Use of fosfomycin as the sole active ingredient in the preparation of drugs for preventing and / or treating amyotrophic lateral sclerosis

Abstract

The present invention discloses the use of fosfomycin as the sole active ingredient in the preparation of a drug for preventing and / or treating amyotrophic lateral sclerosis (ALS). The chemical formula of fosfomycin is C3H7O4P. The drug is used to prevent and / or treat ALS characterized by an abnormal number of GGGGCC sequence repeats in the C9orf72 gene. Using an ALS fruit fly model and phenotypic experiments, the present invention demonstrates that fosfomycin is non-toxic to Drosophila and can attenuate the neurodegenerative phenotype of ALS fruit flies. The application of fosfomycin in the treatment of ALS, particularly ALS characterized by an abnormal number of GGGGCC sequence repeats in the C9orf72 gene, has practical value.

Description

Technical Field

[0001] The present invention relates to the application field of a chemical preparation, and in particular to the use of fosfomycin as the sole active ingredient in the preparation of a drug for preventing and / or treating amyotrophic lateral sclerosis. Background Art

[0002] Amyotrophic lateral sclerosis (ALS), commonly known as Lou Gehrig's disease, is a progressive neurodegenerative disease that affects motor neurons. The main characteristic of ALS patients is the gradual loss of motor neurons, leading to muscle weakness and atrophy. The global prevalence of the disease is approximately 5 / 100,000, the incidence is approximately 2 / 100,000, and the average survival time from onset is 3.5 years. Specific symptoms include: (1) muscle weakness and atrophy: usually starting from a part of the hands, feet, or limbs and gradually spreading to the whole body; (2) motor dysfunction: such as unsteady gait, difficulty holding objects, slurred speech, etc.; (3) muscle twitching and spasms: including spontaneous muscle tremors called fasciculations; (4) swallowing and breathing difficulties: advanced patients often experience breathing difficulties due to respiratory muscle weakness and require mechanical ventilation support; (5) non-motor symptoms: some patients may experience cognitive and behavioral changes. There is currently no cure for this disease. The approved small molecule drug riluzole can delay motor neuron damage by reducing neuronal excitotoxicity. Edaravone, a free radical scavenger, can mitigate oxidative stress damage in neurodegenerative diseases by scavenging reactive oxygen species and inhibiting lipid oxidation. Both drugs can slow disease progression in ALS patients, but these drugs are expensive. Therefore, the hundreds of thousands of ALS patients worldwide urgently need new treatments.

[0003] The cause of ALS is not yet fully understood. Currently known risk factors for the disease include: (1) Gene mutation: About 10% of ALS cases are familial ALS, which is related to the genetic background of gene mutation. Among the many mutant genes such as SOD1, C9orf72, FUS, and TARDBP, C9orf72 is the pathogenic gene with the highest incidence. C9orf72 is part of the guanine nucleotide exchange factor complex, and its specific function is still unclear; (2) Environmental factors: Possible environmental factors include heavy metal exposure, pesticide exposure, smoking, etc., but there is no conclusive evidence; (3) Neuroinflammation and oxidative stress: These factors are believed to play a role in the damage and death of motor neurons. From a molecular pathology perspective, the onset of ALS is highly correlated with the imbalance of intracellular protein homeostasis, aggregation and accumulation of misfolded proteins. Protein aggregation and apoptosis play a significant role in the progression of ALS. Existing studies have reported that abnormal accumulation and aggregation of proteins, including TDP-43, superoxide dismutase 1 (SOD1), Fused in Sarcoma (FUS), TAF15, and UBQLN2, are implicated in the pathogenesis of ALS. The accumulation of these misfolded proteins may lead to a variety of pathological changes, including neuronal dysfunction, protein aggregate formation, mitochondrial dysfunction, oxidative stress, and apoptosis. However, the clinical failure of targeted degradation of aggregated proteins in the nervous system to prevent and treat neurodegeneration suggests that, while the accumulation of aggregated proteins contributes to the pathogenesis of neurodegeneration, the aggregation of pre-synthesized proteins is not the fundamental cause of protein homeostasis imbalance and the development of neurodegeneration. Increasing evidence suggests that protein homeostasis imbalance may arise from the accumulation of nascent polypeptides on stalled ribosomes during translation. Although relevant studies have shown that in C9-ALS, the translation of the (GGGGCC) repeat sequence causes ribosome stalling, the accumulation of nascent polypeptides leads to insufficient function of the ribosome-associated quality control system, which in turn accelerates the course of ALS. Therefore, in-depth research on the role of the ribosome-associated quality control system in ALS will help to better understand the pathophysiology of the disease and delay the progression of ALS by intervening in these abnormal biological processes. Based on this molecular mechanism, screening for the purpose of reducing nascent polypeptide aggregation, improving intracellular protein homeostasis, and promoting protein degradation will become an important direction for the development of new ALS drugs in the future.

