ACYL SULFONAMIDES THAT ARE Bcl FAMILY ANTAGONISTS FOR USE IN CLINICAL MANAGEMENT OF CONDITIONS CAUSED OR MEDIATED BY SENESCENT CELLS AND FOR TREATING CANCER

Abstract

To provide Bcl family antagonists for treating conditions caused or mediated by senescent cells, such as osteoarthritis, eye disease, and lung disease.SOLUTION: For example, a sulfonamide compound of the following formula is shown.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] Priority Application This application claims the benefit of priority of all of U.S. Provisional Patent Application No. 62 / 684,681, filed Jun. 13, 2018, which is hereby incorporated by reference in its entirety for all purposes.

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

Background Art

[0003] Background Senescent cells are cells that no longer have the ability to replicate but remain in the tissue of origin and are characterized as cells that induce the senescence-associated secretory phenotype (SASP). The present disclosure is premised on the fact that many age-related conditions are mediated by senescent cells and that selective removal of such cells from tissues can be clinically used for the treatment of such conditions when in or near such a condition.

[0004] U.S. Patent No. 10,130,628 (Patent Document 1) (Laberge et al.) describes the treatment of certain age-related conditions thought to be at least partially mediated by senescent cells using MDM2 inhibitors, Bcl inhibitors, and Akt inhibitors. US 20170266211 A1 (Patent Document 2) (David et al.) describes the use of specific Bcl inhibitors for the treatment of age-related conditions. U.S. Patent No. 8,691,184 (Patent Document 3), U.S. Patent No. 9,096,625 (Patent Document 4), and U.S. 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 diseases are the pre-grant publications US 2017 / 0056421 A1 (Patent Document 6) (Zhou et al.), WO 2016 / 185481 (Patent Document 7) (Yeda Inst.), US 2017 / 0216286 A1 (Patent Document 8) (Kirkland et al.), and US 2017 / 0281649 A1 (Patent Document 9) (David); as well as the papers by Furhmann-Stroissnigg et al. (Nat Commun. 2017 (Sep 4); 8(1):422) (Non-Patent Document 1), Blagosklonny (Cancer Biol Ther. 2013 Dec; 14(12):1092-7) (Non-Patent Document 2), 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

Patent Document 2

Patent Document 3

Patent Document 4

Patent Document 5

Patent Document 6

Patent Document 7

Patent Document 8

Patent Document 9

Non-Patent Documents

[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] Summary The following disclosure outlines strategies for selectively eliminating senescent cells, provides effective compounds, pharmaceutical compositions, development strategies, and treatment protocols, and describes numerous advantages that follow.

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

[0010] [Invention 1001] A compound of formula (I): TIFF2025089427000002.tif55128, wherein X 1 is -Cl; X 2 is -COOH; X 3is -SO 2 CF 3 、-SO 2 CH 3 、or -NO 2 ; and X 5 is -F or -H; R 1 is -CH(CH 3 ) 2 ; R 2 is -CH 3 ; R 3 and R 4 are both -H; n 1 is 2; and R 6 is -OH, selected from TIFF2025089427000003.tif10128; wherein the hydroxyl group in R 6 may be phosphorylated, provided that when X 3 is -SO 2 CF 3 the hydroxyl group in R 6 must be phosphorylated, compound. [Invention 1002] R 6 is -OR 7 , TIFF2025089427000004.tif10128, and R 7 is -H, -P(O)(OH) 2 , or -(C n H 2n )P(O)(OH) 2 (where n is 1 to 4), provided that when X is -SO 3 CF 2 the R 3 is -P(O)(OH) 7 or -(C 2 H n )P(O)(OH) 2n ) 2 ; The compound of the present invention 1001. [The present invention 1003] X 3 is -SO 2 CF 3 The compound of the present invention 1001 or the present invention 1002. [The present invention 1004] X 3 is -SO 2 CH 3 The compound of the present invention 1001 or the present invention 1002. [The present invention 1005] X 3 is -NO 2 The compound of the present invention 1001 or the present invention 1002. [The present invention 1006] X 5 is -F, the compound of any one of the present inventions 1001 to 1005. [The present invention 1007] X 5 is -H, the compound of any one of the present inventions 1001 to 1005. [The present invention 1008] R 6 is -OH, the compound of any one of the present inventions 1001 to 1007. [The present invention 1009] R 6 is TIFF2025089427000005.tif9128, the compound of any one of the present inventions 1001 to 1007. [The present invention 1010] R 6 is TIFF2025089427000006.tif9128, the compound of any one of the present inventions 1001 to 1007. [The present invention 1011] R 6 in which the hydroxyl group is phosphorylated, the compound of any one of the present inventions 1001 to 1010. [The present invention 1012] R 6 in which the hydroxyl group is phosphorylated with -PO 3 H 2 the compound of the present invention 1010. [Invention 1013] X 3 is -SO 2 CF 3 not, but a compound of any one of Inventions 1001 to 1010 in which the hydroxyl group in R 6 is not phosphorylated. [Invention 1014] X 2 A compound of any one of Inventions 1001 to 1012 in which the carboxyl group in is phosphorylated. [Invention 1015] A compound of Invention 1001 or Invention 1002 selected from the compounds listed in Table 1A. [Invention 1016] The following: A compound of Invention 1001 or Invention 1002 selected from TIFF2025089427000007.tif64154TIFF2025089427000008.tif197155TIFF2025089427000009.tif121153. [Invention 1017] Any compound of the preceding inventions having apoptosis - promoting activity. [Invention 1018] Any compound of the preceding inventions that specifically kills senescent cells defined as non - cancerous cells expressing p16 compared to non - senescent cells. [Invention 1019] Any compound of the preceding inventions that specifically kills cancer cells compared to non - cancerous cells of the same tissue type. [Invention 1020] IC 50 against Bcl - xL is less than 1 nM and / or IC 50 against Bcl - 2 is less than 10 nM, any compound of the preceding inventions. [Invention 1021] LD 50 against irradiated IMR90 cells or HRMEC cells is less than 1 μM, any compound of the preceding inventions. [Invention 1022] LD for irradiated IMR90 cells 50 is less than or equal to one-third of the LD for confluent IMR90 cells 50 or the LD for proliferating IRM90 cells 50 of any compound of the present invention preceding it. [Invention 1023] A pharmaceutical composition comprising any compound of the present invention preceding it in a pharmaceutically compatible excipient. [Invention 1024] A method for selectively removing senescent cells and / or cancer cells from a mixed cell population or tissue, comprising the step of contacting the cells, cell population or tissue with any compound or composition of Inventions 1001 - 1023. [Invention 1025] A method for treating an aging-related condition in a tissue of a subject, wherein the aging-related condition is characterized by being at least partially caused or mediated by senescent cells, or having an excess of senescent cells within or around the tissue compared to a tissue not affected, the method comprising the steps of: administering to the tissue of a subject in need thereof an effective amount of any compound or composition of Inventions 1001 - 1023 for selectively removing senescent cells from the tissue, thereby reducing or alleviating one or more symptoms of the aging-related condition in the subject. [Invention 1026] A unit dose of a pharmaceutical composition comprising a compound in an amount that inhibits Bcl function, configured for use in the treatment of an aging-related condition at least partially caused or mediated by senescent cells, wherein the compound is any compound of Inventions 1001 - 1022, and the composition comprises a formulation of the compound configured for administration to a target tissue in a subject presenting the aging-related condition. The formulation and amount of the compound in the unit dose are configured such that the unit dose is effective to selectively remove senescent cells within or around the tissue in the subject, such that when administered to the tissue as a single dose, it reduces the severity of one or more signs or symptoms of the condition without causing adverse effects in the subject. A unit dose of a pharmaceutical composition. [Inventive concept 1027] A unit dose of Inventive concept 1026, packaged with a package insert describing the use and attendant advantages of the agent in treating senescent cell-related conditions. [Inventive concept 1028] A compound of any of Inventive concepts 1001 - 1022 or a pharmaceutical composition of Inventive concept 1023 for use in selectively eliminating senescent cells from a tissue or mixed cell population or for use in treating an aging-related condition. [Inventive concept 1029] Use of a compound of any of Inventive concepts 1001 - 1022 in the manufacture of a medicament for treating an aging-related condition. [Inventive concept 1030] Any method, product, or use of Inventive concepts 1025 - 1029, wherein the condition is osteoarthritis. [Inventive concept 1031] Any method, product, or use of Inventive concepts 1025 - 1029, wherein the condition is an eye condition. [Inventive concept 1032] Any method, product, or use of Inventive concepts 1025 - 1029, wherein the condition is a lung condition. [Inventive concept 1033] A method for treating cancer, the method comprising the following steps: Administering to the tissue of a subject in need thereof an effective amount of a compound or composition of any of Inventive concepts 1001 - 1023 that is effective to selectively remove cancer cells from the tissue. [Inventive concept 1034] A compound of any of Inventive concepts 1001 - 1022 or a pharmaceutical composition of Inventive concept 1023 for use in selectively eliminating cancer cells from a tissue or mixed cell population or for use in treating cancer. The present invention is described in the following description, in the drawings, and in the appended claims.

