Azithromycin and roxithromycin derivatives as anti-aging drugs

Abstract

The present disclosure describes the use of azithromycin, roxithromycin and telithromycin (including their derivatives) as anti-aging drugs. BrdU was used to induce senescence in a model human fibroblast cell line. A method for screening compounds with anti-aging activity is also disclosed. The SRB assay is used to determine cell viability by protein content. The clinically approved drugs azithromycin, roxithromycin and telithromycin have been found to be anti-aging drugs. However, the closely related parent compound erythromycin does not show anti-aging activity. Azithromycin strongly induces both aerobic glycolysis and autophagy in human fibroblasts, but shows a bidirectional effect, including 50 μM inhibitory activity and 100 μM stimulatory activity on mitochondrial oxygen consumption rate. The xCELLigence real-time assay system showed that azithromycin preferentially targeted senescent cells, removing approximately 97% of senescent cells (a nearly 25-fold reduction in senescent cells).

Description

Technical Field

[0001] The present disclosure relates to senolytic drugs, a class of compounds that selectively induce senescent cell death. Background Art

[0002] As various organisms undergo chronological aging, many genetic, phenotypic, and metabolic defects accumulate. This accumulation includes the onset of senescence in various cell types. The overall view of cumulative defects is consistent with the "accumulated damage" hypothesis of aging.

[0003] Senescence is a clear sign of normal chronological aging. Senescence involves a possible irreversible cell cycle arrest by inducing CDK inhibitors (e.g., p16-INK4A, p19-ARF, p21-WAF, and p27-KIP1) and the initiation of SASP (senescence-associated secretory phenotype) and inducing key lysosomal enzymes (β-galactosidase) and lipofuscin (a known aging pigment). Interestingly, SASP leads to the secretion of large amounts of inflammatory cytokines such as IL-1-β and IL-6, allowing senescent cells to spread the senescence phenotype "infectiously" from one cell type to the whole body through chronic inflammation. This chronic inflammation can also promote the initiation of cancer and drive tumor recurrence and metastasis.

[0004] Using the promoter of p16-IN4KA as a transgenic probe to detect and label senescent cells, several research groups have now established murine models of aging in which senescent cells can be genetically eliminated in a real-time temporal manner. Although this cannot be used as an anti-aging therapy, it can provide us with an indication of whether the removal of senescent cells may have therapeutic benefits for the organism. The results so far show great promise, indicating that genetic removal of senescent cells can indeed extend health span and lifespan.

[0005] Given the results of this genetic data, a large number of pharmaceutical companies are actively working to discover "senolytic" drugs that can target senescent cells. In theory, such anti-aging drugs could produce various excessive effects associated with aging. However, drug development is a long and expensive process that requires a large number of clinical trials and has the risk of failure for one of many possible reasons.

[0006] Therefore, there is a need to identify compounds that are already approved for one or more therapeutic uses that also have anti-aging activity. Summary of the invention

[0007] The present disclosure relates to compounds with anti-aging activity that can be used as anti-aging drugs. Many FDA-approved drugs have anti-aging activity to varying degrees. Identifying such compounds and improving their selectivity for inhibiting senescent cells will greatly accelerate their availability in anti-aging drug trials. The identification of such compounds is described herein, using controlled DNA damage as a tool to induce senescence in human fibroblasts. BrdU treatment (BrdU-treatment), which has a long history of use as a DNA damaging agent, can be used as an effective platform for screening the anti-aging activity of compounds. More specifically, BrdU treatment allows reproducible induction of senescence in cultured cells with high efficiency.

[0008] The inventors used BrdU treatment as a platform for identifying anti-aging activity and identified two macrolide antibiotics from the erythromycin family, particularly azithromycin and roxithromycin, as clinically approved drugs with anti-aging efficacy. The parent macrolide compound (erythromycin itself) did not have anti-aging activity in the screening assay disclosed herein, directly supporting the high specificity of these complex interactions. However, like chemical analogs or derivatives of azithromycin, roxithromycin and telithromycin, telithromycin (another macrolide) also showed anti-aging activity. Note that this specification uses the terms "derivative" and "analog" interchangeably. Screening methods have been used to identify anti-aging agents, such as those described herein. In addition, anti-aging agents can be modified with one or more targeting signals to improve anti-aging activity, and can be used in combination with other therapeutic agents such as mitochondrial biogenesis inhibitors to more effectively eradicate senescent cells.

[0009] This method can take various forms. Some embodiments can take the form of an anti-aging composition, which has a therapeutic amount of at least one anti-aging agent (senolytic agent) selected from azithromycin, roxithromycin, telithromycin, azithromycin analogs, roxithromycin analogs and telithromycin analogs. The disclosure uses the terms "derivative" and "analog" interchangeably, although typical usage may be different. For example, an embodiment can be a pharmaceutically effective amount of a chemical formula selected from one of the following: In such embodiments, R1 and R2 represent functional groups, which may be the same group or different groups, provided that at least one of R1 and R2 is one of a membrane targeting signal and a mitochondrial targeting signal. Otherwise, R1 and R2 are selected from hydrogen, carboxyl, alkane, cyclic alkane, alkane-based derivatives, olefin, cyclic olefin, olefin-based derivatives, alkyne, alkyne-based derivatives, ketone, ketone-based derivatives, aldehyde, aldehyde-based derivatives, carboxylic acid, carboxylic acid-based derivatives, ether, ether-based derivatives, ester, ester-based derivatives, amine, amine-based derivatives, amide, amide-based derivatives, monocyclic aromatic hydrocarbons, polycyclic aromatic hydrocarbons, heteroaromatic hydrocarbons, aromatic hydrocarbon-based derivatives, heteroaromatic hydrocarbon-based derivatives, phenol, phenol-based derivatives, benzoic acid, and benzoic acid-based derivatives. In some embodiments, at least one of R1 and R2 is a membrane targeting signal selected from the following: palmitic acid, stearic acid, myristic acid, oleic acid, short-chain fatty acids, and medium-chain fatty acids. The anti-aging composition of claim 1, wherein at least one of R1 and R2 is a mitochondrial targeting signal selected from the group consisting of triphenylphosphonium (TPP), TPP-derivatives, guanidine salts, guanidine salt derivatives, and 10-N-nonylacridine orange. In some embodiments of the anti-aging composition, at least one of R1 and R2 is a TPP-derivative selected from the group consisting of 2-butene-1,4-bis-TPP; 2-chlorobenzyl-TPP; 3-methylbenzyl-TPP; 2,4-dichlorobenzyl-TPP; 1-naphthylmethyl-TPP; p-xylylenebis-TPP; derivatives of 2-butene-1,4-bis-TPP; derivatives of 2-chlorobenzyl-TPP; derivatives of 3-methylbenzyl-TPP; derivatives of 2,4-dichlorobenzyl-TPP; derivatives of 1-naphthylmethyl-TPP; and derivatives of p-xylylenebis-TPP.

[0010] In some embodiments, the anti-aging compound can be an azithromycin derivative or analog. For example, the anti-aging composition can be a compound having the formula Compounds wherein either R1 is methyl and R2 is one of a membrane targeting signal and a mitochondrial targeting signal, or R1 is one of a membrane targeting signal and a mitochondrial targeting signal and NH-R2 is N(CH3)2. Of course, as described elsewhere herein, the functional groups may be different. Under typical usage of the terms, a single substitution of a base compound (e.g., azithromycin) will be referred to as an analog, and one or more modifications to the base compound may be considered a derivative. For simplicity, the present disclosure uses the terms "analog" and "derivative" interchangeably.

[0011] In some embodiments, the anti-aging compound can be a roxithromycin derivative or analog. For example, the anti-aging composition can be a compound having the formula Compounds wherein either R1 is O-CH2-O-(CH2)2-OCH3 and R2 is one of a membrane targeting signal and a mitochondrial targeting signal, or R1 is one of a membrane targeting signal and a mitochondrial targeting signal and NH-R2 is N(CH3)2. Of course, as described elsewhere herein, the functional groups may be different.

[0012] In some embodiments, the anti-aging compound can be a telithromycin derivative or analog. For example, the anti-aging composition can be a compound having the formula A compound wherein either R1 is and R2 is one of a membrane targeting signal and a mitochondrial targeting signal, or R1 is one of a membrane targeting signal and a mitochondrial targeting signal and NH-R2 is N(CH3)2.

[0013] In a preferred embodiment of the present method, the anti-aging composition has at least one functional group targeting signal to improve mitochondrial uptake and thus enhance the anti-aging activity of the compound. Using the above general formula, at least one of R1 and R2 is either a membrane targeting signal selected from the following: palmitic acid, stearic acid, myristic acid, oleic acid, short-chain fatty acids and medium-chain fatty acids; or a mitochondrial targeting signal selected from the following: 2-butene-1,4-bis-TPP; 2-chlorobenzyl-TPP; 3-methylbenzyl-TPP; 2,4-dichlorobenzyl-TPP; 1-naphthylmethyl-TPP; terephthalene-bis-TPP; derivatives of 2-butene-1,4-bis-TPP; derivatives of 2-chlorobenzyl-TPP; derivatives of 3-methylbenzyl-TPP; derivatives of 2,4-dichlorobenzyl-TPP; derivatives of 1-naphthylmethyl-TPP; and derivatives of terephthalene-bis-TPP.