[0004] Fosfomycin, with a chemical formula of C3H7O4P and a molecular weight of 138.06 g / mol, is a common antibiotic isolated from Streptomyces freundii. It exerts its antibacterial effect by inhibiting bacterial cell wall synthesis by blocking a key step in peptidoglycan synthesis. Fosfomycin exhibits broad-spectrum antibacterial activity against a variety of Gram-positive bacteria, including some multidrug-resistant strains, such as methicillin-resistant Staphylococcus aureus and Enterococcus faecalis, as well as Gram-negative bacteria such as Escherichia coli, Klebsiella, Proteus, and Pseudomonas aeruginosa. It is primarily used to treat intestinal infections, urinary tract infections, respiratory tract infections, bone infections, and skin and soft tissue infections.

[0005] There are currently no reports on the application of fosfomycin in non-antibacterial applications, especially for neurodegenerative diseases. Summary of the Invention

[0006] In order to solve the above technical problems, the present invention targets the molecular pathogenesis of ALS and provides a use of fosfomycin as the sole active ingredient in the preparation of a drug for preventing and / or treating amyotrophic lateral sclerosis.

[0007] The present invention is achieved through the following technical solutions:

[0008] The invention relates to a use of fosfomycin as the sole active ingredient in preparing a drug for preventing and / or treating amyotrophic lateral sclerosis, wherein the chemical formula of the fosfomycin is C3H7O4P.

[0009] Furthermore, the drug of the present invention is a drug for preventing and / or treating amyotrophic lateral sclerosis characterized by an abnormal number of (GGGGCC) sequence repeats in the C9orf72 gene.

[0010] Furthermore, the drug of the present invention is a preparation prepared with fosfomycin as the active ingredient and pharmaceutically acceptable excipients or auxiliary ingredients.

[0011] Furthermore, the preparation of the present invention is an oral preparation.

[0012] Furthermore, the oral preparation of the present invention is a solution, granules, paste, emulsion, pills, capsules or tablets.

[0013] The present invention has the following beneficial effects:

[0014] The present invention discloses the use of fosfomycin as the sole active ingredient in the preparation of a drug for preventing and / or treating amyotrophic lateral sclerosis. It is found that fosfomycin can activate the phosphorylation of eIF2α by inducing ribosome arrest. At a safe concentration, it can activate the ribosome-related quality control pathway in the cell, reduce the accumulation of newly synthesized polypeptides in the cell, alleviate the toxic effects of protein homeostasis imbalance on the body, improve neuronal damage, and delay the progression of amyotrophic lateral sclerosis patients. Through the ALS fruit fly model and its phenotypic experiments, it is confirmed that fosfomycin is not toxic to fruit flies and can attenuate the neurodegenerative phenotype of ALS fruit flies. Therefore, the application of fosfomycin in the treatment of amyotrophic lateral sclerosis, especially amyotrophic lateral sclerosis with an abnormal number of (GGGGCC) sequence repeats in the C9orf72 gene, has practical value. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 In Example 1 of the present invention, a drug capable of inducing phosphorylation of eukaryotic translation initiation factor 2 subunit alpha (eIF2α) was screened from an FDA-approved drug library;

[0016] Figure 2 This is a diagram showing the results of screening for a drug mixture that activates eIF2α phosphorylation in Example 1 of the present invention;

[0017] Figure 3 This is a diagram showing the results of screening for small molecule drugs that activate eIF2α phosphorylation in Example 1 of the present invention;

