Bcl Family Antagonist Acyl Sulfonamides for Use in the Clinical Management of Conditions Caused or Mediated by Senescent Cells and for Treating Cancer - Patent application

Abstract

The arylsulfonamide compounds of the present invention have potent and cell type-specific Bcl activity. Selected compounds of this class promote apoptosis in senescent cells, and therefore are being developed for the treatment of aging-related conditions. Selected compounds of this class promote apoptosis in cancer cells, and therefore can be developed as chemotherapeutic agents.

Description

[Technical field]

[0001] Priority Application This application claims priority to U.S. Provisional Patent Application No. 62 / 684,681, all filed on June 13, 2018, which priority applications are incorporated herein by reference in their entirety for all purposes.

[0002] FIELD OF THEINVENTION The technology disclosed and claimed below relates generally to the field of senescent cells and their role in age-related conditions. In particular, the disclosure provides novel small molecule compounds that inhibit Bcl protein activity. [Background technology]

[0003] background Senescent cells are characterized as cells that no longer have replicative capacity but remain in the tissue of origin and induce senescence-associated secretory phenomena (SASP).The present disclosure is premised on the premise that many age-related conditions are mediated by senescent cells, and that selective removal of such cells from tissues when at or near such a state can be used clinically to treat such conditions.

[0004] US Patent No. 10,130,628 (Laberge et al.) describes the treatment of certain age-related conditions believed to be at least partially mediated by senescent cells with MDM2 inhibitors, Bcl inhibitors, and Akt inhibitors. US 20170266211 A1 (David et al.) describes the use of specific Bcl inhibitors for the treatment of age-related conditions. US Patent No. 8,691,184 (Patent Document 3), US Patent No. 9,096,625 (Patent Document 4), and US Patent No. 9,403,856 (Patent Document 5) (Wang et al.) describe Bcl inhibitors in small molecule libraries.

[0005] Other disclosures related to the role of senescent cells in human disease are found in pre-grant publications US 2017 / 0056421 A1 (Zhou et al.), WO 2016 / 185481 (Yeda Inst.), US 2017 / 0216286 A1 (Kirkland et al.), and US 2017 / 0281649 A1 (David); as well as Furhmann-Stroissnigg et al. (Nat Commun. 2017 (Sep 4); 8(1):422), Blagosklonny (Cancer Biol Ther. 2013 Dec; 14(12):1092-7), and Zhu et al. (Aging Cell. 2015 Aug;14(4):644-58) (Non-Patent Document 3). [Prior art documents] [Patent documents]

[0006] [Patent Document 1] U.S. Patent No. 10,130,628 [Patent Document 2] US 20170266211 A1 [Patent Document 3] U.S. Patent No. 8,691,184 [Patent Document 4] U.S. Patent No. 9,096,625 [Patent Document 5] U.S. Patent No. 9,403,856 [Patent Document 6] US 2017 / 0056421 A1 [Patent Document 7] WO 2016 / 185481 [Patent Document 8] US 2017 / 0216286 A1 [Patent Document 9] US 2017 / 0281649 A1 [Non-patent literature]

[0007] [Non-Patent Document 1] Furhmann-Stroissnigg et al. (Nat Commun. 2017 (Sep 4); 8(1):422) [Non-Patent Document 2] Blagosklonny (Cancer Biol Ther. 2013 Dec; 14(12):1092-7) [Non-Patent Document 3] Zhu et al. (Aging Cell. 2015 Aug;14(4):644-58) Summary of the Invention

[0008] overview The following disclosure reviews strategies for selectively eliminating senescent cells, provides effective compounds, pharmaceutical compositions, development strategies, and treatment protocols, and describes many of the subsequent advantages.

[0009] A new family of Bcl inhibitors has been developed. Some of the Bcl inhibitors in this family are particularly effective senescent cell killing agents. Contacting senescent cells in vitro or in vivo with the compounds and compositions of the present disclosure selectively regulates or eliminates such cells. The inhibitors can be used for administration to target tissues in subjects with age-related conditions, thereby selectively eliminating senescent cells in or around the tissue, and alleviating one or more symptoms or signs of the condition. Alternatively or additionally, selected compounds from the family can be formulated and sold as chemotherapeutic agents.

[0010] [The present invention 1001] Compounds of formula (I): TIFF0007678672000001.tif55128 wherein X 1 is -Cl; X 2 is -COOH; X 3 But -SO 2 CF 3 , -SO 2 CH 3 , or -NO 2 and X 5 is -F or -H; R 1 But -CH(CH 3 ) 2 can be; R 2 But -CH 3 and; R 3 and R 4 are both -H; n 1 is 2; and R 6 But -OH, TIFF0007678672000002.tif10128 More selected; Here, R 6 The hydroxyl group in may be phosphorylated, except for X 3 -SO 2 CF 3 If R 6 The hydroxyl group in must be phosphorylated. compound. [The present invention 1002] R 6 But -OR 7 、 TIFF0007678672000003.tif10128 and R 7 -H, -P(O)(OH) 2 , or -(C n H 2n )P(O)(OH) 2 (where n is 1 to 4); However, X 3 -SO 2 CF 3 If R 7 -P(O)(OH) 2 Or -(C n H 2n )P(O)(OH) 2 That is, 1001 compounds of the present invention. [The present invention 1003] X 3 But -SO 2 CF 3 The compound of the present invention 1001 or 1002, [The present invention 1004] X 3 But -SO 2 CH 3 The compound of the present invention 1001 or 1002, [The present invention 1005] X 3 But -NO 2 The compound of the present invention 1001 or 1002, [The present invention 1006] X 5 The compound of any one of the present inventions 1001 to 1005, wherein is -F. [The present invention 1007] X 5 The compound of any one of the present inventions 1001 to 1005, wherein is -H. [The present invention 1008] R 6 Any of the compounds of the present invention 1001 to 1007, wherein is -OH. [The present invention 1009] R 6 but, TIFF0007678672000004.tif9128 The compound of any one of the present inventions 1001 to 1007, [The present invention 1010] R 6 but, TIFF0007678672000005.tif9128 The compound of any one of the present inventions 1001 to 1007, [The present invention 1011] R 6 The compound according to any one of claims 1001 to 1010 of the present invention, wherein the hydroxyl group is phosphorylated. [The present invention 1012] R 6 The hydroxyl group in is -PO 3 H 2 The compound of the present invention, wherein the compound is phosphorylated at 1010. [The present invention 1013] X 3 -SO 2 CF 3 Instead, R 6 The compound according to any one of claims 1001 to 1010, wherein the hydroxyl group is not phosphorylated. [The present invention 1014] X 2 The compound according to any one of claims 1001 to 1012, wherein the carboxyl group in said compound is phosphorylated. [The present invention 1015] A compound of invention 1001 or invention 1002 selected from the compounds listed in Table 1A. [The present invention 1016] below: TIFF0007678672000006.tif64154TIFF0007678672000007.tif197155TIFF0007678672000008.tif121153 The compound of the present invention 1001 or 1002 selected from the following: [The present invention 1017] Any of the preceding compounds of the invention having pro-apoptotic activity. [The present invention 1018] Any of the preceding compounds of the present invention specifically kill senescent cells, defined as non-cancerous cells that express p16, compared to non-senescent cells. [The present invention 1019] Any of the preceding compounds of the present invention which specifically kill cancer cells as compared to non-cancerous cells of the same tissue type. [The present invention 1020] IC against Bcl-xL 50 <1 nM and / or IC for Bcl-2 50 Any of the preceding compounds of the present invention, wherein [The present invention 1021] LD against irradiated IMR90 or HRMEC cells 50 Any of the preceding compounds of the present invention, wherein [The present invention 1022] LD against irradiated IMR90 cells 50 However, LD against confluent IMR90 cells 50 or LD against proliferating IRM90 cells 50 3-fold or less of any of the preceding compounds of the present invention. [The present invention 1023] A pharmaceutical composition comprising any of the preceding compounds of the present invention in a pharma- ceutically compatible excipient. [The present invention 1024] contacting a cell, a cell population, or a tissue with any of the compounds or compositions of the present invention 1001-1023; 1. A method for selectively removing senescent cells and / or cancer cells from a mixed cell population or tissue, comprising: [The present invention 1025] 1. A method of treating a senescence-associated condition in a tissue in a subject, wherein the senescence-associated condition is characterized as being at least partially caused or mediated by senescent cells or as having an excess of senescent cells in or around the tissue compared to unaffected tissue, the method comprising the steps of: Administering to a tissue of a subject in need thereof an amount of any of the compounds or compositions of the present inventions 1001-1023 effective to selectively remove senescent cells from the tissue, thereby reducing or ameliorating one or more symptoms of a senescence-associated condition in the subject. [The present invention 1026] A compound comprising an amount that inhibits Bcl function and is adapted for use in treating a senescence-associated condition that is at least partially caused or mediated by senescent cells. A unit dose of a pharmaceutical composition comprising: The compound is any one of compounds 1001 to 1022 of the present invention, The composition comprises a formulation of a compound configured for administration to a target tissue in a subject exhibiting a senescence-associated condition; the formulation and amount of compound in the unit dose constitute the unit dose to be effective to selectively eliminate senescent cells in or around a tissue in a subject, thereby reducing the severity of one or more signs or symptoms of a condition without causing adverse effects in the subject when administered as a single dose to a tissue; A unit dose of the pharmaceutical composition. [The present invention 1027] A unit dose of the present invention 1026 packaged with an informational insert describing the use and attendant benefits of the agent in treating a senescence-associated condition. [The present invention 1028] A compound of any of claims 1001 to 1022 or a pharmaceutical composition of claim 1023 for use in selectively eliminating senescent cells from a tissue or mixed cell population or for use in treating a senescence-associated condition. [The present invention 1029] Use of any of the compounds of claims 1001 to 1022 in the manufacture of a medicament for treating an aging-related condition. [The present invention 1030] The method, product, or use of any one of claims 1025 to 1029, wherein the condition is osteoarthritis. [The present invention 1031] The method, product, or use of any one of claims 1025 to 1029, wherein the condition is an ophthalmic condition. [The present invention 1032] The method, product, or use of any of claims 1025 to 1029, wherein the condition is a pulmonary condition. [The present invention 1033] 1. A method of treating cancer, comprising the steps of: Administering to a tissue of a subject in need thereof any of the compounds or compositions of the present inventions 1001-1023 in an amount effective to selectively remove cancer cells from the tissue. [The present invention 1034] A compound of any of claims 1001 to 1022 or a pharmaceutical composition of claim 1023 for use in selectively eliminating cancer cells from a tissue or mixed cell population or for use in treating cancer. The invention is set forth in the following description, in the drawings, and in the appended claims. [Brief description of the drawings]