[0014] In some embodiments, the anti-aging composition may also include one or more additional therapeutic agents. For example, some embodiments may include at least one of a therapeutic amount of tetracycline, chlortetracycline, oxytetracycline, demeclocycline, methacycline, doxycycline, minocycline, and tigecycline. Some embodiments may include at least one of a therapeutic amount of pyrvinium pamoate, atovaquone, bedaquiline, irinotecan, sorafenib, niclosamide, stirpentol, chloroquine, and rapamycin. And some embodiments may include a therapeutic amount of at least one of the following: mitoriboscin, mitoketoscin, mitoflavoscin, 2-butene-1,4-bis-TPP; a derivative of 2-butene-1,4-bis-TPP; 2-chlorobenzyl-TPP; a derivative of 2-chlorobenzyl-TPP; 3-methylbenzyl-TPP; a derivative of 3-methylbenzyl-TPP; 2,4-dichlorobenzyl-TPP; a derivative of 2,4-dichlorobenzyl-TPP; 1-naphthylmethyl-TPP; a derivative of 1-naphthylmethyl-TPP; terephthalenebis-TPP; and a derivative of terephthalenebis-TPP. It should be understood that the TPP derivatives can be conjugated to derivatives or analogs of anti-aging compounds and act as targeting signals and can exist as separate treatment compounds. Some embodiments may include at least one of vitamin C, berberine, caffeic acid phenyl ester, silybin, brutieridin, and melitidin.

[0015] Embodiments in the form of anti-aging compositions can be prepared in various forms, including, for example, cosmetics, pills, lotions, shampoos, creams, soaps, skin cleansers, shaving preparations, aftershave preparations, gels, sticks, pastes, sprays, aerosols, powders, liquids, aqueous suspensions, aqueous solutions, foams, transdermal patches, tinctures and vapors. These forms are intended as non-limiting examples.

[0016] The embodiment of the present method can take the form of a composition for senescence therapy. Such a composition includes a therapeutic amount of an anti-aging agent as described above. For example, the anti-aging agent can be one of an azithromycin analog, a roxithromycin analog, and a telithromycin analog, which has at least one of the following: (i) a membrane targeting signal selected from palmitic acid, stearic acid, myristic acid, oleic acid, short-chain fatty acids, and medium-chain fatty acids; and (ii) a mitochondrial targeting signal selected from triphenylphosphonium (TPP), TPP-derivatives, guanidine salts, guanidine salt derivatives, and 10-N-nonylacridine orange.

[0017] In some embodiments, the composition for aging therapy may also include a therapeutic amount of at least one of tetracycline, chlortetracycline, oxytetracycline, demeclocycline, methacycline, doxycycline, minocycline, and tigecycline. Some embodiments may include a therapeutic amount of at least one of pyralidone pamoate, atovaquone, bedaquiline, irinotecan, sorafenib, niclosamide, stiripentol, chloroquine, and rapamycin. And some embodiments may include a therapeutic amount of at least one of mitoriboscin, mitoketoscin, mitoflavoscin, 2-butene-1,4-bis-TPP; a derivative of 2-butene-1,4-bis-TPP; 2-chlorobenzyl-TPP; a derivative of 2-chlorobenzyl-TPP; 3-methylbenzyl-TPP; a derivative of 3-methylbenzyl-TPP; 2,4-dichlorobenzyl-TPP; a derivative of 2,4-dichlorobenzyl-TPP; 1-naphthylmethyl-TPP; a derivative of 1-naphthylmethyl-TPP; terephthalenebis-TPP; and a derivative of terephthalenebis-TPP. In addition, some embodiments may include at least one of vitamin C, berberine, caffeic acid phenyl ester, silybin, brutieridin, and melitidin.

[0018] The present method can take the form of a method for inducing senescent cell death in a subject. In such an embodiment, a therapeutic amount of an anti-aging agent is administered to the subject. As described above, the anti-aging agent can be at least one of azithromycin, roxithromycin, telithromycin, an azithromycin analog, a roxithromycin analog, and a telithromycin analog. The derivative or analog may include one or more targeting signals, such as a membrane targeting signal and a mitochondrial targeting signal. The method may include administering one or more additional therapeutic agents. For example, at least one of a therapeutic amount of tetracycline, chlortetracycline, oxytetracycline, demeclocycline, methacycline, doxycycline, minocycline, and tigecycline may be administered together with an anti-aging agent. As another example, at least one of a therapeutic amount of pamoate, atovaquone, bedaquiline, irinotecan, sorafenib, niclosamide, stiripentol, chloroquine, and rapamycin may be administered. As another example, a therapeutic amount of at least one of the following substances can be administered: mitoriboscin, mitoketoscin, mitoflavoscin, 2-butene-1,4-bis-TPP; derivatives of 2-butene-1,4-bis-TPP; 2-chlorobenzyl-TPP; derivatives of 2-chlorobenzyl-TPP; 3-methylbenzyl-TPP; derivatives of 3-methylbenzyl-TPP; 2,4-dichlorobenzyl-TPP; derivatives of 2,4-dichlorobenzyl-TPP; 1-naphthylmethyl-TPP; derivatives of 1-naphthylmethyl-TPP; terephthalene bis-TPP; and derivatives of terephthalene bis-TPP. As another example, a therapeutic amount of at least one of vitamin C, berberine, caffeic acid phenyl ester, silybin, brutieridin, and melitidin can be administered. The therapeutic agent can be co-administered with the anti-aging agent, or in some embodiments can be administered before or after the anti-aging agent.

[0019] Some embodiments of the present method can take the form of a method for delaying the onset of age-related diseases in a subject. Age-related diseases can be at least one of atherosclerosis, arthritis, cancer, cardiovascular disease, cataracts, dementia, diabetes, hair loss, hypertension, inflammatory diseases, kidney disease, muscle atrophy, neurological diseases, osteoarthritis, osteoporosis, lung disease, disc degeneration and alopecia. For example, age-related diseases can be neurological diseases, such as mild cognitive impairment, motor neuron dysfunction, Alzheimer's disease, Parkinson's disease and macular degeneration. In such embodiments, an anti-aging agent of a therapeutic amount as described herein can be administered to the subject. In some embodiments, an anti-aging agent can be administered together with another therapeutic agent as described herein. The anti-aging agent can be administered at the time of onset, that is, immediately after or when the age-related disease is diagnosed. Alternatively, the anti-aging agent can be routinely administered after diagnosis, and the frequency and dosage can be determined using techniques known in the art. In some embodiments, the anti-aging agent can be administered before onset, particularly in the case where an age-related disease is expected or may occur in the subject (e.g., due to genetic markers or other biological markers).

[0020] The present method can also take the form of a composition for delaying the onset of age-related diseases in a subject. For example, but not limited to, the present method can be used to delay atherosclerosis, arthritis, cancer, cardiovascular disease, cataract, dementia, diabetes, alopecia, hypertension, inflammatory diseases, nephropathy, muscular atrophy, neurological disease, osteoarthritis, osteoporosis, lung disease, intervertebral disc degeneration and alopecia. In such embodiments, as described herein, a therapeutic amount of an anti-aging agent can be administered, comprising at least one of azithromycin, roxithromycin, telithromycin, an azithromycin analog, a roxithromycin analog and a telithromycin analog. In some embodiments, the anti-aging agent can be administered together with another therapeutic agent as described herein.

[0021] The present method can also take the form of a method for screening the anti-aging activity of a compound. In such an embodiment, a cell population is exposed to a DNA damaging agent to produce a senescent cell population in a first time period. An exemplary DNA damaging agent is bromodeoxyuridine (BrdU), but other DNA damaging agents can be used without departing from the method of the present invention. At least a portion of the senescent cell population is treated with a candidate compound in a second time period to form a treated cell population. The candidate compound is a compound screened for anti-aging activity. At least one anti-aging activity marker of the treated cell population is analyzed. Example markers of anti-aging activity include cell viability, aerobic glycolysis, autophagy, inhibitory activity and reduced cell proliferation. For example, as described herein, autophagy LC3 protein can be quantitatively measured. The determination of sulforhodamine B and the measurement of cell-induced electrical impedance are examples of determinations that can be used to analyze the anti-aging activity of a cell population. The first time period can vary, and is about 8 days in the embodiment described below. The second time period can also vary, and is about 3 to about 5 days in the embodiment discussed below. In some embodiments, BrdU can be washed out before the second time period.

[0022] Other embodiments of the present methods may be recognized by one of ordinary skill in the art upon reviewing the following specific embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 A method for screening and identifying anti-aging drugs according to an embodiment of the present method is shown.