[0018] Figure 4 This is an enzyme-linked immunosorbent assay (ELISA) diagram for validating the activation of eIF2α phosphorylation by small molecule drugs in Example 1 of the present invention;

[0019] Figure 5 This is a diagram showing the polyribosome analysis in Example 2 of the present invention verifying the occurrence of ribosome arrest induced by fosfomycin treatment;

[0020] Figure 6 This is a diagram showing the cytotoxicity determination of fosfomycin in Example 3 of the present invention;

[0021] Figure 7 This is a diagram of the Drosophila model of ALS related to C9orf72 in Example 4 of the present invention;

[0022] Figure 8 This is a statistical graph showing the pupation rate of fruit flies raised on the control medium in Example 5 of the present invention;

[0023] Figure 9This is a statistical graph showing the pupation rate of fruit flies raised in 5 μM fosfomycin medium according to Example 5 of the present invention;

[0024] Figure 10 This is a statistical graph showing the pupation rate of fruit flies raised in 25 μM fosfomycin medium according to Example 5 of the present invention;

[0025] Figure 11 This is a statistical graph showing the emergence rate of fruit flies raised on the control medium in Example 5 of the present invention;

[0026] Figure 12 This is a statistical graph showing the emergence rate of Drosophila grown in 5 μM fosfomycin medium according to Example 5 of the present invention;

[0027] Figure 13 This is a statistical graph showing the emergence rate of Drosophila grown in 25 μM fosfomycin medium according to Example 5 of the present invention;

[0028] Figure 14 This is the figure of Example 6 of the present invention, which shows that feeding ALS fruit flies in 5 μM and 25 μM fosfomycin culture medium can significantly improve the neurodegenerative phenotype. DETAILED DESCRIPTION

[0029] Below in conjunction with the accompanying drawings, specific embodiment of the present invention is described in further detail. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope limited by the appended claims of the application.

[0030] Example 1 Fosfomycin can activate the phosphorylation of eIF2α

[0031] 1. Experimental Principle

[0032] Ribosome stalling during translation triggers the ribosome-associated quality control (RQC) pathway. The RQC system prevents the accumulation of aberrant nascent polypeptides by identifying and isolating stalled ribosomes, clearing mRNA, and ubiquitinizing and degrading aberrant nascent polypeptides. During this process, ribosome stalling recruits the kinase GCN2 (General Control Nonderepressible 2) to phosphorylate eIF2α. Therefore, eIF2α phosphorylation can be used as a marker for ribosome stalling.

[0033] 2. Based on the above principles, this embodiment carries out the following screening experiments

[0034] like Figure 1As shown, 1×10 6 Drosophila S2 cells (Schneider2, a cell line derived from late-stage Drosophila embryos) were cultured overnight at 25°C. The next day, drugs from the FDA-approved drug library were added for screening. A cocktail of 8-10 drugs was added to each well of a 24-well plate, with each drug at a final concentration of 1 μM. After 20 minutes of drug treatment, the cells were harvested by centrifugation (500 g for 5 minutes at 4°C). The supernatant was discarded, and the cells were resuspended in 1 mL of ice-cold 1x phosphate-buffered saline (PBS). Repeat the centrifugation and discard the supernatant. Add 300 μL of cell lysis buffer to lyse the cells, centrifuge at 12,000 rpm and 4°C for 5 minutes, take 100 μL of the cell lysate supernatant, add 25 μL of 5× loading buffer, boil the sample at 95°C for 10 minutes, and perform protein immunoblotting (Western Blotting). Use a commercial antibody against phosphorylated eIF2α to detect the level of phosphorylated eIF2α in each cell sample. At the same time, use a commercial antibody against eIF2α to detect the expression of endogenous eIF2α, which is used as an internal reference. Use Image J to measure the grayscale value of phosphorylated eIF2α and eIF2α protein immunoblotting results, calculate the comparison value of phosphorylated eIF2α and eIF2α, normalize the ratio of the control group, and draw a scatter plot after sorting the ratio from small to large, as shown in the following figure. Figure 2 By analyzing and ranking the relative values ​​of grayscale quantitative p-eIF2α / eIF2α protein immunoblotting results after treating cells with 130 drug cocktails, we found that 18 drug cocktails could increase eIF2α phosphorylation levels (grayscale quantitative relative values ​​greater than 1.25). The top 10 drug cocktails (p-eIF2α / eIF2α relative quantitative values ​​greater than 1.40) were selected for the second round of screening.

