BACKGROUND

(a) Field

[0001] The subject matter disclosed generally relates to senotherapeutic drugs for age-related diseases and cancer.

(b) Related Prior Art

[0002] Cellular senescence is believed to limit the proliferation of aged or damaged cells. However, it has been recently shown that cellular senescence is a physiological and pathological process contributing to embryogenesis, immune response, wound repair, aging and age-related diseases, as well as, as a suppressive mechanism of tumorigenesis.

[0003] Cancer is characterized by the increase in the number of abnormal, or neoplastic, cells derived from a normal tissue which proliferate to form a tumor mass, the invasion of adjacent tissues by these neoplastic tumor cells, and the generation of malignant cells which eventually metastasise via the blood or lymphatic system to regional lymph nodes and to distant sites.

[0004] Senescent cells accumulate in an individual’s tissues and organs as they age. Their presence in an individual contributes to aging and dysfunction associated with aging, which affects millions of people worldwide.

[0005] Senescent cells are also believed to be important in inhibiting the growth of dysfunctional or damaged cells, in particular in suppressing the progression of malignant tumors.

[0006] The mechanisms of senescent cells and their role in aging or in cancer is not fully understood. However, therapy induced senescence could play a critical role in the treatment of age-related diseases and cancer. [0007] Low back pain is another global health condition that is experienced by ~80% of individuals at some point in their lifetime. This problem is the number one single cause of years lived with disability with enormous personnel and health system related costs. Intervertebral disc (IVD) degeneration is a major factor contributing to low back pain. The cellular pathogenesis of IVD degeneration and the mechanisms leading to pain are not fully understood.

[0008] One novel approach to treat painful degeneration is to target cellular senescence, a state of irreversible growth arrest occurring in response to cellular stress. Stress-induced premature senescence is caused by factors such as extensive loading, oxidative and genotoxic stresses. Increasing evidence suggests that accumulation of senescent cells during tissue degeneration contributes directly to initiation and development of musculoskeletal degenerative diseases like osteoarthritis and IVD degeneration. Senescent cells secrete a range of cytokines, chemokines, growth factors, and proteases termed as the senescence- associated secretory phenotype (SASP). These SASP factors are suggested to further induce senescence in a paracrine manner, to promote matrix catabolism and sterile inflammation in IVDs, thereby accelerating the degenerative process.

[0009] Elimination of senescent cells enhances disc tissue homeostasis in genetically modified progeroid Ercc1 ^/A and p16-3MR mice suggesting that senotherapeutic drugs have great potential to treat low back pain resulting from IVD degeneration (Patil, P. et al., Aging Cell 18:e12927. https://doi.Org/10.1111/acel.12927 (2019)). The effect could potentially be mediated by apoptotic (senoptosis) or nonapoptotic (senolysis) mechanisms or by modulating the SASP, indirectly suppressing senescence (senomorphics).

[0010] Further, interest is growing towards the use of natural senotherapeutic compounds such as quercetin, fisetin and piperlongumine, curcumin and o-Vanillin; their key advantages being low toxicity and great potential to be translated into clinical applications (Cherif, H. et al. Curcumin and o-Vanillin Exhibit Evidence of Senolytic Activity in Human IVD Cells In Vitro. J Clin Med 8, doi:10.3390/jcm8040433 (2019)). However, these results are solely in vitro cell culture and there is a major leap to demonstrate ex vivo and in vivo activity.

[0011] Given the high specificity of senolytics with respect to organs and diseases, there is a need for natural senotherapeutic drugs for age-related diseases and cancer.

SUMMARY

[0012] According to an embodiment, there is provided a pharmaceutical composition comprising: a core comprising a purified o-Vanillin or a synthetic biological molecule mimicking o-Vanillin generated by artificial intelligence; and a layer of natural polymeric material enveloping the core.

[0013] According to another embodiment, there is provided a stable aqueous composition comprising: purified o-Vanillin or a synthetic biological molecule mimicking o-Vanillin generated by artificial intelligence; and a solubilizing agent.

[0014] According to an embodiment, the composition further comprising at least one senolytic drug, an antagonist of toll-like receptor 1 , 2, 4 and 6, and combination thereof.

[0015] According to another embodiment, there is provided a method of treating age-related diseases in an individual by administering a therapeutically effective amount of the present composition for a sufficient period of time to selectively eliminate senescent cells in said individual.

[0016] The administration may be a daily dose of purified o-Vanillin to a patient for a period of time from 10 days to 20 days, wherein said daily dose comprises about 0.75 to about 1 .25 teaspoons of o-Vanillin.

[0017] The administration may be a daily dose of a synthetic biological molecule mimicking o-Vanillin generated by artificial intelligence to a patient for a period of time from 10 days to 20 days, wherein said daily dose comprises about 0.75 to about 1 .25 teaspoons of o-Vanillin.

[0018] The age-related diseases may be selected from the group consisting of an adult onset diabetes, diabetes, diabetic ulcers, metabolic syndrome, obesity, arthritis, kidney and bladder health conditions, dementia, Parkinson’s disease, Huntington’s disease, glaucoma, lung disease, cataracts, eczema, psoriasis, hyperpigmentation, nevi, rash, atopic dermatitis, hives, photosensitivity or diseases related to photoaging, wrinkles, pruritus, eczema rash, eosin-favored dermatosis, reactive neutrophilic dermatosis, pemphigus, pemphigoid, immune blistering dermatosis, fibrohistiocytosis of skin, osteoporosis, enlarged prostate, Alzheimer’s disease, macular degeneration, depression, cardiovascular disease, motor neuron dysfunction, an inflammatory or autoimmune disease or disorder selected from the group consisting of osteoarthritis, osteoporosis, oral mucositis, inflammatory bowel infection, lupus, kyphosis, asthma, and a herniated disc.

[0019] The age-related disease may be a painful, degenerate or herniated disc.

[0020] According to another embodiment, there is provided a method of treating non-cancer aging related diseases in an individual by administering a therapeutically effective amount of the present composition for a sufficient period of time to selectively eliminate senescent cells in said individual.

[0021] The non-cancer aging related diseases may be selected from the group consisting of an adult onset diabetes, diabetes, diabetic ulcers, metabolic syndrome, obesity, arthritis, kidney and bladder health conditions, dementia, Parkinson’s disease, Huntington’s disease, glaucoma, lung disease, cataracts, eczema, psoriasis, hyperpigmentation, nevi, rash, atopic dermatitis, hives, photosensitivity or diseases related to photoaging, wrinkles, pruritus, eczema rash, eosin-favored dermatosis, reactive neutrophilic dermatosis, pemphigus, pemphigoid, immune blistering dermatosis, fibrohistiocytosis of skin, osteoporosis, enlarged prostate, Alzheimer’s disease, macular degeneration, depression, cardiovascular disease, motor neuron dysfunction, an inflammatory or autoimmune disease or disorder selected from the group consisting of osteoarthritis, osteoporosis, oral mucositis, inflammatory bowel infection, lupus, kyphosis, asthma, and a herniated disc.

[0022] The non-cancer aging related diseases may be a painful, degenerate or herniated disc.

[0023] According to another embodiment, there is provided a method of treating cancer comprising: administering a therapeutically effective amount of purified o-Vanillin to an individual suffering from cancer for a sufficient period of time to selectively eliminate senescent cells and cancer cells in said individual.

[0024] According to another embodiment, there is provided a method of treating cancer comprising: administering a therapeutically effective amount of a synthetic biological molecule mimicking o-Vanillin generated by artificial intelligence to an individual suffering from cancer for a sufficient period of time to selectively eliminate senescent cells and cancer cells in said individual.

[0025] According to another embodiment, there is provided a use of a therapeutically effective amount of the composition for treating age-related diseases in an individual to selectively eliminate senescent cells in said individual.

[0026] According to another embodiment, there is provided a use of a therapeutically effective amount of the composition for a sufficient period of time for treating non-cancer aging related diseases in an individual to selectively eliminate senescent cells in said individual.

[0027] According to another embodiment, there is provided a use of a therapeutically effective amount of purified o-Vanillin to an individual suffering from cancer for a sufficient period of time for treating cancer to selectively eliminate senescent cells and cancer cells in said individual. [0028] According to another embodiment, there is provided a use of a therapeutically effective amount of a synthetic biological molecule mimicking o- Vanillin generated by artificial intelligence to an individual suffering from cancer for a sufficient period of time for treating cancer to selectively eliminate senescent cells and cancer cells in said individual.

[0029] According to another embodiment, the senolytic drug is selected from the group consisting of inhibitors of the Bcl-2 family of apoptosis regulatory proteins, inhibitors of the p53/MDM2 complex, HSP-90 and PI3K/Akt inhibitors and natural flavonoids.

[0030] According to another embodiment, the senolytic drug is selected from the group consisting of RG-7112, CRX 527, Metformin, Ruxotinilib, Cortisol, Corticosterone, KU-60019, NBD peptide, Dasatinib, ABT-263 (Navitoclax), ABT- 737, A1331852 A1155463, UBX0101 , UBX1325, P5091 , Geldanamycin,

Rapamycin, Resveratrol, Kaempferol, Apigenin, EGCG, Loperamide, niguldipine, Mmu-miR-2910-3p, Quercetin, Fisetin, Piperlongumine, EF24 curcumin analog, Curcumin, o-Vanillin, Ouabain, Digoxin, ABT-263 (Navitoclax), ABT-737, A1331852, UBX1325, A1155463, P5091 , Geldanamycin, Tanespimycin (17-AAG), Alvespimycin (17-DMAG), FOX04-DRI, Panobinostat, and MitoTam

List of Abbreviations:

[0031] IVD = intervertebral disc

[0032] TLR = Toll-like receptors

[0033] SASP = senescence-associated secretory phenotype [0034] Dimethyl-Methylene Blue Assay = DMMB [0035] ICC = immunocytochemistry

[0036] ELISA = enzyme-linked immunosorbent assay

[0037] NP = nucleus pulposus [0038] AF = annulus fibrosus

[0039] sGAG = sulfated glycosaminoglycans

[0040] Senolytic drugs. Synthetic drugs that selectively target and remove senescent cells (senolytic) have recently been identified (Kirkland JL, Tchkonia T. J Intern Med. 2020;288(5):518-536. doi:10.1111/joim.13141). The mode of action and target of some senolytics are well characterized while the mechanism of others is still not clear. There are 4 main groups described to date. 1) Inhibitors of the Bcl-2 family of apoptosis regulatory proteins. 2) Inhibitors of the p53/MDM2 complex, that alleviate resistance to apoptosis. 3) HSP-90 and PI3K/Akt inhibitors, releasing pro-apoptotic transcription factors. 4) Natural flavonoids with a less clear mode of action.

[0041] The drug UBX0101, an inhibitor of the p53/MDM2 complex, known to reduce senescent cell burden in cartilage, is in clinical trials for the treatment of knee osteoarthritis. The compound completed Phase I clinical trials and entered Phase II trials for treatment of moderate to severely painful knee osteoarthritis (UNITY Biotech). The first Phase II results were recently released indicating that the drug failed to demonstrate less pain than placebo after short-term follow-up. Results for long-term follow-up and repeated treatment have not yet been released. RG7112, a drug we will use here, is also a member of the p53/MDM2 complex inhibitors. It is FDA approved for acute myeloid leukemia and underwent clinical trials to treat a range of cancers. The drug lacked efficiency for cancer and the high doses used lead to hematological toxicity. Flowever, in accordance with the present composition and method, a low non-toxic concentration removed senescent IVD cells in vitro, ex vivo and in vivo. Low doses will potentially avoid the described side effects in patients treated for IVD degeneration

[0042] One drawback of using a single senolytic agent is the failure to target multiple senescent anti-apoptotic pathways in the same cell type, or different cell populations within a target tissue. Concurrently targeting multiple and indirectly related anti-apoptotic pathways may result in increased selectivity for senescent cells in the absence of toxicity for normal proliferating or quiescent cells. A successful combination therapy is exemplified by the combination of dasatinib and quercetin that targets antiapoptotic networks, instead of a single target. The lower therapeutic dosages enabled by combinations also decreased side effects associated with single drugs. Clinical Phase II trials were initiated in 2020 with a combination of fisetin, dasatinib and quercetin, results are expected in 2022 and 2023. This is also demonstrated in cancer treatment, where the combination of chemotherapy with metformin, a known inhibitor of the SASP, provided prolonged tumor remission. Combined treatment at lower doses may allow the repurposing of drugs that, were previously discarded due to undesirable side effects and increase success in clinical trials.

[0043] Examples of senomorphics and synthetic senolytics: Metformin, Ruxotinilib, Cortisol, Corticosterone, KU-60019, NBD peptide. Dasatinib, ABT-263 (Navitoclax), ABT-737, A1331852 A1155463, UBX0101 ,UBX1325, P5091 ,

Geldanamycin, Tanespimycin (17-AAG), Alvespimycin (17-DMAG), FOX04-DRI, Panobinostat, MitoTam, Rapamycin, Resveratrol, Kaempferol, Apigenin, EGCG, Ruxotinilib, Cortisol & Corticosterone, Loperamide & niguldipine, Mmu-miR-2910- 3p, Quercetin, Fisetin, Piperlongumine, EF24 curcumin analog, Curcumin, o- Vanillin, Ouabain, Digoxin, ABT-263 (Navitoclax), ABT-737, A1331852, UBX1325, A1155463, P5091, Geldanamycin, Tanespimycin (17-AAG), Alvespimycin (17- DMAG), FOX04-DRI, Panobinostat, MitoTam.

[0044] Features and advantages of the subject matter hereof will become more apparent in light of the following detailed description of selected embodiments, as illustrated in the accompanying figures. As will be realized, the subject matter disclosed and claimed is capable of modifications in various respects, all without departing from the scope of the claims. Accordingly, the drawings and the description are to be regarded as illustrative in nature, and not as restrictive and the full scope of the subject matter is set forth in the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Further features and advantages of the present disclosure will become apparent from the following detailed description, taken in combination with the appended drawings, in which:

[0046] Fig 1. RG-7112 treatment of IVD pellet cultures. (A) Representative images indicating how we deemed positive and negative p16 ^//VK4a (a), Ki-67 (d), and caspase-3 (g) staining (b, e and h) Magnified images of a, d and g. Arrow heads indicate positive (green) and negative (red) staining (c, f and i) no secondary antibody control photomicrographs. Quantification of (B) p16 ^//VK4a (n =7), (C) Ki-67, and (D) caspase-3 expression, (n =8). Scale bars: 20 pm in (A). The cells were from degenerating IVDs as indicated in Table 2. Values are presented as mean ± SEM in (B-D). ^* indicates a significant difference assessed by the two- tailed Student's t-test: p < 0.05; ^** p < 0.01 and ^**** p < 0.0001 .

[0047] Fig 2. Differentially expressed senescence related genes in NP pellets. (A) Venn diagrams of the differentially up and downregulated genes among the different groups. O-Vanillin, RG-7112 treated NP cells in pellets culture. For Upregulated genes odds ratio (OR) = 2.13 and p = 0.39; for Downregulated genes: OR = 0 and p = 1 . (B) Heatmap of the top 44 over and under expressed genes in control (CTRL), RG-7112 and o-Vanillin treated NP cells. All genes shown were first normalized to the housekeeping gene GAPDH. Data shown are relative to the calculated Z scores across the samples (see methods) and ranked by significance adjusted to p < 0.05. Red represents relatively high levels of expression; blue represents relatively low levels of expression. Significantly differentially expressed genes are indicated with green lines for o-Vanillin and in orange lines for RG-7112. Each column represents one individual (for a total of n=5 per group) and each row represents expression of a single gene. Donor ID and gender are indicated for each subject. (C) Volcano plots of mRNA expression of o-Vanillin and RG-7112 treated NP pellets: Plotted along the x-axis is the mean of log2 fold-change, along the y-axis the negative Iog10 of the p-values. Blue circles refer to downregulated genes, red circles refer to upregulated genes and grey circles to non-DEGs in o- Vanillin and RG-7112 treated NP pellets. The horizontal grey line is the negative logarithm of the t-test-adjusted p-value threshold (-Iog10 of p = 0.05). (D) IPA diagrams of differentially expressed genes in RG-7112 and (E-G) o-Vanillin- treated NP pellets within the selected set of 91 genes. Direct and indirect interactions are shown by solid lines and dashed lines respectively. Green indicates gene downregulation; red depicts upregulation and molecules found by the data mining tools of IPA (build tools) are shown in gray. Color intensity represents the average of log2 fold change with brighter colors representing a more significant difference between treated and controls. Symbols for each molecule are presented according to molecular functions and type of interactions. Functional assignations attributed by IPA software. Significant difference set at p < 0.05 was assessed by repeated measures Analysis of Variance (ANOVA) with Turkey’s post hoc test for multiple pairwise comparison in (B-C) and Fisher’s exact test in (A, D-G). The cells were from degenerating IVDs as indicated in Table 2.