Brief Description of the Drawings

[0011]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Mode for Carrying Out the Invention

[0012] Detailed Description Senescent cells no longer have the ability to replicate but remain in the tissue of origin and are characterized as cells that induce the senescence-associated secretory phenotype (SASP). This disclosure is predicated on the fact that many age-related conditions are mediated by senescent cells and that selective removal of such cells from tissues can be clinically used for the treatment of such conditions when a subject is in or near such a condition.

[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 purpose of treating age-related conditions.

[0014] Inhibition of Bcl Protein Activity The Bcl protein family (TC# 1.A.21) includes proteins that are evolutionarily conserved and share Bcl-2 homology (BH) domains. Bcl proteins are best known for their ability to up- or down-regulate apoptosis, a form of programmed cell death, at the mitochondria. The following explanations are provided to help the user understand some of the scientific basis of the compounds of the present disclosure. These concepts are not necessary to practice the invention and do not in any way limit the use of the compounds and methods described herein beyond what is explicitly recited or claimed.

[0015] In the context of the present disclosure, Bcl proteins of particular interest are those that downregulate apoptosis. Anti-apoptotic Bcl proteins contain BH1 and BH2 domains, some of which also contain an additional N-terminal BH4 domain (Bcl-2, Bcl-x(L) and Bcl-w (Bcl-2L2)), and inhibiting these proteins increases the rate or sensitivity of cells to apoptosis. Thus, inhibitors of such proteins can be used to help eliminate cells in which the protein is 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, although ABT-737 is effective against some types of cancer cells, it is prone to dose-limiting thrombocytopenia.

[0017] It has now been discovered that the novel compounds described herein fit into the active site of Bcl proteins, providing potent Bcl inhibition and / or promoting apoptosis of target cells. These compounds can be developed as very potent and specific drugs targeting senescent and cancer cells, as described in the following sections.

[0018] Model Compounds Many of the compounds of the present invention have structures falling within the scope of the formula shown below, TIFF2025089427000010.tif33128wherein, X 1is a halide, preferably -Cl; X 2 is -COOH; X 3 is -SO 2 CF 3 , -SO 2 CH 3 or -NO 2 ; X 5 is -F or -H; R 1 is -CH(CH 3 ) 2 ; R 2 is -H or -CH 3 either, preferably -CH 3 ; R 3 and R 4 are independently either -H or -CH 3 either, preferably both are -H; n 1 is 1 to 3, preferably 2; and R 6 is -OH, selected from TIFF2025089427000011.tif10128; wherein the hydroxyl group in R 6 may be phosphorylated.

[0019] Any of the possible components in the formula, when X 3 is -SO 2 CF 3 is, is accompanied by the condition that the hydroxyl group in R 6 must be phosphorylated. In any combination with any of the foregoing options, the -COOH group of X 2 may optionally be phosphorylated, either with or instead of the hydroxyl group, at the user's discretion.

[0020] The "phosphorylated" form of the compound is such that one or more -OH or COOH groups are -OPO 3 so that the phosphate group can be removed in vivo (e.g., by enzymatic degradation)H 2 or -CnPO 3 H 2 (where n is from 1 to 4), a compound substituted with a phosphate group. The non-phosphorylated or dephosphorylated form does not have such a group.

[0021] Unless explicitly stated or otherwise required, compounds shown without using stereochemistry include racemic mixtures of all stereoisomers and, alternatively, enantiomerically pure preparations by any of the enantiomers. Any of the compounds of Formula I, although not necessarily, usually have the stereochemistry shown in the following Formula I: TIFF2025089427000012.tif43128, which is as follows: TIFF2025089427000013.tif50128 can also be shown as, where each R 3 is independently either -H or -CH 3 of either.

[0022] Many of the compounds of the present invention have structures falling within the scope of Formula II shown below, TIFF2025089427000014.tif43128 which is as follows: TIFF2025089427000015.tif51128 can also be shown as, where X 1 is -Cl; X 2 is -COOH; X 3 is -SO 2 CF 3 ,-SO 2 CH 3 or -NO 2 ; X 5 is -F or -H; R 1 is -CH(CH 3 ) 2 ; R 2 is -CH 3 ; R 3 and R 4 are both -H; n 1 is 2; and R 6 is -OR 7 , TIFF2025089427000016.tif10128, and R 7 is -H, -P(O)(OH) 2 , or -(C n H 2n )P(O)(OH) 2 (where n is from 1 to 4 or from 1 to 8); Provided that when X 3 is -SO 2 CF 3 then R 7 is -P(O)(OH) 2 or -(C n H 2n )P(O)(OH) 2 This includes both the acid form of the indicated R 7 and the salt form such as when R 7 is -P(O)(ONa) 2 or -(C n H 2n )P(O)(ONa) 2 separately and together.

[0023] In the context of a composition or for use, each chemical species and / or each structural formula recited in this disclosure may be optionally used or claimed, provided that they are not explicitly and strictly illustrated or described in any of U.S. 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, provided that they are not explicitly and strictly illustrated or described in US 20170266211 A1 (David et al.).

