RU2748437C2 - Hydrocortisone derivative for alzheimer's disease treatment - Google Patents

Abstract

FIELD: medicine.SUBSTANCE: invention relates to the use of 21-hemisuccinate hydrocortisone (RX624) as Alzheimer's disease treatment, as well as to the use of 21-hemisuccinate hydrocortisone in the production of drugs for the treatment of Alzheimer's disease.EFFECT: use of RX624 in transgenic mice with genetically determined Alzheimer's disease leads to selective inhibition of the formation of the exogenous GAFD-A complex and stops memory impairment in animals according to the results in the Morris water test.2 cl, 3 ex, 4 dwg

Description

The invention relates to medicine, in particular to agents for the treatment of neurodegenerative diseases, namely to an agent for the treatment of Alzheimer's disease.

Alzheimer's disease (AD) is a common neurodegenerative disease associated with the accumulation of neurotoxic peptides beta-amyloid (Aβ) located inside the membranes of neurons or extracellularly, in a soluble form or in plaques, usually serving as a histological marker of AD [Benilova I, Karran E, De Strooper B. The toxic Aβ oligomer and Alzheimer's disease: an emperor in need of clothes // Nat Neurosci., 2012, 29; 15 (3): 349-57]. One of the important properties of Aβ peptides is their ability to form fibrillar aggregates and their neurotoxicity, which is associated with synapse damage, neuronal dysfunction and, ultimately, their death [Mucke L, Masliah E, Yu GQ, Mallory M, et al. High-level neuronal expression of abeta 1-42 in wild-type human amyloid protein precursor transgenic mice: synaptotoxicity without plaque formation // J Neurosci., 2000, 20 (11): 4050-4058; Brouillette J, Caillierez R, Zommer N, et al. Neurotoxicity and memory deficits induced by soluble low-molecular-weight amyloid-pi-42 oligomers are revealed in vivo by using a novel animal model // J Neurosci., 2012; 32 (23): 7852-7861].

Neuronal death triggered by Aβ causes the release of cytosolic proteins, some of which can bind Aβ and affect their function. One of these proteins is the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which, in addition to its role in energy metabolism of cells, performs other functions, including participation in the last stages of apoptosis [Sirover MA, Pleiotropic effects of moonlighting glyceraldehyde-3-phosphate dehydrogenase ( GAPDH) in cancer progression, invasiveness, and metastases // Cancer Metastasis Rev., 2018; 37 (4): 665-676]. Oxidized GAPDH molecules, binding through Cys-148 residues, form aggregates in cells subjected to oxidative stress, leading to their death [Nakajima T, Kinoshita T, Nashimoto A, et al. Randomized controlled trial of adjuvant uracil-tegafur versus surgery alone for serosa-negative, locally advanced gastric cancer // Br J Surg., 2007; 94 (12): 1468-1476; Lazarev VF, Nikotina AD, Semenyuk PI, et al., Small molecules preventing GAPDH aggregation are therapeutically applicable in cell and rat models of oxidative stress // Free Radic Biol Med., 2016; 92: 29-38]. Found that GAPDH binds mutant polypeptides, for example, mutant huntingtin, and forms aggregates with them, insoluble in sodium dodecyl sulfate. Moreover, the formation of such aggregates is catalyzed by tissue transglutaminase (tTG) [Guzhova IV, Lazarev VF, Kaznacheeva AV, at al., Novel mechanism of Hsp70 chaperone-mediated prevention of polyglutamine aggregates in a cellular model of huntington disease // Hum Mol Genet. 2011; 20 (20): 3953-3963].

It has been shown that these aggregates accumulate extracellularly and are more toxic than the components of which they are composed - polyglutamine and GAPDH [Mikhaylova ER, Lazarev VF, Nikotina AD, et al., Glyceraldehyde 3-phosphate dehydrogenase augments the intercellular transmission and toxicity of polyglutamine aggregates in a cell model of Huntington disease // J Neurochem., 2016; 136 (5): 1052-1063]. It was also reported that the formation of aggregates consisting of GAPDH and Aβ causes the death of nerve cells in vitro and in the hippocampus of transgenic mice. It has been shown that aggregates are formed inside nerve cells during the random formation of complexes from a disordered mass of GAPDH with Aβ 1-40 [Itakura M, Nakajima H, Kubo T, et al. Glyceraldehyde-3-phosphate Dehydrogenase Aggregates Accelerate Amyloid-β Amyloidogenesis in Alzheimer Disease // J Biol Chem. 2015; 290 (43): 26072-26087].

According to our unpublished data, the amount of Aβ-GAPDH aggregates insoluble in sodium dodecyl sulfate in the extracellular matrix and in cerebrospinal fluid samples taken from AD patients is proportional to the severity of the pathology stage.

