RU2815482C2 - Treatment of eye diseases such as macular degeneration, glaucoma and diabetic retinopathy with medications that eliminate aging cells - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to the field of medicine, namely ophthalmology, and is intended to inhibit pathological angiogenesis in the eye of a subject in need of it. To inhibit pathological angiogenesis in the subject’s eye, a therapeutically effective dosage of a pharmaceutical composition containing the following compounds or their pharmaceutically acceptable salts is administered.

EFFECT: use of the invention provides effective suppression of pathogenic angiogenesis in the eye due to the selected dose profile and high specificity.

7 cl, 14 dwg, 1 tbl, 12 ex

Description

Related applications

The present invention claims benefit from U.S. Patent Application No. 15/675,171, filed Aug. 11, 2017 (pending), and U.S. Patent Application No. 62/579,793, filed Oct. 31, 2017 (pending). In those jurisdictions where incorporation by reference is permitted, these priority applications are hereby incorporated herein by reference in their entirety for all purposes.

Field of technology to which the invention relates

The technology disclosed and stated below generally relates to the field of eye diseases resulting in vision impairment. In particular, the present invention provides a family of compounds and techniques that can be used to treat eye diseases by eliminating the senescent cells involved in their pathophysiology and symptoms.

State of the art

The prevalence of visual impairment and blindness in adults due to age-related eye diseases is one of the greatest challenges facing modern medicine.

Three eye diseases pose a potential threat to vision in people in middle-income and industrialized countries around the world, according to the World Health Organization. The increasing prevalence of type II diabetes has brought diabetic retinopathy to the top of the WHO priority list. Glaucoma, a disabling eye disease known for centuries, remains on the public health agenda due to the difficulty of early diagnosis and the frequent need for lifelong treatment. Age-related macular degeneration (AMD) is the third leading global cause of visual impairment, with a blindness rate of 8.7%. It is the leading cause of visual impairment in industrialized countries.

In the United States, 2.1 million people have age-related macular degeneration, 7.7 million have diabetic retinopathy, 2.7 million have glaucoma, and 24.4 million have cataracts, according to the National Eye Institute (NEI). This represents an astonishing 26% of the American population over 40 years of age.

Despite the prevalence of visual impairments and all the attention they receive from the medical research community, these diseases remain largely intractable. Many diseases do not have available or disease-modifying therapeutic alternatives. With some exceptions, most drugs currently approved for the treatment of these diseases target late-stage pathophysiology or symptomatic relief rather than the factors that initiate and/or maintain the disease.

Currently available therapies include agents such as vascular endothelial growth factor (VEGF) inhibitors for the treatment of VEGF-related eye diseases (eg, wet AMD, diabetic eye diseases), vitamins and antioxidants for dry AMD, and intraocular pressure-lowering agents for glaucoma. . Laser therapy is available to treat certain conditions: for example, retinal photocoagulation for retinal edema or neovascularization due to diabetes, vein occlusion, and choroidal neovascularization; and laser trabeculoplasty for the treatment of increased intraocular pressure refractory to drug therapy. Many eye diseases, including dry AMD, currently have no approved therapeutic agents. A phase 3 clinical trial of a humanized antibody developed to treat geographic atrophy in dry AMD (lampalizumab) recently failed to meet its primary endpoint of preventing atrophy progression. Even for those eye diseases where therapeutic options are available, treatment regimens are often burdensome and have limited long-term effectiveness.

The invention presented here creates a new paradigm for the treatment of ocular diseases by eliminating senescent cells involved in the pathophysiology of visual system disorders. The following description outlines its implementation and use and describes the many benefits that result from it.

The essence of the invention

The present invention is based on the discovery that many ocular diseases associated with aging are mediated, at least in part, by cells with an aging phenotype. Senescent cells accumulate with age and express factors that contribute to the pathophysiology of age-related diseases. Data show that in age-matched patients, the severity of age-related diseases correlates with the abundance of senescent cells, and removing senescent cells may help eliminate such diseases.

The present invention provides small molecule drugs that remove senescent cells from diseased ocular tissue and are particularly effective in the treatment of eye diseases. They not only inhibit the progression of diseases, but can also reverse some pathophysiological processes such as neovascularization and vascular obliteration, which lead to vision loss. These senolytic agents have an appropriate dose profile and specificity, being effective in the clinical management of previously intractable ocular diseases.

In general, the present invention provides technologies for preventing or treating eye diseases in subjects by removing senescent cells from or around the eyes of the subject, thereby slowing the progression of the disease or reducing the severity of at least one sign or symptom of the disease.

For the purposes of the present invention, eye diseases are classified into six main types of pathophysiology: ischemic or vascular diseases; degenerative diseases; genetic diseases; bacterial, fungal or viral infections; inflammatory diseases; or iatrogenic diseases. Their pathophysiology is informative when implementing a senolytic strategy for the treatment of each disease. The classification of eye diseases within these types is given below in the description.

The invention includes methods of treatment, unit doses and intended use of certain inhibitors and senolytic agents. Effective agents that can be used in the context of this invention for the removal of senescent cells and the treatment of eye diseases include compounds of the following formula or phosphorylated forms thereof:

where: R ₁ and R ₂ independently mean C ₁ –C ₄ -alkyl;

R ₃ , R ₄ and R ₅ are independently -H or -CH ₃ ;

R ₈ is -OH or -N(R ₆ )(R ₇ ), where R ₆ and R ₇ are independently alkyl or heteroalkyl, which is optionally cyclized;

X ₁ means -F, -Cl, -Br or -OCH ₃ ;

X ₂ means -SO ₂ R′ or -CO ₂ R′, where R′ means -H, -CH ₃ or -CH ₂ CH ₃ ;

X ₃ means -SO ₂ CF ₃ , -SO ₂ CH ₃ or -NO ₂ ; And

X ₅ means -F, -Br, -Cl, -H or -OCH ₃ .

In some embodiments, X ₃ is -SO ₂ CF ₃ or -NO ₂ and R ₈ is -N(R ₆ )(R ₇ ), wherein R ₆ and R ₇ are independently alkyl or heteroalkyl, which is optionally cyclized.

Depending on how the technology is implemented, in addition to generally improving symptoms or preventing the progression of a specific eye disease, the technology may have one or more of the following effects in any combination:

• a decrease in the number of p16-positive senescent cells in or around the subject's eyes;

• inhibition or regression of neovascularization in the subject's eyes;

• inhibition or regression of vascular obliteration in the subject's eyes; And

• inhibition or regression of elevated intraocular pressure (IOP) in the subject's eyes.

The invention also provides new screening methods. One such method involves selecting a test compound as a potential pharmaceutical treatment for glaucoma by contacting trabecular meshwork (TM) cells in culture or tissue of non-human test subjects and determining whether the test agent will reduce the number of senescent cells in the culture or tissue. Another such screening method involves administering a test agent to the eyes of non-human test subjects and determining whether the test agent will inhibit or reverse neovascularization or vascular obliteration occurring in an animal model of the disease.

Other features of the technology according to the invention are presented in the following sections and in the accompanying claims.

Brief description of the figures

In fig. 1A is a flow diagram that shows the pathophysiological interactions in the eye that occur during ischemia. In fig. Figure 1B is a flow diagram that shows the multifactorial pathophysiology of glaucoma leading to retinal ganglion cell (RGC) death. In fig. Figure 1C is a flow diagram that shows the pathophysiological cascade in age-related macular degeneration. In fig. Figure 1D is a flow diagram that shows the events leading to cell degeneration and cell death in Leber hereditary optic neuropathy.

In fig. Figure 2A shows histopathology images that show retinal cell loss during the stages of retinitis pigmentosa. In fig. 2B, 2C, 2D, and 2E show fluorescein angiograms of normal retinas and nonperfused and neovascularized retinas in diabetes, sickle cell disease, and inflammatory vasculitis, respectively.

In fig. 3A and 3B show 9 specific compounds selected from the library based on binding to Bcl-2 or Bcl-xL.

In fig. 4A, 4B and 4C show the quantitative binding affinities of 9 compounds to Bcl isoforms: Bcl-xL, Bcl-2 and Bcl-w, respectively. Each of the compounds for which data is given are identified by their assigned BM numbers.

In fig. 5A, 5B, and 5C provide a comparison of compound structure by effectiveness to determine which substructures contribute to the desired properties of the compounds.

In fig. 6 shows the binding affinity for Bcl isoforms and the effective concentration (EC ₅₀ ) to kill senescent fibroblasts (SnCs) in culture.

In fig. 7 shows a concentration-response curve for senescent human retinal microvascular endothelial cells HRMEC and control cells when treated in vivo with a senolytic agent.

In fig. 8 shows a concentration-response curve for senescent retinal pigment epithelial (RPE) cells and control cells when treated in vivo with a senolytic agent. The agent has much greater activity (lower LD ₅₀ ) on senescent cells than on normal proliferating RPE cells. Its selectivity index for senescent RPE cells compared to nonsenescent RPE cells ranges from 10 to 100.

In fig. 9A and 9B show regression of neovascularization and vascular occlusion in a mouse model of oxygen-induced retinopathy (OIR) following intravitreal administration of the senolytic agent UBX1967.

In fig. 10A, 10B, 10C and 10D show decreased expression of senescence-associated markers at the RNA transcript level following treatment with UBX1967.

In fig. 11A and 11B show the streptozotocin (STZ) model of diabetic retinopathy. STZ-induced vascular leak is attenuated by intravitreal administration of UBX1967.

In fig. 12A and 12B show immunohistochemical staining for p16 (a marker of senescent cells) in tissue taken from a patient with primary open angle glaucoma (POAG). p16-positive cells are clearly visible in the trabecular meshwork (TM).

In fig. 13A and 13B show p16 expression in human ocular tissue obtained from donors diagnosed with primary open angle glaucoma (POAG).

In fig. 14A and 14B show immunohistochemical staining for p16 in human retinal tissue from a patient with age-related macular degeneration (AMD).

Disclosure of the invention

Review

It is a premise of the present invention that many or most of those eye diseases that are age-related or associated with cellular defects leading to an accelerated aging phenotype are caused or mediated, at least in part, by senescent cells that accumulate with age and have deleterious effects on eye tissue. Senescent cells are typically those cells that no longer have the ability to replicate but remain in the original tissue, causing the senescence-associated secretory phenotype (SASP). Senescent cells are thought to originate from proliferative cells of various tissue types, including cells found in and around the eyes. SASP factors include various angiogenic, inflammatory, fibrotic, and extracellular matrix-modifying molecules (Acosta et al., 2013). Several factors involved in ocular pathologies are part of a constellation of factors produced by senescent cells. For this reason, eliminating or controlling senescent cells provides a means to treat eye diseases, not only by eliminating senescent cells, but also by reducing their associated SASP factors and effects on surrounding cells.

Various eye diseases present clinically with different signs and symptoms and have different types of pathophysiological mechanisms. The heterogeneity of ocular diseases is consistent with the proposed role of senescent cells in disease pathology, as senescent cells may originate from different cell generations, be induced by different stressors, reside in different ocular tissues, and interact differently with surrounding cells. However, senescent cells in various ocular tissues have an associated secretory phenotype that contributes to disorders of the entire visual system. The specific clearance of senescent cells from tissue is referred to in the present invention as senolysis. Small molecule compounds capable of inducing senolysis are called senolytic agents, which remove senescent cells regardless of the senescence-causing mechanism, SASP profile, or cell origin.

To clarify, the authors found that inhibitors of the Bcl family of proteins triggered apoptosis in senescent cells derived from a type of cell known to reside in the back of the eye, which is represented by cells involved in retinal diseases such as AMD, and also triggered apoptosis in senescent cells. , originating from a type of cell known to be found in the anterior part of the eye and represented by cells involved in diseases such as glaucoma.

Discoveries That Change the Current Drug Development Paradigm

In addition to new insights into the general role of senescent cells in eye disease, the present invention describes other findings that open up new opportunities for drug development.

One such finding is that senescent cells are abundantly present in the trabecular meshwork of glaucoma patients and in related animal models. Increased intraocular pressure is caused either by overproduction of aqueous humor, mainly in the ciliary body, or by a decrease in the outflow of aqueous humor, mainly through the trabecular meshwork. The data presented here are consistent with the idea that at least part of the underlying pathophysiology consists of impaired drainage of aqueous humor through the trabecular meshwork and down Schlemm's canal and episcleral veins into the orbital venous system. Targeting senolytic drugs to trabecular meshwork cells is an important new avenue for drug development, either as monotherapy or in combination with drugs that act by regulating fluid production.

Another finding is that removing senescent cells from the back of the eye in diseases such as diabetic retinopathy not only inhibits disease progression, but actually reverses some of the pathophysiology leading to vision loss, including neovascularization and vascular obliteration. To the best of the authors' knowledge, there is currently no therapy (either in the clinic or in development) that would be able to reverse the course of retinopathy to such an extent. Now, the goal of treating eye diseases may be more ambitious, giving new hope for patients with such diseases to improve their quality of life.

Benefits of treating eye diseases by removing senescent cells

The role of senescent cells in triggering or causing various eye diseases provides a treatment approach with several advantages for the treating physician.

• Because senescent cells are not proliferative, eliminating senescent cells allows for a clinically beneficial effect that lasts long between treatment episodes. The features of the senescent cell-mediated condition resolve at least until senescent cells accumulate again. Because senescent cells can accumulate slowly (given that age-related diseases typically take many years to develop), the effects of a single treatment or treatment cycle can last for weeks, months, or years.

• To the extent that senescent cells exacerbate other types of pathology such as inflammation or tissue destruction, the prolonged effect of senolysis provides a window in which such pathology is contained, potentially allowing the tissue to recover. This means that a drug for senescent cells makes it possible not only to stop the progression of eye diseases, but also to some extent to cause regression of the disease and its symptoms for the benefit of the patient.

• Because senescent cells in different parts of the eye respond to the same senolytic agents, several different eye diseases can be treated simultaneously in the same patient. For example, a patient may see a doctor with several ongoing disease processes such as glaucoma and macular degeneration. This makes it possible to administer a single senolytic agent according to a treatment regimen that takes into account the disease and its symptoms for each of several diseases. In addition to the convenience of this approach, it has the added benefit of reducing the risk of side effects that can occur when multiple drugs are administered in combination to treat each disease separately. It is also possible that factors caused by cells in one part of the eye may affect other parts of the eye, so that treating aging in two locations may have a beneficial effect on both diseases.

• By targeting early pathology in a disease, senolytic medicine can be an important adjunct to other types of therapy used to treat advanced disease or to relieve symptoms that occur with disease. The two treatment regimens may work synergistically or additively, reducing the burden, frequency, and side effects of each regimen when administered separately.

Classification of eye diseases according to their pathophysiology

As a guide for the treatment of eye diseases of the present invention, the corresponding diseases can be classified according to their primary pathophysiology. Diseases that fall under the same classification are suitable for the application of senolytic medicine according to the same principles and for similar purposes.

Eye diseases suitable for treatment are discussed in more detail below under the following classification.

• 1st type. Ischemic or vascular diseases: Occur when blood supply to tissues is limited, causing a deficiency of oxygen and/or essential nutrients required by cellular metabolism to maintain tissue function.

• 2nd type. Degenerative diseases: characterized by progressive deterioration in the quality, function or structure of a tissue or organ, leading to progressive visual impairment.

• 3rd type. Genetic diseases: Caused by mutations, deletions, or insertions in an individual's DNA sequence.

• 4th type. Infectious diseases caused by pathogenic microorganisms such as bacteria, viruses, parasites or fungi; diseases can be transmitted, directly or indirectly, from one person to another.

• 5th type. Inflammatory diseases characterized by a localized response to injury, foreign objects, or tissue destruction that serves to destroy, dilute, or confine both the noxious agent and the damaged tissue by producing proinflammatory mediators and recruiting immune system cells.

• 6th type. Iatrogenic diseases, defined as diseases that arise as a result of diagnostic and therapeutic procedures performed on patients.

This classification is provided to assist the reader in understanding and applying the invention to a particular patient and is not intended to limit the use of this technology. Some diseases may cause more than one of these categories: for example, an inflammatory process may contribute to pathological processes that have other underlying causes. Similarly, SASP can trigger additional pathological processes independent of the primary injury.

Treatment regimen

Senescent cells accumulate with age, so senescent cell-mediated diseases are more common in older people. In addition, various types of stress on ocular tissues may contribute to the appearance of senescent cells and the phenotype they express. Cellular stressors include oxidative stress, metabolic stress, DNA damage (eg, from exposure to ultraviolet light or a genetic disorder), activation of oncogenes, and telomere shortening (eg, from hyperproliferation). Ocular tissue exposed to such stressors may have a higher content of senescent cells, which in turn may lead to the onset of certain eye diseases at an earlier age or in a more severe form. Inherited susceptibility to some eye diseases suggests that the accumulation of pathogenic senescent cells may be directly or indirectly influenced by genetic components, which may lead to earlier presentation.

When treating specific ophthalmic diseases with the senolytic agent of the present invention, the treatment regimen will depend on the location of the senescent cells and the pathophysiology of the disease.

Regarding location: diseases of the visual system are broadly classified as anterior and posterior. Anterior eye diseases affect or affect the front part of the eyes such as the periocular muscles, eyelids, or eye tissues or fluids located in front of the posterior wall of the lens capsule or ciliary muscles. Posterior eye diseases mainly affect or affect the back of the eyes such as the choroid, ciliary body, vitreous body, vitreous chamber, retina, retinal pigment epithelium, Bruch's membrane, optic nerve (i.e. optic disc), optic pathway and those blood vessels vessels and nerves that supply blood vessels or innervate the back of the eyes.

Regarding pathophysiology, senescent cells and the emergence of SASP may contribute to ongoing dysfunction and cell degeneration/death. Senescent cells and their associated SASP factors may contribute to ongoing cell dysfunction, cell loss, and disease progression by blocking angiogenic, inflammatory, fibrotic, and extracellular matrix-modifying proteins present in the pathophysiology.

Thus, eliminating or reducing the number of senescent cells in or around the site of pathology eliminates at least one of the causes or mediators of the disease that manifests itself in the clinic. Senolytic agents are formulated and administered in such a way that they bind to senescent cells in or around the site of pathology, removing them and/or inhibiting their activity to such an extent that the progression of the disease is stopped and/or the signs and symptoms of the disease are reduced.

Different diseases mediated by senescent cells in approximately the same location and/or with similar underlying pathology are often treated in the same way. For example, the senolytic agent can be administered by local topical application to the site affected by anterior segment diseases, eg, the conjunctiva and/or cornea, for example, using eye drops, ointment or using a contact lens. Intraocular administration, intracameral or intravitreal, can also be used for diseases of both the anterior and posterior segments.

Diagnosis and monitoring of visual system disorders

The approach to diagnosing and monitoring all ocular diseases and to assessing therapeutic effects is facilitated by the widespread availability of a standard “battery” of tests on ocular structure and function. These tests can evaluate individual layers of the eye, extending from the eyelids and anterior segment to the vitreous and all cellular layers of the retina, optic nerve and visual cortex.

A standard ophthalmologic examination includes a detailed slit-lamp biomicroscopic evaluation that evaluates the eyelids, appendages, eyelashes, corneal surface, anterior chamber, pupils, lens, vitreous, and central retinal anatomy, including the optic nerve and macula. Gonioscopy allows a detailed examination of the anterior chamber angle, which is important in the diagnosis and monitoring of all forms of glaucoma. Indirect ophthalmoscopy allows assessment of the periphery of the retina, which is important in monitoring diseases of the vitreous and peripheral retina.

Ancillary testing is also widely used in the diagnosis and monitoring of therapeutic response for all visual system disorders, and these tests include the following.

Functional tests of visual acuity (including best-corrected visual acuity, contrast acuity and low-luminance acuity), color vision (including Ishihara and Farnsworth-Munsell tests) and visual field assessment (including automatic perimetry and Humphrey microperimetry), tear production (test Schirmer) and tonometry to measure intraocular pressure (IOP). They are used in conjunction with structural tests including anterior and posterior segment photography, corneal pachymetry, ultrasound, ultrasound biomicroscopy, optical coherence tomography (OCT), intravenous fluorescein angiography (IVFA), and fundus autofluorescence (FAF). Imaging such as computed tomography (CT) or magnetic resonance imaging (MRI) is used to evaluate the ocular, periocular, and orbital structures, as well as the intracranial portion of the optic nerve, optic pathway, and visual cortex. These tests visualize the structural integrity and thickness of the layers of the eye and surrounding structures, as well as assess blood flow and circulation.

Advanced functional testing of the retina, optic nerve, and optic tract/cortex, including electrophysiological tests such as full-field and multifocal electroretinography, visual evoked potentials, and microperimetry, is also used to diagnose and monitor disease progression and the effects of therapy (Mengini and Duncan, 2014). Although currently only available in research laboratories, other imaging technologies may be important additions to the diagnosis and treatment of eye diseases, including those caused by aging, such as adaptive optics and optical coherence angiography.

Clinical examination, structural and functional measurements and correlations can be obtained in both animal models and clinical settings, and are applicable to the conditions and diseases of the visual system described in this application. The battery of tests given above is part of the diagnosis, evaluation and response to treatment for these diseases.