[0048] Fig 3. Profile of SASP factors released from pellet cultures following senolytic treatment. (A) Culture media was analyzed by a RayBio Fluman Cytokine Array. Relative mean densitometry units of the 80 factors were normalized to untreated controls with the most 25 downregulated SASP factors presented: cytokines (A-a), CC-chemokines (A-b), CXC-chemokines (A-c), growth and neurotrophic factors (A-d). Scatter plot showing the distribution in average change of 50 cytokines quantified using cytokine array (A-e).

[0049] (B) Heatmap displaying quantification of 19 selected cytokines (19- plex Luminex® array). Each column represents one individual (n=5). The rows represent expression of a single protein. Data shown are log2 (fold change) relative to the average expression level in each condition. Donors ID and gender are indicated for each subject. (C) Significantly downregulated factors are presented as mean fold difference ± SEM; (n = 5). Culture media was collected from the same NP cells used in Figure 2. ^* lndicates significant difference assessed by repeated measures Analysis of Variance (ANOVA) with Turkey’s post hoc test for multiple pairwise comparison: p < 0.05 and ^indicates p < 0.01 .

[0050] Fig 4. RG-7112 and o-Vanillin effects in ex vivo human IVD culture. (A) Schematic of the ex vivo organ culture experiment. Lumbar spines from organ donors were assessed radiographically for signs of degeneration. Three discs per experiment were isolated from the same spine, cultured for 4-6 days then scanned with MRI and injected with vehicle, o-Vanillin or RG-7112. Discs were then cultured for an additional 28 days, with media changes every 3-4 days. The discs were scanned by MRI again at day 28. Media and tissues were used for SASP factor release and histology respectively. (B) Representative images of mid-axial Ti p slices pre-treatment (a-b, e-f, i-j) and the same location post treatment (c-d, g-h, k- I) with vehicle (CTRL), RG-7112 (5 mM) or o-Vanillin (100 pM). The heatmap correlates the red color with the highest and the blue color with the lowest Tip values. (C) Quantification for NP regions with the graph showing percentage change in Ti p values post- compared to the pre-treatment scans. (D) Representative safranin O/fast green staining of histological sections. (E) Representative images of disc sections stained with antibodies against p16 ^//VK4a and Ki-67. Quantification of (F) p16 ^//VK4a and (G) Ki-67 expression. Scale bars = 150 pm in 4D, 25 pm in 4E (p16 ^INK4a ) and 50 pm in 4E (ki-67); Error bars represent mean ± SEM, Statistical significance was assessed by two-tailed Student's t-test to compare pre and post disc groups (Figure 4C) and by repeated measures Analysis of Variance (ANOVA) with Turkey’s post hoc test for multiple pairwise comparison in (Figure 4F and 4G). Indicates p < 0.05 and ^indicates p < 0.01 , n = 4 for each condition. The tissues were from degenerating IVDs as indicated in Tables 2 and 3.

[0051] Fig 5. Profile of SASP factors released from ex vivo human IVDs cultures following senolytic treatment. Culture media was analyzed by a RayBio Human Cytokine Array. Relative mean densitometry units of the 80 factors were normalized to pre-treatment media of the same IVD. The percentage change (post /pre) of the 15 most affected factors are shown for RG-7112 and o-Vanillin (A) and vehicle (B) treated discs. (C) Heatmap displaying quantification of 19 selected cytokines (19-plex Luminex® array). Each column represents one individual and each row represent expression of a single protein. Data shown are log2 (fold change) in pre or post treated disc media relative to their respective expression level average. (D) 9 analytes (INF-g, TNF-oc, CCL11 , CCL24, CXCL1 , CXCL9, CXCL10, Angiogenin and VEGF) displayed statistically significant differences when measured in post compared to pretreated disc media. Error bars represent mean ± SEM. Statistical significance when comparing pre and post discs groups was assessed by two-tailed Student's t-test (Figure 5A, 5B and 5D): ^* Indicates p < 0.05 and ^** indicates p < 0.01. Data was analyzed by repeated measures Analysis of Variance (ANOVA) with Turkey’s post hoc test for multiple pairwise comparison (Figure 5D) where # Indicates significant difference (p < 0.05) between treated and untreated groups, (n = 4). The analyzed media were collected from same donors used in Figure 4.

[0052] Figure 6. Senolytic activity of RG-7112 on human IVD cells in vitro. (A) Metabolic activity in control and treated NP and AF pellet cultures from degenerate IVDs (n=6). (B) Representative photomicrographs of untreated (a-c) and RG-7112 treated (d-f) NP cells stained for cytocalcein (viable), apopxin (apoptotic) and merged images. (C) Quantification of apoptosis in treated and control groups (n = 3). (D) p16 ^//VK4a separate-staining with Ki-67 (a-c) and colocalization with caspase-3 (d-f) in NP cells (n = 3). (E) RG-7112 (5 mM) selectively induced apoptosis in degenerate NP cells compared to non-mildly- degenerate as measured by the caspase 3/7 activity kit. The results are expressed as a percentage compared to the untreated control (n = 8). (F) Alamar blue assay comparison of the effect of RG-7112 (5 pM) on the viability of degenerate and non degenerate NP cells. Results are presented as fold-change compared to the control (n = 8). (G) Glycosaminoglycan (sGAG) levels in the NP and AF treated pellet media at days 7, 14, 21 and 28 were assessed by DMMB. GAG release was normalized to day 0 GAG concentration and then to the sGAG concentration of untreated culture media (n = 6). Scale bars: 50 m in (B) and 25 Mm (D). Values are presented as mean ± SEM in (A, C, E, F and F). Data was analyzed by Student’s t-tests to compare two groups. ^* indicates a significant difference of p < 0.05, ^** of p < 0.01 and ^*** of p < 0.001.

[0053] Figure 7. Non differentially expressed genes Fleatmap and NP area measure in human IVD. (A) 47 genes did not show significant expression change in RG-7112 (5 pM) and o-Vanillin (100 mM) treated NP cells when compared with control (CTRL). See methods (Bioinformatics analysis) for statistical calculation and genes ranking. Quantification of the average NP (B) area (C) and position in human IVD. Red lines indicate the average ratio (n=11 discs). (D) Schematic representation of the average NP area and position.

[0054] Figure 8. Total cytokine array and Luminex® measures in pellet culture media. (A) Schematic representation of the media analysis by cytokine arrays and Luminex assay. Cytokine/chemokine array quantification of the media in NP cell pellets treated with (B) o-Vanillin (100 mM) and (C) RG-7112 (5 mM). Results present the percentage of change in treated compare to untreated pellet cultures. (D) Nine inflammatory factors in pellet media (TNF-oc, IL-1oc, IL-1 b, CCL5, CCL11 , CCL26, CXCL11 , CX3CL1 and Angiogenin) displaying no statistically significant decrease measured by Luminex® assay. Data is presented as mean ± SEM and was analyzed by repeated measures Analysis of Variance (ANOVA) with Turkey’s post hoc test for multiple pairwise comparison (n = 5).

[0055] Figure 9. Complete profiling and measure of growth factors and cytokines in disc cultures media. Total cytokine/chemokine array quantification of inflammatory factors release in (A) Control, (B) o-Vanillin and (C) RG-7112 discs. Shown in the graphs is the ratio (% change) of the factors detected in the cultures media of post-treated and compared with their respective pre-treated cultures media. (D) Ten analytes in disc media (IL-1 a, IL-1 b, IL-6, IL-8, CCL5, CCL7, CCL26, CXCL5, CXCL11 and CX3CL1 ) displayed no statistically significant decrease when compared with their respective controls. Data is presented as mean ± SEM and was analyzed by t-tests for pre and post groups and by repeated measures Analysis of Variance (ANOVA) with Turkey’s post hoc test for multiple pairwise comparison (n = 4).

[0056] Figure 10 illustrates pain relief in SPARC mice following oral administration of o-Vanillin. Improved behavioral signs of axial and radiating pain after 8 weeks of oral administration. Green: WT-vehicle, black: SPARC-vehicle, blue: SPARC-o-vanillin (100 mg/kg), red: SPARC-RG7112 (5 mg/kg). ^** p<0.01 , ^*** p<0.001 , ^**** p<0.0001 , mixed effects 2- way ANOVA followed by Dunnett’s post-hoc test. Arrows indicating more (red) and less (green) pain.

[0057] Figure 11 illustrates cell activation with a TLR-2/6 agonist, caused an increase in the number of senescent cells and SASP factor release in cells from non-degenerate human IVDs. (A) p16 ^ink4a immunostaining images of untreated cells (a-d) from healthy donor IVDs or treated with either TLR-2/6 agonist (e-h), TLR-1/2 agonist (i-l) or TLR-4 agonist (m-p) cultured for 6, 12, 24 or 48 hours. Scale Bars= 25pm. (B) Quantification of the percentage of p16 ^ink4a positive cells in the control and treated cells for the four time points. Examples of positive cell are indicated by the red arrow and for negative cells by the black arrow. (C, D, E) qPCR was performed using cells from healthy donor IVDs cultured for 48 hours with TLR- 2/6 agonist, TLR-1/2 agonist or TLR-4 agonist. Gene expression for (C) TLR-1 , - 2, -4 and -6), (D) senescence markers p16 ^ink4a and p21 and (E) SASP factors (CCL2, CCL5, CCL7, CCL8, IL-6, IL-8, TNF-oc, NGF and BDNF). Fold changes were normalized relative to the non-treated control (CTRL). (B-E) Two-way ANOVA with Tukey’s multiple comparisons test, n = 5, significance was was evaluated between treated groups compared to control group. Data shown as mean ± SD, ^* p<0.05, ^** p<0.01 , ^*** p<0.001 , ^**** p<0.0001 . [0058] Figure 12 illustrates that O-Vanillin reduced the number of senescent cells and enhanced proteoglycan production in cell pellet cultures from degenerate IVDs. All experiments were performed on day 21. (A) Representative photomicrographs of p16 ^ink4a immunohistochemistry staining in pellet cultures from degenerate IVD cells either treated with o-Vanillin or not (CTRL). Examples of positive cell are indicated by the red arrow and for negative cells by the black arrow. (B) Quantification of the percentage of p16 ^ink4a positive cells in the pellet cultures. n=3 (C) Images for Safranin 0 staining for proteoglycan content in CTRL and o-Vanillin cell pellet cultures from degenerate IVD. (D) sGAG concentration measured using DMMB assay in the culture media of CTRL and o-Vanillin cell pellets. n=5 (E) Percentage of change in concentration of IL-1 b, IL-8, IL-6 and TNFoc in pellet culture media from untreated and o-Vanillin treated degenerate IVD cells measured by ELISAs. Percent difference was evaluated by normalizing the post-treated media to the pretreated media. n=5. ^* p<0.05, ^** p<0.01 , ^**** p<0.0001 . (B, D, E) Mean ± SD, Statistical analysis was done using paired t-test.

[0059] Figure 13 illustrates that o-Vanillin reduced gene expression of p16, TLR-2 and SASP factors following TLR-2 activation in IVD cells from patients with back pain and IVD degeneration. (A-C) Gene expression of (A) senescence markers p16 ^ink4a and p21 , (B) TLR-1 , -2, -4 and -6 and (C) SASP factors (CCL2, CCL5, CCL7, CCL8, GM CSF, BDNF, NGF, TNFoc, TGF-b, CLCX1 , CLCX8, CLCX10) of disc cells from degenerate IVDs cultured for 48 hours with TLR-2/6 agonist with o-Vanillin (TLR-2/6 + VAN) or without o-Vanillin (TLR-2/6) treatment for 6h or no induction with TLR-2/6. Fold changes were normalized relative to non- induced control. ^# p<0.05, ^## p<0.01 , ^### p<0.001 indicate significant difference between the TLR-2/6 treated to the non-induced control and ^* p<0.05, ^** p<0.01 , ^*** p<0.001 indicate significant difference between groups TLR-2/6 + VAN and TLR-2/6. (A-C) Mean ± SD, measured by Two-way ANOVA with Sidak’s multiple comparisons test. Values are expressed in average fold change for n = 5. [0060] Figure 14 illustrates o-Vanillin reduced the protein expression of SASP factors (II_-1b, NGF, IL-8 and TNF-oc) following TLR-2/6 activation of IVD cells from patients with back pain and IVD degeneration. Disc cells from degenerate IVD were cultured for 48 hours with TLR-2/6 with o-Vanillin (TLR-2/6 + VAN) or without o-Vanillin (TLR-2/6) treatment for 6h or no induction with TLR2/6 (CTRL). (A-D) Using Immunocytochemistry, untreated and treated IVD cells were stained for DAPI and either (A) IL-1 b, (B) NGF, (C) IL-8 (D) TNFoc respectively. Scale Bars= 25pm. (E) Quantification of the percentage of the cells that stained positive for IL-1 b, NGF, IL-8 and TNFoc when non-induced, treated with TLR-2/6 agonist or treated with TLR-2/6 agonist and o-Vanillin. n=5 (F) ELISAs were performed to measure the concentration of TNFoc, IL-1 b and IL-8 in cell media and NGF from cell lysate in non-induced, TLR-2/6 agonist treated or the combined treatment TLR-2/6 agonist and o-Vanillin treated samples. Percentage of positive cells in (E) were the average for n = 5. ^# p<0.05, ^## p<0.01 , ^### p<0.001 indicate significant difference between the TLR-2/6 agonist treated to the non-induced control and ^* p<0.05, ^** p<0.01 , ^*** p<0.001 indicate significant difference between the tested groups (E-F) Mean ± SD, statistical analysis was done using paired t- test.

[0061] Figure 15 illustrates that o-Vanillin reduced the number of cells co expressing TLR-2 and p16 ^ink4a in cells exposed to TLR-2/6 agonist. Disc cells from degenerate IVDs were induced with TLR-2/6 agonist for 48 hours with (TLR- 2/6+VAN) or without (TLR-2/6) o-Vanillin treatment for 6 hours or no induction with TLR-2/6 agonist (CTRL). (A) Photomicrographs of IVD cells stained for DAPI (blue) and either p16 ^ink4a (green), TLR-2 (red), or the merge (p16 ^ink4a and TLR-2) as revealed by Immunocytochemistry. DAPI, p16, TLR2 and merge images scale bars= 25pm. Enlarged images scale bar: 10pm. (B-D) Quantification of the percentage of IVD cells that stained positive for (B) TLR-2 (C) p16 ^ink4a or (D) co localized cell for TLR-2 and p16 ^ink4a . Percentage of positive cells in (E) were the average for n = 5. ^# p<0.05, ^## p<0.01 , ^### p<0.001 indicate significant difference between the TLR-2/6 agonist treated to the non-induced control and ^* p<0.05, ^** p<0.01 , ^*** p<0.001 indicate significant difference between the TLR-2/6 agonist with o-Vanillin treated to the TLR-2/6 agonist treated. (B-D) Mean ± SD, statistical analysis was done using paired t-test.

[0062] Figure 16 illustrates the methods used.

[0063] Figure 17 illustrates pro-inflammatory cytokines are significantly reduced in 9-month-old SPARC mouse IVDs treated with senolytics ex-vivo. Mobile SASP factor released from untreated WT and treated, and untreated SPARC IVDs were measured by Luminex.

