Mitochondrially targeted isolevuglandin scavengers and uses thereof

Abstract

Use of novel 2-hydroxybenzylamine derivatives as isolevuglandin scavengers.

Description

[0001] Government support

[0002] This invention was made with government support under grant numbers R01HL124116, P01HL129941, K23GM129662, R01GM112871, and RO1HL144943 granted by the National Institutes of Health. The government owns certain rights in this invention.

[0003] Background and content of the invention

[0004] This application relates to novel compounds and compositions and their uses. The compounds of this invention have been found to be useful in treating diseases and conditions such as inflammation, sepsis, mitochondrial dysfunction, oxidative stress, and hypertension.

[0005] Inflammation is a leading cause of morbidity and mortality in Western societies. Despite the use of various medications, chronic and acute inflammation remains a major health burden. Inflammation produces highly reactive dicarbonyl lipid peroxidation products, such as isolevuglandin, which covalently modify and cross-link proteins through lysine residues. Mitochondrial dysfunction is also associated with inflammation.

[0006] The inventors have discovered that inflammation-induced isolevuglandin promotes mitochondrial dysfunction and death. The inventors have (a) investigated mitochondrial dysfunction in response to synthetically produced 15-E2-isolevuglandin (IsoLG) and its adducts; (b) developed novel mitochondrial-targeting isolevuglandin scavengers comprising (4-(4-aminomethyl)-3-hydroxyphenoxy)butyl)-triphenylphosphonium (mito2HOBA) by conjugating 2-hydroxybenzylamine to a lipophilic cationic triphenylphosphonium; and (c) discovered, using a lipopolysaccharide inflammation model, that the compounds of the present invention, including mito2HOBA, prevent mitochondrial dysfunction and death.

[0007] Acute exposure to IsoLG, or to IsoLG adducts with lysine, ethanolamine, or phosphatidylethanolamine, inhibits mitochondrial respiration and attenuates complex I activity. Complex II is much more resistant to IsoLG. The inventors have demonstrated that the compounds of the present invention, including mito2HOBA, accumulate significantly in isolated mitochondria and are highly reactive with IsoLG.

[0008] To test the role of mitochondrial isoLG, the inventors investigated the therapeutic potential of the compounds of this invention in a lipopolysaccharide (LPS) mouse sepsis model. For example, supplementing the drinking water of LPS-treated mice with mito2HOBA (0.1 g / L) increased survival by 3-fold, improved complex I-mediated respiration, and histopathological analysis supported mito2HOBA-mediated renal cortical protection against cell damage. These data support the role of mitochondrial isoLG in mitochondrial dysfunction and inflammation. Therefore, the inventors were able to demonstrate that reducing mitochondrial isoLG is a therapeutic target for inflammation and conditions associated with mitochondrial oxidative stress and dysfunction.

[0009] Therefore, aspects of the present invention include isolevuglandin and its adducts that inhibit mitochondrial respiration and attenuate the activity of complex I; compounds of the present invention, including 2-hydroxybenzylamine conjugated with triphenylphosphonium, mito2HOBA, which accumulate in mitochondria; mito2HOBA in drinking water that improves complex I-mediated respiration in an LPS sepsis model; and mito2HOBA, an isolevuglandin scavenger targeting mitochondria, that reduces mortality in an LPS model.

[0010] Similarly, hypertension remains a major health problem in Western societies, with one-third of patients experiencing poor blood pressure control despite the use of various medications. Mitochondrial dysfunction is associated with hypertension, and mitochondrial-targeting agents may improve hypertension treatment. The inventors discovered that mitochondrial oxidative stress produces the reactive dicarbonyl lipid peroxidation product isolevuglandin (isoLG), and that the clearance of mitochondrial isoLG improves vascular function and reduces hypertension. To verify this hypothesis, we investigated the accumulation of mitochondrial isoLG-protein adducts in patients with essential hypertension and angiotensin II hypertension models using mass spectrometry and Western blot analysis. The therapeutic potential of targeting mitochondrial isoLG was examined using the novel mitochondrial isoLG scavenger mito2HOBA. Mitochondrial isoLG in the arterioles of hypertensive patients was 250% higher than in normotensive individuals, and in vitro treatment of hypertensive arterioles with mito2HOBA increased the deacetylation of the key mitochondrial antioxidant superoxide dismutase 2 (SOD2). In human aortic endothelial cells stimulated with angiotensin II plus TNFα, mito2HOBA reduced mitochondrial superoxide and cardiolipin oxidation, specific markers of mitochondrial oxidative stress. In mice infused with angiotensin II, mito2HOBA reduced mitochondrial isoLG-protein adducts, increased Sirt3 mitochondrial deacetylase activity, reduced vascular superoxide, increased endothelial nitric oxide, improved endothelium-dependent diastole, and alleviated hypertension. In mice infused with angiotensin II, mito2HOBA protected mitochondrial respiration, protected ATP production, and reduced mitochondrial permeability pore opening. These data support the role of mitochondrial isoLG in endothelial dysfunction and hypertension. The inventors have discovered that the clearance of mitochondrial isoLG has therapeutic benefits in treating vascular dysfunction and hypertension.

[0011] According to recent guidelines, nearly half of all adults have hypertension, and an estimated 1.4 billion people worldwide suffer from it. This disease represents a major risk factor for stroke, myocardial infarction, and heart failure. Despite treatment with various medications, one-third of people with hypertension remain high, possibly because current treatments do not affect its mechanisms. There has long been a need for novel antihypertensive drugs that can improve the treatment of hypertension. Hypertension is a multifactorial disease, and oxidative stress is increased in multiple organs. Oxidative stress contributes to hypertension by increasing sympathetic efferent radiation, promoting renal dysfunction, and increasing systemic vascular resistance. Meanwhile, common antioxidants, such as ascorbic acid and vitamin E, are ineffective in treating cardiovascular disease and hypertension, and have even worsened outcomes in some studies. Endogenous enzymatic antioxidants are more effective at combating oxidative stress than low molecular weight antioxidants, but these endogenous antioxidants may be inactivated in hypertension. Primary hypertension is associated with the inactivation of a key mitochondrial antioxidant, superoxide dismutase 2 (SOD2), through the acetylation of lysine residues at its catalytic center, which is due to reduced activity of the mitochondrial deacetylase sirtuin 3 (Sirt3). However, the exact mechanism of Sirt3 inactivation and the molecular consequences of SOD2 inhibition remain unclear.

[0012] One potential mechanism involves lipid peroxidation, particularly the formation of mitochondrial isoleuglandin (isoLG). IsoLG is a highly reactive and harmful dicarbonyl lipid peroxidation product. It is produced by the peroxidation of arachidonic acid by oxidants such as protonated forms of superoxide and hydroperoxide. IsoLG rapidly combines with primary amines, such as protein lysine residues, promoting cellular dysfunction. In dendritic cells, isoLG promotes the modification of its own proteins, which can act as neoantigens to drive adaptive immune responses. Treatment with the isoLG scavenger 2-hydroxybenzylamine (2HOBA) reduces dendritic cell and T cell activation and attenuates angiotensin II and DOCA-induced hypertension. Notably, 2HOBA is not an antioxidant, but it reduces superoxide production in dendritic cells by scavenging reactive isoLG and decreasing dendritic cell activation. However, the specific sources and targets of isoLG remain unclear. The pathophysiological effects of isoLG are not limited to dendritic cells, as isoLG can be produced in vascular tissue, endothelial cells, epithelial cells, and other cells. Hypertension is associated with mitochondrial oxidative stress, and mitochondria may be a potential source of isoLG, but the role of mitochondrial isoLG in hypertension has not been investigated. The inventors demonstrate that mitochondrial isoLG may promote Sirt3 inactivation and mitochondrial dysfunction in hypertension.

[0013] Treatment of isolated mitochondria with isoLG doses disrupted mitochondrial respiration and promoted the opening of the mitochondrial permeability transition pore (mPTP). To investigate the specific role of isoLG within mitochondria in vivo, the inventors developed a mitochondrial-targeting isoLG scavenger, mito2HOBA, by conjugating 2HOBA to a lipophilic cationic triphenylphosphine. mito2HOBA does not scavenge reactive oxygen species. Unbound by theory or mechanism, it may react with various γ-ketoaldehydes, but exhibits particular reactivity with isoLG. mito2HOBA accumulates in mitochondria, and in a lipopolysaccharide sepsis mouse model, mito2HOBA supplementation increased animal survival by 3-fold, increased complex I-mediated respiration, and prevented renal cortical damage, supporting the role of mitochondrial isoLG in mitochondrial dysfunction.

[0014] Mitochondrial dysfunction promotes the pathogenesis of hypertension and cardiovascular disease; however, despite the central role of mitochondria in human health and disease, there are currently no approved drugs that directly target mitochondria. Mitochondrial dysfunction is characterized by decreased ATP levels and increased oxidative stress, leading to cellular dysfunction and apoptosis. The opening of the mitochondrial permeability transition pore (mPTP) plays a crucial role in mitochondrial dysfunction and end-organ damage in hypertension. The inventors have discovered that the depletion or inhibition of the regulatory subunit of mPTP opening, cyclophilin D (CypD), improves vascular function and alleviates hypertension. Interestingly, CypD acetylation at lysine 166 promotes mPTP opening, and mitochondrial Sirt3 deacetylates CypD-K166.

[0015] Sirt3 is a key node in the regulation of mitochondrial metabolism and antioxidant function. Sirt3 depletion promotes endothelial dysfunction, vascular hypertrophy, vascular inflammation, and end-organ damage. Clinical studies have shown that cardiovascular disease risk factors are associated with decreased Sirt3 levels and activity. The inventors have discovered a novel convergent mechanism underlying the interactions of major cardiovascular risk factors. The inventors demonstrate that mitochondrial isoLG inactivates Sirt3, and that the clearance of mitochondrial isoLG protects Sirt3 activity, improves vascular function, and reduces hypertension.

[0016] The abbreviations used in this article include: IsoLG, isolevuglandin (also known as isoketal); PE, phosphatidylethanolamine; 15-E2-IsoLG, the 15-E2 stereoisomer of isolevuglandin; 15-E2-IsoLG-PE, an adduct of PE and the 15-E2-IsoLG stereoisomer; 2HOBA, 2-hydroxybenzylamine; 4HOBA, the non-scavenger analog of 2HOBA, 4-hydroxybenzylamine; mito2HOBA, 2-hydroxybenzylamine and lipophilic cation. The conjugates of ionic triphenylphosphonium, (4-(4-aminomethyl)-3-hydroxyphenoxy)butyl)-triphenylphosphonium; mitoTEMPO, (2-(2,2,6,6-tetramethylpiperidin-1-oxy-4-ylamino)-2-oxoethyl)triphenylphosphonium; mPTP, mitochondrial permeability transition pore; PUFA, polyunsaturated fatty acid; ROS, reactive oxygen species; LPS, lipopolysaccharide; WT, wild-type C57BL / 6J mouse; SMP, submitochondrial granules.

[0017] Brief description of the attached figures

[0018] Figure 1 A-1C shows that acute treatment with IsoLG or IsoLG-PE impairs mitochondrial respiration. (A) Intact mouse kidney mitochondria were incubated with ethanol (sham surgery), IsoLG (20 µM), or IsoLG-PE (20 µM) as carriers for 5 min, then diluted 20-fold with respiratory buffer, followed by the addition of glutamate and malate, ADP (50 µM), and oxygen consumption in State III was measured. P<0.001 vs sham surgery P < 0.03 vs IsoLG. (B) Oxygen consumption and respiratory control rate (state III / state IV, %) in intact kidney mitochondria treated with the carrier, IsoLG (1.5 µM), or IsoLG-PE (1.5 µM) in the respiratory chamber in the presence of the complex I substrate glutamate + malate and ADP (state III). (C) State III respiration and respiratory control rate (state III / state IV, %) in the presence of the complex II substrate succinate and ADP after the addition of the carrier, IsoLG (1.5 µM), or IsoLG-PE (1.5 µM) in the respiratory chamber. Data are expressed as mean ± STD (N = 4–6). P<0.01 vs. carrier.

[0019] Figure 2The inhibitory effects of IsoLG and IsoLG-adduct on the activities of complex I and complex II were shown. (A, B) Mouse kidney mitochondrial lysates were incubated with DMSO (carrier) or IsoLG for 5 minutes, and then the activities of complex I or complex II were analyzed as previously described [29, 30]. P<0.01 vs sham surgery. P<0.05 vs sham surgery. (C) Mouse kidney mitochondrial lysates were acutely treated with DMSO (control), ethanolamine, spermine, L-lysine, IsoLG (1.5 µM), or IsoLG-modified ethanolamine (IsoLG-ETN, 1.5 µM), IsoLG-sper (IsoLG-Sper, 1.5 µM), IsoLG-L-lysine (IsoLG-Lys, 1.5 µM), or IsoLG-phosphatidylethanolamine (IsoLG-PE, 1.5 µM), followed by measurement of complex I activity, expressed as a percentage of control (100%). Data are presented as mean ± STD (N=3–6). P<0.001 vs sham surgery, #P<0.01 vs IsoLG.

[0020] Figure 3 This study demonstrates the mitochondrial targeting of the IsoLG scavenger mito2HOBA. Due to the significantly higher membrane potential of mitochondria compared to other intracellular organelles, linking 2-hydroxybenzylamine to the lipophilic cationic triphenylphosphonium directs mito2HOBA towards mitochondria, resulting in selective accumulation within the mitochondria. Inflammation and oxidative stress oxidize arachidonic acid to reactive IsoLG, which rapidly reacts with protein lysine residues and phosphatidylethanolamine to produce cytotoxic IsoLG adducts. Incubation of mito2HOBA (0.1 μM) with isolated mitochondria (1 mg / ml) resulted in strong accumulation of mito2HOBA in the mitochondrial fraction. Data are presented as mean ± STD (N=4). Mito2HOBA targeting mitochondria may potentially reduce mitochondrial dysfunction by scavenging IsoLG from the mitochondrial matrix.

[0021] Figure 4Respiration of kidney mitochondria isolated from control sham-operated, mito2HOBA-supplemented, LPS-treated, and LPS-plus-mito2HOBA mice is shown. To investigate mitochondrial function, a combination of glutamate and malate (GM) or succinate was used as a substrate. Since glutamate can be converted to α-ketoglutarate and further to succinate via transamine, the inventors used the complex II inhibitor malonic acid to define specific complex I respiration. Basal respiration was measured in mitochondria supplemented with respiratory substrates (1). ADP was then added to measure coupled respiration (2). Proton leakage was measured after the addition of the complex V inhibitor oligomycin A (3). Decoupled respiration was measured after CCCP supplementation (4). Finally, antimycin A and rotenone were added to assess non-mitochondrial respiration (5), as previously described

[32] . Data are mean ± STD (n=4–6). P<0.001 vs GM, #P<0.01 vs control, §P<0.001 vs control. P<0.01 vs LPS.