[0011] [Figure 1] 1 shows a general synthetic scheme for chemically synthesizing exemplary compounds according to the present invention. [Diagram 2] 2A, 2B, and 2C show the expression of senescent cell markers p16, IL-6, and MMP13, respectively, in an osteoarthritis animal model. The senescent phenotype can be ameliorated by Nutlin-3A, a senolytic agent that inhibits MDM2. The Bcl inhibitors of the present invention can be selected as senolytic agents for the same purpose. [Diagram 3]Figure 3A shows that an effective senolytic agent restores weight bearing to the same level as treated mice in an osteoarthritis model. Figures 3B, 3C, and 3D are images showing the histopathology of the joints in these mice. Treatment with the agent helps prevent or reverse the destruction of the proteoglycan layer. [Figure 4] Figures 4A and 4B show the reversal of both angiogenesis and vascular occlusion in a mouse oxygen-induced retinopathy (OIR) model when a senolytic agent is administered intravitreally. Figures 4C and 4D are from a streptozotocin (STZ) model of diabetic retinopathy. STZ-induced vascular leakage is attenuated by intravitreal administration of a senolytic agent. [Diagram 5] We show that removing senescent cells helps restore oxygen saturation (SPO2) in a mouse model of cigarette smoke (CS)-induced chronic obstructive pulmonary disease (COPD). [Figure 6] Data are shown from a mouse model of atherosclerosis in inbred LDL receptor-deficient mice fed a high-fat diet. The right panel shows plaque staining in the aorta. The middle panel quantitatively shows that the surface area of ​​the aorta covered with plaque was reduced by treatment with a senolytic agent. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] Detailed Description Senescent cells are characterized as cells that no longer have replicative capacity but remain in the tissue of origin and induce senescence-associated secretory phenomena (SASP).The present disclosure is premised on the premise that many age-related conditions are mediated by senescent cells, and that selective removal of such cells from tissues when at or near such a state can be used clinically for the treatment of such conditions.

[0013] The technology described and claimed below represents the first description of a novel class of Bcl inhibitors that can be used to selectively eliminate senescent cells from target tissues for the treatment of age-related conditions.

[0014] Inhibition of Bcl protein activity The Bcl protein family (TC# 1.A.21) includes evolutionarily conserved proteins that share a Bcl-2 homology (BH) domain. Bcl proteins are best known for their ability to up- or down-regulate apoptosis, a form of programmed cell death, at mitochondria. The following explanation is provided to help users understand some of the scientific basis of the compounds of the present disclosure. These concepts are not necessary to practice the invention and are not intended to limit the use of the compounds and methods described herein beyond what is expressly described or required.

[0015] In the context of this disclosure, the Bcl proteins of particular interest are those that downregulate apoptosis.Anti-apoptotic Bcl proteins contain BH1 and BH2 domains, some of which contain an additional N-terminal BH4 domain (Bcl-2, Bcl-x(L) and Bcl-w(Bcl-2L2)), and inhibiting these proteins increases the speed or sensitivity of cells to apoptosis.Therefore, inhibitors of such proteins can be used to help eliminate cells in which the proteins are expressed.

[0016] In the mid-2000s, Abbott Laboratories developed a novel inhibitor of Bcl-2, Bcl-xL and Bcl-w known as ABT-737 (Navitoclax). This compound is part of a group of BH3 mimetic small molecule inhibitors (SMIs) that target these Bcl-2 family proteins but not A1 or Mcl-1. ABT-737 is superior to previous BCL-2 inhibitors due to its higher affinity for Bcl-2, Bcl-xL and Bcl-w. In vitro studies have shown that primary cells from patients with B-cell malignancies are sensitive to ABT-737. In human patients, ABT-737 is effective against several types of cancer cells but is prone to dose-limiting thrombocytopenia.

[0017] The novel compounds described herein have now been discovered to fit into the active site of the Bcl protein to provide potent Bcl inhibition and / or promote apoptosis of target cells. These compounds can be developed as highly potent and specific drugs to target senescent and cancer cells, as described in the next section.

[0018] Model Compounds Many of the compounds of the present invention have a structure that falls within the formula shown below: TIFF0007678672000009.tif33128In formula, X1 is a halide, preferably -Cl; X2 is -COOH; X3 is -SO2CF3, -SO2CH3, or -NO2; X5 is -F or -H; R1 is -CH(CH3)2; R2 is either -H or -CH3, preferably -CH3; R3 and R4 are independently either -H or -CH3, preferably both -H; n1 is 1 to 3, preferably 2; and R6 is -OH, Selected from TIFF0007678672000010.tif10128; Here, the hydroxyl group in R6 may be phosphorylated.

[0019] Any of the possible components in the formula are accompanied by the proviso that if X3 is -SO2CF3, then the hydroxyl group in R6 must be phosphorylated. In combination with any of the preceding options, the -COOH group of X2 may be phosphorylated along with or in place of the hydroxyl group at the user's option.

[0020] A "phosphorylated" form of a compound is one in which one or more -OH or COOH groups have been replaced with a phosphate group, either -OPO3H2 or -CnPO3H2, where n is 1 to 4, such that the phosphate group can be removed in vivo (e.g., by enzymatic degradation). The unphosphorylated or dephosphorylated forms do not have such groups.

[0021] Unless explicitly stated or otherwise required, compounds depicted without stereochemistry include racemic mixtures of all stereoisomers, and alternatively enantiomerically pure preparations of either enantiomer. Any of the compounds of formula I will usually, but not necessarily, be represented by the formula I below: The stereochemistry is shown in TIFF0007678672000011.tif43128, which is as follows: It can also be represented as: TIFF0007678672000012.tif50128, where each R3 is independently either -H or -CH3.

[0022] Many of the compounds of the present invention have a structure that falls within Formula II, shown below: TIFF0007678672000013.tif43128This is the following: It can also be represented as TIFF0007678672000014.tif51128, where: X1 is -Cl; X2 is -COOH; X3 is -SO2CF3, -SO2CH3, or -NO2; X5 is -F or -H; R1 is -CH(CH3)2; R2 is -CH3; R3 and R4 are both -H; n1 is 2; and R6 is -OR7, TIFF0007678672000015.tif10128, and R7 is -H, -P(O)(OH)2, or -(C n H 2n )P(O)(OH)2, where n is 1-4 or 1-8; However, when X3 is -SO2CF3, R7 is -P(O)(OH)2 or -(C n H 2n )P(O)(OH)2. This includes the acid form of R7 shown and the aryl group where R7 is -P(O)(ONa)2 or -(C n H 2n )P(O)(ONa)2, both separately and together.

[0023] Each chemical species and / or each structural formula recited in this disclosure may be optionally used or claimed as a composition or for use in a particular context, provided that it is not explicitly and precisely shown or described in any of US Patents 8,691,184, 9,096,625, and 9,403,856 (Wang et al.). Each chemical species and / or each structural formula recited in this disclosure may be optionally used or claimed as a composition or for use in a particular context, provided that it is not explicitly and precisely shown or described in US 20170266211 A1 (David et al.).

[0024] Exemplary compounds that may be suitable for preparation and / or use in accordance with the present disclosure are shown in Table 1A.

[0025] [Table 1A] TIFF0007678672000017.tif204170TIFF0007678672000018.tif204170TIFF0007678672000019.t if204170TIFF0007678672000020.tif204170TIFF0007678672000021.tif204170TIFF00076786720 00022.tif204170TIFF0007678672000023.tif210170TIFF0007678672000024.tif204170TIFF000 7678672000025.tif140168TIFF0007678672000026.tif207169TIFF0007678672000027.tif153167

[0026] Other compounds that can be tested and developed as senolytic agents or for the treatment of senescence-related diseases according to the present disclosure include the compounds shown in Table 1B.

