Preparations containing apocynin and paeonol

Abstract

The present invention relates to a composition comprising 2-hydroxy-4-methoxyacetophenone (paeonol) or an isomer thereof and 4-hydroxy-3-methoxyacetophenone (apocynin) or an isomer thereof for use in the treatment of aging, e.g., by a senolytic mechanism of action.

Description

[Technical Field]

[0001] The present invention relates to compositions comprising 2-hydroxy-4-methoxyacetophenone (paeonol) or an isomer thereof and 4-hydroxy-3-methoxyacetophenone (apocynin) or an isomer thereof for use in the treatment of aging, e.g., by a senolytic mechanism of action.

[0002] Cellular senescence is important for maintaining tissue homeostasis. Senescence, as a general term, refers to biological aging. Dysregulation of senescence is associated with the aging process, which is a major risk factor for many human diseases and debilitating conditions, including cancer, cardiovascular disease, and neurodegenerative diseases.

[0003] Cellular senescence is a mechanism that permanently arrests cell proliferation and can be induced by stresses such as repeated cell division, mitochondrial deterioration, DNA damage, and telomere erosion resulting from oxidative stress.

[0004] Cellular senescence is characterized by the cessation of cell division, ensuring that senescent or damaged cells do not perpetuate their genomes. While this can prevent carcinogenesis during normal development, senescent cells accumulate and produce pro- and pro-inflammatory cytokines, chemokines, and enzymes, collectively known as senescence-associated secretory phenotype (SASP) factors. SASP factors disrupt tissue structure and function and are thought to play an important role in the development of age-related diseases. These include, but are not limited to, osteoporosis; sarcopenia; pulmonary diseases such as chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), pulmonary hypertension (PH), and pulmonary arterial hypertension (PAH); liver diseases such as acute liver injury, chronic liver disease, and fatty liver; kidney diseases such as acute kidney injury (AKI), chronic kidney disease (CKD), tubulointerstitial fibrosis (TIF), glomerulosclerosis, and angiosclerosis; vascular diseases such as atherosclerosis; type 1 diabetes; age-related macular degeneration; and neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease, frontotemporal dementia, amyotrophic lateral sclerosis, and multiple sclerosis.

[0005] Osteoporosis is an age-related skeletal disorder characterized by low bone mass and progressive deterioration of bone tissue, leading to an increased risk of fracture. It is associated with the accumulation of senescent cells (osteocytes) in bone. Overproduction of pro-inflammatory cytokines by senescent bone cells contributes to chronic inflammation and creates a toxic microenvironment that causes age-related bone loss. Removal of senescent cells and / or SASP holds promise for delaying or preventing age-related osteoporosis.

[0006] Sarcopenia is an age-related musculoskeletal disorder associated with the progressive loss of muscle mass, strength, and muscle function. The aging of muscle stem cells is thought to cause a loss of muscle regeneration capacity, while the increased levels of SASP factors secreted by senescent cells cause chronic inflammation, which can accelerate protein degradation and muscle fiber loss, leading to sarcopenia. Removal of senescent cells and / or SASP has the potential to treat sarcopenia.

[0007] Several lung diseases have been associated with aging, particularly chronic obstructive pulmonary disease (COPD), an inflammatory lung disease that causes progressive airflow restriction to the lungs; idiopathic pulmonary fibrosis (IPF), a chronic and fatal lung disease that scars the tissue surrounding the alveoli of the lungs; and pulmonary hypertension (PH) and pulmonary arterial hypertension (PAH). The accumulation of senescent fibroblasts and lung epithelial cells, as well as SASP, has been strongly associated with the pathology of lung disease. Removal of senescent cells and / or SASP may have potential as a therapeutic strategy for treating COPD, IPF, PH, and PAH.

[0008] Liver conditions, particularly acute liver injury, chronic liver disease, and fatty liver (fat accumulation in the liver unrelated to alcohol consumption), are also associated with aging. A close correlation has been found between markers of aging in hepatocytes and acute liver injury, chronic liver disease, hepatic fat accumulation, and steatosis. Therefore, removal of senescent cells and / or SASP may have the potential to treat liver conditions.

[0009] Renal diseases, especially acute kidney injury (AKI), chronic kidney disease (CKD), tubulointerstitial fibrosis (TIF), glomerulosclerosis, and vascular sclerosis, are strongly associated with cellular senescence. Removal of senescent cells and / or SASP may be a novel therapeutic approach for kidney diseases.

[0010] Atherosclerosis is a chronic disease of the arteries characterized by excessive deposition of fatty substances (plaque, or atheroma) on the inner walls of arteries. Senescence of lipid-laden macrophages ("foam cells") has been shown to be detrimental at all stages of atherosclerosis. The accumulation of senescent foam cells in the subendothelial space during the development of atherosclerosis is thought to promote atheroma formation and maturation via the SASP. Therefore, removal of senescent cells and / or the SASP may have therapeutic potential for atherosclerosis.

[0011] Type 1 diabetes (T1D) is an autoimmune disease characterized by the progressive loss of pancreatic beta cells, leading to hyperglycemia. The pathogenesis of T1D is strongly associated with beta cell senescence. Targeted elimination of senescent beta cells has been shown to prevent T1D and may be a viable approach for the treatment of T1D.

[0012] Age-related macular degeneration (AMD) is an age-related condition in the macula of the eye that affects central vision. Senescence in retinal and choroidal tissue cells has been shown to be an important factor in the onset and development of AMD. Removal of senescent cells and / or SASP may have the potential to treat AMD.

[0013] Senescence in astrocytes, microglia, oligodendrocytes, neurons and neural stem cells is involved in the development of neurodegenerative diseases including Alzheimer's disease, Parkinson's disease, frontotemporal dementia, amyotrophic lateral sclerosis and multiple sclerosis.

[0014] A variety of studies in mice and humans suggest that targeting senescent cells and / or the SASP may be a powerful strategy to delay age-related pathologies, improve healthspan, and potentially improve lifespan.

[0015] Senescence can be targeted by senotherapeutic compounds, which are divided into two classes: senolytic compounds and senoinhibitory compounds. A compound is said to be senolytic if its administration results in the selective elimination of senescent cells, thus resulting in a reduction in both the number of senescent cells and the total number of cells. Senonetically inhibiting compounds are compounds whose administration results in the selective inhibition of SASP and the maintenance or increase of the total number of cells.

[0016] Given the central role of senescent cells and SASP as risk factors for age-related pathologies, there is a need for compositions for use in the treatment of aging.