[0022] Figure 5 Animal survival (A), complex I / complex II activity ratio (B), and histopathological scores are shown for control, mito2HOBA supplementation, and LPS-treated mice. Three-month-old C57BL / 6J mice (25–28 g) were supplemented with mito2HOBA (drinking water, 0.1 g / L) for 72 hours prior to LPS (25 μg / g) administration. The complex I / complex II activity ratio is expressed as a percentage compared to the control (100%). (C) Histopathological scores of kidney injury, as described in the Methods section. Quantitative analysis of cellular damage showed that mito2HOBA treatment resulted in a significant reduction in cellular damage compared to LPS treatment alone. Data are mean ± STD. P<0.01 vs LPS (n=6), P<0.01 vs LPS (n=10).

[0023] Figure 6 AH shows a histological analysis of cellular damage in the kidneys of control, mito2HOBA-supplemented, LPS-injected, and LPS+mito2HOBA-treated mice. Representative sections from control mice show normal glomeruli (g), proximal tubules (P), and distal tubules located in the cortex (A) and medulla (B). Kidney sections from mice treated with mito2HOBA (C&D) alone were very similar to those from control mice. While most tubules appeared normal, a small number of proximal tubule cells in the medulla (D) showed slight signs of cytoplasmic vacuolation. The cortex (C) showed no signs of damage. In contrast, kidney sections from mice treated with LPS showed vacuolation and cellular degeneration in both the cortex (E) and medulla (F) (arrows). In the medulla, many proximal tubule cells stained basophilic (triangles), suggesting altered intracellular metabolism. Distal tubules ( Normal. When mice were treated with LPS and mito2HOBA, cell damage was detected in the medulla (H), while the cortex (G) appeared normal. Overall damage was significantly reduced compared to LPS-treated mice. Within the medulla, small areas of cytoplasmic degeneration (arrows) and basophilic staining (triangles) were visible. Scale bar = 50 µM.

[0024] Figure 7 A and 7B show (A) the synthesis of an example of the mitochondrial-targeting compound of the present invention and (B) that the compound is an effective IsoLG scavenger.

[0025] Figure 8 AD shows Western blots of mitochondrial isoLG in arterioles from normotensive and hypertensive individuals (n=5), (A), (B) formation of the mitochondrial isoLG scavenger mito2HOBA, and (C, D) SOD2 acetylation isolated from normotensive and hypertensive individuals and treated in vitro with mito2HOBA (0.5 µM, 24 h, DMEM). Data are normalized to complex I levels (sham surgery is 100%). Data are mean ± SEM. P<0.01 vs. normal blood pressure sham surgery P<0.01 vs hypertension (n=5).

[0026] Figure 9 A and B demonstrate the effects of mito2HOBA on angiotensin II plus TNFα-induced mitochondrial superoxide and cardiolipin oxidation in human aortic endothelial cells. (A) Mitosomatic superoxide was determined by HPLC analysis of mito-2-hydroxyethidium (Mito-2OH-ET+), a specific product of mitoSOX-superoxide. Mito2HOBA (50 nM) eliminated the stimulation of mitochondrial superoxide, while similar doses of the non-targeted isoLG scavenger 2HOBA (50 nM) or high doses of the isoLG-inactive analog 4HOBA (10 µM) showed no protective effect. P<0.01 vs. control P < 0.001 vs angiotensin II + TNFα. (B) Cardiolipin oxidation induced by angiotensin II + TNFα as measured by LC / MS. mito2HOBA (50 nM) significantly attenuated cardiolipin oxidation, while non-targeted 2HOBA (10 µM) had no effect. Data are mean ± SEM. P<0.01 vs control P<0.01 vs angiotensin II + TNFα (n=4).

[0027] Figure 10 AD shows the effects of mito2HOBA on angiotensin II-induced hypertension and the accumulation of mitochondrial isoLG protein adducts. (A) Tail-cuff measurements of blood pressure in male mice sham-operated or infused with angiotensin II and supplemented with mito2HOBA (0.1 g / L) or an equimolar amount of the non-targeted analog 2HOBA (0.17 mmol / L) in drinking water. (B) Telemetry studies of blood pressure in mice infused with angiotensin II and supplemented with mito2HOBA or purified water as a carrier. (C) Representative LC / MS / MS chromatograms of the IsoLG-lysyl-lactam adduct; (D) IsoLG-Lys-lactam levels in renal mitochondria isolated from sham-operated or infused with angiotensin II and supplemented with mito2HOBA. Results are mean ± SEM. P<0.01 vs sham surgery P<0.01 vs Ang II (n=8).

[0028] Figure 11AD shows a Western blot analysis of mitochondrial acetylation in aortas isolated from mice subjected to sham surgery and angiotensin II infusion treated with mito2HOBA. (A) Typical Western blots of isoLG protein adduct (D11 ab), Sirt3, acetylated lysine, SOD2-K68-acetylation, CypD-acetylation, adducts of isoLG with the NDUFS1 75 kDa subunit of complex I, and mitochondrial complex I; (B) Sirt3 level; (C) mitochondrial protein lysine acetylation; (D) SOD2-K68-acetyl level; and (E) CypD-acetyl level. Mice were administered mito2HOBA (m2H) (0.1 g / L) and angiotensin II (osmotic pump, 0.7 mg / kg / day) in drinking water for 14 days. Data were normalized to complex I levels (sham surgery = 100%). Results are presented as mean ± SEM (n=5). P<0.01 vs sham surgery P<0.01 vs angiotensin II (Ang II).

[0029] Figure 12 AD showed the effects of mito2HOBA supplementation on mitochondrial superoxide (A), vascular superoxide (B), endothelial nitric oxide (C), and endothelial-dependent relaxation (D) in angiotensin II infusion mice. Mitochondrial and vascular O2• were measured by HPLC using either the mitochondrial-targeted superoxide probe mitoSOX (1 µM) or the non-targeted superoxide probe DHE (50 µM). 46 Endothelial nitric oxide was analyzed using the NO spin trap Fe(DETC)2 and ESR. 47 C57BL / 6J mice were infused with AngII and provided with mito2HOBA (0.1 g / L) in drinking water. Results are mean ± SEM. P<0.01 vs sham surgery P<0.01 vs Ang II (n=6).

[0030] Figure 13 AB studies showed that mito2HOBA reduced mPTP opening and prevented mitochondrial dysfunction. C57Bl / 6J mice were infused with Ang II (0.7 mg / kg / ml) and given mito2HOBA (0.1 g / L) in their drinking water. Fourteen days after Ang II infusion, the animals were sacrificed, and the kidneys were dissected for mitochondrial studies. Adding CaCl2 to the mitochondria beyond their Ca2+ retention capacity resulted in mPTP opening and mitochondrial swelling.71 Mitochondria isolated from Ang II-infused mice exhibited a significant decrease in Ca2+ capacity due to increased mPTP opening, and the Ca2+ retention capacity was salvaged by the CypD inhibitor cyclosporine A (CsA) (A). Respiratory control rates (3-state / 4-state) of isolated renal mitochondria were determined using glutamate and malate (B). The control level was 100%. (C) Renal ATP in freshly isolated tissues was measured using a luciferase-based luminescence assay. 72 The results are the mean ± SEM. P<0.01 vs sham surgery P<0.01 vs angiotensin II (n=5).

[0031] Figure 14 This study demonstrates an association between hypertension and Sirt3 inactivation, which leads to hyperacetylation of mitochondrial proteins such as cyclophilin D (CypD) and mitochondrial superoxide dismutase (SOD2). CypD acetylation promotes mPTP opening, which increases mitochondrial superoxide production, while SOD2 acetylation inactivates SOD2. This results in an imbalance between increased superoxide production and decreased SOD2 activity, leading to mitochondrial oxidative stress and the oxidation of polyunsaturated fatty acids (PUFAs) in mitochondria to reactive γ-ketoaldehydes, isolevuglandin (isoLG). Mitochondrial isolevuglandin promotes vascular and mitochondrial dysfunction, while treatment with the mitochondrial-targeting isolevuglandin mito2HOBA reduces Sirt3 inactivation, improves mitochondrial and vascular function, and alleviates hypertension. This invention demonstrates that targeting mitochondrial isolevuglandin prevents Sirt3 inactivation and can improve the treatment of vascular dysfunction in human individuals. Detailed Implementation

[0032] The invention can be more readily understood by referring to the following detailed description of the invention and the embodiments included therein.

[0033] Before disclosing and describing the compounds, compositions, articles, systems, devices, and / or methods of the present invention, it should be understood that they are not limited to specific synthetic methods unless otherwise stated, or to specific reagents unless otherwise stated, as variations are naturally possible. It should also be understood that the terminology used herein is for descriptive purposes only and is not intended to be limiting. Although any methods and materials similar to or equivalent to those described herein may be used to practice or test the invention, exemplary methods and materials are now described.

[0034] As used in the specification and appended claims, the singular forms “a,” “an,” and “the” include a plurality of indicators unless the context clearly specifies otherwise. Thus, for example, references to “functional group,” “alkyl,” or “residue” include mixtures of two or more such functional groups, alkyl groups, or residues, etc.

[0035] This document may express a range as "about" to one specific value and / or "about" to another specific value. When expressing such a range, the other side includes from one specific value and / or to another specific value. Similarly, when a value is expressed as an approximation using the antecedent "about," it should be understood that the specific value forms the other side. It should also be understood that the endpoints of each range are meaningful whether they are related to or not to another endpoint. It should also be understood that this document discloses many numerical values, and each numerical value is also disclosed in this document as "about" that specific numerical value as well as the numerical value itself. For example, if the numerical value "10" is disclosed, then "about 10" is also disclosed. It should also be understood that each unit between specific units is also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0036] As used herein, the terms “optional” or “optionally” mean that the event or situation described below may or may not occur, and the description includes both the scenario in which the event or situation occurs and the scenario in which the event or situation does not occur.

[0037] As used herein, the term "individual" refers to the subject of drug administration. The individual in the methods disclosed herein can be a vertebrate, such as a mammal, fish, bird, reptile, or amphibian. Therefore, the individual in the methods disclosed herein can be a human, non-human primate, horse, pig, rabbit, dog, sheep, goat, cattle, cat, guinea pig, or rodent. This term does not refer to a specific age or sex. Therefore, both male and female adult and newborn individuals, as well as fetuses, are included. A patient is an individual suffering from a disease or ailment. The term "patient" includes both human and mammalian individuals.

[0038] As used herein, the term "treatment" refers to the medical management of a patient aimed at curing, improving, stabilizing, or preventing a disease, pathological condition, or symptom. This term includes active treatment, which is treatment specifically designed to improve a disease, pathological condition, or symptom, and etiological treatment, which is treatment aimed at eliminating the cause of the associated disease, pathological condition, or symptom. Furthermore, the term includes palliative treatment, which is treatment aimed at relieving symptoms rather than curing a disease, pathological condition, or symptom; preventative treatment, which is treatment aimed at minimizing or partially or completely suppressing the development of an associated disease, pathological condition, or symptom; and supportive treatment, which is treatment used in conjunction with another specific therapy aimed at improving the associated disease, pathological condition, or symptom.

[0039] As used herein, the terms “prevent” or “preventing” mean to exclude, prevent, avoid, deter, or hinder something from happening, especially by taking proactive measures. It should be understood that, unless otherwise expressly stated, the use of the other two terms is also explicitly disclosed in the context of the use of “reduce,” “inhibit,” or “prevent” herein. As seen herein, there is overlap in the definitions of treatment and prevention.

[0040] As used herein, the term "diagnosed" means that a person has been examined by someone skilled in the art (e.g., a physician) and found to have a condition that can be diagnosed or treated by the compounds, compositions, or methods disclosed herein. As used herein, phrases such as "identified as requiring treatment" refer to selecting an individual based on the need for treatment of a condition. For example, an individual may be identified as requiring treatment for a condition (e.g., an inflammation-related condition) based on an early diagnosis by someone skilled in the art, and then receive treatment for that condition. In one aspect, it is anticipated that the identification may be performed by someone different from the person making the diagnosis. In another aspect, it is also anticipated that the administration may be performed by the person subsequently administering the medication.

[0041] As used herein, the terms "administering" and "administration" refer to any method of delivering a pharmaceutical preparation to an individual. These methods are well known to those skilled in the art and include, but are not limited to, oral administration, transdermal administration, inhalation administration, nasal administration, topical administration, vaginal administration, ophthalmic administration, intraocular administration, intracerebral administration, rectal administration, and parenteral administration, including injection, such as intravenous administration, intra-arterial administration, intramuscular administration, and subcutaneous administration. Administration can be continuous or intermittent. In various aspects, the preparation can be administered therapeutically; that is, administered to treat an existing disease or condition. In other aspects, the preparation can be administered prophylactically; that is, administered to prevent a disease or condition.

[0042] As used herein, the term "effective amount" refers to an amount sufficient to achieve a desired outcome or to be effective against an undesirable symptom. For example, a "therapeutic effective amount" is an amount sufficient to achieve a desired therapeutic outcome or to be effective against an undesirable symptom, but generally insufficient to cause adverse side effects. The specific therapeutic effective dose level for any particular patient will depend on a variety of factors, including the condition being treated and its severity; the specific composition used; the patient's age, weight, general health, sex, and diet; the time of administration; the route of administration; the rate of excretion of the specific compound used; the duration of treatment; drugs used in combination with or concurrently with the specific compound used; and similar factors well known in the medical field. For example, within the scope of the art, it is permissible to begin with a dose of the compound at a level below that required to achieve the desired therapeutic effect, gradually increasing the dose until the desired therapeutic effect is achieved. It is not necessary to divide the effective daily dose into multiple doses for administration. Therefore, a single-dose composition may contain such an amount or an approximation thereof to achieve the daily dose. In the event of any contraindications, an individual physician may adjust the dose. The dose may vary and may be administered once or multiple times daily for one or several days. For a given class of pharmaceutical products, guidance on appropriate dosages can be found in the literature. In all other respects, a formulation can be administered in a “preventive effective amount”; that is, an amount that effectively prevents disease or ailment.

[0043] This invention relates to mitochondrial-targeting derivatives of 2-hydroxybenzylamine (scavengers of highly reactive lipid dicarbonyl compounds derived from arachidonic acid and other polyunsaturated fatty acids—isolevulin (isoLG, also known as isoacetal or γ-ketoaldehyde), pharmaceutical compositions comprising such compounds, and methods for treating diseases involving mitochondrial dysfunction, oxidative stress (e.g., mitochondrial oxidative stress), hypertension, and sepsis.

[0044] In another embodiment of the invention, a method for treating, preventing, and improving mitochondrial dysfunction in an individual is provided, comprising administering an effective amount of the mitochondrial-targeting scavenger of the invention or a pharmaceutically acceptable salt thereof.

[0045] In another embodiment of the invention, a method for treating, preventing, and improving oxidative stress in an individual is provided, comprising administering an effective amount of the compound of the invention or a pharmaceutically acceptable salt thereof.

