stamos and Rosenson, 1999, Atherosclerosis, 146(1), 161-5) and low blood levels of lipophilic antioxidants are also observed in more common pathologies in the general population such as diabetes (Barnes, 1986, Acta Med. Port.,. 7(5-6), S369) and cardiovascular diseases (Namazi et al., 2014, Cholesterol, Article ID:821686; Simon and Silverstein, 2015, Circulation, 132(20), 1860-2) and may be the source of diabetes complications.

[003] Attempts to simultaneously treat the common features that link these dysregulations affecting SCD, thalassemia, and diabetes have not been made, and vascular and microvascular complications of these diseases are still unmet medical needs.

[004] Several studies have reported decreased levels of high-density lipoprotein cholesterol (HDL-C) in SCD (Seixas et al., 2010, Lipids Health Dis., 9, 91) which may result in an increased risk for endothelial dysfunction in patients suffering from this disease (Yuditskaya et al., 2009, Blood, 113(5), 1122-1128). This association could be related to the release of oxidized fatty acids during lipolysis (Sandor et al., 2016, Br. J. Haematol., 173(1), 145-9), resulting in inflammation of endothelial cells (Yuditskaya et al., 2009, supra).

[005] Although it shares common mechanisms with atherosclerosis (oxidative stress, inflammation, and vascular adhesion), ACS vasculopathy differs clearly in that the accumulation of Petition 870260057648, dated 12 / 06 / 2026, page 18 / 56 / 32 cholesterol in the arterial wall and atheroma have not been reported (Zorca et al., 2010, Br. J. Haematol., 149(3), 436-45). Surprisingly, although total plasma lipid and cholesterol levels are usually lower in individuals with ACS than in healthy individuals, the level of total lipids and cholesterol is higher in the RBC membrane of ACS patients compared to healthy control patients (Westerman et al., 1964, Blood, 23, 200-5), which may be related to the decreased plasma level of Lecithin-Cholesterol Acyltransferase (LCAT) found in patients with SCD (Jain et al., 1982, Biochim. Biophys. Acta., 688:11-15, and Emokpae et al., 2010, Afr. J. Biochem. Res., 4:17-20) and more particularly during vaso-occlusive crises (Homan et al., 2013, Anal. Biochem., 441(1), 80-6).

[006] It has been repeatedly observed that patients with Sickle cell disease and thalassemia present with low plasma levels of HDL-C and low plasma levels of LDL-C, but increased levels of oxidized LDL and increased plasma levels of oxidation markers (Voskou et al., 2015, Redox Biology, 6,226-239; Boudrahem-Addour et al., 2015, Hemoglobin, 39(1):36-41; Unchern et al., 2010, Lipids, 45:627-633). The function of HDL in removing cholesterol from peripheral tissues such as macrophages is well established, in contrast to the potential function of RBCs in removing excess cholesterol, which has been investigated and is still insufficiently investigated and therefore offers very limited therapeutic prospects. As early as 1976, it was observed that the red blood cells of patients with SCD are significantly enriched in cholesterol compared to healthy control subjects (Akinyanju et al., 1976, Ann. Clin. Lab. Sci., 6(6):521-4).

[007] It has been shown that L-4F, a mimetic of ApoA1 (US 6,664,230) improves vasodilation in hypercholesterolemia and in an animal model of Sickle Cell Disease (Ou et al., 2003, Circulation, Petition 870260057648, dated 12 / 06 / 2026, page 19 / 56 / 32 107(18): 2337-41) but not in clinical studies with humans. In fact, treatment with L-4F, delivered either by subcutaneous injection or intravenous infusion to patients with cardiovascular disease, did not improve functional HDL biomarkers despite achieving plasma levels that improved identical biomarkers in ex vivo and animal models (Watson, et al., 2011, 52: 361-373, [sic]).

[008] Sickle cell disease, thalassemia, and glucose-6-phosphate dehydrogenase deficiency are all hereditary disorders with a higher potential for oxidative damage due to chronic redox imbalance in red blood cells, which frequently results in clinical manifestations of mild to severe hemolysis in patients with these disorders. Hamdy et al. (2015, Journal of Advanced Research, 6, 1071-1077) found a decreased level of antioxidants in both SCD and thalassemia and considered accelerated oxidative damage as one of the unmistakable characteristics in both SCD and thalassemia. Most importantly, Tangney et al. (1989, Am. J. Hematol., 32(3), 161-6) observed that although dietary analyses suggested that dietary intakes of individuals with ACS exceeded the recommended daily allowances of all macronutrients and micronutrients (specifically, beta-carotene, alpha-carotene, and cryptoxanthin), all serum values ​​for carotenoids examined, specifically beta-carotene, alpha-carotene, and cryptoxanthin, were greatly decreased when compared with those of healthy control individuals. These results suggest that in individuals with ACS, several micronutrients vital for maintaining antioxidant capacities are present in smaller quantities in plasma / serum.

[009] In addition, Natta et al. (1988, Eur. J. Haematol., 41(2), 1315) measured a decrease in the major carotenoids, beta-carotene, cryptoxanthin, lycopene, lutein, and also alpha-tocopherol and retinol in the plasma of patients with ACS. Petition 870260057648, dated 12 / 06 / 2026, p. 20 / 56 / 32

[0010] It has been consistently verified that plasma vitamin E levels are decreased in patients suffering from thalassemia. For example, Zannos-Mariolea et al. (1974, Br. J. Haematol., 26(2), 193-9) reported that serum vitamin E levels were below normal (<0.5 mg / 100 mL) in 46% of the 56 children with beta-thalassemia major (P <0.001) evaluated in their study.

[0011] Low plasma lipophilic antioxidant levels in SCD and thalassemia may intensify the well-documented pathological manifestations of increased oxidative stress in these diseases.

[0012] It is important to note that attempts to provide only vitamin E as a dietary supplement have not been successful in treating SCD pathologies (Muskiet et al., 1991, Am. J. Clin. Nutr., 54(4): p. 736-44).

[0013] Based on an analysis of therapeutic approaches to treat the oxidative status of SCD, vitamin E and other lipophilic antioxidants alone have not been considered an effective therapeutic treatment for SCD (Vichinsky, 2012, Hematology Am. Soc. Hematol. Educ. Program, 271-5).

[0014] SCD is a disease that worsens over time and there is no available treatment. The use of hydroxyurea is the only disease-modifying treatment currently approved for people affected with SCD. Hydroxyurea is used in the prevention of primary and secondary stroke. Although it has been shown to prevent all organ damage related to SCD, treatment modalities can improve the quality of life of individuals suffering from SCD. Stem cell transplants from bone marrow or blood from healthy donors are also used to treat Sickle Cell Anemia. Complications from hydroxyurea therapy and stem cell transplants are rare, but can be serious and life-threatening. Thalassemia syndrome can be a life-threatening hemolytic anemia. Petition 870260057648, dated 12 / 06 / 2026, page 21 / 56 / 32, requesting blood transfusion and removal of excess iron. The management of transfusion-dependent patients with β-thalassemia major remains a challenge for clinicians, and despite an effective transfusion protocol, transfusion hemosiderosis is a major cause of mortality in these patients.