[0024] Figure 2 Shown are the results of reduced DNA synthesis in MRC-5 fibroblasts.

[0025] Figure 3A Shown are the results of an SRB assay of azithromycin in MRC-5 fibroblasts. Figure 3B and 3C Images of MRC-5 fibroblasts treated with 100 μM azithromycin without and with BrdU pretreatment are shown, respectively.

[0026] Figure 4 Shown are the results of the SRB assay for roxithromycin in MRC-5 fibroblasts.

[0027] Figure 5 Shown are the results of autophagy induction by 50 μM azithromycin in MRC-5 fibroblasts.

[0028] Fig. 6A and 6BShown are the results of extracellular acidification rate from metabolic flux analysis of MRC-5 cells treated with azithromycin for 72 hours.

[0029] Fig. 7A and 7B Shown are the oxygen consumption rate results of metabolic flux analysis of MRC-5 cells treated with azithromycin for 72 hours.

[0030] Figure 8 Shown is the anti-aging activity in senescent human skin cells pretreated with BrdU for 8 days to induce senescence and then exposed to azithromycin for 5 days.

[0031] Fig.9A Shown are representative cell tracks from xCELLigence real-time cell health monitoring for the MRC-5 cell line, comparing BrdU pre-treatment, BrdU pre-treatment plus azithromycin, azithromycin alone, and control. Fig. 9B Final Cell Index results for the same cell lines are summarized.

[0032] Fig. 10A and Fig. 10B Final Cell Index results for the MRC-5 cell line and a representative cell track from the xCELLigence real-time cell health monitor are shown, comparing BrdU pretreatment, BrdU pretreatment plus roxithromycin, roxithromycin alone, and control.

[0033] Fig.11A and 11B Final Cell Index results for the MRC-5 cell line and a representative cell track from the xCELLigence real-time cell health monitor are shown, comparing BrdU pretreatment, BrdU pretreatment plus telithromycin, telithromycin alone, and control. DETAILED DESCRIPTION

[0034] The following description includes currently contemplated modes of carrying out exemplary embodiments of the present method.The following description is not limiting and is merely intended to illustrate the general principles of the invention.

[0035] As described herein, the present methods relate to the development of screening assays for inducing senescence and their use in identifying compounds with anti-aging activity (i.e., selective inhibition of senescent cells and induction of senescent cell death). In some embodiments, the present methods can be used to identify and repurpose clinically approved therapeutic agents with anti-aging activity to treat aging and aging-related disorders. Figure 1The screening methodology according to the present method is shown. In step S101, normal fibroblasts are selected. Next, in step S102, the cells are exposed to DNA damaging agents to induce senescence. For example, bromodeoxyuridine can be used as a damaging agent. In step S103, the senescent cells are treated with candidate treatment compounds. Of course, multiple partial senescent cells can be used to screen multiple candidate treatment compounds, and changes in compound concentrations and treatment time periods can be included in this step. Finally, in step S104, the treated cells are analyzed for anti-aging activity markers. Normal cells and / or untreated senescent cells as controls can also be analyzed. The anti-aging activity markers used can vary and can include, for example, cell viability, aerobic glycolysis, autophagy, inhibitory activity, and reduced cell proliferation.

[0036] Bromodeoxyuridine (5-bromo-2'-methyl-deoxyuridine, also known as BrdU) can be used to induce senescence. BrdU is an analog of thymidine commonly used to identify proliferating cells. BrdU induces controlled DNA damage and drives cell senescence with high efficiency. The BrdU method of the present method requires that normal fibroblasts be cultured for a long time (8 days) in the presence of 100 μM BrdU to induce controlled DNA damage and senescence. In an illustrative embodiment, the inventors used two independent normal non-immortalized human fibroblast cell lines, MRC-5 lung cells (for screening) and BJ skin cells (for verification) in a BrdU-based assay. Then, isogenic matching cultures of normal and senescent fibroblasts can be used for drug screening to identify drugs with anti-aging activity. Anti-aging activity can be detected using the sulforhodamine B assay (also known as the SRB assay in the art). This assay measures the amount of protein residues attached to tissue culture dishes and is a surrogate marker for cell viability. The method can be used to rapidly screen compounds, including clinically approved drugs such as antibiotics. For example, in the embodiments described herein, the method is used to screen members of the erythromycin family, including compounds such as azithromycin and roxithromycin. It should be understood that the method can be used to screen other compounds.

[0037] Mechanistically, the present method directly compares the responses of "normal" fibroblasts and "senescent" fibroblasts in parallel. Drugs that preferentially kill senescent fibroblasts rather than normal fibroblasts can be considered to be positive for anti-aging activity. Using this method in the embodiments discussed herein, the inventors identified two erythromycin family members, azithromycin and roxithromycin, that preferentially target senescent fibroblasts. Table 1 below shows the results of two concentrations of erythromycin, azithromycin and roxithromycin, and shows that azithromycin and roxithromycin have anti-aging activity at 100 μM. Telithromycin (another macrolide) and chemical analogs of azithromycin, roxithromycin and telithromycin also showed beneficial anti-aging activity. However, erythromycin itself did not show any anti-aging activity.

[0038] Viability of senescent MRC-5 cells (percentage relative to control)

[0039]

[0040] Table 1. Results of screening for anti-aging activity of erythromycin family antibiotics

[0041] The exact chemical structures of some of the erythromycin family members screened in the disclosed embodiments are shown below as compounds I-IV. Note that the structures of the compounds in the erythromycin family have significant similarities, but anti-aging activity is only present in azithromycin, roxithromycin, and telithromycin.

[0042]

[0043] Erythromycin

[0044]

[0045] Azithromycin

[0046]

[0047] Roxithromycin

[0048]

[0049] Telithromycin

[0050] Furthermore, the present inventors demonstrated that DNA damage induced by the use of BrdU is sufficient to induce cellular senescence. Figure 2Results showing reduced DNA synthesis in MRC-5 fibroblasts. Treatment with BrdU for 2 days significantly reduced DNA synthesis in MRC-5 fibroblasts by about 70%, as measured using the Muse cell cycle kit. MRC-5 cells were positively stained for β-galactosidase (a biomarker of cellular aging) after 8 days of BrdU treatment. For this data, n=3; and * indicates p<0.05. Figure 2 The inset in Figure 4 shows β-galactosidase positivity in cells treated with BrdU. The results demonstrated that cells treated with BrdU underwent cell cycle arrest, as evidenced by a decrease of approximately 70% in the number of cells in the S phase and the induction of β-galactosidase activity. These results confirm that BrdU treatment of MRC-5 cells effectively inhibits DNA synthesis and induces β-galactosidase, both hallmarks of senescence.

[0051] In one embodiment, azithromycin exhibits anti-aging activity in senescent MRC-5 human lung fibroblasts. MRC-5 cells were pretreated with BrdU for 8 days (to induce senescence), then BrdU was washed out and exposed to azithromycin for another 5 days. Thereafter, an SRB assay was performed to determine the effect of the drug on cell viability. Figure 3A The results of SRB assays of BrdU-treated MRC-5 fibroblasts treated with azithromycin at concentrations of 100 μM and 50 μM relative to controls (BrdU only) are shown. 100 μM azithromycin had no effect on the viability of normal MRC-5 lung fibroblasts, but selectively killed senescent MRC-5 fibroblasts. After 5 days, this concentration of azithromycin effectively and selectively eliminated approximately 50% of senescent cells without affecting control cells. However, 50 μM azithromycin had no effect. These experiments were repeated at least 3 times independently, and the results were very similar. For this data, ** indicates p<0.01. Figure 3B and 3C Images of MRC-5 fibroblasts treated with 100 μM azithromycin without and with BrdU pretreatment. These images show that azithromycin has little effect on normal cells but induces cell death in senescent cells. Figure 3B and Figure 3C The upper right scale bar indicates 20 μm.

[0052] In contrast, the same concentration of roxithromycin can kill senescent MRC-5 fibroblasts more effectively (about 70%), but has little effect on the viability of normal MRC-5 fibroblasts. In one embodiment, MRC-5 cells are pretreated with BrdU for 8 days (to induce senescence), then the BrdU is washed out and exposed to roxithromycin for another 5 days. After 5 days of exposure, SRB assays are performed to determine the effect of the drug on cell viability. Figure 4Results of SRB assays of MRC-5 fibroblasts treated with roxithromycin are shown. The data show that roxithromycin at a concentration of 100 μM has a potent and selective effect on MRC-5 cells as it eliminates more than 50% of senescent cells after 5 days. However, roxithromycin at a concentration of 50 μM had no effect. These experiments were repeated independently at least 3 times with very similar results. Figure 4 In the table, * indicates p < 0.05.

[0053] Figure 3A and 4 The data in show that 50 μM azithromycin and roxithromycin have no significant effect on the viability of senescent cells. This indicates that the anti-aging effect is concentration-dependent. It should be understood that a person with at least ordinary skills in the art can determine the appropriate concentration of a drug with anti-aging activity using methods known and available in the art. Based on the concentrations tested, azithromycin toxicity showed the highest specificity for selectively targeting the senescent cell phenotype.