As an example, structural assessment of retinal nonperfusion and neovascularization following retinal ischemia caused by a variety of different etiologies (eg, diabetic retinopathy, occlusive vascular disease due to atherosclerotic or inflammatory causes, retinopathy of prematurity, and genetic vascular disease) may be performed to diagnose and monitor response to senolytics. diseases such as sickle cell retinopathy) using IVFA and OCT methods. Glaucomatous optic neuropathy with loss of retinal ganglion cells and visual field function may be diagnosed and monitored as a result of optic nerve susceptibility to increased IOP caused by a variety of causes (eg, trabecular meshwork remodeling, primary open angle glaucoma (POAG), pseudoexfoliation, pigment dispersion, treatment steroids, trauma) OCT and visual field testing methods. The role of senescent cells based on molecular markers such as p16 in the trabecular meshwork tissue of glaucoma patients (Example 5) and in the retinal tissue of an AMD donor (Example 6) and the presence of SASP factors that are involved in various stages of these diseases highlight the potential impact of senolytic medicine on these diseases.

An implication of the present invention is that, regardless of the precise manner in which senescent cells accumulate and subsequently express SASP, senolytic therapy may have a beneficial effect on the properties of ocular diseases by restoring homeostasis in the cellular environment. This leads to modification of the disease through changes in the course and outcome of the disease.

Comparison of senolytic medicine with existing therapies

The therapies currently used in the clinic are limited in their ability to modify the course of the disease or potential regression of pathology. The standard of care for the most common eye diseases (glaucoma and retinal and choroidal vascular diseases) are eye drops to lower intraocular pressure (IOP) for glaucoma, intraocular anti-VEGF agents for retinal and choroidal neovascularization, and laser photocoagulation to control both IOP (Stein and Challa , 2007) and vitreoretinal diseases (AAO Retina/Vitreous Panel, 2014).

Topical intraocular pressure (IOP)-lowering agents and drugs targeting VEGF-related ocular diseases are burdened with a frequent administration schedule that must be followed for maximum effectiveness. Even when administered optimally, anti-VEGF drugs are associated with significant rates of incomplete response, disease relapse, and continued progression of non-VEGF-mediated aspects of ocular disease (eg, macular atrophy in the treatment of wet AMD (Bhisitkhul, 2015). These same problems arise with the use of IOP-lowering agents. Glaucoma medications that must be administered at least once daily to lower IOP are also associated with continued progression of glaucoma, even when used correctly and due to IOP lowering (Levin, 2005).

Laser photocoagulation has been another pillar of ocular therapy for a wide range of ocular diseases, over which senolytic administration may have many advantages. Although clinically effective, retinal laser photocoagulation is associated with collateral damage and side effects, including decreased night vision, macular and peripheral scotomas with decreased central and peripheral vision, worsening macular edema, and disruption of retinal anatomy due to scarring (Kozak and Luttrul, 2015). When applied to the trabecular meshwork to lower IOP, laser therapy is associated with IOP peaks, formation of peripheral anterior adhesions, the need for additional laser or surgical procedures, and failure to reduce the need for IOP-lowering drops after the procedure (Damji et al., 2006).

Removal of senescent cells and associated SASP factors by senolytic therapy may positively influence disease progression by modulating many disease-causing factors, including inflammatory, angiogenic, and extracellular matrix-modifying aspects of disease. With little or no damage or destruction to healthy cells needed to maintain visual function, and with a rare dosing regimen with long-lasting therapeutic effects, this invention represents a major advance over existing treatments that do not specifically target senescent cells or multiple factors associated with SASP, which limits their ability to modify many aspects of disease pathophysiology.

As an example, removal of senescent cells in the setting of ischemia may have an impact on visual function by allowing functioning retinal ganglion cells to thrive in a healthier environment free from SASP-associated harmful inflammatory, angiogenic, and extracellular matrix-modifying factors. This can be monitored structurally by measuring retinal thickness using optical coherence tomography (OCT) and functionally by electrophysiological tests (VEP and ERG), which can isolate the function of the retinal ganglion cell layer. Automated perimetry can also be used to assess peripheral visual field function in patients.

Similarly, in the setting of vaso-occlusive diseases and neovascularization, removal of senescent cells and SASP may potentially ameliorate the resulting cellular damage and promote reperfusion of affected vascular beds and reduction of neovascularization. It is possible to evaluate the similarity of ischemic phenotype (vascular obliteration and neovascularization) regardless of etiology and monitor the structural and functional response to therapy using IVFA, OCT and ERG methods. In fig. 2B (image from Carver College of Medicine, University of Iowa website, received October 30, 2017), FIG. 2C (Retina Gallery website image, received October 30, 2017), FIG. 2D (Retina Vitreous Associates of Florida website image, received October 30, 2017) and FIG. Figure 2E (Retina Gallery website image, received October 30, 2017) shows examples of fluorescein angiography of normal retina (Figure 2B) and retinal nonperfusion and neovascularization in diabetes (Figure 2C), sickle cell disease (Figure 2D), and inflammatory vasculitis (Fig. 2E). Response to senolytic therapy can be monitored using intravenous fluorescein angiography (IVFA) and OCT. Factors that are involved in senescent cell SASP can be measured directly in ocular fluids, including tears, aqueous humor, and vitreous humor.

The influence of senolytic medicine on the treatment of eye diseases includes three main concepts. First, once senescent cells are removed, the associated SASP factors derived from senescent cells are also expected to be greatly reduced. It is postulated that in the absence of these inflammatory, angiogenic and fibrotic proteins and extracellular matrix-modifying enzymes, many or most of the ocular disease symptoms described herein may be greatly suppressed. The important thing is that once the senescent cells are removed, if possible, the surrounding cells can regain some functionality. This represents a major pathophysiological advance over existing treatments for ocular diseases. The effects of senolytics and recovery of function can be monitored clinically using structural and functional tests as described above.

Secondly, depending on the circumstances, senolytic agents can be administered as a single dose. If repeated administration is necessary, the time between doses increases significantly. Because macular degeneration, glaucoma, vascular retinopathy and hereditary retinopathy are characterized by slow degradation of the retina over many years, the reaccumulation of senescent cells takes a significant period of time. Further therapy may not be needed for several years. This represents a major improvement over, for example, the dosing schedule of existing anti-VEGF drugs, which require dosing from once a month to every other month and are associated with suboptimal visual and anatomical results when administered on a less frequent schedule (Maguire et al. , 2016; Holz et al., 2014). Topical IOP-lowering agents require daily administration, and a significant percentage of patients demonstrate progression of glaucoma despite lowering IOP (Levin, 2005).

Finally, senolytic therapy for ocular diseases may address the underlying common mechanistic cause of ocular disease rather than only targeting symptoms that occur in downstream signaling pathways. Senolytic agents, for example, may target multiple disease-related cytokines (inflammatory, cytotoxic, angiogenic, fibrotic) rather than specifically inhibiting one factor involved in one aspect of the disease (eg, anti-VEGF therapy for the VEGF-related aspect of neovascularization ).

As an example, senolytic therapy can reduce a number of growth factors involved in various stages of AMD. From fig. Figure 1C (Kumar and Fu, 2014) shows that early deposits in the RPE cause degeneration of the extracellular matrix, including elastin and fibronectin. Increased oxidative stress causes damage to mitochondrial DNA, a known strong inducer of aging. This is followed by additional activation of proinflammatory cytokines and chemokines (IL-1, IL-6) with possible induction of VEGF and metalloproteases (MMPs) and inflammasome activation. Senolytic therapy targeting this range of factors (all identified as components of the SASP) may have multifaceted effects on the pathophysiology of AMD, with the ability to modulate and potentially reverse disease progression. True disease modification has not yet been demonstrated in existing therapies for ocular diseases, underscored by the need for frequent administration to control the disease. The possibility of infrequent dosing and modification of the disease course with senolytic therapy represents a major advance in the treatment of eye diseases.

Removal of senescent cells may be a disease-modifying treatment for ocular diseases arising from ischemia, degeneration, or genetic underlying causes, by stopping progression or potentially allowing endogenous repair systems or improved cellular function to modify outcome.

Suitable senolytic agents

Compounds that may be useful for removing senescent cells in or near the eyes for the treatment of eye diseases of the present invention include Bcl-2 inhibitors, Bcl-xL inhibitors, MDM2 inhibitors and Akt inhibitors. See US Patent Nos. 8,691,184, 9,096,625 and 9,403,856; published applications WO 2015/17159, WO 2015/116740, WO 2016/127135 and WO 2017/008060; and unpublished application PCT/CN2016/110309.

Candidate senolytic agents acting as inhibitors of Bcl-2, Bcl-w and Bcl-xL can be characterized as benzothiazole hydrazone, aminopyridine, benzimidazole, tetrahydroquinoline or phenoxyl compounds. Examples of compounds that inhibit Bcl isoforms include WEHI 539, A 1155463, ABT 737 and ABT 263 (Navitoclax).

Candidate senolytic agents acting as MDM2 inhibitors can be characterized as cis-imidazolines, dihydroimidazothiazoles, spiro-oxindoles, benzodiazepines, or piperidinones. MDM2 inhibitor candidates include Nutlin-1, Nutlin-2, Nutlin-3a, RG-7112, RG7388, R05503781, DS-3032b, MI-63, MI-126, MI-122, MI-142, MI-147, MI -18, MI-219, MI-220, MI-221, MI-773, 3-(4-chlorophenyl)-3-((1-(hydroxymethyl)cyclopropyl)methoxy)-2-(4-nitrobenzyl)isoindolin- 1-on, Serdemetan, AM-8553, CGM097, R0-2443 and R0-5963.

Candidate senolytic agents acting as Akt (protein kinase B) inhibitors include the competitive Akt inhibitors: CCT128930, GDC-0068, GSK2110183 (afuresertib), GSK690693 and AT7867; lipid Akt inhibitors: Calbiochem Akt inhibitors I, II and III, PX-866 and perifosine (KRX-0401); pseudosubstrate inhibitors vKTide-2T and FOXO3-hybrid; allosteric inhibitors of the Akt kinase domain, in particular MK-2206 (8-[4-(1-aminocyclobutyl)phenyl]-9-phenyl-2H-[1,2,4]triazolo[3,4-f][1,6 ]naphthyridin-3-one dihydrochloride); GST antibody against Akt1-MTS; compounds interacting with the PH domain of Akt: triciribine and PX-316; and other compounds such as GSK-2141795, VQD-002, miltefosine, AZD5363, GDC-0068 and API-1.

Exemplary Bcl inhibitors for use in the treatment of ocular diseases of the present invention contain the structure of Formula I below, or a phosphorylated form thereof.

where: R ₁ and R ₂ independently mean C ₁ –C ₄ -alkyl;

R ₃ , R ₄ and R ₅ are independently -H or -CH ₃ ;

R ₈ is -OH or -N(R ₆ )(R ₇ ), where R ₆ and R ₇ are independently alkyl or heteroalkyl, which is optionally cyclized;

X ₁ means -F, -Cl, -Br or -OCH ₃ ;

X ₂ means -SO ₂ R′ or -CO ₂ R′, where R′ means -H, -CH ₃ or -CH ₂ CH ₃ ;

X ₃ means -SO ₂ CF ₃ , -SO ₂ CH ₃ or -NO ₂ ;

X ₅ means -F, -Br, -Cl, -H or -OCH ₃ .

Optionally, R ₈ is -N(R ₆ )(R ₇ ), where R ₆ and R ₇ are independently alkyl or heteroalkyl, which is optionally cyclized.

Optionally, R ₁ and R ₂ are independently C ₁ –C ₄ -alkyl;

R ₃ and R ₄ both mean -H;

R ₅ means -H or -CH ₃ ;

R ₆ and R ₇ are independently alkyl or heteroalkyl, which is optionally cyclized;

X ₁ means -F or -Cl;

X ₂ means -SO ₂ R′ or -CO ₂ R′, where R′ means -H, -CH ₃ or -CH ₂ CH ₃ ;

X ₃ means -SO ₂ CF ₃ , -SO ₂ CH ₃ or -NO ₂ ; And

X ₅ means -F or -H.

Other exemplary Bcl inhibitors for use in the treatment of ocular diseases of the present invention contain the structure of Formula II below, or a phosphorylated form thereof.

where: R ₁ and R ₂ independently mean C ₁ –C ₄ -alkyl;

R ₃ and R ₄ are independently -H or -CH ₃ ;

R ₈ means -OH or ;

X ₁ means -F, -Cl, -Br or -OCH ₃ ;

X ₂ means -SO ₂ R′ or -CO ₂ R′, where R′ means -H, -CH ₃ or -CH ₂ CH ₃ ;

X ₃ means -SO ₂ CF ₃ , -SO ₂ CH ₃ or -NO ₂ ;

X ₄ means -OH, -COOH or -CH ₂ OH;

X ₅ means -F, -Cl or -H; A

n ₁ and n ₂ are independently equal to 1, 2, or 3.

Optionally X ₃ means -SO ₂ CF ₃ or -NO ₂ and R ₈ means where X ₄ means -OH or -COOH.

Optionally, a compound may have one, two, three, more than three, or all of the following in any combination:

R ₁ is isopropyl;

R ₂ means methyl;

R ₃ means -H;

R ₄ means -H;

X ₁ means -Cl;

X ₂ means -SO ₂ CH ₃ ;

X ₃ means -SO ₂ CF ₃ ;

X ₄ means -OH;

n ₁ equals 2; And

n ₂ equals 2.

Other exemplary Bcl inhibitors for use in the treatment of eye diseases of the present invention contain the structure of Formula III below, or a phosphorylated form thereof.

where: R ₁ and R ₂ independently mean C ₁ –C ₄ -alkyl;

R ₃ , R ₄ and R ₅ are independently -H or -CH ₃ ;

R ₆ and R ₇ are independently alkyl or heteroalkyl, which is optionally cyclized;

X ₁ means -F, -Cl, -Br or -OCH ₃ ;

X ₂ means -SO ₂ R′ or -CO ₂ R′, where R′ means -H, -CH ₃ or -CH ₂ CH ₃ ;

X ₃ means -SO ₂ CF ₃ or -NO ₂ ; And

X ₅ means -F, -Br, -Cl, -H or -OCH ₃ ;

or:

R ₁ and R ₂ independently mean C ₁ –C ₄ -alkyl;

R ₃ and R ₄ both mean -H;

R ₅ means -H or -CH ₃ ;

R ₆ and R ₇ are independently alkyl or heteroalkyl, which is optionally cyclized as shown in formula VII;

X ₁ means -F or -Cl;

X ₂ means -SO ₂ R′ or -CO ₂ R′, where R′ means -H, -CH ₃ or -CH ₂ CH ₃ ;

X ₃ means -SO ₂ CF ₃ or -NO ₂ ; And

X ₅ means -F or -H.

Other exemplary Bcl inhibitors for use in the treatment of ocular diseases of the present invention contain the structure of Formula IV below, or a phosphorylated form thereof.

where: R ₁ and R ₂ independently mean C ₁ –C ₄ -alkyl;

R ₃ and R ₄ are independently -H or -CH ₃ ;

X ₁ means -F, -Cl, -Br or -OCH ₃ ;

X ₂ means -SO ₂ R′ or -CO ₂ R′, where R′ means -H, -CH ₃ or -CH ₂ CH ₃ ;

X ₃ means -SO ₂ CF ₃ or -NO ₂ ;

X ₄ means -OH or -COOH;

X ₅ means -F, -Cl or -H; A

n ₁ and n ₂ are independently equal to 1, 2, or 3.

Screening of compounds for senolytic activity

These and other compounds can be screened at the molecular level for their ability to act in a manner that indicates that they are candidate agents for use in the present invention.

For example, if the therapy involves triggering apoptosis of senescent cells via Bcl-2, Bcl-xL or Bcl-w, then compounds can be tested for their ability to inhibit the binding between Bcl-2, Bcl-xL or Bcl-w and the corresponding ligand. Example 1 provides an illustration of a homogeneous assay (an assay that does not require a separation step) to determine binding to Bcl isoforms. Compounds can be screened at the molecular level for their ability to act as MDM2 agonists while stimulating p53 activity and inducing senolysis. Example 2 provides an illustration of an analysis for this purpose.

Cell culture systems for testing senolytic agents

Compounds can be screened for biological activity when assayed in senescent cells. Cultured cells are brought into contact with the compound and the degree of cytotoxicity or cell inhibition is determined. The ability of a compound to kill or inhibit senescent cells can be compared to the effect of the compound on normal cells that divide freely at low densities and normal cells that are quiescent at high densities.

Example 3 provides an illustration of IMR90 fibroblast cells from human lungs. Due to their ease of expansion, IMR90 cells are effective as an early screening tool. Since the present description represents cell types in the eye that generate senescent cells involved in ocular diseases, the compounds selected in the early screen can be rescreened on primary cultures of target eye cells: in particular, trabecular meshwork cells, as shown in Example 4 , and RPE cells, as shown in example 5.

Where technically feasible, it is possible to obtain tissue explants from the eyes of donor patients and measure the reduction in markers of senescent cells and related diseases after incubation with test compounds. This format can measure senolysis and its subsequent effects in intact tissue where appropriate cell types are present and aging is driven by disease pathogenesis. Tissue explants may also provide a means to evaluate disease-associated cell types that are difficult to amenable to standard in vitro cell culture techniques individually (eg, photoreceptors and neurons).

Animal models for testing senolytic agents

Investigational compounds can be evaluated in preclinical animal models to ascertain the recruitment of appropriate cell types and subsequent effects such as SASP reduction or functional efficacy in mechanistic/disease models.

Evidence of target binding can be examined in vivo in models of induced aging. Several senescence induction techniques can be used to understand whether test compounds access disease-associated cell types in the eye. DNA-damaging agents such as doxorubicin, bleomycin, and radiation can induce cellular senescence and can be administered directly (e.g., intravitreal, intracameral, subretinal, etc.) to the eyes of mice (or act locally or throughout the body in case of irradiation) to induce senescence in the trabecular meshwork or retina. Test compounds can then be administered to determine whether the compounds reach the appropriate cell layers (measured by loss of aging markers). In addition, many SASP factors in these tissues can be measured to understand the downstream effects of senescence induction and the influence of senolysis on such mediators.

By way of illustration, administration of the DNA damaging agent bleomycin into the anterior chamber of mouse eyes results in cellular senescence in the trabecular meshwork (TM), as measured by the induction of p16 transcript in the TM (Example 8). 14 days after intracameral (IC) administration of bleomycin, there was an increase in the relative expression of p16 mRNA in the right eye compared to the control left eye. Intracameral administration of a senolytic (UBX1967) on day 7 after bleomycin led to a decrease in p16 mRNA on day 14, indicating clearance of senescent cells by UBX1967 in the TM of mice.

The oxygen-induced retinopathy (OIR) model (Scott and Fruttiger, Eye (2010) 24, 416-421; Oubaha et al., 2016) mimics elements of ischemic retinopathy in humans such as diabetic retinopathy (DR), retinopathy of prematurity (ROP) and diabetic edema macula (DME). Exposure of young mice to a hyperoxic environment results in obliteration of the retinal vasculature followed by pathological angiogenesis (neovascularization) after switching to normal air.

The examples below demonstrate the effectiveness of the model compound UBX1967 in a mouse model of oxygen-induced retinopathy (OIR). Intravitreal (IVT) administration of UBX1967 showed a statistically significant improvement in the extent of neovascularization and vasoobliteration at all dose levels (Example 6A). In addition, the relative abundance of several transcripts associated with aging (p16, pai1) and human disease (VEGF) was measured and found that UBX1967 treatment resulted in a decrease in these transcripts. Also, aging-associated β-galactosidase (SA-βGal) activity was reduced after UBX1967 administration.

The streptozotocin (STZ) rodent model (Feit-Leichman et al., IOVS 46: 4281-87, 2005) reproduces the features of diabetic retinopathy and diabetic macular edema by inducing hyperglycemia through the direct cytotoxic effect of STZ on pancreatic β-cells. Hyperglycemia occurs within days of STZ administration, and phenotypic aspects of diabetic retinopathy occur within weeks, with vascular leakage and decreased visual acuity and contrast sensitivity observed in rodents. This model has been widely used to evaluate therapeutic agents for diabetic eye diseases. The data below shows that UBX1967 corrects vascular leakage in the retina and choroid.

The testing can also use models of retinal ganglion cell damage that are relevant to glaucoma, in which increased intraocular pressure (IOP) causes loss of retinal ganglion cells and damage to the optic nerve. In preclinical studies, increased anterior chamber pressure may lead to loss of retinal neurons, as has been reported in several established models, including magnetic bead occlusion (Ito et al., Vis Exp. 2016 (109): 53731), and in other glaucoma models (Almasieh and Levin, Annu Rev Vis Sci. 2017). In addition, ischemia-reperfusion injury has been shown to cause retinal damage, which can lead to cellular senescence. The presence of retinal aging in such models can be used to monitor the effects of senolysis following intravitreal administration of test compounds.

Methods of administration

Typically, the senolytics of the present invention are administered directly to the outside or inside of a subject's eye or surrounding tissue. Local administration includes topical administration, syringe administration and/or implantable device administration. It covers the treatment of anterior eye diseases (the front of the eye) and posterior eye diseases (the back of the eye).

Anterior eye diseases are those diseases that affect or affect the front part or area of the eyes such as the periocular muscles, eyelids, or ocular tissues or fluids located in front of the posterior wall of the lens capsule or ciliary muscles. Anterior eye diseases affect or affect primarily the conjunctiva, cornea, anterior chamber, iris, posterior chamber (behind the iris but in front of the posterior wall of the lens capsule), lens or lens capsule, and the blood vessels and nerves that supply or innervate the anterior chamber. part of the eye. Examples include dry eye syndrome, conjunctival diseases, conjunctivitis, corneal diseases, presbyopia, cataracts, and refractive errors. Glaucoma can also be considered an anterior eye disease, since the clinical goal of glaucoma treatment may be to reduce aqueous fluid tension in the anterior chamber of the eye (i.e., decrease intraocular pressure).