[0046] In another embodiment of the invention, a method for treating, preventing, and improving hypertension in an individual is provided, comprising administering an effective amount of the compound of the invention or a pharmaceutically acceptable salt thereof.

[0047] In another embodiment of the invention, a method for treating, preventing, and improving sepsis in an individual is provided, comprising administering an effective amount of the compound of the invention or a pharmaceutically acceptable salt thereof.

[0048] Before disclosing and describing the compounds, compositions, articles, systems, devices, and / or methods of the present invention, it should be understood that they are not limited to specific synthetic methods unless otherwise stated, or to specific reagents unless otherwise stated, as variations are naturally possible. It should also be understood that the terminology used herein is for descriptive purposes only and is not intended to be limiting. Although any methods and materials similar to or equivalent to those described herein may be used to practice or test the invention, exemplary methods and materials are now described.

[0049] In one aspect, the present invention relates to compounds or pharmaceutically acceptable derivatives thereof for treating the indications described herein, including inflammation, hypertension, mitochondrial oxidative stress, and mitochondrial dysfunction. Generally, each disclosed derivative is contemplated to be optionally further substituted. It is also contemplated that any one or more derivatives may be optionally omitted from the invention. It should be understood that the disclosed compounds can be provided by the disclosed methods. It should also be understood that the disclosed compounds can be used with the disclosed methods of use. It should also be understood that all disclosed compounds can be used as corresponding pharmaceutical compositions.

[0050] One embodiment of the present invention is a compound of the following formula:

[0051] ;

[0052] in:

[0053] X is a bond, alkyl, alkoxy, methoxy, -O-, or -CH2-;

[0054] Each R is independent and selected from C1-C2. 12 Substituted or unsubstituted alkyl groups; and

[0055] A is , , , and ;

[0056] Each R1 is independent and selected from C1-C1. 12 Substituted or unsubstituted alkyl groups; and optional counterions;

[0057] Its stereoisomers and medicinal salts.

[0058] Another embodiment of the present invention is a compound of the following formula:

[0059] ,

[0060] in:

[0061] X is a bond, alkyl group, -O- or -CH2-; and

[0062] R is C1-C 12 Substituted or unsubstituted alkyl groups;

[0063] Its stereoisomers and medicinal salts.

[0064] Another embodiment of the present invention is a compound of the following formula:

[0065] ,

[0066] in:

[0067] R is C1-C 12 Substituted or unsubstituted alkyl groups;

[0068] Its stereoisomers and medicinal salts.

[0069] Another embodiment of the present invention is a compound of the following formula:

[0070] ,

[0071] in:

[0072] R1 is C1-C 12 Substituted or unsubstituted alkyl groups;

[0073] Its stereoisomers and medicinal salts.

[0074] Another embodiment of the present invention is a compound of the following formula:

[0075] ,

[0076] in:

[0077] R1 is C1-C 12 Substituted or unsubstituted alkyl groups;

[0078] Its stereoisomers and medicinal salts.

[0079] Another embodiment of the present invention is a compound of the following formula:

[0080] ,

[0081] in:

[0082] X is a bond, -O-, or -CH2-;

[0083] R is C1-C 12Substituted or unsubstituted alkyl groups; and

[0084] R1 is C1-C 12 Substituted or unsubstituted alkyl or acetoxymethyl groups;

[0085] Its stereoisomers and medicinal salts.

[0086] Another embodiment of the present invention is a compound of the following formula:

[0087] ,

[0088] in:

[0089] Each R is independent and selected from C1-C2. 12 Substituted or unsubstituted alkyl groups; and

[0090] Each R1 is independent and selected from C1-C1. 12 Substituted or unsubstituted alkyl or acetoxymethyl groups;

[0091] Its stereoisomers and medicinal salts.

[0092] In another embodiment, the mitochondrial-targeting scavenger is a compound of the following formula:

[0093] ,

[0094] in:

[0095] R is C1-C 12 Substituted or unsubstituted alkyl groups;

[0096] R2 is selected from -P-PH3; or ;

[0097] Its stereoisomers and medicinal salts.

[0098] Another embodiment of the present invention is a compound of the following formula:

[0099] , , ,

[0100] , ,

[0101] , , and

[0102] ; and its pharmaceutically acceptable salts.

[0103] Another embodiment of the present invention is a compound of the following formula:

[0104] ,

[0105] Its stereoisomers and medicinal salts.

[0106] General routes

[0107] The synthesis of the isoLG scavenger targeting mitochondria of the present invention is described below.

[0108] Synthetic routes for ether bonds

[0109]

[0110]

[0111]

[0112] Cesium carbonate (4.9 g, 15 mmol) was added to 2,4-dihydroxybenzaldehyde (4.2 g, 30 mmol) and 1,4-dibromobutane (6.6 g, 30 mmol) in acetonitrile (50 ml). The mixture was heated at 80 °C for 5 h under argon, cooled, and then added to 1 M phosphate buffer (30 ml), pH 7, ice, and KH₂PO₄ (2 g) with stirring. The solid was removed by filtration, and the filtrate was extracted with ethyl acetate. Purification by column chromatography (silica, 9:1 hexane-ethyl acetate) yielded 4-(4-bromobutoxy)-2-hydroxybenzaldehyde (4.1 g, 50%). This was mixed with triphenylphosphine (4.2 g) in toluene (75 ml) and refluxed under argon for 15 h. The pink solid was purified by rapid chromatography (0-10% methanol in dichloromethane) to give 4-(4-formyl-3-hydroxyphenoxy)butyl)triphenylphosphonium bromide (4.8 g, 60%). The aldehyde was converted to an oxime by stirring in ethanol (40 ml) for 1 h in the presence of NH₂OH·HCl (0.63 g) and CH₃CO₂Na (0.74 g). The crude product was dissolved in acetic acid (40 ml) and treated with zinc powder (6 g). The reaction mixture was slowly heated to 60 °C in a water bath, maintained at that temperature for 20 m, cooled, and filtered. Acetic acid was removed, and the residue was purified by column chromatography (silica; 5-20% 0.1 M ammonium acetate-acetonitrile). Other compounds were prepared similarly using suitable dibromoalkanes.

[0113] The route of the methylene bond:

[0114]

[0115] 5-(chloromethyl)-2-hydroxybenzaldehyde (4.5 g) was refluxed with triphenylphosphine (5.75 g) in acetonitrile for 5 h. After cooling, the adduct was purified by column chromatography (silica; 0-15% methanol in dichloromethane). The aldehyde was converted to an oxime, reduced with zinc in acetic acid, and purified according to the description of mito2HOBA-C4. The alkyl chain can be extended.

[0116] The preparation of the original mito salicylamine analogue is shown below:

[0117]

[0118] Synthetic routes for esters and acids:

[0119]

[0120]

[0121] Methyl 4-hydroxybenzoate (8.3 g) was refluxed in acetonitrile containing paraformaldehyde (8 g), magnesium chloride (10 g), and triethylamine (28 mL). After 30 m, the reaction mixture was acidified and extracted with ethyl acetate. The crude product was purified on a silica gel column (5:1 hexane-ethyl acetate). The aldehyde was converted to an oxime, which was then heated with 2 equivalents of LiOH in a 1:1 methanol-water mixture at 75–80 °C for 2 h. The oxime acid was separated after cooling and acidification as a white solid. It (2.3 g) was dissolved in DMF (25 mL), chilled, and treated with KHCO3 and bromomethyl acetate (1.7 g). The mixture was stirred for 18 h, and the crude product was purified on silica gel (2:1 hexane-ethyl acetate 0; 2.1 g supernatant). The oxime was reduced with zinc powder (3.2 g) and acetic acid (30 mL) to give AcMo-2HOBA.

[0122] The methyl 3-formyl-4-hydroxybenzoate obtained in the first step was converted to an oxime and then reduced to give MCM-2HOBA. Similarly, ethyl 3-(4-hydroxyphenyl)propionate is the starting compound for ece-2HOBA.

[0123] Synthetic routes for diesters:

[0124]

[0125] 5-(chloromethyl)-2-hydroxybenzaldehyde (8 g) was stirred in THF (40 ml) with diethyl iminodiacetate (5 ml) and triethylamine (5.6 ml) for 2 h. The crude product was subjected to rapid chromatography (silica; 3:1 hexane-ethyl acetate). The aldehyde group was converted to an oxime by treatment with hydroxylamine. The 2HOBA-diester was purified by reduction with zinc and acetic acid in an ethanol solution of HCl and sodium acetate, followed by purification on a silica gel column. The purified product was eluted with 30% methanol-ethyl acetate.

[0126] As used herein, the term "alkyl" refers to a branched or unbranched saturated hydrocarbon group having 1 to 24 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, sec-pentyl, neopentyl, hexyl, heptyl, octyl, nonyl, decyl, dodecyl, tetradecyl, hexadecyl, eicosyl, tetradecyl, etc. Alkyl groups can be cyclic or acyclic. Alkyl groups can be branched or unbranched. Alkyl groups can also be substituted or unsubstituted. For example, an alkyl group can be substituted with one or more groups, including but not limited to optionally substituted alkyl, cycloalkyl, alkoxy, amino, ether, halide, hydroxyl, nitro, silyl, sulfoxy, or thiol groups, as described herein. A "lower alkyl" group is an alkyl group containing 1 to 6 (e.g., 1 to 4) carbon atoms.

[0127] Throughout this specification, "alkyl" is generally used to refer to both unsubstituted and substituted alkyl groups; however, substituted alkyl groups are also specifically referred to herein by identifying the specific substituents on the alkyl group. For example, the term "haloalkyl" specifically refers to an alkyl group substituted with one or more halides (e.g., fluorine, chlorine, bromine, or iodine). The term "alkoxyalkyl" specifically refers to an alkyl group substituted with one or more alkoxy groups as described below. The term "alkylamino" specifically refers to an alkyl group substituted with one or more amino groups as described below, and so on. When "alkyl" is used in one context and a specific term such as "alkyl alcohol" is used in another context, it does not imply that the term "alkyl" refers to a specific term such as "alkyl alcohol," etc.

[0128] As used in this article, the terms "alkoxy" and "alkoxyl" refer to alkyl or cycloalkyl groups bonded by ether bonds; that is, the "alkoxy" group can be defined as -OA. 1 A 1 It is an alkyl or cycloalkyl group as defined above. "Alkoxy" also includes alkoxy polymers as described above; that is, the alkoxy group can be a polyether, such as -OA. 1 -OA 2 or -OA 1 -(OA 2 ) a -OA3 , where “a” is an integer from 1 to 200, and A 1 A 2 and A 3 It is an alkyl and / or cycloalkyl group.

[0129] The compounds described herein may contain one or more double bonds, thus potentially yielding cis / trans (E / Z) isomers and other conformational isomers. Unless otherwise stated, the invention encompasses all such possible isomers, as well as mixtures of these isomers.

[0130] It should also be understood that the compounds described herein may contain optional counterions, if desired or necessary. Examples of such optional counterions include chloride, methanesulfonate, bicarbonate, fluoride, nitrate, bromide, sulfate, citrate, benzoate, saccharin anion, and acetate. For example, if a triphenylphosphonium compound is described, it may be assumed to be triphenylphosphonium bromide.

[0131] Unless otherwise stated, chemical bonds are represented by solid lines rather than wedges or dashed lines to encompass every possible isomer, such as each enantiomer and diastereomer, and mixtures of isomers, such as racemic or scalemic mixtures. The compounds described herein may contain one or more asymmetric centers, thus potentially yielding diastereomers and optical isomers. Unless otherwise stated, the invention includes all such possible diastereomers and their racemic mixtures, their substantially pure resolved enantiomers, all possible geometric isomers, and their pharmaceutically acceptable salts. Mixtures of stereoisomers and isolated specific stereoisomers are also included. In synthetic methods used to prepare such compounds, or in the use of racemization or epimerization methods known to those skilled in the art, the products of such methods may be mixtures of stereoisomers. Furthermore, unless explicitly stated as “unsubstituted,” all substituents may be substituted or unsubstituted.

[0132] In some respects, the structure of a compound can be represented by the following formula:

[0133] ,

[0134] It is understood to be equivalent to the following formula:

[0135] ,

[0136] in n It is usually an integer. That is, R n R is understood to represent five independent substituents. n(a) R n(b) R n(c) R n(d) R n(e) "Independent substituents" means that each R substituent can be defined independently. For example, if in one example R n(a) It's a halogen, so in that example R n(b) It is not necessarily a halogen. Similarly, when group R is defined as having four substituents, R is understood to represent four independent substituents. a R b R c And Rd. Unless otherwise stated, the substituents are not limited to any particular order or arrangement.

[0137] In one aspect, the present invention relates to pharmaceutical compositions comprising the disclosed compounds. That is, pharmaceutical compositions may be provided comprising a therapeutically effective amount of at least one disclosed compound or at least one product of the disclosed method and a pharmaceutically acceptable carrier.

[0138] In some aspects, the disclosed pharmaceutical compositions comprise the disclosed compound as an active ingredient (including pharmaceutically acceptable salts thereof), a pharmaceutically acceptable carrier, and optional other therapeutic ingredients or excipients. The compositions of the present invention include those suitable for oral, rectal, topical, and parenteral (including subcutaneous, intramuscular, and intravenous) administration; however, the most suitable route in any given case will depend on the specific host and the nature and severity of the condition for which the active ingredient needs to be administered. The pharmaceutical compositions can be conveniently provided in unit dosage forms and prepared by any method known in the pharmaceutical field.

[0139] As used herein, the term "pharmaceutically acceptable salt" refers to a salt prepared from a pharmaceutically acceptable non-toxic alkali or acid. When the compounds of the present invention are acidic, their corresponding salts can be readily prepared from pharmaceutically acceptable non-toxic alkalis (including inorganic and organic bases). Salts derived from such inorganic bases include aluminum salts, ammonium salts, calcium salts, copper (divalent and monovalent) salts, ferric salts, ferrous salts, lithium salts, magnesium salts, manganese (trivalent and divalent) salts, potassium salts, sodium salts, zinc salts, etc. Ammonium salts, calcium salts, magnesium salts, potassium salts, and sodium salts are particularly preferred. Salts derived from pharmaceutically acceptable non-toxic organic alkalis include salts of primary amines, secondary amines, and tertiary amines, as well as cyclic amines and substituted amines (e.g., naturally occurring and synthetically produced substituted amines). Other pharmaceutically acceptable non-toxic organic bases that can be used to form salts include ion exchange resins, such as arginine, betaine, caffeine, choline, N,N'-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucosamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucosamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purine, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, etc.