[0024] Exemplary compounds suitable for the preparation and / or use according to the present disclosure are shown in Table 1A.

[0025] [Table 1A] TIFF2025089427000018.tif204170TIFF2025089427000019.tif204170TIFF2025089427000020.tif204170TIFF2025089427000021.tif204170TIFF2025089427000022.tif204170TIFF2025089427000023.tif204170TIFF2025089427000024.tif210170TIFF2025089427000025.tif204170TIFF2025089427000026.tif140168TIFF2025089427000027.tif207169TIFF2025089427000028.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] Evaluating Compounds for Senescent Cell Degradation Activity and Chemotherapeutic Activity These and other compounds described in the present disclosure can be evaluated at the molecular level for their ability to act in a manner indicating that they are candidate agents for use as active agents in the preparation of pharmaceuticals and for use in 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, the compound can be tested for its ability to inhibit the binding between one or more Bcl proteins and their respective cognate ligands. Example 1 provides an illustration of a homogeneous assay (an assay that does not require a separation step) for the purpose of determining binding to Bcl isoforms. The compound can be screened at the molecular level for its ability to interact with the target isoform and thereby cause the death of senescent cells. Examples 2 and 3 provide an illustration of assays designed for this purpose.

[0030] Alternatively or additionally, the compound can be evaluated for its ability to specifically kill senescent cells. Cultured cells are contacted with the compound and the degree of cytotoxicity or cell inhibition is determined. The ability of the compound to kill or inhibit senescent cells can be compared to the effect of the compound on normal cells that freely divide at low density and normal cells that are in a quiescent state at high density. Examples 2 and 3 provide an illustration of senescent cell killing using the 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 following Examples section provide an illustration for osteoarthritis, eye diseases, lung diseases, and atherosclerosis, respectively.

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

[0033] Formulation of the Drug The preparation and formulation of pharmaceuticals for use according to the present disclosure can incorporate standard techniques such as those described, for example, in the current edition of Remington: The Science and Practice of Pharmacy. The formulation is typically optimized for delivery to the target tissue, for example by topical administration, in a manner that improves access of the active agent to the target senescent cells and provides an 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 a Bcl inhibitor with a pharmaceutically acceptable base or carrier, and optionally one or more pharmaceutically acceptable excipients. Depending on the target tissue, it may be appropriate to formulate a pharmaceutical composition for sustained or extended release. Oral extended-release formulations may contain a mixture of isomer variants, binders, or coating agents. Injectable extended-release formulations may contain an active agent combined with a binder, encapsulating agent, 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 containing unit doses of one or more of the agents or compositions described in the present disclosure. Such kits typically contain a pharmaceutical formulation in one or more containers. The formulation may be provided as one or more unit doses (either combined or separate). The kit may include an apparatus such as a syringe for administration of the agent or composition into or around the target tissue of the subject in need thereof. The product may also include or be accompanied by an informational leaflet describing the use and attendant advantages of the agent in treating aging cell-related conditions and, optionally, an instrument or device for the therapeutic delivery of the composition.

[0036] Treatment Design Senescent cells accumulate with age, which is why senescent cell-mediated conditions occur more frequently in the elderly. Additionally, various types of stress on lung tissue can promote the appearance of senescent cells and the phenotypes they exhibit. 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 overproliferation). Tissues exposed to such stressors may have a higher prevalence of senescent cells, which in turn can lead to the development of certain conditions at a younger age or in a more severe form. Genetic susceptibility to certain conditions suggests that the accumulation of senescent cells mediating the disease can be directly or indirectly affected by genetic factors, which may lead to earlier onset.

[0037] One of the advantages of the senescent cell paradigm is that the successful removal of senescent cells can provide a long-term therapeutic effect to the subject. Senescent cells are essentially non-proliferative, which means that the tissue can only be filled with more senescent cells by the conversion of non-senescent cells in the tissue to 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 the target tissue (a single dose or multiple doses administered daily, twice a week, or once a week over a period of, for example, several days, one week, or several months) can provide an effective period during which the senolytic agent is not administered (e.g., two weeks, one month, two months, or more), during which the subject experiences a remission, reduction, or recovery of one or more harmful signs or symptoms of the condition being treated.

[0038] To treat certain age-related conditions with the senolytic agents 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] Aging-Related Conditions Suitable for Treatment The Bcl inhibitors of the present disclosure can be used for the prevention or treatment of various aging-related conditions. Such conditions are typically (but not necessarily) characterized by senescent cells (such as cells expressing p16 and other senescence markers) within or around the site of the condition, or by an overexpression of p16 and other senescence markers compared to the frequency of such cells or the level of such expression in tissues not affected by the frequency or influence of such cells. Non-limiting examples of interest at present include the treatment of osteoarthritis, eye diseases, and lung diseases, as described in the following sections.

[0040] Treatment of Osteoarthritis Any of the Bcl inhibitors listed in the present disclosure can be developed for treating osteoarthritis in accordance with the present disclosure. Similarly, the Bcl inhibitors listed in the present disclosure can be developed for selectively eliminating senescent cells within or around joints in a subject in need thereof, including, without limitation, joints affected by osteoarthritis.

[0041] The degenerative joint disease of osteoarthritis is characterized by high mechanical stress, osteosclerosis, and fibrosis of cartilage at sites of hypertrophy of the synovium and joint capsule. Fibrosis is a local surface disruption accompanied by separation of the surface layer of cartilage. The initial separation occurs tangentially to the cartilage surface along the axis of many collagen bundles. Collagen within the cartilage breaks down and proteoglycans are lost from the cartilage surface. Without the protective and lubricating effects of proteoglycans on the joint, collagen fibers are more susceptible to degradation and mechanical failure occurs. Risk factors contributing to the development of osteoarthritis include aging, obesity, past joint injury, overuse of joints, weakness of the thigh muscles, and genetics. Symptoms of osteoarthritis include pain or stiffness in joints, particularly the hip, knee, and lower back, after inactivity or overuse; stiffness after rest that resolves with movement; and pain that worsens after activity or towards the end of the day.

[0042] The compounds according to the present disclosure can be used to reduce or inhibit the loss or erosion of the proteoglycan layer in joints, reduce inflammation in affected joints, and promote, stimulate, enhance, or induce the production of collagen, such as type II collagen. The compounds can cause a decrease in the amount or level of inflammatory cytokines, such as IL-6, produced in joints, and the inflammation decreases. The compounds can be used to treat osteoarthritis and / or to induce the production of collagen, such as type II collagen, in the joints of a subject. The compounds can also be used to reduce, inhibit, or decrease the production of matrix metalloproteinase 13 (MMP-13) that degrades collagen in joints and to repair the proteoglycan layer or inhibit the loss and / or degradation of the proteoglycan layer. Thereby, treatment with the compounds can also reduce the likelihood of bone erosion, inhibit erosion, or reduce or delay erosion. The compounds can be administered directly to the joints of osteoarthritis, for example, intra-articularly, topically, transdermally, intradermally, or subcutaneously. The compounds can also restore, improve, or inhibit the decrease in joint strength and relieve joint pain.