Thus, GAPDH is an attractive target for the development of a drug against AD. GAPDH-binding substances are known that have potential for treating AD, these are such small molecules as NMC, NAC, GSH, deprenyl, hydroxynonenal, GAI peptide (GAPDH aggregation inhibitor peptide) [Chernorizov KA, Elkina JL, Semenyuk PI, et al., Novel inhibitors of glyceraldehyde-3-phosphate dehydrogenase: covalent modification of NAD-binding site by aromatic thiols // Biochemistry (Mosc)., 2010.75 (12): 1444-9; Guzhova IV, Lazarev VF, Kaznacheeva AV, at al., Novel mechanism of Hsp70 chaperone-mediated prevention of polyglutamine aggregates in a cellular model of huntington disease // Hum Mol Genet. 2011; 20 (20): 3953-3963; Itakura M, Nakajima H, Semi Y, et al., Glyceraldehyde-3-phosphate dehydrogenase aggregation inhibitor peptide: A potential therapeutic strategy against oxidative stress-induced cell death // Biochem Biophys Res Commun., 2015; 467 (2): 373-376] However, the effect of GAPDH-binding substances on the development of AD has not yet been shown.

Currently, two classes of pharmaceuticals are used to treat AD: cholinesterase inhibitors (donepezil, (arisept), rivastigmine, and galantamine) and the N-methyl-D-aspartate receptor antagonist (NMDA) memantine (namenda). But all registered drugs give only a limited in time and symptomatic effect and cannot stop or significantly slow down the destructive course of the disease.

The technical problem to be solved by the present invention was the creation of a medicament for the treatment of Alzheimer's disease.

The technical result was obtained as a result of the discovery of the fact that the known substance that binds GAPDH, 21-hemisuccinate hydrocortisone (RX624), is able to selectively inhibit the formation of the GAPDH-Aβ complex in the intercellular space and doubles the efficiency in the analysis of the Morris water maze in transgenic mice with simulated Alzheimer's disease.

The invention is illustrated by the following graphic materials:

FIG. 1 shows the structural formula of a hydrocortisone derivative - hydrocortisone 21-hemisuccinate (RX624).

FIG. 2 shows an analysis of the memory function of mice using the Morris water maze. The abscissa shows groups of mice (naive animals, animals that did not receive therapy; DMSO, animals that received DMSO injections; RX624, animals that received RX624 injections), and the ordinate shows the platform search time in seconds.

FIG. 3 shows the results of the analysis of the number of aggregates in the hippocampus of mice. Aggregates were identified using antibodies against GAPDH or against Aβ.

FIG. 4 shows the results of a quantitative analysis of the color intensity of the dot blots shown in FIG. 3.

The essence and industrial applicability of the present invention are illustrated by the following examples:

Example 1 Animal Model of Alzheimer's Disease

As an animal model of AD, transgenic mice of the 5xFAD line (Center for Collective Use of the FGBUN IAP RAS, Chernogolovka, Russia) were used, the pathological phenotype of which includes:

- amyloid deposits, neurodegeneration, memory impairment;

- accumulation of intracellular Aβ and pronounced death of neurons.

This line contains a triple mutation in the gene encoding the APP protein and a double mutation in the presenilin gene, found in hereditary forms of AD, and reproduces the main signs of amyloidosis characteristic of AD and is used as model A (342-induced neurodegeneration and formation of amyloid plaques.

Example 2. Morris water maze.

Memory impairment in mice was assessed using a Morris water maze with a diameter of 1.5 m (OpenScience, Russia) 180 days after the injection of 5xFAD into mice. The time required for the experimental animals to find the flooded platform was estimated.

Example 3. Effect of a hydrocortisone derivative on the development of BA It has recently been demonstrated that a hydrocortisone derivative, RX624 (Fig. 1), prevents the aggregation of oxidized GAPDH and reduces the toxicity of extracellular complexes of the enzyme with extended polyglutamine tracts [Lazarev VF, Mikhaylova ER, Dutysheva EA, et al., A hydrocortisone derivative binds to GAPDH and reduces the toxicity of extracellular polyglutamine-containing aggregates // Biochem Biophys Res Commun. 2017, 487 (3): 723-727]. To test whether the drug could be effective in animal models of AD, wild-type and 5xFAD transgenic mice were used. The mice were divided into three groups: (1) without any treatment, (2) treated with 100 μl of DMSO as a solvent, and (3) treated with 100 μl of the GAPDH-binding derivative of hydrocortisone RX624, diluted at 1 mg of substance per 1 kg of body weight animal. Treatment of mice began at 2.5 months of age and continued for the next 5 months with one injection per week. Two weeks after the last injection, the memory function of the mice was assessed using a Morris water maze. 5xFAD mice treated with RX624 showed significant memory recovery: platform seek time was the same as in wild-type mice (22.7 ± 4.4 vs 26.2 ± 6.6 s) and was significantly reduced compared to untreated mice 5xFAD (46.2 ± 12.4 s) (Fig. 2). We then isolated the hippocampus from the three groups of animals and analyzed them using a filter trap assay. Aggregates insoluble in sodium dodecyl sulfate containing both Aβ and GAPDH were found in control animals, while hippocampus of mice treated with RX624 were completely free of aggregates (Figs. 3, 4). This suggests that the hydrocortisone derivative selectively inhibits the formation of the exogenous GAPDH-Aβ complex and can be used in the manufacture of a drug for the treatment of Alzheimer's disease.

The presented results indicate that RX624 blocks the formation of the GAPDH-Aβ complex in the hippocampus and prevents memory dysfunction in mice with genetically determined AD (Fig. 2-4).

Claims (2)

1. The use of hydrocortisone 21-hemisuccinate as a treatment for Alzheimer's disease. 2. The use of hydrocortisone 21-hemisuccinate in the manufacture of drugs for the treatment of Alzheimer's disease.

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