[0017] The present invention provides a composition comprising 2-hydroxy-4-methoxyacetophenone (paeonol) or an isomer thereof and 4-hydroxy-3-methoxyacetophenone (apocynin) or an isomer thereof for use in the treatment of aging, wherein administration of the composition results in a reduction in the number of senescent cells and a concomitant increase in the total number of viable cells. As used herein, the term "viable" refers to cells that are alive and capable of reproduction. Cell viability can be assessed by staining with a viability dye such as trypan blue or calcein-AM, or by staining with Hoechst stain, followed by counting the number of intact nuclei. The composition of the present invention can exert its effect through a senolytic or senolytic mechanism of action. Preferably, the composition of the present invention exerts its effect through a senolytic mechanism of action. Preferably, the composition of the present invention exerts its effect through a senolytic mechanism of action that results in selective inhibition of SASP.

[0018] In a further aspect, the present invention provides a composition comprising paeonol or an isomer thereof and apocynin or an isomer thereof for use in the treatment of aging by a senolytic mechanism of action.

[0019] According to a further aspect, the present invention provides a composition comprising paeonol or an isomer thereof and apocynin or an isomer thereof for use in the treatment of ageing by a senolytic mechanism of action.

[0020] Paeonol is 2-hydroxy-4-methoxyacetophenone and has the following formula: [ka] Paeonol can be found in plant materials and plant extracts. For example, paeonol can be found in peony (Paeonia suffruticosa), peony (Paeonia lactiflora), Paeonia veitchii, mountain peony (Paeonia obovata), rhubarb (Rheum palmatum) (rhizome), and Scutellaria baicalensis (root). The compositions (and medicaments) of the present invention contain 2-hydroxy-4-methoxyacetophenone (paeonol).

[0021] According to the inventors' research, paeonol is a solid with a low melting point of about 49.7°C.

[0022] Preferably, the paeonol is synthesized or extracted and purified from a plant. This may be referred to as isolated paeonol. The amounts and ratios described herein refer to isolated paeonol. Less preferably, paeonol may be present in the compositions (and medicaments) according to the present invention as a direct extract from the plant (i.e., as part of an unseparated mixture of compounds in the form of an unpurified plant or root extract). These are referred to as "natural form" paeonol or "natural paeonol." For example, paeonol present in the compositions (and medicaments) according to the present invention in the form of peony is referred to as "natural paeonol." The terms "natural form" paeonol or "natural paeonol" include glycosides of paeonol, such as those found in the plant species in which paeonol is found. Such glycosides include, for example, paeonin, paeonolide, and paeonoside. When natural paeonol is used, those skilled in the art will readily understand how to adjust the ratios to provide the formulations of the present invention.

[0023] Isolated paeonol provides a more consistent quality of paeonol compared to natural paeonol. It can also be produced on a larger scale than natural paeonol. Therefore, it is preferred to use isolated paeonol in this application.

[0024] Apocynin is a plant phenolic 4-hydroxy-3-methoxyacetophenone and has the formula: [ka] Apocynin is a solid that, according to our studies, has a much higher melting point of about 114.6°C.

[0025] Apocynin is found in plant materials and plant extracts, such as extracts of the plants Picrorrhiza kurroa, Apocynum cannabinium, Apocynum venatum, Apocynum androsaemifolium, and species of the genus Vanilla (e.g., Vanilla planifolia).

[0026] The compositions (and medicaments) of the present invention contain 4-hydroxy-3-methoxyacetophenone (apocynin). Preferably, the apocynin is synthesized or extracted from a plant and purified. This may be referred to as isolated apocynin. The amounts and ratios described herein refer to those of isolated apocynin.

[0027] Apocynin may be present in the compositions (and medicaments) according to the present invention as a direct extract from such plants (e.g., as part of an unseparated mixture of compounds in the form of a crude plant or root extract). These are referred to as "natural form" apocynin or "natural apocynin." For example, apocynin present in the compositions (and medicaments) according to the present invention in the form of picrorrhiza kurroa is referred to as "natural apocynin." The terms "natural apocynin" or "natural form" apocynin also include glycosides of apocynin, such as those found in plant species in which apocynin is found. Such glycosides include, for example, androsin and other iridoid glycosides.

[0028] The composition may contain apocynin as part of an unresolved mixture of compounds in the form of a crude plant or root extract, i.e., "natural" apocynin. Picrorhiza kurroa is a standardized form based on a standardized iridoid glucoside fraction, and such forms are well known. Standardized forms of picrorhiza kurroa contain picrorhiza kurroa standardized to "not less than 4% kutkin." Kutkin is obtained by crystallization and consists of the glucosides picroside I and kutkin in a 1:2 ratio, along with minor amounts of other glycosides (Sing and Rastogi, 1972, Ansari et al., 1988).

[0029] Isolated apocynin provides apocynin of more consistent quality than natural apocynin. It can also be produced on a larger scale than natural apocynin. Therefore, it is preferred to use isolated apocynin in the present invention.

[0030] As noted above, the composition may contain apocynin in its natural form, although this is less preferred. If natural apocynin is used, those skilled in the art will readily understand how to adjust the ratio to produce the formulations of the present invention. However, in this case, it may be necessary to limit the amount of picorrhiza kurroa to prevent side effects (e.g., stomach upset that may occur due to other phytochemical species in picorrhiza kurroa). However, it is noteworthy that most human subjects can ingest up to 2,000 mg of picorrhiza kurroa (2% or more of kutkin) per day without discomfort.

[0031] Isomers of paeonol and apocynin are known in the art and include 2-hydroxy-3-methoxyacetophenone "orthoacetovanillone" (CAS: 703-98-0), 2-hydroxy-5-methoxyacetophenone (CAS: 705-15-7), 3-hydroxy-4-methoxyacetophenone "isoacetovanillone" (CAS: 6100-74-9), and 4-hydroxy-2-methoxyacetophenone "isopaeonol" (CAS: 493-33-4).

[0032] Paeonol and apocynin are isomers of each other. It will be understood that according to the present invention, the composition must contain two different active compounds (for example, paeonol itself and apocynin itself). Preferably, the composition according to the present invention contains paeonol itself and apocynin itself. In this specification, the name "APPA" is used to indicate a composition containing paeonol and apocynin, as described above.

[0033] The applicants have found that compositions of the present invention, such as APPA, have the ability to reduce the number of senescent cells in a human chondrocyte cell line.

[0034] The present applicants have further found that administration of the compositions of the present invention, such as APPA, to a human chondrocyte cell line results in an increase in total cell number, and thus the compositions of the present invention, such as APPA, exhibit a senescent cell-inhibiting effect on the human chondrocyte cell line (see Examples).

[0035] The use of paeonol or its isomers together with apocynin or its isomers as senolytic and / or senolytic drugs can be considered a significant advancement, enabling new therapeutic avenues in eliminating or delaying the deleterious effects of cellular senescence and consequently age-related pathologies.

[0036] The composition according to the present invention may have a weight ratio of paeonol or its isomers to apocynin or its isomers of 3:2 to 9:1. For example, the composition according to the present invention may have a weight ratio of paeonol or its isomers to apocynin or its isomers of 3:2 to 5:1. For example, the composition according to the present invention may have a weight ratio of paeonol or its isomers to apocynin or its isomers of 5:1 to 9:1.