[0043] Treatment of Eye Conditions Any of the Bcl inhibitors listed in the present disclosure can be used to prevent or treat the condition of the eye in a subject in need thereof by removing senescent cells in or around the subject's eye, whereby the severity of at least one sign or symptom of the disease is reduced. Such conditions include both fundus diseases and anterior eye diseases. Similarly, the Bcl inhibitors listed in the present disclosure can be developed to selectively eliminate senescent cells in or around the eye tissue in a subject in need thereof.

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

[0045] Age-related macular degeneration is a neurodegenerative condition that can be characterized as an eye disease of the retina, causing the loss of photoreceptor cells in the central part of the retina called the macula. Age-related macular degeneration may be atrophic or exudative. The atrophic form is more common than the exudative form, and about 90% of patients with age-related macular degeneration (AMD) are diagnosed with the atrophic form. Atrophic AMD is associated with atrophy of the retinal pigment epithelium (RPE) layer, which causes the loss of photoreceptor cells. In exudative AMD, new blood vessels grow under the retina and blood and body fluids can leak out. Abnormally leaky choroidal neovascularization can cause retinal cells to die and create a blind spot in central vision. The formation of exudate, i.e., "drusen," under Bruch's membrane in the macula can be a physical sign that age-related macular degeneration is present. Symptoms of age-related macular degeneration include, for example, distortion and abnormal color vision.

[0046] Another eye disease of the retina 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, in which microaneurysms (tiny blood-filled bulges in the artery wall) can be seen. If there is a decrease in visual acuity, fluorescein angiography can be performed to view the fundus. Narrowing or occlusion of the retinal blood vessels can be clearly seen, which is called retinal ischemia (insufficient blood flow). Macular edema, in which 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 different dark, distorted images in both eyes. Optical coherence tomography can show areas of retinal thickening (due to the accumulation of body fluids) 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), causing the visual field to become blurred. In a fundus examination, the clinician will look for cotton-wool spots, flame-shaped hemorrhages (similar lesions are also caused by the alpha toxin of Clostridium novyi), and dot-blot hemorrhages.

[0047] The advantages of treating fundus diseases with the senescent cell killing agents of the present disclosure may include inhibiting or delaying the harmful characteristics of conditions such as abnormal angiogenesis, pathogenic angiogenesis, vascular occlusion, intraocular hemorrhage, retinal damage, and vision loss. The senescent cell killing agents can be administered into or around the eye, for example, by intravitreal, vitreous, or retrobulbar injection. Optimally, there will be some reversal of pathophysiology such as restoration of functional vasculature, functional angiogenesis, retinal regrowth or restoration, accompanied by a partial improvement in vision.

[0048] Presbyopia is an age-related condition in which the eye shows a progressive decline in the ability to focus on near objects as the normal rate and amplitude of accommodation of the eye decrease with age. Loss of elasticity of the lens and loss of contractility of the ciliary muscle can cause presbyopia. Age-related changes in the mechanical properties of the anterior lens capsule and posterior lens capsule suggest that the mechanical strength of the posterior lens capsule decreases significantly with age as a result of tissue compositional changes. The major structural element of the lens capsule is type IV collagen of the basement membrane organized in a three-dimensional molecular network. Inhibiting cell migration and reducing the risk of PCO can be achieved by attaching type IV collagen, fibronectin, and laminin to the intraocular lens.

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

[0050] Glaucoma and other anterior eye diseases may also be suitable for treatment with the senescent cell killing agents provided in the present disclosure. Normally, a clear fluid enters and exits the front part of the eye known as the anterior chamber. In people with open-angle glaucoma, the drainage of the clear fluid is too slow, leading to an increase in intraocular pressure. If left untreated, the high intraocular pressure can damage the optic nerve and may lead to complete blindness. Loss of peripheral vision is caused by the death of ganglion cells in the retina.

[0051] The possible advantages of the treatment include reduction of intraocular pressure, improvement of aqueous humor drainage through the trabecular meshwork, and inhibition or delay of resulting vision loss. The senolytic agent can be administered into or around the eye, for example, by intraocular or intracameral injection or in a topical formulation. The effect of the treatment can be monitored by automated perimetry, keratometry, imaging techniques, scanning laser tomography, HRT3, laser polarimetry, GDX, optical coherence tomography of the eye, ophthalmoscopy, and pachymeter measurement, which measure the thickness of the central part of the cornea.

[0052] Treatment of Lung Conditions Any of the Bcl inhibitors recited in the present disclosure can be developed for treating lung diseases according to the present disclosure. Similarly, the Bcl inhibitors recited in the present disclosure can be developed to selectively eliminate senescent cells in or around the lungs of a subject in need thereof. Lung 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 a persistent airflow deterioration caused by destruction of lung tissue, emphysema, and obstructive bronchiolitis, which is a dysfunction of the small airways. The main symptoms of COPD include shortness of breath, wheezing, chest tightness, chronic cough, and excessive sputum production. Elastase from neutrophils and macrophages activated by cigarette smoke can disrupt the extracellular matrix of the alveolar structure, leading to dilation of the air cavities and loss of vital capacity. COPD can be caused, for example, by exposure to cigarette smoke, cigar smoke, pipe smoke, secondhand smoke, occupational exposure, dust, fumes, smog, and pollution, and can occur over decades, which is why aging is associated with an increased risk of developing COPD.

[0054] The processes that cause lung injury include, for example, oxidative stress generated by high concentrations of free radicals in tobacco smoke, cytokine release due to the inflammatory response to irritants in the airways, and the dysfunction of anti-protease enzymes by tobacco smoke and free radicals that damage the lung with proteases. Genetic susceptibility can also contribute to the disease. In about 1% of COPD patients, the disease is due to a genetic disorder in which the production of alpha-1-antitrypsin in the liver is at a low level. 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 the hardening and scarring of the lungs that can lead to respiratory failure, lung cancer, and heart failure. Fibrosis is associated with epithelial repair. Fibroblasts are activated, the production of extracellular matrix proteins increases, and the differentiation and conversion into contractile myofibroblasts contribute to wound contraction. A provisional matrix seals the damaged epithelium and provides a scaffold for epithelial cell migration with epithelial-mesenchymal transition (EMT). Blood loss associated with epithelial injury induces platelet activation, the 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 and wound healing remains unresolved. The formation of fibroblast foci is characteristic 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 infections; subjects taking certain drugs including, for example, amiodarone, bleomycin, busulfan, methotrexate, and nitrofurantoin; subjects receiving radiation therapy to the chest; and subjects with a family history of pulmonary fibrosis.

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

[0058] Symptoms of lung diseases can include shortness of breath, wheezing, chest tightness, having to cough up excessive mucus in the lungs in the morning, chronic coughing with sputum that may be clear, white, yellow, or greenish in color, cyanosis, frequent respiratory infections, lack of vitality, and unintentional weight loss. Symptoms of pulmonary fibrosis can include shortness of breath, especially during exercise; dry cough; rapid and shallow breathing; progressive and unintentional weight loss; fatigue; joint pain and muscle pain; and clubbing of the fingers or toes.