[0027] [Table 1B]

[0028] Evaluate compounds for senolytic and chemotherapeutic activity These and other compounds described in this disclosure can be evaluated at the molecular level for their ability to act in a manner that indicates they are candidate agents for use as active agents for use in pharmaceutical preparations and human therapy.

[0029] For example, if the therapy involves inducing apoptosis of senescent cells via Bcl-2, Bcl-xL, Bcl-w, or other Bcl family proteins, compounds can be tested for their ability to inhibit binding between one or more Bcl proteins and their respective cognate ligands. Example 1 provides a description of a homogeneous assay (an assay that does not require a separation step) for the purpose of determining binding to Bcl isoforms. Compounds can be screened at the molecular level for their ability to interact with target isoforms, thereby causing the death of senescent cells. Examples 2 and 3 provide a description of an assay designed for this purpose.

[0030] Alternatively or additionally, compounds can be evaluated for their ability to specifically kill senescent cells. Cultured cells are contacted with the compound and the degree of cytotoxicity or inhibition of cells is determined. The ability of a compound to kill or inhibit senescent cells can be compared with the effect of the compound on normal cells that divide freely at low density and normal cells that are quiescent at high density. Examples 2 and 3 provide a description of senescent cell killing using human target tissue fibroblast IMR90 cell line and HUVEC cells. Similar protocols are known and can be developed or optimized to test the ability of cells to kill or inhibit other senescent cells and other cell types, such as cancer cells.

[0031] Candidate Bcl inhibitors that are effective in selectively killing senescent cells in vitro can be further screened in animal models for specific diseases. Examples 4, 5, 6, and 7 in the Examples section below provide illustrations for osteoarthritis, eye disease, pulmonary disease, and atherosclerosis, respectively.

[0032] Alternatively, or in addition, compounds can be evaluated for their ability to specifically kill cancer or tumor cells. Cultured cells are contacted with the compound to determine the degree of cytotoxicity to the cells and / or the ability to inhibit cell proliferation. The effect on cancer cells can be compared with the effect of the compound on normal cells of the same original tissue type in culture. Compounds can also be tested for their ability to eliminate tumors in established animal models, inhibit the growth of cancer cells, and treat symptoms and signs of cancer. Example 8 provides a description of in vitro and in vivo assays for evaluating the potential of compounds in the present disclosure as chemotherapeutic agents.

[0033] Drug formulation Preparation and formulation of pharmaceutical agents for use in accordance with the present disclosure can incorporate standard techniques, such as those described in the current edition of Remington: The Science and Practice of Pharmacy. Formulations are typically optimized for administration to the target tissue, e.g., by local administration, in a manner that enhances access of the active agent to the targeted senescent cells and provides optimal duration of effect while minimizing side effects or exposure to tissues not involved in the condition being treated.

[0034] Pharmaceutical formulations for use in treating aging-related conditions and other diseases can be prepared by mixing the Bcl inhibitor with a pharma- ceutically acceptable base or carrier, and optionally one or more pharma- ceutically acceptable excipients. Depending on the target tissue, it may be appropriate to formulate the pharmaceutical composition for sustained or sustained release. Oral sustained release formulations may include a mixture of isomeric variants, binders, or coating agents. Injectable sustained release formulations may include an active agent combined with binders, encapsulating agents, or microparticles. For the treatment of joint diseases such as osteoarthritis, the pharmaceutical composition is typically formulated for intra-articular administration. For the treatment of eye diseases such as glaucoma, diabetic retinopathy, or age-related macular degeneration (AMD), the composition may be formulated for intravitreal or intracameral administration. For the treatment of lung diseases, the composition may be formulated as an aerosol or for intratracheal administration.

[0035] The present disclosure provides a commercial product that is a kit that includes a unit dose of one or more agents or compositions described in the present disclosure. Such a kit typically includes a pharmaceutical formulation in one or more containers. The formulation can be provided as one or more unit doses (either combined or separate). The kit can include a device, such as a syringe, for administration of the agent or composition into or around the target tissue of a subject in need thereof. The product can also include or be accompanied by an informational package insert that describes the use and associated benefits of the agent in treating senescent cell-associated conditions, and optionally an instrument or device for therapeutic delivery of the composition.

[0036] treatment design Senescent cells accumulate with age, which is why conditions mediated by senescent cells occur more frequently in older people. In addition, various types of stress on lung tissues can promote the emergence of senescent cells and the phenotypes they manifest. Cellular stressors include oxidative stress, metabolic stress, DNA damage (e.g., as a result of environmental ultraviolet light exposure or genetic disorders), activation of oncogenes, and telomere shortening (e.g., resulting from hyperproliferation). Tissues exposed to such stressors may have a higher prevalence of senescent cells, which in turn may lead to the onset of certain conditions at a younger age or in a more severe form. Genetic susceptibility to certain conditions suggests that the accumulation of disease-mediating senescent cells may be directly or indirectly influenced by genetic factors, which may lead to earlier onset.

[0037] One of the advantages of the senescent cell paradigm is that it can provide long-term therapeutic effects to subjects when senescent cells are successfully removed.Senescent cells are essentially non-proliferative, which means that tissues can only be filled with more senescent cells by converting the non-senescent cells in the tissue into senescent cells, a process that takes much longer than simple proliferation.As a general principle, a treatment period with a senolytic agent sufficient to remove senescent cells from target tissue (single dose or multiple doses given daily, twice weekly, or once weekly for a period of several days, a week, or a few months, for example) can provide the subject with an effective period (for example, 2 weeks, a month, two months, or more) during which the senolytic agent is not administered, and the subject experiences relief, alleviation, or recovery from one or more adverse signs or symptoms of the condition being treated.

[0038] To treat a particular senescence-associated condition with a senolytic agent according to the present disclosure, the treatment regimen will vary depending on the location of the senescent cells and the pathophysiology of the disease.

[0039] Age-related conditions suitable for treatment The Bcl inhibitors of the present disclosure can be used for the prevention or treatment of various senescence-related conditions. Such conditions are typically (but not necessarily) characterized by an excess of senescent cells (such as cells expressing p16 and other senescence markers) or expression of p16 and other senescence markers in or around the site of the condition, compared to the frequency of such cells or the level of such expression in unaffected tissue. Non-limiting examples of current interest include the treatment of osteoarthritis, eye disease, and lung disease, as described in the following sections.

[0040] Treatment of osteoarthritis Any of the Bcl inhibitors listed in this disclosure can be developed to treat osteoarthritis according to the present disclosure. Similarly, the Bcl inhibitors listed in this disclosure can be developed to selectively eliminate senescent cells in or around joints in subjects in need thereof, including but not limited to joints affected by osteoarthritis.

[0041] Osteoarthritis degenerative joint disease is characterized by fibrillation of cartilage at sites of high mechanical stress, osteosclerosis, and thickening of the synovium and joint capsule. Fibrillation is a localized surface breakdown with separation of the superficial layers of cartilage. Initial separation occurs tangentially to the cartilage surface along the axis of the numerous collagen bundles. Collagen within the cartilage breaks down and proteoglycans are lost from the cartilage surface. Without the protective and lubricating effect of proteoglycans in the joint, collagen fibers become susceptible to degradation and mechanical destruction. Risk factors contributing to the development of osteoarthritis include aging, obesity, previous joint injury, overuse of the joint, weak thigh muscles, and heredity. Symptoms of osteoarthritis include pain or stiffness in the joints, especially the hips, knees, and lower back, after inactivity or overuse; stiffness after rest that resolves with movement; and pain that worsens after activity or toward the end of the day.

[0042] The compounds of the present disclosure can be used to reduce or inhibit the loss or erosion of the proteoglycan layer in joints, reduce inflammation in the affected joints, and promote, stimulate, enhance, or induce the production of collagen, for example, type 2 collagen. The compounds can cause a decrease in the amount or level of inflammatory cytokines, such as IL-6, produced in the joints, reducing inflammation. The compounds can be used to treat osteoarthritis and / or induce the production of collagen, for example, type 2 collagen, in the joints of a subject. The compounds can also be used to reduce, inhibit, or reduce the production of metalloproteinase 13 (MMP-13), which degrades collagen, in joints, and to repair or inhibit the loss and / or degradation of the proteoglycan layer. Thus, treatment with the compounds can also reduce the likelihood of, inhibit, or reduce erosion, or slow erosion of bone. The compounds can be administered directly to the osteoarthritic joints, for example, intraarticularly, topically, transdermally, intradermally, or subcutaneously. The compounds may also reverse, ameliorate or inhibit the loss of joint strength and reduce joint pain.

[0043] Treating eye conditions Any of the Bcl inhibitors listed in this disclosure can be used to prevent or treat ocular conditions in a subject in need thereof by removing senescent cells in or around the subject's eye, thereby reducing the severity of at least one sign or symptom of the disease.Such conditions include both fundus diseases and preocular diseases.Similarly, the Bcl inhibitors listed in this disclosure can be developed to selectively eliminate senescent cells in or around ocular tissue in a subject in need thereof.