[0037] The composition according to the present invention may be a liquid formulation. The composition according to the present invention may be a liquid formulation prepared from a eutectic mixture containing the active compounds paeonol or an isomer thereof and apocynin or an isomer thereof, and the liquid formulation further comprises at least one excipient. The excipient may be a glycol or glycol derivative, a polyhydric alcohol, or an ester and / or ether thereof. In one embodiment, the formulation has a weight ratio of total active compounds to excipients of 2:3 to 19:1. Preferably, the weight ratio of total active compounds to excipients is 2:3 to 9:1. Preferably, the weight ratio of total active compounds to excipients is 64:36. Preferably, the formulation is a stable liquid at room temperature (15 to 25°C).

[0038] The compositions according to the invention may be in the form of a solid formulation such as a powder, tablet or pill.

[0039] The compositions according to the present invention may be used to treat age-related diseases.

[0040] The compositions according to the present invention may be used to treat osteoporosis. The compositions according to the present invention may be used to treat sarcopenia. The compositions according to the present invention may be used to treat chronic obstructive pulmonary disease (COPD). The compositions according to the present invention may be used to treat idiopathic pulmonary fibrosis (IPF). The compositions according to the present invention may be used to treat lung diseases such as pulmonary hypertension (PH) and pulmonary arterial hypertension (PAH). The compositions according to the present invention may be used to treat liver diseases such as acute liver injury, chronic liver disease, and fatty liver. The compositions according to the present invention may be used to treat kidney diseases such as acute kidney injury (AKI), chronic kidney disease (CKD), tubulointerstitial fibrosis (TIF), glomerulosclerosis, and angiosclerosis. The compositions according to the present invention may be used to treat vascular diseases such as atherosclerosis. The compositions according to the present invention may be used to treat type 1 diabetes. The compositions according to the present invention may be used to treat age-related macular degeneration (AMD). The compositions according to the invention may be used to treat Alzheimer's disease (AD), Parkinson's disease, frontotemporal dementia, amyotrophic lateral sclerosis or multiple sclerosis.

[0041] The compositions according to the present invention may be administered orally.

[0042] The pharmaceutical compositions of the present invention can be formulated into well-known compositions for any route of drug administration, for example, oral, rectal, parenteral, transdermal (e.g., patch technology or transdermal gel formulations), intravenous, intramuscular, subcutaneous, intracisternal, intravaginal, intraperitoneal, topical (powder, ointment, or drops), buccal, or nasal spray. Typical compositions contain pharmaceutically acceptable carriers, such as aqueous solutions, non-toxic excipients including salts and preservatives, buffers, and the like, as described, inter alia, in Remington's Pharmaceutical Sciences fifteenth edition (Matt Publishing Company, 1975), pages 1405-1412 and 1461-1487, and in National Formulary XIV, fourteenth edition (American Pharmaceutical Association, 1975).

[0043] Examples of suitable aqueous and non-aqueous pharmaceutical carriers, diluents, solvents, or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), carboxymethylcellulose and suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters such as ethyl oleate.

[0044] The compositions of the present invention may also contain additives such as, but not limited to, preservatives, wetting agents, emulsifying agents, and dispersing agents. Antibacterial and antifungal agents may be included to prevent the growth of microorganisms, for example, parabens, chlorobutanol, phenol, sorbic acid, etc. Furthermore, it may be desirable to include isotonic agents, for example, sugars, sodium chloride, etc.

[0045] According to a further aspect, the present invention provides the use of apocynin or an isomer thereof and paeonol or an isomer thereof in the manufacture of a medicament for the treatment of ageing.

[0046] In a further aspect, the present invention provides the use of apocynin or an isomer thereof and paeonol or an isomer thereof in the manufacture of a medicament for the treatment of age-related diseases.

[0047] In a further aspect, the present invention provides use of apocynin or an isomer thereof and paeonol or an isomer thereof in the manufacture of a medicament for the treatment of: osteoporosis and sarcopenia; lung diseases such as chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), pulmonary hypertension (PH), and pulmonary arterial hypertension (PAH); liver diseases such as acute liver injury, chronic liver disease, and fatty liver; kidney diseases such as acute kidney injury (AKI), chronic kidney disease (CKD), tubulointerstitial fibrosis (TIF), glomerulosclerosis, and angiosclerosis; vascular diseases such as atherosclerosis; type 1 diabetes; age-related macular degeneration (AMD); and neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease, frontotemporal dementia, amyotrophic lateral sclerosis, and multiple sclerosis.

[0048] In a further aspect, the present invention provides the use of apocynin or an isomer thereof and paeonol or an isomer thereof in the manufacture of a medicament for the treatment of Alzheimer's disease, Parkinson's disease, frontotemporal dementia, amyotrophic lateral sclerosis or multiple sclerosis.

[0049] According to the present invention, there is provided a method for treating aging, comprising the step of administering to a patient in need thereof a composition comprising apocynin or an isomer thereof and paeonol or an isomer thereof.

[0050] According to the present invention, there is provided a method for treating age-related diseases, the method comprising the step of administering a composition comprising apocynin or an isomer thereof and paeonol or an isomer thereof to a patient in need thereof.

[0051] According to the present invention, in a further aspect, there is provided a method for treating osteoporosis, sarcopenia, pulmonary diseases such as chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), pulmonary hypertension (PH) and pulmonary arterial hypertension (PAH), liver diseases such as acute liver injury, chronic liver disease and fatty liver, kidney diseases such as acute kidney injury (AKI), chronic kidney disease (CKD), tubulointerstitial fibrosis (TIF), glomerulosclerosis and angiosclerosis, vascular diseases such as atherosclerosis, type 1 diabetes, age-related macular degeneration (AMD), Alzheimer's disease (AD), Parkinson's disease, frontotemporal dementia, amyotrophic lateral sclerosis or multiple sclerosis, the method comprising the step of administering a composition comprising apocynin or an isomer thereof and paeonol or an isomer thereof to a patient in need thereof. [Brief explanation of the drawings]

