Methods for minimizing adverse effects mediated by external influences on cells, tissues, organ systems and organisms using the bioadhesive and steric interactions of copolymers having at least two moieties

Abstract

A method for minimizing adverse effects mediated by external influences on cells, tissues, organ systems, and living organisms using the specific bioadhesive and steric interactions of a copolymer having at least two moieties. The copolymer exerts bioadhesion through electrostatic and hydrophobic interactions and inactivation by hydrophilic moieties. The copolymer is useful for reducing viral infection rates in target cells and reducing host morbidity. The copolymer is useful for reducing toxicity associated with ADCs, including corneal epithelial toxicity. Formulations of the copolymer are safe and well tolerated. Treatment of epithelial cells and surfaces, including progenitor or stem cell corneal epithelial cells, with the copolymer provides utility and benefits.

Description

[Technical Field]

[0001] Priority This application claims the benefit of U.S. Provisional Application No. 63 / 021,277, filed May 7, 2020, the entire contents of which are incorporated herein by reference.

[0002] FIELD OF THE INVENTION The present invention provides a method for addressing significant problems currently facing the biomedical field by utilizing bioadhesive and inactivated copolymers (containing charged or hydrophobic moieties and inactivated hydrophilic moieties) to improve the health of cells, tissues, organs, and mammals. Specifically, the field of the present invention relates to the prevention, attenuation, reduction, or treatment of viral infections. And specifically, the field of the present invention relates to the prevention, attenuation, reduction, or treatment of drug-related toxicity associated with antibody-drug conjugates (ADCs) and their toxic payloads. "ADC" is frequently used herein to refer to antibody-drug conjugate therapeutics. ADCs are composite molecules composed of an antibody bound to a biologically active cytotoxic (anticancer) payload or drug. Antibody-drug conjugates can be a type of bioconjugate and immunoconjugate. "ADC" can refer to a host of antibody-drug conjugates herein. ADCs combine the inherent targeting capabilities of monoclonal antibodies with the cancer-fighting capabilities of cytotoxic drugs. ADCs are often designed to distinguish between healthy and diseased cells and tissues.

[0003] The viral infection of particular interest here is the infectivity of SARS-CoV-2. However, a key advance is that this effect also works against novel viruses, where steric and electrostatic interactions can inhibit their infectivity in cells, tissues, or organisms where host immunity has not yet developed or where targeted antibodies or antiviral therapies are not yet available. In other words, the effect is broad, effective, and nonspecific. Novel viruses are well suited to treatment with this approach.

[0004] The method for improving ADC toxicity is particularly related to the off-target entry of ADCs into non-neoplastic cells and adverse events associated with the inhibition of cellular processes mediated by the ADC payload. Neoplastic cells are cancer cells. ADCs are used to treat cancer or neoplastic diseases of one or more organ systems or cell types, including, but not limited to, renal cell carcinoma, leukemia, lymphoma, myeloma, lung cancer, prostate cancer, uterine or cervical cancer, breast cancer, bladder cancer, colon cancer, esophageal cancer, liver cancer, Hodgkin's disease, ovarian cancer, pancreatic cancer, rectal cancer, skin cancer, small intestine cancer, solid tumors, gastric cancer, white blood cell cancer, urethral cancer, and mesenteric lymphadenopathy. Any or all of the cancers named herein and those not named herein may be combined with specific inventive claims of ADCs targeting such cancer cells or tissues.

[0005] Specifically, macropinocytosis-mediated toxicity is inhibited, reduced, and limited by steric and electrostatic interferences that operate at the molecular-cell-tissue (global or isolated) level, where the surface of cells or tissues interacts with their local microenvironment, including, but not limited to, limbal stem cells, transient amplifying cells, transient amplifying cell daughter cells, basal epithelial cells, wing cells, and corneal epithelial cells and differentiated corneal epithelial cells.

[0006] The primary underlying polymer structure that is most useful in both settings (but not the only useful embodiment) is a cationic graft copolymer. This basic structure comprises a cationic backbone and grafted hydrophilic side chains. A representative example of such a polymer is poly(L)lysine grafted (poly)ethylene glycol. Other molecular structures also achieve the interactions necessary to confer benefits. Other polymers can use charge, hydrophobic sites, and inactive sites to achieve these effects, and such polymers are addressed herein. The treatment method involves applying the aforementioned polymers (whether in solution or not) to cells, tissues, organs, living organisms, or mammals in amounts and for periods effective to achieve the intended beneficial effect.

[0007] Therefore, there is a need to reduce the severity and risk associated with novel viral diseases spreading through human populations and affecting human health.

[0008] Furthermore, there is a need to reduce the negative effects associated with antibody-drug conjugates, particularly corneal epithelial toxicity.

[0009] Background of the Invention Cationic graft copolymers have been demonstrated to be useful in vitro for coating nonbiological surfaces, medical device coatings, and the treatment of dry eye. One example of an effective cationic graft copolymer is poly(L-lysine)-grafted poly(ethylene glycol) (PLL-g-PEG). PLL-g-PEG is a water-soluble copolymer consisting of a poly(L-lysine) backbone and poly(ethylene glycol) side chains (Sawhney et al. Biomaterials 1992 13:863-870). The PLL chains carry multiple positive charges and spontaneously adsorb to negatively charged surfaces, while the PEG is a hydrophilic polymer that functions as a non-binding domain. The PEG moieties passivate surfaces, while the PLL moieties attach to charged components of cell membranes, antibodies, viruses, or viral proteins through electrostatic interactions. PLL-g-PEG has been used in in vitro surface passivation, experimental coatings for medical devices, and as a dry eye drop for ocular lubrication and tear film stabilization. Going far beyond the treatment of dry eye, this invention is the first identified approach to directly ameliorate ADC corneal toxicity from tubulin inhibitors, among other cytotoxic payloads.

[0010] The role of cationic graft copolymers, particularly PLL-g-PEG, in inhibiting viral infectivity and / or ADC-associated toxicity has not been considered, studied, or implemented prior to the present invention. Similarly, to the applicant's knowledge, no similarly effective polymer molecules have been studied or implemented in these regards prior to the present invention. There are multiple configurations of cationic graft copolymers that are effective in inhibiting viral infectivity and / or ADC-associated toxicity. These alternative molecular approaches are discussed and addressed herein.

[0011] Summary of the Invention One aspect of the present invention relates to methods for preventing or reducing viral infectivity and viral load exposure, thereby reducing morbidity and mortality from viral infections, particularly novel viruses, such as SARS-CoV-2. The charged graft copolymers are safe and effective when applied to at-risk cells and tissues to prevent or reduce infectivity.

[0012] One aspect of the present invention relates to methods for preventing or reducing ADC-associated drug toxicity to off-target cells and tissues. In particular, the present invention relates to a method for reducing the severity of corneal epithelial cell toxicity in the setting of ADC therapy. Charged graft copolymers are safe and effective when applied to at-risk cells and tissues to prevent or reduce ADC-associated corneal toxicity. Exposure of human corneal epithelial cells, including basal epithelial cells and their precursors, wing cells, and superficial epithelial cells, to the cytotoxic payload carried by ADCs is reduced by treatment with cationic graft copolymers. Exposure of epithelial cells to an effective amount of the graft copolymer in a dissolved state is effective in reducing the severity of corneal epithelial toxicity. Reductions in severity can be manifested by laboratory findings such as a decrease in microcystic epithelial keratopathy, a decrease in the severity of punctate superficial staining, a decrease in the incidence of epithelial abnormalities, a decrease in ocular adverse events, a decrease in the rate of visual acuity loss, and a decrease in complaints such as eye irritation and blurred vision.

[0013] One aspect of the invention disclosed herein is a method for reducing viral infectivity by treating tissue involved in transfection with an effective amount of a graft or block copolymer having either cationic, hydrophobic, or anionic moieties and hydrophilic inactivation moieties. In one embodiment of this method, the copolymer is PLL-g-PEG. In another embodiment of this method, the graft copolymer of the formulation comprises a cationic backbone and water-soluble, non-ionic side chains. In another embodiment of this method, the block copolymer of the formulation comprises at least one cationic block and at least one water-soluble, non-ionic block. In another embodiment of this method, the block copolymer of the formulation comprises at least one block that is hydrophobic and at least one block that is water-soluble and anionic, cationic, or non-ionic. In any of the above embodiments, the biological surface to which the copolymer formulation is administered is a mucosa selected from the group consisting of ocular, oral, nasal, and respiratory tract mucosa, respiratory epithelium, urinary mucosa, and gastrointestinal mucosa of a subject. In any of the above embodiments, the biological surface to which the copolymer formulation is administered is the ocular surface. In any of the above embodiments, the viral infection is selected from coronavirus, influenza virus, Ebola virus, and a novel virus transmitted by mucosal contact, for example, but not limited to, SARS-CoV-2. In any of the above embodiments, the graft copolymer or block copolymer of the formulation comprises 0.001-40% of the formulation. In any of the above embodiments, the graft copolymer or block copolymer of the formulation comprises 0.1-10% of the formulation. In any of the above embodiments, the inactivation effect is based on interference with the SARS-CoV-2 spike protein and the ACE2 receptor on at-risk cells. In any of the above embodiments, the therapeutic effect is general steric inhibition.

[0014] In a second aspect of the present invention, a method for reducing adverse events associated with the use of antibody-drug conjugates is disclosed herein by administering an effective amount of a copolymer having electrostatic and steric mediating properties that are applied to cells affected by the toxicity through off-target uptake pathways that damage non-neoplastic cells. In one embodiment of this aspect, the copolymer is selected from a cationic graft copolymer, a cationic block copolymer, a hydrophobic graft copolymer, a hydrophobic block copolymer, an anionic graft copolymer, and an anionic block copolymer. In one embodiment of this aspect, the copolymer is formulated in one or more of the following approaches: powder, solution, suspension, topical agent, intravenous agent, oral agent, mouthwash, nasal spray, and eye drops. In one embodiment of this aspect, the proportion of the copolymer solution is at least 0.01% by weight. In one embodiment of this aspect, the proportion of the copolymer solution is at most 40% by weight for solutions and suspensions. In one embodiment of this aspect, the copolymer is PLL-g-PEG. In one embodiment of this aspect, the copolymer is selected from the list of combinations described in the present application above.