[0059] Lung function before, during, and after treatment can be determined by measuring, for example, 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 across the alveolar-capillary membrane can be measured using the diffusing capacity of carbon monoxide (DLCO). Exercise capacity can be measured as a surrogate. Peripheral capillary oxygen saturation (SpO 2 ) can also be measured, and normal oxygen levels are typically 95% - 100%. SpO 2 levels below 90% suggest that the subject has hypoxemia. Values below 80% are considered dangerous and require intervention to maintain brain and heart function and avoid cardiac or respiratory arrest.

[0060] Advantages of treatment can also include inhibiting or reversing the progression of any of these effects. Administration of the senolytic agent can be systemic or local at sites inside or around the lungs, for example, by inhalation as an aerosol or powder, or by intubation. Optimally, the agent will improve SpO 2 levels and exercise capacity.

[0061] Treatment of Atherosclerosis The senescent cell death compound can be used for the treatment of atherosclerosis, for example, by inhibiting the formation, enlargement, or progression of atherosclerotic plaques in a subject. The senescent cell death compound can also be used to improve the stability of atherosclerotic plaques present in one or more blood vessels of a subject, thereby inhibiting them from rupturing and occluding blood vessels.

[0062] Atherosclerosis is characterized by atherosclerotic plaques that invade the lumen of medium-sized and large arteries. The plaques contain lipids, inflammatory cells, smooth muscle cells, and connective tissue. Atherosclerosis can affect large and medium-sized arteries, including coronary arteries, carotid arteries, and cerebral arteries, the aorta and its branch vessels, as well as the main trunk arteries of the extremities.

[0063] Atherosclerosis can lead to an increase in arterial wall thickening. Symptoms appear when plaque growth or rupture reduces or obstructs blood flow, and the symptoms may vary depending on which artery is affected. Atherosclerotic plaques can be stable or unstable. Stable plaques grow slowly, sometimes over decades, until they regress, remain static, or cause stenosis or occlusion. Unstable plaques are prone to spontaneous erosion, cracking, or rupture and can cause acute thrombosis, occlusion, and infarction long before they cause hemodynamically significant stenosis. Since clinical events can be caused by unstable plaques that do not appear severe on angiography, plaque stabilization can be a means of reducing morbidity and mortality. Plaque rupture or erosion can lead to serious cardiovascular events such as acute coronary syndrome and stroke. Ruptured plaques may have more lipid and macrophage content and may have a thinner fibrous capsule than intact plaques.

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

[0065] Potential advantages of treatment with senolytic agents include alleviating or arresting 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 by plaque, angina pectoris, and reduced exercise tolerance.

[0066] Definitions "Senescent cells" are generally considered to be typically derived from cell types that replicate, but can no longer replicate as a result of aging or other events that cause changes in the cell state. Depending on the context, senescent cells can be identified as expressing at least one marker selected from p16, or p16, senescence-associated β-galactosidase, and lipofuscin; in some cases two or more of these markers, and non-limitingly other markers of senescence-associated secretory phenotypes (SASP) such as interleukin 6 and inflammatory, angiogenesis and extracellular matrix-modifying proteins. Unless otherwise specified, senescent cells referred to in the claims do not include cancer cells.

[0067] "Senescence associated", "senescence related", or "age related" diseases, disorders, or conditions are physiological states that exhibit one or more symptoms or signs that are detrimental to a subject. A state is "senescence related" if it is "at least partially caused or mediated by senescent cells". This means that the elimination of at least some senescent cells in the affected tissue results in a substantial reduction or alleviation of the detrimental symptoms or signs to the benefit of the patient, such that at least one component of the SASP within or surrounding the affected tissue plays a role in the pathophysiology of the condition. Senescence related disorders that can potentially be treated or managed using the methods and products according to the present disclosure include those disorders in past disclosures mentioned and considered in the present disclosure. 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 functions that the protein or Bcl family member normally performs in the target cell. This results in the elimination of the target cell or the cell becoming more sensitive to the toxicity of another compound or event. The compound is "a Bcl inhibitor" or "inhibits Bcl activity" in the present disclosure if it has an IC 50 of less than 1,000 nM (1.0 μM) when tested in the assay according to Example 1 below. Activity of less than 100 nM or 10 nM, or between 100 nM and 1 nM is often preferred, depending on the circumstances.

[0069] The term "Bcl" or "Bcl protein" refers to the family of Bcl proteins exemplified by Bcl-2, Bcl-xL, and Bcl-w. The Bcl inhibitors of the present disclosure will be able to inhibit at least one of Bcl-2, Bcl-xL, and Bcl-w. Although not necessarily, typically, an inhibitor of one of these Bcl proteins will inhibit the other two to some extent. The compounds provided in the present disclosure can be tested for the activity of any Bcl family member in order to identify compounds that have inhibitory activity and may be specific for Bcl-2, Bcl-xL, or Bcl-w. Such inhibitors have an IC 50 for the target Bcl from this list that is at least 10-fold superior to the IC 50 for the other two Bcl family members in the list.

[0070] A compound, composition or agent is typically referred to as "senolytic" when 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 can be effectively used if it reduces the release of pathological soluble factors or mediators as part of the senescence-associated secretory phenotype that plays a role in the initial symptoms or ongoing pathologies of the condition or inhibits its dissipation. In this regard, the term "senolytic" can be used in a similar manner for compounds (senescence inhibitors) that act primarily by inhibiting rather than eliminating senescent cells, such that they have the resulting advantages. The model senolytic compositions and agents in the present disclosure have an EC 50 of less than 1 μM when tested in the assay according to Example 2 below. Activity of less than 0.1 μM or between 1 μM and 0.1 μM may be preferred. The selectivity index (SI) (EC 50 of senescent cells compared to non-senescent cells of the same tissue type) may be superior to 1, 2, 5, or 10, depending on the situation.

[0071] The selective removal or “exclusion” of senescent cells from a mixed cell population or tissue does not require that all cells with a senescent phenotype be removed, but rather that the proportion of senescent cells remaining in the tissue after treatment, which was initially present in the tissue, is substantially higher than the proportion of non-senescent cells remaining in the tissue after treatment, which was initially present in the tissue.

[0072] A successful “treatment” of a condition according to the present disclosure can be one that has any beneficial effect on the subject being treated. This can include reducing the severity, duration, or progression of the condition, or any adverse signs or symptoms resulting therefrom. There may be cases where the treatment is not successful and as a result there is no improvement in the typical signs and symptoms of the condition. The aim with the therapy is to minimize any adverse effects on the target tissue or elsewhere in the subject being treated. In some cases, for example, due to genetic susceptibility or medical history, a senolytic agent can also be used to prevent or inhibit the symptoms of a condition to which the subject is susceptible.

[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, alleviates, 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 the pre-treatment condition.