[0064] Figure 18 illustrates chemokines are significantly reduced in 9- month-old SPARC mouse IVDs treated with senolytics ex-vivo. Mobile SASP factor released from untreated WT and treated, and untreated SPARC IVDs were measured by Luminex.

[0065] Figure 19 illustrates pro-inflammatory cytokines are significantly reduced in SPARC-null mice following 8-weeks of senolytic treatment. Mobile SASP factor released from untreated WT and treated, and untreated SPARC IVDs were measured by Luminex.

[0066] Figure 20 illustrates chemokines are significantly reduced in SPARC- null mice following 8-weeks of senolytic treatment. Mobile SASP factor released from untreated WT and treated, and untreated SPARC IVDs were measured by Luminex.

[0067] Figure 21 illustrates that preventative treatment improved behavioral signs of axial and radiating pain after 8 weeks of 6-month-old mice. Acetone test; radiating leg pain, Von Frey; Mechanical sensitivity. Blue: SPARC-vehicle, Black: WT-vehicle, Green: SPARC- 100 mg/kg o-Vanillin + 5mg/kg RG-7112, Red: SPARC- 100 mg/kg o-Vanillin + 2.5mg/kg RG-7112, Yellow: SPARC- 50 mg/kg o- Vanillin + 5mg/kg RG-7112, Purple: SPARC- 50 mg/kg o-Vanillin + 2.5mg/kg RG- 7112, Pink: SPARC-100 mg/kg O-Vanillin, Orange: SPARC-5 mg/kg RG-7112. [0068] Figure 22 illustrates that preventative treatment improved behavioral signs of axial and radiating pain after 8 weeks of 6-month-old mice. Grip force; axial pain. Blue: SPARC-vehicle, Black: WT-vehicle, Green: SPARC- 100 mg/kg o-Vanillin + 5mg/kg RG-7112, Red: SPARC- 100 mg/kg o-Vanillin + 2.5mg/kg RG- 7112, Yellow: SPARC- 50 mg/kg o-Vanillin + 5mg/kg RG-7112, Purple: SPARC- 50 mg/kg o-Vanillin + 2.5mg/kg RG-7112, Pink: SPARC-100 mg/kg O-Vanillin, Orange: SPARC-5 mg/kg RG-7112.

[0069] Figure 23 illustrates the histology assessment of mouse lumbar spine. A) Histological scoring of mouse IVDs as described in Millecamps et al. , 2015 ( The Spine Journal 15.12 (2015): 2524-2537). B) Histological assessment of lumbar spines following 8 weeks of oral gavage treatment of senolytics. WT= WT- Vehicle, SPARC= SPARC-vehicle, o-Vanillin= SPARC-o-Vanillin (100 mg/kg), RG- 7112 = SPARC-RG7112 (5 mg/kg).

[0070] Figure 24 illustrates method for: tumour microenvironment model.

[0071] Figure 25 illustrates that chemotherapy (Doxorubicin) treatment induce cell senescence in MDA-MB-231 (breast cancer cell line) and IRM-90 (fibroblast cell line). The percentage of p16 positive senescent MDA-MB-231 and IRM-90 cells were quantified in monolayer cultures treated with Doxorubicin for 72 Flours.

[0072] Figure 26 illustrates preliminary data showing that a combination of chemotherapeutic and senolytic treatment further reduce cancer cell viability and tumor size. Spheroids of MDA-MB-231 cells, representing the tumor are situated in a collagen matrix supplemented with IRM-90 cells representing tumor stroma following chemotherapeutic and senolytic treatment.

[0073] Figure 27 illustrates that RG-7112 and o-Vanillin combination reduced the number of p16 positive cells greater than RG-7112 and o-Vanillin alone. DETAILED DESCRIPTION

[0074] Here we utilized in vitro and ex vivo models, to assess the senotherapeutic effects of RG-7112 compared to o-Vanillin on naturally occurring senescent cells in degenerating human IVDs. o-Vanillin is a natural compound that in addition to its senolytic effect has antioxidant and anti-inflammatory properties. We compared the effect of o-Vanillin to RG-7112, a pure senolytic drug, that we found to efficiently kill senescent AF and NP cells. The objective was to evaluate if o-Vanillin with its dual function could further reduces inflammatory factors released by non-senescent cells thus enhancing the therapeutic effect.

[0075] The main objective is to demonstrate if one or both drugs could reach and kill senescent IVD cells in their native environment of intact human disc with naturally occurring degeneration. RG-7112 known for its senolytic activity was also used as a benchmark comparator to demonstrate the senolytic activity and unexpected effect of o-Vanillin.

[0076] Methods [0077] Human Disc Collection

[0078] Human lumbar IVDs were harvested from organ donors through a collaboration with Transplant Quebec. All procedures are approved by and performed in accordance with the ethical review board at McGill University (IRB#s A04-M53-08B). Familial consent was obtained for each subject. Table 1 provides an overview of donor demographics and Table 2 provides detailed description of the used discs. Lumbar spinal columns were removed from organ donors, they were imaged radiographically and visually, and signs of degeneration were noted. Discs were then dissected from the spinal column and used for cell and organ cultures. Nucleus pulposus (NP) and annulus fibrosis (AF) cells were isolated separately as described previously. All cultures were mycoplasma free as verified by Mycoplasma PCR Detection Kit (ZmTech ^® Scientific).

[0079] Table 1. Donor demographics. [0080] Table 2. Complete donors list per assays and results. [0081] Alamar Blue Metabolic Activity in Monolayer Cell Culture

[0082] 10,000 cells were seeded per well in 96-well tissue culture plates for

12 h. Then, cells were incubated in the presence or absence of RG-7112 (5 mM) for 6 h. Metabolic activity was evaluated by the Alamar blue assay as previously described ⁸² We measured the metabolic activity of treated and untreated NP cells cultured in monolayer or in pellet from degenerate and non-degenerate discs to verify a non cytotoxic window of RG-7112. Results are presented as a percentage of metabolic activity compared to their control. Experiments were performed (3-6) times in triplicate wells for each compound and concentration.

[0083] Pellet Cell Culture & Immunohistochemistry

[0084] 300,000 NP cells/tube were collected by centrifugation at 500* g for

5 min. Pellets were incubated in 1 ml_ DMEM (2.25 g/L glucose, 5% FBS, ascorbic acid (5 pM) (Sigma-Aldrich, Oakville, ON, Canada)) at 37°C and 5% CO2. Culture media was changed every 3 days and collected for analysis. After 4 days, the pellet culture is stabilized and a single dose of the senolytics or vehicle was added to the culture media. Metabolic activity measures on pellet culture media from degenerate NP and AF cells, treated or not with RG-7112, was performed to evaluate a potential cytotoxic effect of RG-7112 by comparing, in the same pellet culture, the effect of the treatment (at day 21 ) to the control before treatment (at day 4). RNA was collected in TRIzol (Thermo Fisher Scientific) for gene expression experiments (as described in Krock et al., J Cell Mol Med 18, 1213-1225, doi: 10.1111/jcmm.12268 (2014)). For immunohistochemistry, pellets were fixed in 4% paraformaldehyde and cryopreserved, 5 pm sections were prepared and stained overnight at 4 °C for p'\6 ^lnk4a and 1 h at room temperature for Ki-67 and caspase-3 primary antibodies as described {J Clin Med 8, doi:10.3390/jcm8040433 (2019)). Coverslips were mounted using Aqua Polymount, and bright-field images were visualized. Ten fields, randomly distributed across the well, were analyzed, and the number of positive (brown stained) and total cells were counted to calculate the percentage of senescent, proliferative and apoptotic cells. Senescence, Apoptosis and proliferation assessments were performed on cells pellet cultured for 21 days by comparing, in the same subject, the senolytics-treated pellet to their respective controls.

[0085] Monolayer Cell Culture & Immunofluorescence

[0086] Isolated cells were expanded to Passage 1 (P1 ) in monolayer cultures. P1 cells were then seeded at 20,000 or 10,000 cells per well in 8-well chamber slides (Nunc™ Lab-Tek™ II Chamber Slide™ System) and 96- well flat clear bottom black microplates (Corning, NY, USA) respectively. Cells were serum- starved in DMEM with ITS (1X) (Thermo Fisher, Waltham, MA, USA) for 2 h prior to treatment with 5 mM RG-7112 (Selleck Chemicals, TX, USA), 100 mM o-Vanillin (Sigma-Aldrich, Oakville, ON, Canada) or vehicle (DMSO (0.01 %, (Sigma-Aldrich, Oakville, ON, Canada) for 6 h. Immunocytochemistry was performed as previously described {J Clin Med 8, doi:10.3390/jcm8040433 (2019)). Apoptosis was detected using a commercial kit (ab176749, Abeam, Cambridge, Ma, USA) according to the manufacturer’s instructions. Photomicrographs were acquired with a fluorescent Olympus BX51 microscope equipped with an Olympus DP71 digital camera (Olympus, Tokyo, Japan).

[0087] Caspase 3/7 Activity in Monolayer Cell Culture

[0088] Caspase 3/7 activity of treated and untreated NP cells from degenerate and non-degenerate IVDs was measured using the Amplite Fluorimetric Caspase 3/7 Assay Kit (AAT Bioquest, Sunnyvale, CA, USA) according to the manufacturer’s protocol. Cells were incubated with the caspase 3/7 assay solution, which contained caspase substrate (Z-DEVD-R110), at room temperature for 1 h in the dark. Fluorescence intensity was then measured at 490 nm excitation and 525 nm emission. The results are expressed as a percentage of the mean of the control group (set at 100%). Each experiment was performed in triplicate and carried out three times from each round of cell isolation. [0089] DMMB in Pellet Cell Culture

[0090] Sulphated glycosaminoglycans (GAGs) were quantified using the DMMB assay in the media of degenerate NP and AF pellets with or without RG- 7112 treatment, performed as previously described ( Methods Mol Med 135, 201- 209 (2007)). Chondroitin sulfate was used to generate the standard curve. Conditioned media samples from days 7, 10, 14 and 21 were evaluated separately in triplicate into clear 96-well plates (Costar, Corning, NY, USA). sGAG release in media was normalized to the sGAG concentration in media at day 0 and then normalized to the untreated group.

[0091] Quantitative Real-Time PCR in Pellet Cell Culture

[0092] Following treatment, RNA was extracted using the TRIzol chloroform extraction method, as previously described ( J Cell Mol Med 18, 1213-1225, doi: 10.1111/jcmm.12268 (2014)). Briefly, 500 ng of RNA was reverse transcribed using a qScript cDNA Synthesis Kit (Quanta Biosciences, Beverly, MA, USA) with an Applied Biosystems Verti Thermocycler (Thermo Fisher, Waltham, MA, USA). RT-qPCRwas performed using an Applied Biosystems StepOnePlus machine with TaqMan® Fast Universal PCR Master Mix (2X) and Custom TaqMan® Array 96- Well Fast Plates (Thermo Fisher, Waltham, MA, USA). The evaluated genes are senescent and apoptotic genes included in the Fluman TaqMan® Array, for Human Cellular Senescence (Thermofisher scientific, Array ID: RPU62T7, Catalog number: 4413255). The Array Plate was customized to include 12 additional recently identified senescence and anti-apoptotic pathway genes ( Int J Oncol 3, 473-476 (1993)). The 96 included genes are described in Cherif H et al. (Elife. 2020 Aug 21 ;9:e54693. doi: 10.7554/e Life.54693. PMID: 32821059; PMCID: PMC7442487). Each well of the TaqMan® Array Plate was reconstituted using a mix of Fast Master Mix and a cDNA sample (20 ng) to a set final volume (10 pi). Five plates were used for each group (CTRL, RG-7112 and o-Vanillin), and fold- change in gene expression was calculated using the 2 ^_DDa method ( Methods 25, 402-408, doi:10.1006/meth.2001 .1262 (2001 )) after normalizing to the housekeeping gene and vehicle-treated cells.

[0093] Bioinformatics Analysis

[0094] We conducted a gene expression study of a pre-specified set of apoptoticand senescence-genes of interest (Custom Taqman 96-Well Fast Plates, Thermofisher scientific) in nucleus pulposus cells. This single-gene approach offers the advantage that highly relevant genes can be identified and tested first. The candidate genes examined with this approach allowed us also to identify the genes within the selected group that together carry out the drug response in disc cells.

[0095] In order to mine the feature genes from different datasets, a Venn diagram analysis was conducted using Venny version 2.1.0 software. Differences in the expression levels of DEGs for treated and untreated groups were obtained, and the number of DEGs upregulated and downregulated were calculated. The odds ratio (OR) was calculated according to OR = (DEGs_o-Vanillin ^* DEGs_RG- 7112 / DEGs_o-Vanillin ^* NonDEGs_RG-7112) / (NonDEGs_o-Vanillin ^* DEGs_RG-71 12 / nonDEGs_o-Vanillin ^* NonDEGs_RG-7112).

[0096] Heatmaps of gene expression pattern were constructed using unsupervised hierarchical clustering using Euclidean distance metric and complete linkage clustering method of the 43 differentially expressed genes in response to either RG-7112 or o-Vanillin (P < 0.05) treatment of degenerate NP cells pellet. Differences in gene expression between o-Vanillin, RG-7112 and control groups were respectively compared via unpaired t-tests using the R package. Genes for which met the P<0.05 cut-off point were selected as DEGs, following which gene expression profiles of DEGs were visualized (heatmaps) via the ‘ggplots’ in R package (Wickham, H. ggplot2: elegant graphics for data analysis. (Springer, 2016)) and were represented in the heatmaps as Z-scores, which is: (expression value - mean expression value across samples) / divided by the standard deviation. Colors, ranging from blue to grey then red, for each treatment represents the average fold change of each subject in that group.

[0097] Differentially expressed genes were subjected to Ingenuity Pathways Analysis (IPA) (Ingenuity Systems, Redwood City, CA) and used as a starting point for building biological networks. This analysis uses cresent in our list of 91 genes) and their interactions with other genes (“non-focused”) in the knowledge base. Scores were calculated for each network according to the fit of the network to the set of focus genes and used to rank networks on the Ingenuity analysis. IPA uses the genes from the highest-scoring network to extract a connectivity pathway that relates candidate genes to each other based on their interactions. The involved function and disease significantly associated with these candidates’ genes were shown. To generate the networks, significant pathways were filtered by p-value (a) < .05 and activation Z-score > -2 or >2, set as the cut-off values and representing a significant deactivation or activation, respectively.

[0098] Colors are based on log2 fold changes on these genes. To rank networks of the IPA, p-scores were calculated from p-values. For example, for n genes in the network and f of them are Focus Genes. The p-value is the probability of finding f or more Focus Genes in a set of n genes randomly selected from the Global Molecular Network calculated using Fisher’s exact test. Since interesting p- values are typically quite low, it is visually easier to concentrate on the exponent and the p-score is defined as p-score = -loglO(p-value). Networks with a score >2 have at least 99% confidence that indicate a 1/100 chance that the focus genes are in a network because of random chance.