[0015] In one aspect of the ophthalmic aspect of the present invention, topical eye treatment with an ophthalmic pharmaceutical product and / or commercial formulation comprising the copolymers described herein can be beneficial, with a frequency of once a day (one drop per eye per day), twice a day (two drops per eye per day), three times a day (three drops per eye per day), four times a day (four drops per eye per day), or even hourly or more frequent administration. Administration may be less frequent, even once a day or less per affected eye. Administration may be as needed or ad hoc. The range of administration frequency requirements may depend on the patient and the ADC.

[0016] In another aspect of the present invention, disclosed herein is a method for reducing corneal epithelial toxicity associated with ADCs by administering a copolymer having bioadhesive and inactivating components to cells at risk of off-target drug uptake. In one embodiment of this aspect, the copolymer is applied to corneal epithelial cells and conjunctival epithelial cells. In one embodiment of this aspect, the copolymer is PLL-g-PEG. In one embodiment of this aspect, the formulation is a solution for delivery to the subconjunctival space.

[0017] In another aspect of the present invention, disclosed herein is a method for reducing adverse events associated with the use of antibody-drug conjugates in humans, comprising administering to cells involved in said adverse events an effective amount of a copolymer having electrostatic and steric mediation properties. In one embodiment of this aspect, the copolymer is selected from a cationic graft copolymer, a cationic block copolymer, a hydrophobic graft copolymer, a hydrophobic block copolymer, an anionic graft copolymer, and an anionic block copolymer. In one embodiment of this aspect, the copolymer is selected from the polymers disclosed herein.

[0018] In another aspect of the present invention, disclosed herein is a method for reducing microcyst-like epithelial toxicity associated with cleaved cytotoxins from ADCs by applying to corneal epithelial cells an effective amount of a copolymer having bioadhesive and inactivating properties, the copolymer comprising a graft or block copolymer having either a cationic, hydrophobic, or anionic moiety and a hydrophilic moiety that can function as an inactivating moiety. In one embodiment of this aspect, the copolymer is PLL-g-PEG.

[0019] In another aspect of the invention, disclosed herein are methods for reducing the rate and severity of ocular adverse events associated with the use of ADCs by delivering to the eye, prior to the initiation of systemic ADC therapy, a copolymer having a bioadhesive and a deactivation site, including a graft or block copolymer having either a cationic, hydrophobic, or anionic site and a hydrophilic deactivation site.

[0020] Ocular adverse events include, but are not limited to, corneal epithelial cell death, superficial punctate keratopathy or keratitis, corneal scarring, corneal infection, microcystic keratopathy, corneal cell damage, corneal cell apoptosis, ocular irritation, ocular foreign body sensation, blurred vision, ocular pain, trouble with vision-related tasks, photophobia, keratopathy, and corneal epitheliopathy.

[0021] In another aspect of the present invention, a method for improving the signs and symptoms of ocular adverse events associated with the use of ADCs by delivering a copolymer having bioadhesive and passivation moieties, including a graft or block copolymer having either cationic, hydrophobic, or anionic moieties and a hydrophilic passivation moiety, to the eye after the initiation of systemic ADC therapy is disclosed herein. The signs and symptoms include, but are not limited to, vision-related symptoms, blurred vision, irritation, redness, and ophthalmic examination findings. Eyes treated with the copolymer formulation exhibit improved visual acuity.

[0022] In another aspect of the present invention, a method for reducing superficial punctate keratopathy or keratitis associated with the use of ADC is disclosed herein by delivering a copolymer having bioadhesive and inactivation sites, including a graft or block copolymer having either cationic, hydrophobic, or anionic sites and a hydrophilic inactivation site, to the eye in conjunction with the initiation of systemic ADC therapy. Delivery may be before, simultaneously with, or after the initiation of ADC therapy. This topical eye treatment has similar benefits when used in combination with other corneal toxic agents described herein. In other words, this specification broadly supports the claims of benefits of this copolymer approach, particularly in addressing systemic or topical agents or pharmaceuticals that contribute to known or anticipated corneal toxicity or ocular adverse events associated with ADC.

[0023] A reduced risk of corneal infection is one of the advantages of copolymer-based topical therapy in this setting.

[0024] In another aspect of the invention, disclosed herein are methods for reducing ADC uptake into corneal epithelial cells (whether in culture, experimental models, or in vivo) by exposing the cells to a copolymer having bioadhesive and passivation moieties, including a graft or block copolymer having either cationic, hydrophobic, or anionic moieties and hydrophilic passivation moieties, prior to exposure to the ADC.

[0025] In another aspect of the invention, disclosed herein is a method of reducing ADC uptake into corneal epithelial cells by exposing the cells to a copolymer having bioadhesive and inactivation moieties, including a graft or block copolymer having either cationic, hydrophobic, or anionic moieties and hydrophilic inactivation moieties, after exposure to the ADC.

[0026] In another aspect of the invention, disclosed herein is a method for reducing uptake of an ADC by corneal epithelial cells via macropinocytosis (or more generally, pinocytosis) by exposing the cells to a copolymer having bioadhesive and passivation moieties, including a graft or block copolymer having either cationic, hydrophobic, or anionic moieties and hydrophilic passivation moieties, either before or after exposure to the ADC, using a formulation having an effective ratio of the copolymer on a weight / weight basis.

[0027] In another aspect of the invention, disclosed herein is a method for reducing ocular adverse events associated with ADCs by treating a patient with an effective amount of a copolymer having bioadhesive and passivation sites, including a graft or block copolymer having either cationic, hydrophobic, or anionic sites and hydrophilic passivation sites. Treatment is topical in some embodiments.

[0028] In another aspect of the invention, disclosed herein are methods of using copolymers that exhibit electrostatic and steric interactions at the cellular level, including graft or block copolymers with either cationic, hydrophobic, or anionic moieties and hydrophilic inactivation moieties, to minimize the adverse effects resulting from exposure of those cells to agents including SARS-Cov-2, novel viruses, epidemic viruses, and ADCs with cytotoxic payloads that can lead to human pathology and morbidity.

[0029] In another aspect of the present invention, disclosed herein is a method for reducing ocular toxicity in humans resulting from systemic exposure to an ADC having a tubulin-disrupting agent as a payload by treating the eye with an effective amount of a cationic graft copolymer formulation comprising a graft or block copolymer having either a cationic, hydrophobic, or anionic moiety and a hydrophilic inactivation moiety. In one embodiment of this aspect, the cationic graft copolymer is PLL-g-PEG. In one embodiment of this aspect, the treatment is administered via an ophthalmic formulation. In one embodiment of this aspect, the formulation is preservative-free. In one embodiment of this aspect, the cationic graft copolymer is PLL-g-PEG at a concentration ranging from 0.01% to 5% by weight in the ophthalmic formulation. In one embodiment of this aspect, the low-toxicity preservative is selected from sodium perborate, stabilized oxychloro complexes, disappearing preservatives, and hydrogen peroxide-based preservatives.

[0030] In another aspect of the present invention, disclosed herein is a method for reducing ADC withdrawal and dose reduction in the treatment of human malignancies by applying an effective amount of a PLL-g-PEG eye drop formulation to the eye in at-risk patients to reduce corneal adverse events and alleviate ocular safety concerns. In another embodiment of the present invention, ocular toxicity, when the toxicity is due to secondary effects of cytotoxic agents on tear secretion and the corneal epithelium, can be reduced by topical treatment with a copolymer containing a graft or block copolymer having either a cationic, hydrophobic, or anionic moiety and a hydrophilic inactivation moiety. Another aspect is the use of the methods described herein to reduce the risk of poor outcomes or poor progress due to corneal drug-related toxicity, selected from the following effects: reduced incidence of epithelial erosion, reduced incidence of corneal ulcers, reduced incidence of keratoepitheliopathy, reduced incidence of punctate epitheliopathy, reduced incidence of superficial corneal changes, reduced incidence of corneal stromal inflammation or other changes, and reduced incidence of secondary bacterial infections.

[0031] As used herein, "adversely affected" can mean a change from normal physiology and function of a cell, tissue, organ, or organism, including, but not limited to, a decrease in cell viability, initiation of apoptosis, a decrease in the proliferative capacity of normally dividing cells, decreased and inappropriate cell adhesion and migration, changes in adhesive and junctional connections, including tight junctions, an inflammatory response to a cell or tissue, changes in cellular metabolism and catabolism, bystander injury to cells, changes resulting in pain or discomfort, or changes in the visual or other function of an organ, tissue, or organism, changes resulting in tear film irregularities, changes resulting in the uptake of fluorescein dye or other vital dyes used to assess epithelial cells (such as Lissamine Green or Rose Bengal), eye damage, and changes affecting the health of normal cells. Adverse events include, but are not limited to, medical problems occurring during treatment with a drug, pharmaceutical, or other therapy, cell health problems, tissue health problems, organ health problems, or organism health problems. As used herein, the terms drug and pharmaceutical may be used interchangeably.

[0032] In this specification, inactivation sites can be, and are typically considered to be, hydrophilic sites, although alternative sites are possible inactivation sites where hydrophilicity is not predominant.

[0033] In another embodiment of the invention disclosed herein, an antibody is designed against an ADC with corneal toxicity, wherein the antibody further comprises an inactivation site and is delivered to the eye in an amount effective to reduce ocular adverse events such that the antibody with the inactivation site specifically and directly interacts with a molecular component of the ADC that interferes with the activity of the ADC and / or binding of the ADC to off-target cells, including corneal cells.

[0034] Another embodiment of the present invention is a method for nonspecifically inhibiting corneal toxicity due to the adverse effects of a drug, wherein the inhibition is mediated by nonspecific (electrostatic or hydrophobic) interactions between a cytotoxic drug and a copolymer described herein, including graft or block copolymers having either cationic, hydrophobic, or anionic moieties and hydrophilic inactivation moieties, thereby reducing the adverse effects of drugs with toxic effects on the cornea through the effect of the inactivation moieties. In some embodiments, the drug associated with corneal adverse events is an ADC, while in other embodiments, the drug or drug is a small molecule. Drugs can be biological agents, antibodies, and antibody-drug conjugates, polymers, or small molecules. Small molecule drugs are any organic compounds that affect biological processes with a relatively low molecular weight of 900 daltons or less. Polymer drugs are organic compounds that affect biological processes with a relatively low molecular weight of 900 daltons or more. Polymers can be organic compounds, PEGylated compounds, proteins, biological agents, or peptides. Peptides can be small or polymeric, depending on the molecular weight of the molecule. A biological substance or biological agent in the pharmaceutical setting herein is a product produced from or containing components of a living organism.