[0140] As used herein, the term "pharmaceutically acceptable non-toxic acid" includes inorganic acids, organic acids, and salts prepared therefrom, such as acetic acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, citric acid, ethanesulfonic acid, fumaric acid, gluconic acid, glutamic acid, hydrobromic acid, hydrochloric acid, hydroxyethanesulfonic acid, lactic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, mucilage, nitric acid, pamoic acid, pantothenic acid, phosphoric acid, succinic acid, sulfuric acid, tartaric acid, p-toluenesulfonic acid, etc. Citric acid, hydrobromic acid, hydrochloric acid, maleic acid, phosphoric acid, sulfuric acid, and tartaric acid are preferred.

[0141] In practice, the compounds of the present invention or their pharmaceutically acceptable salts can be tightly mixed with a pharmaceutical carrier as active ingredients according to conventional pharmaceutical techniques. The carrier can take many forms depending on the desired formulation for administration (e.g., oral or parenteral (including intravenous)). Therefore, the pharmaceutical compositions of the present invention can be provided in the form of separate units suitable for oral administration, such as capsules, pouches, or tablets each containing a predetermined amount of the active ingredient. Furthermore, the compositions can be provided as powders, granules, solutions, suspensions in aqueous liquids, non-aqueous liquids, oil-in-water emulsions, or water-in-oil emulsions. In addition to the common dosage forms described above, the compounds of the present invention and / or their pharmaceutically acceptable salts can also be administered via controlled-release devices and / or delivery devices. The compositions can be prepared by any pharmaceutical method. Typically, such methods involve combining the active ingredient with a carrier constituting one or more essential components. Typically, the compositions are prepared by uniformly and tightly mixing the active ingredient with a liquid carrier or a solid carrier ground into a fine powder, or both. The product can then be conveniently shaped into the desired form.

[0142] Therefore, the pharmaceutical compositions of the present invention may comprise a pharmaceutically acceptable carrier and a compound of the present invention or a pharmaceutically acceptable salt thereof. The compound of the present invention or a pharmaceutically acceptable salt thereof may also be included in the pharmaceutical composition in combination with one or more other therapeutically active compounds. The pharmaceutical carrier used may be, for example, solid, liquid, or gas. Examples of solid carriers include lactose, kaolin, sucrose, talc, gelatin, agar, pectin, gum arabic, magnesium stearate, and stearic acid. Examples of liquid carriers are syrup, peanut oil, olive oil, and water. Examples of gaseous carriers include carbon dioxide and nitrogen.

[0143] When preparing compositions for oral dosage forms, any convenient pharmaceutical matrix can be used. For example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents, etc., can be used to form oral liquid dosage forms, such as suspensions, elixirs, and solutions; while carriers such as starch, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrants, etc., can be used to form oral solid dosage forms, such as powders, capsules, and tablets. Because of their ease of administration, tablets and capsules are preferred oral dosage units, and the carriers used are solid pharmaceutical carriers. Optionally, tablets can be coated using standard aqueous or non-aqueous techniques.

[0144] Tablets containing the compositions of the present invention can optionally be prepared by compression or molding with one or more auxiliary components or excipients. Compressed tablets can be prepared by compressing the active ingredient, which is present in a free-flowing form, such as powder or granules, with a binder, lubricant, inert diluent, surfactant, or dispersant on a suitable machine. Cast tablets can be prepared by shaping a powdered compound moistened with an inert liquid diluent in a suitable machine.

[0145] The pharmaceutical compositions of the present invention may comprise the compound of the present invention (or a pharmaceutically acceptable salt thereof) as an active ingredient, a pharmaceutically acceptable carrier, and optionally one or more other therapeutic agents or adjuvants. The compositions of the present invention include those suitable for oral, rectal, topical, and parenteral (including subcutaneous, intramuscular, and intravenous) administration; however, the most suitable route of administration in any given situation will depend on the specific host and the nature and severity of the condition to which the active ingredient is administered. The pharmaceutical compositions may be readily available in unit dosage forms and prepared by any method well known in the pharmaceutical field.

[0146] The pharmaceutical compositions of the present invention, suitable for parenteral administration, can be formulated as solutions or suspensions of the active compound in water. Suitable surfactants, such as hydroxypropyl cellulose, may be included. Dispersions can also be prepared in glycerol, liquid polyethylene glycol, and mixtures thereof in oil. Furthermore, preservatives may be included to prevent harmful microbial growth.

[0147] The pharmaceutical compositions of the present invention suitable for injection comprise sterile aqueous solutions or dispersions. Furthermore, the compositions may be in the form of sterile powders for the ad hoc preparation of such sterile injectable solutions or dispersions. In any case, the final injectable form must be sterile and must be substantially fluid to facilitate injection. The pharmaceutical compositions must be stable under the conditions of manufacture and storage; therefore, preferred storage should prevent contamination by microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), vegetable oils, and suitable mixtures thereof.

[0148] The pharmaceutical compositions of the present invention can be in forms suitable for topical use, such as aerosols, creams, ointments, lotions, powders, mouthwashes, and gargles. Furthermore, the compositions can be in forms suitable for transdermal devices. These formulations can be prepared using conventional processing methods, utilizing the compounds of the present invention or their pharmaceutically acceptable salts. For example, a cream or ointment is prepared by mixing a hydrophilic substance with water and about 5% by weight to about 10% by weight of a compound to produce a cream or ointment having a desired consistency.

[0149] The pharmaceutical compositions of the present invention can be in a form suitable for rectal administration, wherein the carrier is solid. Preferably, the mixture forms a unit-dose suppository. Suitable carriers include cocoa butter and other substances commonly used in the art. Suppositories can be conveniently formed by first mixing the composition with a softened or melted carrier, and then cooling and shaping it in a mold.

[0150] In addition to the carrier components described above, the pharmaceutical formulations described above may, depending on the circumstances, contain one or more other carrier components, such as diluents, buffers, flavoring agents, binders, surfactants, thickeners, lubricants, preservatives (including antioxidants), etc. Furthermore, other excipients may be included to make the formulation isotonic with the blood of the target receptor. Compositions containing the compounds of the present invention and / or their pharmaceutically acceptable salts may also be prepared in powder or liquid concentrate form.

[0151] In therapeutic situations requiring enhanced activity of metabolite glutamate receptors, appropriate dosage levels are typically 0.01-500 mg / kg of patient body weight per day, and can be administered as a single dose or in multiple doses. Preferably, the dosage level is about 0.1 to about 250 mg / kg per day; more preferably, 0.5 to 100 mg / kg per day. Suitable dosage levels may be 0.01 to 250 mg / kg per day, 0.05 to 100 mg / kg per day, or 0.1 to 50 mg / kg per day. Within this range, the dosage may be 0.05 to 0.5 mg / kg per day, 0.5 to 5.0 mg / kg per day, or 5.0 to 50 mg / kg per day. For oral administration, the composition is preferably provided in tablet form, the tablets containing 1.0-1000 mg of the active ingredient, particularly 1.0, 5.0, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 400, 500, 600, 750, 800, 900, and 1000 mg of the active ingredient, for symptomatic dosage adjustment in the patients being treated. The compound can be administered in a regimen of 1 to 4 times daily, preferably once or twice daily. This dosage regimen can be adjusted to provide an optimal therapeutic response.

[0152] However, it is understandable that a specific dose level for any given patient will depend on a variety of factors. These factors include the patient's age, weight, general health condition, sex, and diet. Other factors include the timing and route of administration, excretion rate, drug combination, and the type and severity of the specific disease being treated.

[0153] The disclosed pharmaceutical compositions may further include other therapeutically active compounds, which are typically used to treat the aforementioned pathological conditions.

[0154] It should be understood that the disclosed compositions can be prepared from the disclosed compounds. It should also be understood that the disclosed compositions can be used in the disclosed methods of application.

[0155] This article also discloses pharmaceutical compositions comprising one or more of the disclosed compounds and a pharmaceutically acceptable carrier.

[0156] Therefore, the pharmaceutical compositions of the present invention include pharmaceutical compositions containing one or more other active ingredients besides the compounds of the present invention.

[0157] The aforementioned combinations include not only combinations of the disclosed compound with one other active compound, but also combinations of the disclosed compound with two or more other active compounds. Similarly, the disclosed compound can be used in combination with other medicaments for the prevention, treatment, control, improvement of diseases or conditions for which the disclosed compound is useful, or reduction of their risk. These other medicaments can be administered simultaneously or sequentially with the compound of the present invention via their usual route of use and dosage. When the compound of the present invention is used simultaneously with one or more other medicaments, it is preferable to have a pharmaceutical composition containing, in addition to the compound of the present invention, such other medicaments. Therefore, the pharmaceutical compositions of the present invention comprise pharmaceutical compositions containing one or more other active ingredients besides the compound of the present invention.

[0158] The weight ratio of the compound of the present invention to the second active ingredient can be varied and will depend on the effective dosage of each ingredient. Typically, the effective dosage of each ingredient is used. Therefore, for example, when the compound of the present invention is combined with another pharmaceutical agent, the weight ratio of the compound of the present invention to the other pharmaceutical agent is typically from about 1000:1 to about 1:1000, preferably from about 200:1 to about 1:200. Combinations of the compound of the present invention with other active ingredients will also generally fall within the above range, but in each case, the effective dosage of each active ingredient should be used.

[0159] In such combinations, the compounds and other active agents of the present invention can be administered alone or in combination. Furthermore, administration of one component can be performed before, simultaneously with, or after administration of other agents.

[0160] Therefore, the compounds of the present invention can be used alone or in combination with other agents known to be beneficial in the indications of the present invention or other drugs that affect receptors or enzymes, which increase the effectiveness, safety, convenience, or reduce the harmful side effects or toxicity of the disclosed compounds. The compounds of the present invention and other agents can be administered concurrently or in a fixed combination.

[0161] In one aspect, the compound may be used in combination with antihypertensive agents, anti-inflammatory agents, and / or antioxidant stress agents. Therefore, the disclosed compound can be used to treat, prevent, control, improve, or reduce the risk of the aforementioned diseases, disorders, and conditions. When the disclosed compound is used concurrently with one or more other drugs, a pharmaceutical composition containing such drugs and the disclosed compound in a unit dosage form is preferred. However, combination therapy may also be administered on overlapping schedules. It is also anticipated that combinations of one or more active ingredients and the disclosed compound will be more effective than either as a single agent.

[0162] In one aspect, the present invention relates to a treatment method in mammals, comprising the step of administering at least one compound of the invention to the mammal, the dosage and amount of said compound being effective in treating an indication of said mammal. In some embodiments, said compound has a structure represented by a compound of the following formula:

[0163] ;

[0164] in:

[0165] X is a bond, alkyl, alkoxy, methoxy, -O-, or -CH2-;

[0166] Each R is independent and selected from C1-C2. 12 Substituted or unsubstituted alkyl groups; and

[0167] A is , , , and ;

[0168] Each R1 is independent and selected from C1-C1. 12 Substituted or unsubstituted alkyl groups; and optional counterions;

[0169] Its stereoisomers and medicinal salts.

[0170] In some aspects, the individual, such as a mammal or human, has been diagnosed with the indication prior to the administration step. In a further aspect, the disclosed method may further include the step of identifying an individual (e.g., a mammal or human) who requires treatment for the indication, disease, disorder, or condition described herein. In a further aspect, the individual, such as a mammal or human, has been diagnosed with the need for treatment prior to the administration step.

[0171] The disclosed compounds can be used as a single agent or in combination with one or more other drugs to treat, prevent, control, improve, or reduce the risks of the aforementioned indications, diseases, disorders, and conditions to which the compounds of the present invention or other drugs are useful, wherein the combination of drugs is safer or more effective than any single drug alone. The other drugs may be administered simultaneously or sequentially with the disclosed compounds via their usual route of use and dosage. When the disclosed compounds are used simultaneously with one or more other drugs, pharmaceutical compositions containing such drugs and the compounds in unit dosage form are preferred. However, combination therapy may also be administered on overlapping schedules. It is also anticipated that combinations of one or more active ingredients and the disclosed compounds may be more effective than any single agent.

[0172] The compounds of this invention can be administered with pharmaceutically acceptable carriers. As used herein, the term "pharmaceutically acceptable carrier" refers to sterile aqueous or non-aqueous solutions, dispersions, suspensions, or emulsions, as well as sterile powders intended for reconstitution into sterile injectable solutions or dispersions prior to use. Suitable aqueous or non-aqueous carriers, diluents, solvents, or loads include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.), carboxymethyl cellulose and suitable mixtures thereof, vegetable oils (e.g., olive oil), and injectable organic esters such as ethyl oleate. Suitable flowability can be maintained, for example, by using coating materials such as lecithin, for dispersants by maintaining the desired particle size, and by using surfactants. These compositions may also contain excipients such as preservatives, wetting agents, emulsifiers, and dispersants. Prevention of microbial action can be ensured by adding various antimicrobial and antifungal agents (e.g., parabens, chlorobutanol, phenol, sorbic acid, etc.). Isotonic agents, such as sugars, sodium chloride, etc., may also be required. Extended absorption of injectable drug formulations can be achieved by incorporating agents that delay absorption, such as aluminum monostearate and gelatin. Injectable "reservoirs" are also formed by creating microcapsule matrices of the drug within biodegradable polymers, such as polylactic-co-glycolic acid, poly(orthoester), and polyanhydride. The drug release rate can be controlled depending on the ratio of drug to polymer and the properties of the specific polymer used. Reservoirs of injectable formulations can also be prepared by encapsulating the drug in tissue-compatible liposomes or microemulsions. Injectable formulations can be sterilized, for example, by filtration with a bacteria-retaining filter, or by incorporating a sterilizing agent in the form of a sterile solid composition soluble or dispersible in sterile water or other sterile injectable media just before use. Suitable inert carriers may include sugars, such as lactose. It is desirable that at least 95% by weight of the active ingredient particles have an effective particle size of 0.01 to 10 μm.

[0173] As used herein, the term "substituted" is intended to include all permissible substituents of an organic compound. In a general sense, permissible substituents include acyclic and cyclic, branched or unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of an organic compound. Exemplary substituents include, for example, those described below. For a suitable organic compound, permissible substituents may be one or more and may be the same or different. For the purposes of this invention, a heteroatom, such as nitrogen, may have a hydrogen substituent and / or any permissible substituent of the organic compound that conforms to the heteroatom valence as described herein. This invention is not intended to be limited in any way by the permissible substituents of organic compounds. Moreover, the terms "substituted" or "replaced" include the implicit condition that such substitution conforms to the permissible valence of the substituted atom and the substituent, and that the substitution forms a stable compound, such as a compound that does not spontaneously transform through rearrangement, cyclization, elimination, etc.

[0174] Of course, one aspect of the invention relates to the use of the compounds of the invention in treating the indications, diseases, disorders and conditions discussed herein.