[0035] right Figure 2 The individual drugs in the drug mixture obtained by screening were screened and confirmed to be small molecule drugs that can phosphorylate eIF2α in S2 cells. Figure 3 A second round of screening was conducted to identify 160 small molecules from the 10 drug mixtures identified in the first round of screening to identify those that effectively induced eIF2α phosphorylation. A total of 21 small molecules were found to effectively induce eIF2α phosphorylation (relative quantitative value of p-eIF2α / eIF2α greater than 1.25).

[0036] Enzyme-Linked Immunosorbent Assay (ELISA) Figure 3The activation effect of the small molecules screened out on eIF2α phosphorylation was further verified.

[0037] In a 24-well plate, 1 × 10 6 S2 cells were cultured at 25°C overnight and the next day, Figure 3 The cells were treated with the small molecule drugs identified in the experiment, with each drug at a final concentration of 1 μM. After 20 minutes of drug treatment, the cells were harvested by centrifugation (500 g for 5 minutes at 4°C). The supernatant was discarded and the cells were resuspended in 1 mL of pre-chilled 1× PBS (phosphate-buffered saline). The cells were centrifuged again and the supernatant discarded. 400 μL of lysis buffer (containing protease / phosphatase inhibitors) was added and incubated on ice for 30 minutes. The cells were centrifuged at 13,000 rpm for 10 minutes at 4°C, and the supernatant was transferred to a fresh centrifuge tube. Samples and controls were distributed according to the experimental design, with duplicate wells for both positive controls and samples. 100 μL of sample or positive control was added to each well, the plate was sealed, and the cells were incubated at room temperature for 2.5 hours. The wells were cleared of sample and washed with 300 μL of wash buffer. This was repeated four times. Subsequently, 100 μL of biotinylated anti-phospho-eIF2α (S52) antibody was added and incubated at room temperature for 1 hour. Add 300 μL of washing solution and wash, repeat washing 4 times, add 100 μL of HRP-labeled streptavidin solution, and incubate at room temperature for 1 hour. After discarding the solution, add 100 μL of TMB colorimetric solution, incubate at room temperature with shaking in the dark for 30 minutes, then add 50 μL of stop solution to terminate the reaction and immediately measure the OD value at a wavelength of 450 nm. Calculate the average OD value of each replicate well and deduct the average OD value of the blank well (zero standard) as shown in Figure 2. Figure 4 . Compared with the untreated cell lysate (OD 450 =0.49) compared to Figure 3 After the small molecule drugs screened in the experiment were treated with S2 cells, OD 450 The values ​​were significantly increased (OD 450 greater than 2.4), indicating that these small molecule drugs can effectively induce the phosphorylation of eIF2α as verified by ELISA.

[0038] Example 2 Fosfomycin induces ribosome arrest

[0039] In a 10 cm cell culture dish, add 10 mL of a 1×10 6S2 cells were cultured at 25°C for 48 hours and treated with 1 μM fosfomycin for 20 minutes. Emetine (EME) was then added to a final concentration of 20 μg / mL for 10 minutes to stabilize ribosomes. The cells were then harvested by centrifugation at 500 g for 5 minutes at 4°C, washed with pre-chilled PBS, and lysed on ice with 600 μL of lysis buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM sodium chloride, 5 mM magnesium chloride, 20 μg / mL EME, 0.5% Triton X-100, and protease and RNase inhibitors. The cells were then mechanically disrupted using a Dounce homogenizer (50 strokes). After the lysate was centrifuged at 1000×g for 10 minutes to remove the cell nuclei, the total RNA content of the lysate was determined using the Qubit RNA HS quantification kit. Then, 300μL of digestion buffer containing 50mM Tris-HCl (pH 7.5), 5mM magnesium chloride, 20μg / mL EME, 0.5% Triton X-100, and 15mM calcium chloride was added. After mixing, micrococcal nuclease (MNase) was added at a ratio of 4U / μg total RNA and incubated at 25°C for 40 minutes. The reaction was terminated by adding EGTA to a final concentration of 6.25mM and placed on ice. The digested sample was applied to a 10%-45% linear sucrose gradient (containing the same buffer) and centrifuged at 35,000 rpm for 3 hours at 4°C using an SW41Ti rotor (Beckman) to achieve ribosome fractionation. Fractions were collected using the Biocomp automated gradient fractionation system, and the A260 absorbance value was simultaneously detected to observe the distribution of ribosomes. Figure 5 The standard polyribosomes showed typical 40S, 60S, and 80S single peaks and a ladder-like distribution of polyribosomes. Fosfomycin treatment caused ribosome arrest, and the arrested ribosomes formed di-ribosome and tri-ribosome complexes with subsequent ribosomes, while the 80S peak height decreased.