[0054] Further experiments were performed using MRC-5 fibroblasts to better understand the phenotypic and metabolic effects of azithromycin mechanistically. MRC-5 cells were treated with 50 μM azithromycin for 72 hours. Autophagy was then monitored by detecting the autophagic LC3 protein using the Muse Autophagy LC3-antibody based kit. Figure 5 The results are presented and demonstrate that azithromycin is a potent inducer of the autophagic phenotype. Figure 5 ** in the figure indicates p < 0.01; as indicated, azithromycin treatment resulted in a more than 3-fold increase in autophagy in MRC-5 cells.

[0055] The inventors next measured the effects of azithromycin on aerobic glycolysis and mitochondrial metabolism using a Seahorse XFe96 metabolic flux analyzer.After treatment with 25 μM to 100 μM azithromycin for 72 hours, metabolic flux analysis was performed on MRC-5 cells using Seahorse XFe96 to measure the extracellular acidification rate (ECAR). Fig. 6A and 6B The ECAR data from this metabolic flux analysis are shown, and it can be seen that azithromycin enhances aerobic glycolysis at all concentrations tested. Fig. 6A At 40 minutes, the data from top to bottom represent the concentrations of azithromycin 100 μM, 50 μM, 25 μM and control (i.e., only solvent). Figure 6B In the figure, the column data represent control, 100 μM, 50 μM and 25 μM from left to right. For this data, n=3; ** represents p<0.01, and *** represents p<0.001.

[0056] The inventors also used metabolic flux analysis to evaluate the effect of azithromycin on oxygen consumption rate (OCR). The results are shown in Fig. 7A and 7B , indicating that azithromycin has a biphasic effect on OCR in MRC-5 cells. These data were generated by metabolic flux analysis of MRC-5 cells using Seahorse XFe96 after 72 hours of treatment with 25 μM to 100 μM azithromycin. At 41 minutes, Fig. 7A The data in each line are from top to bottom for control (i.e., vehicle only) and concentrations of 25 μM, 50 μM, and 100 μM. Note that the highest concentration (100 μM) of azithromycin triggered an increase in mitochondrial respiration for 54 minutes, while the lower concentration (50 μM) significantly reduced mitochondrial respiration. However, 25 μM did not have any significant effect on OCR. For this data, n=3, and * indicates p<0.05.

[0057] Figure 7B OCRs for basal respiration, proton leak, ATP-link respiration, maximal respiration, and spare respiratory capacity are shown. The bar data represent control and concentrations of 100 μM, 50 μM, and 25 μM from left to right. Note that the mitochondrial effects of azithromycin are concentration-dependent and biphasic. At 25 μM, azithromycin did not show a significant effect on OCR. However, at 50 μM, azithromycin significantly inhibited mitochondrial metabolism, particularly affecting maximal respiration and spare respiration. In contrast, at 100 μM, azithromycin stimulated maximal respiration and more than twice the spare respiration. This may represent a cellular compensatory response to azithromycin treatment to overcome its mitochondrial inhibitory effects.

[0058] The selectivity and potency of azithromycin were verified using normal, non-immortalized BJ human fibroblasts. BJ cells were pretreated with BrdU for 8 days to induce senescence, followed by washing out the BrdU and exposure to azithromycin for 5 days. Thereafter, SRB assays were performed to determine the effect of azithromycin on cell viability. Azithromycin had a potent and selective effect on BJ cells, as it eliminated more than 50% of senescent cells after 5 days at 50 μM without reducing the viability of control cells. These experiments were repeated at least 3 times independently, and the results were very similar. The results are shown in Figure 8 . Note that Figure 8 In the table, ** indicates p<0.01. Figure 8As shown, azithromycin is more effective in BJ skin fibroblasts, showing significant anti-aging activity at only 50 μM. Azithromycin also increased the viability of normal BJ skin fibroblasts by more than 25%. Therefore, azithromycin shows equivalent selectivity and anti-aging activity in human fibroblasts derived from two different anatomical sites (lung tissue and skin).

[0059] Screened compounds Test cells Test concentration Doxycycline BJ 25–200 μM Diphenylene iodonium chloride BJ 0.5–10 μM Melatonin BJ 200–400 μM Aspartame BJ 200–400 μM Glucosamine BJ 10–15 μM Quercetin BJ 10–100 μM Dasatinib BJ 0.1–1 μM Chloroquine BJ, MRC-5 100–200 μM Erythromycin BJ, MRC-5 100–200 μM Clarithromycin BJ, MRC-5 100–200 μM Rapamycin MRC-5 50–500 μM Lycopene MRC-5 25–50 μM Alpha-lipoic acid MRC-5 25–50 μM

[0060] Table 2. Anti-aging activity of other compounds tested by this method

[0061] Using this anti-aging assay system using MRC-5 or BJ fibroblasts, the present method was used to test a number of other drug candidates. These compounds are listed in Table 2 above, which also identifies the cell lines used and the compound concentration ranges used for testing. Unfortunately, none of these other drug candidates showed any specific anti-aging activity while retaining their normal fibroblast counterparts.

[0062] Although the erythromycin family as a whole does not show anti-aging activity, azithromycin, roxithromycin and telithromycin show specific and selective anti-aging activity. It should be understood that many chemical analogs and derivatives of azithromycin, roxithromycin and telithromycin also have anti-aging activity. Traditionally, an "analog" has only a single element different from the parent compound, while a "derivative" is a chemical derived from another compound or synthesized by another compound. In the present disclosure, these terms are used interchangeably, although many disclosed derivatives may also be referred to as analogs under the common usage of the term. For example, a derivative in which a substitution is made to connect a targeting signal portion can be considered an analog. It should be understood that derivatives with targeting signals (such as fatty acid membrane targeting signals or TPP-derivative mitochondrial targeting signals) have increased mitochondrial uptake and therefore have increased efficacy. This effect is particularly evident in cells that rely heavily on mitochondrial biogenesis (such as cancer stem cells and senescent cells). The following compounds V-VII are general structural formulas, showing the functional group positions of azithromycin, roxithromycin and telithromycin analogs, respectively.

[0063]

[0064] Azithromycin analogs

[0065]

[0066] Roxithromycin analogs

[0067]

[0068] Telithromycin analogs

[0069] Each of the general structures V to VII has a possible conjugation or substitution position, represented by a numbered "R" and referred to as an R group. Each R group can be selected from: hydrogen, carbon, nitrogen, sulfur, oxygen, fluorine, chlorine, bromine, iodine, carboxyl, alkane, cycloalkane, alkane-based derivatives, olefins, cyclic olefins, olefin-based derivatives, alkynes, alkyne-based derivatives, ketones, ketone-based derivatives, aldehydes, aldehyde-based derivatives, carboxylic acids, carboxylic acid-based derivatives, ethers, ether-based derivatives, esters, ester-based derivatives, amines, amine-based derivatives, amides, amide-based derivatives, monocyclic or polycyclic aromatic hydrocarbons, heteroaromatic hydrocarbons, aromatic hydrocarbon-based derivatives, heteroaromatic hydrocarbon-based derivatives, phenols, phenol-based derivatives, benzoic acid, benzoic acid-based derivatives, membrane targeting signals, and mitochondrial targeting signals. It should be understood that reference to a single atom (e.g., carbon or nitrogen) still requires that the valence be satisfied (e.g., one or more additional bonds or hydrogen atoms may be required). The term "derivative" as used herein refers to a compound derived from a similar compound by a chemical reaction. One or more R groups of the derived anti-aging compound can be replaced by membrane targeting signals and / or mitochondrial targeting signals to enhance the selectivity and effectiveness of the compound for senescent cells. Membrane targeting signals include, for example, palmitic acid, stearic acid, myristic acid, oleic acid, short-chain fatty acids, and medium-chain fatty acids. The resulting conjugate can be synthesized according to techniques known in the art, such as lipidation reactions (e.g., myristoylation, palmitoylation, etc.), to achieve chemical modification of fatty acids. Mitochondrial targeting signals include, for example, lipophilic cations, such as triphenylphosphonium (TPP), TPP- derivatives, guanidine salts, guanidine salt derivatives, and 10-N-nonyl acridine orange. Non-exhaustive examples of TPP-derivatives include 2-butene-1,4-bis-TPP; 2-chlorobenzyl-TPP; 3-methylbenzyl-TPP; 2,4-dichlorobenzyl-TPP; 1-naphthylmethyl-TPP; terephthalene-bis-TPP; derivatives of 2-butene-1,4-bis-TPP; derivatives of 2-chlorobenzyl-TPP; derivatives of 3-methylbenzyl-TPP; derivatives of 2,4-dichlorobenzyl-TPP; derivatives of 1-naphthylmethyl-TPP; and derivatives of terephthalene-bis-TPP. Derivatives can be synthesized using techniques known in the art. It should be understood that these are non-exhaustive lists provided as examples.