[0175] inflammation

[0176] Inflammation is associated with many diseases that are leading causes of morbidity and mortality in Western societies, including cardiovascular disease, acute kidney injury, and lung and heart failure. Despite the use of various medications, chronic and acute inflammation remains a major health burden. In recent years, it has become clear that oxidative stress plays a significant role in the pathophysiology of many inflammation-related diseases, such as cardiovascular disease and sepsis. Increased lipid peroxidation has been shown in hypertension, atherosclerosis, and sepsis using the clinically validated biomarker F2-isoprostaglandin. Lipid peroxidation via the isoprostaglandin pathway produces a series of highly reactive γ-ketoaldehydes, namely isolevuglandin (IsoLG), which react rapidly with primary amines and lead to cellular dysfunction. IsoLG covalently modifies and cross-links proteins by reacting with lysine residues, and this modification can directly inhibit enzyme function, induce inflammation, and cause cytotoxic effects. IsoLG is associated with pro-inflammatory dendritic cell and T cell activation in hypertension. Acute treatment of isolated mitochondria with IsoLG disrupted mitochondrial respiration and promoted the opening of the mitochondrial permeability transition pore (mPTP). However, the role of mitochondrial IsoLG in pathological conditions has not been investigated.

[0177] Sepsis leads to severe multi-organ failure, such as acute kidney injury, which is associated with increased oxidative stress and mitochondrial dysfunction. In experimental models of sepsis, levels of IsoLG adducts are elevated. Inflammation is associated with mitochondrial dysfunction; however, its pathophysiological role and molecular mechanisms remain unclear. Mitochondria are one of the major sources of free radicals that may generate IsoLG. Given the potentially highly damaging nature of IsoLG, the inventors demonstrate that inflammation-induced IsoLG plays a significant role in mitochondrial dysfunction.

[0178] Although oxidative stress is common in a variety of pathological conditions, there are currently no antioxidant treatments available, and common antioxidants such as ascorbic acid and vitamin E have proven ineffective in clinical studies. These drugs are unlikely to reach key sites of free radical production, such as mitochondria. Furthermore, antioxidants may interfere with redox signaling, increasing inflammation and tissue damage by producing cytokines and inhibiting Nrf2 signaling.

[0179] Oxidative stress in aging and inflammation leads to increased peroxidative damage to polyunsaturated fatty acids (PUFAs). The cause of isoprostaglandin-type lipid peroxidation is unclear. It has been proposed that the autoxidation of PUFAs can be caused by peroxidative hydroxyl radicals (HO2). • This process triggers the formation of hydroxyl peroxide radicals, which are protonated forms of superoxide radicals produced in mitochondria. Increased tissue hypoxia and acidification lead to increased HO2. • The inventors demonstrate that the accumulation of oxidatively damaged mitochondrial phospholipids and IsoLG adducts is related to the formation of mitochondrial HO2. • The result of oxidized PUFAs. HO2 from the peroxidation of isoprostaglandin lipids. • The hypothesis is consistent with the known fact that classical antioxidants are ineffective in preventing this type of oxidative stress and aging. Isoprostaglandin lipid peroxidation produces a racemic mixture of various forms of isoprostaglandins and isoleuglandin. Some isoprostaglandins can directly induce inflammatory responses, while reactive isoprostaglandins produce cytotoxic and immunogenic isoprostaglandin-lactam adducts.

[0180] IsoLG is a common downstream product of oxidative stress, and scavenging IsoLG with 2-hydroxybenzylamine (which is not an antioxidant) alleviated endothelial dysfunction, fibrosis, and hypertension. Treatments specifically targeting mitochondria represent a promising strategy for reducing target organ damage. The inventors have demonstrated that the IsoLG scavenger 2-hydroxybenzylamine, by targeting mitochondria through its conjugation with the lipophilic cationic triphenylphosphonium, reduces mitochondrial dysfunction and decreases sepsis-related mortality. This invention investigated (a) mitochondrial dysfunction in response to synthetic IsoLG and its adducts, (b) the development of a novel mitochondrial-targeting IsoLG scavenger, mito2HOBA, and (c) testing whether mito2HOBA prevented mitochondrial dysfunction and death in a lipopolysaccharide (LPS) sepsis model.

[0181] Materials and methods

[0182] reagents

[0183] LPS was obtained from Sigma (St. Louis, Missouri). 2-Hydroxybenzylamine (2HOBA) and its non-scavenger analog 4-hydroxybenzylamine (4HOBA) were prepared according to previous descriptions. 15-E2-IsoLG was synthesized using the method of Amarnath et al. and stored in DMSO stock solution at -80°C. All other reagents were obtained from Sigma (St. Louis, Missouri).

[0184] Animal experiments

[0185] All experimental procedures were approved by Vanderbilt and the Mercer Institutional Animal Care and Use Committees. The use of LPS is a recognized model for bacterial sepsis in rodents (1-3). The concentration of LPS that can be used to induce sepsis in mice depends on many factors (source of LPS, age / size, animal strain, required reaction time, target of interest, etc.) and may vary between different manufacturers. To test the protective properties of mito2HOBA, the inventors used LPS derived from *E. coli* O111:B4 (Sigma L8274). The LD50 of this batch of LPS tested in the preliminary study was [not specified] at 24 hours post-injection. 50 The concentration was 25 µg / g. The same batch of LPS was used throughout the study.

[0186] Forty 3-month-old C57BL / 6J mice were equally divided into four groups: sham-operated group (control), mice injected with LPS (LPS), mice supplemented with mito2HOBA (0.1 g / L) in their drinking water (mito2HOBA), and mice injected with LPS pretreated with mito2HOBA in their drinking water (LPS+mito2HOBA). Sepsis-induced mortality was used to evaluate the protective effect of mito2HOBA. Mortality was assessed several times daily for three consecutive days. In another experiment, mice were sacrificed 24 hours after LPS injection to analyze the activity of mitochondrial complex I and complex II.

[0187] Mitochondrial research

[0188] All methods for mitochondrial isolation, respiratory analysis, and respiratory chain enzymology have been described previously. As mentioned above, the activities of mitochondrial complexes I and II were assessed 24 hours after LPS injection. Mitochondria were isolated from the kidneys of 12–14 week old male C57BL6 / 6J mice. For respiratory studies, electrons entered either complex I (glutamate + malate as substrate) or complex II (succinate as substrate). Mitochondria in certain organs, such as the brain, oxidize up to 50% of pyruvate and glutamate to α-ketoglutarate via transamines, which are further converted to succinate. Since studies on renal mitochondria are far less common than those from other organs, the inventors used malonate (5 mM), a specific inhibitor of complex II, to assess alternative pathways of glutamate oxidation. Complex II-mediated respiration was defined as malonate-inhibited oxygen consumption, while complex I-specific respiration was defined as malonate-resistant oxygen consumption.

[0189] Respiratory rates were measured using a Fluorescence Lifetime Micro Oxygen Monitoring System (Instech Laboratories, Inc.). Oxygen consumption rate measurements were performed twice for each substrate, with each run including the addition of 0.24 mg / ml protein and ADP (125 µM) to stimulate state III and subsequent state IV respiration. OXPHOS-specific enzyme activity in submitochondrial particles (SMPs) was determined using a Varian Cary 300 Bio UV / vis spectrophotometer with a temperature-controlled cuvette holder. Briefly, SMPs were prepared by sonication of the isolated organelle. Complex I activity was monitored at 272 nm in triplicate samples using 15 μg mitochondrial protein and 40 µM NADH, expressed as a reduction to 10 µM decylubiquinone. Using this method, 90–100% of the total Complex I activity was sensitive to inhibition by rotenone. The activity of complex II was determined by monitoring the absorbance at 600 nm during the oxidation of 50 µM DCPIP, which acts as an artificial electron acceptor, with ubiquinone in the presence of 2 mM KCN, 2 μg / ml rotenone, and antimycin A.

[0190] Analysis of renal mitochondrial respiration mediated by complex I and complex II

[0191] To determine specific changes in mitochondrial respiration in a sepsis-associated LPS model and to test the potential protective effect of mito2HOBA, the inventors employed the Seahorse protocol to conduct mitochondrial studies in the presence of mitochondrial substrates glutamate + malate (GM) or succinate. To determine the specific role of complex I-mediated respiration, measurements were performed in the presence of the complex II inhibitor malonate (5 mM). Basal respiration in the presence of the substrate, coupled respiration with the addition of ADP (2 mM), proton leakage with the addition of oligomycin A (2.5 µM), uncoupled respiration with the addition of CCCP (1 µM), and non-mitochondrial respiration with a mixture of antimycin A and rotenone (1 µM) were measured. Mitochondrial studies were independently validated in two laboratories using an Oroboros O2k high-resolution respiration meter and a fluorescence lifetime micro-oxygen monitoring system (Instech Laboratories, Inc.). Kidney mitochondria were isolated from control sham-operated mice, LPS-injected mice (25 μg / g, 16 h post-injection), mice supplemented with mito2HOBA (0.1 g / L drinking water, 4 days), or mito2HOBA plus LPS (0.1 g / L mito2HOBA plus LPS injection for 3 days). One kidney was used for mitochondrial studies, and a second kidney was used for histopathological studies.

[0192] Kidney histological analysis

[0193] Mouse kidneys were harvested and immediately placed in 10% formalin. After fixation, the kidneys were rinsed with physiological saline, placed in 70% ethanol, and treated in the following order: 70% ethanol; 80% ethanol; 95% ethanol; 100% ethanol; 100% xylene. The kidneys were then embedded in POLY / Fin paraffin (ThermoFisher). They were cut into 5 μm sections using a Leitz 1512 microtome and mounted on glass slides. The sections were stained with hematoxylin and eosin and observed under an Olympus IX70 microscope. Images were taken using a Jenoptix Progress C12 digital camera. The histopathological scores of the kidney tissues were as follows: (0) no tubular damage; (1) tubular damage <10%; (2) tubular damage 10-25%; (3) tubular damage 25-50%; (4) tubular damage 50-75%; (5) tubular damage >75%.

[0194] Synthesis of mito2HOBA, an IsoLG scavenger targeting mitochondria (see...) Figure 7 )

[0195] Cesium carbonate (4.9 g, 15 mmol) was added to 2,4-dihydroxybenzaldehyde (4.2 g, 30 mmol) and 1,4-dibromobutane (6.6 g, 30 mmol) in acetonitrile (50 ml). The mixture was heated at 80 °C for 5 h under argon, cooled, and then added with stirring to pH 7 (30 ml) of 1 M phosphate buffer, ice, and KH₂PO₄ (2 g). The solid was removed by filtration, and the filtrate was extracted with ethyl acetate. Purification by column chromatography (silica, 9:1 hexane-ethyl acetate) yielded 4-(4-bromobutoxy)-2-hydroxybenzaldehyde (4.1 g, 50%). This was mixed with triphenylphosphine (4.2 g) in toluene (75 ml) and refluxed under argon for 15 h. The pink solid was purified by rapid chromatography (0-10% methanol in dichloromethane) to give 4-(4-formyl-3-hydroxyphenoxy)butyl)triphenylphosphonium bromide (4.8 g, 60%). The aldehyde was converted to an oxime by stirring in ethanol (40 ml) for 1 h in the presence of NH₂OH·HCl (0.63 g) and CH₃CO₂Na (0.74 g). The crude product (5.6 g) was dissolved in acetic acid (60 ml). Zinc powder (6 g) was added, and the suspension was heated in a water bath (60 °C) for 1 h. The mixture was cooled and filtered through diatomaceous earth. The filtrate was evaporated and co-evaporated with toluene (3 × 10⁻⁶ mL) and ethanol (15 mL). The residue was heated in hot 2-propanol (200 ml), filtered, and cooled to give pure mito₂HOBA; 3.0 g; MS m / z 456 (M + ).

[0196] statistics

[0197] Data were analyzed using Student-Neuman-Keuls post-hoc tests and analysis of variance (ANOVA). A p-value < 0.05 was considered significant.

[0198] result

[0199] Isolevuglandin impairs mitochondrial respiration.

[0200] Complex I is a key component of mitochondrial oxidative phosphorylation. Inactivation of Complex I can lead to reduced ATP production and tissue damage. Adding IsoLG to cells produces protein adducts and IsoLG-phosphatidylethanolamine adducts (IsoLG-PE), both of which can independently cause mitochondrial dysfunction. The inventors tested whether IsoLG or IsoLG-PE could cause mitochondrial dysfunction. Treatment of isolated mitochondria with 15-E2-IsoLG-PE (20 µM) for 5 minutes inhibited state 3 respiration by 41%, while a similar dose of 15-E2-IsoLG reduced state 3 respiration by 74% in the presence of the Complex I substrate glutamate + malate. Figure 1 A). These data support the potential role of IsoLG-PE and IsoLG-protein adducts in mitochondrial dysfunction. To further identify potential targets of IsoLG in mitochondria, the inventors investigated the effects of IsoLG and IsoLG-PE on respiration mediated by complex I and complex II. Acute administration of a low dose of 15-E2-IsoLG (0.5 µM) significantly attenuated respiration mediated by complex I, but respiration mediated by complex II was less affected. Figure 1 (B, C) Treatment of intact mitochondria with a low dose of 15-E2-IsoLG-PE reduced complex I respiration but did not affect complex II respiration. These data directly demonstrate the damaging effects of both IsoLG and IsoLG-PE on mitochondrial respiration.

[0201] IsoLG and IsoLG adducts inhibit the activity of complex I.

[0202] The inventors hypothesized that IsoLG could directly affect the activities of complex I and complex II. To verify this hypothesis, the inventors investigated the activities of complex I and complex II in mitochondrial lysates treated with IsoLG. They found that IsoLG caused a massive 74% inactivation of complex I, while the activity of complex II was only inhibited by 21%. Figure 2 A). These data suggest that complex I respiration is particularly sensitive to IsoLG.

[0203] As shown above, both IsoLG and IsoLG-PE attenuate the respiration mediated by complex I. Therefore, complex I may be directly affected by IsoLG or inhibited by low-molecular-weight IsoLG adducts. The inventors tested the inhibitory effect of complex I by comparing IsoLG adducts with IsoLG dosage. Supplementation with IsoLG-modified ethanolamine (IsoLG-ETN), IsoLG-modified L-lysine (IsoLG-Lys), or IsoLG-modified -PE (IsoLG-PE) inhibited complex I activity by more than 80%, similar to the effect of IsoLG dosage. Figure 2B). Interestingly, IsoLG-modified spermine (IsoLG-spermine) had no effect on complex I, suggesting that natural polyamines may protect complex I from IsoLG-mediated inhibition. These data indicate that complex I is directly inhibited by low-molecular-weight IsoLG adducts such as IsoLG-Lys and IsoLG-PE; therefore, these adducts may mediate I damage induced by direct IsoLG adduct. These data directly confirm that IsoLG-mediated complex I inhibition leads to mitochondrial dysfunction.