[0074] The “phosphorylated” form of a compound is a compound that typically, although not necessarily, has one or more phosphate groups covalently attached to the core structure via an oxygen atom that was present on the molecule prior to phosphorylation. For example, one or more -OH or -COOH groups are replaced by -OPO 3 H 2 or -C n PO 3 H 2It may be substituted with a phosphate group where n is any one of 1 to 4. In some phosphorylated forms, the phosphate group may be removed in vivo (e.g., by enzymatic degradation), and in this case, the phosphorylated form may be a prodrug of the non-phosphorylated form. The non-phosphorylated 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] The "small molecule" Bcl inhibitors according to the present disclosure have a molecular weight of less than 20,000 Daltons, and are often less than 10,000, 5,000, or 2,000 Daltons. The small molecule inhibitors are not antibody molecules or oligonucleotides, and typically do not have more than 5 hydrogen bond donors (total number of nitrogen-hydrogen and oxygen-hydrogen bonds), and more than 10 hydrogen bond acceptors (all nitrogen or oxygen atoms).

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

[0077] Unless otherwise specified or required, each of the compound structures referred to in the present disclosure includes conjugate acids and bases having the same structure, crystals and amorphous forms of those compounds, pharmaceutically acceptable salts, and prodrugs. This includes, for example, polymorphs, solvates, hydrates, non-solvated polymorphs (including anhydrides), as well as phosphorylated and non-phosphorylated forms of the present compound.

[0078] Incorporation by Reference For all purposes in the United States and in other jurisdictions where it is in effect, every publication and patent document cited in this disclosure is hereby incorporated by reference in its entirety for all purposes to the same extent as if each such publication or document was specifically and individually indicated to be incorporated herein by reference.

[0079] U.S. Patent No. 10,130,628 (Laberge et al.) and US 20170266211 A1 (David et al.) are hereby incorporated by reference in their entirety for all purposes including, but not limited to, the identification, formulation, and use of senolytic agents for treating various conditions thought to be mediated at least in part by senescent cells. U.S. Patent Nos. 8,691,184, 9,096,625, and 9,403,856 (Wang et al.) are hereby incorporated by reference in their entirety for all purposes including the characterization, preparation, and use of compounds in a Bcl library. U.S. Patent Application Nos. 15 / 675,171 (filed August 11, 2017) and 62 / 579 / 793 (filed October 31, 2017) are hereby incorporated by reference in their entirety for all purposes 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 eye 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 homogeneous oxygen-channeling-based assay technology commercially available from PerkinElmer Inc., Waltham, Massachusetts; see Eglin et al., Current Chemical Genomics, 2008, 1, 2-10. The test compound is combined with the target Bcl protein and a peptide that 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 decrease luminescence if 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 and then diluted 1:100 in assay buffer. In a 96-well PCR plate, the following are combined in order: 10 μL of peptide (120 nM BIM or 60 nM BIM), 10 μL of test compound, and 10 μL of Bcl protein (0.8 nM Bcl-2 / W or 0.4 nM Bcl-XL). The assay plate is incubated at room temperature in the dark for 24 hours. The next day, the donor beads and acceptor beads are combined and 5 μL is added to each well. After incubating for 30 minutes 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 Death Activity in Fibroblasts The human fibroblast IMR90 cells are available from the American Type Culture Collection (ATCC®) under the name CCL-186. The cells are maintained in DMEM containing FBS and Pen / Strep at less than 75% confluence in an atmosphere of 3% O2, 10% CO 2 , and approximately 95% humidity. The cells are sorted 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, irradiated cells are prepared as follows. Wash the IMR90 cells, place them in a T175 flask at a density of 50,000 cells per mL, and irradiate them with 10 - 15 Gy. After irradiation, seed 100 μL of the cells into a 96-well plate. Aspirate the medium in each well on days 1, 3, 6, 10, and 13 and replace it with fresh medium.

[0084] On day 10, quiescent normal cells are prepared as follows. Wash the IMR90 cells, combine them with 3 mL of TrypLE trypsin-containing reagent (Thermofisher Scientific, Waltham, Massachusetts), and culture for 5 minutes until the cells round up and begin to detach from the plate. Disperse and count the cells, and prepare them in the medium at a concentration of 50,000 cells per mL. Seed 100 μL of the cells into each well of a 96-well plate. Replace the medium on day 13. On day 14, combine the test inhibitor compounds with the cells as follows. Prepare a DMSO dilution series of each test compound at 200-fold the final desired concentration in a 96-well PCR plate. Immediately before use, dilute the DMSO solution 1:200 with pre-warmed complete medium. Aspirate the medium from the cells in each well and add 100 μL / well of the compound-containing medium.

[0085] The candidate senescent cell death agent for testing is cultured with cells for 6 days, and on the 17th day, the medium is replaced with fresh medium at the same compound concentration. The Bcl2 inhibitor is cultured with cells for 3 days. The assay system utilizes the properties of thermostable luciferase to enable reaction conditions that generate a stable luminescence signal while inhibiting the endogenous ATPase released into the cell lysate. At the end of the culture 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 Senescent Cell Death Activity in HUVEC Cells and Other Senescent Cells Human umbilical vein (HUVEC) cells from a single lot were expanded approximately 8-fold in vascular cell basal medium supplemented with Endothelial Cell Growth Kit (TM)-VEGF from ATCC and then cryopreserved. Nine days before the start of the assay, the cells for the senescent population were thawed and seeded at approximately 27,000 cells / cm 2 . All cells were cultured in a humidified incubator with 5% CO 2 and 3% O 2 , and the medium was changed every 48 hours. Two days after seeding, the cells were irradiated with 12 Gy of radiation from an X-ray source. Three days before the start of the assay, the cells for the non-senescent population were thawed and seeded in the same manner as the senescent population. One day before the assay, all cells were trypsinized and seeded at 5,000 senescent cells / well and 10,000 non-senescent cells / well in separate 384-well plates at 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, the compound was diluted from a 10 mM solution into the medium to provide the working solution at the highest concentration, and then a defined aliquot was further diluted with the medium to provide the remaining two working solutions. To initiate the assay, 5 μL of the working solution was added to the cell plates. The final test concentrations were 20, 2, and 0.2 μM. In each plate, for 100 test compounds, the assay was repeated three times at a single concentration, along with 3 wells of positive control and 5 untreated (DMSO) controls. After the addition of the compound, the plates were returned to the incubator for 3 days.

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

[0089] For the dose-response tracking of potential lead compounds, senescent and non-senescent cells in 384-well plates were prepared as described above. The compounds were prepared as a 1:3 dilution series of 10 points in DMSO and then diluted up to 12-fold in the medium. Then, 5 microliters of this working solution was added to the cell plates. After 3 days of culture, cell viability compared to the DMSO control was calculated as described above. All measurements were performed 4 times.

[0090] As an alternative to IMR90 fibroblasts or HUVEC cells, other cell lines and primary cell cultures may be used that match the intended target tissue in vivo. An example is the use of cultured human retinal microvascular endothelial cells (HRMEC) for screening compounds intended for the treatment of eye diseases. The cells are cultured according to known protocols for the selected cell line and irradiated in a similar manner to induce senescence.

[0091] Example 4: Efficacy of a Senescent Cell Death Agent in an Osteoarthritis Model This example describes the testing of an MDM2 inhibitor in a mouse model for the treatment of osteoarthritis. Modifications can be made and adapted as necessary to test and develop a Bcl inhibitor for use in clinical treatment.