Posterior eye diseases are those diseases that primarily affect or affect the back part or area of the eyes such as the sclera, ciliary body, choroid (behind the plane passing through the posterior wall of the lens capsule), vitreous body, posterior chamber, retina, pigment the retinal epithelium, Bruch's membrane, the optic nerve (i.e., the optic disc), and the blood vessels and nerves that supply or innervate the back part or region of the eye. Examples include acute macular neuroretinopathy; choroidal neovascularization; histoplasmosis; infections such as viral infections; non-exudative age-related macular degeneration and exudative age-related macular degeneration; edema such as macular edema, cystoid macular edema and diabetic macular edema; multifocal choroiditis; eye injuries that affect the back or part of the eye; retinal diseases such as central retinal vein occlusion, diabetic retinopathy, proliferative vitreoretinopathy (PVR), retinal artery occlusion, retinal detachment, inflammatory chorioretinal diseases; sympathetic ophthalmia; retinitis pigmentosa and glaucoma. Glaucoma can be considered a posterior eye disease because the goal of treatment is to prevent or reduce the degree of vision loss due to damage or loss of retinal ganglion cells or optic nerve cells (ie, neuroprotection).

In some cases, an effective amount of senolytic is administered into the anterior chamber of the eye by topical administration. The senolytic may be instilled into the front of the eye using eye drops, ointment, or gel (eg, into the conjunctiva and/or cornea) as needed to treat, relieve, and/or prevent a specific eye condition. The senolytic agent may be an ophthalmic preparation in the form of eye drops containing a sufficient amount of the active agent to provide a therapeutically effective concentration at the site of action within the eye.

Local administration to the anterior part of the eye (eg, conjunctiva and/or cornea) can also be achieved using a contact lens carrying a senolytic agent. This may improve bioavailability and prolong the residence time of the active agent.

To increase drug loading and control drug release, the contact lens or hydrogel may include: (i) polymer hydrogels with a controlled hydrophilic/hydrophobic copolymer ratio; (ii) hydrogels for incorporating the drug into a colloidal structure dispersed in contact lenses; (iii) ligand-containing hydrogels; (iv) molecularly imprinted polymer hydrogels; (v) hydrogels with a surface containing a multilayer structure for drug loading and release. Hydrogels are the preferred soft contact lens material due to their biocompatibility and transparency. A hydrogel contact lens can be used to release the active agent onto the front of the eye in a controlled manner upon contact with the thin film of tears covering the eye. Contact lenses may be worn daily according to a dosing schedule to provide topical administration of an effective amount of senolytic for the treatment of ocular diseases (eg, those described herein). Contact lens devices include those described in US Patent No. 6,827,996 and US Patent Publications Nos. 2010/0330146 and 2006/0251696.

Local administration to the anterior part of the eye can also be achieved by subconjunctival injection. Subconjunctival injection can be used to administer a senolytic drug into the subconjunctival space or sub-Tenon's space. Because the subconjunctival space is anterior to the sub-Tenon's space, subconjunctival administration may have a greater effect on drug delivery to the anterior segment, whereas sub-Tenon's administration may have a greater effect on the posterior segment.

Local administration to the anterior and posterior segments of the eyes can also be achieved by intraocular injection, e.g. intracameral or intravitreal. An intracameral injection is an injection that is usually given into the chamber at the front of the eye (eg, in front of the lens). An intravitreal injection is an injection that is given into the vitreous chamber at the back of the eye (eg, behind the lens). Due to the risk of damage to the layers of the retina and optic nerve, when intraocular pressure increases, a volume of no more than 0.1 ml should be administered intracamerally or intravitreally. In some cases, intracameral injection can provide administration without the increase in intraocular pressure that is associated with intravitreal injection. A sudden increase in intraocular pressure can cause discomfort in the patient and lead to the risk of damage to the optic nerve. Administration by injection is usually done in a manner that minimizes ocular exposure to pathogens.

Local administration may also be through an implantable ocular device placed in the eye, for example through a corneal incision. Ocular devices include stents (eg, trabecular stents), organogel implants, and the compositions and devices described in U.S. Patent Nos. 5,501,856, 5,869,079, 5,824,072, 4,997,652, 5,164,188, 5,443,505, and 5,766,242.

US Pat. No. 5,501,856 discloses controlled release pharmaceutical formulations for intraocular implants for intraocular administration following surgery for retinal/vitreous diseases or glaucoma. US Pat. No. 5,869,079 discloses combinations of hydrophilic and hydrophobic elements in biodegradable sustained-release implants and describes a polylactic acid-polyglycolic acid (PLGA) implant containing dexamethasone. US Pat. No. 5,824,072 discloses implants for insertion into the suprachoroidal space or avascular region of the eye and describes a methylcellulose implant containing dexamethasone. US Patent Nos. 4,997,652 and 5,164,188 disclose biodegradable ocular implants containing microencapsulated drugs and describe implantation of microcapsules containing hydrocortisone succinate into the posterior segment of the eye. US Pat. No. 5,164,188 discloses encapsulated agents for administration to the ocular suprachoroids and describes the installation of microcapsules and plaques containing hydrocortisone in the pars plana. US Pat. Nos. 5,443,505 and 5,766,242 disclose implants containing active agents for administration into the suprachoroidal space or avascular region of the eye and describe the placement of microcapsules and plaques containing hydrocortisone in the pars plana.

To provide an implant or depot for sustained release drug delivery, an injectable formulation containing the subject's active agent may be administered to a subject, resulting in the formation of an organogel implant in situ. Upon contact with a biological fluid, the cross-linking agents begin to cross-link the organogel to form a more stable matrix that modulates the release of the active agent into the subject's eye. In some cases, this route of administration may provide a prolonged period of release of the active agent. In some cases, a biodegradable cross-linked matrix is formed in vivo, which includes a non-aqueous aprotic biocompatible solvent system that is immiscible with water (Zhou T. et al., Journal of Controlled Release 55: 281-295, 1998).

Dosage forms

Ophthalmic preparations can be prepared by mixing the senolytic agent with a pharmaceutically acceptable base or carrier and, optionally, one or more pharmaceutically acceptable excipients. Ingredients acceptable in ophthalmic forms are those excipients or carriers that cause little or no eye irritation, provide suitable preservation if desired, and deliver one or more agents in an appropriate volume. Examples of bases or carriers include water; aqueous solvents such as polar solvents; polyalcohols; vegetable oils; and oily bases. Examples of vehicles or carriers for intraocular administration include water for injection and saline.

For ophthalmic delivery, senolytic agents can be combined with suitable excipients for use in or around the eye, such as surfactants, preservatives, cosolvents, flavoring or refreshing agents, antiseptics, bactericidal or antibacterial agents, pH adjusting agents, isotonicity agents, chelating agents, buffering agents, stabilizers, antioxidants, viscosity enhancers, penetration enhancers, sodium chloride and thickeners. In some cases, the composition for intraocular administration may contain one or more of solubilizing agents, suspending agents, isotonicity agents, buffering agents, emollients, stabilizers, and antiseptics. Carriers and excipients for ophthalmic compositions may be combined to form an aqueous, sterile ophthalmic suspension, solution, or viscous or semi-viscous gels or other types of solid or semi-solid ointment-type compositions.

Typical excipients and additives that may be used include the following. Surfactants: for example, nonionic surfactants such as block copolymers of polyoxyethylene (hereinafter sometimes referred to as “POE”) and polyoxypropylene (hereinafter sometimes referred to as “POP”) (e.g., poloxamer 407, poloxamer 235, poloxamer 188), POE-POP block copolymer adducts with ethylenediamine (e.g., poloxamine), POE-sorbitan fatty acid esters (e.g., polysorbate 20, polysorbate 60, polysorbate 80 (TO-10, etc.)), hydrogenated POE castor oils (e.g. hydrogenated castor oil with POE (60) (HCO-60, etc.)), POE castor oils, POE alkyl esters (e.g. polyoxyethylene lauryl ether (9), polyoxyethylene cetyl ether (20 ) and polyoxypropylene (4)) and polyoxyl stearates; amphoteric surfactants such as amphoteric surfactants of the glycine type (for example, alkyl-diaminoethylglycine, alkyl-polyaminoethylglycine), amphoteric surfactants of the betaine type (for example, lauryl dimethylaminoacetic betaine, imidazolinium betaine); cationic surfactants such as quaternary alkyl ammonium salts (for example, benzalkonium chloride, benzethonium chloride); and etc.

Flavorings or fresheners: eg camphor, borneol, terpenes (these can be in d-form, l-form or dl-form); essential oils such as peppermint water, eucalyptus oil, bergamot oil, anethole, eugenol, geraniol, menthol, limonene, peppermint oil, peppermint oil, rose oil, etc.

Antiseptics, bactericidal or antibacterial agents: for example, polydronium chloride, alkyldiaminoethylglycine hydrochloride, sodium benzoate, ethanol, benzalkonium chloride, benzethonium chloride, chlorhexidine gluconate, chlorobutanol, sorbic acid, potassium sorbate, sodium dehydroacetate, methyl paraoxybenzoate, ethyl paraoxybenzoate, propyl- paraoxybenzoate, butyl paraoxybenzoate, hydroxyquinoline sulfate, phenethyl alcohol, benzyl alcohol, biguanide compounds (in particular, polyhexamethylene biguanide or its hydrochloride, etc.), Glokill (Rhodia Ltd.), etc.

pH adjusting agents: e.g. hydrochloric acid, sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, triethanolamine, monoethanolamine, diisopropanolamine, sulfuric acid, phosphoric acid.

Substances for isotonicity: e.g. sodium bisulfite, sodium sulfite, potassium chloride, calcium chloride, sodium chloride, magnesium chloride, potassium acetate, sodium acetate, sodium bicarbonate, sodium carbonate, sodium thiosulfate, magnesium sulfate, disodium hydrogen phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, glycerin, propylene glycol.

Chelating agents: eg ascorbic acid, tetrasodium EDTA, sodium EDTA, citric acid. Buffering agents: for example, phosphate buffering agents; citrate buffers such as citric acid and sodium citrate; acetate buffers such as acetic acid, potassium acetate and sodium acetate; carbonate buffers such as sodium bicarbonate and sodium carbonate; borate buffers such as boric acid and borax; buffer substances such as taurine, aspartic acid and their salts (eg potassium salts, etc.) and ε-aminocaproic acid.

Ophthalmic solution forms can be prepared by dissolving the agent in a physiologically acceptable isotonic aqueous buffer. In addition, ophthalmic solutions may include ophthalmologically acceptable surfactants to assist in dissolution of the agent. For better retention of compounds, viscosity enhancing agents such as hydroxymethylcellulose, hydroxyethylcellulose, methylcellulose, and polyvinylpyrrolidone can be added.

Sterile ophthalmic gel forms can be prepared by suspending the agent in a hydrophilic base prepared, for example, from a combination with Carbopol ^® -940. For intraocular injections, Viscoat ^® (Alcon Laboratories, Inc., Fort Worth, Tex.) can be used. Other compositions of the present invention may contain penetration enhancing materials such as ^Cremophor® (Sigma Aldrich, St. Louis, Mo.) and ^Tween® 80 (polyoxyethylene sorbitan monolaurate, Sigma Aldrich) in case the agents of the present invention have limited penetration into the eye.

The present invention provides commercial kit products containing unit doses of one or more of the agents or compositions described herein. Such kits typically contain a pharmaceutical drug in one or more containers. Drugs can be presented in the form of one or more single doses (combined or separate). The kit may contain a syringe-type device for administering the agent or composition into or around the eye of a subject in need thereof. The product may also contain or be accompanied by an information insert describing the use and associated benefits of the drugs in the treatment of senescent cell-related eye diseases, and optionally an apparatus or device for administering the composition.

Eye diseases suitable for treatment

The following sections provide a discussion of specific ocular diseases, grouped into broad etiological categories (see above), that are candidates for treatment with the senolytic agents of the present invention. The extent to which a particular ocular disease responds to treatment with a senolytic agent will depend on the extent and extent of the role senescent cells play in the pathology or symptomatology of the disease. Treatment and management protocols are at the discretion of the attending physician. The effectiveness of therapy can be determined empirically.

TYPE 1. Ischemic or vascular diseases

These diseases are characterized by a restriction of blood supply to tissues, which causes a deficiency of oxygen and/or essential nutrients required for cellular metabolism to maintain tissue function. Ischemia is usually caused by diseases related to the blood vessels, with subsequent tissue damage or dysfunction. It also includes localized deficits that occur in a given part of the body as a result of problems affecting the blood flow but not the vessels themselves (such as vasoconstriction, thrombosis or embolism).

Examples of ischemic or vascular diseases of the eye include diabetic retinopathy, glaucomatous retinopathy, ischemic arterial optic neuropathy and vascular diseases characterized by arterial and venous occlusion, retinopathy of prematurity and/or sickle cell retinopathy.

The general approach and goals of senolytic therapy for ischemic or vascular diseases are based on the following.

Ischemia causes a well-known series of pathophysiological interactions in the eye. Senolytic therapy targets this pathophysiology by improving multiple known inducers of aging that populate the ischemic pathway. The initial exposure to an ischemic event initiates a cascade that exposes the cells of the affected tissue to inducers of senescence, which include mitochondrial and DNA damage, oxidative stress, inflammation, and lipid peroxidation.

The accumulation of senescent cells and the release of their associated SASP factors have a negative impact on the tissue microenvironment, both for cells with direct and indirect effects. The goal of senolytic therapy for ischemic ocular diseases is to reduce the population of senescent cells present in the affected area and reduce the influence of their associated SASP factors on surrounding cells. This limits ongoing tissue damage after an ischemic event and potentially restores function by improving the properties of the cellular microenvironment.

As an example, elimination of senescent cells under ischemic conditions influences visual function by allowing functioning retinal ganglion cells to thrive in a healthier environment free from SASP-associated harmful inflammatory, angiogenic, and extracellular matrix-modifying factors. Ischemic events in the visual system typically affect the posterior structures of the eye. In some diseases, ischemia may be influenced by features of the anterior segment. Thus, senolytic agents can be administered into the anterior compartment or into the anterior and posterior compartments.

Basic pathophysiology

Retinal ischemia contributes to multiple ocular diseases and occurs secondary to multiple underlying etiologies. Ischemia has been implicated in glaucoma, diabetic retinopathy, retinal and choroidal vascular occlusion, retinopathy of prematurity, and ischemic and traumatic optic neuropathies. The retinal and macular edema associated with these conditions is also secondary to the effects of the ischemic cascade.

In fig. Figure 1A presents the pathophysiological interactions in the eye that result from ischemia. All known retinal ischemic paradigms result in the loss of ganglion cells, which are critical for maintaining functional vision (Osborne N et al., 2004). In addition, neovascularization mediated by multiple growth factors such as vascular endothelial growth factor (VEGF), TNFα, TGFβ, FGF, PKC, angiopoietins, PDGF, among others (Ucuzian et al., 2010) is another effect of end-stage ischemia and is associated with vision-threatening complications from neovascularization of both the retina and choroid (Campochiaro, 2015).

Ischemia due to interruption of the retinal blood supply of any etiology initiates a cascade that can ultimately lead to cell death and is known to include selective neuronal death, cellular edema, and neovascularization. This is described in detail in FIG. 1A, which shows the main stages of the cascade, which include failure of the sodium-potassium ATPase pump, membrane depolarization and accumulation of sodium and calcium ions in the cytoplasm with subsequent formation of destructive free radicals. This process ultimately leads to cell death from necrosis or apoptosis (Osborne N. et al., 2004). Thus, the ischemic pathway is replete with known inducers of aging and supports the hypothesis that senolytic agents may interfere with the effects of many ischemia-induced aging responses.

Glaucoma is another example of an eye disease that illustrates this progression to death of retinal ganglion cells under the influence of a variety of factors, including ischemia (Choi and Kook, 2015), elevated IOP, genetic susceptibility and oxidative stress (Wang et al., 2014), each of which can cause aging and contribute to changes in the environment created by senescent cells and their associated SASP factors.

In fig. Figure 1B shows the multifactorial pathophysiology of glaucoma leading to the death of retinal ganglion cells (RGCs) (Wang et al., 2014).

Glaucoma

Glaucoma has significant associations with age in both prevalence and pathophysiological characteristics. The prevalence of glaucoma increases markedly after age 60 years in Caucasians and after age 40 years in African Americans and Hispanics. Age is the most reliable and least variable risk factor for glaucoma, including IOP. Age is also associated with decreased ocular outflow and increased IOP, scleral hardening with subsequent effects on the biomechanical properties of the optic nerve, and a decrease in the retinal ganglion cell population (Caprioli, 2013).

Glaucoma is a form of optic neuropathy (disease of the optic nerve) that is associated with increased intraocular pressure resulting from an inability to relieve pressure in the anterior chamber of the eyes caused by an abnormal accumulation of clear fluid known as aqueous humor. There are two main types of glaucoma: open-angle glaucoma and closed-angle glaucoma. Glaucoma can be classified based on pathogenesis as primary glaucoma: primary open-angle glaucoma or primary angle-closure glaucoma; and secondary glaucoma caused by other disorders, including steroid glaucoma, pseudoexfoliation glaucoma, or pigmentary dispersion glaucoma. In open-angle glaucoma (also called wide-angle glaucoma), the drainage structure of the trabecular meshwork, or canal of the eye, does not drain fluid secreted by the ciliary body as it should, resulting in increased intraocular pressure. In angle-closure (or narrow-angle) glaucoma, the eye does not drain properly because the angle between the iris and cornea is too narrow, which can cause a sudden increase in intraocular pressure. The manifestation of angle-closure glaucoma can be acute or chronic. Angle-closure glaucoma may be associated with farsightedness and cataracts.

Aqueous humor, which forms in the ciliary body in the posterior chamber of the eye at a rate of about 2.5 μl per minute, enters the anterior chamber through the gap between the anterior part of the lens and the posterior part of the iris through the pupillary opening in the iris. When the eye is functioning normally, aqueous humor flows out of the anterior chamber at the same or nearly the same rate as it enters, causing the pressure in the eye to remain within the normal range of 12 to 22 mm Hg. Art. Fluids are relatively incompressible, so intraocular pressure is distributed relatively evenly throughout the eye. Increased intraocular pressure occurs when there is an imbalance between the production of aqueous fluid from the ciliary body and the outflow of this fluid.

The outflow of aqueous humor from the anterior chamber occurs in two ways. A small amount (about 10%) exits through the “uveoscleral drainage” between muscle fibers in the ciliary body. This outflow is independent of intraocular pressure. However, the main outflow pathway is through the trabecular meshwork (TM) into Schlemm's canal and is pressure dependent. When this pathway becomes obstructed, intraocular pressure may rise because the influx of aqueous humor is not balanced until the pressure in the eye rises enough to overcome the resistance to outflow. The authors' discovery of senescent cells in the TM of patients with glaucoma confirms TM dysfunction in the pathophysiology of this disease. Over time, this can lead to vision loss, both peripheral and central, and ultimately lead to complete blindness through loss of retinal ganglion cells and optic nerve function.

Subjects with open-angle glaucoma and chronic angle-closure glaucoma may not have obvious symptoms in the early stages of the disease. Visual field loss can occur in later stages of glaucoma. A subject in need of treatment for glaucoma may exhibit one or more symptoms, including, but not limited to, increased intraocular pressure, optic nerve abnormalities with associated visual field loss, decreased visual acuity, corneal edema, and closed drainage angle. In certain cases, a subject with fluctuations in intraocular pressure levels may experience blurred vision and see halos around light sources. In some cases, symptoms of acute angle-closure glaucoma include rapid onset of eye pain, headache, nausea, vomiting, and visual blurring. The eyes of patients with acute angle-closure glaucoma may appear red, and the pupil of the eye may be large and unresponsive to light. In some cases, the cornea may appear cloudy to the naked eye.

Subjects to be treated according to the present invention may be selected based on clinical presentation or ophthalmological examination that suggests the presence of glaucoma, using diagnostic methods described previously. These include slit lamp and gonioscopy, tonometry, optic nerve imaging and examination, fundus photography and OCT techniques.

The senolytic agents of the invention can provide a reduction in ocular intraocular pressure in a subject in need of treatment, eg, a reduction from elevated intraocular pressure to normal intraocular pressure by eliminating senescent cells and associated SASP in the TM and anterior chamber. Optic nerve damage can be alleviated and suppressed by significantly reducing intraocular pressure, and in some cases, retinal visual ability can be restored or improved.

In some cases, the senolytic is administered by intracameral injection into the anterior chamber of the eye or intravitreal injection into the posterior segment, vitreous, or vitreous chamber of the eye. The senolytic agents of the invention may be effective in improving the outflow of aqueous humor, thereby reducing intraocular pressure. Intravitreal administration can utilize the natural fluid flow of the eye: the vitreous delivers the active agent into the trabecular meshwork and uveoscleral pathway as it flows into the anterior chamber. This can ensure delivery of the active agent to the site of action in the anterior segment of the eye. Intravitreal delivery can also directly target retinal ganglion cells affected by glaucoma.

Optionally, senolytic agents can be administered in combination with a surgical method for relieving intraocular pressure, such as canaloplasty, laser trabeculoplasty, trabeculectomy, and insertion of shunts or other implantable devices. Generally, surgery provides temporary relief from elevated intraocular pressure. Implantable drainage devices include those described in US Pat. No. 9,468,558, which can be inserted into the eye through an incision and placed, for example, on the sclera behind the limbus to facilitate drainage.