[0015] Therefore, there is an urgent need to discover new treatment approaches for those life-threatening disorders. SUMMARY OF THE INVENTION

[0016] The present invention relates to a novel combination for use as a therapy for the treatment of acute and chronic complications associated with red blood cell dysfunction, increased red blood cell cholesterol and decreased plasma levels of lipophilic antioxidant (sickle cell disease, thalassemia, diabetes).The present invention is based on the surprising finding that the combination of inducing an increase in HDL activity together with a dietary supplement, parenteral, subcutaneous or intravenous administration (as a free or fixed combination) of fat-soluble antioxidants acts synergistically for the treatment of rheological complications of red blood cell disorders, in particular increased cholesterol in red blood cells found in sickle cell disease and also in thalassemia, and will subsequently contribute to the treatment and / or prevention of microvascular damage and associated pathologies (retinopathies, renal failure, leg ulcers, priapism, acute chest syndrome, stroke) such as those found in SCD or thalassemia.Furthermore, the combination according to the invention can advantageously act on the volume of red blood cells, the hydration of red blood cells, the shape of red blood cells, or the rheological properties of red blood cells, by making them more... Petition 870260057648, dated 12 / 06 / 2026, page 22 / 56 / 32 close to the parameters found in healthy patients.

[0017] According to one aspect, the invention provides a combination of at least one agent that increases HDL activity and at least one fat-soluble antioxidant or a mixture thereof for use in the prevention and / or treatment of a disease or disorder distinguished by acute and chronic complications associated with red blood cell dysfunction, increased red blood cell cholesterol and decreased plasma levels of lipophilic antioxidant, in particular for the prevention and / or treatment of acute and chronic complications associated with red blood cell dysfunction in sickle cell disease, thalassemia and diabetes.

[0018] According to another aspect, the invention provides the use of a combination of at least one agent that increases HDL activity and at least one fat-soluble antioxidant or a mixture thereof for the preparation of a pharmaceutical formulation useful in the prevention and / or treatment of a disease or disorder distinguished by acute and chronic complications associated with red blood cell dysfunction, increased red blood cell cholesterol and decreased plasma levels of lipophilic antioxidant, in particular for the prevention and / or treatment of acute and chronic complications associated with red blood cell dysfunction in sickle cell disease, thalassemia and diabetes.

[0019] According to another aspect, the invention provides a pharmaceutical composition comprising at least one agent that increases HDL activity and at least one fat-soluble antioxidant or a mixture thereof and a pharmaceutically acceptable carrier, diluent or excipient thereof.

[0020] According to another aspect, the invention provides an agent that increases HDL activity for use in the prevention and / or treatment of Petition 870260057648, dated 12 / 06 / 2026, page 23 / 56 / 32 a disease or disorder characterized by acute and chronic complications associated with red blood cell dysfunction, increased cholesterol in red blood cells and decreased plasma levels of lipophilic antioxidant, in particular for the prevention and / or treatment of acute and chronic complications associated with red blood cell dysfunction in sickle cell disease, thalassemia and diabetes, said compound being to be administered in combination with at least one fat-soluble antioxidant or a mixture thereof.

[0021] According to another aspect, the invention provides a method of preventing and / or treating a disease or disorder distinguished by acute and chronic complications associated with red blood cell dysfunction in sickle cell disease, thalassemia and diabetes, said method comprising administering at least one agent that increases HDL activity in combination with at least one fat-soluble antioxidant or a mixture thereof to a subject in need thereof. DESCRIPTION OF THE FIGURES

[0022] Figure 1 is a schematic representation of low HDL cholesterol, LCAT activity, fat-soluble antioxidant, and high cholesterol in RBC and oxidized LDL in untreated SCD, where red blood cells in sickle cell disease are enriched in cholesterol and exhibit pathological rheological properties resulting in vascular and microvascular complications. Oxidized inactive Lecithin-Cholesterol-Acyltransferase (LCAT) cannot effectively generate large, cholesterol-rich HDL. The fat-soluble antioxidant is not effectively absorbed through the intestine and is distributed to LDL, resulting in oxidized and highly atherogenic LDL.

[0023] Figure 2 is a schematic representation of high HDL cholesterol, LCAT activity, fat-soluble antioxidant, and low cholesterol in Petition 870260057648, dated 12 / 06 / 2026, page 24 / 56 / 32 RBC in subjects with SCD treated with a combination or method according to the invention, wherein the combined administration of at least one agent that increases HDL activity and at least one fat-soluble antioxidant or a mixture thereof results in an increase in inactive HDL, thus resulting in the effective removal of cholesterol from red blood cells in sickle cell disease and restoring their normal rheological properties, decreasing macrovascular and microvascular complications. Furthermore, fat-soluble antioxidant supplements are more effectively absorbed through the intestine via the HDL pathway and then distributed to LDL, resulting in less atherogenic LDL. In a less oxidative environment, Lecithin-Cholesterol Acyltransferase (LCAT) activity more effectively produces large, atheroprotective, cholesterol-rich HDL. DETAILED DESCRIPTION

[0024] Plasma HDL is formed through the interaction between Apolipoprotein A1 (ApoA1) and ATP-Binding Membrane Cassette Transporter A1 (ABCA1), the latter effluxing numerous lipophilic molecules (such as cholesterol, phospholipids, plant sterols, vitamin E, lutein, and zeaxanthin) into ApoA1, thus forming a nascent HDL particle (discoid particle of pre-beta-1-HDL). LCAT activity will further increase the ability of HDL to capture and transport more of the aforementioned lipophilic molecules. This cargo will be released to cells expressing the HDL receptor (scavenger receptor class B type 1 (SRB-1) (Monty-Krieger et al., 1993, Journal Biological Chemistry 268: 4568-4572)) and / or exchanged with various tissues, including circulating RBCs that are closely adjacent to the plasma HDL pool.

[0025] ApoA1 exhibits an unusual ability to remove cholesterol from charged cell membranes through an action of Petition 870260057648, dated 12 / 06 / 2026, page 25 / 56 / 32 membrane transporters (ABCA1 and ABCG1) or by diffusion following the concentration gradient. The phenomenon called reverse cholesterol transport, whereby peripheral cellular cholesterol is transferred to HDL for release to the liver, has been very well documented using cholesterol-labeled macrophages. It should be noted that the larger plasma pool of cholesterol in RBCs, which has been shown to play an important role in this process (Hung et al., 2012, Arterioscler. Thromb. Vasc. Biol., 32(6): 1460-1465), has not been fully investigated.

[0026] Therefore, to improve the regulation of cholesterol levels in RBCs, agents capable of enhancing HDL activity can be selected from among HDL enhancers, HDL peptide mimetics, or HDL analogs.