[0044] Ocular diseases that can be treated according to the present disclosure include presbyopia, macular degeneration (including wet or dry AMD), diabetic retinopathy, and glaucoma.

[0045] Macular degeneration is a neurodegenerative condition that can be characterized as a fundus disease, causing the loss of photoreceptor cells in the central part of the retina called the macula. Macular degeneration may be of the atrophic or wet type. The atrophic type is more common than the wet type, with approximately 90% of patients with age-related macular degeneration (AMD) diagnosed with the atrophic type. Atrophic AMD is associated with atrophy of the retinal pigment epithelium (RPE) layer, which causes the loss of photoreceptor cells. In wet AMD, new blood vessels can grow under the retina, leaking blood and fluid. Abnormally leaky choroidal neovascularization can cause retinal cells to die, creating blind spots in central vision. The formation of exudates or "drusen" under the Bruch's membrane of the macula can be a physical sign that macular degeneration is present. Symptoms of macular degeneration include, for example, distortions and color vision disorders.

[0046] Another fundus disease is diabetic retinopathy (DR). According to Wikipedia, the first stage of DR is non-proliferative and typically has no substantial symptoms or signs. NPDR is detectable by fundus photography, where microaneurysms (tiny blood-filled bulges in the arterial wall) can be seen. If there is loss of vision, a fluorescein angiogram can be performed to view the fundus. Narrowing or blockage of the retinal blood vessels can be clearly seen, which is called retinal ischemia (lack of blood flow). Macular edema, where the contents of the blood vessels leak into the macular area, can occur at any stage of NPDR. Symptoms of macular edema are blurred vision and a dark, distorted image that is different in both eyes. Optical coherence tomography can show areas of retinal thickening (due to fluid accumulation) in macular edema. In the second stage of DR, as part of proliferative diabetic retinopathy (PDR), abnormal new blood vessels form in the fundus (neovascularization), which can rupture and bleed (vitreous hemorrhage), blurring vision. On fundus examination, clinicians will look for cotton wool spots, flame hemorrhages (similar lesions are also caused by alpha toxin of Clostridium novyi), and dot blot hemorrhages.

[0047] The benefits of treating ocular fundus diseases with the senescent cell killing agent of the present disclosure may include inhibition or delay of adverse characteristics of the condition, such as abnormal angiogenesis, pathogenic angiogenesis, vascular occlusion, intraocular hemorrhage, retinal damage, and vision loss. The senescent cell killing agent may be administered into or around the eye, for example, by intraocular, intravitreal, or retrobulbar injection. Optimally, there will be some reversal of pathophysiology, such as restoration of functional vasculature, functional angiogenesis, retinal regrowth or recovery, with partial improvement of vision.

[0048] Presbyopia is an age-related condition that refers to a gradual decline in the eye's ability to focus on nearby objects as the speed and amplitude of normal ocular accommodation declines with age. Loss of elasticity of the lens and loss of ciliary muscle contractility can cause presbyopia. Age-related changes in the mechanical properties of the anterior and posterior lens capsules suggest that the mechanical strength of the posterior capsule declines significantly with age as a result of changes in the composition of the tissue. The main structural element of the lens capsule is the basement membrane type IV collagen, which is organized into a three-dimensional molecular network. Attachment of type IV collagen, fibronectin, and lamina to intraocular lenses can inhibit cell migration and reduce the risk of PCO.

[0049] The senescent cell killing agents provided by the present disclosure can delay the breakdown of type IV collagen network, reduce or inhibit epithelial cell migration, and delay the onset of presbyopia or reduce or slow the progression of the severity of the condition. They may also be useful after cataract surgery to reduce the likelihood of PCO occurring.

[0050] Glaucoma and other anterior eye diseases may also be suitable for treatment with the senolytic agent provided in the present disclosure. Normally, clear fluid flows in and out of the front of the eye, known as the anterior chamber. In people with wide-angle glaucoma, the clear fluid drains too slowly, leading to increased pressure within the eye. If left untreated, high pressure within the eye can damage the optic nerve and lead to complete blindness. Loss of peripheral vision is caused by the death of ganglion cells in the retina.

[0051] The possible benefits of treatment include lowering intraocular pressure, improving drainage of ocular fluid through the trabecular meshwork, and inhibiting or delaying the resulting loss of vision.Senocyte killing agents can be administered into or around the eye, for example, by intraocular or intracameral injection, or in topical formulations.The effectiveness of treatment can be monitored by automated perimetry, keratoscopy, imaging techniques, scanning laser tomography, HRT3, laser polarimetry, GDX, ocular coherence tomography, ophthalmoscopy, and pachymetry, which measure central corneal thickness.

[0052] Treating lung conditions Any of the Bcl inhibitors listed in this disclosure can be developed to treat pulmonary disease according to the present disclosure.Similarly, the Bcl inhibitors listed in this disclosure can be developed to selectively eliminate senescent cells in or around the lungs of subjects who need it.The pulmonary conditions that can be treated include idiopathic pulmonary fibrosis (IPF), chronic obstructive pulmonary disease (COPD), asthma, cystic fibrosis, bronchiectasis, and emphysema.

[0053] COPD is a lung disease defined by the destruction of lung tissue, emphysema, and persistent airflow impairment due to bronchiolitis obliterans, which is a dysfunction of small airways. The main symptoms of COPD include shortness of breath, wheezing, chest tightness, chronic cough, and excessive phlegm production. Elastase from neutrophils and macrophages activated by cigarette smoke can destroy the extracellular matrix of alveolar structures, leading to enlargement of air spaces and loss of vital capacity. COPD can be caused by exposure to, for example, tobacco smoke, cigarette smoke, cigar smoke, passive smoking, pipe smoke, occupational exposure, dust, smoke, haze, and pollution, and takes decades to develop, which implicates aging as a risk factor for developing COPD.

[0054] Processes that cause lung damage include, for example, oxidative stress generated by high concentrations of free radicals in cigarette smoke, cytokine release due to inflammatory responses to irritants in the airways, and dysfunction of antiprotease enzymes by cigarette smoke and free radicals that cause proteases to damage the lungs. Genetic susceptibility may also contribute to the disease. In about 1% of COPD patients, the disease is due to a genetic disorder that results in low levels of alpha-1-antitrypsin production in the liver. Alpha-1-antitrypsin is normally secreted into the bloodstream to help protect the lungs.

[0055] Pulmonary fibrosis is a chronic progressive lung disease characterized by lung stiffening and scarring that can lead to respiratory failure, lung cancer, and heart failure. Fibrosis is associated with epithelial repair. Fibroblasts are activated, increase production of extracellular matrix proteins, and transdifferentiate into contractile myofibroblasts, contributing to wound contraction. A reserve matrix seals the damaged epithelium and provides a scaffold for epithelial cell migration that involves epithelial-mesenchymal transition (EMT). Epithelial injury and associated blood loss induces platelet activation, production of growth factors, and an acute inflammatory response. Normally, the epithelial barrier heals and the inflammatory response resolves. However, in fibrotic diseases, the fibroblast response continues, leading to unresolved wound healing. The formation of fibroblast nests is a hallmark of the disease and reflects the location of ongoing fibrosis.

[0056] Subjects at risk of developing pulmonary fibrosis include, for example, subjects exposed to environmental or occupational pollutants, such as asbestosis and silicosis; subjects who smoke cigarettes; subjects with connective tissue diseases, such as RA, SLE, scleroderma, sarcoidosis, or Wegener's granulomatosis; subjects with infectious diseases; subjects taking certain medications, including, for example, amiodarone, bleomycin, busulfan, methotrexate, and nitrofurantoin; subjects undergoing chest radiation therapy; and subjects with a family history of pulmonary fibrosis.

[0057] Other pulmonary conditions that may be treated by using compounds related to this condition include emphysema, asthma, bronchiectasis, and cystic fibrosis. Lung diseases may be exacerbated by tobacco smoke; occupational exposure to dust, smoke, or fumes; infections; or pollutants that contribute to inflammation.

[0058] Symptoms of lung disease may include shortness of breath, wheezing, chest tightness, having to clear your throat first thing in the morning because of excess mucus in your lungs, a chronic cough that produces phlegm that may be clear, white, yellowish or greenish in color, cyanosis, frequent respiratory infections, lack of energy, and involuntary weight loss. Symptoms of pulmonary fibrosis may include shortness of breath, especially during exercise; dry cough; rapid, shallow breathing; gradual and involuntary weight loss; fatigue; joint and muscle pain; and clubbing of fingers or toes.

[0059] Pulmonary function before, during, and after treatment can be determined, for example, by measuring the expiratory reserve volume (ERV), forced vital capacity (FVC), forced expiratory volume (FEV), total lung capacity (TLC), vital capacity (VC), residual volume (RV), and functional residual capacity (FRC). Gas exchange through the alveolar-capillary membrane can be measured using the diffusion capacity for carbon monoxide (DLCO). Exercise capacity can be measured as a surrogate. Peripheral capillary oxygen saturation (SpO2) can also be measured, with normal oxygen levels typically being 95%-100%. SpO2 levels below 90% suggest that the subject has hypoxemia. Values ​​below 80% are considered dangerous and require intervention to preserve brain and heart function and avoid cardiac or respiratory arrest.