[0092] The model was created as follows. C57BL / 6J mice were subjected to surgery to induce osteoarthritis in the joints of one hindlimb by cutting the anterior cruciate ligament. During the 3rd and 4th weeks after surgery, mice were treated with 5.8 μg of Nutlin-3A (n = 7) every other day by intra-articular injection for 2 weeks for each surgically treated knee. At the end of 4 weeks after surgery, the joints of the mice were monitored for the presence of senescent cells, evaluated for function, monitored for markers of inflammation, and histologically evaluated.

[0093] The study conducted included two control groups of mice: sham surgery (i.e., surgical procedure performed without cutting the ACL) and a group of C57BL / 6J or 3MR mice (n = 3) that received an intra-articular injection of vehicle in parallel with the GCV (ganciclovir) treatment group; and a group of C57BL / 6J or 3MR mice (n = 5) that underwent ACL surgery and received an intra-articular injection of vehicle in parallel with the GCV-treated group. RNA from the surgically treated joints of the mice from the 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 the mRNA levels.

[0094] Figures 2A, 2B, and 2C show the expression of p16, IL-6, and MMP13 in the tissues, respectively. The OA-induced surgery was associated with an increase in the expression of these markers. Treatment with Nutlin-3A decreased the expression to levels below those of the control. Treatment with Nutlin-3A removed senescent cells from the joints.

[0095] Four weeks after surgery, limb function was evaluated by a weight-bearing test that determines which leg the mouse prefers. Before measurements were taken, the mice were acclimated to the room at least three times. The mice were moved around the room and made to stand on one hind paw on each weighing scale. The weight borne by each hind limb was measured over 3 seconds. At each time point, at least three separate measurements were made for each animal. Results were expressed as the percentage of the weight borne by the operated limb relative to the non-operated limb on the opposite side.

[0096] Figure 3A shows the results of the functional test. Untreated mice that underwent osteoarthritis induction surgery preferred the non-operated hind limb over the operated hind limb (Δ). However, when senescent cells were eliminated with Nutlin-3A, this effect was abolished in the operated mice (▽).

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

[0098] Example 5: Efficacy of a Senescent Cell Death Agent in a Diabetic Retinopathy Model This example describes the testing of a Bcl inhibitor in a mouse model for the treatment of eye diseases, particularly diabetic retinopathy. It can be adapted with the necessary changes to test senolytic agents for use in clinical treatment.

[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 pups and their CD1 foster mothers were placed in a hyperoxic environment (75% O 2) was exposed. In P12, 1 μl of the test compound (200, 20, or 2 μM) prepared in 1% DMSO, 10% Tween-80, and 20% PEG-400 was intravitreally injected into the animals and returned to room air until P17. At P17, the eyeballs were removed and the retinas were dissected for either vascular staining or qRT-PCR. To determine the avascular or neovascular regions, the retinas were laid flat and stained with Isolectin B4 (IB4) diluted 1:100 in 1 mM CaCl 2 and stained with Isolectin B4 (IB4) diluted 1:100 in 1 mM CaCl. qPCR was performed for quantitative measurement of senescence markers (e.g., Cdkn2a, Cdkn1a, Il6, Vegfa). RNA was isolated, cDNA was generated by reverse transcription, and this was used for qRT-PCR of the selected transcripts.

[0100] Figures 4A and 4B show that intravitreal ITT) administration of UBX1967 resulted in a statistically significant improvement in the degree of angiogenesis and vascular occlusion 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). The mice were intraperitoneally injected with STZ (Sigma-Aldrich, St. Louis, MO) at 55 mg / kg for 5 consecutive days. Controls of the same age were injected with buffer only. One week after the last STZ injection, the blood glucose levels were measured again, and mice were considered diabetic if their blood glucose levels outside of fasting were higher than 17 mM (300 mg / L). Diabetic C57BL / 6J mice treated with STZ 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. The retinal Evans blue permeability assay was performed 10 weeks after STZ treatment.

[0102] Figures 4C and 4D show the results of this protocol. Vascular leakage in the retina and choroid after intravitreal (IVT) administration of UBX1967 was improved in vascular permeability at both dose levels.

[0103] Other models of retinal ganglion cell damage associated with glaucoma, where an increase in intraocular pressure (IOP) is thought to cause loss of retinal ganglion cells and optic nerve damage, can be used in the study. In preclinical species, an increase in anterior chamber pressure can result in loss of retinal nerves, 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 can lead to cellular senescence. The presence of retinal senescence in such models can be used to monitor the effect of test compounds on senescent cell death after intravitreal injection.

[0104] Example 6: Efficacy of a Senescent Cell Death Agent in a Lung Disease Model This example describes the testing of an inhibitor in a mouse model for the treatment of lung diseases, specifically a model for idiopathic pulmonary fibrosis (IPF). The necessary modifications can be made and adapted to test and develop Bcl inhibitors for use in clinical treatment. As a model of chronic obstructive pulmonary disease (COPD), mice were exposed to cigarette smoke.

[0105] The effect of a senolytic agent on mice exposed to smoke is evaluated by clearance of senescent cells, lung function, and histopathology.

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

[0107] Other mice used in this study included the INK-ATTAC strain described in US 2015 / 0296755 A1 and Baker et al., Nature 2011 Nov 2;479(7372):232-236. INK-ATTAC mice have 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, whereupon caspase 8 directly induces apoptosis in senescent cells and eliminates them from the mice.

[0108] To conduct the experiment, 6-week-old 3MR (n = 35) or INK-ATTAC (n = 35) mice were chronically exposed to cigarette smoke generated from a Teague TE-10 system. This system is an automated controlled cigarette smoke generator that produces a combination of sidestream and mainstream cigarette smoke within a chamber, which is then transferred to a collection and mixing chamber where various amounts of air are mixed with the smoke mixture. The COPD protocol was adopted from the COPD Core Facility at Johns Hopkins University (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 hours per day, 5 days per week, for 6 months. Lit research cigarettes 3R4F [University of Kentucky, Lexington, KY] (each 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 puffed at a flow rate of 1.05 L / min for 2 seconds each, once per minute, for a total of 8 puffs to make a standard puff of 35 cm. 3 The cigarette smoke generator was adjusted to produce a mixture of sidestream smoke (89%) and mainstream smoke (11%) by burning two cigarettes at a time. The atmosphere in the smoke chamber was monitored for total suspended particles (80 - 120 mg / m3) and carbon monoxide (350 ppm).

[0110] Starting on day 7, (10) INK-ATTAC and (10) 3MR mice were treated with AP20187 (three times a week) or ganciclovir (a 5-day continuous treatment followed by a 16-day drug holiday repeated until the end of the experiment), respectively. The corresponding vehicle was given to an equal number of mice. The remaining 30 mice (15 INK-ATTAC and 15 3MR) were evenly divided, with 5 mice from each transgenic line assigned to three different treatment groups. One group (n = 10) was given Nutlin-3A (dissolved in PBS containing 10% DMSO / 3% Tween-20™ at 25 mg / kg and treated continuously for 14 days, followed by a 14-day drug holiday and repeated until the end of the experiment). One group (n = 10) was given ABT-263 (navitoclax) (dissolved in 15% DMSO / 5% Tween-20 at 100 mg / kg and treated continuously for 7 days, followed by a 14-day drug holiday and repeated until the end of the experiment). The last group (n = 10) was given only the vehicle (15% DMSO / 5% Tween-20) used in place of ABT-263 according to the same treatment regimen as ABT-263. An additional 70 animals not exposed to cigarette smoke were used as controls for the experiment.