[0035] In some embodiments, the drug is selected from the group consisting of cationic amphiphilic drugs, amiodarone, aminoquilon, chloroquine, hydroxychloroquine (Plaquenil), amodiaquine, mepacrine, tafenoquin, thorazine, tamoxifen, NSAIDs, ibuprofen, indomethacin, naproxen, benoquin, atovaquone, suramin, tilorone, perhexiline maleate, gentamicin, tobramycin, clarithromycin, ciprofloxacin, clofazimine, gold salts, vandetanib, and osimertinib, and the small molecule can be selected from, but is not limited to, kinase inhibitors, erlotinib, and cytarabine. This can reduce or prevent keratopathy verticillata, corneal deposits, epitheliopathy, keratomileusis, corneal lines, Hudson-Staley lines, crystalline deposits, stromal inflammation, and corneal opacity. For pharmaceutical agents associated with limbal stem cell dysfunction toxicity, limbal stem cell dysfunction can be prevented. Pannus, corneal neovascularization, and conjunctival epithelialization of the cornea can be reduced or prevented. Without being bound by any particular method, the copolymer inactivates cytotoxic agents found in tears and / or reaching limbal stem cells from the limbal circulation.

[0036] Another aspect is a method of reducing adverse events associated with the corneal toxicity of any drug by treating at-risk cells with the copolymer or a solution applied to those cells.

[0037] In another aspect, a method for reducing adverse events associated with the use of antibody drug conjugates by applying an effective amount of copolymer to a solution that is subjected to non-neoplastic cells in the setting of off-target uptake pathways that damage non-neoplastic cells (or in the absence of such off-target pathways and where toxicity is more direct, either by targeted uptake, similarity of cellular receptors to the ADC binding site, or warhead entry into at-risk cells).

[0038] The fundamental methodology for reducing adverse events in cells, including corneal cells, associated with exposure to antibody-drug conjugates is to limit uptake, including uptake of ADCs by macropinocytosis, by prolonged or acute exposure of ocular and / or corneal cells, including progenitor epithelial cells, transiently amplifying cells, basal epithelial cells, and differentiated epithelial cells, and stem cells (and daughter cells), to (or onto) cationic graft copolymers in solution or suspension, via superficial, topical, or (in certain cases) systemic approaches. PLL-g-PEG is particularly safe and effective in this setting. Other similar approaches are also contemplated.

[0039] The copolymers of the present invention have bioadhesive moieties (cationic, anionic, hydrophobic) as described above, and inert moieties (hydrophilic, sometimes chemically inert, where "chemically inert" means incapable of significantly reacting with cells or proteins, either covalently, electrostatically, or via hydrophobicity), thereby reducing interactions between viruses and ADCs and cells damaged by infection or toxicity. Each moiety can have a variety of MW sizes, and combinations of polymers can be used. Formulations with multiple copolymer structures can be used. The degree of polydispersity between each polymer is known and expected in manufacturing, and does not reduce efficacy.

[0040] Patent documents 9,884,074; 9,295,693; 9,283,248; 9,005,596; 8,802,075 discuss graft copolymers and the use of such multifunctional copolymers, and are incorporated herein by reference. U.S. Patent Application Publication No. 2004 / 0181172 has been considered, which discusses tear collection for tear analysis.

[0041] The method steps of the present invention involve applying a copolymer having bioadhesive and inactivation moieties to cells and tissues at risk.

[0042] A preferred mode of the invention is by applying PLL-g-PEG to cells and tissues at risk, preferably before exposure, although its use after exposure confers benefits in reducing the severity of adverse findings (which can reduce secondary epithelial cell damage or repeated exposure or infection or reinfection) and the extent of adverse events (minimizing continued uptake by the ADC).

[0043] Without being bound by the specific mechanism of action of an ADC, on-target toxicity includes antibody / receptor-mediated epithelial cell uptake, which can lead to adverse events, and this approach can also reduce this on-target toxicity (especially in the local environment). Embodiments of the present invention also address this approach to ameliorate or reduce the toxicity of ADCs. For example, if corneal epithelial cells express a receptor or protein target on the cell surface that initiates cellular uptake, the copolymer locally inactivates the ADC, thereby reducing on-target toxicity.

[0044] In one embodiment of the present invention, a formulation containing the copolymer eye drops can be provided as a kit along with a chemotherapy agent at the initiation or contemplation of ADC therapy, optionally with a one-month or more supply of an eye drop delivery system. The kit and eye drops can be delivered to the patient by mail or similar delivery service, or can be refilled online.

[0045] In some embodiments, the copolymers are supplied as pharmaceutical compositions in eye drop bottles, preservative-free multidose bottles, standard three-piece bottles, and unit doser / blow-fill-seal containers. Eye drop bottles typically have a fill volume of 1 mL to 30 mL. Blow-fill-seal containers with various sizes and fill volumes of 0.1 mL to 1 mL (0.5 mL fill, 0.3 mL fill, 0.4 mL fill, 0.7 mL fill) are examples of unit doser containers. The pharmaceutical ingredients can be delivered to consumer products through aerosols, sprays, mist generators, mechanical or electronic spray bottles, pump spray bottles, mouthwashes, drinking solutions, powders, dilution concentrates, and other commercial systems. Kits containing eye drops, nasal sprays, and mouthwashes can be sold. A supply of eye drops formulated with ADCs for the treatment of oncology and other diseases can be included in the kits.

[0046] The advantages include the fact that there is currently no way to apply a barrier to mucous membranes to reduce the risk of infection, which is needed in the emergency preparedness for viral diseases.

[0047] Advantages include the absence of any treatments that reduce the risk of ADC corneal toxicity based on reducing ADC / epithelial cell interactions, other than supportive care such as warm compresses, contact lens bandages, and ophthalmic lubricants. [Brief explanation of the drawings]

[0048] [Figure 1] Projection of SARS-CoV-2 virions in the presence of airway epithelial cells. 1. Airway epithelial cell nucleus. 2. Airway epithelial cell. 3. Cell villi. 4. ACE2 receptor. 5. SARS-CoV-2 virion RNA. 6. SARS-CoV-2 virion spike protein. 8. Virus binding to ACE2 receptor. 9. Virus entry into the cell. 10. SARS-CoV-2 virus. [Figure 2]Figure 1 shows a projection of an airway epithelial cell and a SARS-CoV-2 virion in the presence of a cationic graft copolymer, with interference imparted by the copolymer preventing the virus from entering the cell through ACE2. While an airway epithelial cell is shown, other epithelial cells that can be infected by SARS-CoV-2 can be substituted. 1. Airway epithelial cell nucleus. 2. Airway epithelial cell. 3. Cell villi. 4. ACE2 receptor. 5. SARS-CoV-2 virion RNA. 6. SARS-CoV-2 virion spike protein. 7. Cationic graft copolymer. 11. Virus entry into the cell is prevented due to interference from the cationic graft copolymer (which in some embodiments is PLL-g-PEG). [Figure 3] Prediction of antibody-drug conjugate in the presence of corneal epithelial cells. 1. ADC. 2. Antibody component. 3. Toxic payload. 4. Corneal epithelial cell. 5. Corneal epithelial cell microvilli. 6. Initiation of macropinocytosis. 7. Completion of macropinocytosis. 8. ADC involved in the macropinocytosis process, showing the ADC being captured by corneal epithelial cells from extracellular fluid. 9. ADC with toxic payload in epithelial cells and lysosomes, where the payload is cleaved from the ADC and released intracellularly, where it damages or kills the cells. [Figure 4] This is a prospective diagram showing an antibody-drug conjugate in the presence of corneal epithelial cells and a cationic graft copolymer (which, in one embodiment, is PLL-g-PEG). Note that the corneal epithelial cells may be transiently amplifying cells, wing cells, basal epithelial cells, or limbal epithelial stem cells. For simplicity, the diagram shows surface epithelial cells. 1. ADC. 2. Antibody component. 3. Toxic payload. 4. Corneal epithelial cells. 5. Corneal epithelial cell microvilli. 6. Initiation of macropinocytosis. 7. Completion of macropinocytosis. 8. PLL-g-PEG on the surface of the corneal epithelial cells. 9. PLL-g-PEG in solution. 10. PLL-g-PEG attached to the ADC at several points. 11. The ADC with the toxic payload is absent from the epithelial cells. Cell injury and death are prevented. [Figure 5]Predictive diagrams demonstrating the ocular benefit of cationic graft copolymer eye drop therapy in the presence of systemically administered ADC. 1. Schematic of the anterior surface of the eye. 2. Schematic of the cornea. 3. Superficial punctate keratitis on the corneal surface. 4. Microcystic changes in the corneal epithelium observed after a patient received systemic ADC therapy for cancer. 5. Eye drops containing PLL-g-PEG administered to the eye of a patient receiving systemic ADC therapy for cancer. 6. PLL-g-PEG in solution in eye drops. 7. The corneal surface of the patient receiving ADC therapy and the patient treated with PLL-g-PEG eye drops appears healthier. 8. Patients treated with PLL-g-PEG eye drops have fewer microcystic changes. 9. Patients treated with PLL-g-PEG eye drops have less superficial punctate keratitis despite the use of systemic ADC.

[0049] Detailed Description of the Invention Graft copolymers with positively charged and hydrophilic sites, or block copolymers with positively charged and hydrophilic sites, have now been shown to be effective in two important aspects of human health.

[0050] First, these polymers are effective in reducing viral infectivity and the severity of infection when infection occurs via contact with epithelial surfaces, as well as other exposure methods. The amount of virus is known to be related to the severity of infection in a dose-response manner. Even if infection does occur, reducing the number of intracellular viral infections can have a beneficial effect on the course of the disease (although 100% effective interventions are rare).