[0070] In a further application of this method, the selectivity of azithromycin for senescent cells was verified using the xCELLigence system. Because senescent cells undergo the so-called senescence-associated secretory phenotype (SASP), which involves a significant increase in the synthesis and secretion of proteins, the inventors evaluated whether the protein measurement assay system underestimated the anti-aging activity of the test compound. The xCELLigence assay system does not rely on proteins, but uses electrical impedance to continuously measure cell proliferation in real time.

[0071] Fig.9A and 9B Representative data from the xCELLigence assay for azithromycin are shown. Fig.9A Representative cell tracking in shows that senescent cells (BrdU-treated / MRC-5 fibroblasts) are effectively killed. At the 308.44 hour mark, and from top to bottom, the curve represents cells treated with BrdU only, cells treated with azithromycin only, controls, and cells treated with BrdU and azithromycin. The normalized cell index of the control is significantly higher immediately after treatment and is consistent with the BrdU curve only until about 235 hours. Fig. 9B A bar graph is shown highlighting the final cell index, expressed as mean ± standard error of the mean. As can be seen, azithromycin targets approximately 97% of senescent MRC-5 cells. In contrast, normal control MRC-5 cells were only transiently affected by azithromycin and recovered rapidly through cell proliferation. The cell lines treated with azithromycin showed a recovery that was only greater than 30% above the level of vehicle control cells alone. This data confirms that azithromycin preferentially targets senescent cells and is highly effective in removing approximately 97% of senescent cells (a reduction of nearly 25-fold). For Fig.9A and 9B Data shown in , **** indicates p < 0.001. Thus, the real-time xCELLigence assay system complements the more static SRB assay and allows for more direct visualization of possible anti-aging effects of compounds during drug screening.

[0072] The xCELLigence assay was also used to confirm the anti-aging activity of roxithromycin and telithromycin. Fig. 10A Final cell index is shown for control, roxithromycin-treated control, BrdU-treated control, and BrdU-treated fibroblasts subjected to 90 μM roxithromycin, expressed as mean ± standard error of the mean. These assays were performed using MRC-5 fibroblasts. Roxithromycin treatment had minimal effect on the viability of normal fibroblasts compared to controls, but targeted 82% of fibroblasts treated with BrdU to induce senescence. Senescent MRC-5 cells were pretreated with BrdU for 8 days to induce senescence, then the BrdU was washed out and exposed to roxithromycin for another 5 days. These experiments were repeated at least 3 times independently and the results were very similar. Note that in Fig. 10A In the table, * indicates p < 0.01. Representative data of the xCELLigence assay for roxithromycin are shown in Fig. 10BAt time 249 hours, from top to bottom, the lines represent control, BrdU only, control and roxithromycin (90 μM), and BrdU and roxithromycin. It can be seen that roxithromycin causes a sustained decrease in the cell index for senescent fibroblasts. These data confirm that roxithromycin has a strong anti-aging activity.

[0073] The same method was used to confirm the anti-aging activity of telithromycin. Fig.11A The final cell index is highlighted and is presented as the mean ± standard error of the mean, and Fig. 11B Representative xCELLigence data are shown. The concentration of telithromycin treatment was 90 μM. Fig.11A Telithromycin targets 100% of senescent cells and has negligible effects on normal MRC-5 cells. Fig. 11B Control, control and telithromycin treatment, control and BrdU, and BrdU and telithromycin treatment are shown. These data demonstrate that telithromycin has a strong anti-aging activity.

[0074] Therefore, the present method provides an anti-aging screening methodology to systematically identify compounds that target the senescent phenotype of human fibroblasts. As shown in the data disclosed above and the accompanying drawings, the present method can be used to screen clinically approved drugs, and it should be understood that the scope is not limited and can be used to screen other compounds. Results were generated using MRC-5 and / or BJ cells (two mature non-immortalized human fibroblast cell lines). It should be understood that other human cells can be used as long as the induction of senescence is confirmed. To induce cell cycle arrest and senescence, fibroblasts were exposed to 100 μM concentration of BrdU (DNA damaging agent) for 8 days. It should be understood that other DNA damaging agents can be used without deviating from the present method. Concentrations and exposure times can vary, but it should be understood that senescence induction should be verified. After BrdU treatment and washing, fibroblasts were exposed to test drugs or compounds. In the above results, the test compound was exposed for 5 days. However, the time and concentration of the test compound can vary. After drug treatment, cell attachment can be assessed using a microplate reader using the SRB assay system, allowing high-throughput analysis. The above-mentioned xCELLigence assay can also be used.

[0075] Using the screening methodology of the present method, the inventors confirmed that three clinically approved macrolide antibodies, azithromycin, roxithromycin and telithromycin, have highly selective anti-aging activity against senescent cells. In contrast, erythromycin, a parent compound generally considered to be chemically similar, did not show any toxicity to senescent fibroblasts. Metabolic analysis of the chemical effects of azithromycin showed that it induced the initiation of autophagy and glycolysis. In addition, azithromycin increased mitochondrial activity at high doses (100 μM), but had the opposite effect at lower doses (50 μM), indicating a clear bidirectional effect. These metabolic effects can give azithromycin highly specific anti-aging activity.

[0076] In summary, the present method provides a screening method to identify compounds with anti-aging activity, such as pre-existing clinically approved antibiotics and other drugs. The present method can be used for drug reuse, for example, as an anti-aging drug that can be used to target aging fibroblasts. The present method is used to demonstrate the selective anti-aging activity of azithromycin, roxithromycin and telithromycin. Chemical analogs of these compounds, such as those that can be formed using the general chemical formula above, also have anti-aging activity. In particular, derivatives that include the addition of membrane targeting signals and / or mitochondrial targeting signals have enhanced anti-aging effects.

[0077] Therefore, the present method can take the form of a method for screening the anti-aging activity of a compound. A cell population can be exposed to bromodeoxyuridine (BrdU) to produce or induce senescence in the cell population. In some embodiments, for example, as described above, it can be exposed to a BrdU concentration of 100 μM for about 8 days, although a person of ordinary skill in the art can determine the appropriate exposure time based on the cell population and the BrdU concentration. A senescent cell population can be treated with a candidate compound to produce a treated cell population. For some embodiments, the treatment time can be about 3 to 5 days, but can vary. In some embodiments, a portion of a senescent cell population can be treated, an untreated control portion is allowed, and, if desired, multiple portions are allowed to be treated with different treatment compounds at different compound concentrations and / or at different treatment times. BrdU can be washed off before treatment. It should be understood that treatment compounds, concentrations, and treatment times can vary. After treatment, indicators or markers of the anti-aging activity of the cell population can be analyzed. For example, assays described herein and / or known in the art can be used to analyze cell viability, aerobic glycolysis, autophagy, inhibitory activity, quantitative measurement of autophagic LC3 proteins, and reduced cell proliferation of the cell population. As another example, one or both of the sulforhodamine B assay and measuring cell-induced electrical impedance can be used to analyze anti-aging activity. It should be understood that one of ordinary skill in the art can assess the anti-aging activity of treatment compounds using assays other than those described herein.

[0078] The method of the present invention can also take the form of an anti-aging composition having a therapeutic amount of at least one anti-aging agent. The anti-aging agent can be azithromycin, roxithromycin, telithromycin, azithromycin analogs, roxithromycin analogs and telithromycin analogs. It should be understood that the therapeutic amount can be determined by a person of ordinary skill in the art using the present disclosure and methods known and available in the art. In some embodiments, the anti-aging agent can be replaced by at least one targeting signal, which can be (i) a membrane targeting signal selected from palmitic acid, stearic acid, myristic acid, oleic acid, short-chain fatty acids and medium-chain fatty acids; or (ii) a mitochondrial targeting signal selected from triphenylphosphonium (TPP), TPP-derivatives, guanidine salts, guanidine salt derivatives and 10-N-nonyl acridine orange. Targeting signal substitution enhances anti-aging activity by increasing the mitochondrial uptake of the anti-aging agent. For example, the R group shown in any of the above compounds IV-VI can include a targeting signal. In some embodiments, the targeting signal can be a TPP derivative connected to the anti-aging compound at one of the substitution positions. Examples of TPP-derivatives include, but are not limited to, 2-butene-1,4-bis-TPP; 2-chlorobenzyl-TPP; 3-methylbenzyl-TPP; 2,4-dichlorobenzyl-TPP; 1-naphthylmethyl-TPP; p-xylylenebis-TPP; derivatives of 2-butene-1,4-bis-TPP; derivatives of 2-chlorobenzyl-TPP; derivatives of 3-methylbenzyl-TPP; derivatives of 2,4-dichlorobenzyl-TPP; derivatives of 1-naphthylmethyl-TPP; and derivatives of p-xylylenebis-TPP. It should be understood that substitution with a TPP-derivative may require covalent bonding.