[0060] The benefits of treatment may include inhibiting or reversing the progression of any of these effects. Administration of the senolytic agent may be systemic or localized at sites in or around the lungs, for example, by inhalation as an aerosol or powder, or by intubation. Optimally, the agent will improve SpO2 levels and exercise capacity.

[0061] Treatment of Atherosclerosis Senescent cell killing compounds can be used to treat atherosclerosis, for example by inhibiting the formation, expansion, or progression of atherosclerotic plaques in a subject. Senescent cell killing compounds can also be used to increase the stability of atherosclerotic plaques present in one or more blood vessels of a subject, thereby inhibiting them from rupturing and occluding the blood vessels.

[0062] Atherosclerosis is characterized by atheromas, patchy intimal plaques that invade the lumen of medium and large arteries; the plaques contain lipids, inflammatory cells, smooth muscle cells, and connective tissue. Atherosclerosis can affect large and medium-sized arteries, including the coronary, carotid, and cerebral arteries, the aorta and its branches, and the major arteries of the hands and feet.

[0063] Atherosclerosis can lead to increased thickening of the arterial wall. Symptoms appear when plaque growth or rupture reduces or impedes blood flow, and symptoms may vary depending on which artery is affected. Atherosclerotic plaques can be stable or unstable. Stable plaques regress, remain stationary, or grow slowly, sometimes over decades, until they can cause stenosis or occlusion. Unstable plaques are prone to spontaneous erosion, cracking, or rupture, causing acute thrombosis, occlusion, and infarction long before they cause hemodynamically significant stenosis. Because clinical events can result from unstable plaques that do not appear severe on angiograms, plaque stabilization can be an approach to reduce morbidity and mortality. Plaque rupture or erosion can lead to serious cardiovascular events, such as acute coronary syndromes and stroke. Disrupted plaques can have more lipid, macrophage content, and a thinner fibrous cap than intact plaques.

[0064] The diagnosis of atherosclerosis and other cardiovascular diseases can be based on the patient's symptoms, such as angina, chest pressure, numbness or weakness in arms or legs, difficulty in speaking or slurred speech, drooping facial muscles, leg pain, high blood pressure, renal failure and / or erectile dysfunction, medical history, and / or physical examination. The diagnosis can be confirmed by angiography, ultrasound, or other imaging tests. Subjects at risk of developing cardiovascular disease include those with any one or more predisposing factors, such as a family history of cardiovascular disease, those with other risk factors, such as high blood pressure, dyslipidemia, high cholesterol, diabetes, obesity and smoking, sedentary lifestyle, and predisposing factors including hypertension. The condition can be evaluated, for example, by angiography, electrocardiography, or stress test.

[0065] Potential benefits of treatment with a senolytic agent include mitigating or halting the progression of one or more signs or symptoms of the condition, such as the frequency of plaque formation, the surface area of ​​blood vessels covered with plaque, angina, and reduced exercise tolerance.

[0066] definition "Senescent cells" are generally considered to be derived from a cell type that typically replicates, but can no longer replicate as a result of senescence or other events that cause a change in cell state. Depending on the context, senescent cells can be identified as expressing p16, or at least one marker selected from p16, senescence-associated β-galactosidase, and lipofuscin; possibly two or more of these markers, as well as other markers of senescence-associated secretory phenomena (SASP), including, but not limited to, interleukin 6 and inflammatory, angiogenic, and extracellular matrix-modifying proteins. Unless otherwise specified, senescent cells referred to in the claims do not include cancer cells.

[0067] A "senescence associated", "senescence related" or "age related" disease, disorder, or condition is a physiological condition that presents one or more symptoms or signs that are deleterious to a subject. A condition is "senescence associated" if it is "at least partially caused or mediated by senescent cells". This means that at least one component of the SASP in or around the affected tissue plays a role in the pathophysiology of the condition, such that elimination of at least some senescent cells in the affected tissue results in a substantial alleviation or reduction of the deleterious symptoms or signs to the benefit of the patient. Senescence-associated disorders that may be treated or managed using the methods and products of the present disclosure include disorders in the previous disclosures mentioned in this disclosure and in the discussion. Unless otherwise specified, the term does not include cancer.

[0068] An inhibitor of protein function or Bcl function is a compound that substantially prevents a target protein already expressed in a target cell from performing the enzymatic, binding, or regulatory function that the protein or Bcl family member normally performs in the target cell. This results in the elimination of the target cell or in the cell becoming more susceptible to the toxicity of another compound or event. The compound has an IC of less than 1,000 nM (1.0 μM) when tested in the assay according to Example 1 below. 50 A compound qualifies as a "Bcl inhibitor" or "inhibits Bcl activity" in the present disclosure if it has an activity of less than 100 nM or 10 nM, or between 100 nM and 1 nM, depending on the circumstances, is often preferred.

[0069] The term "Bcl" or "Bcl protein" refers to the family of Bcl proteins exemplified by Bcl-2, Bcl-xL, and Bcl-w. A Bcl inhibitor of the present disclosure will be able to inhibit at least one of Bcl-2, Bcl-xL, and Bcl-w. Typically, but not necessarily, an inhibitor of one of these Bcl proteins will inhibit the other two to some degree. Compounds provided in the present disclosure can be tested for activity of any Bcl family member to identify compounds that have inhibitory activity and may be specific for Bcl-2, Bcl-xL, or Bcl-w. Such inhibitors will be evaluated by measuring the IC for a target Bcl from this list. 50 However, the IC for the other two Bcl family members listed 50 It's at least 10 times better than

[0070] A compound, composition or agent is typically referred to as "senocidal" if it eliminates senescent cells, preferentially replicating cells of the same tissue type, or quiescent cells lacking SASP markers. Alternatively or additionally, a compound or combination may be effectively used if it reduces the release of pathological soluble factors or mediators as part of a senescence-associated secretory phenotype that plays a role in or inhibits the resolution of early symptoms or ongoing pathology of a condition. In this regard, the term "senocidal" refers to functional inhibition, such that compounds that act primarily by inhibiting rather than eliminating senescent cells (senolytic inhibitors) can be used in a similar manner with resulting benefits. Model senocidal compositions and agents in the present disclosure have an EC of less than 1 μM when tested in the assay according to Example 2 below. 50 Activity below 0.1 μM, or between 1 μM and 0.1 μM, may be preferred. Selectivity index (SI) (EC50 of senescent cells compared to non-senescent cells of the same tissue type) 50 ) may be better than 1, 2, 5, or 10 depending on the situation.

[0071] Selective removal or "elimination" of senescent cells from a mixed cell population or tissue does not require that all cells having a senescent phenotype are removed, merely that the proportion of senescent cells originally present in the tissue that remain after treatment is substantially higher than the proportion of non-senescent cells originally present in the tissue that remain after treatment.

[0072] Successful "treatment" of a condition according to the present disclosure may have any beneficial effect on the subject being treated. This includes reducing the severity, duration, or progression of the condition, or any adverse signs or symptoms resulting therefrom. Treatment may be unsuccessful, resulting in no improvement in the typical signs and symptoms of the condition. The goal with therapy is to minimize adverse effects on the target tissue or elsewhere in the subject being treated. In some cases, senolytic agents may be used to prevent or inhibit symptoms of a condition to which the subject is susceptible, for example, by genetic susceptibility or by medical history.

[0073] A "therapeutically effective amount" is an amount of a compound of the present disclosure that (i) treats a particular disease, condition, or disorder; (ii) reduces, ameliorate, or eliminates one or more symptoms of a particular disease, condition, or disorder; (iii) prevents or delays the onset of one or more symptoms of a particular disease, condition, or disorder described herein; (iv) prevents or delays the progression of a particular disease, condition, or disorder; or (v) at least partially reverses the damage caused by a pre-treatment condition.

[0074] A "phosphorylated" form of a compound is a compound that has one or more phosphate groups covalently attached to a core structure, typically, but not necessarily, via an oxygen atom that was present on the molecule prior to phosphorylation. For example, one or more -OH or -COOH groups are replaced with -OPO3H2 or -COOH instead of hydrogen. nThe phosphorylated forms may be substituted with a phosphate group, which may be any of PO3H2 (where n is 1-4). In some phosphorylated forms, the phosphate group may be removed in vivo (e.g., by enzymatic degradation), in which case the phosphorylated form may be a prodrug of the unphosphorylated form. The unphosphorylated form does not have such a phosphate group. The dephosphorylated form is a derivative of the phosphorylated molecule after at least one phosphate group has been removed.

[0075] A "small molecule" Bcl inhibitor according to the present disclosure has a molecular weight of less than 20,000 daltons, and often less than 10,000, 5,000, or 2,000 daltons. Small molecule inhibitors are not antibody molecules or oligonucleotides, and typically do not have more than five hydrogen bond donors (total number of nitrogen-hydrogen and oxygen-hydrogen bonds) and more than ten hydrogen bond acceptors (all nitrogen or oxygen atoms).

[0076] "Prodrug" refers to a derivative of an active agent that requires a transformation in the body to release the active agent. The transformation may be an enzymatic transformation. In some cases, the transformation is a cyclization transformation or a combination of an enzymatic transformation and a cyclization transformation. Prodrugs are often, but not necessarily, pharmacologically inactive until they are converted to an active agent.