[0051] Second, these polymers are effective in reducing antibody-drug conjugate (ADC) toxicity to non-neoplastic cells. These polymers can interfere with off-target cellular uptake of ADCs bearing cytotoxic payloads to cells that do not express the ADC's target receptor. Topical administration can also reduce uptake by locally treating tissue and inactivating the ADC. (The copolymers can be present in the lining mucosal fluid or extracellular space to interfere with off-target ADC uptake.) Specifically, with regard to off-target uptake, these cationic grafts or cationic block copolymers interfere with macropinocytosis of ADCs by human corneal epithelial cells, including basal epithelial cells, limbal stem cells, basal stem cells, wing cells, or superficial epithelial cells. This reduction in exposure to internal cell bodies or cytoplasm, including lysosomes, and in some embodiments, tubulin-forming components, provides benefits to the cells and the organism.

[0052] Without being bound by theory, the method of interference is that the graft or block copolymer inactivates the affected cells and / or the surface of the ADC or virus, preventing or reducing cellular uptake. The cationic graft copolymer or other polymer embodiments interact with biological surfaces and / or the surface of the virus or ADC.

[0053] A key finding is the ability of cationic graft copolymers such as PLL-g-PEG to be effective in this setting. They are safe, well-tolerated, and highly efficacious. PLL-g-PEG has multiple effective embodiments, and these polymers are tunable.

[0054] definition "Off-target uptake" means that the ADC is taken up into cells using a mechanism that is different from, or completely independent of, the antibody-cell receptor interaction for which the targeted therapy was designed.

[0055] "Novel virus" means one that is new to the host (human or otherwise) or one that humans have not encountered before.

[0056] Host refers to the living organism in which a bacterium, virus, protozoan, or other disease-causing microorganism normally resides.

[0057] "Biological surface" refers to the surface of a cell, tissue, or bodily organ, whether exposed to the external environment or located inside the body. For example, the surface of the eye includes the cornea, conjunctiva, posterior Tenon's capsule, and sclera, which are covered by epithelial cells; the epithelial layer of the digestive tract and skin includes membranes such as mucous membranes, including oral mucosa, nasal mucosa, respiratory tract mucosa, and vaginal mucosa. Other surfaces include the capsules of organs such as the spleen and liver, as well as the outermost surfaces of bone, cartilage, and muscle. The copolymers described herein can also interact with and benefit the surfaces of viruses and ADCs.

[0058] "Formulation" refers to a solution, suspension, powder, spray, irrigation solution, or eye drop administered to a cell, tissue, organ, or mammal to be treated, containing the necessary components to enable the beneficial effects of the graft or block copolymer to occur. The formulation may or may not contain a pharmaceutically active ingredient. As used herein, the graft copolymer may or may not be considered a pharmaceutically active ingredient in regulatory terms. Eye drop formulations can have various copolymer ratios (effective ranges), a pH of 3.9 to 9.9, an osmolality of 150 to 400, and a viscosity of 1.0 to 15 cP. Viscosity may be even higher in some embodiments. The formulation is safe for subconjunctival injection.

[0059] "Microtubule disruption" is considered herein to be a particularly important cytotoxic effect. Examples of these "cytotoxic agents" include, but are not limited to, MMAF (monomethyl auristatin F (MMAF) is an antitubulin agent that inhibits cell division by inhibiting tubulin polymerization), MMAE (monomethyl auristatin E), DMF (dimethylformamide), maytansine, aurastatin, DM4 (ravtansine), and DM1 (mertansine). Other cytotoxic agents are listed elsewhere, some in combination with antibodies. All are included, as are unknown cytotoxic agents, in various embodiments.

[0060] A "cytotoxic payload" is sometimes also called a "warhead." A linker is used to connect the antibody and the warhead. The linker is cleaved by intracellular enzymes and, in some cases, extracellular enzymes. In particular, the linker is cleaved in the lysosome, releasing the warhead intracellularly. Enzymes in extracellular fluid or tears may cleave the linker, releasing the warhead. In this way, the warhead may enter the cell directly, or the warhead may leak from the cell and cause a bystander effect (damaging nearby cells that did not take up the ADC). Any linker and many (known and unknown) linkers are contemplated in embodiments of the present invention. The linker may be specific to the cell and the therapeutic situation.

[0061] As used herein, the term "pharmaceutical composition" refers to a composition in which an active agent is formulated with one or more pharmaceutically acceptable carriers. The composition is suitable for administration to a human or animal subject. The active agent is present in a unit dose suitable for administration in a therapeutic regimen that exhibits a statistically significant probability of achieving a desired therapeutic effect when administered to a relevant population.

[0062] Formulations of the copolymers of the present invention include embodiments using many types of formulations, including gels, lotions, creams, ointments, sprays, wipes, and salves. Formulations may or may not contain preservatives. Formulations may be monophasic or multiphasic. Without limitation, multiphasic formulations have different amounts of different components to affect efficacy and duration of action. Some formulations exhibit long-term activity, while others exhibit short-term activity. Overall, a single application provides protection for a meaningful duration. As used herein, "meaningful duration" refers to up to 0.1 minutes, up to 0.5 minutes, up to 1 minute, up to 15 minutes, up to 30 minutes, up to 1 hour, up to 2 hours, up to 3 hours, up to 4 hours, or typically up to 6-12 hours. In some embodiments, the duration is longer than 12 hours. Sustained-release formulations extend the duration of action. Duration of activity values ​​may be shorter or longer for viral protection and reduction of ADC toxicity as determined during development. Regarding methods for attenuating and mitigating ADC toxicity, administration can range from daily to hourly, depending on the formulation and / or ADC payload, the patient's condition and underlying circumstances, and the formulation. The use of these approaches in vitro (and in vivo) has practical value and may be shorter or longer depending on the in vitro model.

[0063] Animal data from other uses with small interfering RNA molecules have shown that PLL-g-PEG can be tolerated intravenously. PLL-g-PEG is well tolerated locally and therefore particularly valuable here. "Tunable" means that the cationic graft copolymer can be varied and still maintain its usefulness. For example, the PLL chain length, graft ratio relative to the number of monomer linkages in the polymer, and length of hydrophilic side chains can be adjusted. By way of example, PLL-g-PEG molecules utilized in experimental proof-of-concept or reduction to practice of the present invention include PLL (15,000-30,000 daltons)-graft (3.5 ratio)-PEG (5,000). Alternative molecular weights and graft ratios may be equally, more, or slightly less effective. All PLL-g-PEG variations are effective and are referred to herein as PLL-g-PEG. Mimetics can be similarly adjusted. Because PEG is a hydrophilic molecule, it has been used to deactivate microscope slides. Polyethylene glycol has low toxicity and is used in a variety of products. This polymer is used as a lubricating coating on a variety of surfaces in aqueous and non-aqueous environments.

[0064] Regarding the properties of PLL-g-PEG, polylysine (PLL) promotes the attachment of proteins and cells to surfaces. PLL can be greater than 30,000 daltons, up to 60,000 daltons, or smaller than 15,000 daltons. A range of polydispersities is acceptable. Polydispersities of 0.3, 0.5, 0.8, 1, or 1.2 are considered acceptable (endpoints included), as are ranges from 0.1 to 3 or greater. PLL is sometimes reported as a single size (e.g., 20,000 daltons). Larger or smaller PLL molecules are also effective in this context. A preferred configuration is an average PLL size of 10,000 to 40,000, although larger or smaller PLL sizes are also effective. The polydispersity can vary and still be effective. The number of lysine monomers (including, but not limited to, L-lysine, D-lysine, α- or ε-polylysine) can range from 50 to 200 in some embodiments, and can be longer or shorter (fewer or more monomers). The grafting ratio is optimally 3.5 or 4 PLL:PEG, with a reasonable preferred range of 2 to 6. Grafting ratios greater than 6, such as 7, 8, 9, or 10, are acceptable in some embodiments. The upper limit is simply the point at which tolerability decreases. While efficacy is expected to decrease at grafting ratios below 2, a lower limit of 1.1 is likely where optimal benefit is obtained from the mechanism of charge-utilizing cationic PLL chains for bioadhesion. Success rates may vary and can be tailored with hydrophobic chains or other configurations. The hydrophilic portion, in this case PEG, can be of different sizes or lengths. PEG 5,000 daltons is a preferred embodiment, but PEG 2,000 is also effective. The range of PEG molecular weights useful for this particular molecule includes PEG 1000 to PEG 20000. Ranges and variable polydispersities are acceptable (0.1 to 2 or greater). The monomer bond number of ethylene glycol can be similarly counted as an alternative to the molecular weight reported in Daltons. Here, a reasonable range of monomer bond number is 22 to 250. In some embodiments, the monomer bond number is 50 to 150.In some embodiments, this is 250 or greater. These factors can be adjusted to achieve the same or similar effects while maintaining the PLL size, grafting ratio, and PEG size. Optimal configurations are described, but are not limited to these. Mixtures of different base PLL-g-PEG copolymers are acceptable. Multiple different linkers of mPEG to PLL are acceptable. Mimicry as outlined herein can be similarly addressed by grafting ratio, backbone size, and size of hydrophilic / deactivation sites.

[0065] Thus, the present invention provides methods for inhibiting, reducing, or preventing the infectivity of a virus (e.g., SARS-CoV-2) and reducing the severity of the disease process in a subject by topically or locally administering a formulation comprising a graft copolymer having a positively charged moiety and a hydrophilic moiety or a block copolymer having a positively charged moiety and a hydrophilic moiety to the subject's biological surface. Because charge dynamics are complex, negative charge-based inactivation methodologies may also be identified, and these discoveries are also discussed and utilized herein. Formulations comprising the polymers claimed herein can be topically or locally administered to a subject's biological surface, including, but not limited to, the skin, mucous membranes, oral mucosa, nasal mucosa, and ocular surfaces, according to the methods of the present invention. As used herein, "subject" is meant to include all animals, particularly mammals such as, but not limited to, humans and dogs, as well as agricultural animals such as cattle, sheep, and pigs.

[0066] The graft copolymers used in the methods and formulations of the present invention are polymers having a linear section of repeating units, called the "backbone," with at least one side chain (called a "graft"), usually of a different chemical repeating unit, branching off from points along the backbone. In one embodiment, the graft copolymer comprises a cationic backbone and water-soluble, non-ionic side chains. In another embodiment, the graft copolymer comprises a water-soluble, non-ionic backbone and cationic side chains. In another embodiment, negative or anionic graft copolymers are utilized.

[0067] The block copolymers used in the methods and formulations of the present invention are polymers in which a linear section of an initial section of repeating units is joined at both ends to a linear section of a subsequent repeating unit that is chemically dissimilar to the initial one.