[0079] In some embodiments, the anti-aging agent may include tetracycline compounds, including tetracycline, chlortetracycline, oxytetracycline, demeclocycline, methacycline, doxycycline, minocycline and tigecycline. These mitochondrial biogenesis inhibitors can be used to inhibit oxidative metabolism and further enhance anti-aging activity. Data and further examples are in the international application PCT / US2018 / 028587 filed on April 20, 2018, which is incorporated herein by reference in its entirety. In some embodiments, the anti-aging agent may include a glycolysis inhibitor, such as pamoate, atovaquone, bedaquiline, irinotecan, sorafenib, niclosamide, stiripentol, chloroquine and rapamycin. As described above, senescent cells treated with anti-aging agents are converted to a glycolytic phenotype. The introduction of glycolysis inhibitors causes these cells to lose functional metabolic pathways, thereby enhancing the induction of cell death in senescent cell populations.

[0080] Under the present method, many mitochondrial biogenesis inhibitors can be used in combination with anti-aging agents. Other examples of mitochondrial inhibitors include: mitoriboscin, oxidative metabolism inhibitors and glycolytic metabolism inhibitors, repurposcin, antimitoscin, mitoketoscin, mitoflavoscin, mitoflavin, TPP- derivatives, MDIVI-1 derivatives, chloramphenicol, puromycin and other inhibitors of protein synthesis (including, for example, aminoglycosides and rapamycin analogs), antiparasitic drugs (for example, pamoate promethazine and niclosamide), chloroquine, stiripentol, caffeic acid phenyl ester (CAPE), vitamin C, 2-deoxy-glucose (2-DG), MCT1 inhibitors (AZD3965 and AR-C155858), D-glucosamine, quercetin and carvedilol. The following paragraphs describe certain classes of mitochondrial biogenesis inhibitor therapeutic agents. For the sake of brevity, the related co-pending applications are incorporated by reference as if fully described herein.

[0081] The first class of mitochondrial biogenesis inhibitors is as described in International Application No. PCT / US2018 / 022403 filed on March 14, 2018, and is incorporated by reference in its entirety. The cited references include data for selecting mitoriboscin compounds. Typically, mitoriboscin is a mitochondrial inhibitor compound that has anticancer activity and generally has antimicrobial activity, chemotherapy sensitization, radiosensitization, and photosensitization, as well as anti-aging effects. These compounds bind to the large or small subunits of the mitochondrial ribosome (or in some cases, both bind) and inhibit mitochondrial biogenesis. Examples of mitoriboscin groups as well as general chemical structures and specific compounds are described in the incorporated applications, and include mitoribocycline, mitoribomycin, mitoribosporin, and mitoribofloxin. Illustrative mitoriboscins are shown below as compounds VIII-XVII.

[0082]

[0083] Mitoriboscin compound 23 / E9

[0084]

[0085] Mitoriboscin compound 23 / G4

[0086]

[0087] Mitoriboscin compound 24 / B10

[0088]

[0089] Mitoriboscin compound 24 / D4

[0090]

[0091] Mitoriboscin compound 24 / F9

[0092]

[0093] Mitoriboscin compound 24 / H6

[0094]

[0095] Mitoriboscin compound 24 / H9

[0096]

[0097] Compound 25 / B3

[0098]

[0099] Mitoriboscin compound 25 / E3

[0100]

[0101] Mitoriboscin compound 26 / H4

[0102] Mitoketoscin is another class of mitochondrial biogenesis inhibitors that can be used to enhance anti-aging activity. These are non-carcinogenic compounds that bind to at least one of ACAT1 / 2 and OXCT1 / 2 and inhibit mitochondrial ATP production. These compounds are more fully described in International Application PCT / US2018 / 039354 filed on June 25, 2018, and are incorporated herein by reference in their entirety. In general, mitoketoscin targets mitochondrial enzymes that are responsible for ketone recycling and have anticancer and antibiotic properties. These compounds bind to one or both active catalytic sites in OXCT1 / 2 and ACAT1 / 2 to inhibit mitochondrial function.

[0103] Repurposcin and antimitoscin are a third class of mitochondrial biogenesis inhibitors that can be used in conjunction with the present method. International patent application PCT / US2018 / 062956, filed on November 29, 2018 and incorporated herein by reference in its entirety, more fully describes repurposcin. Generally, a "repurposcin" is a compound with inherent anti-mitochondrial properties that has been chemically modified to target the compound to mitochondria. Antimitoscin is a class of repurposcins, more fully described in International patent application PCT / US2018 / 033466, filed on May 18, 2018, the entire contents of which are incorporated herein by reference. Existing antibiotics with inherent anti-mitochondrial properties can be chemically modified to target mitochondria and inhibit mitochondrial biogenesis. The term "antimitoscin" refers generally to antibiotics with inherent anti-mitochondrial properties that have been chemically modified to target the antibiotic to mitochondria. Previously, inherent anti-mitochondrial activity in antibiotics was considered a harmful side effect. In fact, due to excessive anti-mitochondrial properties, some potential antibiotics have been excluded from the test, and researchers have regarded anti-mitochondrial activity as a potential disadvantage. However, under this method, the inherent anti-mitochondrial activity of antibiotics can become the basis of a completely new treatment. Antimitochondrial antibiotics (antimitoscin) can be an antibiotic with inherent anti-mitochondrial properties, which has a chemical modification of a mitochondrial targeting signal (e.g., a chemical moiety). Chemical modification can be, for example, by covalent or non-covalent bonds. In some embodiments, the antibiotic is one of the members of the tetracycline family, the erythromycin family, chloramphenicol, pamoate, atovaquone and bedaquiline. The mitochondrial targeting signal can be at least one compound or part selected from the group including a membrane targeting signal and a mitochondrial ribosome targeting signal. Examples of membrane targeting signals include short-chain (e.g., less than 6 carbon atoms in the chain) fatty acids and medium-chain (e.g., 6-12 carbon atoms in the chain) fatty acids, palmitic acid, stearic acid, myristic acid and oleic acid. Examples of mitochondrial targeting signals include triphenylphosphonium (TPP)-based and guanidine-based parts. TPP and guanidine salts are non-toxic chemical moieties that functionally behave as mitochondrial targeting signals (MTS) in living cells. Alternatively, they can be conjugated to antibiotics, usually by using carbon spacer arms or linker chains.

[0104] Mitoflavoscin and mitoflavin are fourth class mitochondrial biogenesis inhibitors that can be used in the present method. These compounds are more fully described in International Patent Application PCT / US2018 / 057093 filed on October 23, 2018, and are incorporated herein by reference in their entirety. Mitoflavoscin is a compound that binds to an enzyme containing flavin and inhibits mitochondrial ATP production. Diphenylene iodonium chloride (DPI) is an example of mitoflavoscin. It should be understood that mitoflavoscin can be modified with a mitochondrial targeting signal, such as discussed above for antimitoscin. Mitoflavin is a riboflavin derivative that inhibits mitochondrial function and can also be chemically modified with a mitochondrial targeting signal. For example, rose yellow pigment [8-demethyl-8-(dimethylamino)-riboflavin or 8-dimethylaminoriboflavin)] is a naturally occurring antibacterial compound that is a riboflavin derivative that can be chemically modified to optimize its potential to target CSCs and inhibit mitochondrial biogenesis. Photochrome (7,8-dimethylalloxazine) is a fluorescent photoproduct of riboflavin degradation, which can also be chemically modified to optimize its potential to target CSCs. Other common derivatives of riboflavin include: Alloxazine, Lumiflavine, 1,5-dihydroriboflavin, and 1,5-dihydroflavin. Each of these riboflavin derivatives can be chemically modified with a mitochondrial targeting signal to form mitoflavin and can be used as a mitochondrial biogenesis inhibitor according to the present method.

[0105] The sixth class of mitochondrial biogenesis inhibitors is TPP-derived compounds, which not only show a strong preference for uptake in cancer cells (bulk cancer cells, cancer stem cells and high-energy cancer stem cells), but also destroy mitochondrial biogenesis in these cells. These TPP-derived compounds are more fully described in the international patent application PCT / US2018 / 062174 filed on November 21, 2018, and are incorporated herein by reference in their entirety. As used with respect to TPP-derivatives, derivatives known in the art are compounds that can be synthesized from a parent compound by replacing one atom with another atom or atom group. For example, a derivative of TPP is 2-butene-1,4-bis-TPP, which includes two phosphonium groups connected by butene. Then, a derivative of 2-butene-1,4-bis-TPP may include replacing one or more phenyl groups with another compound such as a halogen or an organic compound. For the sake of brevity, the present disclosure does not identify all possible derivatives, because the description should be sufficient for those of ordinary skill in the art. Other examples of TPP-derived compounds that can be used as mitochondrial biogenesis inhibitors according to the present methods include 2-butene-1,4-bis-TPP; derivatives of 2-butene-1,4-bis-TPP; 2-chlorobenzyl-TPP; derivatives of 2-chlorobenzyl-TPP; 3-methylbenzyl-TPP; derivatives of 3-methylbenzyl-TPP; 2,4-dichlorobenzyl-TPP; derivatives of 2,4-dichlorobenzyl-TPP; 1-naphthylmethyl-TPP; derivatives of 1-naphthylmethyl-TPP; terephthalenebis-TPP; and derivatives of terephthalenebisTPP. Of course, it should be understood that the above list is not an exhaustive list of TPP-derivatives.