Diabetic retinopathy

The prevalence of diabetes and diabetic retinopathy increases with age and is the most common cause of blindness in people over 50 years of age. It is a multifactorial disease with hyperglycemia causing toxic effects on cells and inflammatory cytokines implicated in many aspects of diabetic eye disease (Lutty, 2013).

Diabetic eye disease develops in patients with diabetes due to changes in the cells lining blood vessels and involves both vascular and nerve dysfunction. Patients with diabetes often develop ophthalmological complications such as corneal abnormalities, glaucoma, iris neovascularization, cataracts and neuropathy. Diabetic retinopathy is a common and potentially serious complication of diabetes.

The duration and severity of hyperglycemia is a factor associated with the development of diabetic retinopathy. When glucose levels are high, as in diabetes, glucose can cause damage in a variety of ways. For example, glucose or a glucose metabolite binds to the amino groups of proteins, resulting in tissue damage. In addition, excess glucose enters the polyol pathway, leading to sorbitol accumulation. Sorbitol cannot be metabolized by retinal cells and may contribute to high intracellular osmotic pressure, intracellular edema, impaired diffusion, tissue hypoxia, capillary cell damage, and capillary weakening. Diabetic retinopathy involves thickening of capillary basement membranes and prevents pericytes from contacting capillary endothelial cells. Loss of pericytes increases capillary leakage and can lead to breakdown of the blood-brain barrier. Weakened capillaries can lead to aneurysm formation and further leakage. These effects of hyperglycemia can also impair neuronal function in the retina. This is an early stage of diabetic retinopathy called non-proliferative diabetic retinopathy.

Diabetic retinopathy is also a degenerative disease of the neural retina associated with changes in neuronal function even before the onset of clinical vascular disease. Retinal capillaries can become blocked in diabetes, causing ischemic areas in the retina. Nonperfused tissue responds by causing new blood vessels to grow from existing vessels (ie, angiogenesis). These new blood vessels can also cause vision loss, a condition called proliferative diabetic retinopathy, because the new blood vessels are fragile and tend to leak blood into the eyes. In progressive proliferative diabetic retinopathy, a VEGF-mediated angiogenic response occurs with retinal neovascularization, placing the eye at additional risk of severe vision loss due to the development of vitreous hemorrhage or retinal traction. Without treatment, irreversible vascular or neuronal damage is possible, highlighting the need for early intervention.

Symptoms of diabetic retinopathy include loss of central vision, inability to see colors, blurry vision, floaters, distortions, holes or black spots in vision. In the initial stages of diabetic retinopathy, in some cases the subject may be asymptomatic. However, microaneurysms in the eye may be an early clinical sign of diabetic retinopathy. In some cases, microaneurysm occurs secondary to capillary wall prolapse due to loss of pericytes and may appear as small red dots in the superficial layer of the retina.

Diagnostic modalities include a complete ophthalmologic examination as previously described, with fluorescein angiography, optical coherence tomography (OCT), and B-type ultrasound scanning, which typically use ancillary tests to determine staging and monitor response to therapy. The severity of nonproliferative diabetic retinopathy can be assessed by the presence, number and location of microaneurysms and hemorrhages. Microaneurysms may appear as punctate, hyperfluorescent lesions in the early stages of the angiogram and usually leak in the later phases of the fluorescein angiographic test. Proliferative diabetic retinopathy can be assessed in part by the presence of neovascularization, preretinal hemorrhages, vitreous hemorrhages, fibrovascular tissue proliferation, and traction retinal detachments. OCT can be used to determine retinal thickness and the presence of intraretinal edema and associated vitreomacular traction.

Other signs and symptoms of diabetic retinopathy include: 1) pinpoint bleeding, which may appear similar to microaneurysms if small, and may result from rupture of a microaneurysm in the deeper layers of the retina (inner nuclear and outer plexiform layers); 2) hemorrhages in the form of flames, which are fragment hemorrhages that occur in the more superficial layer of nerve fibers; 3) retinal edema and hard exudates caused by the breakdown of the blood-brain barrier, which promotes the leakage of serum proteins, lipids and protein from the blood vessels; 4) cotton wool spots, which are infarcts of the nerve fiber layer from occlusion of precapillary arterioles; they are often limited by microaneurysms and vascular hyperpermeability; 5) venous loops and venous branches, which can arise near areas of non-perfusion; they reflect increased retinal ischemia and their appearance may be a predictor of the progression of proliferative diabetic retinopathy (PDR); 6) intraretinal microvascular disorders, which include reconstructed capillary beds without proliferative changes; they usually occur at the borders of the non-perfused retina; and 7) macular edema, causing blurred vision.

Other vascular diseases of the eyes

Other vascular eye diseases: arterial and/or venous occlusion of the retina and/or optic nerve and retinopathy (for example, retinopathy of prematurity and/or sickle cell retinopathy).

Retinal vein occlusion (RVO) is a blockage of the small veins that carry blood away from the retina. Blockage of smaller veins (eg, branched veins) in the retina can occur in areas where retinal arteries that have been thickened or hardened by atherosclerosis cross and put pressure on a retinal vein. When the capillaries become blocked, pressure increases, which leads to hemorrhage, leakage of fluid and blood. This can lead to macular edema with leakage near the macula. Macular ischemia occurs when these capillaries, which supply oxygen to the retina, exhibit leakage and nonperfusion. Next occurs neovascularization, new abnormal growth of blood vessels, which can lead to neovascular glaucoma, vitreous hemorrhage and, in late or severe cases, retinal detachment. The visual impairment and blindness that occurs with RVO may result from macular edema, retinal hemorrhage, macular ischemia, and/or neovascular glaucoma.

A related event is central or branched retinal artery occlusion (RAO), which can occur when plaque (eg, a blood clot or fatty deposit) blocks a blood vessel or artery in the eye. This can lead to sudden and permanent loss of vision, usually in only one eye. In some cases of central RAO, fundoscopic examination of the affected eye may reveal a pale retina with a cherry-red macula (i.e., cherry-red spot), which results from obstruction of retinal blood flow from the retinal artery, causing pallor, and continued flow of blood to the choroid from the ciliary artery, which results in a bright red coloration in the thinnest part of the retina (i.e., the macula). It typically develops only after an embolism and may resolve within a few days of the acute event. In some cases, by this time, vision loss is already permanent and primary optic nerve atrophy develops. In some cases, when the ciliary artery supplies the macula, a cherry red patch is not observed. Branch RAO may occur when plaques lie in a more distal branch of the retinal artery and may involve temporary retinal vessels. In some cases, a subject at risk of retinal artery blockage is identified and treated with a senolytic agent before severe blockage occurs.

Retinopathy of prematurity (ROP) (also called retrolental fibroplasia (RLF)) is a vascular disease of the eye caused by fibrovascular proliferation, e.g. disorganized growth of retinal blood vessels, which can lead to scarring and retinal detachment. Oxygen toxicity and relative hypoxia may contribute to the development of ROP. Various stages of ROP disease have been defined (Committee for the Classification of Retinopathy of Prematurity, Arch. Ophthalmol. 102(8): 1130-1134, 1984).

Sickle cell retinopathy (SCR) can develop in subjects with sickle cell disease, in some cases during the second decade of life. The ocular manifestations of sickle cell disease (SCD) result from vascular blockages that can occur in the conjunctiva, iris, retina, and choroid. SCR is caused by vascular occlusion of the ocular microvasculature, in contrast to diabetic retinopathy, which may be due to excessive exposure of vascular tissue to hyperglycemia. SCR can cause vision impairment depending on its location and the tissue affected. SCR can be classified as nonproliferative or proliferative depending on the presence or absence of neovascularization in the eye.

In the nonproliferative form of SCR, clinical findings may include salmon spot, iridescent spot, and sunburst hemorrhages that may be seen in the peripheral retina. The central retina may also exhibit venous tortuosity, dilatation of the foveal avascular zone, central retinal artery obstruction, and occlusion of the peripapillary and perimacular arterioles.

Proliferative complications of SCR can lead to deterioration or loss of vision in 10-20% of affected eyes. In some cases, a subject may be diagnosed by a history of spontaneous regression after the initial development of proliferative SCR. Neovascularization of the peripheral retina can develop after vaso-occlusion of the peripheral retina, which can grow anteriorly from the perfused to the non-perfused retina. Initially, these new vessels were flat and resembled sea blades. Neovascularization can cause vitreous hemorrhage due to the constant penetration of blood components into the vitreous through the fragile neovascular tissue. Recurrence of this hemorrhagic phenomenon leads to worsening of vitreoretinal traction, which can lead to rhegmatogenous or tractional retinal detachment.

Senolytic therapy can be applied to a range of ischemic ocular disease phenotypes, which include RGC loss in glaucoma, diabetic and vascular occlusive retinopathy (eg, arterial and venous occlusion, retinopathy of prematurity, sickle cell retinopathy, inflammatory and infectious retinopathy, radiation retinopathy, etc. ) and neovascular AMD (known to be mediated by VEGF and other known SASP factors such as IL-6). These phenotypes have well-defined clinical features that can be diagnosed and monitored using a variety of clinical testing procedures, including examination and tests of retinal structure (eg, fluorescein angiography for areas of nonperfusion, optical coherence tomography for retinal cell layer structure, presence of fluid, thickness ) and function (visual field testing, electrophysiology, which can specifically measure retinal ganglion cell function).

In support of this invention, the photographs of FIGS. 12A and 12B show the presence of senescent cells in the trabecular meshwork tissue of patients with glaucoma. There is an association of senescent cells with the loss of retinal ganglion cells in glaucoma (Skowronska-Krawcyzyk, 2015; Li et al., 2017).

TYPE 2. Degenerative diseases

These diseases are characterized by a progressive deterioration in the quality, function or structure of the eyes, resulting in a progressive decrease in visual acuity.

Examples of degenerative eye diseases include chalasoderma, ptosis, keratitis sicca, Fuchs corneal dystrophy, presbyopia, cataracts, wet age-related macular degeneration (wet AMD), dry age-related macular degeneration (dry AMD); degenerative diseases of the vitreous, including vitreomacular traction syndrome (VMT), macular hole, epiretinal membrane (ERM), retinal breaks, retinal detachments and proliferative vitreoretinopathy (PVR).

The general approach and goals of senolytic therapy for degenerative diseases are based on the following.

Degenerative eye diseases affect all anatomical locations of the visual system and are associated with a pathophysiological cascade that includes features of aging and SASP-related factors that are caused by a variety of cellular stressors. These include environmental factors such as UV exposure and smoking, oxidative stress and inflammatory factors, mitochondrial and DNA damage, and extracellular matrix degradation. These stressors are also associated with the secretion and accumulation of SASP-like factors, which continue to disrupt the local tissue microenvironment and cause ongoing degenerative changes.

The goal of senolytic therapy is to disrupt this cycle by eliminating senescent cells and their associated SASPs. This could limit ongoing tissue damage and potentially restore function by improving the properties of the cellular microenvironment. For example, senolytic therapy can reduce a range of growth factors known to be involved in various stages of AMD. Senolytic agents that block the production of SASP factors may have a multifaceted effect on the pathophysiology of AMD and may modulate and potentially reverse the course of the disease.

Degenerative diseases of the visual system affect anterior and posterior structures, so the approach to senolytic delivery for these diseases will involve both anterior and posterior delivery mechanisms, depending on the site of the primary pathology.

Chalasoderma

Chalazoderma is an age-related change of the upper and lower eyelids characterized by loose, redundant skin and orbicularis muscle, often with bulging orbital fat pockets. This may be due to lowering of the eyebrows and relaxation of the forehead. When the upper eyelids are severely affected, it can limit peripheral vision and obscure the central visual axis. The location, severity, and frequency of visually significant ptosis increases with age.

Systemic diseases such as thyroid-related orbitopathy, renal failure, trauma, lax skin, Ehlers-Danlos syndrome, amyloidosis, hereditary angioedema, and xanthelasma may predispose a subject to chalazoderma. In some cases, chalazoderma is associated with a hereditary condition. Chalazoderma is caused by a loss of elasticity in the connective tissue that supports the structure of the front of the eyelid. The pathophysiology of chalazoderma is consistent with normal age-related skin changes. This includes loss of elastic fibers, thinning of the epidermis, skin redundancy and lymphatic dilatation. Subjects with chalazoderma may also have blepharitis, a condition caused by a blockage of the glands in the eyes that produce lubricating fluid (meibomian glands).

Chalasoderma can be so severe that it pushes the eyelashes into the eyes, causing entropion (entropion). Eyelid deformities, such as entropion of the upper eyelid and ectropion or retraction of the lower eyelid, can occur when there is excess skin of the upper or lower eyelid. Excess skin of the upper eyelid hangs over the eyelashes, causing drooping eyelashes and entropion with subsequent keratitis. Patients with severe lower eyelid chalazoderma develop lower eyelid weakness with resultant eyelid retraction or ectropion.

Chalasoderma can lead to loss of visual field. In severe cases of chalazoderma, the patient may lose 50% or more of their upper visual field. Patients with a purely aesthetic deformity may not experience any visual field defects. In some cases, patients with moderate to severe chalazoderma experience blepharitis. Blepharitis is characterized by swelling of the eyelid skin and erythema; dandruff; inflammation and blockage of the meibomian glands; and sometimes barley.

Local administration (eg, by injection or topical administration to the skin and/or eyelid) of a senolytic agent can provide a subject with an effective dose for the treatment of chalazoderma. The senolytic agents of the invention can stop the progression of chalazoderma in a subject. In some cases, this invention can stop or reverse at least some of the damage that occurred before treatment.

Ptosis

Ptosis refers to the abnormal position of the eyelid margins, the most common cause of which is aging. Acquired ptosis is a condition in which patients develop normal eyelids at birth and develop drooping eyelids in adulthood. The condition can affect one eye or both eyes and is more common in older people as the eyelid muscles may begin to deteriorate. In some cases, a person may be born with congenital ptosis and experience early onset of the condition, including drooping of either eyelid.

Ptosis occurs due to dysfunction of the muscles that lift the eyelid or their innervation. Depending on the cause, ptosis can be classified into several types: neurogenic ptosis, which includes oculomotor nerve palsy, Horner's syndrome, Marcus-Gunn syndrome, misdirection of the third cranial nerve; myogenic ptosis, which includes muscular purulent dystrophy, myasthenia gravis, myotonic dystrophy, ocular myopathy, simple congenital ptosis, blepharophimosis syndrome; aponeurotic ptosis, which can be involutional or postoperative; mechanical ptosis resulting from edema or tumors of the upper eyelid; neurotoxic ptosis; and pseudoptosis.

The main symptom of ptosis is visible drooping or sagging of the upper eyelid in one or both eyes. Ptosis usually gives the face a tired or serious appearance. Ptosis can also lead to dry eyes or watery eyes as the eyelids no longer function effectively to keep the eyes moist. Severe ptosis can lead to loss of visual field. In some cases, the subject experiences fatigue and pain around the eyes as the eyebrows are constantly raised to see properly.

Topical administration may be by injection or topical application to the skin and/or eyelids. In some cases, a senolytic drug can reverse or stop at least some of the damage caused by the disease.

Dry keratoid conjunctivitis

Keratoid conjunctivitis sicca (keratitis sicca) is a disease characterized by dryness of the conjunctiva and/or cornea. Keratoid conjunctivitis sicca is also referred to as keratoconjunctivitis sicca, dry eye, dry eye syndrome, dry eye disease (DED), or dysfunctional tears syndrome. Specific subtypes that fall within the scope of keratitis sicca include water deficiency DED, evaporative DED, Sjögren's syndrome, lacrimal gland deficiency, and meibomian gland disease.

In some cases, keratoconjunctivitis sicca occurs due to inadequate tear production and may be referred to as tear water deficiency dry eye or water deficiency DED. Dry eyes with tear water deficiency may occur in postmenopausal women or in patients with an autoimmune disorder or a disease such as rheumatoid arthritis, systemic lupus erythematosus, or Sjögren's syndrome. In some cases, keratitis sicca occurs due to an abnormality in the composition of tears, which leads to rapid evaporation of tears and may therefore be called evaporative dry eye. Dry eyes can also result from partially opening the eyes for a period of time at night (eg, in subjects suffering from nocturnal lagophthalmos) or due to insufficient blink rate (eg, as may occur in patients with Parkinson's disease). Damage to the surface of the eye that may occur with keratitis sicca may include increased discomfort and sensitivity to bright light.

Symptoms of keratitis may include eye irritation, tingling, burning, pain or tenderness, itching, tightness, pressure behind the eye; feeling of dryness, scratching, feeling of roughness or sand in the eye; foreign body sensation, sensitivity to bright light, blurred vision, increased blinking, eye fatigue, photophobia, redness, mucus, contact lens intolerance and excessive reflex tearing. Foreign body sensation means a sensation or sensation as if something is in the eye. In certain cases, the subject may experience blurred vision or eye irritation that is severe, frequent and/or prolonged in duration. In some patients with severe eye dryness, the surface of the cornea may thicken and/or ulcers and scars may occur. In some cases, blood vessels can grow throughout the cornea and impair vision.

Not all subjects suffering from keratitis sicca exhibit all symptoms. The subject may complain of discomfort or burning in the eyes. Photophobia or blurred vision may be present even in severe cases. The patient's medical history may also indicate dry eyes, such as in a patient with a pre-existing diagnosis of rosacea, radiation therapy, rheumatoid arthritis, systemic lupus erythematosus, scleroderma, or other autoimmune disorders. Slit-lamp biomicroscopic examination may be performed to identify meibomitis, conjunctival dilatation, decreased tear meniscus, increased tear debris, mucus filaments, or a staining profile consistent with keratitis sicca. You can also set tear breakup time to less than 10 seconds and perform a Schirmer test to identify subjects who will benefit from treatment.

Senolytics can be administered by topical injection into the site affected by keratitis sicca, e.g. into the conjunctiva and/or cornea: for example, through eye drops, ointment, gel, or through controlled sustained release from a contact lens placed on the outer surface of the eye. Delivery via a punctal plug system or direct injection into the meibomian or lacrimal glands to deliver the senolytic agent may also be considered. Improvement may include decreased fluorescein staining in the cornea, improvement in the Schirmer test, or improvement in the signs and/or symptoms of dry eye syndrome. Improvements can be measured using the Ocular Disease Surface Index or Ocular Comfort Index.

Fuchs corneal dystrophy (FCD)

Fuchsian corneal dystrophy is the leading cause of corneal transplantation and affects 5% of the US population over the age of 40 years. It is characterized by progressive loss of endothelial cells and deposition of abnormal cellular matrix in the cornea. The cornea gradually swells and becomes cloudy because the remaining endothelial cells are not enough to dehydrate the cornea and keep it clear. Increased protein expression of known aging-associated genes CDKN1A and CDKN2A has been demonstrated (Matthaei et al., 2014). Symptoms of FCD include blurred and blurred vision and may be associated with pain and blistering of the cornea. Diagnosis is made using standard ophthalmic tests as above, and corneal thickness is measured through corneal pachymetry. Currently available treatments include topical drops and surgery with partial or complete penetrating keratoplasty. Administration of the senolytic agent may be accomplished by local delivery to the cornea or anterior chamber adjacent to the cornea in the eye, for example, by intracameral injection or through an ocular implantable device. This may stop progression or reverse at least some of the damage that occurred before treatment.

Presbyopia

Presbyopia is a condition directly associated with aging, and it is almost universal after middle age. Presbyopia is associated with the aging of the eyes and leads to a gradual decline in the ability to focus clearly on close objects. This occurs due to the hardening and loss of flexibility of the lens of the eye, causing the eye to focus light behind the retina rather than on the retina when looking at close objects. Hardening of the lens may result from decreased levels of α-crystallin in the lens. Weakening of the muscle fibers of the ciliary body may also contribute to the inability of the ciliary muscle to deform the lens and cause presbyopia. Administration of a senolytic may strengthen the ciliary muscle by eliminating senescent cells and subsequently reducing muscle fibrosis that occurs with age.

Symptoms include refractive errors, difficulty reading small print (eg, in low light), holding reading material away, blurring of close objects, eye strain, headaches, and fatigue when performing tasks that require near vision. Diagnosis can be made through an eye examination by an ophthalmologist, including tests to evaluate vision and the ability to focus on and see objects.

Administration may be by local delivery to the lens or a chamber adjacent to the lens in the eye, for example, by intracameral injection, intravitreal injection, or by an ocular implantable device. This may stop the progression or reverse at least some of the damage that has occurred.

Cataract

Cataracts remain the largest cause of total blindness, accounting for 18 of the 39 million blind people worldwide. It is known that the incidence increases with age and there is no region in the world that is immune to the onset of age or the effects of vision-threatening lens opacity.

A cataract is a clouding of the lens of the eye that causes progressive, painless loss of vision. Cataracts can be classified by their location. Subcapsular cataracts occur at the back of the lens. People with diabetes or taking high doses of steroid medications may be at greater risk of developing subcapsular cataracts. Nuclear cataracts form deep in the nucleus (eg central zone) of the lens and may be associated with aging. Cortical cataracts are characterized by white, wedge-shaped opacities that begin at the periphery of the lens and move toward the center in a spoke pattern. Cortical cataracts can occur in the cortex of the lens, which surrounds the central nucleus.

The normal lens inside the eye works much like a camera lens, receiving and focusing light onto the retina for clear vision. The lens also adjusts the focus of the eye, allowing us to see things both close and far. The lens is composed primarily of water and protein, which are assembled into a highly ordered, interactive macrostructure essential for lens transparency and refractive index. Disruption of intra- or interprotein interactions can alter this structure, exposing hydrophobic surfaces and causing protein aggregation associated with cataract formation. Over time, the cataract can enlarge and cloud much of the lens, blocking some light from passing through the lens and scattering it, preventing it from focusing clearly on the retina.