[0099] Ex-vivo Organ Culture and MRI Analysis

[00100] Intact lumbar spines were x-rayed, and discs were selected for the study based on the grading system described by Wilke et at. ( Eur Spine J 15, 720- 730, doi: 10.1007/s00586-005-1029-9 (2006)). Discs with a Wilke grade of 2 were included for this study. Three IVDs from the same spine (n = 4 spines, 12 discs) were isolated and cultured as previously described ( Spine (Phila Pa 1976) 36, 1835-1842, doi:10.1097/BRS.0b013e3181f81314 (2011 ), J Cell Mol Med 18, 1213-1225, doi: 10.1111/jcmm.12268 (2014), Eur Cell Mater 31, 26-39 (2016)). Disc characteristics are described in Table 3. Isolated discs were scanned by MRI before and after treatment at day 28 as described by Rosenzweig et al. ( Eur Cell Mater 36, 200-217, doi:10.22203/eCM.v036a15 (2018)). Images were obtained on a 7T Bruker BioSpec 70/30 USR (Bruker Biospin, Milton, ON, Canada) with the high-performance mini-imaging kit gradient upgrade AVI 11 electronics (Bruker) and a Bruker-issued T1 p-RARE pulse sequence, as previously established ( Eur Cell Mater 36, 200-217, doi:10.22203/eCM.v036a15 (2018), Eur Spine J 24, 2395- 2401 , doi: 10.1007/s00586-014-3582-6 (2015)). Briefly, 3D images were acquired and Tip values were quantified using the MIPAV software (NIH Center for Information Technology, Bethesda, MD, USA). Ti p values of the same region of interest (ROI) of 'before' and 'after' treatment scans of each disc were normalized to the surrounding culture medium using editing features in MIPAV software. After 4 days of culture and one media change, single injection of the discs with vehicle, RG-7112 (5 pM/g disc) or 100 pM o-Vanillin (100 pM/g disc) in a total volume of 200 pi PBS was performed as previously described ( J Cell Mol Med 18, 1213-1225, doi: 10.1111/jcmm.12268 (2014), Eur Cell Mater 31, 26-39 (2016)). Discs were then cultured in DMEM supplemented with 1x Glutamax, 50 pg/ml gentamicin and 1 % FBS for 28 days. Media was changed and collected every 4 days. On day 28 and after MRI imaging, discs were prepared for immunohistochemistry as described below (See Intact IVD Tissue Immunohistochemistry). Conditioned media was collected at each change and frozen as individual samples at -80 °C for protein analysis.

[00101] Preliminary measures to delimitate the NP region of interest (ROI) were calculated using 11 -disc images from random organ donors. A contour was drawn around the disc using a Wacom Intuos Pro tablet and stylus. (Wacom, Japan). This polygon was used to measure total disc voxel intensity. The NP area was created by reshaping the original disc contour to 30% of its frontal size and 40% of its sagittal size centered around the middle of NP area, which was calculated to be 10% shift below the center of the disc (Supplemental Figure 7B- D). The T 1 p images were manually cropped around the perimeter of the IVDs. The average of the Ti p values was calculated within the ROIs for the vehicle and the injected discs slices per image. Heat maps representing signal intensity were created using the MIPAV software.

[00102] Table 3. Characteristics of discs injected with each treatment.

[00103] Age of an individual donor is specified in years. The level of the disc injected with either DMSO, O-Vanillin, and RG-7112 from the lumbar region is indicated. Discs were graded based on Wilke et al. 2006 ( Eur Spine J 15, 720-730, doi:10.1007/s00586-005-1029-9 (2006)). Disc height was determined by averaging the dorsal, ventral and midsection disc height. No differences in grade or height were found between groups prior to treatment.

[00104] Human Cytokine Array of Pellet and Intact IVD Culture Media

[00105] Media from the Degenerate NP Pellet used in gene expression analysis was collected from day 4 to day 21 and pooled separately for each group (vehicle, o-Vanillin and RG-7112) and subject. Then, they were analyzed and compared to evaluate the effect of the two senolytics on SASP release. Disc media collected from day 4 (pre-treatment) and day 28 (post-treatment) were analyzed separately for each disc and each factor. The Human Cytokine Antibody Array C5 (RayBiotech, Inc.) was used for semi quantitative detection of 80 proteins according to manufacturer’s instructions and as previously described {J Cell Mol Med 18, 1213-1225, doi:10.1111/jcmm.12268 (2014)). Intensity units were detected by the chemiluminescence using an ImageQuant LAS4000 Image Analyzer (GE Healthcare, Baie d'Urfe, QC, Canada) and analyzed with ImageQuant TL array analysis software (GE Healthcare). The relative quantity of each factor present in each media sample was normalized to the positive and negative controls included on the array. Mean relative concentration of each factor of treated and control groups were then calculated. Data was normalized to secretion of vehicle injected discs from the same donor spine. A list of included cytokines is provided in Cherif H et al. (Elife. 2020 Aug 21 ;9:e54693. doi: 10.7554/eLife.54693. PMID: 32821059; PMCID: PMC7442487).

[00106] Luminex ^® Mulitplex Assay of Pellet and Intact IVD Culture Media

[00107] 19 proteins were selected for analysis by Luminex ^® multiplex assay according to manufacturer's instructions. A limitation in the number of factors we could measure was the incompatibility of some factors to be measured simultaneously as indicated by the supplier. Concentrations (pg/mL) (INF-g, TNF- oc, IL-1oc, IL-1 b, IL6, IL8, CCL5, CCL7, CCL11 , CCL24, CCL26, CXCL1 , CXCL5, CXCL9, CXCL10, CXCL11 , CX3CL1 , VEGF-A and Angiogenin) were measured in 40 pi media. Median fluorescence intensity (MFI) from microspheres was acquired with a BD FACSCanto II and analyzed in FlowCytomix Pro2.2.1 software (eBioscience). Concentration of each analyte was obtained by interpolating fluorescence intensity to a 7-point dilution standard curve supplied by the manufacturer.

[00108] Intact IVD Tissue Immunohistochemistry [00109] For intact IVDs, post-MRI analysis, a 2 mm wide sagittal tissue segment from the center of the IVD was fixed in periodate lysine paraformaldehyde (PLP) fixative overnight at 4 °C. Samples were then washed in PBS and decalcified using Shandon™ TBD1 ™ Decalcifier solution (ThermoFischer Scientific) over 72 h at 4 °C, changing solution each day. Tissue segments were washed in PBS and placed in 70% ethanol prior to paraffin embedding. Sections of 5 pm were cut and mounted on glass slides. All sections were heated on a hot plate at 55 °C for 45 min and deparaffinized and rehydrated. Next, sections were stained with safranin- 0/ fast green (Sigma-Aldrich, Oakville, ON, Canada) and with antibodies against p1 Qink4a _anc | «i-67 and counter stained using the DAB detection IHC Kit (ab64264, Abeam, Cambridge, Ma, USA) following the manufacturer’s instructions. All images were acquired using a Zeiss Axioskop 40 and an AxioCam MR (Zeiss) and processed using AxioVision LE64 software (Zeiss).

[00110] Statistical Analysis

[00111] The data was analyzed using Graph Prism 8 (Graph Pad, La Jolla, CA, USA). Analysis was performed by two-tailed Student's t test for comparison between two groups and a multiple pairwise comparison (Analysis of Variance (ANOVA) was used to evaluate the variance between multiple groups with Turkey’s post hoc test. A p value <0.05 was considered statistically significant.

[00112] Determination of the senotherapeutic activity of RG-7112

[00113] We aimed to compare the senotherapeutic activity of the natural senolytic o-Vanillin a natural senolytic with antioxidative and anti-inflammatory properties with a pure synthetic senolytic drug. We chose the commercially available FDA approved drug RG-7112 as a candidate. A concentration of 5 mM RG-7112, shown as a safe and effective dose for human IMR90 lung fibroblasts, was used to evaluate its effect on senescent and non-senescent human IVD cells. Pellet cultures of IVD cells from degenerate nucleus pulposus (NP) and annulus fibrosus (AF) regions were exposed to RG-7112 for 4 days, the drug was removed, and the treated pellets were maintained for 21 days in standard media. Then, evaluated for cytotoxicity, senolytic and therapeutic activity. No cytotoxicity was observed following treatment. In contrast, both NP (9.87%; p < 0.05) and AF (11.87%; p < 0.05) cells showed a significant increase in metabolic activity (Fig. 6A). Pellet cultures were treated as before and the presence of p'\6 ^lnk4a , Ki-67 and caspase-3 were analyzed after21 days (Fig. 1A). RG-7112 significantly decreased the percentage of p'\6 ^lnk4a positive NP (11.48%; p < 0.001 ) and AF (20.03%; p < 0.0001 ) cells compared to untreated control cultures (Fig. 1 B). Similarly, RG-7112 significantly increased the percentage of Ki-67 positive NP (23.07%; p < 0.05) and AF (34.92%; p < 0.0001 ) cells (Fig. 1C). Caspase-3 positive AF cells increased significantly (17.88%; p < 0.01 ) while a non-significant increase (2.94%; p = 0.51 ) was observed in NP cells following treatment (Fig. 1 D). Caspase 3 and caspase 3/7 activity was confirmed by fluorescence microscopy and activity assays respectively (Fig. 6B-C and 6D(d-f)). Confocal immunofluorescence confirmed co localization of p'\6 ^lnk4a and caspase-3 whereas proliferating (Ki-67-positive) cells did not co-localize with p'\6 ^lnk4a (Figure 6D(a-c). RG-7112 selectively increased apoptosis (Fig. 6E) while it maintained comparable metabolic activity (Fig. 6F) in NP cells from both degenerate and non-degenerate IVDs. Finally, we investigated proteoglycan synthesis using the DMMB assay following treatment. A significant increase in proteoglycan release in conditioned media was observed after 14 days of treatment (Fig. 6G).

[00114] Gene expression analysis revealed the potential pathways by which RG-7112 and o-Vanillin mediate their senolytic effect

[00115] To identify molecular pathways affected by RG-7112 and o-Vanillin in human disc cells, gene expression analysis of 96 selected cellular senescence genes was performed and 91 of the 96 genes were expressed at a detectable level. Pellet cultures were treated with RG-7112 (5 mM) or o-Vanillin (100 pM) for 4 days. The relationship between the differentially up and downregulated genes are depicted in Figure 2A. Compared with the control group, the number of differentially expressed genes for o-Vanillin were 40 (30 upregulated and 10 downregulated) and 8 for RG-7112 (6 upregulated and 2 downregulated). In upregulated DEGs, three are common to both drugs, mitogen-activated protein kinase 14 (MAPK14), cell division cycle 25c (CDC25c) and cyclin dependant kinase 2D (CDKN2D or p'\9 ^ARF ). No down-regulated DEGs were common to the drugs. Although cyclin B1 (CCNB1) is significantly expressed following the treatment with the two compounds, it is upregulated in RG-7112 and downregulated in o-Vanillin (Figure 2A). Of the 91 genes identified, 44 were differentially expressed with a P<0.05 in one or both treatments (Figure 2B). The 47 genes that did not meet the significance criteria of p < 0.05 are shown in (Fig. 7A). Next, we compared gene expression profiles of the o-Vanillin group with the RG-7112 group. Of the 91 evaluated, only 8 genes were significantly affected by RG-7112 treatment, 4 were common to o-Vanillin (MAPK14, CCNB1 , CDC25c and CDKN2D (r19 ^L * ), and four were exclusive to RG-7112 (MDM2, CDKN1A {p2'\ ^ARF ), E2F1 and RBL1). In contrast, 40 genes were significantly affected by o- Vanillin treatment. Cell cycle and senescence genes were significantly downregulated including cyclin dependent kinase 2A (CDK2A or p'\6 ^lnk4a ), cyclin dependent kinase 2C (r18 ^LK/= ), Cyclin A2, CCNB1 , CDC25c, Vimentin, Mitogen- activated protein kinase 6 (MAPK6) and Checkpoint kinase 1 (CFIEK1 ) (Fig. 2C). Examples of apoptotic and proliferative genes significantly upregulated by o- Vanillin include B-cell lymphoma 2 (Bcl-2), Bcl-2-like 1 , Bcl-2-like 2, interferon regulatory factor 5 (IRF5), IRF7 and receptor tyrosine-protein kinase ERBB2. Interestingly, MAPK14, CDC25c and CDKN2D (p'\9 ^ARF ) were the only genes significantly upregulated after the treatment with both compounds, while CCNB1 showed opposite regulation patterns. In conjunction, apoptotic pathways are significantly upregulated to kill senescent cells while proliferation related pathways are activated in non-senescent cells.

[00116] To gain a further insight into a potential mechanism of action, the differentially expressed genes were mapped to networks in the Ingenuity Pathways Analysis (IPA) database. The scores consider the number of focus genes and the size of the network to approximate the relevance of the network to the original list of focus genes. The IPA core analysis features allowed identification and determination of one network connecting the cell cycle, cell death and survival, connective tissue development and function pathways (RG-7112, network 1 ) of NP cells treated with RG-7112 (Fig. 2D). In the o-Vanillin treated pellets, the highest- scoring network revealed a significant link with cell death and survival, neurological disease and organismal injury and abnormalities (o-Vanillin, network 1 ) (Fig. 2E). Furthermore, connective tissue development and function, cell cycle (o-Vanillin, network 2), cancer, cellular movement (o-Vanillin, network 3), were shown to be influenced in the other two networks (Fig. 2F-G). All networks were identified and ranked by the score of the calculated p-value of the IPA assay within the selected set of 91 genes, the scores and molecules used to order these networks are shown in Cherif H et al. (Elife. 2020 Aug 21 ;9:e54693. doi: 10.7554/e Life.54693. PMID: 32821059; PMCID: PMC7442487). These results confirmed the expected mode of action for RG-7112 and provide new insights to the predicted pathways involved in senescence and the responses to treatment with o-Vanillin.

[00117] RG-7112 and o-Vanillin reduced inflammatory factors

[00118] Transcriptomic results following the treatment with the two senolytics predicted the activation of apoptotic (in senescent cells) and proliferative (in non- senescent cells) pathways which suggest a reduction in the senescence burden likeley affecting SASP factor release. To verify an effect on SASP, media of untreated NP cell pellets, RG-7112 and o-Vanillin treated pellets were used. The media collected were from the same donors used for gene expression analysis in Figure 2. Samples were analyzed using an antibody array that simultaneously screen 80 factors. Out of the 80 factors present, 50 were detected in at least four donors and were included in further analysis (Figure S3A). The 25 most affected SASP factors are divided into 4 classes: Cytokines (Figure 3A-a), chemokines CC (Figure 3A-b), chemokines CXC (Figure 3A-c), growth and neurotrophic factors (Figure 3A-d). The most reduced factors following RG-7112 treatment compared with control were for cytokines (TNF-oc; -78.05 ± 4.69%) (p = 0.71 ), CC-chemokines (CCL26; -66.69 ± 7.09%) (p = 0.93), CXC-chemokines (CXCL13; -29.02 ± 17.26%) (p = 0.68), growth and neurotrophic factors (FIGF; -65.24 ± 5.9%) (p = 0.87). The top four factors decreased by o-Vanillin treatment were: IL-7 (-60,37 ± 11 .46%) (p = 0.5), CCL26 (-40,14 ± 3.41 %) (p = 0.99), CXCL10 (-18,11 ± 6,15%) (p = 0.62) and FIGF (-52,42 ± 5,45%) (p = 0.96) (Fig. 3A(a-d)). To better visualize the overall effect of the two drugs on SASPs release, a scatter plot of average changes was generated demonstrating a similar overall effect of the two senolytics (Figure 3A- e). The trends for all 80 factors are visualized in (Fig. 8B-C). The semi-quantitative cytokine array results were used to select 19 factor that were quantified with the Luminex ^® assay. The 19 factors were selected based on the following criteria: only factors detected in all conditions and in at least four donors were chosen. These factors were selected to represent all the SASP classes and to include the most variably expressed cytokines, chemokines and growth factors. The 19 factors selected for further investigation were (INF-g, TNF-oc, IL1 -oc, IL1 -b, IL-6, IL-8, CCL5, CCL7, CCL11 , CCL24, CCL26, CXCL1 , CXCL5, CXCL9, CXCL10, CXCL11 , CX3CL1 , Angiogenin and VEGF-A). The concentrations were measured and expression heatmap showed an overall decrease in the level of proinflammatory factors from both RG-7112 and o-Vanillin when compared to non-treated cultures. (Fig. 3B). The levels of INF-g, IL-6, IL-8 and CCL24 were significantly decreased following both treatments whereas the decrease in CCL7, CXCL1 , CXCL5, CXCL9, CXCL10 and VEGF significance was reached only following o-Vanillin treatment (Fig. 3C). However, significance was not reached for TNF-oc, IL1 -oc, IL1- b, CCL5, CCL11 , CCL26, CXCL11 , CX3CL1 and Angiogenin in treated cultures compared to untreated cultures (Fig. 8D). Although, o-Vanillin reduced more SASP factors than RG-7112 at a significant level when compared to untreated cultures, there was no significant difference in concentrations between o-Vanillin and RG- 7112 treated cultures, validating the similar overall effect the two drugs have on SASP factor release observed by cytokine array in Figure 3A-f. These results demonstrate that both RG-7112 and o-Vanillin decreased the overall inflammatory environment in pellet cultures of degenerating tissue and suggest a broader anti inflammatory effect of o-Vanillin treatment.