[0027] Therapeutic approaches targeting HDL were recently analyzed by Uehara et al., 2015, Circ. J., 79(12), 2523-8. In particular, LCAT activators, CETP (Cholesteryl Ester Transfer Protein) inhibitors and modulators, ApoA1 mimetics, full-length ApoA1, ApoA1 mimetic peptides such as 5A, D-4F, L-4F, FAMP (Fukuoka University ApoA-I Mimetic Peptide) as described in Uehara et al., 2015, Circ. J., 79(12), 2523-8 and analogues, reconstituted HDL, including ApoA1-phospholipid complexes, ApoA1 Milano, ApoE-phospholipid complexes are considered as HDL enhancers.

[0028] The term “HDL enhancer” refers to agents that are capable of increasing plasma HDL / ApoA1 levels, agents capable of enhancing the production or accumulation of ApoA1 in plasma, or increasing ApoA1 turnover. Examples of such enhancers include hormone receptor activators that have been shown to increase ApoA1 production, such as PPAR agonists. Petition 870260057648, dated 12 / 06 / 2026, p. 26 / 56 / 32 (Peroxisome Proliferator-Activated Receptors) as analyzed by Lamers et al, 2012, Journal Expert Opinion on Therapeutic Patents, 22(7), 802-841, in particular PPAR-alpha agonists. For example, the enhancement of HDL formation has been achieved by increasing ApoA1 production by tissues such as the liver and intestine with PPAR-alpha agonists (fibrates), Thyroid Receptor agonists, and Niacin and analogues. Therefore, those agents can be used as agents capable of enhancing HDL activity according to the invention. Alternatively, plasma HDL can be elevated by increasing ABCA1 activity through activation of the Liver X Receptor (LXR), which is a direct regulator of ABCA1 gene expression, for example by using Liver X Receptor (LXR) activators such as TO901317 (US 6,316,503).Furthermore, recombinant human LCAT protein can be infused into animal and human HDL enhancers, according to the invention, to increase plasma HDL apolipoproteins and lipids. Similarly, agents that increase plasma LCAT levels (Shamburek et al., 2016, Journal of Clinical Lipidology, 10, 356-367), that increase LCAT activity (Gunawardane et al., 2016, J. Biol. Chem., 291(6), 2799-811), and modified LCAT protein with enhanced enzymatic activity, which are under development, increase plasma HDL and can be used as HDL enhancers in the context of the invention.

[0029] The term “HDL peptide mimetic” or “HDL mimetic peptide” or “HDL peptidomimetic” refers to agents that mimic the properties of ApoA1 in the formation of HDL particles by binding to ABCA1 and increasing the efflux of cholesterol, phospholipids, and other lipophilic substances. A large number of peptides that mimic the 22-amino acid multiple repeats (22-mer) of human ApoA1 and are capable of forming α-helices have been synthesized and exhibit some of the Petition 870260057648, dated 12 / 06 / 2026, page 27 / 56 / 32 properties of ApoA1 and especially with regard to their ability to remove cholesterol from the cell membrane. For example, HDL peptide mimetics include 5A, D-4F and L-4F, all mimetics of ApoA1. FAMP differs from other ApoA1 mimetics because it is designed to specifically interact with human ABCA1 without participating in the non-specific passive efflux pathway. Therefore, it functions similarly to human ApoA1. Furthermore, FAMP greatly increases pre-ε-HDL particles (nascent HDL particles) and also the total efflux of cholesterol from peripheral tissues. Therefore, ApoA1 mimetics, full-length ApoA1, ApoA1 mimetic peptides such as D-4F, L-4F, FAMP and analogs can be used as agents capable of enhancing HDL activity according to the invention. According to another specific aspect, peptide 5A and analogs thereof, as described in Sethi et al., 2008, J. Biol.Chem., 283 (47): 32273-32282, US 8,148,323; US 8,835,378; US 8,936,787, can be used as agents capable of enhancing HDL activity according to the invention.

[0030] The term “HDL analogue” refers to any particle containing at least one HDL-like particle associated with an apolipoprotein (ApoAl, ApoA2, ApoE, ApoCl, ApoC3, ApoL, ApoM, ApoAIV, etc.) alone or prepared as a lipoprotein complex. Reconstituted HDL (US 9,125,943), including ApoAl-phospholipid complexes such as ApoA1 Milano (Franceshini et al., 1980, J. Clin. Investig., 66, 892900) or ApoA1 Paris (Bruckert et al., 1997, Atherosclerosis, 128, 121-128), ApoE-phospholipid complexes can, in particular, be used as agents capable of enhancing HDL activity according to the invention.

[0031] The term “ApoA1 mimetics” refers to substances, in particular peptides, that mimic the properties of ApoA1, especially its ability to immobilize cellular lipoprotein lipids and transport and release said lipids into cells and tissues. ApoA1 mimetics Petition 870260057648, dated 12 / 06 / 2026, p. 28 / 56 / 32 ApoA1 includes analogs of ApoA1, in particular but not limited to those specifically defined herein, i.e., peptides or peptidomimetics having a sequence analogous to the ApoA1 sequence. In addition, the term “ApoA1 mimetics” also refers to non-peptidic substances such as cyclodextrin or cyclodextrin derivatives, which mimic the properties of ApoA1, in particular its ability to mobilize cellular lipoprotein lipids and transport and release said lipids into cells and tissues (Kilsdonk et al., 1995, J. Biol. Chem., 270(29):17250-6; Coisne et al., 2016, Front Physiol., 7:185). Examples of cyclodextrins or cyclodextrin derivatives include, but are not limited to, beta-cyclodextrins such as sulfobutyl ether-beta-cyclodextrin, 2-hydroxypropyl beta-cyclodextrin, methyl beta-cyclodextrin, and 2,6-di-O-methyl beta-cyclodextrin. Preferred cyclodextrins are sulfobutyl ether-betacyclodextrin and 2-hydroxypropyl beta-cyclodextrin.

[0032] The term “ApoA1 inducer” refers to inducers of ApoA1 gene expression such as Nuclear Hormone Receptor Agonists. Examples of ApoA1 inducers include, but are not limited to, LXR (Liver Receptor X), RXR (Retinoid X Receptor), ROR (RAR-related Orphan Receptors), and PPAR agonists. The ability of an agent to act as an ApoA1 inducer can be tested by standard methods as described in Chinetti et al., 2001, Nat. Med. 7(1):53-8.

[0033] The term a “Lecithin-Cholesterol-Acyltransferase (LCAT) activator” refers to an agent capable of increasing the level or enzymatic activity of LCAT. Examples of LCAT activators include, but are not limited to, peptides such as LAP-20 (Buchko et al., 1996, J. Biol. Chem., 271(6): 3039-3045), but also include antibodies that bind to active LCAT such as those described in Gunawardane et al., 2016, J. Petition 870260057648, dated 12 / 06 / 2026, p. 29 / 56 / 32 Biol. Chem., 291(6):2799-811, and small molecules such as those described in Freeman et al., 2017, J. Pharmacol. Exp. Ther., 362 (2), 306-318. The ability of an agent to act as an LCAT activator can be tested by standard methods as described in Vaisman et al., 2013 Methods Mol. Biol., 1027: 343-52 and Homan et al., 2013, Anal. Biochem., 441(1):80-6.