A number of risk factors are associated with the formation of cataracts, including, but not limited to, exposure to ultraviolet radiation, diabetes, hypertension, obesity, smoking, long-term use of corticosteroid drugs, statins used to lower cholesterol, previous eye damage or inflammation, previous eye surgery, hormone replacement therapy , significant alcohol consumption, severe myopia, family history. In some cases, cataracts are associated with oxidative disorders. Senile cataract is an age-related visual impairment characterized by gradual progressive thickening of the lens of the eye. As set forth herein, all of these risk factors may be associated with the induction of aging and SASP factors contributing to the development of diseases of the visual system.

Symptoms that may be associated with cataracts include decreased visual acuity, glare (eg, seeing halos and starbursts around lights), myopic shift, and monocular diplopia. In some cases, symptoms are caused by one or more of the following: difficulty reading due to decreased ability to distinguish the contrast between light and dark printed letters on a page, requiring more light to see clearly, trouble distinguishing dark blue from black, blurred vision, colors appear yellower and less vibrant, and slight double vision (also called ghosting). In some cases, cataracts can swell and increase pressure in the eyes, causing glaucoma.

Cataracts are detected by examining the eyes as described previously. Diagnostic methods specific to the detection of cataracts include: 1) examination of the ocular appendages and intraocular structures, which can provide insight into the etiology of the patient's cataract, concomitant disease and possible visual prognosis; 2) a swinging light test to detect a Marcus-Gunn pupil or relative afferent pupillary defect (RAPD), indicating optic nerve damage or diffuse macular damage, which may be an alternative cause of decreased vision; 3) study of nucleus size and brunescence (after dilatation, nucleus size and brunescence as indicators of cataract density can be determined before phacoemulsification surgery); 4) direct and indirect ophthalmoscopy – to assess the integrity of the posterior segment; or 5) genetic testing for a disease that predisposes the subject to developing cataracts.

Senolytic agents may be effective in preventing the development of cataracts, regression of the patient's cataracts, reducing the severity of the cataract, or increasing lens clarity. Administration may include local delivery to the lens or an ocular chamber adjacent to the lens, via intraocular or intracameral injection, or through an ocular implantable device. This may eliminate the need for standard cataract surgery in the eyes. As a rule, the method is performed without surgical removal of the lens or part of it.

Age-related macular degeneration (AMD)

Macular degeneration refers to a family of diseases that are characterized by progressive loss of central vision associated with abnormalities of Bruch's membrane, choroid, neural retina, and/or retinal pigment epithelium. AMD is divided into two types: dry macular degeneration and wet macular degeneration. Dry AMD is defined by the gradual loss of retinal pigment epithelial (RPE) and photoreceptor cells in the macula. Wet AMD is characterized by the growth of abnormal blood vessels beneath the macular epithelium. Age-related changes in the development of retinal morphology and energy metabolism, as well as the cumulative effects of environmental exposures, may render the neural and vascular retina and retinal pigment epithelium more susceptible to damage in late adult life. The pathogenesis of age-related macular degeneration may be related to various processes associated with aging, including oxidative stress, mitochondrial dysfunction, and inflammatory processes.

Dry AMD (also called non-exudative AMD) is a broad term that covers all forms of AMD that are not neovascular, including early and intermediate forms of AMD and the advanced form of dry AMD known as geographic atrophy or atrophic AMD. In geographic atrophy, the progressive and irreversible loss of retinal cells leads to loss of visual function. AMD-like pathology is characterized by the progressive accumulation of characteristic yellow deposits called drusen (eg, accumulation of extracellular proteins and lipids) in the macula between the retinal pigment epithelium and the underlying choroid, which are believed to damage the retina over time.

Patients with dry AMD may have minimal symptoms in the early stages of the disease. In approximately 10-20% of patients, dry AMD progresses to wet AMD. Patients who have dry AMD have a gradual loss of central vision due to the death of photoreceptor cells and their close partners, the retinal pigment epithelial (RPE) cells, with the deposition of drusen. Photoreceptors, that is, the cells in the retina that actually “see” light, are necessary for vision. RPE cells are essential for photoreceptor survival, function, and renewal. RPE cells in the eye act as macrophages that phagocytose and recycle components of the photoreceptor outer segment membranes. If mitochondria in RPE cells are damaged, photoreceptor recycling is blocked, leading to the accumulation of drusen. The accumulation of drusen causes physical displacement of the RPE from its immediate vascular supply, the choriocapillaris. This displacement creates a physical barrier that can interfere with the normal diffusion of metabolites and cellular waste between the choriocapillaris and the retina.

Symptoms of dry AMD include drusen, visual distortions such as straight lines appearing bent, decreased central vision in one or both eyes, need for brighter light when reading, loss of contrast sensitivity, increased blurriness of printed words, decreased color intensity or brightness, difficulty recognizing faces.

The “wet” form of progressive AMD (wet AMD), also called neovascular or exudative AMD, results in vision loss due to abnormal growth of blood vessels (eg, choroidal neovascularization) in the choriocapillaris through Bruch's membrane. Wet AMD is preceded by dry AMD, and both forms usually coexist in the same individual—thus, both processes can be affected by simultaneous treatment with a senolytic drug. As the photoreceptors and RPE cells in Bruch's membrane slowly degenerate and break down, there is a tendency for blood vessels to grow from their normal location in the choroid to an abnormal position under the retina. Choroidal neovascularization (CNV) is stimulated by vascular endothelial growth factor (VEGF). The abnormal blood vessels can be fragile, eventually causing blood and protein to leak under the macula. Bleeding, leaking, and scarring from these blood vessels ultimately leads to permanent damage (eg, hemorrhage, swelling, and scar tissue) and severe loss of central photoreceptor vision and rapid vision loss. Without treatment, wet AMD is responsible for approximately 90% of all cases of blindness caused by AMD.

Symptoms of wet AMD include decreased central vision in one or both eyes, eg, a dark spot (or spots) in the center of their vision, normal side or peripheral vision, straight lines appear bent, decreased intensity or brightness of colors, general blurring of all vision , a distinct blurred or bleeding spot in the visual field, abnormal neovascularization, leakage of blood into the eye, eg behind the macula, sudden onset or rapid worsening of symptoms.

Eligible subjects can be identified by visual examination of the subject's eyes or by risk factor criteria. Testing may include fundus photography, dark adaptation test, Pelly-Robson test, quality of vision tests (eg, Snellen chart, Amsler grid, Farnsworth 100-shade test and maximum color contrast sensitivity test), perimetric predominant test. hyperacuity, electroretinography, optical coherence tomography (OCT), and angiography (eg, fluorescein angiography) in any combination.

AMD is divided into three stages: early, middle and late, partially
based on the grade (size and number) of drusen. Early AMD is diagnosed based on the presence of moderate-sized drusen and may be asymptomatic. Intermediate AMD is diagnosed by large drusen and/or any retinal pigment abnormalities. In some cases, intermediate AMD can cause some vision loss, but it is also usually asymptomatic. At the end of AMD, enough damage to the retina occurs that it results in symptomatic loss of central vision in addition to drusen. The risk of developing symptoms is higher when drusen are large and numerous and are associated with disruption of the layer of pigmented cells under the macula. Large, soft drusen are thought to be associated with elevated cholesterol levels.

Diagnosis of macular degeneration relies on signs in the macula itself, regardless of visual acuity. Diagnosis of AMD may involve various methods and tests, as previously described.

Relevant features of drusen include the number, area, and volume of drusen identified; the shape of the identified drusen; drusen density; and reflectivity of drusen. You can analyze data from retinal images obtained using spectral domain optical coherence tomography (SD-OCT). The image data contains a cross-section of the retina and an en face image of the retina. The image data is processed to produce an accurate structure showing the location, shape, size and other details of the drusen. This structural information can provide quantitative features of drusen that indicate the risk of developing dry to wet AMD in a given subject and over a given period of time (US Patent No. 9,737,205).

Optical coherence tomography may also be used in diagnosis and subsequent assessment of response to treatment. In wet AMD, angiography (eg, fluorescein angiography) can identify and localize abnormal vascular processes and can be used to visualize blood leakage behind the macula.

Senolytic therapy can be used for degenerative retinal diseases such as age-related macular degeneration (AMD) as follows. Deposits of abnormal compounds such as lipofuscin in the retinal pigment epithelium (RPE) result in a persistent accumulation of debris that makes it difficult to support photoreceptors in the outer retina. In AMD, this occurs due to age-related changes in Bruch's membrane, choriocapillaris, and retinal pigment epithelium (RPE). As with ischemic ocular diseases, a complex pathophysiological cascade is initiated, again with multiple senolytic therapy pathways to influence disease stabilization and potential regression based on the ability to eliminate senescent cells and associated SASPs.

In fig. Figure 1C (adapted from Kumar and Fu, 2014) presents factors that influence many points along the pathophysiological cascade of AMD. In fig. 14A and 14B show the presence of senescent cells in AMD. In fig. 8 shows the effect of a senolytic agent on senescent RPE cells in accordance with the invention. The potential for senolytic therapy to influence many features of both wet and dry AMD is new and unprecedented.

Degenerative diseases of the vitreous body

The vitreous is a clear, gel-like substance that fills the cavity at the back of the eye behind the lens. It is composed of a network of collagen fibers and helps stabilize the different layers of the retina and the retinal vasculature. Over time, the vitreous gel breaks down due to degradation of the collagen fiber network, placing patients at risk for traction forces on the underlying retina. These forces are associated with many vitreoretinal diseases that increase with age and vitreous syneresis, which occurs in 70% of patients under 70 years of age. These vitreoretinal diseases are described below.

Vitreomacular traction syndrome

Vitreomacular traction syndrome (VMT) is a disorder of the vitreoretinal interface characterized by: (i) incomplete posterior vitreous detachment (PVD), (ii) abnormally strong adherence of the posterior facet of the hyaloid to the macula; and/or (iii) anteroposterior traction due to syneretic stretch of the vitreous at its attachment sites on the macula, which causes morphological and often functional effects. With age, the vitreous gel undergoes liquefaction, forming pockets of fluid in the vitreous, which leads to contraction or condensation (syneresis) of the vitreous. When the volume of the vitreous body is lost, traction tension occurs in the places of vitreoretinal and vitreopapillary attachment under the influence of the thickening dense cortex of the vitreous body. At the same time, weakening of these attachments between the vitreous and the internal limiting membrane (ILM) may occur, leading to detachment of the posterior hyaloid. Symptoms include blurred or decreased vision, metamorphopsia, micropsia, scotomas, and difficulty performing daily vision-related tasks such as reading.

Macular hole

A macular hole (hole) is a tear in the retina that usually affects the fovea. Macular holes are associated with aging and usually occur in people over 60 years of age. Macular holes can be caused by tangential traction as well as anteroposterior stretching of the posterior hyaloid at the parafovea. Macular holes can occur as complications of posterior vitreous detachment in the earliest stages. Risk factors for macular hole include age, nearsightedness, injury, and eye inflammation.

Macular holes may begin gradually and develop in three stages: foveal detachment (stage I), which may progress without treatment; Part-thickness tears (stage II), which may progress without treatment; and complete ruptures (stage III). The size of the tear and its location on the retina can determine how much vision will be affected. When a stage III macular hole occurs, most central and detailed vision may be lost. If left untreated, macular holes can lead to vision-threatening retinal detachment.

In the early stages of a macular hole, a subject may notice slight distortion or blurring of their central vision. Straight lines or objects may appear curved or wavy. Reading and performing other routine tasks becomes difficult with the affected eye. Symptoms of macular holes include blurred vision and distorted central vision.

Epiretinal membrane

Epiretinal membrane (ERM, also called cellophane maculopathy or macular pucker) is an avascular fibrocellular tissue that can develop on the inner surface of the retina. There are many cytokines associated with the development and proliferation of ERM, the increase in this condition with age, and vitreous syneresis.

The tissue is translucent and proliferates on the surface of the internal limiting membrane. When ERM occurs, residual cortical vitreous due to posterior vitreous detachment or partial separation of the posterior hyaloid may contribute to the proliferation of glial cells in the retina. In some cases, ERM is an idiopathic condition. In some cases, inflammatory mediators promote fibrocellular growth along with the formation of secondary ERM. Secondary ERM can occur in association with retinal vascular disease, inflammatory ocular disease, trauma, intraocular surgery, intraocular tumors, and retinal tear or detachment. Other risk factors include age, posterior vitreous detachment, and history of ERM in the fellow eye. Symptoms include painless vision loss, metamorphopsia (visual distortion, such as in which shapes may appear wavy or curved), double vision, light sensitivity, and images appearing larger or smaller than usual.

In some cases, subjects with ERM have few symptoms and are diagnosed with ERM incidentally during retinal dilatation testing or retinal imaging such as ocular coherence tomography (OCT). OCT imaging technique can be used to assess the severity of ERM. Fluorescein angiography can also be used to determine whether ERM is caused by other underlying retinal problems.

Retinal tear and detachment

Retinal breaks can occur as a result of extension of the vitreous to the site of existing vitreoretinal adhesion, most often associated with age-related syneresis of the vitreous. In some cases, the retina often appears completely normal before an acute tear occurs. A retinal tear can occur with or without subsequent retinal detachment. The vitreous fills the space in the eye between the lens and the retina. Usually the vitreous moves away from the retina without causing problems, but in some cases the movement is so severe that it tears the retina in one or more places. Fluid may pass through the hole in the retina, lifting or tearing the retina away from the back of the eye.

Retinal detachment occurs when subretinal fluid accumulates between the neurosensory retina and the retinal pigment epithelium. This process can happen in three ways. One mechanism is by creating a break in the retina that allows the vitreous to directly penetrate the subretinal space and is known as rhegmatogenous retinal detachment. Rhegmatogenous retinal detachment is often associated with retinal tears associated with posterior vitreous detachment or trauma. The second mechanism involves proliferative membranes on the surface of the retina or vitreous. These membranes can pull back on the neurosensory retina, causing a physical separation between the neurosensory retina and the retinal pigment epithelium, called tractional retinal detachment.

Tractional retinal detachment can occur with proliferative retinopathy caused by diabetes, sickle cell anemia and other pathological processes leading to retinal neovascularization. Traction retinal detachments can also be caused by proliferative vitreoretinopathy after injury or surgery. The third mechanism of retinal detachment is based on the accumulation of fluid under the retina due to inflammatory mediators or exudation of fluid from the lesion mass. This mechanism is known as exudative or serous retinal detachment and can be caused by a number of inflammatory or exudative processes of retinal diseases such as sarcoidosis or choroidal neoplasms. Rhegmatogenous retinal detachment has a characteristic appearance that distinguishes it from traction or serous detachment. Rhegmatogenous retinal detachment has a wavy appearance and bends in a wave-like manner when the eyes move. A traction release has a smooth, concave surface with minimal movement with eye movements. Serous detachments exhibit a smooth retinal surface and fluid movement depending on the patient's position.

Retinal tears and rhegmatogenous and tractional detachments require correction by any or all of laser therapy, cryotherapy, pneumatic retinopexy, scleral indentation, and/or vitrectomy.

Proliferative vitreoretinopathy

Proliferative vitreoretinopathy (PVR) occurs when a scar forms under or on the retina after a retinal detachment, preventing the retina from healing and returning to its original place. PVR is associated with failed repair of retinal detachment. When there is a hole in the retina, cells that are normally found underneath the retina enter the eyeball and settle in the inner layer of the eye on top of the retina. These cells multiply and form a scar on the surface (and in some cases under) the retina. This scar tissue then shrinks and separates the retina from the innermost walls of the eye, resulting in a second retinal detachment.

The senolytic agents of the present invention can be administered by intravitreal injection into the vitreous body of the eye or by local administration.

TYPE 3. Genetic diseases

Genetic eye diseases are characterized as diseases that are caused by a mutation, deletion, or insertion in a person's DNA sequence.

Genetic diseases can be divided into three main categories. 1. Single gene diseases: diseases caused by defects in one specific gene, often with simple and predictable patterns of inheritance such as dominant, recessive and X-linked. 2. Chromosomal diseases: diseases resulting from changes in the number or structure of chromosomes. 3. Multifactorial diseases (complex diseases): diseases caused by changes in many genes, often in complex interaction with environmental and lifestyle factors such as diet or cigarette smoke.

Examples of genetic eye diseases include retinitis pigmentosa, Stargardt disease, Best disease, and Leber hereditary optic neuropathy (LHON).

The general approach and goals of senolytic therapy for genetic conditions are based on the following.

Genetic diseases of the visual system can affect all anatomical layers of the eye and are associated with cellular defects that can lead to an accelerated aging phenotype caused or mediated, at least in part, by senescent cells. Inherited susceptibility to certain eye diseases suggests that the accumulation of pathogenic senescent cells may be directly or indirectly influenced by genetic components, which again may lead to earlier presentation. Genetic disorders exhibit a multifactorial cascade with senescent cells and SASP production contributing to ongoing dysfunction and cell degeneration/death.

These diseases may benefit from senolytic therapy because senescent cells and their associated SASP factors mediate associated contributions to ongoing cell dysfunction, cell loss, and disease progression by blocking angiogenic, inflammatory, fibrotic, and extracellular matrix-modifying proteins present in the pathophysiology . Genetic disorders of the visual system affect anterior and posterior structures. Senolytic agents are administered either to the anterior or posterior region of the eye, or a combination of both, depending on the location of the primary pathology.

Basic pathophysiology

Complex monogenic retinal diseases such as retinitis pigmentosa, Stargardt disease, or mitochondrial DNA disorders such as Leber hereditary optic neuropathy are diseases with a known genetic basis that may accelerate the accumulation of senescent cells. Diagnosis, clinical monitoring and response to therapy are monitored in diseases such as ischemic or degenerative diseases through a combination of functional and structural tests including visual acuity, visual field, intravenous fluorescein angiography (IVFA), OCT scanning and electrophysiology including electroretinography (ERG). ) and visual evoked potential (VEP).

Figure 1D shows the events leading to degeneration and cell death in Leber hereditary optic neuropathy (Mann et al., 2002). In fig. Figure 2A shows the loss of retinal cells in the clinical stages of retinitis pigmentosa (Dryja, 1986). These diseases may benefit from the use of a senolytic agent due to the elimination of senescent cells and the elimination of SASP.

In Stargardt disease, lipofuscin accumulates due to a mutation in the ABCA4 gene. Diagnosis, clinical monitoring and response to therapy are monitored in these diseases, as in ischemic diseases, through a combination of functional and structural tests, including visual acuity, visual field, intravenous fluorescein angiography (IVFA), OCT scanning and electrophysiology, including electroretinography (ERG). ) and visual evoked potentials (VEP).

Retinitis pigmentosa

Retinitis pigmentosa is a group of inherited diseases that result from harmful changes in any of more than 200 genes and lead to retinal degeneration, which results in the destruction and loss of cells in the retina. Retinitis pigmentosa is characterized by progressive loss of peripheral vision and difficulty with night vision (nyctalopia), which can lead to loss of central vision. In general, the result of the inherited gene is damage to the photoreceptor cells in the retina. Symptoms of retinitis pigmentosa include nyctalopia, vision loss such as loss of peripheral vision, tunnel vision, and photopsia (light flashes).

There is variability in physical symptoms depending on the specific form of retinitis pigmentosa, and not all subjects exhibit all symptoms at the same time. Diagnostic methods include visual field testing, fundoscopic examination, electroretinography (ERG) (measuring the electrical activity of photoreceptor cells), and genetic testing. The eye examination may include assessment of visual acuity and pupillary response, as well as anterior segment, retinal, and fundoscopic evaluation. Subjects with retinitis pigmentosa may have decreased electrical activity, reflecting decreased photoreceptor function. To exclude the possibility of diagnosing other diseases, additional methods that are used in diagnosing related diseases may be used.

In some cases, DNA samples from a subject can be used to genetically diagnose retinitis pigmentosa. Genetic typing of specific forms of the disease can be carried out. Genetic testing is available through the National Eye Genotyping and Phenotyping Network (eyeGENE).

Stargardt disease

Stargardt's disease (also called Stargardt's macular dystrophy, juvenile macular degeneration, and fundus flavimaculatus) is an inherited disease of the retina. Stargardt disease is one of several genetic diseases that cause macular degeneration. Stargardt disease is characterized by excessive accumulation of lipofuscin in the retina, which causes progressive damage or degeneration of the macula, the small central region of the retina responsible for central visual acuity.

Vitamin A is used to create light-sensitive molecules inside photoreceptors. Mutations in a gene called ABCA4 are the most common cause of Stargardt's disease. This gene produces a protein that normally removes potentially harmful vitamin A byproducts inside photoreceptors. Affected cells accumulate clumps of lipofuscin, a fatty substance that forms yellowish spots. As lipofuscin builds up in the area around and around the macula, central vision can deteriorate. Ultimately, these fatty deposits cause the photoreceptors to die and vision becomes even worse.

Symptoms of Stargardt's disease include gray, black, or cloudy spots in the center of vision; lipofuscin deposits; color blindness, sensitivity to bright light, long adjustment times when transitioning from light to dark. The progression of symptoms in Stargardt's disease may vary for each subject. Subjects with earlier onset of the disease typically experience more rapid vision loss. Vision loss may decrease slowly at first and then quickly worsen until it levels off. Subjects with Stargardt disease may have vision of 20/200 or worse. People with Stargardt's disease may also begin to lose some of their peripheral vision as they age.

