Use of nitrone compounds for the preparation of products for the prolongation of life

Abstract

The application discloses application of a nitro compound in preparation of products prolonging life, and the nitro compound has the structure shown in the following general formula (I). The application takes Caenorhabditis elegans as an object, and studies influence of TBN on life and health indexes of the Caenorhabditis elegans. Experimental results show that the TBN prolongs the life of the Caenorhabditis elegans, significantly improves pharyngeal pumping rate and movement ability of the Caenorhabditis elegans, and significantly reduces lipofuscin accumulation degree of the Caenorhabditis elegans.

Description

TECHNICAL FIELD

[0001] The present application relates to a new use of nitrones or pharmaceutically acceptable salts thereof, in particular to the use of nitrones or pharmaceutically acceptable salts thereof in the preparation of products for prolonging life. BACKGROUND

[0002] For thousands of years, longevity has been an eternal topic pursued by mankind. Among the factors related to the prolongation of life, the most effective one is caloric restriction. Caloric restriction refers to reducing the total daily intake of calories while providing the necessary nutrients for the organism. Caloric restriction is considered the most reliable and reproducible method to prolong life except for genetic modification, which has been verified in experiments on a variety of animals (worms, fruit flies, mice, rhesus monkeys, etc.) (Cao Ke et al. Research progress of nutritional signaling pathways related to caloric restriction for life extension. Chinese Journal of Obesity and Metabolic Diseases [J]. 2021, 7(4): 270). However, caloric restriction requires reducing daily food intake, and thus is not a practical method. SUMMARY

[0003] To solve the problems in the prior art, one of the purposes of the present application is to provide the use of nitrones or pharmaceutically acceptable salts thereof in the preparation of products for prolonging life.

[0004] The second purpose of the present application also relates to the use of nitrones or pharmaceutically acceptable salts thereof in the preparation of products related to increasing exercise capacity.

[0005] The third purpose of the present application also relates to the use of nitrones or pharmaceutically acceptable salts thereof in the preparation of products related to preventing or reducing the production of lipofuscin.

[0006] To achieve the above-mentioned purposes, the technical solution of the present application is as follows:

[0007] In some embodiments, the present application provides the use of nitrones or pharmaceutically acceptable salts thereof in the preparation of products for prolonging life. In the present application, "prolonging life" means prolonging the life of a human or an animal compared to the natural state. For example, "prolonging life" can mean prolonging the life of a subject determined by innate factors such as genetic factors or acquired factors. For example, prolonging life can mean delaying the natural death time of a human or an animal.

[0008] In other embodiments, the present application also provides the use of nitrones or pharmaceutically acceptable salts thereof in the preparation of products related to increasing exercise capacity. The increase in exercise capacity described in the present application can be an increase in exercise capacity at different ages, for example, increasing the exercise capacity of a healthy living being (such as a human or an animal).

[0009] In other embodiments, the present application also provides the use of the nitrone compound or pharmaceutically acceptable salt thereof in the preparation of a product for preventing or reducing lipofuscin production. The lipofuscin production described in the present application can include, but is not limited to, the production of lipofuscin caused by various factors such as age, ultraviolet light, stress, pollution, etc.

[0010] The nitrone compound described in the present application has the following general formula (I):

[0011]

[0012] wherein:

[0013] R1is hydrogen, methyl or; ;

[0014] R2, R3are the same or different, each independently selected from hydrogen or C1-C6 alkyl; R4is sec-butyl, iso-butyl, tert-butyl, cyclopentyl or cyclohexyl, and R5is sec-butyl, iso-butyl, tert-butyl, cyclopentyl or cyclohexyl.

[0015] Further, the C1-C6 alkyl described in the present application is selected from methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, n-pentyl, preferably methyl, ethyl or propyl.

[0016] Further, the nitrone compound described in the present application is selected from TBN or TN2:

[0017] .

[0018] According to one embodiment of the present application, the "pharmaceutically acceptable salt" of the nitrone compound described in the present application can be a salt formed with an inorganic acid, such as hydrochloric acid (HCl), hydrobromic acid (HBr), hydroiodic acid, sulfuric acid, pyrosulfuric acid, phosphoric acid or nitric acid; or a salt formed with an organic acid, such as methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, acetic acid, pyruvic acid, trifluoroacetic acid, propionic acid, hexanoic acid, benzoic acid, salicylic acid, cinnamic acid, cyclopentanepropionic acid, dodecylsulfic acid, 2-naphthalenesulfonic acid, citric acid, tartaric acid, stearic acid, lactic acid, oxalic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, D-gluconic acid, aspartic acid.