[0204] Mito2HOBA, an IsoLG scavenger targeting mitochondria

[0205] To test the hypothesis that specific clearance of IsoLG in mitochondria improves mitochondrial function, the inventors developed a mitochondrial-targeting IsoLG scavenger, mito2HOBA, by conjugating a lipophilic cationic triphenylphosphonium with 2-hydroxybenzylamine. Figure 3 The membrane potential of mitochondria in living cells is negative (-150 mV). Because this membrane potential is much higher than that of other organelles in the cell, lipophilic cations such as triphenylphosphonium selectively accumulate in mitochondria. Therefore, molecules conjugated with triphenylphosphonium target mitochondria. For example, mitoTEMPO is concentrated more than 500-fold in the mitochondrial matrix.

[0206] Mito2HOBA is a water-soluble compound that can be provided in culture media and in drinking water for animals. In animal studies, mito2HOBA was well tolerated at doses of 0.1–0.3 g / L. Mass spectrometry analysis of kidney and heart mitochondria isolated from mice given mito2HOBA (0.1 g / L) in drinking water for 5 days confirmed that mito2HOBA accumulated primarily at µM levels in the mitochondrial fraction (80%). Similarly, incubation of isolated mitochondria with mito2HOBA (0.1 µM) resulted in a strong accumulation of 400–600 times mito2HOBA in the mitochondrial particles. Figure 3 ).

[0207] To verify the IsoLG scavenging performance of mito2HOBA, the inventors investigated its reaction with the IsoLG analog 4-oxopentanal, as previously described. Mito2HOBA exhibits high reactivity with 4-oxopentanal, with a reaction rate constant approximately 50% of that of mito2HOBA itself. The slight decrease in reaction rate is likely due to the steric hindrance of the large triphenylphosphonium group. Under physiological conditions, the overall IsoLG scavenging rate depends on the rate constant and the local concentration of the scavenger. It is worth noting that 2HOBA analogs do not scavenge oxidizing agents, such as O2. • And peroxynitrite. The inventors' research indicates that supplementation with mito2HOBA at low submicromolar levels leads to low cytoplasmic levels but significant mitochondrial accumulation (…). Figure 3 As previously described by the inventors regarding mitoTEMPO targeting mitochondria, this results in low levels of mito2HOBA in the cytoplasm but high mitochondrial accumulation, leading to specific clearance of IsoLG from mitochondria. Figure 3 ).

[0208] Complex I and Complex II-mediated renal respiration in LPS and mito2HOBA-treated mice

[0209] The kidneys have a high energy requirement, and renal mitochondria may oxidize glutamate to α-ketoglutarate via transamination, which is further converted to succinate. The inventors analyzed mitochondrial respiration in the presence of glutamate + malate (GM) and succinate, and used the complex II inhibitor malonate to measure complex I-mediated specific respiration. Malonate inhibited 58% of glutamate-driven respiration (…). Figure 4 A), which supports the metabolic plasticity of renal mitochondria. Interestingly, LPS injection reduced GM-mediated respiration and succinate-mediated respiration, and significantly reduced complex I-specific oxygen consumption in the presence of GM+malonate. Figure 4 B). LPS significantly increased mitochondrial protein leakage from both substrates, indicating decoupling of mitochondrial respiration. In the presence of GM+ malonate, mito2HOBA alone slightly reduced succinate-driven respiration and improved complex I-specific oxygen consumption (B). Figure 4 C). Furthermore, in the presence of GM+ malonate, mito2HOBA supplementation significantly prevented LPS-induced damage to GM-mediated respiration and complex I-specific oxygen consumption, but did not affect succinate-mediated respiration or mitochondrial protein leakage. Figure 4 D).

[0210] mito2HOBA reduces mitochondrial dysfunction and decreases mortality in an LPS sepsis model.

[0211] To test the role of IsoLG in mitochondrial dysfunction, the inventors supplemented mice with a novel mitochondrial-targeting IsoLG scavenger, mito2HOBA (0.1 g / L). LPS treatment (25 µg / g body weight) resulted in severe mortality, but treatment with the mitochondrial-targeting IsoLG scavenger mito2HOBA increased animal survival by 3-fold at 96 hours post-injection. Figure 5 A). Further studies have shown that, compared to load-treated mice, the activity ratio of complex I / complex II was significantly reduced in mitochondria isolated from the kidneys of LPS-treated mice. Figure 5(B) Even after LPS treatment, mice supplemented with mito2HOBA maintained the complex I / II activity ratio completely. These data support the role of mitochondrial IsoLG in sepsis-related mitochondrial dysfunction and death.

[0212] mito2HOBA prevents LPS-induced kidney injury

[0213] Histological analysis of the kidneys was performed to provide direct evidence of mito2HOBA protection. Figure 6 The control group's kidneys showed normal cortex and medulla with no signs of damage. In contrast, the kidneys of mice treated with LPS showed significant cellular damage. Numerous areas of vacuolation and cellular degeneration were identified in the cortex and medulla of LPS-injected mice (arrows). In the medulla, many proximal tubules stained basophilic (triangles), suggesting alterations in intracellular metabolic processes. Similar to control mice, the kidneys of mice supplemented with mito2HOBA appeared normal. A few small areas of cellular degeneration were sparsely scattered in the medulla (not photographed). When mice were treated with both LPS and mito2HOBA, the cortex appeared normal, while significant cellular damage was observed in the renal medulla. Small areas of cellular degeneration and basophilic staining were visible in the medulla, but the extent and severity of the damage were smaller than in the kidneys of mice treated with LPS alone. Figure 5 As shown by C, mito2HOBA has a protective effect against LPS-induced cell damage.

[0214] discuss

[0215] The inventors have demonstrated that IsoLG or its stable adduct IsoLG-PE impairs mitochondrial respiration, particularly complex I-mediated respiration in the presence of malate and glutamate. Figure 1 Experiments in mitochondrial lysates showed that IsoLG and its adducts specifically inhibited complex I activity. Figure 2 Furthermore, the inventors have shown that the known IsoLG scavenger 2HOBA (targeting mitochondria) Figure 3 It has a significant protective effect against kidney injury and animal death in an LPS-induced sepsis model. Figure 5 , 6 ).

[0216] Impaired oxidative phosphorylation is a significant cause of organ damage in sepsis. Recent findings suggest that mitochondrial dysfunction in sepsis is associated with damage to complex I, and targeted protection of complex I has been proposed as a therapeutic approach for sepsis. Previous studies have shown that sepsis increases the production of IsoLG, and that IsoLG can induce mitochondrial permeability shifts. The inventors demonstrate that IsoLG may also mediate the mitochondrial dysfunction observed in sepsis. In fact, data indicate that complex I activity is particularly sensitive to IsoLG and IsoLG adducts, suggesting that sepsis-induced IsoLG may inhibit complex I activity, thereby promoting mitochondrial dysfunction. Therefore, this pathway may have significant implications for sepsis-induced multiple organ dysfunction syndrome.

[0217] It is well known that for NADH and NAD-dependent substrates, the respiratory rate is limited by the FAD-dependent NADH dehydrogenase of complex I. Therefore, all downstream sites in the respiratory chain remain oxidized, producing very little reactive oxygen species. Organs requiring rapid ATP production utilize the oxidation of succinate, produced via pyruvate or glutamate transamination, to accelerate mitochondrial respiration and ATP production. The inventors have previously shown that brain mitochondria utilize this pathway; now, for the first time, they demonstrate that kidney mitochondria also adapt to high energy demands by diverting mitochondrial metabolite flux to succinate to supply complex II-mediated respiration. Complex II is a much simpler protein and is more abundant than complex I. Interestingly, in the LPS model, it appears to be less sensitive to damaging effects of inflammation. Figure 4 , 5 Meanwhile, it is important to note that the higher respiration rate of complex II also has significant drawbacks, as it drives excessive production of mitochondrial reactive oxygen species (ROS) via reverse electron transport. Succinate-mediated oxidant production leads to brain and heart damage, and succinate-driven reverse electron transport is considered a novel therapeutic target. Data indicate that LPS induces a shift from complex I respiration to complex II respiration, but this maladaptation can promote kidney damage and inflammation, similar to previously reported succinate-driven cardiac damage. Specific substrate utilization analysis showed a significant reduction in complexes I, II, and IV, as well as fatty acid-mediated respiration, in renal mitochondria during LPS-induced sepsis. Data suggest that mito2HOBA protects mitochondria and reduces cellular damage; however, the specific target of mitochondrial IsoLG remains unclear. IsoLG can reduce the function of multiple mitochondrial complexes and decrease mitochondrial fatty acid metabolism. Further research is needed to elucidate the specific pathophysiological role of mitochondrial IsoLG. Sepsis causes kidney inflammation, renal tubular cell damage, apoptosis, and mitochondrial swelling; treatment with the mitochondrial-targeting lipid peroxidation inhibitor SS-31 can alleviate sepsis-induced organ dysfunction. These data support the protective effect of mito2HOBA against pathological changes in sepsis.

[0218] Recent findings suggest that mitochondrial dysfunction in sepsis is associated with damage to complex I, and the targeting of complex I in sepsis has been proposed. In this work, the inventors demonstrate the potential role of IsoLG and IsoLG adducts in inhibiting complex I, as well as the therapeutic effect of mito2HOBA on complex I function. The inventors show that targeting mitochondrial IsoLG improves mitochondrial respiration and rescues mitochondrial dysfunction in conditions associated with inflammatory damage.

[0219] To demonstrate the role of IsoLG in inflammation-induced mitochondrial dysfunction, the inventors developed mito2HOBA, an IsoLG scavenger targeting mitochondria. Driven by its lipophilic triphenylphosphonium moiety, mito2HOBA accumulates extensively in the mitochondria of multiple organs, such as the kidney, heart, and liver. Mice tolerated mito2HOBA well at 0.1 g / L in drinking water. mito2HOBA showed significant protective effects in a sepsis LPS model. In fact, mito2HOBA supplementation reduced immediate and long-term mortality in animals by 3-fold, and mito2HOBA completely maintained the complex I / II activity ratio in LPS-treated mice. Mito2HOBA treatment also eliminated damage to the renal cortical tubules and significantly reduced cellular degeneration and damage to the medullary tubules. The rapid effect of mito2HOBA may improve outcomes in clinical settings, as it can give healthcare professionals additional time to perform additional life-saving procedures for patients with sepsis. In summary, these data support the role of mitochondrial IsoLG in mitochondrial dysfunction and the therapeutic potential of the mitochondrial IsoLG scavenger mito2HOBA.

[0220] The inventors have also demonstrated that targeting mitochondrial IsoLG may be more effective than simply targeting mitochondrial ROS production. Kozlov and colleagues proposed that mitochondrial ROS accelerates inflammatory responses and promotes end-organ damage, thus targeting mitochondrial ROS could be an effective treatment for inflammation. Indeed, treatment of LPS-treated rats with the mitochondrial-targeting antioxidants mitoTEMPO and SkQ1 reduced the expression of inducible nitric oxide synthase and decreased markers of organ damage. However, these mitochondrial-targeting antioxidants also increased markers of organ damage at earlier time points, suggesting that antioxidants may interfere with cellular signaling required to activate protective responses. Furthermore, recent studies in cecal ligation and puncture sepsis models have also shown a lack of survival benefit from mitochondrial antioxidants. Whether these seemingly contradictory effects observed in previous studies can be attributed to specific sepsis models, animal species used, or the extent of sepsis damage remains unclear. NADPH oxidase is a major non-mitochondrial source of ROS. Gene excision of NADPH oxidase in P47phox-deficient mice exacerbates LPS-induced NF-κB activation, increases the expression of pro-inflammatory cytokines in the lungs, increases neutrophilic alveolitis, and maintains greater lung damage compared to wild-type mice. Notably, 2HOBA derivatives do not scavenge ROS such as O2. • Unlike many previously described antioxidants, mito2HOBA does not interfere with cellular redox signaling, nor does it contain peroxynitrites. These data suggest a potentially diverse role for ROS in sepsis, and the importance of targeting specific cellular and subcellular compartments such as mitochondria.

[0221] The inventors used the novel mitochondrial-targeting IsoLG scavenger mito2HOBA to test the potential role of IsoLG in sepsis-related mitochondrial dysfunction and death (see figure summary and figure). Figure 5 (Route). The inventors have also demonstrated that IsoLG can be produced through a variety of enzymatic and non-enzymatic pathways, and that clearing mitochondrial IsoLG can specifically alleviate inflammation-related mitochondrial dysfunction and cellular damage. Interestingly, the mitochondrial-targeting antioxidants MitoQ and MitoE reduced mitochondrial lipid peroxidation, decreased interleukin-6, improved mitochondrial function, and reduced organ dysfunction markers in an LPS-induced sepsis rat model, consistent with the pathophysiological effects of IsoLG produced via lipid peroxidation in the isoprostaglandin pathway. Therefore, this invention demonstrates the potential therapeutic benefits of specifically targeting mitochondrial isoLG.

[0222] hypertension

[0223] To test the role of mitochondrial isoLG in hypertension, the inventors used mass spectrometry and Western blot analysis to investigate the accumulation of mitochondrial isoLG-protein adducts in normotensive and hypertensive human individuals and angiotensin II-induced hypertension mouse models. The therapeutic potential of the novel mitochondrial-targeting isoLG scavenger mito2HOBA was tested. Results showed that significant accumulation of mitochondrial isoLG-protein adducts occurred in vascular and renal tissues in hypertension. Furthermore, treatment with mito2HOBA from small arteries of hypertensive patients increased SOD2 deacetylation and decreased mitochondrial superoxide in human aortic endothelial cells. In mice, mito2HOBA prevented the accumulation of mitochondrial isoLG-protein adducts, reduced SOD2 and CypD acetylation, protected mitochondrial respiration, maintained ATP production, blocked mitochondrial permeability pore opening, reduced vascular superoxide, protected endothelial nitric oxide, improved endothelium-dependent relaxation, and alleviated hypertension. These data suggest that mitochondrial isoLG promotes mitochondrial and endothelial dysfunction, and scavenging mitochondrial isoLG may have therapeutic potential in treating vascular dysfunction and hypertension.

[0224] Materials and methods

[0225] reagents

[0226] Dihydroethidium (DHE) and the MitoSOX superoxide probe were supplied by Invitrogen (Grand Isle, NY). Sirt3 (54905) antibody was purchased from Cell Signaling. Acetyl-K68-SOD2 (ab137037), complex I NDUFS175 kDa subunit (ab22094), CypD (ab110324), and GAPDH (ab8245) antibodies were purchased from Abcam. SOD2 (sc30080) antibody was purchased from Santa Cruz Biotechnology. Acetyllysine antibody (ab3839) was supplied by Millipore-Sigma. D11 is an isoLG-lysyl adduct-specific scFv antibody, previously characterized. All antibodies were used at 1000-fold dilution. As previously described, 2-hydroxybenzylamine (2HOBA), the mitochondrial-targeting isoLG scavenger mito2HOBA, and the isoLG-inactive analog 4-hydroxybenzylamine (4HOBA) were synthesized. All other reagents were purchased from Sigma (St. Louis, Missouri).

