COMPOSITIONS COMPRISING STEROL DERIVATIVES FOR USE IN THE TREATMENT OF NEURONAL PATHOLOGY ASSOCIATED WITH HYPOXIA, HYPOGLYCEMIA AND / OR HYPERGLYCEMIA - Patent application

Abstract

A composition comprising a sterol derivative for use in treating neuronal pathologies associated with hypoxia, hypoglycemia and / or hyperglycemia affecting cells of the central nervous system.

Description

[Technical Field]

[0001] The present invention relates to the field of neuronal pathologies associated with glucose and / or oxygen deprivation. More specifically, the present invention relates to a composition comprising a compound derived from a sterol of formula (I) or a pharmaceutically acceptable salt of such a compound for use in the treatment and / or prevention of stroke, cranial trauma, brain lesions, diabetes-related hypoglycemia, cerebral ischemia caused by hyperglycemia, or respiratory disorders caused specifically by bacterial or viral infections. [Background technology]

[0002] Ischemia is a reduction in blood supply to an organ, and thus cerebral ischemia is a reduction in blood supply to at least part of the brain, resulting in a simultaneous interruption of the supply of oxygen, the supply of nutrients such as glucose, and the removal of toxic molecules resulting from anaerobic metabolism.

[0003] Cerebral ischemia is specifically involved in stroke, commonly referred to as "brain attack," and cranial trauma.

[0004] Stroke is the third leading cause of death after cardiovascular disease and cancer. However, stroke is the most common cause of serious physical disability and the second leading cause of dementia throughout the Western world. Disruption of cerebral blood perfusion causes stroke. Nearly 85% of strokes are ischemic in nature, i.e., caused by blockage of a blood vessel by a blood clot, reducing blood perfusion in the brain region. The clot may form locally within a small artery, originate from the heart, or arise from a lesion in the wall of one of the large carotid arteries. This blockage therefore prevents the supply of oxygen and metabolic substances such as glucose sufficient to meet the metabolic and energy needs of the central nervous system, causing cerebral ischemia. The consequences are even more serious because the brain cannot switch from aerobic to anaerobic metabolism to generate the energy it requires. Therefore, certain brain cells may die.

[0005] The outcome of a stroke depends on many factors: the rate at which blood supply is restored, the duration of the deprivation of oxygen and / or metabolic substances such as glucose, and / or the location of the stroke in the brain. Depending on the affected area, clinical symptoms can be more or less widespread paralysis, loss of speech or language, or coma with the risk of other debilitating sequelae.

[0006] Furthermore, respiratory failure, specifically due to bacterial or viral infection, can also cause cerebral ischemia. For example, ischemic stroke can be triggered when vascular inflammation (vasculitis) or infection (e.g., caused by herpes simplex virus) narrows the blood vessels supplying the brain. Furthermore, for example, Covid-19's adverse effects on the nervous system are increasingly being demonstrated by a growing number of factors. Indeed, various studies have demonstrated a correlation between Covid-19 infection and loss of taste and smell, confusion, headache, transient dizziness or vertigo attacks, encephalopathy, or other myelitis (Ling Mao et al., Neurological Manifestations of Hospitalized Patients with COVID-19 in Wuhan, China: a retrospective case series study and Neo Poyiadji et al., COVID-19-associated Acute Hemorrhagic Necrotizing Encephalopathy: CT and MRI,<doi:https: / / doi.org / 10.1101 / 2020.02.22.20026500> ). In fact, the virus appears to be able to enter the nervous system via the nasal cavity and olfactory bulb. The Covid-19 receptors found in large numbers in the lungs are also found on the blood-brain barrier and nerve endings. Once the virus enters the nervous system, it can destroy neurons responsible for proper respiratory function. Destruction of autonomic nervous system neurons in the medulla oblongata can cause and / or worsen respiratory distress in patients with Covid-19. Conversely, low blood oxygen levels resulting from acute respiratory symptoms in patients with Covid-19 may themselves be the cause of neurological problems. Because neurons are extremely sensitive to oxygen deprivation, prolonged and significant reductions in blood oxygen levels can damage neurons and ultimately lead to death.

[0007] Another cause of cerebral ischemia is cranial trauma, which can range from benign to severe, including all possible intermediate states. Its severity depends on the presence of intracerebral lesions or extracerebral hematomas, which are located between the skull and the brain and cause bleeding. Cranial trauma can be accompanied by contusion, neuronal lesions, edema, intracerebral hemorrhage, and / or ischemia.

[0008] Cerebral ischemia is a major threat affecting the functional and anatomical outcomes of traumatic brain injury. It is a diffuse or multicentric global ischemia affecting the cerebral cortex, similar to anoxia or cardiac arrest. Ischemia affects all of the gray matter, the part of the central nervous system with the greatest metabolic oxygen and glucose demands. Therefore, all brain functions are threatened. Cell loss can also be massive, manifested by visible brain atrophy.

[0009] Ischemia can take other, more localized forms. The microcirculation of tissues located around the center of a contusion or hemorrhage is threatened by vasoconstriction, tissue compression effects, capillary microthrombosis, or other disturbances in cellular metabolism. Energy production by cells is impaired because their oxidative metabolism is disrupted. Toxic products, such as free radicals, are released. In turn, even minor respiratory or circulatory events can cause cells to fall below the viability threshold.

[0010] The consequences of cranial trauma can be physical attacks such as paraplegia, hemiplegia, visual impairment, etc. They can also be neuropsychological attacks that affect memory, attention, and communication skills. Ultimately, they alter the behavior and personality of the injured individual and their daily life, as well as the behavior, personality, and daily life of those close to them.