[0034] The term an “ATP-Binding Cassette Transporter A1 (ABCA1) inducer” refers to an agent capable of increasing ABCA1 activity, for example by activating Liver X Receptor (LXR), which is a direct regulator of ABCA1 gene expression. An agent’s ability to act as an ABCA1 inducer can be tested by standard methods as described in Costet et al., 2000, J. Biol. Chem., 275(36):28240-5. Examples of ABCA1 inductors include, but are not limited to, LXR activators such as TO901317, and LXR activators such as those described in US 6,316,503, US 9,000,022, US 7,579,504, or US 8,993,628.

[0035] The term “niacin analog” refers to compounds that interact with the Niacin receptor (Soudijn et al., 2007, Med. Res. Rev., 27(3):417-33). Examples of niacin analogs include, but are not limited to, those described by Semple et al., 2006, J. Med. Chem., 49(4):122730.

[0036] The term “fat-soluble vitamin” or “fat-soluble antioxidant” refers to lipophilic agents having antioxidant properties such as tocopherols, tocotrienols, and xanthophyll carotenoids.

[0037] The term “carotenoids” refers to one of the most widespread groups of pigments, with more than 600 identified in nature. In plants, the cyclization of lycopene results in the formation of either beta-carotene or alpha-carotene and their derivative xanthophylls, beta-cryptoxanthin, zeaxanthin, astaxanthin, violaxanthin, and lutein. Carotenes are distinguished by cyclization at one or both ends, while Petition 870260057648, dated 12 / 06 / 2026, page 30 / 56 / 32, states that xanthophylls are formed by the introduction of oxygen. According to a specific aspect, carotenoids can be used as fat-soluble vitamins according to the invention. In another specific embodiment, the carotenoids are selected from xanthophylls, lutein, zeaxanthin (e.g., meso-zeaxanthin), beta-cryptoxanthin, astaxanthin, and violaxanthin.

[0038] The terms “tocopherols” and “tocotrienols” refer to agents having vitamin E activity. There are eight naturally occurring isoforms of vitamin E: alpha-tocopherol, beta-tocopherol, gamma-tocopherol, and delta-tocopherol, and alpha-tocotrienol, beta-tocotrienol, gamma-tocotrienol, and delta-tocotrienol. All are potent fat-soluble antioxidants capable of neutralizing free radicals directly by donating hydrogen from their chromanol ring. Alpha-tocopherol is considered the dominant form of vitamin E because the alpha-tocopherol transfer protein in the liver binds predominantly to alpha-tocopherol, thus preventing its degradation. According to a specific aspect, “tocopherols” and “tocotrienols” can be used as fat-soluble vitamins according to the invention.

[0039] It will be recognized that water-soluble antioxidants such as vitamin C, glutathione, N-acetylcysteine, and ions such as Zn, etc., can regenerate fat-soluble antioxidants and can be used to increase or maintain the antioxidant potential of fat-soluble antioxidants.

[0040] As used herein, “treatment” and “to treat” and the like generally mean the attainment of a desired pharmacological and physiological effect. The effect may be prophylactic in terms of prevention or partial prevention of a disease, a condition, a symptom or an adverse effect attributed to the disease. The term “treatment,” as used herein, encompasses any treatment of a disease in a mammal, particularly in a human, and includes: (a) prevention of the occurrence of the disease in a subject who may be predisposed to the disease but has not yet been pre-diagnosed. Petition 870260057648, dated 12 / 06 / 2026, p. 31 / 56 / 32 as having it; (b) inhibition of the disease, that is, interruption of its development; or relief of the disease, that is, causing the regression of the disease and / or its symptoms or conditions.

[0041] According to a specific aspect, the effectiveness of a treatment or use according to the invention can be monitored by the decrease or abolition of acute and chronic complications associated with red blood cell dysfunction, such as a decrease in red blood cell cholesterol or a prolonged increase in plasma levels of lipophilic antioxidants, by measuring the level of oxidative stress biomarkers (e.g., antioxidants, oxidized lipids, Reactive Oxygen Species (ROS), malondialdehyde (MDA), glutathione, catalase, etc.) in total RBCs and plasma. It is also assumed that the rheological properties of RBCs are enhanced.

[0042] The term “subject,” as used herein, refers to mammals. For example, the mammals considered by the present invention include humans and similar animals. Combinations of the invention

[0043] According to a specific aspect, a combination is provided of at least one agent that increases HDL activity (e.g., HDL-enhancing agent, HDL mimetic, or HDL analogue) and at least one fat-soluble antioxidant or a mixture thereof for use in the prevention and / or treatment of a disease or disorder distinguished by acute and chronic complications associated with red blood cell dysfunction, increased red blood cell cholesterol, and decreased plasma levels of lipophilic antioxidant.

[0044] In addition, according to a specific aspect, a combination of at least one HDL-enhancing agent is provided, which is selected from an ApoA1 inducer, a mimetic of or Petition 870260057648, dated 12 / 06 / 2026, p. 32 / 56 / 32 (compound that mimics) HDL or ApoAl, an analog of ApoAl and ApoE, a Lecithin-Cholesterol-Acyl-Transferase (LCAT) activator, a CETP inhibitor or modulator, a Peroxisome Proliferator-Activated Receptor (PPAR) agonist, and an ATP-Binding Cassette-Containing A1 Transporter (ABCA1) inducer.

[0045] According to another specific embodiment, a combination according to the invention is provided in which said at least one HDL-enhancing agent is selected from a recombinant Lecithin-Cholesterol-Acyltransferase (LCAT) protein as described in (US 2008 / 0096900 and US 2014 / 0023631 A1) (e.g., ACP-501, Shamburek et al. 2016, Journal of Clinical Lipidology, 10, 356-367).

[0046] According to another additional specific embodiment, a combination according to the invention is provided in which said at least one agent that increases HDL activity is an LCAT activator, for example as the LAP-20 peptide (Buchko et al., 1996, supra), or an antibody that binds to and activates LCAT.

[0047] According to another additional specific embodiment, a combination according to the invention is provided in which said at least one HDL-enhancing agent is selected from wild-type apolipoprotein A1 (ApoA1) (US 8,436,152); mutant ApoA1 (US 7,439,323) in particular as ApoA1 Milano and ApoA1 Paris and oxidation-resistant ApoA1 mutants (US 8,541,236). Branched ApoA1 (US 6,602,854), multimeric ApoA1 (US 6,753,313; US 7,307,058, 2), and an ApoA1 analogue or an ApoA1 mimetic peptide (US 4,643,988, US 6,004,925; US 8,748,394) such as L-4F. Orally bioavailable ApoA1 mimetic peptides such as D-4F (US 6,664,230; US 6,933,279). Another class of ApoA1 mimetic peptides could be the Fukuoka University APOA-I Mimetic Peptides (FAMP) (Uehara Y. et al, 2013, J. Am. Heart Assoc.; 2(3):e000048, and Yang et al, 2016, Int. J. Cardiol., Petition 870260057648, dated 12 / 06 / 2026, p. 33 / 56 / 32 222:1059-60).