Diagnostic methods that can be used to evaluate symptoms of vision loss in Stargardt disease include examination and ancillary tests as described above and include visual field testing, color testing, fundus examination, electroretinography (ERG), optical coherence tomography (OCT), and genetic testing. The number, size, color and appearance of lipofuscin deposits may vary from subject to subject. Lipofuscin deposits may appear as yellowish spots in the macula. The spots are usually irregular in shape and extend outward from the macula in the shape of a ring.

Accumulation of lipofuscin may be a direct cause of death of the RPE and photoreceptors in the retina. The senolytic agents of the invention can preserve vision in patients with Stargardt's disease suffering from excessive lipofuscin accumulation.

Best's disease

Best's disease (also called vitelliform macular degeneration) is a form of early-onset macular degeneration that is characterized by the appearance of lipofuscin deposits, e.g. in the retinal pigment epithelium layer of the macula. Lipofuscin deposits may appear as yellow or orange yolk-shaped or ovoid lesions. Ocular disorders result from disruption of the retinal pigment epithelium (RPE). Dysfunction of the bestrophin protein results in abnormal transport of fluid and ions across the RPE. Lipofuscin can accumulate in RPE cells and in the RPE subspace, especially in the fovea. A subject's RPE may degenerate over time and in some cases cause secondary loss of photoreceptor cells. In some cases, destruction of the RPE/Brooks' membrane can lead to the development of choroidal neovascularization.

Symptoms of Best's disease include bilateral large yellow ovoid macular spots, mild early-stage vision loss, late-stage moderate vision loss, choroidal neovascular membrane (CNVM), and subretinal hemorrhages that occur with minor eye injuries.

Diagnostic modalities include those previously described, such as electrooculography (EOG), electroretinography (ERG), optical coherence tomography (OCT), angiography (eg, fluorescein angiography), and fundus autofluorescence imaging. Fundus autofluorescence (FAF) can be used to detect hyperautofluorescence in the early vitelliform stages of the disease. Such hyperfluorescence may be observed during the pseudohypopyon stage, become patchy with areas of hypoautofluorescence during the vitelleruptive stage, and eventually become hypofluorescent during the atrophic stage of the disease. The changes seen with FAF may precede or appear greater than those seen with ophthalmoscopy.

The use of senolytic agents may stop or reverse the effects of lipofuscin-related damage.

Leber hereditary optic neuropathy (LHON)

Leber hereditary optic neuropathy (LHON) is a mitochondrial genetic disease characterized by bilateral central vision loss due to focal degeneration of the optic nerve. The onset of vision loss ranges from 8 to 60 years of age, but occurs mostly between 15 and 30 years of age. However, vision deterioration can occur within the first seven years of life. The disease predominantly affects men.

Symptoms in subjects suffering from LHON include blurred or cloudy vision, loss of visual acuity, loss of central vision, impaired color vision, centrocecal scotomas, transient optic disc pallor, circumpapillary telangiectatic microangiopathy, poor pupillary responses to light, swelling of the surrounding retinal nerve fiber layer disc (pseudoedema) and optic nerve atrophy. Subjects with LHON may remain asymptomatic until they experience blurred or blurred vision in one eye, with some cases affecting the other eye after a delay of about six to eight weeks. Patients with LHON may experience a rapid and painless loss of central vision accompanied by fading of colors, especially in the green/red field. Visual acuity can reach 20/400, for example, after a few months. Optic atrophy is a characteristic of the disease and can occur after about 6 months.

Diagnostic methods include fundoscopic examination, fluorescein angiography and optical coherence tomography (OCT). When examining the fundus, characteristic signs of the disease are tortuosity of the central retinal vessels, circumpapillary telangiectatic microangiopathy and swelling of the retinal nerve fiber layer around the disc. Fluorescein angiography may be performed to exclude true papilledema. In some cases, there is no dye leakage along the borders of the swollen optic nerve. OCT of the optic nerve may show elevation in the initial phases of the disease or atrophy in the later stages of the disease.

TYPE 4: Infectious eye diseases

These diseases are caused by pathogenic microorganisms such as bacteria, viruses, parasites or fungi; diseases can be transmitted directly or indirectly from one person to another.

Infectious eye diseases can be caused by bacterial agents, fungal agents or viruses. Etiologic agents include herpes zoster varicella virus (HZV), herpes simplex virus, cytomegalovirus, and human immunodeficiency virus (HIV).

The general approach and goals of senolytic therapy for infectious diseases may be based on the following.

Infectious diseases of the visual system affect all anatomical locations of the eyes and are closely related to age and aging. Infectious agents may contribute to the induction of senescence and a multifactorial cascade with senescent cells and SASP production contributing to ongoing cell dysfunction. Once found in senescent cells, they can in turn affect the ability to fight infection.

Senescent cells have an impaired ability to control viral replication (Kim et al., 2016), consistent with the known increased susceptibility to infection that occurs with age. Aging and the ability to respond to infectious agents are a category of eye diseases that can be significantly impacted by senolytic therapy. Elimination of senescent cells and their associated SASP factors may reduce damage to the cellular microenvironment. Infectious diseases of the visual system affect the anterior and posterior structures. In this case, senolytic agents are administered into the anterior or posterior compartment or a combination of both, depending on the location of the primary pathology.

Eye diseases associated with herpes simplex

Senolytic agents may be used in treating subjects for viral age-related eye diseases associated with herpes simplex. Herpes simplex viruses (eg, herpes simplex 1) are present in most adults. Viruses in the herpes family typically reside around nerve fibers in humans. Herpes viruses can infect the eyes, causing inflammation and scarring of the cornea. Eye herpes can be transmitted through close contact with an infected person who has an active virus. Ranging from a simple infection to a condition that can cause blindness, there are several forms of ocular herpes infection. Herpetic keratitis is a viral infection of the cornea that usually affects the corneal epithelium. Stromal keratitis occurs when the infection penetrates deeper into the layers of the cornea and can lead to scarring and/or vision loss. Stromal keratitis may be associated with a late immune response to the initial infection. Iridocyclitis is a serious form of infection in which the iris and surrounding tissues inside the eye become inflamed, causing severe sensitivity to light, blurred vision, pain and redness of the eyes. When the infection occurs in the retina or the inner lining of the back of the eye, it can be called retinitis herpeticum.

Symptoms include pain in and around one eye only, headache and fever, redness, rash or sores on the eyelids and around the eyes, especially in the forehead or tip of the nose, red eyes, blurred vision, inflammation or swelling and/or cloudiness of the cornea, a feeling of dirt or sand in the eye, tearing, pain when looking at a bright light. Diagnostic methods include those described earlier. In some cases, a Tzanck smear or Wright's stain can be used to determine the presence of lesions containing a herpes-type virus. Viral culture, direct immunofluorescence assay, and/or PCR can also be used to confirm the diagnosis of infection with a specific virus.

Senolytic agents can be administered by topical administration to an infected site, eg, the conjunctiva and/or cornea, to treat, improve and/or prevent ocular disease. This can be accomplished by applying a contact lens that delivers the senolytic drug to the patient's eye(s) at the site of action; or by intraocular injection into a specific site of infection in the eye. Optionally, senolytic agents can be administered in combination with an antiviral agent, which may include oral agents such as acyclovir, famciclovir or valacyclovir.

Ocular herpes zoster

Varicella zoster virus (HZV) or varicella zoster virus (VZV) is a latent virus of the herpes family that usually resides around nerve fibers in humans. It is strongly associated with aging. Reactivation of the virus in the ocular division of the trigeminal nerve causes the eye disease herpes zoster (herpes zoster ophthalmicus (ZHOU)).

Symptoms include severe chronic pain, vision loss, skin rash on the forehead, painful inflammation of all tissues in the front and in some cases the back structure of the eye, pain in and around one eye only, headache and fever, red eye, blurred vision, inflammation or swelling and/or clouding of the cornea, a feeling of dirt or sand in the eye, watery eyes, pain when looking at bright lights.

Subjects to be treated according to the present invention may be selected based on clinical presentation or ophthalmological examination indicating the presence of HZO. Diagnostic methods include external examination, visual acuity, visual field, other eye movements, pupillary response, fundoscopy, intraocular pressure, anterior chamber slit lamp examination, and corneal examination with and without staining. In some cases, a Zanck smear or Wright's stain can be used to determine whether the lesions contain a herpes-type virus. Viral culture, direct immunofluorescence assay, and/or PCR can also be used to confirm the diagnosis of infection with a specific virus.

Senolytics can be administered by topical injection into the site of infection, e.g. into the conjunctiva and/or cornea, for example, through eye drops, through a contact lens, or through an intraocular injection into a specific site of infection in the eye. Senolytic agents can be administered in combination with a second agent such as a corticosteroid or an antiviral agent, eg, against the varicella zoster virus such as acyclovir, famciclovir or valacyclovir. In some cases, the antiviral agent is given orally.

Cytomegalovirus retinitis

Cytomegalovirus (CMV)-associated eye diseases include cytomegalovirus retinitis. CMV retinitis is a common opportunistic eye infection that most often occurs in immunocompromised individuals (HIV, organ transplants), which leads to inflammation of the retina and can affect the light-sensitive cells of the retina, leading to loss of vision and, in some cases, blindness. Following primary infection, CMV establishes latent infection in myeloid progenitor cells and intermittent viral reactivation from activated macrophages or dendritic cells, which is controlled by strong virus-specific CD4 ⁺ T cell and CD8 ⁺ T cell responses. In some cases, patients with HIV become more susceptible to CMV-related eye disease when the CD4 count drops below 50 cells/µL. Subjects who are HIV positive or undergoing immunosuppressive chemotherapy may be susceptible to cytomegalovirus ocular disease.

Symptoms of cytomegalovirus retinitis include infection in one or both eyes, floaters, flashes, blind spots, blurred vision, loss of peripheral vision, and retinal detachment. The diagnosis of cytomegalovirus retinitis can be confirmed by PCR amplification of viral DNA from the subject's sample. Counting CD4 ⁺ T cells in a subject's sample can be used to predict or diagnose the onset of an eye infection. Patients with low CD4 ⁺ T cell counts should undergo regular ophthalmologic examinations for symptoms of retinal disease.

Senolytic agents can be administered to the front of the eye using eye drops, through a contact lens, or by intraocular injection into a specific site of infection in the eye. In some cases, a senolytic agent is administered in combination with an antiviral agent such as ganciclovir, valganciclovir, foscarnet, or cidofovir.

Eye diseases associated with human immunodeficiency virus (HIV)

Human immunodeficiency virus (HIV) can affect the eye directly or indirectly through noninfectious microvascular disorders or various opportunistic infections. Ophthalmological manifestations of HIV infection can affect the anterior or posterior segment of the eye. The anterior segment may develop periocular tissue tumors and various external infections, and the posterior segments may develop HIV-associated retinopathy or opportunistic infection of the retina and/or choroid. Ophthalmic manifestations of HIV infection include molluscum contagiosum, ocular herpes zoster, Kaposi's sarcoma, squamous cell carcinoma of the conjunctiva, trichomegaly, dry eyes, anterior uveitis, retinal microvasculopathy, cytomegalovirus retinitis, acute retinal necrosis, progressive outer retinal necrosis, toxoplasmic retinochoroiditis, with philic retinitis and endophthalmitis , caused by Candida albicans.

Symptoms may include loss of visual acuity, retinal damage, and a number of other symptoms associated with these opportunistic infections. Diagnostic methods include those described earlier.

In some cases, counting CD4 ⁺ T cells in a subject's sample is used to predict and/or diagnose the onset of an eye infection in HIV-positive patients. In some cases, ocular syndromes associated with immunosuppression are rare in patients with early HIV infection (CD4 count >300 cells/μL).

Senolytic agents can be administered by intravitreal injection to a specific site of infection in the eye, optionally in combination with an antiretroviral against HIV or specific antivirals such as valganciclovir when administered systemically by injection or orally.

TYPE 5. Inflammatory diseases

They are characterized by a localized response, triggered by injury, foreign object, or tissue destruction, that serves to destroy, dilute, or confine both the noxious agent and the damaged tissue through the production of proinflammatory mediators and the recruitment of immune system cells. The inflammatory response can be provoked by physical, chemical and biological agents, including mechanical trauma, exposure to excessive sunlight, x-rays and radioactive materials, harsh chemicals, extreme temperatures and cold, or infectious agents such as bacteria, viruses and other pathogens. Inflammation may be acute or chronic and associated with a known trigger or idiopathic (i.e., not associated with an obvious triggering agent). Autoimmune eye diseases occur and may be associated with significant visual impairment.

Examples of idiopathic posterior inflammatory ocular diseases include punctate choroiditis (PIC), multifocal choroiditis (MIC), and serpiginous choroidopathy. Inflammation may also play a role in ocular diseases associated with other etiologies.

The general approach and goals of senolytic therapy for inflammatory diseases are based on the following.

Primary and secondary inflammatory diseases of the eye affect all anatomical areas of the visual system, from the eyelids and cornea to the optic nerve and optic pathway, and can be acute or chronic. They are associated with a pathophysiological cascade that is densely populated with signs of aging and factors associated with SASP. The goal of senolytic therapy is to disrupt the inflammatory cycle by eliminating senescent cells and their associated SASPs. This limits ongoing damage to the tissue and potentially restores function by improving the properties of the cellular microenvironment.

Chronic inflammation is a feature of age and aging and numerous diseases associated with aging. Therefore, it plays a role in all the disease categories presented so far. Inflammatory diseases of the visual system affect the anterior and posterior structures and are treated accordingly, depending on the location of the primary pathology.

Punctate choroiditis (PIC)

PIC is an idiopathic inflammatory disease of the choroid, which is characterized by multifocal, well-circumscribed, small lesions of the choroid when an infectious cause has been excluded. Changes in the blood supply to the choroid associated with inflammation may contribute to the pathogenesis of PIC. Symptoms of PIC include blurred vision, photopsia, central and/or peripheral scotomas, and metamorphopsia.

Diagnostic methods include those described earlier. Fundoscopy may reveal multiple, small, round, yellowish-white dotted lesions, with no signs of intraocular inflammation. SD-OCT can provide structural characterization of PIC lesions. Autofluorescence images may show hypofluorescence in scarred lesions and autofluorescence in active lesions.

Senolytic agents can be administered by intravitreal injection or by intravitreal implant in the vitreous of the subject's eye (eg, bioerodible implant). Senolytic agents can be administered in combination with corticosteroids such as cortisone, dexamethasone, fluocinolone, hydrocortisone, methylprednisolone, prednisolone, prednisone, and triamcinolone; immunosuppressants such as rapamycin; VEGF-A antagonists such as bevacizumab and ranibizumab; and antibiotics.

Multifocal choroiditis (MFC)

Multifocal choroiditis (MFC) is a chronic inflammatory disease characterized by uveitis (inflammation of the middle layer of the eye) and multiple lesions in the choroid. MFC most often occurs in female patients aged 6 to 69 years.

Symptoms of MFC include decreased visual acuity, blurred vision, pop-up sensations, photosensitivity, blind spots, ocular discomfort, photopsia, visible flashes of light, photophobia, inflammation in the anterior, middle and/or posterior layer of the eye, posterior uveitis, multiple scattered yellow/gray -white spots in the choroid and/or retina. Lesions can range in size from 50 to 1000 μm and are distributed in the peripapillary region, arcades, and midperiphery of the eye. In some cases, the subject may develop macular and peripapillary choroidal neovascularization or choroidal neovascular membranes (CNVM), new blood vessels that can cause more severe vision loss.

Diagnostic methods include previously described methods, as well as blood tests to rule out the possibility of a specific disease associated with the virus.

Senolytics can be administered by intravitreal injection or by intravitreal implant. Senolytic agents can be administered in combination with a second active agent, which is administered topically or by intraocular implant or intraocular injection. Suitable second agents include corticosteroids such as cortisone, dexamethasone, fluocinolone, hydrocortisone, methylprednisolone, prednisolone, prednisone and triamcinolone; immunosuppressants such as rapamycin; VEGF-A antagonists such as bevacizumab and ranibizumab; and antibiotics.

Serpiginous choroidopathy

Serpiginous choroidopathy (also called geographic choroiditis or helicoid peripapillary choroidopathy) is an inflammatory eye disease characterized by progressive destruction of the retinal pigment epithelium (RPE) and choriocapillaris with secondary involvement of the outer retina. Lesions may form in the eyes that last for weeks or months, usually recur, and may involve scarring of the eye tissue. If centrifugally expanding lesions bypass the fovea, the subject's central visual acuity may be preserved. In the macular variant of serpiginous choroidopathy, patients may first develop geographic involvement of the macula, causing early and profound vision loss without previous peripapillary activity. The disease typically occurs in subjects between 30 and 70 years of age, and patients may experience an increased incidence of HLA-B7 and retinal S antigen association.

Symptoms of serpiginous choroidopathy include painless unilateral visual loss, blurred vision, metamorphopsia, photopsia, scotomas, central scotomas, or yellowish-white chorioretinal lesions with a serpentine or pseudopodal appearance radiating centrifugally from the optic disc.

Diagnostic methods include imaging techniques as described above.

Senolytic agents may optionally be administered in combination with other agents such as cortisone, dexamethasone, fluocinolone, hydrocortisone, methylprednisolone, prednisolone, prednisone and triamcinolone; immunosuppressants such as rapamycin; VEGF-A antagonists such as bevacizumab and ranibizumab, if indicated for associated choroidal neovascularization; and antibiotics.

TYPE 6. Iatrogenic diseases

Iatrogenic eye diseases are characterized as diseases that result from diagnostic and therapeutic procedures performed on the patient. Examples of iatrogenic ocular diseases include post-vitrectomy cataracts or radiation retinopathy following treatment of tumors.

The general approach and goals of senolytic therapy for iatrogenic diseases are based on the following.

The aging process when exposed to procedural or therapeutic stressors produces a pathophysiological cascade similar to that observed in other forms of the aging process. When exposed to such stressors, ocular tissues may have a greater prevalence of senescent cells, which in turn may cause some eye diseases to appear at an earlier stage or in a more severe form. The goal of senolytic therapy is to disrupt the effects of senescent cells on tissues by eliminating senescent cells and their associated SASPs. This prevents or limits ongoing damage to the tissue and potentially restores function by improving the properties of the cellular microenvironment. Given that in some cases an iatrogenic effect can be predicted, prophylactic therapy can be implemented.

Iatrogenic diseases of the visual system affect anterior and posterior structures. In this case, the senolytic agent is injected into the anterior or posterior compartment or into both, depending on the location of the primary pathology, the nature of the procedure and the senolytic agent chosen for use.

Cataract after vitrectomy

After intraocular surgery, cataract formation or acceleration of its formation may occur. Vitrectomy is a microsurgical technique used to gain access to the vitreous cavity and retina and is used to treat diseases affecting the posterior segment of the eye. Vitrectomy is associated with comorbidities that may impair visual acuity, such as retinal detachment, corneal decompensation, and cataract formation or progression. In some cases, the type of cataract that forms or accelerates after vitrectomy is nuclear sclerotic cataract. In some cases, when the eyes undergo a vitrectomy, the lens is also removed. Cataracts that develop after vitrectomy can limit visual acuity to such an extent that it will require surgical removal of the lens in the normal eye. The formation or acceleration of cataracts after vitrectomy may be associated with light toxicity, oxidation of lens proteins, exposure to intraocular gas, the duration of the operation itself, and increased retrolental oxygen levels.

Symptoms of cataracts after vitrectomy include decreased visual acuity despite the anatomical and/or functional success of the previous vitrectomy surgery, glare, halos, etc. Individuals who have undergone vitrectomy may have a lower level of baseline (pre-cataract) visual acuity due to the hidden nature of their retinal pathology; therefore, patients with cataracts after vitrectomy are more likely to have worse vision than patients with typical senile cataracts.

Radiation retinopathy

Radiation retinopathy is a radiation dose-dependent complication of exposure to radiation such as external beam radiation therapy or capsule brachytherapy. External radiation therapy is used to treat tumors of the nasopharynx, paranasal sinuses, or orbits where ocular protection is limited, and may result in clinically significant radiation retinopathy. Capsule brachytherapy for the treatment of intraocular tumors can also cause damage to the nearby retina and choroid. Exposure to radiation may cause preferential loss of vascular endothelial cells with relative sparing of pericytes. Differences in sensitivity between endothelial cells and pericytes may result from direct exposure of endothelial cells to high levels of oxygen and iron in the blood, which generate free radicals and cause cell membrane damage, capillary occlusion, and microaneurysm formation. Retinal ischemia in areas of retinal nonperfusion ultimately leads to macular edema, neovascularization of microaneurysms, macular edema, vitreous hemorrhage, and tractional retinal detachment.

Symptoms of radiation retinopathy include blurred vision, floaters, telangiectasia, neovascularization, vitreous hemorrhages, hard exudates, cotton wool spots, and/or macular edema.

Diagnostic methods include ophthalmological examination of the subject's eyes, such as angiography and optical coherence tomography (OCT). Fluorescein angiography can be used to identify and isolate microvascular features of radiation retinopathy. Indocyanine green angiography can reveal precapillary arteriolar occlusion and areas of choroidal hypoperfusion. Optical coherence tomography (OCT) may have utility in the evaluation of macular edema.

Combining senolytic agents with other standard treatments

Senolytic agents for the treatment of ocular diseases can be combined with other pharmaceutical agents that are approved for clinical use. Because senescent cell removal works by a different mechanism than current treatments, these two agents may act synergistically or additively, minimizing the administration schedule and improving results. Senolytics remove senescent cells in the eye that contribute to the persistence and progression of disease-related pathophysiology.