[0011] Some cases of diabetes present with hypoglycemia combined with hypoxia, the consequences of which are detrimental to the affected patient.

[0012] Hypoglycemia is the most common clinical complication in the daily management of insulin-treated diabetic patients and continues to be a limiting factor in diabetic glycemic control. Severe hypoglycemia affects 40% of insulin-treated diabetic patients and can cause brain lesions, particularly in vulnerable neurons in the cortex and hippocampus. For example, learning and memory impairments are a direct result of this hippocampal neuronal lesion caused by severe hypoglycemia.

[0013] It is known that hyperglycemia is one of the symptoms indicating diabetes. In fact, diabetes systematically leads to hyperglycemia. In addition, hyperglycemia unrelated to diabetes also exists, and the causes are various, such as the ingestion of sugary foods or beverages or the side effects of medication. Hyperglycemia, whether diabetes-related or not, induces adverse effects at the nervous system level.

[0014] Furthermore, the combination of diabetes and hyperglycemia exacerbates neuronal damage following other forms of attack on the central nervous system, such as stroke. Empirical studies of the relationship between hyperglycemia, diabetes, and neuronal damage have been published, specifically, Yazi Li et al. (“Autophagy impairment mediated by S-nitrosation of ATG4B leads to neurotoxicity in response to hyperglycemia”, <doi:https: / / doi.org / 10.1080 / 15548627.2017.<1320467>), Ruchi Sharma et al. (“Hyperglycemia Induces Oxidative Stress and Impairs Axonal Transport Rates in Mice”, Published October 18, 2010), and Wenjuan Zhou et al. (“TIGAR Attenuates High Glucose-Induced Neuronal Apoptosis via an Autophagy Pathway”, <doi:https: / / doi.org / 10.3389 / fnmol.2019.00193>).

[0015] Currently, as technologies being developed to treat stroke, there are ultra-rapid interventions within 4 to 5 hours after stroke, and chemical removal of blood clots via recombinant tissue plasminogen activator rtPA, or mechanical removal by thrombectomy to enable reperfusion. However, these two intervention methods for dealing with stroke do not show maximum efficacy and are targeted at extremely limited patient inclusion criteria.

[0016] <​​​​The effects of Ganoderma total sterol (GS) and its main component (GS1) on cultured rat cortical neurons exposed to hypoxia / reoxygenation were also reported by Zhao HB et al. ("Ganoderma total sterol (GS) and GS1 protect rat cerebral cortical neurons from hypoxia / reoxygenation injury").<doi:https: / / doi.org / 10.1016 / j.lfs.2004.08.013> ) is known from

[0018] Additionally, strategies to combat hearing loss by targeting cholesterol homeostasis have been described by Brigitte Malgrange et al. ("Targeting Cholesterol Homeostasis to Fight Hearing Loss: A New Perspective")<doi:10.3389 / fnagi.2015.00003> ) Furthermore, it has been pointed out that the role of cholesterol and its metabolites is unclear.

[0019] The above three documents do not disclose or suggest a composition comprising at least one compound of formula (I) for use in treating a neuronal pathology in a subject, the neuronal pathology being related to hypoxia and / or hypoglycemia affecting cells of the central nervous system.

[0020] Therefore, there is a need to develop compounds that make it possible to treat patients suffering from oxygen and / or glucose deprivation, caused for example by cerebral ischemia, hypoglycemia or hypoxia, in order to promote their recovery. Summary of the Invention [Means for solving the problem]

[0021] One idea forming the basis of the present invention is to provide a prophylactic and / or therapeutic composition for use in the treatment of neurological disorders involving hypoxia, hypoglycemia and / or hyperglycemia, in particular those affecting neurons of the central nervous system.

[0022] To that end, the present invention provides compositions comprising at least one compound of formula (I): [ka] A compound of the formula: where: R1 = OH, F, OC n H 2n+1 , R-COO, R-OCOO, RHN-COO or OPO(OR)2, where R=H or C n H 2n+1 , where n≦16, R2=H or OH, R3 = -NR5R6, where R5 is H or —(CH2)3NH2; R6 is -(CH2)3NH(CH2)4NHR7, -(CH2)4NH(CH2)3NHR7, -(CH2)3NH(CH2)4NH(CH2)3NHR7, -(CH2)3NHR7, -(CH2)3NHR7, -(CH2)4NHR7, where R7 = H, or COCH3, -(CH2)2-imidazol-4-yl , to is taken from a group formed by R4 = H or OH at positions 20, 22, 24, 25, 26 or 27, aligned to create an asymmetric center of configuration R or S; Z1 and Z2 represent the number of double bonds (either 0 or 1) between carbon atoms C7 and C8, and between carbon atoms C22 and C23, respectively; T1T2 and T3 = independently of each other H or CH3; T4 = H, CH3, C2H5, positioned to achieve an asymmetric center of configuration R or S at position 24, and / or at least one pharmaceutically acceptable salt of at least one compound of formula (I), wherein the neuronal pathology is associated with hypoxia and / or hypoglycemia affecting cells of the central nervous system.

[0023] The compound of formula (I) defined by Z1 = Z2 = 0, R1 = R2 = OH, R4 = H, R5 = H, R6 = -(CH2)3-NCOOC(CH3)3-(CH2)4-NHCOOC(CH3)3, T1 = T2 = T3 = T4 = H is designated DX243BOC and is shown in Table 1.

[0024] The COOC(CH3)3 substituent is also known as the tert-butoxycarbonyl group or Boc functional group.