[00119] Senotherapeutic treatment improves disc matrix and reduces senescent cells in intact human disc.

[00120] RG-7112 or o-Vanillin was injected into the central region of intact human IVDs to verify that the drugs can reach and kill the target cells in native tissue. Discs were pre-cultured for 4-6 days. The IVDs were treated with a single injection of vehicle, o-Vanillin, or RG-7112, delivered to the centre of the NP and were cultured for another 28 days as outlined in Figure 4A. An adapted MRI protocol ( Eur Cell Mater 36, 200-217, doi:10.22203/eCM.v036a15 (2018)) was used to assess T1 p-weighted MRI signal that directly correlates with proteoglycan content ( Eur Cell Mater 36, 200-217, doi:10.22203/eCM.v036a15 (2018), Eur Spine J 24, 2395-2401 , doi:10.1007/s00586-014-3582-6 (2015)). The method used to assess uniform NP regions of interest is shown in (Fig. 7B-D). The same NP regions of interest were scanned in the same orientation pre and post treatment. Increased Tip values were found in RG-7112 and o-Vanillin treated discs while vehicle treated discs displayed decreased Tip values. (Fig. 4B(a-d)) Quantification of pre- versus post- treatment intensity values showed a non significant decrease of 13.2 ± 4.7% (p = 0.058) in vehicle treated control IVDs. In RG-7112 treated discs, there was a significant 6.8 ± 1 .5% (p = 0.024) increase in the Tip value post-treatment. Discs that were treated with o-Vanillin, displayed a significant increase of 11.1 ± 1.2% (p = 0.001 ) in Ti p values following treatment (Fig. 4C). The red dye safranin-O, binds negatively charged molecules, which are primarily represented by proteoglycan in the NP. Histological evaluation post treatment using Safranin-O/ fast green staining showed strong (intense red) staining in treated discs (Fig. 4D). Finally, p'\6 ^lnk4a and Ki-67 immunohistochemistry were performed to verify senolytic activity of the two compounds (Fig. 4E). Indeed, immunohistochemical assessment of p'\6 ^lnk4a showed a significant decrease in the number of senescent cells of 17.38 ± 1.6% (p = 0.0017) in RG-7112 and 22.65 ± 3.6% (p = 0.008) in o-Vanillin treated IVDs compared with vehicle treated control discs (Fig. 4F). Quantification of Ki-67 staining showed a non-significant increase in both RG-7112 and o-Vanillin 3.05 ± 3.4% (p = 0.4) treated IVDs (Fig. 4G). The data suggest that both senolytics can reach and kill naturally occurring senescent human IVDs cells situated in their native environment and at the same time promote tissue repair and regeneration.

[00121] RG-7112 and o-Vanillin decreased SASP factor release from intact human IVDs.

[00122] Culture media from treated and untreated discs (Figure 4) was analyzed using cytokine arrays and were compared to their respective pre treatment media. Interestingly, a single injection with RG-7112 strongly decreased secretion of several proinflam matory cytokines and chemokines including IL-7 (- 60.63% (p = 0.01 )), IL-6 (-59.39% (p = 0.14)), CXCL1 (-36.72% (p = 0.26)), GRO- abg (-32.84% (p = 0.21 )) and CCL24 (-30.57% (p = 0.15)). o-Vanillin also strongly decreased IL-7 (-83.37% (p = 0.09)), CXCL1 (-65.45% (p = 0.12)), IL-6 (-58.68% (p = 0.10)), CXCL6 (-52.47% (p = 0.009)), IGFBP-2 (-44.51 % (p = 0.15)), GRO(^y (-41 .83% (p = 0.03)) and CCL2 (-39.91 % (p = 0.05)). Both compounds significantly decreased IL-8 by 18.72% (p = 0.004) for RG-7112 and by 11.75% (p = 0.04) for o-Vanillin. We also detected a moderate increase of CXCL9, CCL22, NT3 and EGF for o-Vanillin and only in CCL2 and OPN for RG-7112 treated media (Fig. 5A). The level of the SASP factors released from vehicle treated discs showed an increase in IL-6 (530.85% (p = 0.15)), CXCL6 (196.89% (p = 0.07)), CXCL1 (99.83% (p = 0.18)), OPN (92.36% (p = 0.01 )), CCL2 (53.37% (p = 0.18)), IGFBP-2 (39.26% (p = 0.13)), IL-7 (23.52% (p = 0.53), GRO-abg, (14.25% (p = 0.91 )), CCL24 (9.64% (p = 0.97)), EGF (3.18% (p = 0.85)), TNSF-14 (2.09% (p = 0.66)), IL-8 (1.61 % (p = 0.82)) and a decrease in CXCL9 (-35.23% (p = 0.26)), CCL22 (-11.95% (p = 0.56)) and NT3 (-13.46% (p = 0.25))(Fig. 5B). The complete set of factors from all groups are presented in Fig 9 (A-C).

[00123] Luminex ^® quantification of the same 19 selected SASP factors that were analyzed in pellet cultures showed an overall decrease in inflammatory mediators in culture media from both RG-7112 or o-Vanillin treated discs compared with that of vehicle (Fig. 5C). All factors decreased in RG-7112 and o- Vanillin treated discs (Fig. 5D and Fig. 9D). Six factors in RG-7112 and five in o- Vanillin treated discs were significantly decreased in the post-treatment media compared with their respective pre-treatment media. For RG-7112, these proteins were TNF-oc (mean 12.4 pg/ml in pre, 6.8 pg/ml in post, p = 0.03), CCL11 (mean

13.1 pg/ml in pre, 7.8 pg/ml in post, p = 0.009), CCL24 (mean 201.7 pg/ml in pre,

101.1 pg/ml in post, p = 0.04), CXCL1 (mean 261.8 pg/ml in pre, 148.2 pg/ml in post, p = 0.001), CXCL10 (mean 197.1 pg/ml in pre, 158.7 pg/ml in post, p = 0.04) and Angiogenin (mean 51.7 pg/ml in pre, 37.6 pg/ml in post, p = 0.02). o-Vanillin significantly reduced the levels of INF-y (mean 1.6 pg/ml in pre, 1.1 pg/ml in post, p = 0.04), CCL24 (mean 232.7 pg/ml in pre, 73.7 pg/ml in post, p = 0.02), CXCL1 (mean 313.9 pg/ml in pre, 130.2 pg/ml in post, p = 0.01), Angiogenin (mean 47.4 pg/ml in pre, 35.5 pg/ml in post, p = 0.02) and VEGF-A (mean 24802.1 pg/ml in pre, 20905.5 pg/ml in post, p = 0.04) (Fig. 5D). Finally, we found a significant increase in the release of four factors in vehicle treated discs: INF-y(mean 1 pg/ml in pre, 1.45 pg/ml in post, p = 0.02), CCL11 (mean 7 pg/ml in pre, 19.2 pg/ml in post, p = 0.04), CXCL1 (mean 104.3 pg/ml in pre, 476.9 pg/ml in post, p = 0.03) and CXCL9 (mean 60.4 pg/ml in pre, 162.4 pg/ml in post, p = 0.01).

[00124] Discussion

[00125] In this study we aimed to compare the effects of a synthetic pure senolytic compound with a natural anti-inflammatory and senolytic compound, to determine a potential enhanced therapeutic effect with the latter. We used o- Vanillin instead of curcumin, as o-Vanillin has higher specificity and better bioavailability. For a synthetic and pure senolytic we chose RG-7112, a drug with documented senotherapeutic effects in fibroblasts. Cells from degenerate discs or intact degenerate discs that correspond with the tissue targeted for treatment were used. Cells from degenrating tissue was used as it would be difficult to evaluate an effect of removing senescent cells in non-degenerate discs since they have have very few senescent cells. Here, we demonstrated that RG-7112 has a potent senotherapeutic and a strong proliferative effect on human IVD cells in vitro. Metabolic activity and extracellular matrix production were also increased in treated cultures. We verified that treatment specifically targets senescent cells by activating the caspase-3 apoptotic pathway. This is similar to the effect of UBX0101 , an analogue of RG-7112 that triggers apoptosis of senescent chondrocytes in a murine osteoarthritis model. Currently, phase I and II clinical trials are in progress to assess safety, tolerability and clinical effects of single dose (NCT03513016 and NCT04129944) and both single and repeated doses (NCT04229225) of intra-articular administration of UBX0101 in patients with moderate to severe painful knee osteoarthritis.

[00126] Cellular senescence can be induced by replicative senescence or stress-induced premature senescence. The pathway and resulting changes in the microenvironment surrounding the cells depends on inducing factor. In this study we found gene expression modulation for several inflammatory and cell cycle genes following treatment with the two senolytics, o-Vanillin and RG-7112. We evaluated differential gene expression in 96 pre-selected genes to determine the mechanisms by which the compounds mediate their effects. Our data demonstrate that 91 of the genes were expressed at a detectable level and a significant effect was found on 50% of the expressed genes, supporting the senolytic activity of the two compounds in vitro. RG-7112 decreased gene expression of CDK1A and MDM2 and increased expression of the E2F1 , RB factors, MAPK-14, CDK2D, CCNB1 and CDC25c. o-Vanillin modulated gene expression of 40 genes including upregulation of cell cycle genes such as CDK6, CDK2C, CDK2D, and CDC25c while expression levels of CDK2A, Cyclin A2, Cyclin D1 and CCNB1 were decreased.

[00127] Collectively, the data support the mechanistic action of RG-7112 to stabilize p53 and p21 by attenuating the MDM2-p53 interaction. Also, it confirms regulation of the RB-E2F1 pathway that releases E2F1 and activates genes involved in cell cycle regulation, DNA synthesis, and cell proliferation. Another example of a senolytic drug that interferes with the E3 ubiquitin ligase-MDM2-p53 mechanism is UBX0101 , which triggers apoptosis of senescent cells in articular cartilage and synovium in a murine osteoarthritis model. o-Vanillin treatment affected multiple pathways suggesting that o-Vanillin eliminates senescent cells both by apoptosis and non-apoptotic means. Recently, p16 deletion in NP cells from mouse discs with induced degeneration was shown to also upregulate the expression of cyclin-dependent kinases 4/6, phosphorylated retinoblastoma protein, and transcription factor E2FI/2.

[00128] Clinical trials with RG-7112 for cancer treatment were limited by the high incidence of hematological toxicities. Flere we used a lower concentration (5 mM) and short exposure time of RG-7112, in contrast to the high and toxic doses used in cancer therapy (20-1400 mg/m ² administrated daily for 10 days). This may prevent these side effects in patients treated for IVD degeneration. This hypothesis was verified by the clinical study of the RG-7112 analogue UBX0101 in patients diagnosed with painful osteoarthritis of the knee. An administered dose of 4 mg was safe and well-tolerated, and it improved pain scores, reduced SASP factors and disease-related biomarkers after a single dose. The activation of multiple pathways following o-Vanillin treatment and within the selected set of 91 genes implies that diverse biological processes are affected, which was also reported for other natural senolytics like Quercetin, Fisetin, and Dasatinib. Although, it is difficult to determine how these natural senolytics eliminate senescent cells and drive their therapeutic effects, the activation of several mechanisms increases their capacity to target the heterogeneous cellular states acquired by senescent cells after the initial growth arrest.

[00129] We further sought to determine if the two senolytics had apparent effects on the SASP secretome. Conditioned media of pellet cultures was analyzed by cytokine array following treatment. Although donor variation was observed, notable changes between treatment groups were found indicating an overall decrease in the majority of the SASP factors analyzed. Similar decrease in the SASP factors was observed following p16 deletion in NP cells in mice with induced disc degeneration. An anti-inflammatory effect was expected for o-Vanillin from previous studies. However, our study documents, for the first time, anti inflammatory properties of RG-7112 in human IVD cells. We selected 19 factors from the initial screening associated with SASP and measured their concentrations using a Luminex ^® immunoassay. In general, concentrations were decreased in response to treatment. RG-7112 significantly reduced the concentrations of IFN-y, IL-6 and CCL24 while o-Vanillin in addition reduced factors IL-8, CCL7, CXCL1 , CXCL5, CXCL9, CXCL10 and VEGF-A. These factors are known for their implication in painful IVD degeneration and for promoting senescence of surrounding cells. For example, IL-8 and chemokines binding to the C-X-C motif chemokine receptors is needed for the establishment and maintenance of senescence. CCL7 expression has been reported to be concordant with IVD degeneration and it has previously been described as a SASP factor involved in IVD cell senescence. Finally, the decrease of VEGF-A indicates a possible role of o-Vanillin to block the mechanism of neovascularization. VEGF and its receptors have been proposed to be closely correlated with inflammation, chronic back pain and accelerated IVD degeneration. These results consolidate the senolytic effect of the two compounds and suggest a potential role in reducing degenerative factors in human discs with o-Vanillin having a possible stronger and broader anti inflammatory effect. [00130] Animal models that replicate human disc pathology are limited due to the differences in anatomy, disc size, cell type, and loading. Moreover, notochord cells are retained longer in most animal species, further increasing the difference between humans and animals. Thus, we previously developed and validated an ex vivo intact human disc culture system to study the potential for biologic repair and regeneration. Matrix differences were quantified pre and post treatment by Ti p weighted MRI that directly correlates with proteoglycan content in the IVDs. When comparing the intensity in RG-7112 and o-Vanillin-treated IVDs, we observed an increased intensity post-treatment, while a decrease was observed in vehicle treated control. This suggests that treatment with senolytics could increase proteoglycan content in human patients. It also validates our results from the pellet cultures where the proteoglycan content also increased significantly in the treated cultures. The improvement of Tip MRI values was validated by the consistent high levels of proteoglycan content in the treated IVDs. We further verified that the improved matrix was linked to removal of senescent cells. Indeed, both compounds significantly reduced the number of p'\6 ^lnk4a positive cells in treated discs. Moreover, we observed a slight but non-significant increase in proliferating cells. These findings correlate with the negative impact previously reported between the number of senescent cells and cell proliferation during IVD degeneration. Furthermore, culture media from control discs, showed no change in cytokine release whereas both compounds decreased levels of IL-6, IL-7, IL-8, CXCL1 , CXCL6, CCL2, CCL22, GROa/b/g, IGFBP-2, TNSF-14 and OPN. Moreover, we observed a significant increase of EGF in the media from o-Vanillin treated discs. The positive effects of EGF on proteoglycan synthesis were first reported by Thompson et ai\ these effects were more pronounced in the NP. Together the results demonstrate an overall decrease in SASP factors following treatment. The difference in SASP compounds affected by the two drugs could be explained by the difference in selectivity and the specificity of the drugs. [00131] Finally, we compared the SASP factors release in pellet and disc culture media. The common SASP factors that were downregulated include INF-g, IL-6, CCL24, CXCL1 , CXCL10 and Angiogenin. Interestingly, o-Vanillin also significantly reduced the levels of IL8, CCL7, CXCL5, CXCL9 and VEGF-A. This could be due to the effect on the neighboring non-senescent cells. Consistent with prior literature, this decrease highlights the antioxidant and anti-inflammatory properties of o-Vanillin. Also, these decreases have been reported in several other natural compounds such as quercetin, fisetin, and piperlongumine.

[00132] Our findings suggest that both o-Vanillin and RG-7112 have the potential to be translated to treatment of painful IVD degeneration. As well, this study can be used as a base for the development of additional senolytic agents for lower back pain.

[00133] The present invention will be more readily understood by referring to the following examples which are given to illustrate the invention rather than to limit its scope.