[0048] According to another additional specific embodiment, a combination according to the invention is provided wherein said at least one agent that increases HDL activity is an agent that mimics ApoA1, such as selected peptides derived from ApoE (US 9,422,363), ApoA2 (US 6,743,778), ApoJ (US 6,930,085; US 8,568,766) and any amphipathic helical peptide derived therefrom, such as peptide 5A and analogues thereof (Sethi et al., supra; US 8,148,323; US 8,835,378), such as those derived from ApoA1 and ApoA2 and also from ApoC1, ApoC3, ApoJ, ApoL, ApoM, ApoAIV (US 4,643,988).

[0049] According to another additional specific embodiment, a combination according to the invention is provided in which said at least one HDL-enhancing agent is an HDL mimetic, such as a natural or synthetic ApoA1 / phospholipid containing a wild-type apolipoprotein A1 or a cysteine ​​mutant thereof (Zhu et al., 2005, J. Lipid Res., 46:1303-131), including ApoA1 Milano (Franceshini et al, 1980, J. Clin. Investig., 66, 892-900) or ApoA1 Paris (Bruckert et al., 1997, Atherosclerosis, 128, 121-128).

[0050] According to another additional specific embodiment, a combination according to the invention is provided in which said at least one HDL activity-enhancing agent is a natural or synthetic HDL particle analogue (e.g., an ApoA1 / phospholipid mixture) such as CSL-111, CSL-112 (US 9,125,943, US 8,999,920 or US 9,439,946) or CER-001 (US 6,287,590 and US 9,187,551).

[0051] According to another specific embodiment, a combination according to the invention is provided in which said at least one HDL-enhancing agent is one of the Peroxisome Proliferator-Activated Receptor (PPAR) activators (PPAR-alpha, PPAR-gamma or PPAR-alpha-gamma coactivators) such as fenofibrate, pioglitazone or Petition 870260057648, dated 12 / 06 / 2026, p. 34 / 56 / 32 aleglitazar. According to another additional specific embodiment, a combination according to the invention is provided in which said at least one agent that increases HDL activity includes PPARalpha / gamma coagonists.

[0052] According to another additional specific embodiment, a combination according to the invention is provided in which said at least one agent that increases HDL activity is an agent that enhances the expression of the ATP-binding cassette-containing transporter A1 (ABCA1) as a Liver X Receptor (LXR) modulator (US 9,416,135) or LXR activator as described in US 6,316,503, US 9,000,022, US 7,579,504 or US 8,993,628.

[0053] According to another additional specific embodiment, a combination according to the invention is provided in which said at least one agent that increases HDL activity is a protein inhibitor with bromodomains and an extraterminal domain (BET) such as those described in US 8,114,995, for example as RVX-208 (Picaud et al., 2013, PNAS, 110, 49, 19755).

[0054] According to another additional specific embodiment, a combination according to the invention is provided in which said at least one HDL activity-enhancing agent is an inhibitor of Cholesterol Ester Transfer Protein (CETP), for example as analyzed by Shinkai et al. (2009, Expert Opin. Ther. Patents, 19(9):1229-1237) such as anacetrapib and in particular a modulator of Cholesterol Ester Transfer Protein (CETP) such as those described in WO 2004 / 020393, WO 98 / 35937 and defined by Niesor et al., 2010, JLR, 51: 3443-3454, for example such as dalcetrapib.

[0055] According to another specific embodiment, a combination according to the invention is provided in which said at least one agent that increases HDL activity is an agent that upregulates the Petition 870260057648, dated 12 / 06 / 2026, p. 35 / 56 / 32 expression of ApoAl (US 8,242,144).

[0056] According to another additional specific embodiment, a combination according to the invention is provided in which said at least one HDL-enhancing agent or a mixture thereof is selected from Niacin and Niacin analogs that are compounds that interact with the Niacin receptor (Soudijn et al., 2007, supra), for example HM74 receptor activators, such as those described in US 8,394,808 and US 8,703,783 and niacin analogs as described by Semple et al. 2006, above.

[0057] According to another additional specific embodiment, a combination according to the invention is provided wherein said at least one fat-soluble antioxidant or a mixture thereof is a xanthophyll such as lutein, zeaxanthin, meso-zeaxanthin, astaxanthin, beta-cryptoxanthin and combinations thereof as described for example in US 6,218,436, US 6,582,721 and US 6,663,900.

[0058] According to another specific embodiment, a combination according to the invention is provided in which said at least one fat-soluble antioxidant or a mixture thereof is an isomer of tocopherol or a derivative thereof such as alpha-tocopherol, beta-tocopherol, gamma-tocopherol and delta-tocopherol.

[0059] According to another additional specific embodiment, a combination according to the invention is provided in which said at least one fat-soluble antioxidant or a mixture thereof is a tocotrienol isomer or a derivative thereof, such as alpha-tocotrienol, beta-tocotrienol, gamma-tocotrienol and delta-tocotrienol.

[0060] In a particular respect, the combination of the present invention is useful in the prevention and / or treatment of acute and chronic complications associated with red blood cell dysfunction, elevated red blood cell cholesterol / plasma levels. Petition 870260057648, dated 12 / 06 / 2026, p. 36 / 56 / 32 decreased lipophilic antioxidant, such as those present in sickle cell disease, thalassemia or diabetes. Pharmaceutical compositions

[0061] According to another aspect, the invention provides a pharmaceutical composition comprising at least one compound that increases HDL activity and at least one fat-soluble antioxidant or a mixture thereof and a pharmaceutically acceptable carrier, diluent or excipient thereof.

[0062] The pharmaceutical compositions of the invention may contain one or more agent(s) of the invention in any form described herein. The compositions of this invention may further comprise one or more additional pharmaceutically acceptable ingredient(s), such as alum, stabilizers, antimicrobial agents, buffers, coloring agents, flavoring agents, adjuvants, and the like.

[0063] The agents of the invention, together with a commonly used adjuvant, carrier, diluent or excipient, can be transformed into pharmaceutical compositions and unit dosage forms thereof, and in such form can be used as solids, such as tablets or filled capsules, or liquids such as solutions, suspensions, emulsions, elixirs, or capsules filled therewith, all for oral use, or in the form of sterile injectable solutions for parenteral use (including subcutaneous). Such pharmaceutical compositions and unit dosage forms thereof may comprise ingredients in conventional proportions, with or without additional active ingredients or agents, and such unit dosage forms may contain any effective amount of the active ingredient in accordance with the intended dosage range to be used. The compositions according to the invention are preferably oral.