[0025] The compound of formula (I) belongs to the steroid group. Therefore, the numbering of the carbon atoms of the compound of formula (I) follows the nomenclature defined by IUPAC in the 1989 edition of Pure & Appl. Chem., Vol. 61, No. 10, pp. 1783-1822. The numbering of the carbon atoms of the compound belonging to the steroid group according to IUPAC is as follows: [ka]

[0026] The method for preparing the compound of formula (I) has already been described, and is specifically described in DE MEDINA et al., Synthesis of New Alkylaminooxysterols with Potent Cell Differentiating Activities: Identification of Leads for the Treatment of Cancer and Neurodegenerative Diseases, Journal of Medicinal Chemistry, 52(23), 2009, pp. 7765-7777.

[0027] Additionally, the composition may have one or more of the following properties, considered independently or in combination:

[0028] According to one embodiment, the compound of formula (I) has Z2=0, R1 =R 2=OH, R 4 =H、 Defined by R5=H and T1=T2=T3=T4=H.

[0029] According to one embodiment, the compound of formula (I) is defined by Z1=0 and R5=H.

[0030] According to one embodiment, the compound of formula (I) is defined by R6 = -(CH2)4NH(CH2)3NHR7, where R7 = COCH3.

[0031] According to one embodiment, the compound of formula (I) is defined by R6 = -(CH2)2-imidazol-4-yl.

[0032] According to one embodiment, the compound of formula (I) is defined by R6 = -(CH2)3NH(CH2)4NHR7, -(CH2)4NH(CH2)3NHR7, -(CH2)3NH(CH2)4NH(CH2)3NHR7, or -(CH2)4NHR7, and R7 = H.

[0033] According to one embodiment, the compound of formula (I) is defined by Z1=1 and R5=H.

[0034] According to one embodiment, the compound of formula (I) is R1=F,OC n H 2n+1 , R-COO, R-OCOO, RHN-COO or OPO(OR)2, where R=H or C n H 2n+1 , where n≦16, R2=OH, R5=H, R6=-(CH2)4NH(CH2)3NHR7, -(CH2)3NH(CH2)4NHR7, -(CH2)3NH(CH2)4NH(CH2)3NHR7, -(CH2)4NHR7, Z1=0, or Z1=1, Defined by Z2=0.

[0035] According to one embodiment, the compound of formula (I) is defined by R6 = -(CH2)3NH(CH2)4NHR7, -(CH2)4NH(CH2)3NHR7, or -(CH2)3NH(CH2)4NH(CH2)3NHR7, and R7 = H.

[0036] According to one embodiment, at least one compound of formula (I) has Z1=Z2=0, R 1 =R 2 =OH, R 4 =H、 The compound is defined by R5 = H, R6 = (CH2)3NH(CH2)4NH2, T1 = T2 = T3 = T4 = H. In this embodiment, the compound is called DX243. The results achieved with this compound are particularly advantageous. In fact, the therapeutic effect of this compound on the pathophysiological phenomena of hypoxia, hypoglycemia and / or hyperglycemia is observed at extremely low concentrations.

[0037] According to one embodiment, at least one compound of formula (I) is selected from the group consisting of Z1 = Z2 = 0, R1 =R 2=OH, R 4 =H、 The compound is defined by R5 = H, R6 = (CH2)4NH(CH2)3NH2, T1 = T2 = T3 = T4 = H. In this embodiment, the compound is called DX245. The results achieved with this compound are particularly advantageous. Indeed, with this compound, a greater therapeutic effect than that of DX243 is observed against the pathophysiological phenomena of hypoxia, hypoglycemia and / or hyperglycemia.

[0038] According to one embodiment, at least one compound of formula (I) has Z1=1, Z2=0, R1 =R 2=OH, R 4 =H、The compound is defined by R5 = H, R6 = (CH2)3NH(CH2)4NH2, T1 = T2 = T3 = T4 = H. In this embodiment, the compound is called DX242. The results achieved with this compound are particularly advantageous. Indeed, with this compound, a greater therapeutic effect than that of DX243 is observed against the pathophysiological phenomena of hypoxia, hypoglycemia and / or hyperglycemia.

[0039] According to one embodiment, at least one compound of formula (I) has Z1=1, Z2=0, R1 =R 2=OH, R 4 =H、 The compound is defined by R5 = H, R6 = (CH2)4NH(CH2)3NH2, T1 = T2 = T3 = T4 = H. In this embodiment, the compound is called DX244. The results achieved with this compound are particularly advantageous. Indeed, with this compound, a greater therapeutic effect than that of DX243 is observed against the pathophysiological phenomena of hypoxia, hypoglycemia and / or hyperglycemia.

[0040] According to certain embodiments, the neuronal pathology of the central nervous system is taken from the group consisting of brain trauma and stroke.

[0041] According to certain embodiments, the neuronal pathology of the central nervous system is a brain lesion resulting from ischemia.

[0042] According to certain embodiments, the neuronal pathology of the central nervous system is a brain lesion resulting from respiratory failure. The term "respiratory failure" is intended to mean obstructive and restrictive respiratory failure. Such respiratory failure may result from a bacterial or viral lung infection, for example, following coronavirus infection.

[0043] According to certain embodiments, the hypoglycemia is due to diabetes.

[0044] According to certain embodiments, the hyperglycemia results from diabetes.

[0045] According to certain embodiments, the present invention also provides a composition for use in treating a neuronal pathology in a subject, the neuronal pathology being associated with hypoxia, hypoglycemia and / or hyperglycemia affecting cells of the central nervous system, the composition being in the form of an aqueous solution, the composition comprising a compound of formula (I) in an amount of 1 pmol.L. -1 ~1 mmol.L -1 of, preferably 10 pmol.L -1 ~0.1 mmol.L -1 of 0.1 nmol.L, more preferably 0.1 nmol.L -1 ~1 μmol.L -1 For example, the concentration of the compound of formula (I) is 1 nmol.L -1 ~150nmol.L -1 is.