[0052] The present invention will now be described in detail with reference to the following examples and accompanying drawings. Example 1 demonstrates the senescent cell inhibitory activity of APPA in the chondrocyte cell line TC28a2. Through the examples, * " means "comparison with base measurement," and "#" means "comparison with etoposide only measurement." [Figure 1] 1 shows the effect of APPA on the levels of the senescence-associated secretory phenotype (SASP) marker β-galactosidase in TC28a2 chondrocytes when induced by exposure to IL-6 or etoposide (Eto). [Figure 2] 1 shows the effects of APPA, AP, and PA on chondrocyte viability using DRAQ7 in human TC28a2 chondrocytes. [Figure 3] 1 shows the effect of APPA on the number of viable TC28a2 cells exposed to etoposide (Eto). [Figure 4a] 1 shows the effect of APPA in TC28a2 cells exposed to IL-6 or etoposide (Eto), and early and late TC28a2 apoptosis. 2 shows the effects of APPA, AP, and PA on early apoptosis in human TC28a2 chondrocytes. [Figure 4b] 1 shows the effect of APPA in TC28a2 cells exposed to IL-6 or etoposide (Eto), and early and late TC28a2 apoptosis. 2 shows the effects of APPA, AP, and PA on late apoptosis in human TC28a2 chondrocytes. [Figure 5] Example 1 shows the effect of APPA and its components AP and PA on the levels of the senescence-associated secretory phenotype (SASP) marker β-galactosidase in TC28a2 chondrocytes when induced by exposure to etoposide. Example 2 shows the senescent cell inhibitory activity of APPA in primary human chondrocytes. [Figure 6a] Figure 1 shows the effect of APPA on SA-β-Gal activity and CDKN1A gene expression levels in primary human chondrocytes exposed to etoposide and OSM. Figure 2 shows the effect of various concentrations of etoposide (+OSM) on the levels of the senescence-associated secretory phenotype (SASP) marker β-galactosidase. [Figure 6b] Figure 1 shows the effect of APPA on SA-β-Gal activity and CDKN1A gene expression levels in primary human chondrocytes exposed to etoposide and OSM. Figure 2 shows the effect of various concentrations of etoposide (+OSM) on CDKN1A gene expression in primary human chondrocytes. [Figure 6c] Figure 1 shows the effect of APPA on SA-β-Gal activity and CDKN1A gene expression levels in primary human chondrocytes exposed to etoposide and OSM. Figure 2 shows the effect of APPA on the increase in the level of β-galactosidase, a senescence-associated secretory phenotype (SASP), induced by 20 μM etoposide (Eto) + 10 ng / ml oncostatin M (OSM). "+" means "presence" and "-" means "absence." [Figure 6d]Figure 1 shows the effect of APPA on SA-β-Gal activity and CDKN1A gene expression levels in primary human chondrocytes exposed to etoposide and OSM. Figure 2 shows the effect of APPA on the increase in relative gene expression of CDKN1A induced by 20 μM etoposide (Eto) + 10 ng / ml oncostatin M (OSM). "+" means "presence" and "-" means "absence." [Figure 7a] Figure 1 shows the effects of APPA, AP, and PA on SA-β-Gal activity, CDKN1A gene expression levels, BECN-1 gene expression levels, and BCL2L13 expression levels in primary human chondrocytes exposed to etoposide and OSM. Figure 2 shows the effect of 20 μM etoposide (Eto) + 10 ng / ml oncostatin M (OSM) on the level of the senescence-associated secretory phenotype (SASP) marker β-galactosidase. [Figure 7b] Figure 1 shows the effects of APPA, AP, and PA on SA-β-Gal activity, CDKN1A gene expression levels, BECN-1 gene expression levels, and BCL2L13 expression levels in primary human chondrocytes exposed to etoposide and OSM. Figure 2 shows the effects of APPA and its components on the increase in the level of the senescence-associated secretory phenotype (SASP) marker β-galactosidase induced by 20 μM etoposide (Eto) + 10 ng / ml oncostatin M (OSM). [Figure 7c] Figure 1 shows the effects of APPA, AP, and PA on SA-β-Gal activity, CDKN1A gene expression levels, BECN-1 gene expression levels, and BCL2L13 expression levels in primary human chondrocytes exposed to etoposide and OSM. Figure 2 shows the effects of APPA and its components on the increase in CDNK1A gene expression induced by 20 μM etoposide (Eto) + 10 ng / ml oncostatin M (OSM). [Figure 7d]Figure 1 shows the effects of APPA, AP, and PA on SA-β-Gal activity, CDKN1A gene expression levels, BECN-1 gene expression levels, and BCL2L13 expression levels in primary human chondrocytes exposed to etoposide and OSM. Figure 2 shows the effect of APPA and its components on the increase in BECN-1 gene expression induced by 20 μM etoposide (Eto) + 10 ng / ml oncostatin M (OSM). [Figure 7e] The effects of APPA, AP, and PA on SA-β-Gal activity, CDKN1A gene expression levels, BECN-1 gene expression levels, and BCL2L13 expression levels in primary human chondrocytes exposed to etoposide and OSM are shown. The effects of APPA and its components on the increase in BCL2LK13 gene expression induced by 20 μM etoposide (Eto) + 10 ng / ml oncostatin M (OSM) are shown. Drug Preparation: APPA was prepared in vials (AP:PA ratio of 2:7), while AP and PA were obtained from Sigma (Sigma-Aldrich, St. Louis, MO, USA). All compounds were dissolved in dimethyl sulfoxide (DMSO) (Sigma-Aldrich) to a final working concentration of 1 g / ml, and serial dilutions were developed.

[0053] Chondrocyte isolation Written informed consent was obtained from all subjects, and approval was obtained from the local ethics committee of the Galician Health Administration (CEIC). All procedures were performed in accordance with the principles stated in the 1975 Declaration of Helsinki, as revised in 2000.

[0054] Chondrocytes from human OA hip cartilage were isolated from 18 patients (5 male and 13 female patients, with a mean ± SD age of 86 ± 7.17 and 79.71 ± 13.56 years, respectively) undergoing total hip arthroplasty as described in Maneiro E, Martin MA, de Andres MC, Lopez-Armada MJ, Fernandez-Sueiro JL, del Hoyo P, et al. Mitochondrial respiratory activity is altered in osteoarthritic human articular chondrocytes. Arthritis and rheumatism. 2003;48(3):700-8). T / C28a2 chondrocytes The immortalized human juvenile chondrocyte cell line T / C28a2 (25) was used in some experiments.

[0055] Human articular chondrocytes and the T / C28a2 cell line were equilibrated overnight at 37°C in a humidified 5% CO2 atmosphere in Dulbecco's modified Eagle's medium (DMEM) (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS), penicillin (100 U / ml), and streptomycin (100 μg / ml) (Gibco).

[0056] Cell culture chondrocytes and T / C28a2 cell line: Cells were plated at 5 × 10 in Corning® CellBIND® Multiwell 96 Plates MW 96 (Corning, NY, USA) for analysis. 4 at a density of cells / well or 1.8 × 10 in MW 12 plates (Corning, NY, USA). 5 Cells were plated at a density of 1000 cells / well. Cells were equilibrated overnight at 37°C in DMEM with 5% or 2% FBS in 5% CO. Cells were stimulated with 20 μM etoposide for chondrocytes and 5 μM for cell lines (Sigma-Aldrich). [Example]

[0057] Example 1: Demonstration of senescent cell inhibitory activity in the chondrocyte cell line TC28a2 We found that APPA was able to reduce the level of the SASP marker β-galactosidase in the human chondrocyte cell line TC28a2, indicating a decrease in the number of senescent cells, but an increase in the total number of cells. Thus, APPA demonstrated a senescent cell-inhibitory effect on the human chondrocyte cell line TC28a2.