[0068] Formulations for use in the methods of the present invention include, but are not limited to, block or graft copolymers having one section, either a scaffold, graft, or block, that adheres to biological surface tissues such as cells, epithelial cells, mucosa, mammalian tissue, airway cells, alveoli, tracheal and bronchial tissue, nasal mucosa, oral mucosa, and ocular surfaces including corneal and conjunctival epithelial cells, as well as marginal stem cells, marginal cells, marginal epithelial stem cells, basal stem cells, basal cells, early transient amplifying cells, transient amplifying cells, and corneal epithelial cells in general, as well as the glycocalyx and microvilli associated with these cells. In addition to bioadhesion via electrostatic forces, another, chemically distinct section, either a scaffold, graft, or block, is hydrophilic, which, in one embodiment, induces inactivation and reduction of interactions between viruses and other ADCs. This effectively reduces interactions, resulting in benefits for human health. This can reduce viral and contagious disease transmission, particularly in those with insufficient host or population immunity to prevent epidemics or pandemics. One embodiment is a method for reducing the infectivity of novel viruses for which humans have not developed herd immunity, which helps reduce the severity of disease and epidemics and pandemics.

[0069] The patient is a human, but may be any mammal.

[0070] Human health is improved by reducing the overall toxicity of ADCs, particularly to the eye and cornea. Specifically, the health of the corneal epithelium is promoted. Patients can better tolerate ADCs for the treatment of malignant diseases or other conditions while maintaining a healthier corneal epithelium, resulting in less severe loss of vision / visual acuity and fewer ocular symptoms. The tissue-adhesive section of the graft or block copolymer in the formulation used in the method of the present invention can be cationic, in which case the polymer adheres to the biological surface by electrostatic attraction. The interaction can be anionic or hydrophobic using hydrophobic moieties in the combination herein, with both hydrophobic and anionic interactions being promising.

[0071] Examples of cationic polymer sections of grafts or block copolymers of formulations useful in the methods of the present invention include, but are not limited to, poly(L-lysine) (PLL), poly(2-vinylpyridine), and poly(4-vinylpyridine) and vinyl copolymers containing these repeating units, as well as homo- and copolymers of poly(aminoethyl methacrylate) containing N,N-dimethylaminoethyl methacrylate (N,N-dimethylaminoethyl methacrylate) repeating units. Other cationic polymer sections that can be used include chitosan (a copolymer of glucosamine and N-acetylglucosamine, in which 5-100% of the repeating units are glucosamine) and its synthetic derivatives. Examples of hydrophobic polymer sections of grafts or block copolymers of formulations useful in the methods of the present invention include, but are not limited to, long-chain aliphatic hydrocarbons, polyethylene, poly(propylene oxide), polystyrene, poly(methyl methacrylate), and poly(butylene oxide). The hydrophilic section of the polymer may be non-ionic if the tissue adhesive section is cationic, or anionic if the tissue adhesive section is non-ionic (and hydrophobic).

[0072] Examples of anionic polymer sections of grafts or block copolymers of formulations useful in the methods of the invention include, but are not limited to, polyacrylic acid (PAA), polymethacrylic acid, sodium polystyrene sulfonate, carboxylated cellulosics such as carboxymethylcellulose (CMC), polyitaconic acid, polymaleic acid, polyaspartic acid, polyglutamic acid, polyphosphates, polynucleic acids, polyacrylamidopropanesulfonic acid, anionic natural gums, anionic carbohydrates, carrageenan, alginates, and hyaluronic acid.

[0073] Examples of non-ionic hydrophilic polymer sections of formulations useful in the methods of the present invention include, but are not limited to, polyethylene glycol (PEG), polyvinyl alcohol, polyvinylpyrrolidinone, etc. Examples of anionic hydrophilic polymer sections include homopolymers and copolymers containing, for example, acrylic acid, methacrylic acid, itaconic acid, maleic acid, styrene sulfonic acid, carboxymethyl cellulose, carboxyethyl cellulose, succinylated chitosan, cellulose sulfate, etc.

[0074] By block or graft copolymer, it is intended to refer to the architecture of the polymer.

[0075] "Mimetic polymers" refer to alternative chemical / molecular approaches to create the same behavior and attributes as a validated and tested polymer. For example, multiple mimics exist, such as PLL-g-PEG, which is effective. Their effectiveness is primarily due to the inactivation of the polymer.

[0076] "Inactivation" refers to reducing the ability of a cell or biological surface to interact with a virus or antibody-drug conjugate, thereby reducing infection, macropinocytosis, or cellular internalization of the virus or ADC. This phenomenon is a steric interference, which also reduces the ability of charge interactions involved in the interaction of the virus or ADC. "Inactivation" also refers to reducing the ability of the surface of the virus or ADC to interact with at-risk cells, thereby reducing infection, macropinocytosis, or cellular internalization of the virus or ADC.

[0077] Inactivation can occur with an ADC, a cytotoxic agent, or a virus. Inactivation can also occur through interactions on the cell that make the ADC, agent, or virus less susceptible to uptake.

[0078] The graft copolymer may have a cationic (or nonionic, hydrophobic, or anionic) backbone composed of a polymer selected from the list above and hydrophilic grafts, or a hydrophilic backbone and cationic (or nonionic, hydrophobic) grafts selected from the list above. In the case of a graft copolymer, the grafts may occur from every repeat unit of the backbone or may be spaced intermittently (at uniform or random frequency) along the backbone. For example, a useful polymer in a formulation for use in the methods of the present invention is PLL-g-PEG, in which the backbone is the cationic polymer poly(L-lysine) and the grafts are composed of the hydrophilic polymer polyethylene glycol. The PLL backbone may be from three repeat units to several thousand repeat units long, and the PEG grafts may be from one repeat unit to several thousand repeat units long. PEG grafts can be attached to every PLL repeat unit, every other PLL repeat unit, every third PLL repeat unit, or less frequently. In one embodiment, there is an average of one PEG graft for every third PLL repeat unit. Similar properties can be applied to mimetic polymers.

[0079] Block copolymers comprising at least one block that is cationic and at least one block that is water-soluble and non-ionic are also useful in formulations for use in the methods of the present invention. In one embodiment, the block copolymer comprises at least one block that is hydrophobic and at least one block that is water-soluble and anionic, cationic, or non-ionic. Anionic blocks are contemplated.

[0080] Examples of water-soluble, non-ionic copolymer blocks include, but are not limited to, polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyhydroxyethyl methacrylate (pHEMA), polyacrylamide, polyvinylpyrrolidone (PVP), polyethyloxazoline (PEOX), polysaccharides, and copolymers of any two or more thereof.

[0081] Examples of water-soluble and anionic copolymer blocks or backbones include, but are not limited to, polyacrylic acid (PAA), polymethacrylic acid, polysodium styrene sulfonate, carboxylated cellulosics such as carboxymethyl cellulose (CMC), polyitaconic acid, polymaleic acid, polyacrylamidopropane sulfonic acid, anionic natural gums, anionic carbohydrates, carrageenan, alginates, and hyaluronic acid.

[0082] Examples of water-soluble cationic copolymer blocks include vinylpyridine, N,N-dimethylaminoethyl acrylate, N,N-dimethylaminoethyl methacrylate, allyltri(alkyl)ammonium halides, polyaminostyrene, chitosan, polyethyleneimine, polyallylamine, polyetheramine, polyvinylpyridine, polysaccharides with positively charged functionality, polyamino acids, such as, but not limited to, poly-L-histidine, polybenzyl-L-histidine, poly-D-lysine, poly-DL-lysine, poly-L-lysine, poly-ε-CBZ-D-lysine, poly-ε-CBZ-DL-lysine, poly-ε-CBZ-L-lysine, poly-DL-ornithine, poly-L-ornithine, poly- These include, but are not limited to, Δ-CBZ-DL-ornithine, poly-L-arginine, poly-DL-alanine-poly-L-lysine, poly(-L-histidine, L-glutamic acid)-poly-DL-alanine-poly-L-lysine, poly(L-phenylalanine, L-glutamic acid)-poly-DL-alanine-poly-L-lysine, and poly(L-tyrosine, L-glutamic acid)-poly-DL-alanine-poly-L-lysine, copolymers of L-arginine with tryptophan, tyrosine, or serine, copolymers of D-glutamic acid and D-lysine, copolymers of L-glutamic acid with lysine, ornithine, or a mixture of lysine and ornithine, and polymers based on poly(L-glutamic acid).

[0083] Examples of hydrophobic copolymer blocks include, but are not limited to, alkanes, alkenes, alkynes, polyisobutylene, polyesters such as polycaprolactone (PCL), polylactic acid (PLA), polyglycolic acid (PGA) and their copolymers (PLGA), polyamides such as nylon (6,6) and nylon (12), polyurethanes, polypropylene oxide, polytetramethylene oxide, polyethylene, polypropylene, polystyrene, polyacrylates such as polymethyl acrylate (PMA), polymethacrylates such as polymethyl methacrylate (PMMA), polysulfones, polyether ether ketones (PEEKs), polyphosphazines, polycarbonates, polyacetals, and polysiloxanes.

[0084] In the above description of the present specification, if a molecular entity is applicable to another paragraph but is omitted, it can be considered to be included where applicable. Similarly, each term can be moved to the indicated location to support the final claim.

[0085] As will be understood by one of skill in the art upon reading this disclosure, grafts and blocks as well as triblocks and dendrimers are contemplated and may also be used as embodiments of the present invention.