[0046] The present invention will be more clearly understood, and other objects, details, features and advantages of the present invention will become more apparent, through the following description of some particular embodiments thereof, given by way of non-limiting example only, with reference to the accompanying drawings, in which: [Brief explanation of the drawings]

[0047] [Figure 1] 1 outlines a protocol for examining the efficacy of compounds of formula (I), specifically DX243, on an in vitro model of ischemia. [Figure 2] 1 shows the results of immunocytochemical studies to assess neuronal survival in an in vitro model of ischemia / reperfusion in the presence of DX243. [Figure 3] 1 shows the results of an MTT neuron survival assessment study in an in vitro model of ischemia / reperfusion in the presence of DX243. [Figure 4] 1 shows the results of a test to assess neuronal survival by trypan blue in an in vitro model of ischemia / reperfusion in the presence of DX243. [Figure 5] 1 shows the results of a study to assess neuronal survival by trypan blue in an in vitro model of ischemia / reperfusion in the presence of compounds of formula (I) in Table 1 other than DX243. [Figure 6] 1 shows the results of an MTT neuronal survival assessment study in an in vitro model of ischemia / reperfusion in the presence of the compounds in Table 1. [Figure 7] In Table 1 the results of a second study to assess neuronal survival by trypan blue in an in vitro model of ischemia / reperfusion in the presence of compounds of formula (I) are shown. [Figure 8] 1 shows the results of an in vivo stroke study in mice to assess neuronal survival after stroke followed by reperfusion with DX243 treatment. [Figure 9] 1 is a series of graphs showing the results of an in vivo stroke study in mice to assess neuronal survival after stroke followed by reperfusion with DX243 treatment. DETAILED DESCRIPTION OF THE INVENTION

[0048] In Figures 2-4, 6, and 7, stars indicate the power of the results. One star indicates 95% certainty that the results are not random. The presence of two stars means that the results are 99% certain that the results are not random, and the presence of three stars means that the results are 99.9% certain that the results are not random.

[0049] Below are described several experimental protocols that demonstrate the protective effects of the compounds of formula (I) shown in Table 1, including compound DX243, against hypoxia and hypoglycemia.

[0050] The concentration or molarity of a compound is expressed in units of moles per liter, and its symbol is mol.L -1 Or M.

[0051] Example 1: Obtaining cortical neurons

[0052] Referring to Figure 1, the first step in the protocol for studying the neuroprotective effects of compounds of formula (I), specifically those listed in Table 1, consists in obtaining primary cultures of cortical neurons from cells harvested from wild-type mouse embryonic brains under appropriate culture conditions (Step 1). More specifically, the harvested cells are grown in Neurobasal™ medium (see ThermoFisher Scientific, 21103049) supplemented with L-glutamine and B27 supplement 50X (see ThermoFisher Scientific, 17504044) (Step 2). Next, neurons from the primary culture are isolated and purified. It should be noted that it is the culture conditions themselves that allow for obtaining purified cultures of neurons from dissociated embryonic cortices. Next, oxygen and glucose deprivation (OGD) is performed on these neurons to mimic as closely as possible what occurs in vivo during stroke, i.e., the reduction in oxygen and glucose supply due to reduced blood perfusion to the cells (Step 3). To perform this oxygen and glucose deprivation, which mimics ischemia, neurons are placed in an incubator for 4 hours in an atmosphere with an oxygen content of approximately 1% and in which the culture medium is replaced with glucose-free medium (step 3). Since the ischemia model is an "ischemia / reperfusion" model, the neurons are then placed in glucose-containing medium under normoxic conditions for 24 hours (reperfusion) following the ischemia step (step 4). Neuron group A is a group of neurons treated with a solution of the compound of formula (I) during OGD and reperfusion, i.e., a group of neurons to which the molecules in Table 1 are added to the glucose-free medium as soon as oxygen and glucose deprivation (step 3) begins. The concentration of the compound of formula (I), designated DX243, is 1 nmol.L in glucose-free medium. -1 ~1 μmol.L -1 The concentrations of the other molecules in Table 1 are 100 nmol / L in glucose-free medium. -1Neuron group B is a group of neurons that are treated with DX243 solution during reperfusion only (step 4) to mimic what occurs clinically, i.e., treatment that would occur only after a few hours with conventional treatment. DX243 solution is added to glucose-free medium as soon as reperfusion (step 4) begins. The concentration of DX243 is 1 nmol.L in glucose-free medium. -1 ~1 μmol.L -1 is.

[0053] The negative control consisted of neurons undergoing OGD (step 3) followed by reperfusion (step 4) in the absence of DX243. This neuronal group is referred to as "Ctrl" in Figures 2-4. The normal control consisted of neurons derived from primary cultures placed under normoxic conditions for 28 hours. This normal control is referred to as "normox" in Figures 2-4. The positive control consisted of neurons derived from primary cultures undergoing OGD for 4 hours (step 3) followed by reperfusion for 24 hours (step 4) in the presence of roscovitine, a cell cycle inhibitor that provides neuronal protection. This positive control is referred to as "ROSCO" in Figures 2-4.

[0054] At the end of reperfusion (step 4), 24 hours after OGD, neuronal survival is assessed by three different tests: immunocytochemistry, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), and trypan blue-based cell membrane integrity. The results are then compared with those of three control survival tests.

[0055] Example 2: Immunocytochemical study of neuronal survival with DX243

[0056] Figure 2 shows the results of the first neuronal survival test when using DX243. Graphs A and C show the results obtained for group A, with the y-axis representing the percentage of live cells relative to the normox group. Graphs B and D show the results obtained for group B, with the y-axis representing the percentage of cells undergoing apoptosis relative to the normox group. This first test is an immunocytochemical test that allows for the demonstration of live and dead cells.