[0064] The compositions of this invention may be liquid formulations, including, but not limited to, aqueous or oily suspensions, Petition 870260057648, dated 12 / 06 / 2026, page 37 / 56 / 32 aqueous or oily solutions, aqueous or oily emulsions, syrups, and elixirs. Liquid forms suitable for oral administration may include a suitable aqueous or non-aqueous vehicle with buffers, suspending and dispersing agents, colorants, flavorings, and the like. Compositions may also be formulated as a dry product for reconstitution with water or another suitable vehicle before use. Such liquid preparations may contain additives, including, but not limited to, suspending agents, emulsifying agents, non-aqueous vehicles, and preservatives. Suspending agents include, but are not limited to, sorbitol syrup, methylcellulose, fructose, glucose / sugar syrup, gelatin, hydroxyethylcellulose, carboxymethylcellulose, aluminum stearate gel, and edible hydrogenated fats. Emulsifying agents include, but are not limited to, lecithin, sorbitan monooleate, and acacia.Non-aqueous vehicles include, but are not limited to, edible oils, almond oil, fractionated coconut oil, oleyl esters, propylene glycol, and ethyl alcohol. Preservatives include, but are not limited to, methyl p-hydroxybenzoate or propyl p-hydroxybenzoate and sorbic acid. Other materials and processing techniques and the like are described in Part 5 of Remington's "The Science and Practice of Pharmacy," 22nd Edition, 2012, University of the Sciences in Philadelphia, Lippincott Williams & Wilkins, which is incorporated herein by reference. The solid compositions of this invention may be in the form of tablets or rhombic lozenges formulated in a conventional manner. For example, tablets and capsules for oral administration may contain conventional excipients including, but not limited to, binding agents, fillers, lubricants, disintegrants, and humectants.Binding agents include, but are not limited to, syrup, acacia, gelatin, sorbitol, tragacanth, starch mucilage, and polyvinylpyrrolidone. Fillers include, but are not limited to, lactose, sugar, microcrystalline cellulose, corn starch, and phosphate. Petition 870260057648, dated 12 / 06 / 2026, page 38 / 56 / 32 of calcium, and sorbitol. Lubricants include, but are not limited to, magnesium stearate, stearic acid, talc, poly(ethylene glycol), and silica. Disintegrants include, but are not limited to, potato starch and sodium starch glycolate. Humectants include, but are not limited to, sodium lauryl sulfate. Tablets may be coated according to methods well known in the art.

[0065] Injectable compositions are typically based on sterile injectable saline solution or phosphate-buffered saline solution or other injectable carriers known in the art.

[0066] The compositions of this invention may also be formulated as suppositories, which may contain suppository bases including, but not limited to, cocoa butter or glycerides. The compositions of this invention may also be formulated for inhalation, which may be in a form including, but not limited to, a solution, a suspension, or an emulsion that may be administered as a dry powder or in the form of an aerosol using a propellant such as dichlorodifluoromethane or trichlorofluoromethane. The compositions of this invention may also be formulated as transdermal formulations comprising aqueous and non-aqueous vehicles including, but not limited to, creams, ointments, lotions, pastes, medicinal plasters, medicinal bandages, or medicinal membranes.

[0067] The compositions of this invention may also be formulated for parenteral administration, including, but not limited to, by injection or continuous infusion. Formulations for injection may be in the form of suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulation agents including, but not limited to, suspending agents, stabilizers, and dispersants. The compositions may also be provided in a powder form for reconstitution with a suitable vehicle including, but not limited to, sterile, pyrogen-free water. Petition 870260057648, dated 12 / 06 / 2026, page 39 / 56 / 32

[0068] The compositions of this invention can also be formulated as a depot preparation, which can be administered by implantation or intramuscular injection. The compositions can be formulated with suitable polymeric or hydrophobic materials (such as, for example, an emulsion in an acceptable oil), ion exchange resins, or moderately soluble derivatives (such as, for example, a moderately soluble salt).

[0069] The compositions of this invention can also be formulated as a liposome preparation. The liposome preparation may comprise liposomes that penetrate the cells of interest or the stratum corneum and fuse with the cell membrane, resulting in the release of the liposome contents into the cell. Other suitable formulations may utilize niosomes. Niosomes are lipid vesicles similar to liposomes, with membranes consisting predominantly of non-ionic lipids, some forms of which are effective for transporting agents through the stratum corneum.

[0070] The agents of this invention can also be administered in extended-release forms or from extended-release drug delivery systems. A description of representative extended-release materials can also be found in “Remington's Pharmaceutical Sciences”. In addition, analogs and mimetics of ApoA1 can be formulated to produce an extended-release delivery system according to US 9,173,890.

[0071] Various fat-soluble antioxidant formulations, as described above, may be used, in particular as described in US 8,765,186 or US 8,912,237. Patients

[0072] In one embodiment, subjects according to the invention are subjects suffering from acute and chronic complications associated with Petition 870260057648, dated 12 / 06 / 2026, page 40 / 56 / 32 red blood cell dysfunction such as increased cholesterol in red blood cells (Cooper and Jandl, 1969, J. Clin. Invest., 48(4):736-44 and 48(5):906-14; Akinyanju and Akinyanju, 1976, Ann. Clin. Lab. Sci., 6(6):521-4; Westerman et al., 1964, Blood, 2:3, Nayak et al., 2008, Vascular Health and Risk Management, 4(4) 893-899).

[0073] In another embodiment, subjects according to the invention are subjects suffering from decreased plasma levels of lipophilic antioxidant (Ama Moor et al., 2016, BMC Clinical Pathology, 16:15-20, Hermann et al, 2016, J. Pediatr. (Rio J) in press, Alassane et al., 2014, African Journal of Biochemistry Research, 8(2), 39-42; Natta et al., 1988, Eur. J. Haematol., 41:131-135, Voskou et al., 2015, supra).

[0074] In another embodiment, subjects according to the invention are subjects suffering from microvascular complications due to pathological red blood cell functions, hemorrhagic abnormalities associated with high cholesterol in the red blood cell membrane, and low plasma levels of HDL antioxidant.

[0075] In another embodiment, subjects according to the invention are subjects suffering from sickle cell disease.

[0076] In another specific embodiment, subjects according to the invention are subjects suffering from sickle cell disease, wherein the subjects are distinguished by the presence of methylenetetrahydrofolate reductase (MTHFR) polymorphisms, such as a single point mutation in the MTHFR gene, for example 677C>T (677T mutation in the MTHFR gene). Patients with the 677T mutation in the MTHFR gene are distinguished by a depletion of antioxidant activity, according to decreased catalase activity, and a reduction of approximately 30% in glutathione levels (da Silva et al., 2017, Free Radic Biol. Med., 106: 53-61).

[0077] In another embodiment, subjects, according to the invention, are subjects suffering from Thalassemia. Petition 870260057648, dated 12 / 06 / 2026, page 41 / 56 / 32

[0078] In another embodiment, subjects, according to the invention, are subjects suffering from diabetes. Use of combinations of the invention

[0079] According to another aspect, the invention provides an agent that increases HDL activity for use in the prevention and / or treatment of a disease or disorder distinguished by acute and chronic complications associated with red blood cell dysfunction, increased red blood cell cholesterol and decreased plasma levels of lipophilic antioxidant, in particular for the prevention and / or treatment of acute and chronic complications associated with red blood cell dysfunction in sickle cell disease, thalassemia and diabetes, wherein said agent is to be administered in combination with at least one fat-soluble antioxidant or a mixture thereof.