The current standard of care for many vascular diseases of the eye is an agent that inhibits vascular endothelial growth factor (VEGF), namely aflibercept (marketed as Eylea ^® by Regeneron Pharmaceuticals). Aflibercept is a recombinant fusion protein consisting of the VEGF binding regions from the extracellular domains of the human VEGF-1 and -2 receptors fused to the Fc portion of human IgG1 immunoglobulin.

For neovascular (wet) age-related macular degeneration (AMD), the recommended dose for EYLEA is 2 mg (0.05 mL or 50 μL) administered by intravitreal injection every 4 weeks (monthly) for the first 12 weeks (3 months), with followed by 2 mg (0.05 ml) by intravitreal injection once every 8 weeks (2 months). For macular edema following retinal vein occlusion (RVO), the recommended dose for EYLEA is 2 mg (0.05 mL or 50 microliters) administered by intravitreal injection once every 4 weeks (monthly). For diabetic macular edema (DME), the recommended dose for EYLEA is 2 mg (0.05 mL or 50 microliters) administered by intravitreal injection every 4 weeks (monthly) for the first 5 injections, followed by 2 mg (0.05 mL) via intravitreal injection once every 8 weeks (2 months). Although EYLEA can be administered as frequently as 2 mg every 4 weeks (monthly), additional benefit has not been demonstrated when EYLEA is administered every 4 weeks compared to 8 weeks in most patients. Some patients may need dosing every 4 weeks (monthly) after the first 20 weeks (5 months). For diabetic retinopathy (DR) in patients with DME, the recommended dose of EYLEA is 2 mg (0.05 mL or 50 microliters) administered by intravitreal injection every 4 weeks (monthly) for the first 5 injections, followed by 2 mg (0.05 ml) by intravitreal injection once every 8 weeks (2 months).

The use of a senolytic agent in combination with anti-VEGF therapy is a new paradigm that may influence disease characteristics in significant ways. Anti-VEGF therapy, given alone, requires frequent administration to prevent disease progression. Combining anti-VEGF therapy with a senolytic agent may reduce the rate of progression or relapse rate of active disease. It is possible that anti-VEGF may acutely block potential leakage and bleeding, and a senolytic agent may target CNV in a subacute and then chronic manner - with the possibility that the need for combination therapy will be reduced or eliminated, or that it will be used to a greater extent effectiveness in a wider group of patients. This, in turn, may allow the clinician to prescribe anti-VEGF therapy less frequently, with better visual outcomes.

The combination is expected to improve response rates, improve outcomes, and reduce treatment burden and overall cost of therapy. It may also affect aspects of underlying dry AMD that are not affected by anti-VEGF therapy. This is reflected in long-term outcomes in patients with wet AMD treated with anti-VEGF therapy who develop vision loss over time in the absence of active neovascularization, but rather due to atrophy or progression of underlying dry AMD (Bhisitkul et al., 2015).

Senolytic agents can be combined with other agents that target the underlying pathophysiology or symptomatology of the disease.

A non-pharmaceutical intervention that may be performed in conjunction with senolytic administration includes punctal occlusion to reduce the drainage of tears from the eyes. Another example is fitting a subject with scleral or semi-scleral contact lenses, which create a fluid-filled layer over the cornea. In some cases, the administration of a senolytic is carried out in combination with a punctal plug device, blocking the lacrimal canal that drains tears from the eye.

In some cases, the eye disease is associated with other inflammatory diseases and the senolytic agent is administered in combination with antihistamines (such as pheniramine, emedastine or azelastine), decongestants (such as tetrahydrozoline hydrochloride or naphazoline) or non-steroidal anti-inflammatory drugs (such as nepafenac or ketorolac), corticosteroids ( such as fluorometholone or loteprednol), mast cell stabilizers (such as azelastia, cromal, emedastine, ketotifen, iodoxamine, nedocromil, olopatadine or pemirolast) or steroids. In some cases, the pharmaceutical composition is administered in combination with a second active agent, for example, a macrolide immunosuppressant such as cyclosporine, tacrolimus, pimecrolimus, everolimus, sirolimus, deforolimus, temsirolimus and zotarolimus, abetimus, gusperimus or mycophenolic acid.

If the eye disease is associated with an infectious bacterial disease (such as a meibomian gland infection or corneal infection), then eye drops or ointment may be administered with or in a combination composition containing an antibiotic such as ciprofloxacin, erythromycin, gentamicin, ofloxacin, sulfacetamine, tobramycin, or monofloxacin. If the dry eye disease is associated with a viral infection, the senolytic agent may be administered with or in combination with an antiviral agent such as trifluridine or idoxuridine. In another example, a subject comes with a pre-existing autoimmune disease. The subject may also be treated with systemic (eg) oral therapy for the associated disease.

In some cases, the senolytic agent is administered in combination with one or more different biologically active agents, which may belong to the same or different classes of drugs. Biologically active agents or preparations are selected from: anesthetics and analgesics, antiallergic agents, antihistamines, anti-inflammatory agents, anticancer agents, antibiotics, anti-infectives, antibacterial agents, antifungal agents, antiviral agents, anti-transport/motility agents, anti-glaucoma drugs, antihypertensive agents, decongestants, immunological response modifiers, immunosuppressive agents, peptides and proteins, steroid compounds (steroids), low solubility steroids, carbonic anhydrase inhibitors, diagnostic agents, antiapoptotic agents, gene therapy agents, sequestering agents, reducing agents, antipermeabilizing agents, antisense compounds, antiproliferative agents, antibodies and antibody conjugates, blood flow enhancers, antiparasitics, non-steroidal anti-inflammatory drugs, nutrients and vitamins, enzyme inhibitors, antioxidants, anti-cataract drugs, aldose reductase inhibitors, cytoprotectors, cytokines, cytokine inhibitors and cytokine protectors, UV blockers , mast cell stabilizers and antineovascular agents such as antiangiogenic agents such as matrix metalloprotease inhibitors and vascular endothelial growth factor (VEGF) modulators, neuroprotectors, miotic agents and anticholinesterase agents, mydriatic agents, artificial tears/dry eye therapy, anti-TNFα, IL-1 receptor antagonists , protein kinase C-13 inhibitors, somatostatin analogues and sympathomimetics.

Other combinations included in the invention can be found elsewhere in this specification.

Definitions

It is generally believed that “senescent cells” come from a type of cell that normally replicates but, as a result of aging or another event that causes a change in the cell's state, can no longer replicate. They remain metabolically active and typically adopt an aging-associated secretory phenotype (SASP), which includes chemokines, cytokines, extracellular matrix and fibrosis-modifying proteins, and enzymes. The nucleus of senescent cells is often characterized by aging-associated patches of heterochromatin and DNA segments with chromatin changes that enhance aging. Without imposing any limitations on the practice of what is claimed in the present invention that is not expressly stated or required, the invention is based on the hypothesis that senescent cells cause or mediate certain diseases associated with tissue damage or aging. For purposes of practicing aspects of the present invention, senescent cells can be identified as expressing at least one of the markers p16, senescence-associated β-galactosidase and lipofuscin; sometimes two or more of these markers and other SASP markers, such as, but not limited to, interleukin-6 and inflammatory, angiogenic and extracellular matrix-modifying proteins.

An “aging-related” disease, disorder or condition is a physiological condition that is manifested by one or more symptoms or signs and a subject with the disease requires or would benefit from amelioration of such symptoms or signs. A disease is associated with aging if it is caused or mediated in part by senescent cells, which can be induced by many etiological factors, including age, DNA damage, oxidative stress, genetic defects, etc. Lists of aging-related diseases that can potentially be treated or managed with the methods and products disclosed herein include those discussed in this specification and the prior disclosures to which this application claims priority.

A compound is generally referred to as “senolytic” if it eliminates senescent cells compared to replicating cells of the same tissue type or quiescent cells without SASP markers. On the other hand, or in addition, a compound or combination can be used effectively in accordance with the present invention if it reduces the release of pathological soluble factors or mediators as part of the aging-associated secretory phenotype that play a role in the initial manifestation or ongoing pathology of the disease or inhibit his permission. However, the term “senolytic” is illustrative in the sense that compounds that act primarily by inhibiting rather than eliminating senescent cells (senescent cell inhibitors) can be used in the same manner with all the attendant advantages.

Successful “treatment” of an eye disease according to the present invention may mean any effect beneficial to the subject being treated. This includes reducing the severity, duration or progression of the disease or any adverse signs or symptoms resulting therefrom. In some cases, senolytic agents may also be used to prevent or inhibit the manifestation of a disease to which the subject is susceptible, for example, due to hereditary predisposition or due to medical history.

“Therapeutically effective amount” means that amount of a compound of the present invention that (i) treats a specified disease, condition or disorder, (ii) reduces, improves or eliminates one or more symptoms of a specified disease, condition or disorder, (iii) prevents or delays the occurrence of one or more symptoms of a particular disease, condition or disorder described herein (iv) prevents or slows the progression of the particular disease, condition or disorder, or (v) at least partially reverses the damage caused by the disease prior to treatment.

The terms “eye infection” and “ocular infection” refer to infections caused by a microorganism or microorganisms in or around the eyes or in structures of the eyes that include the eyelids and lacrimal apparatus, conjunctiva, cornea, uveal tract, vitreous, retina and optic nerve. .

The “phosphorylated” form of a compound means one in which one or more -OH or -COOH groups are replaced by a phosphate group, which is -OPO ₃ H ₂ , -OC _n H _2n PO ₃ H ₂ or -OC(=O)C _n H _2n PO ₃ H ₂ (where n is from 1 to 4) so that the phosphate group or groups can be removed in vivo (eg by enzymolysis). The unphosphorylated or dephosphorylated form does not have these phosphate groups.

Unless otherwise indicated or required, all structures of the compounds provided in the invention include conjugate acids and bases with the same structure, crystalline and amorphous forms of these compounds, pharmaceutically acceptable salts and their dissolved and solid forms, including, for example, polymorphs, solvates, hydrates, unsolvated polymorphs (including anhydrous), conformational polymorphs and amorphous forms of compounds, as well as mixtures thereof.

Ophthalmic or eye diseases are defined for the purposes of the present invention as diseases of the visual system. Anterior eye diseases are those diseases, ailments or conditions that affect or involve the front part or area of the eye such as the periocular muscles, eyelid, or ocular tissue or fluid located in front of the posterior wall of the lens capsule or ciliary muscles. Posterior eye diseases are those diseases, ailments or conditions that primarily affect or affect the back part or area of the eye such as the choroid or sclera (behind the plane passing through the posterior wall of the lens capsule), vitreous body, posterior chamber, retina, pigment the retinal epithelium, Bruch's membrane, the optic nerve (i.e., the optic disc), the optic pathway, and the blood vessels and nerves that supply or innervate the back part or region of the eye.

Unless otherwise stated or required, other terms used in the specification have their normal meanings.

Incorporation by reference

For all purposes in the United States and other jurisdictions where applicable, each individual publication and patent document cited in this specification is hereby incorporated herein by reference in its entirety for all purposes to the same extent as if each such publication or document has been specifically and individually stated to be incorporated herein by reference.

US 2016/0339019 A1 (Laberge et al.) and WO 2016127135 (David et al.) are hereby incorporated herein by reference for all purposes, including, without limitation, the identification, formulation and use of compounds capable of eliminating or reducing the activity of senescent cells and treating eye diseases. US 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 of compounds in the Bcl library, their preparation, and use.

EXAMPLES

Example 1 Measurement of Bcl Inhibition

The ability of candidate compounds to inhibit Bcl-2 and Bcl-xL activity can be measured at the molecular level by direct binding. This method uses homogeneous oxygen channeling assay technology sold by PerkinElmer Inc., Waltham, Massachusetts: see Eglin et al., Current Chemical Genomics, 2008, 1, 2-10. The test compound is mixed with the target Bcl protein and a peptide representing the corresponding cognate ligand labeled with biotin. The mixture is then combined with streptavidin-containing luminescent donor beads and luminescent acceptor beads, whose luminescence is proportionally reduced if the compound inhibits the binding of the peptide to the Bcl protein.

Bcl-2, Bcl-xL and Bcl-w were obtained from Sigma-Aldrich Co., St. Louis, Missouri. Biotinylated peptide BIM (ligand for Bcl-2) and peptide BAD (ligand for Bcl-xL) are described in US 2016/0038503 A1. AlphaScreen ^® Streptavidin donor beads with streptavidin and AlphaLISA ^® Anti-6XH acceptor beads were obtained from PerkinElmer.

To perform the assay, a series of dilutions of the compound were prepared in DMSO 1:4 and then diluted 1:100 in assay buffer. In a 96-well PCR plate, the following order was mixed: 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 was incubated in the dark at room temperature for 24 hours. The next day, the donor and acceptor beads were combined and 5 μl was added to each well. After incubation in the dark for 30 min, luminescence was measured on a reader and the affinity or degree of inhibition for each test compound was determined.

Example 2 Measurement of MDM2 Inhibition

MDM2 (mouse double small-2 homologue, also known as E3 ubiquitin protein ligase) is a negative regulator of the tumor suppressor p53. Inhibition of MDM2 stimulates p53 activity, thereby conferring senolytic activity. The ability of compounds to act as MDM2 agonists in cells can be measured indirectly by monitoring their effect on p53.

A stable p53 luciferase reporter RKO cell line was obtained from Signosis Inc., Santa Clara, CA. In cells with p53-luciferase, luciferase activity is specifically coupled to p53 activity. The cell line was established by transfecting a p53 luciferase reporter vector together with a G418-expressing vector, followed by selection for G418.

The analysis was carried out as follows. The reporter cell line cells were treated for 24 hours with the candidate compound. Then the medium was removed, the cells were washed with PBS, and 20 μl of lysis buffer was added to the wells. The cells were shaken for 10 sec on a plate shaker. Luciferase buffer was prepared and added to the wells. p53 activity was then read on a Victor™ multilabel reader (Perkin‌Elmer, San Jose, CA).

Example 3 Measurement of senolytic activity on senescent fibroblasts

IMR90 human fibroblast cells were obtained from the American Type Culture Collection (ATCC ^® ) under the designation CCL-186. Cells were maintained at <75% confluency in DMEM containing FBS and Pen/Strep in an atmosphere of 3% O ₂ , 10% CO ₂ at ~95% humidity. Cells were divided into three groups: irradiated cells (cultured after irradiation for 14 days before use), proliferating normal cells (cultured at low density for 1 day before use), and quiescent cells (cultured at high density for 4 days before use). .

On day 0, irradiated cells were prepared as follows. IMR90 cells were washed, added to T175 flasks at a density of 50,000 cells per ml and irradiated at 10-15 Gy. After irradiation, cells were seeded in 100 μl volumes into 96-well plates. On days 1, 3, 6, 10, and 13, the media in the wells was aspirated and replaced with fresh media.

On day 10, resting healthy cells were prepared as follows. IMR90 cells were washed, mixed with 3 ml of trypsin-containing TrypLE reagent (Thermo‌Fisher Scientific, Waltham, Massachusetts) and incubated for 5 min until the cells rounded and began to detach from the plate. Cells were dispersed, counted, and adjusted to a concentration of 50,000 cells per ml with medium. 100 μl of cells were seeded into the wells of a 96-well plate. On the 13th day, the medium was changed.

On day 13, a population of proliferating healthy cells was prepared as follows. Healthy IMR90 cells were washed, mixed with 3 ml TrypLE and incubated for 5 min until the cells rounded and began to detach from the plate. Cells were dispersed, counted, and adjusted to a concentration of 25,000 cells per ml with medium. 100 μl of cells were seeded into the wells of a 96-well plate.

On day 14, study Bcl-2 inhibitors or MDM2 inhibitors were mixed with cells as follows. A series of dilutions in DMSO of each test compound was prepared at 200 times the desired final concentration in a 96-well PCR plate. Immediately before use, stock solutions in DMSO were diluted 1:200 with warmed complete medium. The medium was aspirated from the cells in the wells and 100 μl per well of medium containing the compound was added.

For testing, candidate senolytic agents were cultured with cells for 6 days, replacing the culture medium with fresh medium containing the same compound concentration on day 17. Bcl-2 inhibitor type 001967 was cultured with cells for 3 days. The assay system utilizes the properties of thermostable luciferase to provide reaction conditions that produce a stable luminescent signal while inhibiting the endogenous ATPase released upon cell lysis. At the end of culture, 100 μl of CellTiter-Glo ^® reagent (Promega Corp., Madison, Wisconsin) was added to each well. Plates with cells were placed on a circular rocker for 30 sec, and then luminescence was measured.

Example 3A. Screening a library of compounds for Bcl antagonists

The discovery of senolytic agents useful for implementation according to the present invention is based on the fact that senescent cells can be destroyed by inhibiting one or more regulatory proteins from the Bcl family, which are anti-apoptotic. Molecules with high affinity and selectivity for the Bcl isoform are predicted to effectively induce apoptosis in senescent cells, but not in proliferating or nonsenescent cells of the same tissue type. Compounds with such properties would be candidates for development as therapeutic agents for clinical medicine.

First, a library containing several hundred compounds was built. The synthesis and use of such a library are described in US Patents 8,691,184, 9,096,625 and 9,403,856. The library was first screened elsewhere for compounds capable of binding or inhibiting Bcl-xL and/or Bcl-2. During the initial screening, 15 compounds were selected for further analysis. In fig. 1A, 1B and 1C show 9 of the fifteen compounds.

The selected compounds were further analyzed to quantify the actual affinity for Bcl-xL, Bcl-2 and Bcl-w to identify potential senolytic agents for use in the treatment of age-related diseases. In fig. 2A, 2B and 2C show the results of the binding assay, with the compounds on the left side of each graph having the highest affinity.

Individual compounds in the library had similar basic structures. As shown in FIG. 3A, 3B and 3C compared the structures of each of the most promising compounds to identify those substituents in the molecular structure that contribute to the desired effect.

Example 3B. Screening of high-affinity Bcl antagonists for senolytic activity and specificity

The ability to bind Bcl regulatory proteins does not necessarily mean that the compound is suitable for inducing apoptosis in a clinical setting. Moreover, even if a compound is active, it may not be suitable for use as a therapeutic agent unless it preferentially kills senescent cells with a high degree of selectivity. Accordingly, compounds in the library exhibiting high affinity binding to Bcl were further screened for their ability to kill irradiated fibroblasts compared to replicating fibroblasts or quiescent (due to confluence) but not senescent fibroblasts.

In fig. 4A, 4B and 4C present data obtained for nine model compounds: binding affinity for each Bcl isoform and effective concentrations (EC ₅₀ ) to kill senescent fibroblasts (SnCs) in culture. In fig. Figure 6 shows the binding affinity for Bcl isoforms and effective concentrations (EC ₅₀ ) to kill senescent fibroblasts (SnCs) in culture. The data is shown in the table below. 1.

ID Source ID Mol. weight Binding affinity (K _i ± SD) EC ₅₀ for SnCs (µM) S.I. Bcl-xL (pM) Bcl-2 (pM) Bcl-w (pM) UM/APG-901 BM-501 639.1 n/a n/a n/a n/a n/a UM/APG-902 BM-983 967.5 3307 4037 >250000 5,000 1.0 UM/APG-903 BM-782 1274.4 885.55 10923 17694 11,000 1.3 UM/APG-904 BM-788 1148.2 5292 121045 23132.5 n/a n/a UM/APG-905 BM-792 1165.7 64.125 3324 1295.5 1,000 3.3 UM/APG-906 BM-957 1179.7 45.3 3333 659.95 0.300 11.7 UM/APG-907 BM-962 1193.7 57.41 3389.5 538.15 0.200 18.5 UM/APG-908 BM-1075 1113.7 5.7285 178.95 161.45 0.300 15.3 UM/APG-909 BM-1192 1215.3 n/a n/a n/a 0.050 60.0 UM/APG-910 BM-1195 1229.3 148,565 666.55 174.7 0.006 128.7 UM/APG-911 BM-1197 1245.8 295 447.95 291.6 0.005 366.7 UM/APG-912 BM-1244 1273.8 134.52 450.75 356.75 0.300 66.7 UM/APG-913 BM-1261 1409.9 106.89 274.45 239.45 27,000 n/a UM/APG-914 BM-1252 1395.9 81,085 5.85 123.95 6,000 n/a

Data indicate that high binding affinity to any Bcl isoform does not necessarily predict the effectiveness of a senolytic agent. The compounds designated BM-1075, BM-1195, BM-1197, BM-1244, BM-1261, and BM-1252 all had binding affinities (K _i ) for Bcl isoforms in the nanomolar to picomole range. However, when analyzed to determine the effective concentration to kill cells (EC ₅₀ ), some of these molecules such as BM-1244, BM-1261 and BM-125 were 60-5000 times less effective than the compounds ultimately selected for development.

The compounds with the best senolytic activity, BM-1195 and BM-1197, were effective in the nanomolar range. A wide range of specificity for senescent cells (SI) of various compounds was determined, ranging from 1.0 (non-specific) to over 300. The best compound in terms of both potency and specificity was BM-1197, and BM-1195 is also of interest. BM-1244, BM-1105 and BM-1075.

Based on these data, the following conclusions regarding the chemical structure were made. Effective connections were based on this substructure:

and the groups R ₃ , R ₄ and F are optional. In BM-1197 the -SO ₂ CF ₃ group is important, but can be replaced by groups with similar properties such as -NO ₂ in BM-1075. The -SO ₂ R' group is also important, although R' can range from -CH ₃ to other short-chain alkyl groups. The aryl-SC ₆ H ₅ group is also important, although it could potentially have neutral substituents. Regarding the next part of the structure:

then R ₁ may represent some short-chain alkyl groups, and X ₁ may vary (Cl in BM-1197; F in BM-1195). Next part of BM-1197:

,

has proven to leave some freedom as far as shape is concerned, so that there are a number of alternative substructures effective for many of the purposes of the present invention.