[0057] The first series of images is taken by fluorescent chemical labeling. This labeling is performed using DAPI (4',6-diamidino-2-phenylindole), which can strongly bind to the adenine (A) and thymine (T) bases of DNA. This allows for the detection of live cells. The second series of images is taken by fluorescent labeling using the CC3 antibody. The CC3 antibody allows for the detection of activated caspase 3 and, therefore, apoptotic cells.

[0058] To visualize the proportion of healthy neurons in various culture conditions, the TUJ1 antibody is used. TUJ1 reacts with beta-tubulin III, a structural protein that constitutes tubulin and is unique to neurons. Beta-tubulin III is widely used as a marker to distinguish neurons from other cell types.

[0059] Based on these images, the percentage of viable cells (cell survival) was calculated by determining the ratio of viable cells to the total number of cells. The percentage of cells undergoing apoptosis was determined using the ratio of the number of cells detected by CC3 antibody to the total number of cells. The obtained values ​​were related to those obtained in the normox group.

[0060] It can be seen that OGD induces a decrease in the percentage of live cells in the Ctrl group (Figure 2, graphs A and B) and an increase in the percentage of cells undergoing apoptosis in the Ctrl group (Figure 2, graphs C and D). Meanwhile, treatment with DX243 both during OGD and reperfusion, and only during reperfusion, partially prevents neuronal death. Indeed, compared to the Ctrl group, an increase in the percentage of live cells and a decrease in the percentage of cells undergoing apoptosis is observed following treatment with DX243. 1 nmol.L -1 ~100nmol.L -1 The DX243 concentrations correspond to the ideal concentration range for maximizing neuroprotective effects.

[0061] Furthermore, 24 h treatment with DX243 after OGD appears to protect neurons even more efficiently.

[0062] Example 3: MTT Neuron Viability Assessment Test with DX243

[0063] To confirm the results obtained with DX243, we performed a neuronal metabolic activity-based assay to assess neuronal survival in an alternative way. This assay is based on the use of the tetrazolium salt MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). Tetrazolium is reduced to formazan, a purple precipitate, by mitochondrial succinate dehydrogenase in live, viable cells. The amount of precipitate formed is proportional to the amount of viable cells but also to the metabolic activity of each cell. Therefore, a simple spectrophotometric assay of the optical density at 550 nm allows the relative amount of viable and metabolically active cells to be determined. Given that the greater the number of viable cells, the greater the colorimetric intensity. Consequently, the colorimetric intensity was quantified and related to the intensity observed under normoxic conditions. The results obtained are reported in Figure 3, graph A for Group A and graph B for Group B.

[0064] In Figure 3, graphs A and B, it can be seen that OGD leads to a significant decrease in metabolically active cells in the Ctrl group. Meanwhile, treatment of neurons with DX243 partially prevents this decrease in survival after OGD. This effect is even more pronounced in the group of neurons that benefited from DX243 only during the 24-hour reperfusion period, as shown in Figure 3, group B. In these two graphs, the concentration of 10 nmol / L -1 ~100nmol.L -1 Treatments containing DX243 appear to be the most effective.

[0065] Example 4: Trypan Blue-Mediated Neuronal Survival Assessment Assay Using DX243

[0066] A third cell viability test using DX243 was performed, based on cell membrane integrity being disrupted in dead cells. This test uses trypan blue, which stains dead cells blue. Consequently, the percentage of viable cells was calculated by counting the percentage of blue and non-blue cells and correlating it with the percentage of viable cells observed in the normox group. The results are reported in Figure 4, graph A for group A and in Figure 4, graph B for group B.

[0067] Whether in Figure 4, graph A or Figure 4, graph B, it can be observed that OGD induces a significant decrease in the number of cells that retain membrane integrity. When neurons are treated with DX243, mainly at 1 nmol L -1 ~100nmol.L -1 At a concentration of 0.01, the percentage of viable cells is restored to a level close to that observed in the normox group.

[0068] Therefore, DX243 is 1 nmol.L -1 ~1 μmol.L -1 It is effective at a concentration of 10 nmol.L. -1 ~100nmol.L -1 In the case of , the results are more stable and the protective effect of DX243 is statistically greater, which can be explained by the vulnerability of primary cultures outside their native environment.

[0069] Example 5: Trypan Blue-Mediated Neuronal Survival Assessment Assay Using Molecules from Table I

[0070] A third cell viability test was performed for all molecules in Table I except for DX243 used in Group A. The results obtained, along with those obtained for DX243, are plotted in the graph in Figure 5. The symbol NT means untreated. The unhatched bars NT correspond to neurons derived from primary cultures placed under normoxic conditions for 28 hours. The hatched bars represent neurons derived from primary cultures undergoing OGD and reperfusion without (hatched bars designated NT) or in the presence of a molecule from Table 1. The percentage of viable cells was calculated by counting the percentage of blue and non-blue cells and relating them to the percentage of viable cells observed in the NT group under normoxic conditions, i.e., the NT group not subjected to OGD (not shown).

[0071] For all compounds tested, except for DX243BOC, the survival rates of the neurons were greater than those of the NT neurons (hatched bars NT) undergoing OGD and reperfusion, thus demonstrating neuroprotective effects. Specifically, it should be noted that DX245, DX244, and DX242 had higher survival rates than DX243.

[0072] As for the survival rate of the group treated with DX243BOC, it was lower than that of the group treated with DX243 and lower than that of the NT group subjected to OGD and reperfusion, thus demonstrating the importance of the R3 group in the activity of the compound of formula (I).