[0058] A widely used marker of senescence is increased levels of senescence-associated β-galactosidase (SA-β-Gal) activity, as indicated by quantification of fluorescein di-β-D-galactopyranoside (FDG) using flow cytometry.

[0059] Therefore, the effect of APPA on the number of senescent cells was assessed by quantifying β-galactosidase activity using flow cytometry. β-Galactosidase activity was detected by flow cytometry using fluorescein di-β-D-galactopyranoside (FDG, Thermo Fisher, Waltham, MA, USA). Non-fluorescent FDG is sequentially hydrolyzed by β-galactosidase, first to fluorescein monogalactoside and then to highly fluorescent fluorescein. Enzyme-mediated hydrolysis of FDG can be followed by an increase in fluorescence.

[0060] Cell cultures were pretreated with 5 μM etoposide or 20 ng / mL interleukin 6 (IL-6) for 72 hours (Sigma-Aldrich Merck KGaA, Darmstadt, Germany) to induce DNA damage as a genotoxic stress leading to cellular senescence, and then with 10 nM bafilomycin A (Sigma-Aldrich) for 1 hour to adjust intracellular pH. These conditions were evaluated with and without 10 μg / mL APPA for 24 hours. Fluorescein di-β-D-galactopyranoside (10 μM) was then added to the pretreatment medium. At the end of the incubation period, cultures were washed with PBS, resuspended by trypsinization, and immediately analyzed using a FACScalibur flow cytometer (Becton Dickinson). Data were acquired and analyzed using Cellquest software (Becton Dickinson). Each fluorescein signal was measured with an FL1 detector, and β-galactosidase activity was estimated using the median fluorescence intensity (arbitrary units) of the cell population.

[0061] The median FDG fluorescence (median ± SEM) of human TC28a2 chondrocytes treated with APPA and a comparison with senescent controls (20 ng / ml IL-6 or 5 μM etoposide) are shown in Figure 1 and Table 1. Figure 1 demonstrates that APPA reduced the number of senescent cells, as indicated by a decrease in SA-β-Gal activity, in both IL-6-exposed and etoposide-exposed TC28a2 cells. [Table 1]

[0062] FIG. 2 shows the effects of APPA, AP, and PA on the viability of chondrocytes using DRAQ7.

[0063] The novel cell viability assay uses DRAQ7, a marker of apoptosis, necrosis, and dead cells, which stains the nucleus in dead and permeabilized cells, but not in intact, live cells.

[0064] To determine the effects of APPA (10 μg / ml), AP (2.3 and 10 μg / ml), and PA (7.7 and 10 μg / ml) on chondrocyte viability, an assay based on DRAQ7™ (Thermo Fisher Scientific) was used. DRAQ7™ is a dye that can be used to indicate apoptosis, necrosis, and dead cells; the assay involves staining of nuclei in dead and permeabilized cells, but not staining of intact, live cells.

[0065] Because a larger number of cells was required for the assay, cells were grown in flasks. After 24 hours of different treatments, cells were trypsinized and centrifuged at 1500 rpm for 10 minutes. The collected cells were suspended in 3 μM fluorescent dye DRAQ7™ (Thermo Fisher) and incubated in the dark at room temperature (rt) for 10 minutes. Samples were analyzed using a CytoFLeX flow cytometer (Beckman Coulter Inc., California, USA). At least 1 × 10 cells were used per assay. 4 Cells were measured. Data were analyzed using CyExpert V 2.5 software (Beckman Coulter Inc.). Positive control cells were treated with 5 μM etoposide (Sigma-Aldrich), and negative control (basal) cells were grown in fresh DMEM with 5% FBS. To establish the positions of the DRAQ7- and DRAQ7+ gates, one of each negative and positive control was stained with DRAQ7 dye, and the other was stained with PBS only (data not shown).

[0066] Figure 2 shows the effects of APPA (10 μg / ml), AP (2.3 and 10 μg / ml), and PA (7.7 and 10 μg / ml) on the percentage of DRAQ7-positive (nonviable) cells, which is used to estimate cell viability in the presence of APPA, AP, or PA. The percentage of nonviable cells never exceeded 5.05%. This value was obtained in the 10 μg / ml AP condition. The DRAQ7 data indicate that, at the concentrations used, APPA and its components AP and PA do not affect cell viability, thus ruling out the possibility that the reduction in cell senescence is the result of cytotoxicity.

[0067] FIG. 3 shows the effect of APPA on the total viable cell number in the chondrocyte cell line TC28a2.

[0068] 8×10 4 After culturing the cells and reaching 70%-80% confluence, the cells were pretreated with 5 μM etoposide for 72 hours. The culture medium was then replaced with Dulbecco's modified Eagle's medium (DMEM) supplemented with 2% fetal bovine serum (FBS), and the cells were cultured under these conditions for 48 hours. Finally, APPA (10 μg / ml) was added for an additional 24 hours. After incubation, the cells were fixed in 4% paraformaldehyde (Sigma-Aldrich) for 10 minutes at room temperature, followed by incubation in 0.2% Tween 20 (Sigma-Aldrich) for 5 minutes. This step was followed by incubation with the nuclear dye 2'-(4-ethoxyphenyl)-5-(4-methyl-1-piperazinyl)-2,5'-bi-1H-benzimidazole trihydrochloride (Hoechst 33258) (Sigma-Aldrich) for 5 minutes. After washing with phosphate buffer (PB), coverslips were mounted on microscope chamber slides using Prolong Gold Antifade Reagent Mountant (Thermo Fisher Scientific). Fluorescence was visualized and photographed under an Olympus BX61 fluorescence microscope. All samples were analyzed in duplicate with 3–5 fields per well, and the mean and standard deviation were calculated.

[0069] The results are shown in Figure 3. When TC28a2 cells were incubated with 5 μM etoposide and 10 μg / ml APPA, the number of cells present in the culture increased in a statistically significant manner. Therefore, APPA was shown to exert its effect through a senescent cell-inhibitory mechanism of action, i.e., it reduced the level of the SASP marker SA-β-Gal (indicating a reduction in the number of senescent cells) but increased the total number of viable cells. The cells' susceptibility to apoptosis was then analyzed (Figure 4). Cells were incubated for 72 hours in the presence of 2 μM etoposide or 20 ng / ml IL-6 (Sigma-Aldrich), with or without 10 μg / ml APPA for 24 hours. Cells were harvested by trypsinization and resuspended in 1x annexin binding buffer, followed by the addition of 5 μL of annexin V-fluorescein isothiocyanate (FITC) and 5 μL of propidium iodide (PI; ImmunoStep, Salamanca, Spain). After 15 min of incubation, cells were analyzed on a FACsCalibur flow cytometer (Becton Dickinson, NJ, USA). Cells (1 × 10) per assay were 4 ) were measured. Data were analyzed using CellQuest software (Becton Dickinson). Apoptosis was analyzed by counting cells simultaneously stained with Annexin V-FITC and PI. This allowed us to distinguish intact cells (Annexin V-FITC and PI negative) from cells in early apoptotic states (Annexin V-FITC positive and PI negative), late apoptotic states (Annexin V-FITC and PI positive), and necrotic states (Annexin V-FITC negative and PI positive). Results are expressed as the percentage of positive cells for each dye and represent the mean ± standard error of the mean (SEM) of three independent experiments.