[0019] The product described in the present application is suitable for use in humans or animals.

[0020] According to one embodiment of the present application, the product described above can be a pharmaceutical product or a skin care product.

[0021] The pharmaceutical products described in this invention can be administered orally or parenterally, for example, rectally, topically, transdermally, intravenously, intramuscularly, intraperitoneally, or subcutaneously. Dosage forms for oral administration may include, but are not limited to, tablets, pills, soft or hard capsules, granules, powders, micropowders, liquids, emulsions, or microgranules. Dosage forms for parenterally administration may include, but are not limited to, eye drops, injections, pharmaceutical drops, lotions, ointments, gels, creams, suspensions, emulsions, suppositories, patches, or sprays.

[0022] The dosage of the nitroketone compound or its pharmaceutically acceptable salt described in this invention can be calculated as nitroketoneazine (TBN) at 10-3000 mg / person / dose. The specific dosage may vary depending on the subject's age, sex, weight, specific pathological condition and its severity, route of administration, or diagnosis. The dosage based on the above factors can be determined according to the skill of a person skilled in the art. The specific administration method and frequency can follow the conventional administration method for nitroketoneazine (e.g., TBN), for example, once or multiple times daily.

[0023] The nitrone compounds of the present invention, or their pharmaceutically acceptable salts, can be used as ingredients in the preparation of skin care products. Beneficial effects

[0024] This invention uses *C. elegans* as the target organism to study the effects of TBN on the lifespan and health indicators of the nematode. Experimental results show that TBN not only prolongs the lifespan of *C. elegans*, but also significantly improves its motility and significantly reduces the accumulation of lipofuscin in the nematode. Attached Figure Description

[0025] Figure 1 This is a graph showing the results of TBN extending the healthy lifespan of nematodes;

[0026] Figure 2 This is a graph showing the effect of TBN in delaying age-related kinetic decline in wild-type Caenorhabditis elegans; ** express P <0.01, *** express P <0.001;

[0027] Figure 3 This is a graph showing the results of TBN enhancing the pharyngeal sucking ability of nematodes; ** express P <0.01, *** express P <0.001;

[0028] Figure 4 This is a graph showing the results of TBN reducing lipofuscin accumulation in nematodes. ns indicates no statistical significance. * express P<0.05. DETAILED DESCRIPTION

[0029] The following examples facilitate a better understanding of the present application, but do not limit the present application. The experimental methods in the following examples are all conventional methods unless otherwise specified. The test materials used in the following examples are all obtained from conventional biochemical reagent stores unless otherwise specified.

[0030] Caenorhabditis elegans has a short life cycle, fast reproduction, clear genetic background, and no harm to researchers, and has been widely used in the field of life span research. Caenorhabditis elegans is the first multicellular organism with a complete genome sequence, and 60-80% of human genes have homologous genes in the nematode genome. The functions of genes found in nematodes may be directly applicable to humans.

[0031] The present application takes Caenorhabditis elegans as the experimental object. In nature, nematodes usually feed on microorganisms. In the laboratory, they usually feed on non-pathogenic strains of Escherichia coli OP50 Escherichiacoli OP50, E.coli OP50 Caenorhabditis elegans is usually cultured at 20℃ under laboratory conditions, with a development cycle of about 3 days and a lifespan of about 2-3 weeks. The life cycle of Caenorhabditis elegans includes embryonic period, four larval stages (L1-L4) and adult stage, which is much shorter than that of other organisms. Taking the egg as the starting point of life, when the nematode egg successfully hatches, the nematode begins to gradually experience four larval stages, and then becomes an adult within 40-50 h.

[0032] The preparation method of M9 buffer in the embodiment of the present application is as follows: respectively take NaCl 0.125 g, MgSO4·7H2O 0.0625 g, KH2PO4 0.75 g, Na2HPO4·12H2O 3.78 g, add deionized water 250 mL and dissolve thoroughly, sterilize by high pressure and cool to about 55℃, and then cool to room temperature.

[0033] LB liquid medium: respectively take NaCl 10 g, peptone 10 g, and yeast powder 5 g, dissolve in 1000 mL deionized water, sterilize by high pressure and cool to about 55℃, and then cool to room temperature, and prepare in a clean bench.

[0034] In the following examples, all data analysis is expressed as mean ± SD except for the lifespan experiment. GraphPad Prism 7 is used for analysis, and log-rank (Mantel-Cox) test is used for lifespan; t test is used for comparison between two groups; and 1-way ANOVA or 2-way ANOVA is used for evaluation of multiple groups. P< ​0.05 was considered statistically significant.