[0073] Example 6: MTT neuronal survival assay using molecules from Table 1

[0074] In relation to the graph in Figure 6, a similar test to that in Table Graph A in Figure 3 was performed: an MTT test to evaluate the neuroprotective effects of molecules treated with OGD for 4 hours followed by 24 hours of reperfusion. This test was also based on the protocol for obtaining cortical neurons described in Example 1 above. Unlike the example in Figure 3A, the roscovitine positive control was replaced with a dizocilpine (MK801) positive control. This positive control also consisted of neurons derived from primary cultures that underwent 4 hours of OGD in the presence of dizocilpine, followed by 24 hours of reperfusion. In Figures 6 and 7, the positive control is referred to as "MK801." The y-axis in Figures 6 and 7 represents the percentage of treated viable cells relative to viable cells under normoxic conditions; viable cells under normoxic conditions are not shown in the graphs because this is equivalent to 100%. MK801 is a noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist known to have neuroprotective effects. The test was performed on all compounds listed in Table 1. Figure 6 shows that significant neuroprotection is observed with DX243 and DX245 at 100 nM treatment. In this test, neuroprotective effects were observed with DX101, DX242, DX244, DX301, DX302, DX401, and DX249.

[0075] For all compounds tested in this example, the survival rate of the neuronal population was greater than that of the NT neuronal population, thus demonstrating a neuroprotective effect.

[0076] Example 7: Trypan Blue-Mediated Neuronal Survival Assessment Assay Using Molecules from Table 1

[0077] Further cell viability tests were performed on various compounds in Table 1, and Figure 7 shows the results. This test was similar to that performed in Figure 4. It is based on the disruption of cell membrane integrity in dead cells. This test uses trypan blue, which stains dead cells blue. This was performed in a similar manner to the test in Figure 4A, i.e., based on the protocol for obtaining cortical neurons described in Example 1 above. Unlike the example in Figure 4A, the roscovitine positive control was replaced with a dizocilpine (MK801) positive control. The purpose of this test was to evaluate the neuroprotective effects of the molecules after 4 hours of OGD followed by 24 hours of reperfusion, i.e., a total of 28 hours of experiment time. Tests were performed several times for all compounds listed in Table 1. In addition, DX243 and DX245 were tested at various doses, as shown in Figure 7.

[0078] At the end of this experiment, significant neuroprotection was observed with treatment with 100 nM DX245 and DX243 in the concentration range of 1-100 nM. Neuroprotective effects were also observed with 10 nM DX245, 1000 nM DX245, 1000 nM DX243, DX242, and DX244.

[0079] Thus, in a pharmaceutically acceptable aqueous solution containing a compound of formula (I): 1 pmol .L -1 ~1 mmol.L -1 , preferably 10 pmol .L -1 ~ 0.1 mmol .L -1 , more preferably 0,1 nmol.L -1 ~ 1 μmol .L -1 Concentrations of the compound of formula (I) are expected to be effective.

[0080] Example 8: In vivo neuroprotective effect study in a mouse model In vivo studies were performed to demonstrate the neuroprotective effects of the DX243 molecule in a male mouse model of stroke called middle cerebral artery occlusion (MCAO), shown in Figure 8, where the male mice weighed approximately 20.5 grams (g) to 22 g. This in vivo model, traditionally used to study the effects of molecules on stroke, involves placing a catheter in the anterior cingulate cortex (ACC) and inserting a filament into it at the level of the common carotid artery (CCA), passing the filament through the common carotid artery and the internal carotid artery (ICA) up to the middle cerebral artery (MCA), resulting in a significant reduction (>80%) in blood flow in the brain region irrigated by this artery (Estelle Rousselet et al., Modele murin d'MCAO intraluminale: Evaluation infarctus cerebral par coloration au violet de cresyl [Munine model of intraluminal MCAO: evaluation of cerebral infarction by cresyl violet staining], Journal of Visualized Experiments and Carl Zeiss). After 1 hour, the filament is removed to allow for reperfusion and a follow-up dose of DX243. DX243 is diluted in water to a concentration of 50 mg / kg. 24 hours after the filament is inserted into the middle cerebral artery, the results are observed, whether they are tissue-related (triphenyltetrazolium chloride (TTC) labels live tissue red) or behavioral (neuroscore). The neuroscore is an assessment of the neurological deficits of the mice, allowing for the evaluation of the success of MCAO. The scale used includes the following five points: 0: Normal state 1: slight fencing behavior with or without incoherent rotations when grasped by the tail. <50% rotation attempts towards the contralateral site. 2: Slight, constant rotation. >50% attempts to rotate toward the contralateral side. 3: Regular, strong, immediate circular motion. The mouse holds the circular position for more than 1-2 seconds, with the nose almost reaching the tail. 4: Rolling over from violent turning. Loss of walking or righting reflex. 5: Comatose or near death.

[0081] The in vivo results of the neuroprotective effect of DX243 are shown in Figures 8 and 9. Figure 8(A) shows two images of TTC staining, with areas of necrotic tissue shown in white, demonstrating the reduction in infarct size in mice treated with DX243. Graph B in Figure 9 shows the reduction in the percentage of infarct size between treated and untreated (Ctrl) mice, with the percentage of infarct size relative to the total brain size of the mouse plotted along the y-axis. Graph C shows behavioral improvement (neuroscore) in treated mice. Graph D shows the reduction in the percentage of dead mice among DX243-treated mice. Graphs E and F show the disappearance of the correlation between the reduction in blood flow and histological and behavioral outcomes after DX243 treatment. The reduction in blood flow was measured by laser Doppler flowmetry (LDF). Figure 9 demonstrates that DX243 treatment can improve stroke outcomes in mice at 24 hours. Indeed, graph D shows a reduction in the proportion of dead mice in the group of mice treated with DX243. Graph B similarly shows a reduction in the size of the lesion (infarct area), and graph C shows a reduction in behavioral deficits (neuroscore) in living mice. Furthermore, via laser Doppler, the results of which are shown in graphs E and F, it is observed that the greater the reduction in blood flow, the more severe the deficit. Meanwhile, in treated mice, there appears to be no correlation between the reduction in blood flow and the outcome of the stroke. These in vivo results demonstrate the neuroprotective effect of the molecules observed according to the present invention.