[0070] Figure 4 shows the effect of APPA on early (Figure 4a) and late (Figure 4b) apoptosis in the cell line TC28a2 exposed to either 20 ng / ml IL-6 or 2 μM etoposide. APPA had no clear effect on either early or late apoptosis. Increased apoptosis would have been an alternative pathway for reducing the levels of SASP markers, but this would have been accompanied by a decrease in viable cell number. As shown by Figure 3 above, the fact that APPA increases viable cell number indicates that the decrease in senescence does not result from increased apoptosis, consistent with the apoptosis data.

[0071] Figure 5 and Table 2 show the effects of AP, PA, and APPA on etoposide-induced TC28a2 cell senescence, as determined by SA-β-Gal levels. SA-β-Gal levels were assessed via quantification of fluorescein di-β-D-galactopyranoside (FDG) using flow cytometry, as in Figure 1. When AP and PA were tested in parallel with APPA in etoposide-exposed TC28a2 cells, only APPA produced a significant decrease in SA-β-Gal activity, although PA at 7.7 μg / ml approached significance (p=0.054). The effect produced by PA at 10 μg / ml was not significant. APPA reduced the number of senescent TC28a2 cells more than its individual components, AP or PA (as indicated by lower median FDG fluorescence). The effect of APPA at 10 μg / ml was greater than expected based on (i) the effect of 10 μg / ml AP or 10 μg / ml PA, and (ii) the additive effect of 2.3 μg / ml AP and 7.7 μg / ml PA, suggesting a synergistic effect between AP and PA in the combined product.

[0072] [Table 2]

[0073] Example 2 - Demonstration of senescent cell inhibitory activity in primary human chondrocytes To confirm the above data for the chondrocyte cell line TC28a2, we evaluated the senescent cell inhibitory ability of APPA using primary human chondrocytes.

[0074] To determine the optimal concentration of etoposide in human primary chondrocytes, etoposide at concentrations of 2, 5, and 20 μM was combined with 10 ng / ml OSM and incubated for 48 hours. The results are shown in Figures 6a and 6b. The optimal etoposide concentration was determined to be 20 μM.

[0075] The effect of APPA on the number of senescent cells, as indicated by SA-β-Gal levels, is shown in Figure 6c and Table 3. SA-β-Gal levels were assessed via quantification of fluorescein di-β-D-galactopyranoside (FDG) using flow cytometry, as in Example 1.

[0076] [Table 3]

[0077] CDKN1 (p21) is a genetic marker of cellular senescence that encodes cyclin-dependent kinase inhibitor 1A, a regulator of cell cycle progression. Expression of this gene is tightly controlled by the tumor suppressor protein p53. Activated p53 increases p21 expression, contributing to senescence. Studies in OA have suggested that cell cycle-related proteins play a role in its pathology. Cell cycle arrest, a change that occurs in senescent cells due to increased expression of genes that inhibit proliferation, can also result in increased production of cytokines, growth factors, and matrix metalloproteinases (Loeser RF. Aging and osteoarthritis: the role of chondrocyte senescence and aging changes in the cartilage matrix. Osteoarthritis Cartilage. 2009 Aug;17(8):971-9).

[0078] Evidence of cellular senescence in tissues from aged adults can be obtained by examining for the presence of senescence markers, including histological staining for senescence-associated (SA) β-galactosidase (SA-β-Gal), SA heterochromatin, increased p53, p21, and p16, and decreased Wnt21 (Campisi J, d'Adda di Fagagna F. Cellular senescence: when bad things happen to good cells. Nat Rev Mol Cell Biol. 2007 Sep;8(9):729-40. doi:10.1038 / nrm2233. PMID:17667954).

[0079] The effect of APPA on the expression level of CDKN1 is shown in Figure 6d and Table 4. The relative mRNA expression level of CDKN1 was detected by quantitative real-time (RT)-PCR in the presence of positive stimulation + / - 10 μg / ml APPA. Data were obtained from eight independent donors. Values ​​are expressed as mean ± SEM and were calculated using the Mann-Whitney test ( * p≦0.05).

[0080] RNA extraction was performed using TRIzol® (Sigma-Aldrich) according to the manufacturer's protocol. 0.5 μg of RNA was reverse transcribed into cDNA using SuperScript VILO (Thermofisher Scientific, Waltham, Massachusetts, USA) according to the manufacturer's instructions. RT-PCR was performed using TaqMan Universal Master Mix (Roche) on a LightCycler 480-II Instrument (Roche, Mannheim, Germany). Analysis of results was performed using Qbase+ version 2.5 software (Biogazelle, Ghent, Belgium). Gene expression was calculated relative to the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

[0081] [Table 4]

[0082] In primary human chondrocytes stimulated with 20 μM etoposide and 10 ng / ml oncostatin M, APPA significantly reduced the level of SA-β-galactosidase, indicating a decrease in the number of senescent cells. APPA also reduced the expression level of CDKN1A (p21), a gene marker of cellular senescence. This is consistent with the results for TC28a2 in Example 1.

[0083] FIG. 7 and Tables 5 and 6 show the effects of AP, PA, and APPA on the levels of SA-β-Gal and the expression of the CDKN1A (p21), BECN-1, and BCL2L13 genes in human chondrocytes.

[0084] The effects of AP, PA, and APPA on the number of senescent cells, as indicated by SA-β-Gal levels, are shown in Figure 7b and Table 5. SA-β-Gal levels were assessed by quantification of fluorescein di-β-D-galactopyranoside (FDG) using flow cytometry, as in Example 1 and Figure 6.

[0085] [Table 5]

[0086] When AP, PA, and APPA were tested in human chondrocytes, only APPA significantly reduced SA-β-Gal levels. AP and PA individually had no significant effect (see Figure 7b). The effect of APPA at 10 μg / ml was greater than expected based on (i) the effect of 10 μg / ml AP or 10 μg / ml PA, and (ii) the additive effect of 2.3 μg / ml AP and 7.7 μg / ml PA. This again suggests a synergistic effect between AP and PA in the combined product.

[0087] The effects of APPA, AP, and PA on CDKN1A gene expression are shown in Figure 7c and Table 6. The relative mRNA expression levels of cyclin-dependent kinase inhibitor 1A (CDKN1) were detected by quantitative real-time (RT)-PCR in the same manner as above. Data were obtained from six independent donors. Values ​​are expressed as mean ± SEM and analyzed by Mann-Whitney test ( * p ≤ 0.05; #p ≤ 0.05). * : Comparison with base condition, #: Comparison with 20 μM etoposide + 10 ng / ml oncostatin M.