[0077] Unless otherwise stated or required, each of the compound structures referred to in this disclosure includes conjugate acids and bases of the same structure, crystalline and amorphous forms of those compounds, pharma- ceutically acceptable salts, and prodrugs, including, for example, polymorphs, solvates, hydrates, nonsolvated polymorphs (including anhydrous forms), and phosphorylated and nonphosphorylated forms of the compounds.

[0078] INCORPORATION BY REFERENCE For all purposes in the United States and in any other jurisdiction where effective, all publications and patent documents cited in this disclosure are incorporated by reference in their entirety for all purposes to the same extent as if each such publication or document was specifically and individually indicated to be incorporated by reference herein.

[0079] U.S. Patent No. 10,130,628 (Laberge et al.) and US 20170266211 A1 (David et al.) are incorporated herein for any purpose, including but not limited to, the identification, formulation, and use of senolytic agents to treat various conditions believed to be at least in part mediated by senescent cells. U.S. Patent Nos. 8,691,184, 9,096,625, and 9,403,856 (Wang et al.) are incorporated herein by reference in their entirety for any purpose, including the characterization, preparation, and use of compounds in the Bcl library. U.S. Patent Application Nos. 15 / 675,171 (filed August 11, 2017) and 62 / 579 / 793 (filed October 31, 2017) are incorporated herein for any purpose, including but not limited to the identification, formulation, and use of compounds that can eliminate or reduce the activity of senescent cells and treat various ocular conditions. EXAMPLES

[0080] Example 1: Measuring Bcl Inhibition The ability of a candidate compound to inhibit Bcl-2 and Bcl-xL activity can be measured at the molecular level by direct binding. This assay uses a homogeneous assay technology based on oxygen channeling, which is commercially available from PerkinElmer Inc., Waltham, Massachusetts; see Eglin et al., Current Chemical Genomics, 2008, 1, 2-10. A test compound is combined with a target Bcl protein and a peptide, which is the corresponding cognate ligand, labeled with biotin. The mixture is then combined with streptavidin-containing luminescent donor beads and luminescent acceptor beads, which proportionally reduce luminescence when the compound inhibits the binding of the peptide to the Bcl protein. Bcl-2, Bcl-xL, and Bcl-w are available from Sigma-Aldrich Co., St. Louis, Missouri. Biotinylated BIM peptide (ligand for Bcl-2) and BAD peptide (ligand for Bcl-xL) are described in US2016 / 0038503 A1. AlphaScreen® streptavidin donor beads and Anti-6XHis AlphaLISA® acceptor beads are available from PerkinElmer.

[0081] To perform the assay, a 1:4 dilution series of the compound is prepared in DMSO, then diluted 1:100 in assay buffer. In a 96-well PCR plate, the following are combined in order: 10 μL peptide (120 nM BIM or 60 nM BIM), 10 μL test compound, and 10 μL Bcl protein (0.8 nM Bcl-2 / W or 0.4 nM Bcl-XL). The assay plate is incubated in the dark for 24 hours at room temperature. The next day, donor and acceptor beads are combined and 5 μL is added to each well. After 30 minutes of incubation in the dark, luminescence is measured using a plate reader to determine the degree of affinity or inhibition by each test compound.

[0082] Example 2: Measuring senescent cell killing activity in fibroblasts Human fibroblast IMR90 cells are available from the American Type Culture Collection (ATCC®) under the name CCL-186. Cells are maintained at less than 75% confluency in DMEM with FBS and Pen / Strep in an atmosphere of 3% O2, 10% CO2, and approximately 95% humidity. Cells are divided into irradiated cells (cultured for 14 days after irradiation before use) and quiescent cells (cultured at high density for 4 days before use).

[0083] On day 0, prepare irradiated cells as follows: Wash IMR90 cells, place them in a T175 flask at a density of 50,000 cells per mL, and irradiate at 10-15 Gy. After irradiation, seed the cells in 100 µL into a 96-well plate. On days 1, 3, 6, 10, and 13, aspirate the medium in each well and replace with fresh medium.

[0084] On day 10, quiescent normal cells are prepared as follows: IMR90 cells are washed and combined with 3 mL of TrypLE trypsin-containing reagent (Thermofisher Scientific, Waltham, Massachusetts) and incubated for 5 minutes until the cells begin to round up and detach from the plate. Cells are dispersed, counted, and prepared in media at a concentration of 50,000 cells per mL. 100 μL of cells are seeded into each well of a 96-well plate. Media is changed on day 13. On day 14, test inhibitor compounds are combined with the cells as follows: A DMSO dilution series of each test compound is prepared in a 96-well PCR plate at 200 times the final desired concentration. Immediately prior to use, the DMSO solution is diluted 1:200 in pre-warmed complete media. Media is aspirated from the cells in each well and compound-containing media is added at 100 μL / well.

[0085] Candidate senolytic agents for testing are incubated with the cells for 6 days, and the medium is replaced with fresh medium at the same compound concentration on day 17. Bcl2 inhibitors are incubated with the cells for 3 days. The assay system utilizes the properties of thermostable luciferase to allow reaction conditions that generate a stable luminescent signal while inhibiting endogenous ATPase released into the cell lysate. At the end of the incubation period, 100 μL of CellTiter-Glo® Reagent (Promega Corp., Madison, Wisconsin) is added to each well. The cell plate is placed on an orbital shaker for 30 seconds and luminescence is measured.

[0086] Example 3: Measuring senolytic activity in HUVEC cells and other senescent cells Human umbilical vein (HUVEC) cells from a single lot were allowed to double in population approximately 8 times in vascular cell basal medium supplemented with Endothelial Cell Growth Kit™-VEGF from ATCC and then cryopreserved. Cells for the senescent population were thawed 9 days prior to the start of the assay and seeded at approximately 27,000 cells / cm2. All cells were cultured in a humidified incubator with 5% CO2 and 3% O2, and medium was changed every 48 hours. Two days after seeding, cells were irradiated with 12 Gy of radiation delivered from an X-ray source. Cells for the non-senescent population were thawed 3 days prior to the start of the assay and seeded similarly to the senescent population. One day prior to the assay, all cells were trypsinized and seeded into separate 384-well plates at 5,000 cells / well of senescent cells and 10,000 cells / well of non-senescent cells in a final volume of 55 μL / well. In each plate, the central 308 wells contained cells and the outer wells were filled with 70 μL / well of deionized water.

[0087] On the day of the assay, compounds were diluted from a 10 mM solution into medium to provide the highest concentration working solution, and then an aliquot of this was further diluted with medium to provide the remaining two working solutions. To initiate the assay, 5 μL of working solution was added to the cell plate. Final test concentrations were 20, 2, and 0.2 μM. On each plate, the assay was repeated three times at a single concentration for 100 test compounds, along with three wells of positive controls and five untreated (DMSO) controls. After compound addition, the plates were returned to the incubator for three days.

[0088] Cell viability was indirectly assessed by measuring total ATP concentration using CellTiter-Glo™ reagent (Promega). The resulting luminescence was quantified using an EnSpire™ plate reader (PerkinElmer). The relative cell viability for each concentration of compound was calculated as a percentage of the untreated control on the same plate.

[0089] For follow-up dose response of potential lead compounds, 384-well plates of senescent and non-senescent cells were prepared as described above. Compounds were prepared as a 10-point 1:3 dilution series in DMSO and then diluted up to 12-fold in culture medium. Five microliters of this working solution was then added to the cell plates. After 3 days of culture, cell survival compared to DMSO control was calculated as described above. All measurements were performed in quadruplicate.

[0090] Other cell lines and primary cell cultures may be used as alternatives to IMR90 fibroblasts or HUVEC cells that match the intended target tissue in vivo.One example is the use of cultured human retinal microvascular endothelial cells (HRMEC) to screen compounds intended to treat ocular diseases.Cells are cultured according to the known protocol for the selected cell line and irradiated and aged in the same manner.

[0091] Example 4: Efficacy of senolytic agents in osteoarthritis models This example describes the testing of MDM2 inhibitors in a mouse model for the treatment of osteoarthritis. It can be adapted mutatis mutandis to test and develop Bcl inhibitors for use in clinical therapy.

[0092] The model was generated as follows: C57BL / 6J mice underwent surgery to sever the anterior cruciate ligament of one hind limb to induce osteoarthritis in the joint of that limb. During weeks 3 and 4 post-surgery, mice were treated with 5.8 μg Nutlin-3A (n=7) per operated knee by intra-articular injection every other day for 2 weeks. At the end of the 4 weeks post-surgery, mouse joints were monitored for the presence of senescent cells, assessed for function, monitored for markers of inflammation, and histologically evaluated.

[0093] The performed study included two control groups of mice: one group (n=3) containing C57BL / 6J or 3MR mice that underwent sham surgery (i.e., surgical procedures performed except for the transection of the ACL) and intra-articular injections of vehicle in parallel with the GCV (ganciclovir) treatment group; and one group (n=5) containing C57BL / 6J or 3MR mice that underwent ACL surgery and received intra-articular injections of vehicle in parallel with the GCV treatment group. RNA from the surgical joints of mice from Nutlin-3A-treated mice was analyzed for the expression of SASP factors (mmp3, IL-6) and senescence markers (p16). qRT-PCR was performed to detect mRNA levels.