[0082] All these results indicate that compounds DX101, DX243, DX242, DX245, DX244, DX249, DX301, DX302 and DX401 in Table 1 have neuroprotective effects against cerebral ischemia and, by extension, against neuronal pathologies associated with hypoxia, hypoglycemia and / or hyperglycemia, such as brain trauma and stroke.

[0083] Although experiments were performed with 10 different compounds of formula (I), it is clear that similar results are expected for compounds of formula (I) other than those listed in Table 1.

[0084] Furthermore, in the context of hypoxia alone or hypoglycemia alone, Kato et al. ("Recurrent short-term hypoglycemia and hyperglycemia induce apoptosis and oxidative stress via the ER stress response in immortalized adult mouse Schwann (INMS32) cells," Neuroscience Research, 13 November 2018, retrieved from <https: / / www.sciencedirect.com / science / article / pii / S0168010218304371?via%3Dihub><doi:https: / / doi.org / 10.1016 / j.neures.2018.11.004> ("Apoptotic Cell Death Under Hypoxia", Physiology, 29, pp. 168-176, 2014), or Y. Xu et al. ("Protective effect of lithium chloride against hypoglycemia-induced apoptosis in neuronal PC12 cells", Neuroscience, 330, 25 August 2016, pp. 100-108). Therefore, the above-mentioned results observed in the ischemia / reperfusion model demonstrate the protection afforded by the compound of formula (I) despite this apoptotic component. Furthermore, hypoglycemia has the ability to induce oxidative stress in neuronal cells, which is present in conditions of oxygen and glucose deprivation and is a major component of neuronal death induction. Consequently, neuronal death induced by hypoglycemia and hypoxia, by hypoglycemia alone, and by hypoxia alone share a common mechanism of neuronal death induction, such as apoptosis. In conclusion, the neuroprotective effect of the compounds of formula (I) according to the invention extends to cases of hypoxia alone or hypoglycemia alone.

[0085] Use of the verbs "contain", "comprise" or "include" and their conjugations does not exclude the presence of elements or steps other than those stated in a claim.

[0086] In the claims, any reference signs placed between parentheses shall not be construed as limiting the claim.

[0087] [Table 1]

Claims

1. 1. A composition for use in treating a neuronal pathology in a subject, comprising: The composition comprises at least one compound of formula (I): 【Chemistry 1】 and where: R １ =OH、 R ２ =OH、 R ３ =-NR ５ R ６ ,however, R ５ is H, R ６ is -(CH ２ ) ３ NH (CH ２ ) ４ NHR ７ , -(CH ２ ) ４ NH (CH ２ ) ３ NHR ７ , -(CH ２ ) ３ NH (CH ２ ) ４ NH (CH ２ ) ３ NHR ７ , -(CH ２ ) ３ NHR ７ , -(CH ２ ) ４ NHR ７ , but R ７ =H, or COCH ３ , and -(CH ２ ) ２ -imidazol-4-yl, R ４ = H in position 20, 22, 24, 25, 26 or 27, which is aligned to create an asymmetric center of configuration R or S, Z １ and Z ２ represent the number of individual double bonds between carbon atoms C7 and C8 and between C22 and C23, respectively; Z １ is either 0 or 1, Z ２ =0、 T １ =T ２ =T ３ =T ４ =H、 T ４ is aligned to achieve an asymmetric center of configuration R or S at position 24, and / or at least one pharmaceutically acceptable salt of at least one compound of formula (I), wherein said neuronal pathology is caused by cerebral ischemia associated with hypoxia and / or hypoglycemia affecting cells of the central nervous system.

2. The compound of formula (I) is Z １ 2. The composition for use according to claim 1, wherein R is defined by:

3. The compound of formula (I) is R ６ =-(CH ２ ) ４ NH (CH ２ ) ３ NHR ７ , but R ７ =COCH ３ 3. The composition for use according to claim 2, wherein said composition is defined by:

4. The compound of formula (I) is R ６ =-(CH ２ ) ２ 3. The composition for use according to claim 2, wherein the compound is defined by -imidazol-4-yl.

5. The compound of formula (I) is R ６ =-(CH ２ ) ３ NH (CH ２ ) ４ NHR ７ , -(CH ２ ) ４ NH (CH ２ ) ３ NHR ７ , -(CH ２ ) ３ NH (CH ２ ) ４ NH (CH ２ ) ３ NHR ７ , or -(CH ２ ) ４ NHR ７ , and R ７ 3. The composition for use according to claim 2, wherein said compound is defined by: =H.

6. The compound of formula (I) is Z １ 2. The composition for use according to claim 1, wherein R is defined by:

7. The compound of formula (I) is R ６ =-(CH ２ ) ３ NH (CH ２ ) ４ NHR ７ , -(CH ２ ) ４ NH (CH ２ ) ３ NHR ７ , or -(CH ２ ) ３ NH (CH ２ ) ４ NH (CH ２ ) ３ NHR ７ , and R ７ 7. The composition for use according to claim 6, wherein said compound is defined by =H.