[0086] An exemplary block copolymer having a triblock architecture is PLURONIC® F127, which includes a polyethylene oxide hydrophilic block ("PEO"), a polypropylene oxide hydrophobic block ("PPO"), and another PEO block, and is also known as Poloxamer 407. Other block copolymers for use in the present invention may include only one hydrophilic block and one hydrophobic block, or may include several alternating blocks, such as a PPO-PEO-PPO block copolymer (PLURONIC®, a block copolymer based on ethylene oxide and propylene oxide, BASF, Florham Park, NJ). Additional exemplary PLURONIC block copolymers useful in the present invention include PLURONIC 10R5, PLURONIC 17R2, PLURONIC 17R4, PLURONIC 25R2, PLURONIC 25R4, PLURONIC 31R1, PLURONIC F 108 Cast Solid Surfactant, PLURONIC F 108 Pastille, PLURONIC F 108 Prill, PLURONIC F 108NF Prill Poloxamer 338, PLURONIC F 127 Prill, PLURONIC F 127 NF, PLURONIC F 127 NF 500 BHT Prill, PLURONIC F 127 NF Prill Poloxamer 407, PLURONIC F 38, PLURONIC F 38 Pastille, PLURONIC F 68, PLURONIC F 68 Pastille, PLURONIC F 68 LF Pastille, PLURONIC F 68 NF Prill Poloxamer 188, PLURONIC F 68 Prill, PLURONIC F 77, PLURONIC F 77 Micropastille, PLURONIC F 87, PLURONIC F 87 NF Prill Poloxamer 237, PLURONIC F 87 Prill, PLURONIC F 88 Pastille, PLURONIC F 88 Prill, PLURONIC F98, PLURONIC F 98 Prill, PLURONIC L 10, PLURONIC L 101, PLURONIC L 121, PLURONIC L 31, PLURONIC L 35, PLURONIC L 43, PFLURONIC L 44, PLURONIC L 44 NF Polaxamer 124, PLURONIC L 61, PLURONIC L 62, PLURONIC L 62 LF, PLURONIC L 62D, PLURONIC L 64, PLURONIC L 81, PLURONIC L 92, PLURONIC L44 NF INH surfactant Polaxamer 124, PLURONIC N 3, PLURONIC P 103, PLURONIC P 104, PLURONIC P 105, PLURONIC P 123 Surfactant, PLURONIC P Examples of suitable block copolymers include, but are not limited to, PLURONIC P 65, PLURONIC P 84, and PLURONIC P85. Where appropriate, all particle sizes of block copolymers are included, for example, PLURONIC F127 and PLURONIC F87, which are available as prill and microprill products. For example, nonionic surfactants comprising a hydrophobic segment and a PEO block are considered block copolymers herein.

[0087] Additional exemplary block or graft copolymers that can be used in the present invention are disclosed in U.S. Pat. Nos. 5,578,442 and 5,834,556, the teachings of each of which are incorporated herein by reference in their entirety.

[0088] The block or graft copolymer is included in formulations for use in the methods of the invention as a component of the formulation at a concentration ranging from 0.001% to 40%, more typically 0.01% to 25%, on a weight / weight basis. The formulation, in some embodiments, is a powder, such as a lyophilized powder, for delivery to tissues or for re-formulation and dissolution, which may be 0.001% to 100% block or graft copolymer. The copolymer may, in some embodiments, be water-soluble.

[0089] Copolymer combinations are contemplated and included herein, but the formulation percentages are applicable to each of the different components.

[0090] In the human examples of the formulations described herein, when delivered as a solution or suspension, the amount of copolymer is about 0.1% to 5%. Further, the amount of copolymer can be 0.01% to 3%, 0.1% to 2.5%, or 0.5 to 4%.

[0091] Additional exemplary components that may be incorporated into pharmaceutical formulations and coatings for use in the present invention include, but are not limited to, PLURONIC gelling agents, such as, but not limited to, F127, F108, and the additional PLURONIC agents listed above. Furthermore, in one embodiment, these components are used in amounts equal to or less than that required for gelling activity.

[0092] Other ingredients (either active or inactive) that may be included in these pharmaceutical formulations include, but are not limited to, lipids, oils, surfactants, water, lubricating polymers, typical surfactants, buffers, salts, physical ions, proteins, topical emollients, excipients typically used in oral, topical, and mucosal formulations, dermal and ophthalmic formulations, lubricants such as PEG 400, carboxymethylcellulose, hydroxypropylmethylcellulose, mineral oil, propylene glycol, glycerin, hypromellose, white petrolatum, polyvinyl alcohol, liposomes, mannitol, hydroxypropyl guar, dextran 70, viscoelastics, and hyaluronic acid, and combinations thereof. Additional ingredients may include those typically found in mouthwashes, nasal sprays, shampoos, soaps, and conditioners. Such ingredients may be included in the formulations in various proportions ranging from less than 0.1% to 99% by weight, more preferably from less than 1% to 10% by weight. Other ingredients that may be included in these pharmaceutical formulations include, but are not limited to, preservatives such as polixetonium, polyquaternium-42, polyquaternium-1, polyquat, alkylhydroxybenzoate preservatives, parabens, hydrogen peroxide, benzalkonium chloride, cetylpyridimine chloride, cetalkonium chloride, sodium perborate, Purite, dissolving preservatives, polyhexamethylene biguanide (PHMB), chlorobutanol, benzododecinium bromide, "ionic buffer systems," povidone, silver, silver sulfate, betadine, and other disinfectants, as well as proprietary and non-proprietary preservatives. PLL-g-PEG may also act as a preservative. Antibiotics and antivirals (whether small molecules or biologics) may also be included in the formulation. In some embodiments, preservative-free formulations are preferred.

[0093] Additionally, in some embodiments, the formulations and coatings can include one or more additional active pharmaceutical ingredients. Examples include, but are not limited to, anesthetics, antibiotics, antivirals, anti-inflammatory agents, intraocular pressure-reducing agents, artificial tears, lubricants, dilatants, immunosuppressants, antiangiogenic agents, monoclonal antibodies, proteins, peptides, neuroprotective agents, small molecules, and antibodies. In some embodiments, the formulations are delivered prior to use of the personal protective equipment.

[0094] Some exemplary, but not limited to, additional drugs that may be included in these formulations include antivirals, including remdesivir, antiretrovirals, rimantadine, and others selected from this list, as indicated for human benefit: abacavir used for HIV, acyclovir used for herpes, e.g., chickenpox, adefovir used for chronic hepatitis B, amantadine used for influenza, ampligen, amprenavir (agenase), arbidol, atazanavir, atotsugil, and rivaroxaban used to inhibit HIV. Ripla (fixed-dose drugs), Baravir, baloxavir marboxil (Xofluza), Biktarvy, boceprevir (Victrelis), cidofovir, cobicistat (Tyvost), Combivir (fixed-dose drugs), daclatasvir (Daclinza), darunavir, derevirdine, Descovy, didanosine, docosanol, dolutegravir, doravirine (Pifeltro), Ecoliva, edoxudine, efavirenz, elvitegravir, emtricitabine, enfuvirtide, entecavir, etravirine (Intelence), famciclovir, fixed-dose combination drugs (antivirals) Retroviral agents), fomivirsen, fosamprenavir, foscarnet, fosfonet, fusion inhibitors, ganciclovir (Cytoven), ibacitabine, ibalizumab (Trogarzo), idoxuridine, imiquimod, Immunovir, indinavir, inosine, integrase inhibitors, type I interferon, type II interferon, type III interferon, interferon, lamivudine, letermovir (Prebymis), lopinavir, loviride, maraviroc, methisazone, moroxydine, nelfinavir, nevirapine, Nexavir ru, nitazoxanide, norvir, nucleoside analogues, oseltamivir (Tamiflu), peginterferon alfa-2a, peginterferon alfa-2b, penciclovir, peramivir (rapivab), pleconaril, podophyllotoxin, protease inhibitors (pharmacology), pyramidine, raltegravir, remdesivir, reverse transcriptase inhibitors, ribavirin, rilpivirine (Edurant), rimantadine, ritonavir, saquinavir, simeprevir (Olicio), sofosbuvir, stavudine, synergistic enhancers (antiretrovirals), telaprevir,The formulations include telbivudine (Taizeka), tenofovir alafenamide, tenofovir disoproxil, tenofovir, tipranavir, trifluridine, trizivir, tromantadine, Truvada, valacyclovir (Valtrex), valganciclovir, vicriviroc, vidarabine, viramidine, zalcitabine, zanamivir (Relenza), and zidovudine. The formulations may also include hydroxychloroquine, chloroquine, and azithromycin. Potential formulations include antibody products, antibodies, proteins, and biomolecules selected from polyclonal or monoclonal antibody products, antibodies, proteins, and biomolecules that bind to antigens. Antibody fragments, trap molecules, and biomolecules that bind to other antibodies, cell receptors, and proteins relevant to the conditions addressed herein, whether known, in development, undeveloped, or in conception, are also included. Importantly, the claimed copolymers can be combined in formulations with other active agents in the management of the disease being addressed to enhance performance or efficacy, including synergistic therapies. Biomolecules are produced by living organisms. They include synthetic antibodies, recombinant proteins, and other large and small molecules, including those for which new technologies have led to new nomenclatures. ACE2 receptor blockers, steroids (glucocorticoids, androgens, estrogens, etc.) are also potential pharmaceutical components and are contemplated within various embodiments of the present invention.

[0095] The pH of the formulations of the invention is in the physiological range depending on the site of administration and the location of the biological surface or membrane to be modified. Typically, the pH is greater than or equal to 3, for example greater than or equal to 5.6, and less than or equal to 9.

[0096] The formulations can contain new or established dry eye or corneal treatments, including, but not limited to, cyclosporine, lifitiglast, LFA antagonists, steroids such as loteprednol, dexamethasone, flucinolone, difluprednate, aldehyde scavengers, fonaldepal, varenicline, visomitine, kinases, silk-derived proteins, voclosporin, omega-3 fatty acids, and any emollients or lubricants from the OTC monographs referenced in full herein. Importantly, some formulation embodiments have very low viscosity (lower than most artificial tear products on the market). The advantages of this are that vision is not blurred upon application, the spray functions easily through a small nozzle, and the mouthwash is completely and easily rinsed off and is well tolerated. Electrospray and other microelectronic and mechanical delivery systems, high-precision multi-jet systems, and other approaches to microdose release are among the possible delivery systems. The copolymers are present in the following forms: The remaining bioadhesive polymers, of course, are already bound to the cells they protect. For example, one test formulation had a viscosity of 2.7 cP. Furthermore, the benefits conferred by the present invention demonstrate the value of graft or block copolymers as a mechanism of action.

[0097] This approach is effective in reducing viral infectivity, as described in the in vitro experiments in Example 1. Other examples demonstrate reduced ADC entry into epithelial cells and reduced ocular adverse events.

[0098] Furthermore, as described in the Examples herein, the performance of these block and graft copolymer formulations is evaluated in the eyes of human volunteers. In previous experiments, the eye drops were well tolerated. For example, no subjects reported irritation or discomfort during multiple initial human exposures. Furthermore, no blurring was reported, even after single or repeated instillations of 50 microliters or less. Therefore, one embodiment of the present invention is a product with a lower viscosity.