EXAMPLE 1

Low Back Pain and Disc Degeneration are Decreased Following Chronic Toll-like Receptor 4 Inhibition in a Mouse Model

[00134] Intervertebral disc degeneration is a leading cause of chronic low back pain, but current treatment is limited. Toll-like receptors (TLR) on disc cells are activated by endogenous extracellular matrix fragments and modulate degeneration in vitro. This study investigated whether inhibiting TLR4 slows disc degeneration and reduces behavioral signs of LBP in vivo.

[00135] Design: 7-9-month-old wild-type and SPARC-null (a model of disc degeneration and LBP) male mice were treated with TAK-242 (TLR4 inhibitor) once, and following a 10-day washout, mice were treated 3 times/week for 8 weeks. Behavioral signs of axial discomfort and radiating leg pain were assessed weekly with the grip force assay and acetone test, respectively. Following treatment, pain-related spinal cord changes were evaluated, and lumbar discs were excised and cultured. Cytokine secretion from discs was evaluated with protein arrays.

Results: SPARC-null mice displayed elevated signs of axial and radiating pain at baseline compared to wild-type. Chronic, but not acute, TLR4 inhibition reduced behavioral signs of pain compared to vehicle. SPARC-null mice have increased CGRP- and GFAP-immunoreactivity (astrocyte marker) in the dorsal horn compared to wild-type, which is reduced by chronic TLR4 inhibition. Ex vivo degenerating discs from SPARC-null mice secrete increased levels of many pro- inflammatory cytokines, which chronic TLR4 inhibition reduced. Using the same pre-clinical animal model (SPARC ) we have now been able to demonstrate that oral administration of o-Vanillin provides pain relief /V? vivo, Figure 10. We used the SPARC null mouse model to evaluate if the o-Vanillin and RG7112 can relieve back pain. The animals were treated weekly with oral gavage. They were about 5- 6 months when the treatment started and were sacrificed around 7- 8 months. We are currently evaluating if the drug also reduced the release of inflammatory mediators and if it effected pain signalling in the spinal cord very similar to the described procedure.

[00136] Conclusion: Chronic TLR4 inhibition decreased behavioral signs of LBP, pain-related neuroplasticity and disc inflammation in SPARC-null mice. TAK- 242 inhibits TLR4 activation within discs, as evidenced by decreases in cytokine release. Therefore, TLRs are potential therapeutic targets to slow disc degeneration and reduce pain plotted on a log-scale y-axis; statistical testing was conducted with non-transformed data. EXAMPLE 2

Toll-Like Receptor 2 Induced Senescence in Intervertebral Disc Cells of Patients with Back Pain can be Attenuated by o-Vanillin

[00137] There are an increased level of senescent cells and Toll Like

Receptor-1 -2 -4 and -6 (TLR) expression in degenerating intervertebral discs

(IVDs) from back pain patients. However, it is currently not known if the increase in expression of TLRs is related to the senescent cells or if it is a more general increase on all cells. It is also not known if TLR activation in IVD cells will induce cell senescence.

[00138] Methods: Cells from non-degenerate human IVD were obtained from spine donors and cells from degenerate IVDs came from patients undergoing surgery for low back pain. Gene expression of TLR-1 ,2,4,6, senescence and senescence-associated secretory phenotype (SASP) markers was evaluated by RT-qPCR in isolated cells. Matrix synthesis was verified with safranin-0 staining and Dimethyl-Methylene Blue Assay (DMMB) confirmed proteoglycan content. Protein expression of p'\6 ^INK4a , SASP factors and TLR-2 was evaluated by immunocytochemistry (ICC) and/or by enzyme-linked immunosorbent assay (ELISA).

[00139] Results: An increase in senescent cells was found following 48h induction with a TLR-2/6 agonist in cells from both non-degenerate and degenerating human IVDs. Higher levels of SASP factors, TLR-2 gene expression and protein expression were found following 48h induction with TLR-2/6 agonist. Treatment with o-Vanillin reduced the number of senescent cells, and increased matrix synthesis in IVD cells from back pain patients. Treatment with o-Vanillin after induction with TLR-2/6 agonist reduced gene and protein expression of SASP factors and TLR-2. Co-localized staining of p16 ^//VK4a and TLR-2 demonstrated that senescent cells have a high TLR-2 expression.

[00140] Taken together our data demonstrate that activation of TLR-2/6 induce senescence and increase TLR-2 and SASP expression in cells from non- degenerate IVDs of organ donors without degeneration and backpain and in cells from degenerating human IVD of patients with disc degeneration and backpain. The senescent cells showed high TLR-2 expression suggesting a link between TLR activation and cell senescence in human IVD cells. The reduction in senescence, SASP and TLR-2 expression suggest o-Vanillin as a potential disease modifying drug for patients with disc degeneration and backpain.

[00141] Tissue collection and cell isolation

[00142] All procedures performed were approved by the ethical review board at McGill University (IRB#s A04-M53-08B and A10-M113-13B). Non-degenerate IVDs from humans with no history of back pain were obtained through a collaboration with Transplant Quebec. Degenerate IVDs were obtained from patients with chronic low back pain that received discectomies to alleviate pain. Donor information is presented in Table 4. IVD cells were isolated, as previously described (Gawri R, et al. Arthritis Res Ther. 2014; 16(1 ): R21 . https://doi.org/10.1186/ar4449). Briefly, samples were washed in phosphate- buffered saline solution (PBS, Sigma-Aldrich, Oakville, ON, Canada) and Hank’s- buffered saline solution (HBSS, Sigma-Aldrich, Oakville, ON, Canada) supplemented with Primocin™ (InvivoGen, San Diego, CA, USA) and Fungiozone (Sigma-Aldrich, Oakville, ON, Canada). Then, the matrix was minced and digested in 0.15% collagenase type II (Gibco) for 16 hours at 37°C. Cells were passed through both a 100-pm filter and 70-pm filter, before being re-suspended in Dulbecco’s Modified Eagle Media (DMEM, Sigma-Aldrich, Oakville, ON, Canada) supplemented with 10% fetal bovine serum (FBS, Gibco), Primocin™, Glutamax (Oakville, ON, Canada), and maintained in a 5% CO2 incubator at 37°C. Table 4

[00143] Characteristics of the donors utilized for the study.

[00144] In Vitro Cell Culture and Treatment (ICC): Immunocytochemistry including p'\6 ^INK4a , immunofluorescence for NGF, IL-1 b, TNF-a, IL-8, TLR-2 and p Ί Q ^iNK4a j _{n a} monolayer culture. (IHC): Immunohistochemistry for p16 ^//VK4a and Safranin-0 in pellet culture sections. (RT-qPCR): Real-time Quantitative Polymerase Chain Reaction (ELISA): Enzyme-linked immunosorbent assays. (DMMB): Dimethyl methylene blue (DMMB) assays.

[00145] Monolayer culture: Experiments were performed with NP cells from non-degenerate IVDs and degenerate IVDs (NP and AF cells) within passage 1 to 2. 20,000 cells were seeded in 8-well chamber slides (Nunc™ Lab-Tek™ II Chamber Slide™ System) for immunocytochemistry experiments following treatment. 300,000 cells were seeded in 6-well plates (Sarstedt, TC plate 6-well, Cell+, F) for ELISA and RNA extraction following treatment. All cells were left to adhere for 12 to 24 hours and then serum-starved in DMEM with 1X insulin- transferrin selenium (ITS, Thermo Fisher, Waltham, MA, USA) for 6 hours prior to treatment. To examine the effects of different treatments, healthy cells were treated with either 100 ng/ml Pam2CSK4 (TLR-2/6 agonist, Invivogen), 100 ng/mL Pam3CSK4 (TLR-1/2 agonist, Invivogen) or 5pg/mL lipopolysaccharide (LPS) (TLR-4 agonist, Invivogen) for 6, 12, 24 and 48 hours. Cells were either left untreated (negative control) or treated with 100 ng/mL of Pam2CSK4 for 48 hours of which treatment with 100 mM o-Vanillin (Sigma-Aldrich, Oakville, ON, Canada), 5 pM RG-7112 and 100 pM o-Vanillin + 5 pM RG-7112 was initiated in the last 6 hours of incubation. Results are shown in Figure 27. Figure 27 illustrates that RG- 7112 and o-Vanillin combination reduced the number of p16 positive cells greater than RG-7112 and o-Vanillin alone. Disc cells from degenerate IVDs were induced with TLR-2/6 agonist PAM2CSK4 for 42 hours then treated with senolytics (RG+VAN, RG, VAN) or with vehicle (CTRL) for 6 hours. Quantification of the percentage of IVD cells that stained positive for p16ink4a. Percentage of positive cells were the average for n = 5. ^** p<0.01 , ^*** p<0.001 indicate significant difference between RG-7112 and o-Vanillin combination treatment to the single o- Vanillin and RG-7112 treatment respectively.

[00146] Pellet culture: 300,000 cells/tube were collected by centrifugation at 1500 rpm for 5 minutes. Pellets were incubated in 1 mL DMEM, 2.25g/L glucose (Sigma-Aldrich, Oakville, ON, Canada), 5% FBS, 5 pM ascorbic acid, 1 % GlutaMAX, 0.5% Gentamicin (Thermo Fisher, Waltham, MA, USA) at 37°C and 5% CO2. Pellets were left in DMEM for four days to form and stabilize (in pretreatment media) and then treated with 100 pM o-Vanillin (Sigma-Aldrich, Oakville, ON, Canada) for four days, meanwhile pellets in the control group stayed in DMEM with vehicle 0.01% DMSO (Sigma-Aldrich, Oakville, ON, Canada). Following the treatment period, pellets from both groups were cultured for 21 days and their culture media was collected every 4 days and pooled as post-treatment media.

[00147] Immunofluorescence

[00148] Monolayer cultures (20,000 cells/well in 8-well chambered slide) were washed with PBS, fixed with 4% paraformaldehyde (Thermo Fisher, Waltham, MA, USA), and blocked in PBS with 1% BSA (Sigma-Aldrich, Oakville, ON, Canada), 1% goat serum, and 0.1% Triton X-100 (Sigma-Aldrich, Oakville, ON, Canada) for 1 hour. Pam2CSK4 treated cells were stained with primary antibodies specific to NGF (Santa Cruz, Dallas, Tx, USA), p16 ^lnk4a (Cintec-Roche, Laval, Qc, CAN), Iί1b, TNF-a, IL8 and TLR-2 (Abeam, Cambridge, Ma, USA) overnight at 4 °C. Healthy cells were treated with p16 ^INK4a and TLR-2 only. After washing, cells were incubated with the appropriate Alexa Fluor ^® 488 or 594- conjugated secondary antibody (Thermo Fisher, Waltham, MA, USA) for 2 hours at room temperature, and then counterstained with DAPI for nuclear staining. Photomicrographs were acquired with a fluorescent Olympus BX51 microscope equipped with an Olympus DP71 digital camera (Olympus, Tokyo, Japan). Ten images of each condition per donor were analyzed and positive cell percentage was quantified by Fiji ImageJ (version: 2.1.0/1 53c). Briefly, the number of cells stained positive for one of the target proteins (NGF, IL-1 b, TNF-a, and IL-8) were counted and compared to the total number of cells positive for DAPI staining. For the double staining (TLR-2 and p16 ^INK4a ) the percentage of positive cells represents the ratio of the number of cells positively stained for either one of the 2 markers (TLR-2 & p16 ^INK4a ) divided by the total number of cells positively stained for DAPI. [00149] Immunohistochemistry

[00150] Safranin-0 staining: Pellet culture samples were heated on an iron heater at 50°C for 30 minutes and rehydrated with PBS. Samples were stained with 0.1 % Safranin-0 (Sigma-Aldrich, Oakville, ON, Canada) for 5 minutes at room temperature and rinsed with water, 75% ethanol (15s), and 95% ethanol (15s). Coverslips were mounted with Permount™ Mounting Medium (Fisher Scientific). Samples were imaged with Olympus DP70 digital camera (Olympus) pre-fixed to a Leica microscope (Leica DMRB) under visible light.

[00151] p16 ^INK4a staining: p16 ^INK4a staining was performed for both monolayer cultures and pellet samples. Only the pellet samples were heated on an iron heater at 50°C for 30 minutes and rehydrated by PBS-T (0.1% Triton X- 100) for 10 minutes. Both healthy monolayer cultures and pellet samples were blocked with hydrogen peroxide for 10 minutes, washed three times, and saturated with 1 % BSA, 1 % goat serum, and 0.1 % Triton X-100 for 10 minutes. All samples were incubated at 4°C overnight for p16 ^INK4a antibody (CINTec Kit, Roche) and PBS-T for negative control. The HRP/DAB Detection IHC Kit (Abeam, ab64264) was used for detection. Counting staining was applied with Meyer’s hematoxylin (Sigma-Aldrich, Oakville, ON, Canada) for 2 minutes. Samples were rinsed with water (30s), 75% ethanol (15s), and 95% ethanol (15s) afterwards and coverslips were mounted with Permount™ Mounting Medium (Fisher Scientific). Images were captured as described (Cherif H, et al. , J Clin Med. 2019;8(4)) for Safranin-0 staining and analyzed with Fiji Image J (version 2.1.0/1.53c).

[00152] Real-time Quantitative Polymerase Chain Reaction (RT-qPCR)

[00153] RNA was extracted using the TRIzol chloroform extraction method previously described (Krock E, et al., J Cell Mol Med. 2014; 18(6): 1213-25). 500 ng of RNA was then reverse transcribed using a qScript cDNA Synthesis Kit (Quanta Biosciences, Beverly, MA, USA) with an Applied Biosystems Verti Thermocycler (Thermo Fisher, Waltham, MA, USA). RT-qPCR was performed using an Applied Biosystems StepOnePlus machine (Thermo Fisher, Waltham, MA, USA) with PerfecCTa SYBR Green Fast Mix (Quanta Biosciences, Beverly, MA, USA). Primer sequences for TLRs, senescent markers, pain and inflammatory markers (IL-6, IL-8, p16, p21 , TNF-oc, CXCL-10, CXCL-1 , GM-CSF, TGF-b, CCL-2, CCL-5, CCL-7, CCL-8, NGF, BDNF, IL-8, TLR-1 ,2,4,6) and the housekeeping gene (GAPDH) can be found in Table 5. All reactions were conducted in technical triplicate, and fold-changes in gene expression were calculated by using the 2 ^_DDa method, after normalizing to actin and non-treated samples.

Table 5 qRT-PCR Primer Sequences [00154] Protein analysis

[00155] To determine the concentration of NGF, IVD cells were cultured in monolayer (250,000 cells/sample) and then lysed using 300 pl_ of Cell Lysis buffer (RayBiotech, Norcoss, GA, USA). Cell lysates were incubated for 48 hours at room temperature and protein concentrations were determined using ELISA kits, according to the manufacturer’s instructions (RayBiotech, Norcoss, GA, USA). Cell culture media from degenerate IVD cells cultured in monolayer and in pellets was used to assess the concentrations of IL-6, IL-8, IL-1 b and TNF-oc. 150 pL of monolayer culture media and pellet pre-treated and pooled post-treated media was used. ELISAs were performed as per the manufacturer’s instructions (RayBiotech, Norcoss, GA, USA). Colorimetric absorbance was measured with a Tecan Infinite M200 PRO (Tecan, Mannedorf, Switzerland) spectrophotometer and analyzed with i-control 1.9 Magellan software (Tecan, Mannedorf, Switzerland). Protein levels of the treated conditions and controls were then compared.

[00156] Dimethylmethylene Blue Assay

[00157] Dimethylmethylene Blue (DMMB) assays were conducted as previously described to quantify sulfated glycosaminoglycans (sGAG) in the conditioned media of IVD pellets with or without o-Vanillin treatment. Chondroitin sulfate was used to generate the standard curve. Pooled post-treatment media samples from treated and untreated pellets were used. All samples were ensured to fall into the linear portion of the standard curve. Each sample was placed in triplicate into clear 96-well plates (Costar, Corning, NY, USA). DMMB dye was then added to the wells. The absorbance was measured immediately at room temperature using Tecan Infinite T200 spectrophotometer (Mannedorf, Switzerland).