8. 1. A composition for use in treating a neuronal pathology in a subject, comprising: The composition comprises at least one compound of formula (I): 【Chemistry 2】 and where: R １ = F, OC ｎ H ２ｎ＋１ , R-COO, R-OCOO, RHN-COO or OPO(OR) ２ , where R=H or C ｎ H ２ｎ＋１ , where n≦16, R ２ =OH、 R ３ =-NR ５ R ６ 、 R ４ = H in position 20, 22, 24, 25, 26 or 27, which is aligned to create an asymmetric center of configuration R or S, R ５ =H、 R ６ is -(CH ２ ) ３ NH (CH ２ ) ４ NHR ７ , -(CH ２ ) ４ NH (CH ２ ) ３ NHR ７ , -(CH ２ ) ３ NH (CH ２ ) ４ NH (CH ２ ) ３ NHR ７ , -(CH ２ ) ４ NHR ７ , but R ７ =H, or COCH ３ , and -(CH ２ ) ２ -imidazol-4-yl, R ４ = H in position 20, 22, 24, 25, 26 or 27, which is aligned to create an asymmetric center of configuration R or S, Z １ and Z ２ represent the number of individual double bonds between carbon atoms C7 and C8 and between C22 and C23, respectively; Z １ is either 0 or 1, Z ２ =0、 T １ =T ２ =T ３ =T ４ = H, T ４ is aligned to achieve an asymmetric center of configuration R or S at position 24, and / or at least one pharmaceutically acceptable salt of at least one compound of formula (I), wherein said neuronal pathology is caused by cerebral ischemia associated with hypoxia and / or hypoglycemia affecting cells of the central nervous system.

9. The composition for use according to one of claims 1 to 8, wherein said neuronal pathology of the central nervous system is selected from the group consisting of brain trauma and stroke.

10. The composition for use according to one of claims 1 to 8, wherein said neuronal pathology of the central nervous system is a brain lesion resulting from ischemia.

11. The composition for use according to one of claims 1 to 8, wherein said neuronal pathology of the central nervous system is a brain lesion resulting from respiratory failure.

12. The composition for use according to one of claims 1 to 8, wherein the hypoglycemia is due to diabetes.

13. in the form of an aqueous solution and containing the compound of formula (I) at 1 pmol. L －１ ~1 mmol.L －１ of 10 pmol. L, preferably 10 pmol. L －１ ~0.1 mmol. L －１ , more preferably 0.1 nmol. L －１ ~1 μmol.L －１ 9. The composition for use according to claim 1, having a concentration of:

14. 1. Use of a composition comprising at least one compound of formula (I) for the manufacture of a medicament for the treatment of a neuronal pathology in a subject, comprising: 【Transformation 3】 where: R １ =OH、 R ２ =OH、 R ３ =-NR ５ R ６ ,however, R ５ is H, R ６ is -(CH ２ ) ３ NH (CH ２ ) ４ NHR ７ , -(CH ２ ) ４ NH (CH ２ ) ３ NHR ７ , -(CH ２ ) ３ NH (CH ２ ) ４ NH (CH ２ ) ３ NHR ７ , -(CH ２ ) ３ NHR ７ , -(CH ２ ) ４ NHR ７ , but R ７ =H, or COCH ３ , and -(CH ２ ) ２ -imidazol-4-yl, R ４ = H in position 20, 22, 24, 25, 26 or 27, which is aligned to create an asymmetric center of configuration R or S, Z １ and Z ２ represent the number of individual double bonds between carbon atoms C7 and C8 and between C22 and C23, respectively; Z １ is either 0 or 1, Z ２ =0、 T １ =T ２ =T ３ =T ４ =H、 T ４ is aligned to achieve an asymmetric center of configuration R or S at position 24, and / or at least one pharmaceutically acceptable salt of at least one compound of formula (I), wherein said neuronal pathology is caused by cerebral ischemia associated with hypoxia and / or hypoglycemia affecting cells of the central nervous system.

15. 1. Use of a composition comprising at least one compound of formula (I) for the manufacture of a medicament for the treatment of a neuronal pathology in a subject, comprising: 【Chemistry 4】 where: R １ = F, OC ｎ H ２ｎ＋１ , R-COO, R-OCOO, RHN-COO or OPO(OR) ２ , where R=H or C ｎ H ２ｎ＋１ , where n≦16, R ２ =OH、 R ３ =-NR ５ R ６ 、 R ４ = H in position 20, 22, 24, 25, 26 or 27, which is aligned to create an asymmetric center of configuration R or S, R ５ =H、 R ６ is -(CH ２ ) ３ NH (CH ２ ) ４ NHR ７ , -(CH ２ ) ４ NH (CH ２ ) ３ NHR ７ , -(CH ２ ) ３ NH (CH ２ ) ４ NH (CH ２ ) ３ NHR ７ , -(CH ２ ) ４ NHR ７ , but R ７ =H, or COCH ３ , and -(CH ２ ) ２ -imidazol-4-yl, R ４ = H in position 20, 22, 24, 25, 26 or 27, which is aligned to create an asymmetric center of configuration R or S, Z １ and Z ２ represent the number of individual double bonds between carbon atoms C7 and C8 and between C22 and C23, respectively; Z １ is either 0 or 1, Z ２ =0、 T １ =T ２ =T ３ =T ４ = H, T ４ is aligned to achieve an asymmetric center of configuration R or S at position 24, and / or at least one pharmaceutically acceptable salt of at least one compound of formula (I), wherein said neuronal pathology is caused by cerebral ischemia associated with hypoxia and / or hypoglycemia affecting cells of the central nervous system.

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