[0227] Animal experiments

[0228] As previously described, hypertension was induced with angiotensin II (0.7 mg / kg / min). To investigate the therapeutic potential of clearing mitochondrial isoLG, wild-type C57Bl / 6J male and female mice (Jackson Labs) were given saline or placed with angiotensin II micropumps and provided with purified water (carrier) or mito2HOBA (0.1 g / L) in drinking water. Blood pressure was monitored by telemetry and tail cannulation, as previously described. The procedure was approved by the Vanderbilt Institutional Animal Care and Use Committee (Protocol M1700207). Simple randomization was used to select animals for sham surgery, angiotensin II, or mito2HOBA groups to ensure equal opportunities for assignment to treatment groups.

[0229] Kidney mitochondrial separation

[0230] C57Bl / 6J mice were euthanized with carbon dioxide, and their kidneys were harvested. Adipose tissue was removed, and the kidneys were placed in ice-cold separation medium. In a cold environment, the kidneys were minced, washed with separation medium, and homogenized using a Polytron homogenizer with two 2-second pulses each. The homogenate was diluted 7-fold (w / v) and mitochondria were separated by differential centrifugation and purified using a Percoll gradient. The separation medium contained 75 mM mannitol, 175 mM sucrose, 20 mM MOPS (pH 7.2), and 1 mM EGTA. Mitochondrial protein concentrations were determined using the Bradford method.

[0231] Mitochondrial isoLG was determined by mass spectrometry.

[0232] The isoLG-lysine yl-lactam adduct in mitochondria isolated from the kidneys of sham-operated or angiotensin II-infused mice was determined by mass spectrometry. Mitochondrial proteins were completely enzymatically digested into individual amino acids. [Added] 13 The internal standard [C6] was used to separate and purify the isoLG-lysine adduct by solid-phase extraction and HPLC, followed by isotopic dilution and quantification by liquid chromatography-tandem mass spectrometry (LC / ESI / MS / MS) as previously described.

[0233] Cardiolipin oxidation assay

[0234] Cardiac phospholipid oxidation in human aortic endothelial cells was determined by liquid chromatography-mass spectrometry (LC / MS), as previously described. 44The extracted lipid fractions were separated online by UPLC using a Waters Acquity UPLC system (Waters Corp., Milford, MA). Mass spectrometry analysis was performed on a Thermo Quantum Ultra triple quadrupole mass spectrometer (Thermo Scientific Inc., San Jose, CA, USA).

[0235] Mitochondrial respiration assay

[0236] As previously described, renal mitochondrial respiration was measured using a fluorescence lifetime micro-oxygen monitoring system (Instech Laboratories, Inc.). The following respiration medium (mM) was used: 125 mM KCl, 10 mM MOPS (pH 7.2), 2 mM MgCl2, 2 mM KH2PO4, 10 mM NaCl, 1 mM EGTA, 0.7 mM CaCl2, 10 mM glutamate, and 2 mM malate. Subsequently, renal mitochondria (0.2 mg / ml), ADP (125 µM), and CCCP (0.2 µM) were added to the respiration chamber. Respiratory control rate was calculated as the ratio of 3-state to 4-state respiration, where 4-state respiration was the ratio after ADP phosphorylation.

[0237] ATP levels in kidney tissue

[0238] ATP concentration in kidney tissue was determined using a luminescent ATP assay kit (ABCAM; Cat#ab113849). The luminescent signal was read using a Biotek Synergy H1 microplate reader. The luminescent units (µmol / mg protein) were calculated based on the ATP calibration curve and the protein concentration determined by the Bradford method.

[0239] Estimation of calcium retention capacity

[0240] Calcium retention capacity (CRC) is the amount of calcium that can be loaded into mitochondria before the permeability transition pore opens. CRC is expressed as nanomoles of calcium per milligram of renal mitochondrial protein. 2+ We used the previously described pH method. This method is based on the use of H+ in the presence of 1 mM Pi. + / Ca 2+ The ratio remained relatively stable, and pH changes clearly reflected the added Ca. 2+The time of consumption. Mitochondrial CRC values ​​were estimated in a medium containing 210 mM sucrose, 20 mM KCl, 3 mM glycyl-glycine (pH 7.2), 1 mM KH₂PO₄, and 0.5 mg / m mitochondria in a final volume of 2.0 ml. The substrates were 10 mM glutamate and 2 mM malate. CaCl₂ titration was performed by adding 5 μL aliquots of 10 mM CaCl₂ to the mitochondria.

[0241] Cell culture

[0242] Human aortic endothelial cells (HAECs) were purchased from Lonza (Chicago, IL) and cultured in EGM-2 medium supplemented with 2% FBS but without antibiotics. The FBS concentration was reduced to 1% the day before the study.

[0243] Determination of superoxide by HPLC

[0244] In a tissue culture incubator at 37°C, DHE (50 µM) or mitochondrial-targeting mitoSOX (1 µM) in KHB buffer was loaded onto mouse aortic segments and incubated for 30 min. The aortic segments were then homogenized in methanol (300 μL) using a glass homogenizer. The tissue homogenate was passed through a 0.22 µM syringe filter, and the methanol filtrate was analyzed by HPLC according to a previously published protocol. The superoxide-specific product 2-hydroxyethidium was detected using a C-18 reversed-phase column (Nucleosil column 250–4.5 mm) and a mobile phase containing a gradient of 0.1% trifluoroacetic acid and acetonitrile (37%–47%) at a flow rate of 0.5 ml / min. 2-hydroxyethidium was quantified using a fluorescence detector as previously described, with an emission wavelength of 580 nm and an excitation wavelength of 480 nm.

[0245] Nitric oxide determination by electron spin resonance (ESR)

[0246] As previously described, aortic nitric oxide production was quantified by ESR and colloidal Fe(DETC)₂. All ESR samples were placed in quartz Dewar flasks filled with liquid nitrogen (Corning, New York, NY). ESR spectra were recorded using an EMX ESR spectrometer (Bruker Biospin Corp., Billerica, MA) and an ultra-high Q microwave cavity. The ESR settings were as follows: field scan, 160 Gauss; microwave frequency, 9.42 GHz; microwave power, 10 mW; modulation amplitude, 3 Gauss; scan time, 150 ms; time constant, 5.2 s; and receiver gain, 60 dB. n =4 scans).

[0247] Vasodilation study

[0248] Isometric tension studies were performed on 2 mm mouse aortic rings excluding perivascular fat, dissected from C57B / 6J mice. The studies were conducted using horizontal wire kinesiometers (DMT, Aarhus, Denmark, models 610M and 620M) containing a saline solution of 130 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO4, 1.2 mM KH2PO4, 15 mM NaHCO3, 5.5 mM glucose, and 1.6 mM CaCl2. Isometric tension of each vessel was recorded using LabChart Pro V7.3.7 (ADInstruments, Australia). The aortic rings were equilibrated over 2 hours by heating and stretching the vessels to achieve an optimal baseline tension of 36 mNewtons, followed by 3 cycles of 60 mM KCl saline to induce contraction. Endothelium-dependent and non-endothelium-dependent vasodilation were tested after pre-contraction with 1 µM phenylephrine. Once the blood vessels reach steady-state vasoconstriction, administer acetylcholine at gradually increasing concentrations and record the response to each concentration.

[0249] Human Research

[0250] Small arteries (100-200 µm in diameter) were obtained from human mediastinal fat, which was acquired during cardiac surgery from patients with essential hypertension (BP > 140 / 90 mmHg) and normotensive individuals who participated in randomized clinical trials of oxygen risk during cardiac surgery (ROCS), as described in previous Western blot analyses of SOD2 and SOD2 acetylation. Full informed consent was obtained for all tissue specimens.

[0251] statistics

[0252] Data are presented as mean ± SEM. To compare the response to angiotensin II infusion with and without mito2HOBA, a two-way ANOVA followed by a Bonferroni post-hoc test was performed. For comparisons between more than two groups, one-way ANOVA and a Bonferroni post-hoc test were used. For telemetry blood pressure measurements over time, repeated measures two-way ANOVA was performed using GraphPad Prism 7. A p-value <0.05 was considered significant.

[0253] result

[0254] Mitochondrial isoLG accumulation and SOD2 acetylation in arterioles from hypertensive patients

[0255] Mitochondria are a major source of superoxide radicals and are rich in polyunsaturated fatty acids. The peroxidation of arachidonic acid can produce highly reactive isoLG, which can rapidly form protein adducts with lysine residues. To detect mitochondrial isoLG accumulation, we performed Western blotting using antibody D11, which detects isoLG-adducted proteins in mitochondria isolated from human arterioles, regardless of amino acid sequence. We observed a 250% increase in mitochondrial isoLG-lysyl-lactam protein adducts in mitochondria isolated from hypertensive patients compared to normotensive individuals. Figure 8 A).

[0256] Hypertension is associated with the inactivation of the mitochondrial deacetylase Sirt3 and the high acetylation of mitochondrial superoxide dismutase (SOD2). To investigate the potential role of mitochondrial isoLG in Sirt3 inactivation, we developed a mitochondrial-targeted isoLG scavenger, mito2HOBA (…). Figure 8 B). Mito2HOBA selectively accumulates in mitochondria due to its lipophilic cationic triphenylphosphonium. We tested whether treatment of human arterioles in organelle cultures with low doses of mito2HOBA stimulated SOD2 deacetylation. Indeed, mito2HOBA supplementation (0.5 µM, 24 h, DMEM) significantly reduced SOD2 acetylation ( Figure 8 (C, D) Since SOD2 is deacetylated by Sirt3, these data suggest that mitochondrial isoLG inhibits Sirt3 function. Furthermore, since SOD2 acetylation inactivates SOD2 and contributes to mitochondrial oxidative stress, clearing mitochondrial isoLG may reduce mitochondrial oxidative stress.

[0257] Mito2HOBA, an IsoLG scavenger targeting mitochondria, reduces oxidative stress in endothelial cells.

[0258] The inventors previously demonstrated that angiotensin II (Ang II) and TNFα promote hypertension and reduce endothelial Sirt3 activity. We tested whether mito2HOBA reduces mitochondrial oxidative stress in human aortic endothelial cells (HAECs) stimulated with angiotensin II plus TNFα for 4 hours. Figure 9In fact, supplementing HAECs with mito2HOBA (50 nM) reduced the production of mitochondrial superoxide stimulated by TNFα and Ang II, as measured by the accumulation of the specific superoxide-MitoSOX product 2-OH-Mito-ethidium. Importantly, supplementing cells with the same concentration of the non-targeted isoLG scavenger 2HOBA (50 nM) did not affect mitochondrial superoxide levels. Treatment with the isoLG-inactive analog 4HOBA (which cannot scavenge isoLG due to hydroxyl site rearrangement) did not prevent mitochondrial oxidative stress in endothelial cells.

[0259] Cardiolipin selectively localizes to the matrix side of the mitochondrial inner membrane, and cardiolipin oxidation is a specific marker of mitochondrial oxidative stress. We examined whether mito2HOBA reduced cardiolipin oxidation in human aortic endothelial cells stimulated with angiotensin II plus TNFα. Indeed, low-dose mito2HOBA (50 nM) supplementation to HAECs inhibited cardiolipin oxidation, while the non-targeted isoLG scavenger 2HOBA was ineffective. These data support the role of mitochondrial isoLG in the formation of mitochondrial oxidative stress associated with SOD2 acetylation.

[0260] Effects of mito2HOBA on mitochondrial isoLG protein adduct accumulation and hypertension

[0261] To test the functional role of mitochondrial isoLG in hypertension, we used an Ang II hypertension model and employed a tail cannula ( Figure 10 A) and telemetry Figure 10 B) Monitoring blood pressure. Mito2HOBA alone did not affect blood pressure in control mice. Infusion of Ang II (0.7 mg / kg / day) into wild-type C57Bl / 6J mice increased systolic blood pressure to 162 mm Hg. Treatment of mice with drinking water containing mito2HOBA (0.1 g / L) significantly reduced Ang II-mediated hypertension to 140 mm Hg, as measured by tail cannulation and telemetry. It is important to note that supplementing mice with the same molar dose of the non-targeted analogue 2HOBA did not alleviate Ang II-mediated hypertension. Figure 10 A).

[0262] To provide definitive evidence of the clearance of mitochondrial isoLG, we determined the accumulation of isoLG-lysyl-lactam adducts by liquid chromatography-tandem mass spectrometry (LC / MS) after proteolytic digestion of proteins extracted from isolated mitochondria. Hypertension was associated with a 4-fold increase in mitochondrial isoLG-lysyl-lactam adducts, and mito2HOBA eliminated the formation of isoLG-lysyl-lactam adducts in renal mitochondria. Figure 10C, D).

[0263] Effects of mito2HOBA on mitochondrial deacetylation of CypD and SOD2 in angiotensin II-infused mice

[0264] In another experiment, the inventors found that Ang II-induced hypertension was associated with significant hyperacetylation (420%) of mitochondrial proteins in the aorta, and that this hyperacetylation was normalized by co-treatment of animals with mito2HOBA. Figure 11 (A, B). Since Sirt3 is the major (if not the only) deacetylase in mitochondria, this suggests that mitochondrial isoLG reduces Sirt3 activity. Sirt3 activates SOD2 through the deacetylation of specific lysine residues, and hypertension is associated with high SOD2 acetylation. The inventors tested whether scavenging mitochondrial isoLG reduced SOD2 acetylation. Indeed, SOD2 acetylation was increased by 260% in the aorta isolated from hypertensive mice, while mito2HOBA supplementation significantly reduced SOD2 acetylation (by 147% compared to control mice). Figure 11 C, D).

[0265] The inventors have previously reported that the loss of cyclin D (CypD), the regulatory subunit of the mitochondrial permeability transition pore (mPTP), improves vascular function and alleviates hypertension. Sirt3-mediated CypD deacetylation weakens mPTP opening. The inventors sought to determine whether Ang II-induced hypertension induces hyperacetylation of CypD and whether mito2HOBA attenuates CypD acetylation. Indeed, in the aorta isolated from hypertensive mice, CypD acetylation was increased by 356%, while mito2HOBA supplementation significantly reduced CypD acetylation (by 156% compared to the control group). Figure 11 E).

[0266] Hypertension is associated with the accumulation of isoLG-lysine lactam protein adducts in aortic mitochondria. Mito2HOBA inhibits the formation of mitochondrial isoLG adducts and reduces the level of isoLG-complex I NDUFS1 subunit adducts, accompanied by a decrease in mitochondrial acetylation. Figure 11 ).

[0267] Effects of mito2HOBA on aortic superoxide, endothelial nitric oxide, and endothelium-dependent relaxation

[0268] Mito2HOBA prevents hyperacetylation of SOD2, suggesting that mito2HOBA can reduce mitochondrial superoxide. In fact, hypertension following Ang II infusion was associated with a two-fold increase in aortic mitochondrial superoxide, an increase that was completely blocked by mito2HOBA supplementation. Figure 12 A). Hypertension is associated with increased vascular superoxide in mitochondria and cytoplasm, a phenomenon facilitated by the interaction between mitochondria and NADPH oxidase. We used the non-targeted cellular superoxide probe DHE to examine whether mito2HOBA reduced cytoplasmic superoxide levels in mice infused with Ang II. Hypertension infused with Ang II was associated with a 217% increase in aortic cell superoxide, an increase that was significantly reduced by mito2HOBA supplementation (152% compared to the sham-operated control). Figure 12 B).