[0035] Example 1 Life span experiment

[0036] 1. Experimental method

[0037] (1) Effect of drugs on growth rate E. coli OP50

[0038] The drugs were diluted to the required concentrations (concentrated E. coli OP50 to 100-200 times with freshly prepared LB liquid) and added to each group of solutions for culture in 96-well plates. After the initial absorbance value was detected by a microplate reader (OD = 595 nm), the plates were incubated at 37 °C on a horizontal shaking incubator, and the absorbance was measured every 1 h. E. coli OP50

[0039] (2) Life span experiment

[0040] Before the formal life span experiment, all nematodes needed to be cultured for at least two generations. All life span experiments were performed at 20 °C with E. coli OP50 as the food source. The C. elegans were synchronized to the L4 stage, and 30 worms were randomly selected and transferred to the blank control group and the drug administration group with the addition of FUDR (inhibition of egg production). Specifically, wild-type C. elegans were cultured in TBN solutions with final concentrations of 0, 10, 30, and 100 μM, which was recorded as day 0. The culture medium was replaced every 24 h, and the number of surviving nematodes was recorded. The remaining nematodes in each group were transferred to freshly prepared solutions in each group, and the number of surviving nematodes was recorded until the last nematode in each group died. The criteria for determining nematode death were that the nematodes did not respond when touched with a nematode picking needle and remained straight and motionless.

[0041] 2. Experimental results

[0042] (1) TBN does not affect the growth rate of C. elegans E. coli OP50

[0043] As the food source for C. elegans, the proliferation of bacteria in the diet or the metabolic products produced by the bacteria during culture may affect the life span of the nematodes, thereby interfering with the experimental results. E. coli OP50 To exclude the effect of TBN on the life span of nematodes, the inventors detected whether TBN had possible growth inhibition or proliferation on the growth rate of C. elegans. The experimental results showed that different concentrations of TBN (10, 30, and 100 μM) had no effect on the growth rate of C. elegans within 0-4 h compared with the Ctrl group

[0044] E. coli OP50 E. coli OP50 E. coli OP50 ​​​​​The rising trend of the growth rate curve is completely consistent, and the growth rate of each concentration of TBN group within 4-10 h E. coli OP50 The growth rate is slightly decreased compared with the Ctrl group. The statistical analysis results of GraphPad Prism 7 show that the decrease of the growth rate of each administration group of TBN has no statistical significance compared with the Ctrl group. E. coli OP50

[0045] (2) TBN mediates the life extension of wild-type nematodes

[0046] The experimental results are shown in Figure 1 Compared with the Ctrl group, each administration dose group of TBN (10, 30, 100 μM) can prolong the life of C. elegans, and the medium dose administration group of TBN (30 μM) has the most significant effect on prolonging the life of C. elegans.

[0047] Example 2: Determination of motor ability

[0048] (1) Determination of motor ability

[0049] The wild-type nematodes N2 synchronized to the L4 stage were randomly selected and transferred to the Ctrl group (30) and the TBN group (30 μM) for continuous culture, which was recorded as day 0 at this time. When N2 was cultured to day 5 (young), day 10 (middle-aged), and day 15 (old), respectively, the nematodes in each group were randomly selected and transferred to the blank medium containing M9 buffer. After the nematodes were adapted in the liquid, the body swing times of the nematodes in each group per 30 s were observed and recorded by dissection microscope. The effect of TBN on the motor ability of nematodes at day 5 (young), day 10 (middle-aged), and day 15 (old) was studied.

[0050] The experimental results show that when the nematodes treated with TBN are at day 5, the average body swing times of the nematodes in the Ctrl group and the TBN group per 30 s are 44.8 and 54.2, respectively, and the body swing times of the nematodes in the TBN group per 30 s are increased by 20.9% compared with the Ctrl group; when the nematodes treated with TBN are at day 10, the average body swing times of the nematodes in the Ctrl group and the TBN group per 30 s are reduced to 29.9 and 38.9, respectively, and the body swing times of the nematodes in the TBN group per 30 s are increased by 19.0% compared with the Ctrl group; when the nematodes treated with TBN are at day 15, the average body swing times of the nematodes in the Ctrl group and the TBN group per 30 s are further reduced to 22.2 and 20.6, respectively, and the body swing times of the nematodes in the TBN group per 30 s are still increased by 17.8% compared with the Ctrl group Figure 2 . The above results show that TBN has a significant effect on increasing the motor ability of young, middle-aged, and old nematodes.