[0088] [Table 6]

[0089] Figure 7c and Table 6 show that APPA 10 μg / ml significantly reduced the level of CDKN1A (p21) expression (1.893 ± 0.48 vs. 0.904 ± 0.07, p = 0.05), as did PA 10 μg / ml (1.893 ± 0.48 vs. 0.869 ± 0.17, p = 0.05), but neither the concentration of AP nor PA 7.7 μg / ml had a significant effect.

[0090] Figure 7d shows the effects of APPA, AP, and PA on the gene expression of BECN-1. BECN-1 is a regulator of autophagy. The encoded protein, beclin 1, is a component of the phosphatidylinositol-3-kinase (PI3K) complex, which mediates the vesicular trafficking process. Autophagy is a self-degradation process important for balancing energy sources at critical times during development and in response to cellular stress. Although autophagy is not a form of cell death, it is important for cartilage homeostasis. Generally, autophagy promotes cell survival by allowing cells to adapt to stressful conditions, but this process is also considered a non-apoptotic cell death program (Almonte-Becerril M, Navarro-Garcia F, Gonzalez-Robles A, Vega-Lopez MA, Lavalle C, Kouri JB. Cell death of chondrocytes is a combination between apoptosis and autophagy during the pathogenesis of osteoarthritis within an experimental model. Apoptosis. 2010 May;15(5):631-8. doi:10.1007 / s10495-010-0458-z. PMID:20091349).

[0091] The reduction in BECN-1 expression by etoposide and OSM was significantly counteracted by APPA at 10 μg / ml and PA at 7.7 μg / ml, as shown in Figure 7d, indicating an effect on autophagy. Interestingly, APPA alone reduced BECN-1 expression levels below those of unstimulated chondrocytes (p=0.051, Figure 7d, column 8). Figure 7e shows the effects of APPA, AP, and PA on the gene expression of BCL2L13. BCL2L13 encodes a mitochondrial-localized protein with a conserved B-cell lymphoma 2 homology motif. Overexpression of the encoded protein results in apoptosis. Apoptosis is positively correlated with the severity of cartilage destruction and matrix depletion in human osteoarthritis tissue specimens (doi:10.3390 / ijms16036093). Anti-apoptotic proteins Bcl-2 and Bcl-XL inhibit cytochrome c (cyt-c) release (Musumeci G,Castrogiovanni P,Trovato FM,Weinberg AM,Al-Wasiyah MK,Alqahtani MH,Mobasheri A.Biomarkers of Chondrocyte Apoptosis and Autophagy in Osteoarthritis.Int J Mol Sci.2015 Aug 31;16(9):20560-75.doi:10.3390 / ijms160920560.PMID:26334269;PMCID:PMC4613218.).

[0092] APPA does not appear to regulate BCL2L13 expression, which is consistent with the apoptosis results in Figure 4, indicating that APPA has no apparent effect on either early or late apoptosis, and therefore, the reduction in senescence as a result of APPA administration does not occur via increased apoptosis.

[0093] Example 3 - Liquid Formulation To prepare 1000 mg of APPA formulation, which is a stable liquid at room temperature, 777.8 mg of paeonol (available from Sigma-Aldrich Gillingham) is mixed with 222.2 mg of apocynin (available from Sigma-Aldrich Gillingham). The mixture is heated, and 555.6 mg of PEG 400 is added while stirring the mixture. A stable mixture is produced that does not solidify when stored overnight at approximately 3°C.

[0094] Example 4 - Capsule Formulation - 400 mg APPA Capsules To prepare a formulation containing approximately 400 mg of APPA, 311.1 mg of paeonol (available from Sigma-Aldrich Gillingham) is mixed with 88.9 mg of apocynin (available from Sigma-Aldrich Gillingham). The mixture is heated, and 222.2 mg of PEG 400 (available from Sigma-Aldrich Gillingham) is added while stirring. This results in 0.533 ml of a liquid formulation of paeonol and apocynin that is stable at room temperature. This can be encapsulated in a soft gel capsule by methods known in the art to obtain a pharmaceutical product in capsule form.

[0095] Example 5 - Solid Formulation To prepare a solid formulation, the following preparation can be used to make "O" shaped capsules (474.5 mg total).

[0096] [Table 7] Compositions and uses defined in the following numbered statements have been disclosed hereinbefore: 1. A composition comprising apocynin or an isomer thereof and paeonol or an isomer thereof for use in the treatment of aging, wherein administration of the composition results in a reduction in the number of senescent cells and a concomitant increase in the number of total viable cells. 2. A composition comprising apocynin or an isomer thereof and paeonol or an isomer thereof for use in the treatment of aging by a senolytic mechanism of action. 3. The composition of paragraph 1 or 2, wherein administration of the composition results in an increase in cell number. 4. A composition comprising apocynin or an isomer thereof and paeonol or an isomer thereof for use in the treatment of aging by a senolytic mechanism of action. 5. A composition described in any one of paragraphs 1 to 4, comprising apocynin and paeonol. 6. A composition described in any one of paragraphs 1 to 5, wherein the ratio (by weight) of paeonol to apocynin is 3:2 to 9:1. 7. A composition according to any one of paragraphs 1 to 6, wherein the formulation is a liquid formulation. 8. The composition of any one of paragraphs 1 to 6, wherein the formulation is a solid formulation, such as a pill, tablet, or capsule. 9. A composition according to any one of paragraphs 1 to 8 for use in treating an age-related disease. 10. A composition according to any one of paragraphs 1 to 9 for use in treating osteoporosis. 11. A composition described in any one of paragraphs 1 to 10 for use in treating sarcopenia. 12. A composition according to any one of paragraphs 1 to 11 for use in treating chronic obstructive pulmonary disease. 13. A composition described in any one of paragraphs 1 to 12 for use in the treatment of idiopathic pulmonary fibrosis. 14. A composition according to any one of paragraphs 1 to 13 for use in the treatment of pulmonary diseases such as pulmonary hypertension and pulmonary arterial hypertension. 15. A composition according to any one of paragraphs 1 to 14 for use in treating fatty liver. 16. The composition of any one of paragraphs 1 to 15 for use in treating kidney diseases such as acute kidney injury (AKI), chronic kidney disease (CKD), tubulointerstitial fibrosis (TIF), glomerulosclerosis, and vascular sclerosis. 17. A composition according to any one of paragraphs 1 to 16 for use in the treatment of atherosclerosis. 18. A composition according to any one of paragraphs 1 to 17 for use in treating type 1 diabetes. 19. A composition described in any one of paragraphs 1 to 18 for use in the treatment of age-related macular degeneration. 20. A composition according to any one of paragraphs 1 to 19 for use in the treatment of Alzheimer's disease, Parkinson's disease, frontotemporal dementia, amyotrophic lateral sclerosis and / or multiple sclerosis. 21. A composition described in any one of paragraphs 1 to 20, which is administered orally. 22. Use of apocynin or an isomer thereof and paeonol or an isomer thereof in the manufacture of a medicament for the treatment of aging. 23. Use of apocynin or an isomer thereof and paeonol or an isomer thereof in the manufacture of a medicament for the treatment of age-related diseases. 24. Use of apocynin or an isomer thereof and paeonol or an isomer thereof in the manufacture of a medicament for the treatment of osteoporosis, sarcopenia, lung diseases such as chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), pulmonary hypertension (PH) and pulmonary arterial hypertension (PAH), liver diseases such as acute liver injury, chronic liver disease and fatty liver, kidney diseases such as acute kidney injury (AKI), chronic kidney disease (CKD), tubulointerstitial fibrosis (TIF), glomerulosclerosis and angiosclerosis, vascular diseases such as atherosclerosis, type 1 diabetes, age-related macular degeneration (AMD), Alzheimer's disease (AD), Parkinson's disease, frontotemporal dementia, amyotrophic lateral sclerosis or multiple sclerosis. 25. A method for treating an age-related disease, the method comprising the step of administering to a patient in need thereof a composition comprising apocynin or an isomer thereof and paeonol or an isomer thereof; or A method for treating aging, comprising administering to a patient in need thereof a composition comprising apocynin or an isomer thereof and paeonol or an isomer thereof; or 1. A method for treating osteoporosis, sarcopenia, pulmonary diseases such as chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), pulmonary hypertension (PH) and pulmonary arterial hypertension (PAH), liver diseases such as acute liver injury, chronic liver disease and fatty liver, kidney diseases such as acute kidney injury (AKI), chronic kidney disease (CKD), tubulointerstitial fibrosis (TIF), glomerulosclerosis and vascular sclerosis, vascular diseases such as atherosclerosis, type 1 diabetes, age-related macular degeneration (AMD), Alzheimer's disease (AD), Parkinson's disease, frontotemporal dementia, amyotrophic lateral sclerosis or multiple sclerosis, A method comprising the step of administering a composition comprising apocynin or an isomer thereof and paeonol or an isomer thereof to a patient in need thereof.