[0080] According to another aspect, the invention provides a combination, according to the invention, wherein at least one of said agent (which increases HDL activity or the fat-soluble antioxidant or a mixture thereof) of said combination is to be administered orally.

[0081] According to another aspect, the invention provides a combination, according to the invention, wherein at least one of said agent (which increases HDL activity or the fat-soluble antioxidant or a mixture thereof) of said combination is to be administered by injection.

[0082] According to another aspect, the invention provides a combination, according to the invention, wherein said combination is to be administered orally.

[0083] According to another aspect, the invention provides a combination, according to the invention, wherein said combination is to be administered by injection, in particular by parenteral administration, Petition 870260057648, dated 12 / 06 / 2026, p. 42 / 56 / 32 subcutaneous or intravenous.

[0084] According to another aspect, the invention provides a method of preventing and / or treating acute and chronic complications associated with red blood cell dysfunction in sickle cell disease, thalassemia and diabetes, said method comprising administering at least one compound that increases HDL activity in combination with at least one fat-soluble antioxidant or a mixture thereof to a subject in need thereof.

[0085] The invention includes the administration of an HDL-enhancing agent, according to the invention, or a pharmaceutical formulation thereof, wherein an HDL-enhancing agent or a pharmaceutical formulation thereof is administered to an individual before, simultaneously or sequentially with, at least one fat-soluble antioxidant or a mixture thereof, in an effective amount. HDL-enhancing agents or pharmaceutical formulations thereof that are administered simultaneously with said at least one fat-soluble antioxidant or mixtures thereof may be administered in the same composition or in different composition(s) and by the same route or by different route(s) of administration.

[0086] According to another aspect, the invention provides a method or use, according to the invention, whereby a pharmaceutical composition, according to the invention, is to be administered.

[0087] According to one aspect, a combination of the invention advantageously prevents or decreases or treats the microvascular complications of sickle cell disease and thalassemia and restores the functions of red blood cells.

[0088] According to another aspect, a combination of the invention advantageously normalizes the cholesterol concentration in the red blood cell membrane and decreases oxidative stress, Petition 870260057648, dated 12 / 06 / 2026, p. 43 / 56 / 32 both parameters being pathologically affected in SCD, thalassemia and diabetes resulting from low plasma HDL levels and decreased absorption of dietary lipophilic antioxidant. EXAMPLES

[0089] The following abbreviations refer respectively to the definitions below: Apo (Apolipoprotein); HDL (High Density Lipoprotein); LCAT (Lecithin-Cholesterol Acyltransferase); LDL (Low Density Lipoprotein); LXR (Liver X Receptor); RBC (Red Blood Cell); ROS (Reactive Oxygen Species). Example 1: Administration of a combination of recombinant human LCAT and a lutein-zeaxanthin mixture.

[0090] A combination of the invention comprising recombinant human LCAT as an agent that increases HDL activity and a lutein-zeaxanthin combination were used in the treatment of SCD using a mouse SCD model (Fabry, 1993, Cell Mol. Life Sci., 49:28-36).

[0091] Recombinant human LCAT rhLCAT (ACP-501) (US 2014 / 0023631) is infused intravenously over 1 hour on 3 occasions in a dose optimization phase (0.3, 3.0, and 9.0 mg / kg), then 3.0 or 9.0 mg / kg every 1 to 2 weeks for 7 months in a maintenance phase, with daily oral administration of 5, 10, or 20 mg of lutein-zeaxanthin as Floraglo® (Kemin Industries, USA) described in US 6,663,900. Several parameters are measured, such as increased plasma LCAT activity, HDL particle count, HDL-cholesterol, ApoA1, plasma levels of fat-soluble antioxidants, and decreased levels of oxidative stress markers (such as MDA, lipid peroxides, diene conjugates). The levels of Petition 870260057648, dated 12 / 06 / 2026, p. 44 / 56 / 32 Tissue and cellular fat-soluble antioxidants are also measured, especially in RBCs. RBC count and hematocrit, along with RBC rheological properties, are monitored. Example 2: Administration of a combination of ApoA1 or ApoA1 mimetics and a lutein-zeaxanthin mixture.

[0092] A combination of the invention comprising at least one ApoA1 mimetic (including ApoA1 mimetic peptides) such as D-4F and / or full-length ApoA1 prepared as described in US 9,187,551 or US 8,436,152 as an agent that increases HDL activity and a lutein-zeaxanthin combination has been used in the treatment of SCD (Ryan et al., 1997, Science, 278, 5339, 873-876). Properly formulated ApoA1 mimetics and / or full-length ApoA1 (US 9,125,943, US 8,999,920, US 9,439,946, US 6,287,590 and US 9,187,551) are administered subcutaneously, intravenously or orally in effective therapeutic doses with simultaneous daily oral administration of 5, 10 or 20 mg of lutein-zeaxanthin such as Floraglo® (Kemin Industries, USA). Parameters are monitored as described in Example 1. Example 3: Administration of a combination of apolipoproteins or peptide mimetics thereof and a lutein-zeaxanthin mixture.

[0093] A combination of the invention comprising at least one apolipoprotein selected from ApoA1, ApoA2, ApoE, ApoC1, ApoC3, ApoL, ApoM, ApoJ, ApoAIV or at least one of the alpha-helix mimetic peptides thereof prepared as described in US 9,125,943, US 8,999,920, US 9,439,946, US 6,287,590 or US 9,187,551 as an agent that increases HDL activity and a lutein-zeaxanthin combination were used in the treatment of SCD validated using a mouse SCD model as described by Ryan et al., 1997, Science, 278, 5339, 873-876. ApoA1, ApoA2, ApoE proteins or peptides Petition 870260057648, dated 12 / 06 / 2026, p. 45 / 56 / 32 Properly formulated ApoCl, ApoC3, ApoL, ApoM, ApoJ, and ApoAIV are administered subcutaneously, intravenously, or orally in effective therapeutic doses with simultaneous daily oral administration of 5, 10, or 20 mg of lutein-zeaxanthin such as Floraglo® (Kemin Industries, USA). Parameters are monitored as described in Example 1. Example 4: Administration of a combination of fenofibrate and lutein-zeaxanthin mixture.

[0094] Patients are treated daily with a therapeutically active oral dose of micronized fenofibrate (e.g., 150 mg per day) as an HDL-enhancing agent with the simultaneous daily oral administration of 5, 10, or 20 mg of lutein-zeaxanthin as Floraglo® (Kemin Industries, USA). Parameters are monitored as described in Example 1. Example 5: Administration of a combination of an LXR agonist and a lutein-zeaxanthin mixture.