In fig. 5A, 5B and 5C provide a comparison of the structures of effective compounds to determine which substructures contribute to the desired properties of the compounds. These and other considerations provide a blueprint for the general structure previously presented as Formula VI and Formula VII.

Example 4 Effect of senolytic agents on aging retinal vascular cells

The ability of the candidate substances to eliminate senescent cells or senescent endothelial cell-like retinal cells was measured directly by the following method.

Human retinal microvascular endothelial cells (HRMEC) were obtained from Neuromics under the designation HEC09. Cells were first maintained and expanded to <75% confluency in ENDO growth medium (Neuromics, Edina, MN) at 3% O ₂ , 10% CO ₂ and ~95% humidity. Cells were divided into three groups: irradiated cells (cultured after irradiation for 7 days before use), proliferating normal cells (cultured at low density for 1 day before use), and quiescent cells (cultured to confluence for more than 4 days).

On day 0, irradiated cells were prepared as follows. HRMEC cells were coated with 3 ml of trypsin-containing TrypLE reagent (ThermoFisher Scientific, Waltham, Massachusetts) and incubated for 5 min until the cells rounded and began to detach from the plate. Cells were dispersed, counted, and adjusted to a concentration of 100,000 cells per ml with medium. This cell suspension was added to T175 flasks at a density of 100,000 cells per ml and irradiated at 10-15 Gy. After irradiation, cells were seeded in 100 μl volumes into 96-well plates. On days 1, 3, 6, the medium in the wells was aspirated and replaced with fresh medium.

On day 3, resting healthy cells were prepared as follows. Healthy, nonsenescent HRMEC cells were released from the culture flask by incubation with 3 ml of trypsin-containing TrypLE reagent (ThermoFisher Scientific, Waltham, Massachusetts) and incubated for 5 min until the cells rounded and began to detach from the plate. Cells were dispersed, counted, and adjusted to a concentration of 80,000 cells per ml with medium. 100 μl of cells were seeded into the wells of a 96-well plate. On days 1, 3, 6 and 10 the medium was changed.

On day 6, a population of proliferating healthy cells was prepared as follows. Healthy, nonsenescent HRMEC cells were washed, overlaid with 3 ml TrypLE and incubated for 5 min until the cells rounded and began to detach from the plate. Cells were dispersed, counted, and adjusted to a concentration of 40,000 cells per ml with medium. 100 μl of cells were seeded into the wells of a 96-well plate.

On day 7, candidate senolytic agents were mixed with the cells as follows. A series of dilutions in DMSO of each test compound was prepared at 200 times the desired final concentration in a 96-well PCR plate. Immediately before use, stock solutions in DMSO were diluted 1:200 with warmed complete medium. The medium was aspirated from the cells in the wells and 100 μl per well of medium containing the compound was added.

Candidate senolytic agents were cultured with cells for 3 days. The assay system utilizes the properties of thermostable luciferase to provide reaction conditions that produce a stable luminescent signal while inhibiting the endogenous ATPase released upon cell lysis. At the end of culture, 100 μl of CellTiter-Glo ^® reagent (Promega Corp., Madison, Wisconsin) was added to each well. Plates with cells were placed on a circular rocker for 30 sec, and then left at room temperature for 10 minutes and luminescence was measured. Luminescence values were normalized to determine % cell survival/growth and plotted against test compound concentration, and potency (IC ₅₀ values) was determined by constructing nonlinear curves in GraphPad Prism ^® .

The results are presented in Fig. 7. A concentration-response curve for a representative test compound represents the survival sensitivity of senescent HRMEC cells to incubation with a senolytic molecule. These data indicate that senolytic agents are capable of eliminating senescent HRMEC cells in culture.

Example 5. Effect of senolytic agents on senescent eye cells

The ability of the candidate substances to eliminate senescent cells or senescent cell type cells was directly measured by the following method.

Human retinal pigment epithelial (RPE) cells were obtained from Lonza as 194987. Cells were first maintained and expanded to <75% confluency in RPE medium defined in Sonoda et al., 2009, with 5% FBS and Pen/Strep in an atmosphere with 3% O ₂ , 10% CO ₂ and ~95% humidity. Cells were divided into three groups: irradiated cells (cultured after irradiation for 12 days before use), proliferating normal cells (cultured at low density for 2 days before use), and quiescent cells (cultured to confluence for more than 12 days).

On day 0, irradiated cells were prepared as follows. RPE cells were coated with 3 ml of trypsin-containing TrypLE reagent (ThermoFisher Scientific, Waltham, Massachusetts) and incubated for 5 min until the cells rounded and began to detach from the plate. Cells were dispersed, counted, and adjusted to a concentration of 100,000 cells per ml with medium. This cell suspension was added to T175 flasks at a density of 100,000 cells per ml and irradiated at 10-15 Gy. After irradiation, cells were seeded in 100 μl volumes into 96-well plates. On days 1, 3, 6, and 10, the media in the wells was aspirated and replaced with fresh media.

On day 0, resting healthy cells were prepared as follows. Healthy, nonsenescent RPE cells were released from the culture flask using trypsin, coated with 3 ml of trypsin-containing TrypLE reagent (ThermoFisher Scientific, Waltham, Massachusetts), and incubated for 5 min until the cells rounded and began to detach from the plate. Cells were dispersed, counted, and adjusted to a concentration of 100,000 cells per ml with medium. 100 μl of cells were seeded into the wells of a 96-well plate. On days 1, 3, 6 and 10 the medium was changed.

On day 10, a population of proliferating healthy cells was prepared as follows. Healthy, nonsenescent RPE cells were washed, overlaid with 3 ml TrypLE and incubated for 5 min until the cells rounded and began to detach from the plate. Cells were dispersed, counted, and adjusted to a concentration of 30,000 cells per ml with medium. 100 μl of cells were seeded into the wells of a 96-well plate.

On day 12, candidate senolytic agents were mixed with the cells as follows. A series of dilutions in DMSO of each test compound was prepared at 200 times the desired final concentration in a 96-well PCR plate. Immediately before use, stock solutions in DMSO were diluted 1:200 with warmed complete medium. The medium was aspirated from the cells in the wells and 100 μl per well of medium containing the compound was added.

Candidate senolytic agents were cultured with cells for 3 days. The assay system utilizes the properties of thermostable luciferase to provide reaction conditions that produce a stable luminescent signal while inhibiting the endogenous ATPase released upon cell lysis. At the end of culture, the plates were removed from the incubator and equilibrated at room temperature for 20 min, and then 100 μl of CellTiter-Glo ^® reagent (Promega Corp., Madison, Wisconsin) was added to each well. Plates with cells were placed on a circular rocker for 30 sec, and then left at room temperature for 10 minutes and luminescence was measured. Luminescence values were normalized to determine % cell survival/growth and plotted against test compound concentration, and potency (IC ₅₀ values) was determined by constructing nonlinear curves in GraphPad Prism ^® .

The results are presented in Fig. 8. A concentration-response curve for a typical test compound represents the sensitivity of the survival of senescent RPE cells to incubation with a senolytic molecule. In contrast, IC ₅₀ values for proliferating, non-senescent cells were RPE higher than for senescent cells. These data indicate that senolytic agents are capable of eliminating senescent RPE cells in culture.

Example 6. Efficacy of compounds in an animal model of ischemic retinopathy

The efficacy of the model compound UBX1967 was studied in a mouse model of oxygen-induced retinopathy (OIR), which is an in vivo model of retinopathy of prematurity (ROP) and diabetic retinopathy.

C57BL/6 mouse pups and their CD1 foster mothers were exposed to a high oxygen environment (75% O ₂ ) from postnatal day 7 (P7) to P12. On day P12, animals were injected intravitreally with 1 μl of the test compound (200, 20 or 2 μM) in a composition of 1% DMSO, 10% Tween-80, 20% PEG-400 and were not returned to room air until day P17. Eyes were enucleated at P17 and retinas were dissected for vascular staining or qRT-PCR. To identify avascular or neovascular areas, the retina was flattened and stained with isolectin B4 (IB4) at a dilution of 1:100 in 1 mM CaCl ₂ . qPCR was performed to quantify senescence markers (eg, Cdkn2a, Cdkn1a, Il6, Vegfa). RNA was isolated and cDNA was obtained by reverse transcription, which was used for qRT-PCR of selected transcripts.

From fig. 9A and 9B show that intravitreal (IVT) administration of UBX1967 resulted in statistically significant improvements in neovascularization and vascular occlusion at all dose levels.

In fig. 10A and 10B show the relative abundance of several transcripts associated with aging (p16, pai1) and human disease (vegf). Treatment with UBX1967 reduced p16, pai1, and vegf by 58, 35, and 24%, respectively. Aging-associated β-galactosidase (SA-βGal) activity was reduced by 17% following UBX1967 administration.

These results indicate that a single ocular injection of UBX1967 can functionally inhibit pathogenic angiogenesis and promote vascular repair in this key OIR disease model. We believe that the effectiveness of UBX1967 in the OIR model is due to the elimination of senescent cells and their associated SASPs, which enhance senescence in retinal cells and promote neovascularization of retinal vessels.

Example 7. Efficacy of compounds in an animal model of diabetic retinopathy

The efficacy of UBX1967 was studied in a mouse model of diabetic retinopathy following a single administration of streptozotocin (STZ).

6-7 week old C57BL/6J mice were weighed and baseline blood glucose levels were measured (Accu-Chek, Roche). Mice were intraperitoneally injected with STZ (Sigma-Aldrich, St. Louis, MO) for 5 consecutive days at 55 mg/kg. Age-matched controls received buffer only. Blood glucose levels were measured again one week after the last STZ injection, and mice were considered diabetic if the nonfasting glucose level was above 17 mM (300 mg/dL). STZ-treated diabetic C57BL/6J mice were injected intravitreally with 1 μl of UBX1967 (2 μM or 20 μM 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. Evans Blue retinal penetration assay was performed 10 weeks after STZ treatment.

In fig. 11A and 11B show preliminary results for this technique. Vascular leakage in the retina and choroid after intravitreal (IVT) administration of UBX1967 was improved by vascular permeability at both dose levels.

Example 8. Efficacy of compounds in an animal model of glaucoma

Administration of bleomycin, a DNA-damaging substance, into the anterior chamber of the eyes of mice or rabbits leads to cellular senescence, which is determined by the induction of the p16 transcript in the trabecular meshwork.

To induce a senescent phenotype in the trabecular meshwork in vivo, C57BL/6 mice (8–10 weeks old) were intracamerally injected with 2 μl of 0.0075 U bleomycin sulfate. White New Zealand rabbits were intracamerally injected with 30 μl of bleomycin sulfate, 0.0075 units. 14 days after bleomycin injury, eyes were enucleated and TM-enriched samples were microdissected. To determine changes in senescent cells, RNA was isolated from TM and qPCR analysis was performed to assess the effect of bleomycin on p16 mRNA levels.

In fig. 12A and 12B show an increase in the relative expression of p16 14 days after intracameral (IC) administration of bleomycin in the right (OD) eye compared to the PBS-treated left (OS) eye of the study animals. This model can also be used to evaluate whether a test compound will be pharmacologically capable of reducing or attenuating elevated intraocular pressure, which is a feature of primary open-angle glaucoma (POAG).

Example 9: Senescent Cells in the Trabecular Meshwork of Tissue Samples from Individuals with POAG

To obtain evidence of the presence of senescent cells in patients with primary open-angle glaucoma (POAG), tissue samples obtained from affected patients were stained for p16, a marker of senescent cells.

Donor eyeballs were obtained prospectively from eye banks and immediately placed in Davidson's reagent for fixation for at least 48 hours. After fixation, the solution was replaced with 70% ethanol. The tissue was then placed into histology cassettes and processed for infiltration and paraffin embedding. Paraffin blocks were mounted on a microtome and sections with a thickness of 5-9 μm were made. Tissue sections were placed in a 45°C water bath and sections were mounted on Superfrost Plus™ microscope slides, removing excess water with a Kimwipe™. The slides were then placed upright for 30 min to dry at room temperature, followed by incubation overnight in an incubator at 37°C. The glasses were baked for 15–20 min at 60°C and then left to cool to room temperature.

Slides were deparaffinized by incubation in BOND dewaxing solution at 70°C for 30 sec. The slides were then rehydrated through a series of incubations at decreasing ethanol concentrations as follows: twice in 100% ethanol for 5 min, twice in 90% ethanol for 2 min, twice in 75% ethanol for 2 min, twice in 50% ethanol for 2 min. 2 minutes and then washed with water for 4 minutes. The slides were then washed twice with Tris-buffered saline supplemented with 0.1% Triton-X100 (TBST). Antigen retrieval was performed by incubation in BOND ER1 solution (sodium citrate buffer, pH 6.0) at 95°C for 20 minutes. Slides were incubated in 3% H ₂ O ₂ for 10 min at room temperature and then washed twice in TBST.

Slides were incubated for 30 min at room temperature with anti-p16 primary antibody (Roche, #9517) diluted 1:2 in TBST. After incubation with the primary antibody, the slides were washed twice with TBST before incubation with the secondary rabbit anti-mouse antibody for 20 min at room temperature. The slides were washed twice with TBST and incubated for 20 min at room temperature with HRP-conjugated secondary antibody. After incubation with antibodies, slides were washed twice with TBST, and AEC chromogen prepared according to the manufacturer's procedure was used for detection and color was developed for approximately 20 min. The slides were then washed three times with water and then incubated in Harris hematoxylin for 20 sec. The slides were thoroughly rinsed with water, coverslipped with Vectashield™ mounting medium (Vector Labs, Burlingame, CA), and allowed to dry until images were obtained.

In fig. 13A and 13B are representative images of p16 immunohistochemistry on human ocular tissue obtained from donors diagnosed with POAG. Cells positive for p16 expression appear with dark cell staining in and around the trabecular meshwork (circled).

Example 10: Senescent cells in tissue from people with age-related macular degeneration (AMD)

To obtain evidence for the presence of senescent cells in AMD, tissue samples from affected patients were stained for p16.

Donor eyeballs were obtained prospectively from eye banks and immediately placed in Davidson's reagent for fixation for at least 48 hours. After fixation, the solution was replaced with 70% ethanol. The tissue was then placed into histology cassettes and processed for infiltration and paraffin embedding. Paraffin blocks were mounted on a microtome and sections with a thickness of 5-9 μm were made. Tissue sections were placed in a 45°C water bath and sections were mounted on Superfrost Plus™ microscope slides, removing excess water with a Kimwipe. The slides were then placed upright for 30 min to dry at room temperature, followed by incubation overnight in an incubator at 37°C. The glasses were baked for 15–20 min at 60°C and then allowed to cool to room temperature. Slides were deparaffinized with xylene or a xylene substitute (eg, Histoclear™) for 4 min, repeating a total of 3 incubations. The slides were then rehydrated through a series of incubations at decreasing ethanol concentrations as follows: twice in 100% ethanol for 5 min, twice in 90% ethanol for 2 min, twice in 75% ethanol for 2 min, twice in 50% ethanol for 2 min. 2 minutes and then washed with water for 4 minutes.

Antigen retrieval was carried out by incubation in acidic sodium citrate buffer at 120°C for 3 minutes (or at 95°C for 20 minutes) in a steamer or pressure cooker. The slides were cooled to room temperature for 15 min and then washed twice with Tris-buffered saline supplemented with 0.1% Triton-X100 (TBST) for 2 min each. The slides were washed twice with TBST for 5 min each and then blocked for 1 h at room temperature in TBST containing 5% normal serum from the species from which the secondary antibody was derived. After blocking, the glasses were dried and the excess solution was wiped off. The slides were incubated overnight at 4°C (or for 2 h at room temperature) with the primary anti-p16 antibody diluted 1:2 in TBST. After incubation with the primary antibody, the slides were washed twice with TBST for 5 min each and then treated with 3% H ₂ O ₂ in TBST for 15 min. The slides were then washed twice with TBST for 5 min each before incubation with the secondary antibody. The slides were incubated for 1 hour at room temperature with HRP-conjugated secondary antibody. After incubation with antibodies, the slides were washed three times with TBST for 5 min each. For detection, AEC chromogen prepared according to the manufacturer's procedure was used and color was developed for approximately 20 min. Then the slides were washed with water for 5 minutes, after which counterstaining with hematoxylin was performed. The slides were covered with coverslips over Vectashield mounting medium (Vector Labs, Burlingame, CA) and allowed to dry until images were taken.

In fig. 14A and 14B are representative images of p16 immunohistochemistry on human ocular tissue obtained from donors diagnosed with AMD. Cells positive for p16 expression appear darkly colored in areas of apparent disease pathology (circled).

Example 11: Clinical Assessment

This case study presents a design for a preclinical or clinical trial to evaluate the safety and efficacy of senolytic agents for the treatment of ocular diseases.

Test subjects are administered the senolytic drug using a standard intravitreal (ITV) technique, with the eyes washed and draped in the usual sterile manner after IOP measurement before injection. Local anesthesia is applied and an eyelid speculum is placed to ensure adequate treatment. The attending physician selects a quadrant for injection and measures the injection site at a distance of 3-4 mm behind the corneoscleral limbus. A 28-32 gauge needle is used to inject 0.05 to 0.1 ml of the compound. Once the injection procedure is complete, the eyelid expander is removed. Depending on the nature of the disease, potentially suitable intra- or periocular routes of administration include intravitreal, intracameral, posterior juxtascleral, subconjunctival, or suprachoroidal injections.

Following treatment, subjects are assessed to determine whether symptoms or signs of the ocular disease improve compared to subjects in the control group using publicly available tests of ocular structure and function (see above).

Example 12 Combination therapy

This example illustrates the clinical use of a senolytic agent in combination with standard anti-VEGF therapy for the treatment of VEGF-associated ocular disease.

The study is enrolling subjects with wet AMD who are already receiving anti-VEGF therapy or are about to begin anti-VEGF therapy. The first administration of a senolytic compound is given on the same day as one of the regular administrations of the anti-VEGF drug. No contraindications to the use of ophthalmic therapy in combination with a senolytic compound are expected. First, the senolytic agent is administered via standard intravitreal (ITV) administration. Intraocular pressure (IOP) is measured 15 and 30 minutes after administration. Once the IOP has normalized to pre-injection levels, an anti-VEGF drug (eg, aflibercept 2.0 mg in 0.05 ml) is administered. The eye is washed and draped in the usual sterile manner, as this is a completely different sterile procedure than the first injection. Local anesthesia is administered and an eyelid speculum is placed for adequate treatment. The attending physician selects a quadrant for injection and measures the injection site at a distance of 3-4 mm behind the corneoscleral limbus. To administer 0.05 ml of the compound, a 28-32 gauge needle is used. Once the injection procedure is complete, the eyelid expander is removed. IOP is measured 5, 15 and 30 minutes after the second ITV injection.

The safety and effectiveness of combination therapy is assessed as follows. Immediately after the injection, examine the eye for any acute vision loss or increased intraocular pressure. 30 minutes after the injection, if vision and IOP are stable, the patient is discharged from the clinic with standard recommendations from the attending physician after the procedure. The patient is advised to contact the patient immediately if any changes in vision or pain or redness occur in the injected eye.

For this particular disease, wet AMD, follow-up is scheduled ranging from 1 week to 1 month from treatment. Follow-up includes assessment of visual acuity and IOP, monitoring for any improvement or deterioration after therapy. Examination of the anterior and posterior segments of the eye and standard ancillary testing are performed, including, but not limited to, photography, intravenous fluorescein angiography, and optical coherence tomography (OCT).

Response to senolytic is documented using this combination of clinical examination, functional and structural testing described above. Based on the patient's response, further observation is planned. If a senolytic was administered in combination with an anti-VEGF agent, the physician continues to follow the appointment and treatment schedule associated with that agent.

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Several hypotheses presented in this specification provide background by which the reader can understand the invention. These premises are intended for the reader's information and understanding. The practice of invention does not require detailed understanding or application of hypotheses. Unless otherwise stated, the features of the hypotheses presented in this description do not limit the use or practice of the claimed invention. For example, except in cases where elimination of senescent cells is clearly required, the compounds of the invention can be used to treat the described diseases regardless of their effect on senescent cells. Although many of the eye diseases described herein occur predominantly in elderly patients, the invention can be practiced on patients of any age with the specified disease, unless expressly stated or required otherwise.

Although the invention has been described with reference to specific examples and illustrations, modifications and equivalents may be made to adapt to a particular context or purpose as a matter of routine development and optimization and within the skill of those of ordinary skill in the art, thereby obtaining the benefits of the invention without departing from the scope of the claims.

Claims (15)

1. A method of inhibiting pathological angiogenesis in the eye of a subject in need thereof, comprising administering a therapeutically effective dosage of a pharmaceutical composition containing a compound selected from the following: its phosphorylated forms and pharmaceutically acceptable salts; And and pharmaceutically acceptable salts thereof. 2. The method according to claim 1, wherein the compound is selected from the following: its phosphorylated forms and pharmaceutically acceptable salts. 3. The method according to claim 1, wherein the compound is selected from the following: and pharmaceutically acceptable salts thereof. 4. The method according to claim 2 or 3, in which the pharmaceutical composition is administered intravitreally. 5. The method of claim 4, wherein the therapeutically effective dosage is administered as a single intravitreal dosage. 6. The method of claim 1, wherein the pathological angiogenesis in the eye of the subject is retinal neovascularization. 7. Method according to any one of paragraphs. 1-6, in which the subject has diabetic retinopathy.

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