[0094] Figures 2A, 2B, and 2C show the expression of p16, IL-6, and MMP13, respectively, in the tissues. OA-inducing surgery was associated with increased expression of these markers. Treatment with Nutlin-3A reduced expression to below control levels. Treatment with Nutlin-3A cleared senescent cells from the joints.

[0095] Limb function was assessed 4 weeks after surgery by weight-bearing testing to determine which leg the mouse preferred. Mice were acclimated at least three times before measurements were taken. Mice were moved around the room and stood on each scale with one hind paw on each scale. Weight applied to each hind paw was measured over a 3-second period. At least three separate measurements were taken for each animal at each time point. Results were expressed as a percentage of weight applied to the operated limb relative to the contralateral unoperated limb.

[0096] Figure 3A shows the results of the functional test. Untreated mice that underwent osteoarthritis-inducing surgery preferred the unoperated hindlimb over the operated hindlimb (Δ). However, depletion of senescent cells with Nutlin-3A abolished this effect in the operated mice (▽).

[0097] Figures 3B, 3C, and 3D show the histopathology of the joint tissues from these experiments. Osteoarthritis induced by ACL surgery caused the destruction of the proteoglycan layer. Removal of senescent cells with Nutlin-3A completely abolished this effect.

[0098] Example 5: Efficacy of senescent cell killing agents in a diabetic retinopathy model This example describes the testing of Bcl inhibitors in a mouse model for the treatment of ocular diseases, specifically diabetic retinopathy. It can be adapted mutatis mutandis to test senolytic agents for use in clinical therapy.

[0099] The efficacy of the model compound UBX1967, a Bcl-xL inhibitor, was tested in a mouse oxygen-induced retinopathy (OIR) model (Scott and Fruttiger, Eye (2010) 24, 416-421, Oubaha et al, 2016). C57Bl / 6 mouse pups and their CD1 foster mothers were exposed to a hyperoxic environment (75% O2) from postnatal day 7 (P7) until P12. At P12, animals were intravitreally injected with 1 μl of test compound (200, 20, or 2 uM) prepared in 1% DMSO, 10% Tween-80, 20% PEG-400 and returned to room air until P17. At P17, eyes were removed and retinas were dissected for either vascular staining or qRT-PCR. To determine avascular or neovascular areas, retinas were flat-laid and stained with isolectin B4 (IB4) diluted 1:100 in 1 mM CaCl2. qPCR was performed for quantitative measurement of senescence markers (e.g., Cdkn2a, Cdkn1a, Il6, Vegfa). RNA was isolated and reverse transcribed to generate cDNA, which was used for qRT-PCR of selected transcripts.

[0100] Figures 4A and 4B show that intravitreally administered (ITT) UBX1967 produced statistically significant improvements in the extent of neovascularization and vascular obstruction at all dose levels.

[0101] The efficacy of UBX1967 was also tested in the streptozotocin (STZ) model. Six- to seven-week-old C57BL / 6J mice were weighed and their baseline blood glucose levels were measured (Accu-Chek™, Roche). Mice were injected intraperitoneally with STZ (Sigma-Alderich, St. Louis, MO) at 55 mg / kg for five consecutive days. Age-matched controls were injected with buffer only. Blood glucose levels were measured again one week after the last STZ injection, and mice were considered diabetic if nonfasting blood glucose levels were higher than 17 mM (300 mg / L). STZ-treated diabetic C57BL / 6J mice were intravitreally injected with 1 μl of UBX1967 (2 μM or 20 μM, prepared as a suspension in 0.015% polysorbate-80, 0.2% sodium phosphate, 0.75% sodium chloride, pH 7.2) at 8 and 9 weeks after STZ administration. Retinal Evans blue permeation assays were performed 10 weeks after STZ treatment.

[0102] Figures 4C and 4D show the results of this protocol: retinal and choroidal vascular leakage following intravitreal (IVT) administered UBX1967 improved vascular permeability at both dose levels.

[0103] Other models of retinal ganglion cell damage, relevant to glaucoma, where elevated intraocular pressure (IOP) is believed to cause retinal ganglion cell loss and optic nerve damage, can be used for testing. In preclinical species, elevated anterior chamber pressure can result in retinal nerve loss, as reported in several established models, including magnetic microbead occlusion (Ito et al., Vis Exp. 2016 (109): 53731) and other glaucoma models (Almasieh and Levin, Annu Rev Vis Sci. 2017). Furthermore, ischemia-reperfusion has been demonstrated to cause retinal damage that may result in cellular senescence. The presence of retinal senescence in such models can be used to monitor the impact of senescent cell death following intravitreal injection of test compounds.

[0104] Example 6: Efficacy of senolytic agents in lung disease models This example describes the testing of inhibitors in mouse models for the treatment of lung disease, specifically for idiopathic pulmonary fibrosis (IPF).It can be adapted with necessary modifications to test and develop Bcl inhibitors for use in clinical treatment.As a model of chronic obstructive pulmonary disease (COPD), mice are exposed to cigarette smoke.

[0105] The effect of senolytic agents on smoke-exposed mice is assessed by senescent cell clearance, lung function, and histopathology.

[0106] Mice used in this study include the 3MR strain described in US 2017 / 0027139 A1 and Demaria et al., Dev Cell. 2014 December 22; 31(6): 722-733. 3MR mice carry a transgene encoding a thymidine kinase that converts the prodrug ganciclovir (GCV) into a compound that is lethal to cells. The enzyme in the transgene is under the control of the p16 promoter, which causes it to be expressed specifically in senescent cells. Treatment of mice with GCV eliminates senescent cells.

[0107] Other mice used in this study include the INK-ATTAC line, described in US 2015 / 0296755 A1 and Baker et al., Nature 2011 Nov 2;479(7372):232-236. The INK-ATTAC mice carry a transgene encoding switchable caspase 8 under the control of the p16 promoter. Caspase 8 can be activated by treating the mice with the switch compound AP20187, which then directly induces apoptosis in senescent cells, eliminating them from the mice.

[0108] To conduct the experiments, 6-week-old 3MR (n=35) or INK-ATTAC (n=35) mice were chronically exposed to cigarette smoke generated from the Teague TE-10 system, an automated cigarette smoke generator that produces a combination of sidestream and mainstream cigarette smoke in a chamber that is then transferred to a collection and mixing chamber where variable amounts of air are mixed with the smoke mixture. The COPD protocol was adapted from the Johns Hopkins University COPD Core Facility (Rangasamy et al., 2004, J. Clin. Invest. 114:1248-1259; Yao et al., 2012, J. Clin. Invest. 122:2032-2045).

[0109] Mice were exposed to cigarette smoke for a total of 6 h per day, 5 days per week, for 6 months. Lighted cigarettes (3R4F research cigarettes [University of Kentucky, Lexington, KY], containing 10.9 mg total particulate matter (TPM), 9.4 mg tar, 0.726 mg nicotine, and 11.9 mg carbon monoxide per cigarette) were each puffed for 2 s, once per minute, for a total of 8 puffs, at a flow rate of 1.05 L / min, with standard puffs delivered from 35 cm away. 3 The cigarette smoke generator was adjusted to produce a mixture of sidestream smoke (89%) and mainstream smoke (11%) by smoking two cigarettes at a time. The smoke chamber atmosphere was monitored for total suspended particles (80-120 mg / m3) and carbon monoxide (350 ppm).

[0110] Starting from day 7, (10)INK-ATTAC and (10)3MR mice were treated with AP20187 (three times a week) or ganciclovir (five consecutive days of treatment followed by 16 days of rest, repeated until the end of the experiment). An equal number of mice received the corresponding vehicle. The remaining 30 mice (15 INK-ATTAC and 15 3MR) were divided equally, five of each transgenic line, into three different treatment groups. One group (n=10) received Nutlin-3A (dissolved at 25 mg / kg in PBS with 10% DMSO / 3% Tween-20™, treated for 14 consecutive days, followed by a 14-day rest period, repeated until the end of the experiment). One group (n=10) received ABT-263 (navitoclax) (dissolved at 100 mg / kg in 15% DMSO / 5% Tween-20, treated for 7 consecutive days, followed by a 14-day washout, and repeated until the end of the experiment). The last group (n=10) received only the vehicle (15% DMSO / 5% Tween-20) which was used instead of ABT-263 following the same treatment regimen as ABT-263. An additional 70 animals that were not exposed to cigarette smoke served as controls for the experiment.

[0111] After 2 months of exposure to cigarette smoke (CS), pulmonary function was assessed by monitoring oxygen saturation using a MouseSTAT PhysioSuite™ pulse oximeter (Kent Scientific). Animals were anesthetized with isoflurane (1.5%) and fitted with toe clips. Mice were monitored for 30 seconds and average peripheral capillary oxygen saturation (SpO2) measurements over this period were calculated.

[0112] The results are shown in Figure 5. Clearance of senescent cells via AP2018, ganciclovir, ABT-263 (navitoclax), or Nutlin-3A led to a statistically significant increase in SpO2 levels in mice after 2 months of cigarette smoke exposure compared to untreated controls.