[00158] Statistical analysis:

[00159] Data was analyzed using Graph Prism 8 (Graph Pad, La Jolla, CA,

USA). Analysis was performed using two-tailed Student's t test or Two-way ANOVA. Specific tests are indicated in the figure legends with the corrections. A p- value <0.05 was considered statistically significant. Data are presented as mean ± SD.

[00160] Results

[00161] Cell activation with a TLR-2/6 agonist, caused an increase in the number of senescent cells and SASP factor release in cells from non-degenerate human IVDs

[00162] We have previously reported that TLR expression and the number of senescent cells is positively correlated with level of IVD degeneration. Moreover, we have seen that treating cells from degenerating human IVDs with TLR agonists PAM2csk4 (TLR-2/6), PAM3csk4 (TLR- 1/2) and LPS (TLR-4) increased expression of pain mediators and pro-inflammatory cytokines when compared to vehicle control (23). Here we aimed to determine the effect and the relation of TLR activation and cell senescence in human IVD cells from non-degenerate IVDs. Monolayer cultures were treated with TLR agonists activating TLR-2/6, 1/2 and 4 for 6, 12, 24 and 48 hours. p16 ^4a was used to identify senescent cells and the number of senescent cells were significantly increased following TLR-2/6 activation with a 11 % ± 1.732 increase at 24 hours (p < 0.001 ) and a 22.67% ±4.163 (p < 0.0001 ) increase at 48 hours (Fig.11A-B). Furthermore, using RT- qPCR the gene expression levels of TLRs, senescence markers and SASP factors were evaluated in the treated cell pellets. The expression of the TLR-1 ,2,4,6 in pellets treated with TLR agonists for 48 hours was investigated. A significant increase (7.24-fold ± 3.458, p < 0.001 ) in expression of the TLR-2 in the cells exposed to the TLR-2/6 agonist was observed when normalized to the untreated control. Significance was not reached for TLR-1 (p = 0.9995), TLR-4 (p = 0.9974) and TLR-6 (p = 0.1080) (Fig. 11 C). Moreover, a significant increase (2.56-fold ± 0.288, p < 0.001 ) of p'\6 ^ink4a gene expression only in cells exposed to the TLR-2/6 agonist when compared to control was observed. Of note, no significant difference was found in p21 gene expression following exposure to TLR2/6 agonist (p = 0.8557) (Fig. 11 D). When evaluating SASP factors after 48 hours of exposure to the TLR agonists, the most significant increase was observed following TLR2/6 exposure (Fig. 11 E). Comparing to the control, an increase in CCL2 (12.70 -fold ± 2.541 , p < 0.05), CCL5 (55.11- fold ± 2.696, p < 0.01), CCL7 (13.78-fold ± 2.440, p < 0.05), CCL8 (12.68-fold ± 1.534, p < 0.05), IL-6 (1079.12-fold ±43.135, p < 0.01), IL-8 (1890.28-fold ± 85.617, p < 0.01), TNF-oc (5.30-fold ± 0.214, p < 0.01), NGF (3.49-fold ± 0.250, p < 0.05) and BDNF (3.31 -fold ± 0.051 , p < 0.01 ) was seen following TLR2/6 activation (Fig. 11 E). Altogether, these results validate that activation of TLR-2/6 increases both the number of senescent cells and SASP factors produced in cells from non-degenerate IVDs.

[00163] O-Vanillin reduced the number of senescent cells and enhanced proteoglycan production in cell pellet cultures from degenerate IVDs

[00164] O-Vanillin senolytic activity and effect on matrix production has never been assessed on cells from patients with back pain and degenerating IVDs. Flere we evaluated o-Vanillin’s senolytic activity in 3D pellet cultures of IVD cells back pain patients. The pellet cultures were treated with o-Vanillin (100mM) or vehicle (DMSO 0.01%) for 4 days. At the end of the treatment period, the pellets were maintained in standard culture media for 21 days with the post treatment media collection occurring every 4 days. The senolytic activity was evaluated by immunostaining for the senescence marker p'\6 ^ink4a (Fig. 12A). The percentage of p'\Q ⁱ n ^k4 a positive cells decreased significantly from 14.66% ± 2.758 in the untreated control to 6.38% ± 0.4973 in the o-Vanillin treated pellets (p < 0.05) (Fig. 12B). Safranin-0 staining was used to evaluate proteoglycan content. A more intense red staining was observed, indicating higher proteoglycan content in the o-Vanillin treated IVD cell pellets compared to the control sample (Fig. 12C). Furthermore, a DMMB assay was performed to assesses sGAG content in the culture media (33). Pooled media from all post-treatment time points in the o-Vanillin treated cell pellets (1.62 pg/ml ± 0.4134) had significantly higher sGAG content then the untreated control pellets (0.33pg/ml ± 0.2876) (p < 0.05) (Fig. 12D). We then evaluated o-Vanillin’s ability to reduce SASP factors (IL-1 b, IL-8, IL-6 and TNF-oc) that are commonly produced by senescent IVD cells (18). Using ELISA immunoassay, we compared the percent difference of the pooled post-treated media over the pre-treated media. A significant decrease was observed in all evaluated SASP factors measured in the media of o-Vanillin treated compared to the untreated controls. The percentage of difference in post compared to pre treatment and measured in o-Vanillin and control groups were respectively for IL- 1 b (13.75 % ± 3.473 vs 30.63 % ± 3.279, p < 0.01 ), IL-8 (38.38 % ± 12.903 vs 61.5 % ± 18.821 , p < 0.05), IL-6 (13.38 % ± 5.867 vs 25.75 % ± 1.652, p < 0.05) and TNF-oc (19.38 % ± 0.408 vs 46 % ± 0.750, p < 0.0001) (Fig. 12E).

[00165] o-Vanillin reduced gene expression of p16, TLR-2 and SASP factors following TLR-2 activation in IVD cells from patients with back pain and IVD degeneration.

[00166] IVD cells from patients with back pain and IVD degeneration were exposed the TLR-2/6 agonist for 48 hours in the presence or absence of o-Vanillin (100mM) during the last 6 hours of the treatment. We first assessed gene expression of senescence markers p16 and p21 . Similar to the effect observed in cells from non-degenerate IVDs, there was a significant increase in p16 ^4a expression (3.83-fold ± 1.055, p < 0.001 ) and no significant difference in the expression of p21 (p = 0.4279) following TLR-2/6 exposure when compared to the untreated control (Fig. 13A). Interestingly, treatment with o-Vanillin significantly decreased p'\6 ^ink4a expression (1.07-fold ± 0.308, p < 0.001) while no significant change was found for p21 expression (p = 0.244) (Fig. 13A). When assessing TLRs expression in the cells from patients with back pain and IVD degeneration, a significant increase in TLR-2 gene expression (9.17-fold ± 1.594, p < 0.001 ) following TLR-2/6 exposure, compared to the control was seen. However, there was no significant increase in TLR-1 (p = 0.2420), TLR-4 (p = 0.9985) or TLR-6 (p = 0.3491 ) (Fig. 13B). These samples, when treated with o-Vanillin showed a significant decrease (1.67-fold ± 0.565, p < 0.001 ) in TLR-2 expression (Fig. 13B). Exposure of IVD cells from patients with back pain and IVD degeneration to TLR- 2/6 agonist significantly increased the expression of SASP factors CCL2 (42.32- fold ± 11 .337, p < 0.001 ), CCL5 (49.03-fold ± 11 .487, p < 0.001 ), CCL7 (9.30-fold ± 1 .430, p < 0.01 ), CCL8 (28.40-fold ± 4.936, p < 0.001 ), GM-CSF (118.55-fold ± 10.067, p < 0.001 ), BDNF (1.77-fold ± 0.126, p < 0.01 ), NGF (2.75-fold ± 0.586, p < 0.01 ), TNF-oc (7.36-fold ± 2.361 , p < 0.001 ), CLCX1 (594.16-fold ± 44.718, p < 0.001 ), IL-8 (594.5-fold ± 98.644, p < 0.001 ) and CLCX10 (745.23-fold ± 107.787, p < 0.001) when compared to the untreated control (Fig. 13C). o-Vanillin significantly reduced this increase, CCL2 (5.89-fold ± 2.075, p < 0.001 ), CCL5 (6.21 -fold ± 2.156, p < 0.001), CCL7 (3.51 -fold ± 1.521 , 0.001 ), CCL8 (2.8-fold ± 2.281 , p < 0.01 ), GM-CSF (6.33-fold ± 2.39, p < 0.001 ), BDNF (0.85-fold ± 0.368, p < 0.01 ), NGF (0.62-fold ± 0.135, p < 0.01 ), TNF-oc (1 .09-fold ± 0.656, p < 0.001 ), CLCX1 (24.67-fold ± 5.132, p < 0.01 ), IL-8 (75.49-fold ± 18.608, p < 0.01 ) and CLCX1 0 (10.8-fold ± 3.087, p < 0.001 ) (Fig. 13C).

[00167] o-Vanillin reduced the protein expression of SASP factors (IL-1 b, NGF, IL-8 and TNF-oc) following TLR-2/6 activation of IVD cells from patients with back pain and IVD degeneration

[00168] Protein expression of (TNF-oc, IL-1 b, IL-8 and NGF) was evaluated by immunohistochemistry following a 48 hrs exposure to TLR-2/6 agonist. (Fig. 14A-D). Quantification of the percentage of positive cells for each SASP factor was compared to untreated controls. A significant increase of TNF-oc (56.6% ± 2.881 vs 95% ± 1.155, p < 0.001 ), IL-Ib (56.4% ± 3.050 vs 94.25% ± 0.957, p < 0.001 ), IL-8 (44.2% ± 1.924 vs 84% ± 2.236, p < 0.001 ) and NGF (63.6% ± 2.408 vs 92.15% ± 1.388, p < 0.001 ) positive cells was observed (Fig. 14E). When evaluating the effect of o-Vanillin, a significant decrease was observed in protein expression in the treated samples for TNF-oc (74.5% ± 3.109, p < 0.01 ), IL-1 b (75% ± 0.816, p < 0.01), IL-8 (48.4% ± 5.550, p < 0.01) and NGF (70.46% ± 2.416, p < 0.01 ) (Fig. 14E). Additionally, to measure the concentrations of SASP factors affected by TLR-2/6 activation and o-Vanillin treatment we performed ELISAs immunoassay of the culture media for TNF-oc, IL-1 b, IL-8 and on the cell lysate for NGF. A significant increase was found in the SASP factors in the culture media following TLR-2/6 activation, TNF-oc (72.03 pg/ml ± 9.044 vs 284.25pg/ml ± 30.972, p < 0.001 ), IL-1 b (10.97 pg/ml ± 1.09 vs 42.11 pg/ml ± 3.022, p < 0.01 ), IL-8 (92.95 pg/ml ± 8.385 vs 406.25 pg/ml ± 39.891 , p < 0.001 ) and in the cell lysate for NGF (186 pg/ml ± 5.957 vs 355.03 pg/ml ± 27.086, p < 0.001 ) when compared to the control and that this induction was significantly decreased for all evaluated SASP factors following treatment with o-Vanillin (TNF-oc: 104.5 pg/ml ± 28.831 (p < 0.001), IL-1 b: 21.15 pg/ml ± 2.123 (p < 0.05), IL-8: 117.8 pg/ml ± 36.944 (p < 0.001 ) and NGF: 262.83 pg/ml ± 5.208 (p < 0.001 )) (Fig. 14F).

[00169] o-Vanillin reduced the number of cells co-expressing TLR-2 and p 1 Q ^{n 4} a j _{n ce} || _s exposed to TLR-2/6 agonist

[00170] Based on our findings that exposure to TLR-2/6 agonist caused a significant increase in p16 ^4a and TLR-2 gene expression in both cell and pellet cultures from non-degenerate and degenerate IVDs, we investigated the possibility that senescent IVD cells have an elevated TLR-2 expression. Protein expression of TLR-2 and p16 ^4a was assessed by immunohistochemistry in IVD cells from patients with back pain and IVD degeneration following a 48 hrs exposure to TLR- 2/6 agonist. (Fig. 15A). Quantification of TLR-2 and p16 ^4a was done by measuring the percent of cells positive from the total cell population for the two markers. Following TLR-2/6 activation it was found that there was a significant increase in the expression of TLR-2 (53.17% ± 8.684, p < 0.001 ) and p16 ^4a (47.19% ± 7.951 , p < 0.001 ) when compared to untreated controls; TLR-2 (29.92% ± 9.448) and p'\6 ^ink4a (25.95% ± 6.071 ) (Fig. 15B-C). Furthermore, treatment with o-Vanillin for the final 6 hrs significantly reduced this increase for TLR-2 (36.3% ± 8.057, p < 0.001) and p16 ^,7/<4a (31 .07% ± 3.854, p < 0.001 ) (Fig. 15B-C). Finally, to verify the link between TLR-2 and cell senescence in IVD cells, we assessed the percentage of cells co-expressing p'\6 ^ink4a and TLR-2 by determining the percentage of senescent cells (p16 ^4a positive cell) that express TLR-2. In the untreated control, 26% ± 1.611 of the senescent cells expressed TLR-2 while following TLR2/6 exposure the percentage of senescent cells expressing TLR-2 increased significantly to 61.05% ± 6.946 (p < 0.001) (Fig. 15D). The most noteworthy finding was that o-Vanillin significantly reduced the number of senescent cells expressing TLR-2 to 27.57% ± 2.509 (p < 0.001 ) when exposed to TLR-2/6 agonist. (Fig. 15D). These findings indicate a link between TLR-2 expression, cell senescence and SASP factor production that contribute to IVD degeneration and pain. This deleterious role of TLR-2 is blocked by the dual senolytic and anti-inflammatory effects of o-Vanillin.

[00171] Several studies including our own have demonstrated that senescent cells accumulate in degenerating IVDs and suggested that an elevated SASP factor release and increased expression of TLRs contribute to IVD degeneration. Here we have shown a potential link between the accumulation of senescent cells and TLR activation. As well, we show that o-Vanillin, a TLR antagonist and senolytic compound, has regenerative and anti-inflammatory effects on cells from degenerating IVDs.

[00172] In chondrocytes and IVD cells, TLRs are, in addition to molecules derived from pathogens, activated by exposure to intracellular proteins such as HSP60, HSP70, S100A8/9, HMGB1 released in response to stress and extracellular matrix fragments such as fibronectin, aggrecan, biglycan and other by-products of tissue degeneration (Minguzzi M, et al. Oxid Med Cell Longev. 2018;2018:3075293). As well, it has been reported that synthetic TLR-2 and 4 agonists can induce IVD degeneration, increase inflammatory environment and increase in expression of TLRs. The present study demonstrates that TLR-2 activation, in addition to inducing an inflammatory environment, caused IVD cells from non-degenerate IVDs to become senescent. We used cells of IVDs from organ donors with no signs of degeneration or history of back pain. These IVDs have a low number of senescent cells and low levels of SASP factor release compared symptomatic degenerating IVDs (Cherif H, et al. J Clin Med. 2019;8(4):433. https://doi.Org/10.3390/jcm8040433). Our results demonstrate that the synthetic TLR2 agonist (Pam2CSK4), caused the greatest increase in senescent cell number, TLR-2 expression and SASP factor release in cells from non-degenerate IVDs after 48 h exposure. Our previous study using TLR-1 , 2, and 4 agonists found the cytokines (IL-1 b, 6, 8), chemokines, proteases (MMP3, MMP13) and TLR-2 expression were greatest following exposure to the same TLR-2/6 agonist in NP cells of non-degenerate IVDs. Other studies have shown that continuous stimulation of TLR-4 promotes cellular senescence in mesenchymal stem cells (Feng G et al. Cytotechnology. 2018;70(3): 1023-35. https://doi.org/10.1007/s10616-017-0180-6). Moreover, TLR-2 and 10 have been found to be key mediators of senescence in IMR90 cells, a human diploid fibroblast cell line.