[0099] However, as will be understood by those skilled in the art upon reading this disclosure, alternative ophthalmic delivery means can be used, including, but not limited to, intraocular, periocular, conjunctival, subconjunctival, transconjunctival, periocular, retrobulbar, sub-Tenon, transscleral, topical gel, topical dispersion, intraorbital, intrascleral, intravitreal, subretinal, transretinal, choroidal, uveal, intracavitary, transcorneal, intracorneal, intralental (including phakic and pseudophakic), and intra- or adjacent to the optic nerve. The present invention can be used with other delayed-release formulations of polymers to extend drug delivery. The present invention can be used with depot formulations. The present invention can be administered intravenously to treat other ADC toxicities, such as thrombocytopenia.

[0100] Importantly, for the use of ADCs in toxicity and viral infection, in vitro and in vivo opportunities exist that are of commercial and product development value. For example, these copolymers with bioadhesive and inactivation sites have been shown to be effective in these conditions both in vitro and in humans, making their use valuable in development. Cells can be treated with copolymer formulations and then ADCs added. Tissue culture models, including corneal epithelial models, can also be used. Known approaches to improve off-target or macropinocytotic uptake allow for selection of cells with low uptake in the presence of copolymers for development. Models include epithelial cell lines, harvested epithelial cells, megakaryocytes, other cells, and human umbilical vein endothelial cells. Cell types at risk for toxicity are used in some laboratory development procedures. Similarly, antiviral efficacy in therapeutic and prophylactic strategies can be evaluated in laboratory / development settings with virus and copolymer exposure to select synergistic and optimal molecules and formulations for human development. Therefore, the claims to treat cells broadly are relevant and useful inventions for advancing therapies for human health addressed herein.

[0101] COVID-19, an infectious disease caused by SARS-CoV-2, is a devastating disease for many patients, with a mortality rate of up to 3% and many requiring hospitalization and intensive care unit (ICU) treatment. Millions of patients have been infected, and the mortality rate continues to increase by hundreds of thousands. More than 3 million patients worldwide have been infected, with more than 200,000 dying within six months. Elderly patients and those with pre-existing conditions are at higher risk for morbidity and mortality. High viral load exposure also poses a significant risk for morbidity. SARS-CoV-2 has caused a global pandemic, necessitating the need for better alternative therapies. The emergence of new pandemics is also possible. The SARS-CoV-2 spike protein can be inactivated using graft and block copolymer approaches, but these approaches are not specific to a single virus strain.

[0102] "Viral infectivity" refers to the exposure of at-risk host cells to pathogenic viruses. Using protein receptors and other uptake mechanisms, viral particles enter cells, release their RNA and DNA, and hijack the cell's protein and nucleotide manufacturing machinery or other metabolic processes to produce more viral particles, which can then be released to infect other cells or other organisms. Without being limited to a specific mechanism, formulations containing the above-mentioned graft copolymers, such as PLL-g-PEG (which may be cationic, hydrophobic, or anionic and have hydrophilic side chains), adhere to the biological surfaces of viral particles (including the spike of SARS-CoV-2) or other cell-entry mediating proteins on the viral lipid membrane shell or on viral particles in general. Charged, hydrophobic, or anionic moieties, such as those of PLL, create bioadhesive properties. Hydrophilic (e.g., PEG) moieties prevent and / or reduce interaction with target cells. When applied as droplets, spray mist, or rinse, the graft copolymer can migrate from surface tissues to viral particles through physicochemical on-off binding associated with electrostatic interactions. The inactivation site can be nonionic, nonionic, and inert, as well as hydrophilic. The graft copolymer also protects at-risk cells, potentially directly interfering with receptor proteins (e.g., ACE2) to enhance their activity. Combined exposure of viral particles and at-risk cells significantly reduces their interaction and viral infectivity. Reduced interaction benefits cells, tissues, and organisms, including humans, by reducing exposure to pathogens or toxins, thereby reducing the morbidity and mortality associated with these agents. For example, lower viral loads reduce the severity of subsequent infections and increase the opportunity for host defenses to function. Reduced exposure to the virus reduces transmission rates and, in some cases, the severity of viral infections. Therefore, the methods for reducing SARS-CoV-2 infection described herein will be an important additional approach for safely protecting subjects.

[0103] Similar formulations have already been used in the eye without adverse events and are safe for use in the oral cavity, respiratory tract, and nasal cavity. PLL-g-PEG is composed of amino acids and PEG. Extensive studies have shown this formulation to be safe for use in humans and animals.

[0104] These formulations, in accordance with the present invention, can also be widely used to reduce the transmission of SARS-CoV-2 and other novel viruses via nasal and inhaled applications in settings including, but not limited to, hospitals, emergency departments, intensive care units, aircraft, kindergartens and schools, homes of virus-affected individuals, and nursing homes and chronic care facilities. Healthcare workers and first responders may also benefit. A "new" or "novel" virus refers to a virus that has mutations or general characteristics that indicate that humans, and generally the majority of the population, are not resistant to prior exposure or vaccines. "Transfection" refers to the method by which a virus enters a host. "Epidemic" and "pandemic" are often determined by health authorities. A pandemic is a disease that is prevalent in a country or the world at a particular time. An epidemic is a widespread outbreak of infectious disease in a community at a particular time. The present invention particularly relates to viral pandemics and COVID-19. COVID-19 is a viral disease caused by SARS-CoV-2. Herd immunity is the resistance to the spread of contagious disease in a population when a sufficiently high percentage of individuals are immune to the disease through antibody production, either through vaccination or prior exposure to the virus. Herd immunity requires resistance to a significant number of viruses. There is significant utility for the formulation in children and frail adults whose immune systems are compromised, chronically ill, and have other chronic health conditions, such as cystic fibrosis, that make the host more susceptible to common illnesses.

[0105] Corneal epithelial microcystoid epitheliopathy is a currently identified problem associated with and / or attributed to ADC therapy and has recently become clinically significant as ADCs with corneal epithelial toxicity have become commercially available after years of development. ADCs are being developed as effective treatments for many forms of oncology. These are costly and resource-intensive development programs, and ocular toxicity creates significant limitations on their use. Human toxicity may not manifest until clinical trials. It is important to ensure patients maintain ADC therapy toward optimal oncology (or other indication) disease outcomes (survival or progression-free response, beneficial effect), and corneal epithelial toxicity is an adverse event that can limit treatment or cause patients to discontinue ADC-related therapy. Serious ophthalmic and other types of adverse events associated with ADCs are problematic for patients and treating physicians. It is believed that once inside a cell, the payload or warhead causes adverse events (in the eye, or in some embodiments, other cells or cells in culture or tissue culture) following its release from the ADC (linker cleavage).

[0106] Without being bound by any particular theory or mechanism of toxicity, beneficial findings are demonstrated in vitro and in vivo as a way of illustrating the putative utility of the present invention.

[0107] Based on clinically observed toxicity, ADCs can gain ocular exposure through one of two ways: First, ADCs can be released from the limbal circulation (including but not limited to the palisades of Vogt, which have a distinct vasculature with thin, barely visible arterial and venous components with radially oriented hairpin loops) into the extracellular space, followed by nonspecific uptake by basal cells and their migration to the cornea, where they can reach daughter cells of limbal stem cells and ultimately basal stem cells and basal epithelial cells.

[0108] "Macropinocytosis" has been described as a mechanism by which ADCs enter cells, such as corneal cells, which are susceptible to toxicity because they typically lack the specific receptors that the antibody moiety of the ADC uses to enter the cell. Thus, ADC entry into cells can be considered nonspecific and off-target. Embodiments of the invention herein ameliorate or reduce ADC-induced corneal cell toxicity in off-target cells, such as limbal stem cells, daughter cells, transiently amplifying cells, wing cells, basal cells, corneal epithelial cells, and terminal epithelial differentiated cells.

[0109] ADCs can also be exposed to the cornea through tears. Drugs have been reported to be secreted into tears. Tears have been identified to contain a variety of cytokines, and secretions from the lacrimal system are suggested as a pathway for their presence. The tear film contains hundreds of proteins and / or enzymes, and the mechanism is also understood to be secretion from the lacrimal gland. Leakage from plasma across the blood / tear barrier or leakage from tissue interstitial fluid are also explained as pathways for the presence of proteins in tears. Antibodies have been found in tears.

[0110] Because superficial corneal epithelial cells do not have a blood supply, they must obtain fluid and nutrients through some means independent of a direct blood supply. Epithelial cells utilize macropinocytosis as a method of nutrition. The plasma membrane of cells contains a combination of glycosphingolipids, cholesterol, and protein receptors, which are organized into glycolipid-protein microdomains called lipid rafts. Lipid raft internalization is a documented process of internalization of extracellular material in corneal epithelial cells. Macropinocytosis is a type of endocytosis in which extracellular material is captured by the cell (macropinocytosis is endocytosis in which extracellular material is taken up into the cell). (Macropinocytosis is a means by which eukaryotic cells take up extracellular fluids and dissolved molecules. Pinocytosis may also be used as a term in embodiments herein. Pinocytosis is the uptake of fluids into the cell by budding of vesicles from the cell membrane. Micropinocytosis is contemplated herein and used as a term in embodiments, and can be used in any specific embodiment where either macropinocytosis or pinocytosis is used generically. Micropinocytosis is the uptake of macromolecules or other chemicals into the cell by membrane invasion and pinching with relatively small vesicles.) Toxins and pathogens use endocytosis and macropinocytosis to enter many types of cells. ADCs can enter cells by macropinocytosis.

[0111] Macropinocytosis occurs in many cell types and has been shown to be one of the methods by which ADCs enter corneal epithelial cells. While this is a natural method for cellular uptake of extracellular components, inhibiting it can be beneficial in certain circumstances. Macropinocytosis is thought to be the method by which surface epithelial cells internalize ADCs. It is the least specific pathway and is directed by actin-driven membrane protrusions, which form large endocytic vesicles known as macropinosomes. Ultimately, these vesicles fuse with lysosomes. Once ADCs enter the lysosome, payloads, particularly maytansinoids and auristatins in the context of ADV toxin, are associated with toxicity. However, tubulin inhibitors in general can cause corneal toxicity. In general, the most adverse effects of toxic payloads, or tubulin inhibitors, occur when cells are exposed to either mitotic (microtubule-dependent mitotic activity) or migrating (also involving microtubules).