[0040] Example 3 Fosfomycin does not affect cell viability

[0041] In a 96-well plate, 1 × 10 5 Drosophila S2 cells were cultured at 25°C for 24 hours, and then fosfomycin was added to a final concentration of 25-800 μM using S2 medium (Gibco). The cells were cultured at 25°C for 12 hours. Cell lysis buffer (Vazyme) from the Cell Viability Assay kit was added to a 96-well plate at an equal volume to the cells. The cells were incubated on a shaker for 10 minutes, and cell viability was measured using a microplate reader. Figure 6After treatment with 25-800 μM fosfomycin for 12 hours, the viability of S2 cells did not decrease significantly compared with the control group (all greater than 90%), indicating that fosfomycin had no significant cytotoxicity.

[0042] Example 4 ALS Drosophila Model and Its Phenotype

[0043] Using the Drosophila UAS-gal4 system, GMR-gal4 is specifically used to drive the expression of polyPR in the optic nerve of the Drosophila compound eye. Overexpression and accumulation of polyPR in the optic nerve of the Drosophila compound eye will destroy the structure of the Drosophila compound eye, resulting in the appearance of black spots. The degree of damage to the compound eye can represent the severity of neurodegenerative diseases. Figure 7 The compound eye structure of normal fruit flies in the control group was clear and complete. ALS model fruit flies were divided into three types according to the compound eye phenotype: Type I had the same compound eye phenotype as the control group; Type II had scattered, erosive black spots in the compound eyes, and the compound eye structure was basically intact, representing a moderate degree of neurodegenerative disease; Type III had clustered, black patches in the compound eyes, and the compound eye structure was destroyed, representing a severe neurodegenerative disease phenotype.

[0044] The specific experimental operations are as follows:

[0045] Standard culture medium: Prepare 2L of culture medium. First, thoroughly dissolve 120g brown sugar, 14.5g white sugar, 16g agar, and 49g yeast in 1L of water. Bring to a boil in a pot. Add 98g cornstarch, previously dissolved in 500ml of water, to the pot. Rinse off any remaining cornstarch and other residue with another 500ml of water. Stir constantly until boiling, then reduce the heat and time the mixture for 10 minutes. During this time, dissolve 3.4g methylparaben in 34ml of ethanol and prepare 7ml of propionic acid. After 10 minutes, turn off the heat and pour in the fully dissolved methylparaben and propionic acid. Stir thoroughly. Pour into a vial.

[0046] Cross GMR-gal4 flies with UAS-PolyPR flies. Remove old flies from the tubes on the fifth day. When the offspring begin to eclode, select flies expressing GMR-gal4 and UAS-PolyPR every evening at 8:00 PM and feed them with fresh culture medium. Count flies for seven days, changing the culture medium every two days. At 8:00 PM on the seventh day, collect flies from the tubes into 2 ml EP tubes and freeze at -80°C. Photograph approximately 100 flies for phenotyping. All flies were maintained in a 25°C incubator.

[0047] Example 5 Determination of the toxicity of fosfomycin to fruit flies

[0048] Through drug screening, it was found that fosfomycin can induce phosphorylation of eIF2α in S2 cells, and it has no obvious toxicity in Drosophila cells. The safety of fosfomycin was further evaluated by measuring its toxicity to Drosophila embryos.

[0049] Drug culture medium: Cool the standard culture medium to approximately 55°C. Pour 100ml of the medium into a 200ml conical flask, add fosfomycin to the desired concentration, and shake well. Pour into a vial (keep it cool and away from light).