[00173] We then verified that these findings were also seen in cells isolated from degenerating IVDs of patients undergoing surgery to reduce low back pain. TLR-2 activation of cells from symptomatic IVDs induced expression of SASP factors (CCL-2,-5,-7,-8, IL-6,-8, GM-CSF,TNF-oc,NGF,BNDF,CLCX-1 ,10), a senescence marker (p16 ^4a ) and of the TLR-2 receptor itself. Moreover, we confirmed that protein expression of SASP factors (NGF, IL-1 b, TNF-a and IL-8) was higher in the TLR-2 activated cells. These proteins were chosen since they have been associated to be IVD degeneration and TLR-2 induction and have been reported to be highly expressed in degenerate human and mice IVDs. Taken together our results validate that IVD cells from patients with back pain and IVD degeneration at both gene and protein level respond to TLR-2 activation. [00174] The use of synthetic antagonists aimed towards TLR-2 and TLR-4 has been evaluated in a variety of inflammatory diseases. Antagonists such as TAK-242, a TLR-4 antagonist, has been shown to diminish LPS-induced TLR-4 signaling and inflammation in peritoneal macrophages. Furthermore, our own previous study demonstrated that TAK-242 reduced pain but did not provide tissue regeneration in a mouse model of back pain. Similar to our study, anti inflammatory properties of o-Vanillin was reported previously in NP cells from patients undergoing surgery for disc herniation or spinal stenosis following induction by high mobility group box-1. Furthermore, the capability of o-Vanillin to reduce SASP factors has been previously depicted in IVD cell pellet cultures o- Vanillin has also been shown to reduce cytokines, chemokines and proteases in vitro by in human HEK-TLR2 and THP-1 cells and to reduce a tumor-promoting phenotype of microglia in vivo. It has also previously been shown that o-Vanillin incorporated to Poly (Lactic-co-Glycolic Acid) scaffolds elicited more proteoglycan production and decreased inflammatory response of annulus fibrous cells compared to cells in un-supplemented scaffolds _^ As well, o-Vanillin has been shown to significantly decrease the production of pro-inflammatory cytokines and significantly attenuated UVB irradiation-induced cytotoxicity in human keratinocyte stem cells.

[00175] Senolytic drugs target selective signaling pathways involved in cell survival and apoptosis. These drugs could potentially be used therapeutically to treat disc degeneration, recover loss of disc height in already degenerate discs, or prophylactically to prevent future degeneration either in individuals at risk or following fusion for adjacent disc disease. Our previous study demonstrated that o-Vanillin, reduced senescent cells and enhanced matrix production in cell pellet cultures generated from organ donor IVDs without known history of backpay Here we show that o-Vanillin was able to reduce inflammation, remove senescent cells and enhance proteoglycan production in cell pellets from surgically removed symptomatic IVDs of patients with low back pain. [00176] We further demonstrated that by targeting TLRs and senescent cells with o-Vanillin we can decrease inflammatory processes found in IVD cells from patients with back pain and IVD degeneration. Interestingly, our study demonstrates that both gene and protein expression of SASP factors (CCL2,5,7,8, GM-CSF, BDNF, NGF, TNF-oc, CLCX1 , CLCX8 and CLCX10, IL-1 b, IL-8) were significantly reduced following TLR activation and o-Vanillin treatment.

[00177] The higher expression of TLR-2 in IVD cells from patients with back pain and IVD degeneration leads us to evaluate its expression level in senescent cells and investigate its role in disc cell senescence and associated SASP factors release. We found TLR-2 activation increased the expression of TLR-2 in the senescent cells. Also, treatment with o-Vanillin significantly reduced the number of senescent cells expressing TLR-2.

[00178] Conclusions:

[00179] We showed that TLR-2/6 activation increased TLR-2 expression and senescent cells in IVD cells from both organ donors without degeneration and backpain and patients with disc degeneration and backpain. Further, o-Vanillin reduced the number of senescent IVD cells and the release of SASP factors. This data suggests a possible regulatory effect between TLR-2 and IVD cell senescence IVD. This phenomenon could be explained either by the induction of non-senescent neighbouring cells by senescent cells in a paracrine manner or alternatively that senescent cells retain SASP factor production through TLR-2 activation in an autocrine manner. The detrimental effect of senescent cells can be inhibited by blocking TLR-2 activity with o-Vanillin.

[00180] The present data demonstrate that RG7112 and o-vanillin individually significantly reduce the presence of senescent IVD cells and significantly improve tissue homeostasis. However, some senescent cells remain after either treatment, which may be due to the heterogenous nature of senescent cells where, the anti-apoptotic pathway responsible for senescence varies with cell population and inducer. We therefor combined the 2 drugs and found a synergistic effect (Figure 16-23). The combination of 2 or more senotherapeutic compounds targets heterogeneous populations of senescent cells throughout the body by modulating separate pathways.

EXAMPLE 3

Low Back Pain and Disc Degeneration are Decreased Following senolytic treatment in a Mouse Model

[00181] One of the drawbacks of senolytic agents currently under investigation is their failure to target the same cell type in different species, or different cell types within a species. It is therefore imperative to test novel agents for effectiveness against senescent cell burden, and for potential deleterious side effects, in pre-clinical models with clinically relevant cells. Mice with targeted deletion of the gene encoding secreted protein acidic and rich in cysteine (SPARC) have been characterized as a useful model of pain associated with IVD degeneration. The SPARC-null mice have progressive, age-dependent, intervertebral disc degeneration and back pain presenting a clinically relevant model of low back pain. The ability to prevent or even slightly delay onset of low back pain would have a tremendous socio-economic impact. The overall objective of this project is determining if senolytic drugs reduce pain and improve tissue homeostasis in middle-aged SPARC null mice with back pain in vivo.

[00182] Ex Vivo Mouse IVD isolation and culture. SPARC-null mice on a C57BL6 background, age 9 months (middle aged with an accumulation of senescent cells, established IVD degeneration and back pain) and age-matched wild type C57BI6 mice (WT) will be used as non-degenerate controls. IVDs of 4-5 female and 4-5 male SPARC-null and wild-type C57BL6 animals will be pooled and treated together for each type of analysis as described in Krock et al, 2018 & 2019 (Osteoarthritis and cartilage 26.9 (2018): 1236-1246; EBioMedicine 43 (2019): 487-500). All mice will be bred in-house. We are not anticipating sex differences at the IVD level, but the groups will be extended if such differences become apparent. Culture media from mouse IVDs was collected and analyzed for up to 15 SASP factors with Luminex® multiplex assays (Invitrogen) described in Cherif et al. (Elife. 2020 Aug 21 ;9:e54693. doi: 10.7554/el_ife.54693. PMID: 32821059; PMCID: PMC7442487).

[00183] Treatment: Signs of illness will here be monitored by change in body weight, motor activity and distance travelled in open field. A post-mortem will be done by CMARC to determine cause of death if applicable. Treatment was administered by oral gavage, weekly for 8 weeks. Treatment: T1; 100mg/kg o- vanillin T2; 5mg/kg RG7112, T3;100mg/kg o-vanillin + 5mg/kg RG7112, T4; 100mg/kg o-vanillin + 2.5mg/kg RG7112; T5; 50mg/kg o-vanillin + 5mg/kg RG7112, T6; 50mg/kg o-vanillin + 2.5mg/kg RG7112, T7; 0.01 % DMSO in saline as control.

[00184] Pain behavior: Testing (all animals) will be carried out in a dedicated behavioral testing room with regular indoor lighting between 8:00 AM and 12:00 PM. Mice will be allowed to habituate to the room for 1 hour and to Plexiglas testing boxes on a metal grid for another hour (when applicable). Grip strength (axial discomfort), acetone-evoked behavior and mechanical sensitivity to von Frey filaments (radicular pain) were assessed on non-treatment days bi-weekly. The tail suspension test (axial discomfort and depression-like behavior) and distance travelled in open field (motor ability and anxiety-like behavior) was measured every 4 weeks.

[00185] Release of mobile SASP: IVDs of 6-10 animals per treatment group, were processed at termination of the 8-weeks of treatment, to evaluate mobile senescence associated factors produced by the tissue. Culture media from mouse IVDs was collected and analyzed for up to 15 SASP factors with Luminex® multiplex assays (Invitrogen) described in Cherif et al. (Elife. 2020 Aug 21 ;9:e54693. doi: 10.7554/eLife.54693. PMID: 32821059; PMCID: PMC7442487). [00186] Histologic analysis: Spinal columns were dissected and decalcified by immersion in 4% EDTA (ethylenediamine tetra-acetic acid) at 4°C for 14days. Samples were then cryoprotected in 30% sucrose in phosphate-buffered saline for 4 days at 4°C and embedded in OCT cutting medium (Tissue-Tek). Sixteen-micron sections were cut with a cryostat (Leica CM3050S) in the sagittal plane and thaw- mounted onto gelatin-coated slides for subsequent staining. Staining was performed by the FAST protocol developed by Leung et al. (Society vol. 57,3 (2009): 249-56. doi:10.1369/jhc.2008.952184) for IVDs. After drying, slides were mounted with DPX (Sigma-Aldrich, St. Louis, MO). Sections per animal were carefully examined by a blinded observer to identify degenerating IVDs. Criteria for degeneration included the loss of clear compartmentalization between the nucleus pulposus (NP) and the annulus fibrosus (AF), annular tear, and dorsal bulging or herniation, all of which are characteristic of degenerating discs (Millecamps, Magali, et al., The Spine Journal 15.12 (2015): 2524-2537). Images were obtained with a 10* objective.

[00187] Results: As illustrated in Figures 16 to 23, SPARC-null mice displayed elevated signs of axial and radiating pain at baseline compared to wild- type. We have seen that the treatment with senolytic drugs significantly reduces behavioral signs of low back pain and lower the release of inflammatory senescence associated (SASP) factors from the IVDs. During pain behavior analysis, we have seen that radicular pain, axial discomfort and cold allodynia have been significantly reduced as of 4 weeks of treatment in SPARC-null senolytic treated groups compared to the SPARC-null non-treated group. Furthermore, we observe a greater behavioral effect of the senolytic treatments when compared to the non-treated group at the 8-week time point. Furthermore, our demonstrates that biochemical analysis of the cultured isolated IVDs either ex vivo or following in vivo treatment has shown that SASP factors have been significantly reduced in treated groups compared to the levels of the non-treated groups. [00188] Conclusion: We have demonstrated that senolytic drugs reduce pain and improve tissue homeostasis in middle-aged SPARC null mice with back pain in vivo. There are many shared mechanisms in SPARC-null mice and human patients with IVD degeneration and pain symptoms including, age-related disease onset, degeneration severity, loss of SPARC expression, pain symptoms and accumulation of senescent cells in degenerating IVDs. By 6-months of age, most SPARC-null mice have several lumbar IVDs that are at least moderately degenerated while IVD degeneration is almost never observed in age-matched wild-type mice. Furthermore, the model is also clinically relevant as the condition evolve overtime, and like humans show strong behavioral pattern with axial and radiating back pain. Senolytics have been shown in our in vitro work to have anti inflammatory and antioxidant properties in chondrocytes and human IVD cells. Our goal was to demonstrate that these compounds reduce the expression of pain behaviours, pain mediators and improve tissue homeostasis in a clinically relevant mouse model of lower back pain and IVD degeneration.

Example 4 Cancer treatment

[00189] Standard treatments for cancer include resection surgery followed adjuvant chemotherapy to reduce risk of recurrence. Chemotherapy, such as doxorubicin, is also a major treatment for advanced/metastatic cancer, which often metastasize to bone and/or spine. Doxorubicin inhibits DNA synthesis which triggers cell apoptosis. Cancer cells that escape apoptosis often become senescent. Chemotherapy also induces cellular senescence in normal cells throughout the body. While senescent cells undergo cell cycle arrest, they may adopt a phenotypic shift with secretion of pro-inflammatory and pro-tumorigenic factors called senescence-associated secretory phenotype (SASP). Although chemotherapy-induced cancer senescence was originally thought be beneficial, senescence in surrounding cells/tissues and tumor itself has been linked to advanced cancer growth and metastasis through associated factor secretion. Induced senescence in unrelated tissues also accelerates ageing and degenerative pathways. In addition, the SASP has immune-suppressive effects. Senolytic drugs specifically target senescent cells and induce apoptosis only in these cells. Combining chemotherapeutic and senolytic drugs which target senescent and non-senescent tumor and stromal cells could improve cancer patient outcomes.

[00190] 3D in vitro model: Bringing a new drug to market costs approximately 2 billion dollars and takes over 10 years. One study revealed that about 75% of newly developed drugs failed to pass clinical trials. This is particularly important for cancer patients, where cancer accounted for 30% of all deaths in Canada (CCSRI 2019). The CCSRI has estimated more than 225,800 new cancer cases and over 83,300 deaths from cancer in 2020. The poor clinical translation may be attributed to lack of efficacy, which is often determined by in vitro cell lines in standard 2D polystyrene dishes or in small/large animal models which do not recapitulate human physiology, pathology or immunity. 3D tumor models such as spheroids or organoids have emerged to counter this. These models can determine more physiological responses to therapeutics, opening doors for personalized medicine. Organoid and spheroid models can lack the cellular, biomolecular and biomechanical heterogeneity of the tumor microenvironment in vivo. New screening platforms that better mimic the complex tumor physiological microenvironment are necessary to enhance regulatory approval and clinical translation. Here we will combine our expertise in 3D tissue/tumor modeling, senolytics, orthopaedic oncology and cancer genetics to model the tumor microenvironment and evaluate efficacy of combined therapeutics in reducing tumor growth/invasion, SASP factor secretion and stabilizing associated cell phenotypes within the microenvironment. [00191] Methods

[00192] p16-positive senescent cells were quantified in monolayer cultures of tumor (MDA-MB-231 ) and stroma (IRM90) cells as described in Cherif et al. (Cherif H, et al., J Clin Med. 2019;8(4)). Figure 25.

[00193] MDA-MB-231 breast cancer cell line spheroids are suspended in collagen hydrogels using ultra-low adhesion well-plates, which promote tumor spheroid growth and cell migration/invasion. IRM90 (fibroblasts) are suspended in the collagen gel surrounding the tumor spheroids mimicking the tumor stroma (figure 24). Doxorubicin dose-response curves are generated over 72 hours to optimize exact doses required to reduce spheroid growth while also inducing senescence in the spheroid and surrounding stroma. Doxorubicin [0, 0.1 , 0.25, 0.5-mM] Alamar blue assay is performed to measure cell viability (metabolic activity) following treatments (Fig 26 graph). Phase-contrast imaging was used to quantify spheroid growth. Tumor cell migration to periphery was measured using Image J (Fig 26 green spheroids). Doses, RG-7112 [5mM] o-Vanillin [100pM, Doxorubicin [0.25pM]

[00194] Results

[00195] Complex in vitro 3D culture models consisting of human tumor spheroids surrounded by stroma cell-seeded matrix was used to represent tumor microenvironment. The results demonstrate that a combination of chemotherapeutic and senolytic drugs further reduce tumor size and tumor cell migration compared to either treatment alone.

[00196] Cancer is common disease often associated with bone/spine metastases. Chemotherapy is often used for treatment, which can increase tumor and normal tissue senescence. The associated SASP and systemic inflammation can drive cancer cell proliferation and migration. Targeting and eradicating senescent cells may lead to reduced tumor burden and subdued tumor reactivation and metastasis. The described treatment may prevent chemo-resistance, chemo- sensitization, enhanced growth and metastasis providing improved treatment and outcomes for patients.

[00197] The present data demonstrate that RG7112 and o-vanillin individually significantly reduce the presence of senescent IVD cells and significantly improve tissue homeostasis. However, some senescent cells remain after either treatment, which may be due to the heterogenous nature of senescent cells where, the anti-apoptotic pathway responsible for senescence varies with cell population and inducer. We therefor combined the 2 drugs and found a synergistic effect (Figure 16-23). The combination of 2 or more senotherapeutic compounds targets heterogeneous populations of senescent cells throughout the body by modulating separate pathways.

[00198] While preferred embodiments have been described above and illustrated in the accompanying drawings, it will be evident to those skilled in the art that modifications may be made without departing from this disclosure. Such modifications are considered as possible variants comprised in the scope of the disclosure.