Claims

1. A composition comprising apocynin or an isomer thereof and paeonol or an isomer thereof for use in the treatment of aging, wherein administration of said composition results in a reduction in the number of senescent cells and a concomitant increase in the number of total viable cells.

2. A composition comprising apocynin or an isomer thereof and paeonol or an isomer thereof for use in the treatment of aging by a senolytic mechanism of action.

3. A composition comprising apocynin or an isomer thereof and paeonol or an isomer thereof for use in the treatment of aging by a senolytic mechanism of action.

4. A composition according to any one of claims 1 to 3, comprising apocynin and paeonol.

5. 5. A composition according to any one of claims 1 to 4, wherein the ratio (by weight) of paeonol to apocynin is from 3:2 to 9:

1.

6. The composition according to any one of claims 1 to 5, wherein the formulation is a liquid formulation.

7. The composition according to any one of claims 1 to 6, wherein the formulation is a solid formulation, such as a pill, tablet or capsule.

8. A composition according to any one of claims 1 to 7 for use in the treatment of age-related diseases.

9. A composition according to any one of claims 1 to 8 for use in the treatment of osteoporosis.

10. A composition according to any one of claims 1 to 9 for use in the treatment of sarcopenia.

11. A composition according to any one of claims 1 to 10 for use in the treatment of chronic obstructive pulmonary disease.

12. A composition according to any one of claims 1 to 11 for use in the treatment of idiopathic pulmonary fibrosis.

13. A composition according to any one of claims 1 to 12 for use in the treatment of pulmonary diseases such as pulmonary hypertension and pulmonary arterial hypertension.

14. A composition according to any one of claims 1 to 13 for use in the treatment of fatty liver.

15. 15. The composition of any one of claims 1 to 14 for use in the treatment of kidney diseases such as acute kidney injury (AKI), chronic kidney disease (CKD), tubulointerstitial fibrosis (TIF), glomerulosclerosis and vascular sclerosis.

16. A composition according to any one of claims 1 to 15 for use in the treatment of atherosclerosis.

17. A composition according to any one of claims 1 to 16 for use in the treatment of type 1 diabetes.

18. A composition according to any one of claims 1 to 17 for use in the treatment of age-related macular degeneration.

19. 19. A composition according to any one of claims 1 to 18 for use in the treatment of Alzheimer's disease, Parkinson's disease, frontotemporal dementia, amyotrophic lateral sclerosis and / or multiple sclerosis.

20. The composition of any one of claims 1 to 19, which is administered orally.

21. Use of apocynin or an isomer thereof and paeonol or an isomer thereof in the manufacture of a medicament for the treatment of aging.

22. Use of apocynin or an isomer thereof and paeonol or an isomer thereof in the manufacture of a medicament for the treatment of age-related diseases.

23. Use of apocynin or an isomer thereof and paeonol or an isomer thereof in the manufacture of a medicament for the treatment of osteoporosis, sarcopenia, lung diseases such as chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), pulmonary hypertension (PH) and pulmonary arterial hypertension (PAH), liver diseases such as acute liver injury, chronic liver disease and fatty liver, kidney diseases such as acute kidney injury (AKI), chronic kidney disease (CKD), tubulointerstitial fibrosis (TIF), glomerulosclerosis and angiosclerosis, vascular diseases such as atherosclerosis, type 1 diabetes, age-related macular degeneration (AMD), Alzheimer's disease (AD), Parkinson's disease, frontotemporal dementia, amyotrophic lateral sclerosis or multiple sclerosis.

24. A method for treating an age-related disease, the method comprising administering to a patient in need thereof a composition comprising apocynin or an isomer thereof and paeonol or an isomer thereof; or A method for treating aging, comprising administering to a patient in need thereof a composition comprising apocynin or an isomer thereof and paeonol or an isomer thereof; or 1. A method for treating osteoporosis, sarcopenia, pulmonary diseases such as chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), pulmonary hypertension (PH) and pulmonary arterial hypertension (PAH), liver diseases such as acute liver injury, chronic liver disease and fatty liver, kidney diseases such as acute kidney injury (AKI), chronic kidney disease (CKD), tubulointerstitial fibrosis (TIF), glomerulosclerosis and vascular sclerosis, vascular diseases such as atherosclerosis, type 1 diabetes, age-related macular degeneration (AMD), Alzheimer's disease (AD), Parkinson's disease, frontotemporal dementia, amyotrophic lateral sclerosis or multiple sclerosis, A method comprising the step of administering a composition comprising apocynin or an isomer thereof and paeonol or an isomer thereof to a patient in need thereof.

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