[0269] In hypertension, increased vascular superoxide leads to endothelial dysfunction. It reduces endothelial nitric oxide levels, promotes vasoconstriction, and increases systemic vascular resistance. Therefore, decreased nitric oxide bioavailability is a hallmark of endothelial oxidative stress in hypertension. We tested whether treatment of mice with the mitochondrial-targeting isoLG scavenger mito2HOBA protected endothelial nitric oxide and improved endothelial-dependent vasodilation. Aortic nitric oxide production was quantified by electron spin resonance and the specific nitric oxide spin trap Fe(DETC)2. Figure 12 As shown, Ang II-induced hypertension was associated with a two-fold reduction in endothelial nitric oxide and impaired endothelial-dependent relaxation. Notably, mito2HOBA supplementation completely prevented the decrease in nitric oxide in mice receiving Ang II infusion and maintained endothelial-dependent relaxation. Figure 12 (C, D). These data demonstrate the previously unrecognized role of mitochondrial isoLG in endothelial dysfunction.

[0270] mito2HOBA affects mitochondrial respiration, renal ATP levels, and calcium levels. 2+ Impact of Reservation Capacity

[0271] Hypertension is associated with mitochondrial dysfunction characterized by impaired respiratory function and reduced ATP production, which can be mediated by mPTP opening and lead to end-organ damage in hypertension. In the current study, mito2HOBA reduced the Ca2+ of mPTP. 2+ Dependent regulation of acetylation of the subunit CypD. We tested whether mito2HOBA supplementation reduced mPTP opening (e.g., via mitochondrial Ca2+). 2+ (Measured by retention volume), it improved mitochondrial respiration and ATP production. In fact, Ang II-induced hypertension was associated with a 50% decrease in renal mitochondrial Ca2+ retention volume, and this was normalized in vitro by supplementation with the CypD inhibitor cyclosporine A. Treatment of mice infused with Ang II with mito2HOBA completely blocked Ca2+ retention. 2+ Decrease in retention capacity ( Figure 13A). Furthermore, mito2HOBA also maintains mitochondrial respiration supported by glutamate and malate as substrates (A). Figure 13 B). Ang II-mediated hypertension is also associated with a 50% decrease in renal ATP levels, and this is blocked by mito2HOBA ( Figure 13 C). These data suggest that mitochondrial isoLG in CypD-dependent mPTP opening in hypertension can inhibit mitochondrial respiration, reduce ATP levels, and promote end-organ damage in hypertension.

[0272] discuss

[0273] This embodiment demonstrates for the first time the accumulation of mitochondrial isoLG in the small arteries of patients with essential hypertension and mice with Ang II-mediated hypertension. Compared to normotensive individuals, mitochondrial isoLG was significantly increased in mitochondria isolated from the small arteries of hypertensive patients, and significantly increased in mitochondria isolated from the aorta and kidneys of mice after an Ang II-induced hypertension episode. The formation of the mitochondrial isoLG-lysyl-lactam protein adduct was confirmed by two independent methods: D11 antibody assay and mass spectrometry. These methods have been previously robustly validated and provide clear support for the accumulation of isoLG-protein adducts in mitochondria. Furthermore, the mitochondrial-targeting isoLG scavenger mito2HOBA prevented the accumulation of isoLG-protein adducts in mitochondria, and mito2HOBA increased SOD2 deacetylation in the small arteries of hypertensive patients, reduced mitochondrial superoxide in human aortic endothelial cells, inhibited vascular oxidative stress, improved endothelial function, and reduced Ang II-induced hypertension. Furthermore, supplementing Ang II-induced mice with mito2HOBA increased renal ATP levels, protected mitochondrial respiration, and reduced mPTP opening, supporting the role of mitochondrial isoLG accumulation in the formation of mitochondrial dysfunction in hypertension. Western blot studies revealed that hypertension is associated with decreased Sirt3 deacetylase activity and mitochondrial hyperacetylation, while mito2HOBA increased Sirt3-mediated deacetylation of mitochondrial proteins, particularly SOD2 and CypD. These findings support the role of mitochondrial isoLG in SOD2 inactivation and CypD-dependent mPTP opening (see [link to study]). Figure 14 ).

[0274] Hypertension is a multifactorial disease associated with mitochondrial oxidative stress; however, the precise targets of mitochondrial oxidative stress in hypertension remain unclear. We have previously shown that in animal models of hypertension, the production of mitochondrial superoxide is increased and the activity of mitochondrial SOD2 is decreased. 12An imbalance between increased mitochondrial superoxide and decreased SOD2 activity leads to mitochondrial oxidative stress. Mitochondria are a major source of superoxide radicals and are rich in unsaturated fatty acids, such as arachidonic acid. Radical oxidation of arachidonic acid produces highly reactive lipid dicarbonyl compounds, including isoLG. These rapidly adduct to protein lysine residues and can induce cellular dysfunction. Our data indicate a significant accumulation of isoLG-lysyl-lactam protein adducts in mitochondria isolated from hypertensive vascular and renal tissues. Supplementation with a low dose of the mitochondrial-targeting isoLG scavenger mito2HOBA (50 nM) prevented mitochondrial oxidative stress in human aortic endothelial cells, while the non-targeting analogue 2HOBA was ineffective. It is important to note that 2HOBA and mito2HOBA do not react with superoxide, pernitrite, or hydrogen peroxide, and therefore do not act directly through ROS scavenging. Conversely, the mito2HOBA-mediated reduction of mitochondrial, cellular, and aortic superoxide observed in Ang II-infused mice and HAECs is likely due to enhanced SOD2 scavenging of this free radical. This is a plausible explanation for our findings, as we observed a significant reduction in Ang II-induced hyperacetylation of SOD2 in animals treated with mito2HOBA, and SOD2 is the only mitochondrial superoxide dismutase.

[0275] Endothelial dysfunction is associated with increased vascular superoxide, leading to nitric oxide inactivation, reduced endothelial nitric oxide production, and impaired endothelium-dependent relaxation. mito2HOBA reduces vascular superoxide, protects endothelial nitric oxide, and improves endothelium-dependent relaxation. In endothelial cells, mito2HOBA inhibits superoxide production and reduces oxidative stress. These effects of mito2HOBA are associated with increased Sirt3-mediated deacetylation of SOD2 and CypD. Sirt3 damage leads to vascular inflammation, thickening, and endothelial dysfunction. Our new data support the important role of mitochondrial isoLG in Sirt3 inactivation, endothelial dysfunction, and vascular dysfunction.

[0276] Mitochondrial dysfunction leads to target organ damage in hypertension. Hypertension is a major cause of kidney disease, and it is associated with metabolic and mitochondrial dysfunction. In this study, we found that Ang II-induced hypertension was associated with a 4-fold increase in renal mitochondrial isoLG, increased mPTP opening, and impaired renal mitochondrial respiration. These events were associated with a 2-fold decrease in renal ATP levels. Notably, mito2HOBA supplementation prevented the accumulation of renal mitochondrial isoLG, reduced mPTP opening, maintained mitochondrial respiration, and protected renal ATP production. These data strongly support the role of mitochondrial isoLG in hypertensive kidney injury. These data are consistent with our previous findings, which showed that mito2HOBA supplementation in lipopolysaccharide-treated mice improved renal mitochondrial respiration and protected the renal cortex from cellular damage.

[0277] Therefore, the inventors discovered that Sirt3 inactivation serves as a novel convergence mechanism supporting the interaction of major cardiovascular risk factors. Sirt3 damage inhibits lipid metabolism and inactivates the key mitochondrial antioxidant superoxide dismutase 2 (SOD2) due to the high acetylation of specific lysine residues. Consequently, Sirt3 inactivation increases the levels of polyunsaturated fatty acids and superoxide in mitochondria, which react together to produce highly reactive isoLG. IsoLG covalently binds to lysine residues, generating cytotoxic and pro-inflammatory isoLG adducts. We found a 4-fold increase in mitochondrial isoLG in hypertension. Mitochondrial isoLG is a mechanistic link between mitochondrial oxidative stress and disease progression. Previous studies have confirmed adducts of isoLG with the F1Fo subunit of complex V, and we report adducts of isoLG with the NDUFS1 subunit of mitochondrial complex I. It is conceivable that mitochondrial isoLG can directly... 64 Indirect interactions lead to Sirt3 inactivation. Meanwhile, the causal relationship between mitochondrial isoLG and Sirt3 inactivation remains unclear. Clearly, isoLG exposure inhibits Sirt3; however, it is also possible that Sirt3 damage promotes mitochondrial isoLG formation. In fact, treatment with small arteries isolated from hypertensive patients rescued Sirt3 activity and increased Sirt3-mediated SOD2 deacetylation. We show that the feedforward cycle between Sirt3 inactivation and mitochondrial isoLG promotes vascular dysfunction, and that clearing mitochondrial isoLG breaks this cycle and improves vascular function (see [link to relevant documentation]). Figure 14 Therefore, isoLG appears to be both upstream and downstream of Sirt3 inactivation.

[0278] The pathophysiological role of isoLG in various conditions such as vascular inflammation, hypertension, and heart failure has been reported. Supplementation with the non-targeted isoLG scavenger 2HOBA reduces vascular inflammation, tissue fibrosis, aortic stiffness, myocardial hypertrophy, hypertension, and heart failure. In these conditions, stimulation of NADPH oxidase promotes the formation of cytoplasmic isoLG, which can be cleared by 2HOBA. Meanwhile, mitochondria are both a source and a potential target of isoLG; therefore, isoLG produced in the cytoplasm may also lead to mitochondrial dysfunction. In fact, our experiments show that 2HOBA partially attenuates the excessive production of mitochondrial superoxide in cultured human aortic endothelial cells. Figure 2 This indicates that both intramitochondrial isoLG and extramitochondrial isoLG promote mitochondrial oxidative stress.

[0279] Therefore, this invention demonstrates the role of mito2HOBA in cultured endothelial cells, in organelle cultures supplemented with human arterioles and whole animals. Further research is needed to determine the specific role of mitochondrial isoLG in endothelial cells, smooth muscle, and other cells. We demonstrate that the compounds of this invention effectively block mitochondrial isoLG, rescue Sirt3 deacetylase activity, restore mitochondrial metabolic and antioxidant functions, reduce vascular oxidative stress, and improve endothelial function. Therefore, mito2HOBA can improve the treatment of vascular dysfunction and hypertension.

[0280] Hypertension is highly prevalent with age, and 75% of adults develop hypertension by age 70 and older. Sirt3 function declines with age, and Sirt3 depletion accelerates vascular aging and induces age-dependent hypertension associated with mitochondrial oxidative stress. Sirt3 expression is associated with human lifespan, and Sirt3 overexpression can prevent vascular dysfunction and hypertension. Interestingly, it is hypothesized that Sirt3 damage and mitochondrial isoLG can promote age-dependent vascular changes and hypertension, and therefore, clearing mitochondrial isoLG could slow down and reverse these age-related changes. Indeed, our human tissue studies showed that mito2HOBA partially salvaged Sirt3 activity in patients with essential hypertension. Notably, while most oxidants have short lifespans (seconds), isoLG produces fairly stable adducts (several days lifespan), which can accumulate with age and thus contribute to the development of age-related conditions.

[0281] Besides hypertension, mitochondrial oxidative stress can contribute to many other conditions, including aging, atherosclerosis, diabetes, inflammation, and degenerative neurological disorders. The accumulation of mitochondrial isoLG can affect these conditions. It is conceivable that the use of mitochondrial-targeting isoLG scavengers such as mito2HOBA would be beneficial in these conditions. The ability to protect mitochondria at relatively low doses compared to non-targeting agents such as 2HOBA may also limit potential adverse effects.

[0282] All publications mentioned herein, particularly those referred to below, are incorporated herein by reference to disclose and describe methods and / or materials cited in those publications. The publications discussed herein are provided only for their disclosure prior to the filing date of this application. Nothing herein should be construed as an admission that the invention is not entitled to any prior invention prior to such publications. Furthermore, the publication dates provided herein may differ from actual publication dates and require independent verification.

[0283] References

[0284] [1] WG McMaster, A. Kirabo, MS Madhur, DG Harrison. Inflammation, immunity, and hypertensive end-organ damage. Circ Res 116:1022-1033; 2015.

[0285] [2] SS Davies, V. Amarnath, CJ Brame, O. Boutaud, LJ Roberts,2nd. Measurement of chronic oxidative and inflammatory stress by quantification of isoketal / levuglandin gamma-ketoaldehyde protein adducts using liquid chromatography tandem mass spectrometry. Nat Protoc 2:2079-2091;2007.

[0286] [3] Sullivan CB, Matafonova E, Roberts LJ, 2nd, Amarnath V, Davies SS. Isoketals form cytotoxic phosphatidylethanolamine adducts incells. J Lipid Res 51:999-1009; 2010.

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[0304] It is obvious that the invention described in this way can be varied in many ways. Such variations, which will be apparent to those skilled in the art, should be considered as offshoots of this disclosure.

[0305] Unless otherwise stated, all figures used in this specification to indicate the amount of components, the nature of reaction conditions, etc., should be understood to be modified by the term "approximately" in all cases. Therefore, unless otherwise indicated, the numerical parameters described in the specification and claims are approximate values ​​that may vary according to the desired performance sought according to the invention.

[0306] While the wide range of numerical values ​​and parameters described in this invention are approximate, the values ​​given in the experimental or example sections are reported as accurately as possible. However, any numerical value inherently contains a certain degree of error, which is necessarily caused by the standard deviation present in their respective experimental measurements.

Claims

1. Use of a compound or a pharmaceutical salt thereof in the preparation of a medicament for the treatment, prevention and improvement of sepsis in an individual, said compound being selected from: and , The compound contains a counterion selected from chloride, methanesulfonate, fluoride, nitrate, bromide, sulfate, citrate, benzoate, saccharin anion, and acetate.

2. The use according to claim 1, wherein the compound has the following formula: 。 3. The use according to claim 1, wherein the compound has the following formula: 。 4. The use according to claim 1, wherein the compound has the following formula: 。 5. The use according to any one of claims 1-3, wherein the counterion is selected from chloride ions, fluoride ions, and bromide ions.

6. The use according to claim 1 or 3, wherein the counterion is a chloride ion.

7. Use of the composition in the preparation of a medicament for treating, preventing and improving sepsis in an individual, wherein the composition comprises a compound or a pharmaceutical salt thereof as described in any one of claims 1-6, and a pharmaceutically acceptable carrier.

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