[0050] All fruit flies were cultured in a 25°C incubator. First, select a number of wild-type male fruit flies and virgin fruit flies and feed them separately for 24 hours. The flies were then transferred to standard culture medium and drug-treated culture medium, with the phosphatase drug concentrations at 5 μM and 25 μM, respectively. Twelve tubes of each type of culture medium were prepared, with four virgin flies and four male fruit flies per tube. After two days, discard any old fruit flies. Statistical analysis of pupation and hatching rates was performed.

[0051] Statistics of pupation rate: When the larvae begin to pupate, count the newly added fruit fly pupae in each tube at 8 pm every day and mark them with a black marker. Repeat this process until no new pupae are added for 2 consecutive days. Figure 8 The fruit fly embryos cultured in the control group in conventional culture medium basically completed pupation in 4-6 days. Figure 9 and Figure 10 In the culture medium containing 5μM and 25μM fosfomycin, the pupation time of Drosophila embryos was basically the same as that of the control group (4-6 days), indicating that fosfomycin had no obvious effect on the pupation of Drosophila embryos.

[0052] Emergence rate statistics: When the pupae in the tube begin to emerge, count the number of fruit flies that emerge in each tube at around 8 o'clock every night and discard them. Repeat this process until no new fruit flies are added for two consecutive days. Figure 11-13 In the control group, Drosophila embryos cultured in regular culture medium essentially completed eclosion within 4-6 days. In culture medium containing 5μM and 25μM fosfomycin, the eclosion time of Drosophila embryos was essentially the same as that of the control group (4-6 days), indicating that fosfomycin has no significant effect on Drosophila embryonic eclosion. These results indicate that fosfomycin has no significant toxicity to Drosophila embryonic development.

[0053] Example 6 Fosfomycin can attenuate the neurodegenerative phenotype of ALS Drosophila

[0054] The GMR-gal4 fruit flies were hybridized with the UAS-PolyPR fruit flies and cultured at 25°C. When the offspring began to emerge, they were picked at 8 o'clock every night. The ALS model fruit flies were picked and fed with ordinary culture medium and drug culture medium respectively. Fosfomycin needs to be protected from light. The culture medium was placed in a black plastic bag with holes for ventilation. The culture medium was placed in a 25°C incubator and counting began for 7 days. The culture medium was changed every two days. At 8 o'clock in the evening of the 7th day, the fruit flies in the tube were collected into 2ml EP tubes and frozen at -80°C. 100 fruit flies were collected and the statistical phenotypes were taken. Figure 14 In ALS model fruit flies, without drug treatment, the proportions of type I, type II, and type III phenotypes were 27.91%, 13.95%, and 58.14%, respectively. After treatment with 5μM fosfomycin, the proportions of the three phenotypes were 45.78%, 15.67%, and 38.55%, respectively. After treatment with 25μM fosfomycin, the proportions of the three phenotypes were 36.78%, 22.99%, and 40.23%, respectively. Compared with the control group, fosfomycin treatment significantly increased the proportions of the normal phenotype (type I) and mild phenotype (type II), while significantly decreased the proportion of the severe phenotype (type III), indicating that fosfomycin can significantly ameliorate the neurodegenerative phenotypes of ALS fruit flies.

[0055] The above embodiments are merely descriptions of preferred implementations of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should fall within the scope of protection determined by the claims of the present invention.

Claims

1. Use of fosfomycin as the sole active ingredient in the preparation of a drug for treating amyotrophic lateral sclerosis, wherein the chemical formula of the fosfomycin is C3H7O4P.

2. The use according to claim 1, characterized in that: The drug is a drug for treating amyotrophic lateral sclerosis characterized by an abnormal number of GGGGCC sequence repeats in the C9orf72 gene.

3. The use according to claim 1 or 2, characterized in that: The medicine is a preparation prepared by taking fosfomycin as the active ingredient and adding pharmaceutically acceptable excipients or auxiliary ingredients.

4. The use according to claim 3, characterized in that: The preparation is an oral preparation.

5. The use according to claim 4, characterized in that: The oral preparation is a solution, granule, paste, emulsion, pill, capsule or tablet.

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