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

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

[0113] Example 7: Efficacy of a Senescent Cell Death Agent in Atherosclerosis upon Systemic Administration This example describes the testing of an MDM2 inhibitor 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 deficient in the low-density lipoprotein receptor. The experiments described herein can be adapted with the necessary changes to test and develop other types of inhibitors for use in clinical therapy.

[0114] Two groups of LDLR− / − mice (10 weeks old) 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 old) are fed a normal diet (−HFD). From weeks 0 to 2, one group of HFD mice and one group of −HFD mice are treated with Nutrin-3A (25 mg / kg, intraperitoneally). One treatment cycle is 14 days of treatment followed by 14 days of drug withdrawal. 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 and the presence of senescent cells within plaques is evaluated. For some of the remaining mice, administration of Nutrin-3A and vehicle is repeated from weeks 4 to 6. At week 8 (time point 2), the mice are sacrificed and the presence of senescent cells within plaques is evaluated. The remaining mice are treated with Nutrin-3A or vehicle from weeks 8 to 10. At week 12 (time point 3), the mice are sacrificed and the levels of plaques and the number of senescent cells within plaques are evaluated.

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

[0116] At the end of time point 1, LDLR− / − mice fed HFD and treated with Nutrin-3A or vehicle were sacrificed (n = 3, all groups), and the aortic arch was dissected for RT-PCR analysis of SASP factors and senescent cell markers. Values were normalized to GAPDH and represented as fold change relative to vehicle-treated normal diet LDLR− / − mice of the same age. Data showed that clearance of senescent cells by Nutrin-3A in LDLR− / − mice fed HFD decreased 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, LDLR− / − mice fed an HFD and treated with Nutlin-3A or vehicle (n = 3 in all groups) were sacrificed, and the aortic arch was dissected for RT-PCR analysis of SASP factors and senescence cell markers. Values were normalized to GAPDH and represented as fold change relative to age-matched vehicle-treated LDLR− / − mice on normal chow. The data show the expression of several SASP factors and senescence cell markers in the aortic arch within HFD-fed mice. Clearance of senescent cells by multiple treatment cycles of Nutlin-3A in HFD-fed LDLR− / − mice decreased the expression of most markers.

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

[0119] The results are shown in Figure 6. Treatment with Nutlin-3A decreased the surface area covered by plaques in the descending aorta by approximately 45%. The numbers of platelets and lymphocytes were equivalent between mice treated with Nutlin-3A and vehicle. Treatment with Nutlin-3A also decreased the body weight and body fat composition of mice fed a high-fat diet.

[0120] Example 8: Measuring Cytotoxicity against Cancer Cells In Vitro and In Vivo The cellular activity of the compound can be evaluated in the interleukin-3 (IL-3)-dependent pre-lymphocytic FL5.12 mouse cell line. In the absence of IL-3, FL5.12 apoptosis is induced by the upregulation of the pro-apoptotic factors Bim and Puma. Overexpression of Bcl-2 (FL5.12-Bcl-2) or Bcl-xL (FL5.12-Bcl-xL) protects from the effects of the absence of IL-3 by sequestering Bim and Puma. The compound reverses the protection afforded by overexpression of Bcl-2 or Bcl-xL. The compound has no effect on inducing cell death in the presence of IL-3 where FL5.12 cells are not subjected to apoptosis-promoting stimuli. The ability of the compound 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 assays can be performed to determine whether the cytotoxicity induced by the BH3 mimetic can be attributed to disruption of intracellular Bcl-2 family protein interactions. The compound induces a dose-dependent decrease in the Bim:Bcl-xL interaction in FL5.12-Bcl-xL cells. Similar results are observed for the disruption of the Bim:Bcl-2 complex in FL5.12-Bcl-2 cells, indicating that the compound restores IL-3-dependent cell death by weakening the ability of Bcl-xL and Bcl-2 to sequester pro-apoptotic factors such as Bim.

[0122] The test of the ability of the compounds listed in the present disclosure to specifically kill cancer cells can be tested in a similar assay using other established cell lines. These include HeLa cells, OVCAR-3, LNCaP, and any Authenticated Cancer Cell Lines available from Millipore Sigma, Burlington MA, U.S.A. The compound specifically kills cancer cells if it is lethal to the cells at a concentration at least one-fifth, preferably one-twenty-fifth or one-hundredth, that of non-cancer cells of the same tissue type. Control cells have morphological features and cell surface markers similar to the cancer cell line 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 blood (RS4;11) cell lines or 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 a rapid and complete tumor response (CR) that persists for several weeks after the end of treatment in all animals bearing H889 (SCLC) or RS4;11 (ALL) tumors. Similar treatment of mice bearing H146 SCLC tumors can induce rapid regression in the animals.

[0124] Example 9: Synthesis The compounds of the present invention can be prepared using or adapted to the synthetic scheme shown in Figure 1.

[0125] Example 10: Biochemical and Cellular Activities of Model Compounds The compounds were evaluated for inhibition of ligand binding to Bcl-2 in in vitro assays. Inhibition of Bcl-xL activity in a direct binding assay, a homogeneous assay for determining inhibition of binding of a peptide ligand to a Bcl isoform, was evaluated according to the method described in Example 1. The EC 50 values obtained for the selected compounds 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] The LD 50 values obtained for the selected compounds in three different cell lines are shown in Table 3A and Table 3B.

[0130]

Table 3A

[0131]

Table 3B

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

[0133] For example, except where the elimination of senescent cells is explicitly required, the compounds can be used to treat the described conditions regardless of their effects on senescent cells. Many of the age-related conditions referred to in this disclosure occur primarily in elderly patients, but the generation of senescent cells and the pathophysiology they mediate can result from other events such as radiation, other types of tissue damage, other types of disease, genetic abnormalities, and the like. The present invention can be practiced with respect to patients of any age having the indicated conditions, unless specifically indicated or required otherwise.

[0134] Considerations regarding the mechanism of action of the disclosed compounds are also provided to enhance the reader's knowledge and do not imply any limitation. Unless specifically stated otherwise, the compounds can be used to remove senescent or cancer cells or for the treatment of medical conditions as claimed below, regardless of how they act inside the target cells or in the subject being treated.

[0135] The compounds and compositions referred to in this disclosure are described in the context of eliminating senescent cells and treating age-related conditions and cancer, but the novel compounds and their derivatives described herein can be prepared for any purpose, including but not limited to laboratory use, treatment of age-related conditions, handling of poisons, and for diagnostic purposes.

[0136] The invention has been described with reference to specific examples and drawings, but modifications can be made and equivalents can be substituted in accordance with normal development and optimization matters and within the scope of those skilled in the art, to adapt to specific circumstances or intended uses, 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 can be incorporated into the claims to provide further distinctive features.

Claims

[Claim 1] The invention described herein.

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