[0051] (2) Determination of pharyngeal pumping rate​

[0052] The pharynx is a neuromuscular organ that can rhythmically contract to facilitate food uptake. When the muscle function and structure of the pharynx of C. elegans declines, the number of contractions also decreases, which can be observed under a dissecting microscope.

[0053] Wild-type worms N2 were synchronized to the L4 stage, and randomly picked worms were transferred to the Ctrl group (20) and the TBN group (20) for further culture, which was recorded as day 0. When N2 was cultured to day 3 (young) and day 8 (middle-aged), respectively, worms in each group were randomly picked into clean blank medium, and after about 1 min of adaptation, the number of pharyngeal pump contractions within 30 s was observed and recorded under a dissecting microscope.

[0054] The results are shown in Figure 3 The pharyngeal pumping rate of the Ctrl group of worms on day 8 was significantly reduced relative to day 3, indicating that the pharyngeal pump muscle structure had atrophied. The average pharyngeal pumping rate of the TBN-treated worms within 30 s on day 3 was 51.6 times, and the average pharyngeal pumping rate of the Ctrl group of worms within 30 s was 47.2 times, and the average pharyngeal pumping rate of the TBN group of worms within 30 s was increased by 9.3% relative to the Ctrl group. The average pharyngeal pumping rate of the TBN-treated worms within 30 s on day 8 was 38.4 times, and the average pharyngeal pumping rate of the Ctrl group of worms within 30 s was 32.2 times, and the average pharyngeal pumping rate of the TBN group of worms within 30 s was increased by 13.1% relative to the Ctrl group. The above results show that TBN can significantly increase the pharyngeal pumping rate of wild-type worms.

[0055] Example 3 Effect on lipofuscin accumulation

[0056] An important property of lipofuscin is its extensive autofluorescence, which can be observed by fluorescence microscopy under the action of excitation light of different wavelengths.

[0057] Wild-type worms N2 were synchronized to the L4 stage, and randomly picked worms were transferred to the Ctrl group (15) and the TBN group (15) for further culture, which was recorded as day 0. When N2 was cultured to day 3 (young) and day 8 (middle-aged), respectively, the residual bacterial solution on the surface of the worms was washed off with M9 buffer, and then the worms were transferred one by one to an agar pad, anesthetized with levamisole hydrochloride, and the effect of TBN on lipofuscin fluorescence intensity was observed and imaged under an inverted fluorescence microscope.

[0058] The results are shown in Figure 4As shown, at day 3, both Ctrl and TBN groups showed weak lipofuscin fluorescence. Statistical results showed that there was no significant difference in the intensity of lipofuscin fluorescence between Ctrl and TBN groups. At day 8, the intensity of lipofuscin fluorescence in Ctrl group increased, and there was a significant difference in the intensity of lipofuscin fluorescence between day 3 and day 8, indicating that lipofuscin accumulated significantly in the worms at this stage. Compared with Ctrl group, TBN (30 μM) could significantly reduce the accumulation of lipofuscin in the worms.

Claims

1. Use of a nitro compound or a pharmaceutically acceptable salt thereof in the preparation of a product for preventing or reducing the production of lipofuscin; the product is a pharmaceutical product or a skin care product; the nitro compound is TBN: ###0001### TBN; the "pharmaceutically acceptable salt" of the nitro compound is a salt formed with an acid selected from the group consisting of hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, pyrosulfuric acid, phosphoric acid, nitric acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, acetic acid, pyruvic acid, trifluoroacetic acid, propionic acid, hexanoic acid, benzoic acid, salicylic acid, cinnamic acid, cyclopentanepropionic acid, dodecylsulfic acid, 2-naphthalenesulfonic acid, citric acid, tartaric acid, stearic acid, lactic acid, oxalic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, D-gluconic acid or aspartic acid. 。 2. Use according to claim 1, characterized in that: The product is for use in a human or an animal.

3. Use according to claim 1 or 2, characterized in that: The pharmaceutical product is for oral administration or parenteral administration; the parenteral administration is rectal administration, transdermal administration, intravenous administration, intramuscular administration, intraperitoneal administration or subcutaneous administration.

4. Use according to claim 1, characterized in that: The dosage form of the pharmaceutical product includes a tablet, a pill, a soft capsule, a hard capsule, a granule, a powder, an eye drop, an injection, a medicinal drop, a lotion, an ointment, a gel, a suspension, an emulsion, a suppository, a patch or a spray.

5. Use according to claim 4, characterized in that: The pill is a micro-pill.

6. Use according to claim 5, characterized in that: The powder is a micro-powder.

7. Use according to claim 5, characterized in that: The ointment is a cream.

8. Use according to claim 5, characterized in that: ​

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