[0106] Another class of mitochondrial biogenesis inhibitors that can be used in the present method is MDIVI-1 derivatives, described in International Patent Application PCT / US2018 / 066247 filed on December 18, 2018, and incorporated herein by reference in its entirety. Mitochondrial fission inhibitor-1 (mDIVI-1) is a small molecule that selectively and reversibly inhibits DRP1. MDIVI-1 has been shown to target DRP1 by binding to and inhibiting DRP1 self-assembly into a ring structure around mitochondria and its ability to catalyze GTP hydrolysis. The present method can take the form of a mitochondrial fission inhibitor 1 (mDIVI-1) derivative having the general formula XVIII as shown below:

[0107]

[0108] or a pharmaceutically acceptable salt thereof, wherein each of each R1-R8 can be selected from the group consisting of hydrogen, carbon, nitrogen, sulfur, oxygen, fluorine, chlorine, bromine, iodine, carboxyl, alkane, cycloalkane, alkane-based derivatives, olefins, cyclic olefins, olefin-based derivatives, alkynes, alkyne-based derivatives, ketones, ketone-based derivatives, aldehydes, aldehyde-based derivatives, carboxylic acids, carboxylic acid-based derivatives, ethers, ether-based derivatives, esters, ester-based derivatives, amines, amine-based derivatives, amides, amide-based derivatives, monocyclic or polycyclic aromatic hydrocarbons, heteroaromatic hydrocarbons, aromatic hydrocarbon-based derivatives, heteroaromatic hydrocarbon-based derivatives, phenols, phenol-based derivatives, benzoic acid, benzoic acid-based derivatives, and mitochondrial targeting signals. In some embodiments, at least one R group is a targeting signal, such as those described above.

[0109] Examples of other mitochondrial biogenesis inhibitors that can be used in the present methods to enhance the activity of anti-aging agents include vitamin C, berberine, caffeic acid phenyl ester, silybin, brutieridin, and melitidin.

[0110] It should be understood that the anti-aging composition has a wide range of advantageous uses. For example, the anti-aging composition can be used for aging treatment, which constitutes the treatment of senescent cells by, for example, inducing the death of senescent cells and / or inhibiting the proliferation of senescent cells. The anti-aging composition can be used to delay the onset and / or progression of age-related diseases, such as atherosclerosis, arthritis, cancer, cardiovascular disease, cataract, dementia, diabetes, alopecia, hypertension, inflammatory diseases, nephropathy, muscular atrophy, osteoarthritis, osteoporosis, lung disease, intervertebral disc degeneration and alopecia. Neurological diseases such as mild cognitive dysfunction, motor neuron dysfunction, Alzheimer's disease, Parkinson's disease and macular degeneration can also be treated or delayed by using the anti-aging composition. In some cases, the anti-aging composition can be used to treat age-related diseases.

[0111] Anti-aging agents can take various forms. For example, anti-aging agents can take the following forms: cosmetics, pills, lotions, shampoos, creams, soaps, skin cleansers, shaving preparations, aftershaving preparations, gels, patches, pastes, sprays, aerosols, powders, liquids, aqueous suspensions, aqueous solutions, foams, transdermal patches, tinctures and steam. For example, the anti-aging composition for the treatment of hair loss can take the form of topical application for hair, scalp and / or skin. It should be understood that those of ordinary skill in the art are familiar with the use of methods known in the art and available that do not need to be repeated herein to select the form of anti-aging agents.

[0112] As described above, derivatives of azithromycin, roxithromycin and telithromycin can be used as anti-aging agents without departing from the present method. In some embodiments, the derivative may involve one or more substituted targeting signals. Adding a membrane targeting signal or a mitochondrial targeting signal to azithromycin, roxithromycin or telithromycin significantly increases the mitochondrial uptake of the resulting conjugate, and thus improves the anti-aging activity. For example, compounds XIX-XXI, shown below, demonstrate derivatives in which one or more functional R groups are targeting signals.

[0113] First, compound XIX shows the general formula of an azithromycin derivative according to some embodiments, wherein the functional group R1 or R2 may be the same or may be different, and one or both of them may be a targeting signal. For example, R1 and / or R2 may be a membrane targeting signal selected from the following: palmitic acid, stearic acid, myristic acid, oleic acid, short-chain fatty acids and medium-chain fatty acids. (Of course, the conjugate will include a portion in which the terminal hydrogen is removed for bonding, such as palmitate). R1 ​​and / or R2 may be a mitochondrial targeting signal selected from triphenylphosphonium (TPP), TPP-derivatives, guanidine salts, guanidine salt derivatives and 10-N-nonylacridine orange. Non-exhaustive examples of TPP-derivatives include 2-butene-1,4-bis-TPP; 2-chlorobenzyl-TPP; 3-methylbenzyl-TPP; 2,4-dichlorobenzyl-TPP; 1-naphthylmethyl-TPP; terephthalene-bis-TPP; derivatives of 2-butene-1,4-bis-TPP; derivatives of 2-chlorobenzyl-TPP; derivatives of 3-methylbenzyl-TPP; derivatives of 2,4-dichlorobenzyl-TPP; derivatives of 1-naphthylmethyl-TPP; and derivatives of terephthalene-bis-TPP. In some embodiments, only one of R1 and R2 deviates from the parent compound, and thus the derivative is an analog. For example, R1 can be a methyl group and R2 can be a targeting signal. As another example, R1 can be a targeting signal and NH-R2 can be -N(CH3)2. In some embodiments, R1 or R2 can be a targeting signal, and the other of R1 and R2 can be selected from the following: hydrogen, carboxyl, alkane, cycloalkane, alkane-based derivatives, olefins, cyclic olefins, olefin-based derivatives, alkynes, alkyne-based derivatives, ketones, ketone-based derivatives, aldehydes, aldehyde-based derivatives, carboxylic acids, carboxylic acid-based derivatives, ethers, ether-based derivatives, esters, ester-based derivatives, amines, amine-based derivatives, amides, amide-based derivatives, monocyclic or polycyclic aromatic hydrocarbons, heteroaromatic hydrocarbons, aromatic hydrocarbon-based derivatives, heteroaromatic hydrocarbon-based derivatives, phenols, phenol-based derivatives, benzoic acid, and benzoic acid-based derivatives.

[0114]

[0115] Azithromycin derivatives

[0116] Compound XX shows the general formula of the roxithromycin derivative according to some embodiments, wherein the functional group R1 or R2 can be the same or can be different, and one or both of them can be a targeting signal. For example, as mentioned above, R1 and / or R2 can be a membrane targeting signal or a mitochondrial targeting signal. In some embodiments, only one of R1 and R2 deviates from the parent compound, and therefore the derivative is an analog. For example, R1 can be a methoxy group, such as the O-CH2-O-(CH2)2-OCH3 present in roxithromycin, and R2 can be a targeting signal. As another example, R1 can be a targeting signal and NH-R2 can be N(CH3)2. In some embodiments, R1 or R2 can be a targeting signal, and the other of R1 and R2 can be selected from the following: hydrogen, carboxyl, alkane, cycloalkane, alkane-based derivatives, olefin, cyclic olefin, olefin-based derivatives, alkyne, alkyne-based derivatives, ketones, ketone-based derivatives, aldehydes, aldehyde-based derivatives, carboxylic acids, carboxylic acid-based derivatives, ethers, ether-based derivatives, esters, ester-based derivatives, amines, amine-based derivatives, amides, amide-based derivatives, monocyclic or polycyclic aromatic hydrocarbons, heteroaromatic hydrocarbons, aromatic hydrocarbon-based derivatives, heteroaromatic hydrocarbon-based derivatives, phenols, phenol-based derivatives, benzoic acid and benzoic acid-based derivatives.

[0117]

[0118] Roxithromycin derivatives

[0119] Compound XXI shows the general formula of a telithromycin derivative, wherein the functional groups R1 or R2 may be the same or different, and one or both may be a targeting signal. For example, as described above, R1 and / or R2 may be a membrane targeting signal or a mitochondrial targeting signal. In some embodiments, only one of R1 and R2 deviates from the parent compound, and thus the derivative is an analog. For example, R1 may be an alkyl-aryl group present on the telithromycin carbamate ring, such as And R2 can be a targeting signal. As another example, R1 can be a targeting signal and -NH-R2 can be -N(CH3)2. In some embodiments, R1 or R2 can be a targeting signal, and the other of R1 and R2 can be selected from the following: hydrogen, carboxyl, alkane, cycloalkane, alkane-based derivatives, olefin, cyclic olefin, olefin-based derivatives, alkyne, alkyne-based derivatives, ketones, ketone-based derivatives, aldehydes, aldehyde-based derivatives, carboxylic acids, carboxylic acid-based derivatives, ethers, ether-based derivatives, esters, ester-based derivatives, amines, amine-based derivatives, amides, amide-based derivatives, monocyclic or polycyclic aromatic hydrocarbons, heteroaromatic hydrocarbons, aromatic hydrocarbon-based derivatives, heteroaromatic hydrocarbon-based derivatives, phenols, phenol-based derivatives, benzoic acid, and benzoic acid-based derivatives.