[0095] Patients are treated daily with a therapeutically active oral dose of an LXR agonist as described in US 7,579,504, as an agent that increases HDL activity with the simultaneous daily oral administration of 5, 10, or 20 mg of lutein-zeaxanthin as lutein-zeaxanthin as Floraglo® (Kemin Industries, USA). Parameters are monitored as described in Example 1. Example 6: Administration of a combination of oral CETP modulator / inhibitor and lutein-zeaxanthin mixture

[0096] Patients are treated daily with a therapeutically active oral dose of a CETP modulator / inhibitor, for example 150, 300, 450 or 600 mg of dalcetrapib as an agent that increases HDL activity, with the simultaneous daily oral administration of 5, 10 or 20 mg of lutein-zeaxanthin such as Floraglo® (Kemin Industries, USA). Petition 870260057648, dated 12 / 06 / 2026, page 46 / 56 / 32, monitored parameters as described in Example 1. Example 7: Resistance to cholesterol depletion of red blood cells in patients with SCD

[0097] Blood was collected from four healthy control subjects using citrate as an anticoagulant. After low-speed centrifugation, the RBC pellet was washed with isotonic phosphate-buffered saline (PBS) and resuspended in PBS (hematocrit 20%).

[0098] To confirm the difference in cholesterol in the membrane of RBCs from subjects with SCD compared to healthy subjects, the sensitivity of RBCs to the cholesterol-depleting agent, methyl-beta-cyclodextrin, was evaluated. RBCs were incubated for 20 min at 37°C in the presence of 2.5 mM or 10 mM methyl-beta-cyclodextrin (Sigma, ref. C4555-5G) as described by Motayama et al., 2009, Biol. Pharm. Bull., 32(4) 700-705. The deformability of the RBC membrane was evaluated by ectacytometry (Mechatronics) as described by Renoux et al., 2016, Clin. Hemorheol. Microcirc., 62(2):173-9, at shear stresses of 3 Pa and 30 Pa.

[0099] It was confirmed that 10 mM methyl-beta-cyclodextrin produced intense hemolysis and a significant decrease in RBC deformability only in the RBCs of healthy control subjects, while the RBCs of 4 subjects with SCD appeared to be quite resistant to the cholesterol-depleting effect caused by 10 mM methyl-beta-cyclodextrin, resulting in hemolysis. This confirms not only the hypothesis that a higher cholesterol content in the RBC membranes of patients with SCD is the cause of hemolysis, but also that the use of a cholesterol-depleting agent alone is not capable of reducing the complications of SCD due to RBC dysfunction. Example 8: Effect of a combination of HDL and lutein on RBCs

[00100] The RBCs were prepared as described in Example 7 and Petition 870260057648, dated 12 / 06 / 2026, p. 47 / 56 / 32 The effect of HDL with or without lutein on the oxidative stress of RBCs was determined using RBCs from healthy subjects incubated for 20 min in the presence of 100 μM HDL isolated from human plasma (Sigma, ref. L-1567) in the presence or absence of 1 μM or 10 μM lutein (Bertin Pharma, ref. 1001081). Reactive oxygen species (ROS) were measured as Mean Fluorescence Intensity (MFI) by flow cytometry similarly to Amer et al., 2003, Eur. J. Haematol., 70(2):84-90. While HDL decreased ROS by 7.4% and lutein 1 μM decreased ROS by 9.2%, the combination of HDL with lutein 1 μM resulted in a ROS decrease of 22.6%, suggesting a synergistic effect of their combination (Table 1) on RBCs. Table 1 Mean MH Standard Error of Mean % Control Control 42.2 11.6 HDL 100 μM 39.0 12.4 -7.4 Lutein 1 μM 38.3 7.5 -9.2 Lutein 10 μM 26.8 8.0 -36.4 HDL 100 μM + Lutein 1 μM 32.6 9.6 -22.6

[00101] These data suggest that such a combination would be beneficial for patients with SCD because they suffer from an accelerated oxidative damage status in RBCs. Petition 870260057648, dated 12 / 06 / 2026, pages 48 / 56

Claims

1 / 2 CLAIMS 1. Use of a combination of at least one agent that increases HDL activity and at least one fat-soluble antioxidant or a mixture thereof, characterized in that it is in the preparation of a medicament for the prevention and / or treatment of a disease or disorder defined by acute and chronic complications associated with red blood cell dysfunction, increased red blood cell cholesterol and / or decreased plasma levels of lipophilic antioxidant, wherein said at least one fat-soluble antioxidant is selected from a xanthophyll, lutein, zeaxanthin, meso-zeaxanthin, astaxanthin, beta-cryptoxanthin, a tocopherol isomer or derivative thereof selected from alpha-tocopherol, beta-tocopherol, gamma-tocopherol and delta-tocopherol and a tocotrienol isomer or derivative thereof selected from alpha-tocotrienol, beta-tocotrienol,gamma-tocotrienol and delta-tocotrienol, wherein said disease or disorder is selected from sickle cell disease, diabetic red blood cell dysfunction, and thalassemia, wherein said at least one HDL-enhancing agent is selected from a Cholesterol Ester Transfer Protein (CETP) modulator and an HDL mimetic selected from a natural or synthetic HDL particle.

2. Use according to claim 1, characterized in that said disease or said disorder is sickle cell disease or thalassemia.

3. Use according to claim 1, characterized in that said disease or disorder is a diabetic dysfunction of red blood cells.

4. Use in accordance with any of claims 1 to 3, characterized in that said fat-soluble antioxidant is a xanthophyll. Petition 870260057648, dated 12 / 06 / 2026, page 49 / 56 2 / 2 5. Use in accordance with any of claims 1 to 4, characterized in that said fat-soluble antioxidant is lutein.

6. Use in accordance with any of claims 1 to 3, characterized in that said fat-soluble antioxidant is an isomer of tocopherol or a derivative thereof.

7. Use in accordance with any one of claims 1 to 6, characterized in that said at least one HDL-enhancing agent is a natural or synthetic HDL particle selected from CSL-111, CSL-112 and CER-001.

8. Use in accordance with any one of claims 1 to 6, characterized in that said at least one agent that increases HDL activity is a Cholesterol Ester Transfer Protein (CETP) modulator / inhibitor, such as dalcetrapib or anacetrapib.

9. Pharmaceutical composition, characterized in that it comprises at least one compound that increases HDL activity and at least one fat-soluble antioxidant or a mixture thereof and a pharmaceutically acceptable carrier, diluent or excipient thereof, wherein said at least one fat-soluble antioxidant is selected from a xanthophyll, lutein, zeaxanthin, meso-zeaxanthin, astaxanthin, beta-cryptoxanthin, a tocopherol isomer or derivative thereof selected from alpha-tocopherol, beta-tocopherol, gamma-tocopherol and delta-tocopherol and a tocotrienol isomer or derivative thereof selected from alpha-tocotrienol, beta-tocotrienol, gamma-tocotrienol and delta-tocotrienol and wherein said at least one HDL activity-enhancing agent is selected from a Cholesterol Ester Transfer Protein (CETP) modulator and an HDL mimetic selected of a particle of natural or synthetic HDL. Petition 870260057648, dated 12 / 06 / 2026, pp. 50 / 56

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