[0113] Example 7: Efficacy of senolytic agents in atherosclerosis when administered systemically This example describes the testing of MDM2 inhibitors in a mouse model for the treatment of atherosclerosis. The test compound is administered systemically rather than locally. The model is performed in LDLR- / - strain mice that lack the receptor for low-density lipoprotein. The experiments described here can be adapted with necessary modifications to test and develop other types of inhibitors for use in clinical therapy.

[0114] Two groups of LDLR- / - mice (10 weeks) are fed a high-fat diet (HFD) (Harlan Teklad TD.88137) with 42% calories from fat starting at week 0 and throughout the study. Two groups of LDLR- / - mice (10 weeks) are fed a normal chow diet (-HFD). Starting at weeks 0-2, one group of HFD and one group of -HFD mice are treated with Nutlin-3A (25 mg / kg, i.p.). One treatment cycle is 14 days of treatment, 14 days of rest. Vehicle is administered to one group of HFD mice and one group of -HFD mice. At week 4 (time point 1), one group of mice is sacrificed to evaluate the presence of senescent cells within the plaques. For some of the remaining mice, administration of Nutlin-3A and vehicle is repeated starting at weeks 4-6. At week 8 (time point 2), mice are sacrificed and the presence of senescent cells within the plaques is assessed. The remaining mice are treated with Nutlin-3A or vehicle from weeks 8-10. At week 12 (time point 3), mice are sacrificed and the level of plaques and the number of senescent cells within the plaques are assessed.

[0115] Plasma lipid levels were measured in HFD-fed and Nutlin-3A- or vehicle-treated LDLR- / - mice compared to -HFD-fed mice at time point 1 (n=3 per group). Plasma was collected in the mid-afternoon and circulating lipids and lipoproteins were analyzed.

[0116] At the end of time point 1, HFD-fed Nutlin-3A or vehicle-treated LDLR- / - mice were sacrificed (n=3, all groups) and aortic arches were dissected for RT-PCR analysis of SASP factors and senescent cell markers. Values ​​were normalized to GAPDH and expressed as fold change relative to age-matched vehicle-treated LDLR- / - mice on normal chow. Data show that senescent cell clearance by Nutlin-3A in HFD-fed LDLR- / - mice reduced the expression of several SASP factors and senescent cell markers MMP3, MMP13, PAI1, p21, IGFBP2, IL-1A, and IL-1B after one treatment cycle.

[0117] At the end of time point 2, HFD-fed Nutlin-3A or vehicle-treated LDLR- / - mice (n=3 in all groups) were sacrificed and aortic arches were dissected for RT-PCR analysis of SASP factors and senescent cell markers. Values ​​were normalized to GAPDH and expressed as fold change relative to age-matched vehicle-treated normal chow LDLR- / - mice. Data show expression of several SASP factors and senescent cell markers in the aortic arch in HFD mice. Clearance of senescent cells by multiple treatment cycles of Nutlin-3A in HFD-fed LDLR- / - mice reduced expression of most markers.

[0118] At the end of time point 3, HFD-fed Nutlin-3A or vehicle-treated LDLR- / - mice (n=3 in all groups) were sacrificed and aortas were dissected and stained with Sudan IV to detect the presence of lipids. Mice were analyzed for body composition by MRI and circulating blood cells were counted by Hemavet™.

[0119] The results are shown in Figure 6. Treatment with Nutlin-3A reduced the surface area covered by plaque in the descending aorta by approximately 45%. Platelet and lymphocyte counts were similar between Nutlin-3A and vehicle treated mice. Nutlin-3A treatment also reduced body weight and body fat composition in mice fed a high-fat diet.

[0120] Example 8: Measuring cytotoxicity against cancer cells in vitro and in vivo The cellular activity of the compounds can be evaluated in the interleukin-3 (IL-3)-dependent prolymphocytic FL5.12 mouse cell line. Withdrawal of IL-3 induces FL5.12 apoptosis by upregulation of the proapoptotic factors Bim and Puma. Overexpression of Bcl-2 (FL5.12-Bcl-2) or Bcl-xL (FL5.12-Bcl-xL) protects against the effects of IL-3 absence by sequestering Bim and Puma. The compounds reverse the protection afforded by overexpression of Bcl-2 or Bcl-xL. The compounds are ineffective in inducing cell death in the presence of IL-3, where FL5.12 cells receive no proapoptotic stimuli. The ability of the compounds to kill FL5.12-Bcl-2 or FL5.12-Bcl-xL cells in the absence of IL-3 can be attenuated in the presence of the caspase inhibitor ZVAD, indicating that cell death is caspase-dependent.

[0121] Co-immunoprecipitation studies can be performed to determine whether the cytotoxicity induced by BH3 mimetics can be attributed to the disruption of intracellular Bcl-2 family protein-protein interactions. The compounds induce a dose-dependent decrease in Bim:Bcl-xL interactions in FL5.12-Bcl-xL cells. Similar results were observed for the disruption of the Bim:Bcl-2 complex in FL5.12-Bcl-2 cells, indicating that the compounds restore IL-3-dependent cell death by attenuating the ability of Bcl-xL and Bcl-2 to sequester proapoptotic factors such as Bim.

[0122] The ability of the compounds listed in this disclosure to specifically kill cancer cells can be tested in similar assays using other established cell lines. These include HeLa cells, OVCAR-3, LNCaP, and any Authenticated Cancer Cell Lines available from Millipore Sigma, Burlington MA, USA. A compound specifically kills cancer cells if it is at least 5-fold, preferably 25-fold or 100-fold less lethal to cells than non-cancerous cells of the same tissue type. Control cells have similar morphological characteristics and cell surface markers as the cancer cell lines being tested, but do not have signs of cancer.

[0123] In vivo, the compounds are evaluated in flank xenograft models established from sensitive SCLC (H889) and hematological (RS4;11) cell lines or with other tumor-forming cancer cell lines, depending on the type of cancer of particular interest to the user. When administered orally or intravenously, the compounds induce rapid and complete tumor responses (CR) in all animals bearing H889 (SCLC) or RS4;11 (ALL) tumors, which persist for several weeks after the end of treatment. Similar treatment of mice bearing H146 SCLC tumors can induce rapid regression in the animals.

[0124] Example 9: Synthesis The compounds of the invention can be prepared using or adapting the synthetic scheme shown in FIG.

[0125] Example 10: Biochemical and Cellular Activities of Model Compounds Compounds were evaluated for inhibition of ligand binding to Bcl-2 in an in vitro assay. Compounds were evaluated for inhibition of Bcl-xL activity in a direct binding assay, a homogeneous assay for determining inhibition of binding of peptide ligands to Bcl isoforms, according to the method described in Example 1. The EC values ​​obtained for selected compounds were: 50 The values ​​are shown in Tables 2A and 2B.

[0126] [Table 2A]

[0127] [Table 2B]

[0128] Compounds were evaluated for senolytic activity in human cells according to the methods described in Examples 2 and 3. The cell lines were human bronchial epithelial (HBE) cells, small airway epithelial (SAE) cells, and human retinal microvascular endothelial (HRMEC) cells. Such cell types are available from the American Type Culture Collection (ATCC) under the accession numbers CRL-2741, PCS-301-010, and PCS-1101-010, respectively.

[0129] LDs obtained for selected compounds in three different cell lines 50 The values ​​are shown in Tables 3A and 3B.

[0130] [Table 3A]

[0131] [Table 3B]

[0132] Some hypotheses presented in this disclosure provide a premise for the reader to understand various aspects of the invention. The premise is provided to enhance the reader's knowledge. The practice of the invention does not require a detailed understanding or application of the hypotheses. Unless otherwise specified, the characteristics of the hypotheses presented in this disclosure do not limit the application or practice of the claimed invention.

[0133] For example, unless elimination of senescent cells is expressly required, the compounds may be used to treat the described conditions regardless of their effect on senescent cells. Although many of the aging-associated conditions referred to in this disclosure occur primarily in elderly patients, the development of senescent cells and the pathophysiology they mediate may result from other events, such as radiation, other types of tissue damage, other types of disease, genetic abnormalities, etc. The present invention may be practiced on patients of any age having the indicated conditions, unless otherwise expressly indicated or required.

[0134] The discussion of the mechanism of action of the disclosed compounds is also provided to educate the reader and no limitations are to be implied. Unless otherwise stated, the compounds may be used to eliminate senescent or cancerous cells or to treat disease conditions as claimed below, regardless of how they act within the target cell or in the subject of treatment.

[0135] Although the compounds and compositions referred to in this disclosure are described in the context of eliminating senescent cells and treating senescence-associated conditions and cancer, the novel compounds and their derivatives described herein can be prepared for any purpose, including, but not limited to, laboratory uses, treating senescence-associated conditions, toxicological handling, and for diagnostic purposes.

[0136] While the invention has been described with reference to specific embodiments and drawings, as a matter of routine development and optimization and within the purview of those skilled in the art, modifications can be made and equivalents substituted to adapt to a particular situation or intended use, thereby achieving the benefits of the invention without departing from the scope of what is claimed and their equivalents.

[0137] Other technical aspects of the invention described in the specification may be incorporated into the claims to provide further distinctive features.

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