[0120]

[0121] Telithromycin derivatives

[0122] The following example (Compound XXII) shows an azithromycin derivative in which R1 is a methyl group and R2 is a fatty acid membrane targeting signal. In this example, R2 is a fatty acid moiety from the membrane targeting signal myristic acid. It should be understood that, as an example, compound XXII can be formed by myrostoylation, a lipidation technique known in the art. This example of an anti-aging compound has increased mitochondrial uptake relative to azithromycin, and as a result, anti-aging activity is increased. Compound XXII is an example of an azithromycin derivative with anti-aging activity under the present method. It should be understood that many other derivatives with anti-aging activity can be prepared under the present method.

[0123]

[0124] The following paragraphs describe the materials and methods used in connection with the experiments and data disclosed herein. It should be understood that these materials and methods are illustrative and can be varied by one of ordinary skill in the art without departing from the present methods. MRC-5 ( CCL-171) human lung fibroblasts and BJ ( CRL2522) human skin fibroblasts were purchased from ATCC (American Type Culture Collection). Gibco brand cell culture medium (MEM) was purchased from Life Technologies. Bromodeoxyuridine, azithromycin, roxithromycin, and erythromycin were purchased from Sigma-Aldrich. Azithromycin (from Pfizer) is FDA-approved. Roxithromycin (from GSK and Sandoz) is not available in the United States but is clinically approved in New Zealand, Australia, and Israel.

[0125] MRC-5 or BJ cells were seeded in 24-well plates. The next day, half of the plates were treated with 100 μM BrdU, while the control wells were treated with vehicle (DMSO) only and incubated at 37°C in a humidified atmosphere of 5% CO2 for 8 days. After 8 days of BrdU treatment, cells were treated with various test compounds or drugs (e.g., azithromycin, roxithromycin, telithromycin, erythromycin, etc.) for another 3-5 days. BrdU was washed off before drug treatment.

[0126] Sulforhodamine B assay: After incubation of the plates, cell viability was measured by the sulforhodamine B assay (SRB assay). This assay is based on the measurement of cellular protein content. The cells were fixed with 10% trichloroacetic acid (TCA) at room temperature for 1 hour and dried overnight at room temperature. The plates were then incubated with SRB for 30 minutes, washed twice with 1% acetic acid and air-dried for at least 1 hour. Finally, the protein-binding dye was dissolved in 10 mM Tris, pH 8.8 solution and read at 540 nm using a microplate reader.

[0127] Autophagy and cell cycle assays: Autophagy was performed according to the manufacturer's instructions (using Muse TM Autophagy LC3 antibody-based kit, Merck Millipore) and cell cycle (Muse TM Cell cycle kit (Merck Millipore) was used for the experiment.

[0128] β-Gal staining: β-galactosidase staining was performed on BrdU-treated MRC-5 cells by the Senescence β-galactosidase staining kit (#9860, Cell Signalling Technology Inc.) according to the manufacturer's protocol.

[0129] Seahorse XFe96 metabolic flux assay: The extracellular acidification rate (ECAR) and real-time oxygen consumption rate (OCR) of MCF7 cells were determined using the Seahorse extracellular flux (XF96) analyzer (Seahorse Bioscience, MA, USA). MRC-5 cells were maintained in MEM supplemented with 10% FBS (fetal bovine serum), 2mM Glutamax and 1% Pen-Strep. 40,000 cells per well were seeded into XF96 well cell culture plates and cultured overnight at 37°C in a 5% CO2 humidified atmosphere. The next day, cells were treated with azithromycin for 72 hours. Prior to the experiment, the plates were washed in preheated XF assay medium (for OCR measurements, XF assay medium was supplemented with 10mM glucose, 1mM pyruvate and adjusted to pH 7.4). Cells were subsequently stored in 175 μL / well XF assay medium for 1 hour at 37°C in a non-CO2 incubator. During the incubation period, 25 μL of 80mM glucose, 9 μM oligomycin, 1M 2-deoxyglucose (for ECAR measurement) and 25 μL of 10 μM oligomycin, 9 μM FCCP, 10 μM rotenone, 10 μM antimycin A (for OCR measurement) in XF assay culture medium were loaded into the injection port of XFe-96 sensor box. During the experiment, the instrument injected these inhibitors into the wells at a given time point while continuously measuring ECAR / OCR. ECAR and OCR measurements were normalized by protein content (sulforhodamine B assay). Data sets were analyzed by XFe-96 software, using one-way ANOVA and Student's t-test calculations. All experiments were performed at least in triplicate to obtain the results disclosed herein.

[0130] xCELLigence Analysis System: xCELLigence RTCA System (ACEA Biosciences Inc.) Briefly, MRC-5 lung fibroblasts (vehicle alone and / or vehicle treated with 100 μM BrdU) were seeded in each well and the efficacy of azithromycin was assessed by RTCA (real-time cell analysis) using measurements of cell-induced electrical impedance. This method allows for the quantification of the onset and kinetics of cellular responses. The experiment was repeated several times independently using quadruplicate samples for each condition.

[0131] Statistical analysis: Statistical significance was determined using the Student's t-test; values ​​less than 0.05 were considered significant. Data are shown as mean ± SEM.

[0132] The terms described herein are used only for the purpose of describing specific embodiments and are not intended to limit the present method. As used herein, the singular forms "a, an" and "the" are also intended to include the plural forms, unless the context clearly indicates otherwise. It will also be understood that when used in this specification, the term "comprises and / or comprising" specifies the presence of stated features, integers, steps, operations, elements and / or components, but does not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components and / or groups thereof.

[0133] Without departing from the spirit or essential features of the present invention, the present invention may be implemented in other specific forms. Therefore, the present embodiment should be considered in all aspects as illustrative rather than restrictive, the scope of the present invention is indicated by the claims of the present application rather than by the preceding description, and all changes within the meaning and scope of the equivalents of the claims are therefore intended to be included therein.

Claims

1. An anti-aging composition comprising: Compounds with the following chemical formula 2. The anti-aging composition according to claim 1 further comprises at least one of chlortetracycline, oxytetracycline, demeclocycline, methacycline, doxycycline, minocycline and tigecycline.

3. The anti-aging composition according to claim 1, further comprising tetracycline.

4. The anti-aging composition according to claim 1, further comprising at least one of procambaryl pamoate, atovaquone, bedaquiline, irinotecan, sorafenib, niclosamide, stiripentol, chloroquine and rapamycin.

5. The anti-aging composition according to claim 1, further comprising at least one of the following: mitoriboscin; mitoketoscin; mitoflavoscin; 2-butene-1,4-bis-TPP; 2-chlorobenzyl-TPP; 3-methylbenzyl-TPP; 2,4-dichlorobenzyl-TPP; 1-naphthylmethyl-TPP; and terephthalenebis-TPP.

6. The anti-aging composition according to claim 1, further comprising at least one of vitamin C, berberine, caffeic acid phenyl ester, silybin, brutieridin and melitidin.

7. The anti-aging composition according to any one of claims 1 to 6, wherein the anti-aging composition is in the form of at least one of: a pill, a lotion, a cream, a soap, a gel, a patch, a paste, a spray, a powder, a foam and a tincture.

8. The anti-aging composition according to any one of claims 1 to 6, wherein the anti-aging composition is a shampoo.

9. The anti-aging composition according to any one of claims 1 to 6, wherein the anti-aging composition is a skin cleanser.

10. The anti-aging composition according to any one of claims 1 to 6, wherein the anti-aging composition is a shaving preparation.

11. The anti-aging composition according to any one of claims 1 to 6, wherein the anti-aging composition is an after-shave preparation.

12. The anti-aging composition according to any one of claims 1 to 6, wherein the anti-aging composition is an aerosol.

13. The anti-aging composition according to any one of claims 1 to 6, wherein the anti-aging composition is a liquid.

14. The anti-aging composition according to any one of claims 1 to 6, wherein the anti-aging composition is an aqueous suspension.

15. The anti-aging composition according to any one of claims 1 to 6, wherein the anti-aging composition is an aqueous solution.

16. The anti-aging composition according to any one of claims 1 to 6, wherein the anti-aging composition is a transdermal patch.

17. Use of the anti-aging composition according to any one of claims 1 to 16 in the preparation of a medicament for use in aging therapy.

18. Use of the anti-aging composition according to any one of claims 1 to 16 in the preparation of a medicament for delaying the onset of an age-related disease in a subject, wherein the age-related disease is a lung disease.

19. The use according to claim 18, wherein the anti-aging composition comprises at least one of an oral composition, a topical composition and an intravenous injection composition.

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