[0112] The payload is a cytotoxic molecule linked to an antibody, which is then cleaved at the linker by an intracellular enzyme. While this is how ADCs deliver toxins to target (neoplastic) cells, other cells can also be adversely affected, even if they do not express the receptor / protein that interacts with the ADC. Another means by which ADCs enter cells is pinocytosis. Payloads other than tubulin inhibitors can also be toxic to the corneal epithelium, and this paper explores these alternative cytotoxic payloads. Once cleaved from the ADC, the payload actively exerts its chemical function, such as inhibiting the polymerization of tubulin into microtubules, which are necessary for cell division and, in some cases, cell migration. The presence of tubulin inhibitors in dividing cells can induce cell death (followed by apoptosis and pycnosis). Apoptosis and pycnosis have been observed in corneal epithelial cells in ADC toxicity. It is believed that these pycnotic cells are the cause of the microcystic keratopathy observed on examination. Slit-lamp examination reveals dead and dysfunctional cells in the epithelial layer. The extent of this is quantifiable.

[0113] Once cleaved by a cell that has taken up the ADC, the cytotoxic molecule may be released from that cell, potentially causing a bystander effect. Therefore, reducing cellular uptake and cleavage may be an intervention with an excessive relative reduction (bystander death may protect more cells than actual uptake of the ADC in a single cell). Bystander death means that once cleaved, the cytotoxin is released into the extracellular space, where it enters subsequent cells. Therefore, if taken up by more superficial cells, it may migrate within the corneal epithelial layer. It may also be taken up by stem cells and then released at the periphery, where it may be released to other nearby stem cells and diffuse inward to basal stem cells. The method of the present invention can reduce this diffusion of cytotoxic drugs. This approach not only reduces uptake and cleavage, but also limits local cytotoxin migration and reuptake using polymers. Regardless of the mechanism, the effect is real and valuable.

[0114] Toxicity in humans may be limited to the epithelial layer (not involving the stroma or endothelium) in some cases. (Note that the stroma or endothelium may be involved secondarily or in some ADC designs.) Normal replenishment of superficial epithelial cells is disrupted, resulting in abnormal superficial epithelial layers that can exhibit punctate staining, corneal epithelial defects, and abnormal refractive surfaces. This often adversely affects vision. Patients may experience symptoms such as blurred vision, dry eyes, corneal foreign body sensation, ocular discomfort, and ocular irritation. Corneal infection, stromal keratitis, and ulcerative keratitis have been reported with ADC therapy. These are all serious adverse events, and these toxicities often require treatment interruptions or delays. Histopathological examination of the eye with cytarabine (similar to what is encountered with some ADCs) reveals extensive degeneration of rapidly dividing basal epithelial cells, leading to the formation of epithelial microcysts.

[0115] Eye irritation is a toxicity or adverse event associated with ADCs, but it is not the only symptom. Corneal toxicity from ADCs can or potentially can result in superficial punctate corneal epitheliopathy, corneal erosions and epithelial defects, corneal ulcers, corneal infection, and corneal perforation. Foreign body sensation and decreased vision may occur. This life-saving treatment may require drug interruption, dose delay, and dose reduction. Patients require vision for driving and reading. Vision is an important factor in determining quality of life. An advantage of the present invention is that it allows patients to maintain a more regular dosing schedule and minimize ocular symptoms. Patients can experience better outcomes with fewer serious adverse events without interrupting ADCs.

[0116] As shown herein, corneal toxicity associated with ADCs has no mechanism-directed treatment. The only available intervention is symptom management. Over-the-counter lubricating eye drops, punctal occlusions, and bandage contact lenses can help symptoms.

[0117] In one aspect, a method for reducing adverse events associated with the use of antibody drug conjugates in the setting of off-target uptake pathways that damage non-neoplastic cells, including but not limited to, by administering an effective amount of a copolymer having electrostatic and steric mediation properties that are applied to cells affected by said toxicity.

[0118] Without being bound by any particular theory, the utility of the present invention may be manifested by the interference, reduction and inhibition described herein.

[0119] Another related approach to managing ADC toxicity caused locally by off-target uptake is the use of antibodies against the ADC itself. Antibodies directed against specific ADCs can be engineered with inactivation moieties, such as PEGylation, to reduce the ADC's ability to enter cells by macropinocytosis. ADCs are delivered locally to inhibit uptake by pinocytosis, for example. Antibodies can be PEGylated or have other inactivation moieties, as described herein, using modification of antibodies to ADCs by conjugation to hydrophilic polymers for cell culture, laboratory, and in vivo human use. In embodiments where copolymers or antibodies are shown to reduce ADC-associated toxicity or corneal toxicity of other drugs, their use in the laboratory is important because they allow clinical development to be pursued with a reduced risk of ocular adverse events that complicate treatment.

[0120] The cationic graft copolymer interferes with cellular (corneal epithelial cell) uptake of ADCs. This unique methodological approach improves corneal epithelial health, improves laboratory and symptomatic findings, and reduces ocular risks. Reducing ADC-associated cell death (e.g., toxicity) has many advantages. Intravenous administration and other options exist for reducing toxicity, and the claimed subject matter is therefore broad. Megakaryocytes can use macropinocytosis to internalize ADCs, and therefore options for reducing this known adverse event associated with ADCs are addressed in the present invention.

[0121] Epithelial cells have a negative charge (3.6 × 10 -4) are known to have negative charges. Therefore, cationic graft and block copolymers inactivate these negative charges, which are thought to be involved in ADC uptake. There is evidence that positively charging ADCs increases toxicity, while negatively charging them reduces uptake. An important aspect of the present invention is the discovery that inactivation through electrostatic (or in some embodiments, hydrophobic) interactions is beneficial. Reducing ADC interactions with cell surfaces reduces ADC entry in one embodiment. Protecting cells in general also reduces ADC toxicity. Other (non-epithelial) cells similarly possess charged regions (and hydrophobic regions) that allow for interference with reducing these interactions. Copolymers can also act on key protein components on cells and ADCs to limit uptake.

[0122] It is also important to have multiple methods for delivering cationic graft copolymers (or other effective block or graft copolymers) to target tissues. PLL-g-PEG has been shown to be safe for intravenous injection in mammals. Eye drops and topical exposure have also been shown to be safe. This polymer can reach target limbal cells via intravenous injection. Subconjunctival delivery may also be a valuable approach to ameliorate toxicity because it provides both a reservoir and access to limbal stem cells and their daughter cells, including basal epithelial cells. The conjunctiva is also somewhat permeable to macromolecules. The intercellular spaces of the conjunctival epithelium are wider than those of the cornea, making it more permeable to larger molecules. Therefore, locally delivered PLL-g-PEG can reach stem cells (progenitor cells of superficial corneal epithelial cells) and reduce toxicity by interfering with ADC corneal toxicity. Topical delivery to the cornea (or eye) can interfere with any ADC's penetration into the superficial corneal or conjunctival epithelium. This reduces payload release and subsequent migration through the five or six layers of the cornea to the basal cells. Larger molecules, by definition, can penetrate the superficial epithelium. Therefore, PLL-g-PEG or other identified and claimed graft or block copolymers can protect basal epithelial cells deeper in the epithelial layer. They reach this space. Similarly, ADCs can be exposed to deeper corneal epithelial cells. Therefore, the copolymers can interfere with ADCs to block their interaction with the cell-trapping process and with the corneal cell surface to prevent ADC entry into the cells. These effects provide an effective approach to reducing ADC-associated corneal toxicity. In the case of corneal cells, basal cells and other potentially vulnerable cells are protected, resulting in less toxicity to some extent, and patients experiencing fewer signs and symptoms of ADC corneal toxicity. This option has significant implications and applies to other approaches to minimize off-target ADC toxicity. Corneal cells do not typically display the receptors targeted by ADCs, so toxicity is off-target.In some embodiments, these copolymers may be beneficial in topical applications if there is a component of on-target uptake based on the presence of receptors on corneal cells.

[0123] Delivery can be localized, regionalized, or systemic. Many formulations exist. Copolymers (PLL-g-PEG) can reduce corneal tissue exposure to ADC and reduce uptake. The interference is steric and charge-based. Inactivating the ADC and inactivating the cell surface reduces uptake. Charged or hydrophobic active sites adhere to cells or ADC. The hydrophilic components of the polymer reduce ADC-corneal interactions and reduce uptake. Binding to the conjunctiva or cornea by the copolymer can ultimately serve as a reservoir for interference and inactivation of the ADC as it moves from the tear film to the tear drainage system. The selected copolymer is nontoxic. Concentrations of eye drops can range from 0.01 to 10% by weight, but are more commonly 0.5 to 3% by weight. Subconjunctival formulations can be at higher concentrations.

[0124] The graft copolymer tested was PLL(20)-g[3.5]-PEG(5) in one experiment, but other molecular sizes and grafting ratios are also safe and effective.

[0125] The formulations described herein also provide a point of safety, and it may be beneficial to include other drugs or polymer components in the eye drops to reduce ADC toxicity or protect against viruses. Subconjunctival delivery may require less frequent administration. Topical delivery is compatible with long-term use. In many embodiments, preservatives are avoided to help protect the corneal surface and avoid damaging epithelial cells. Exemplary additional active pharmaceutical ingredients for ophthalmic use include, but are not limited to, lubricants, emollients, and sterile water, as well as other standard excipients. In some embodiments, it may be beneficial to combine with another active agent, including novel protective agents that may have a synergistic effect with the copolymer.Also, antibiotics (fluoroquinolones, vancomycin, cephalosporins, gentamicin, erythromycin, azithromycin, sulfa drugs, bacitracin, gatifloxacin, levofloxacin, moxifloxacin, ofoxacin), acetazolamide, antazoline, aspirin, atropine, azelastine, bacitracin, betaxolol, bimatoprost, botanicals such as zeaxanthin, lutein, riboflavin ... Copinbrimonodine, brinzolamide, carbachol, carteolol, ciprofloxacin, ofloxacin, cromalin, cyclosporine, dapiprazole, dexamethasone, diclofenac, dipibifren, dorzolamide, epinephrine, erythromycin, fluoromethalone, flurbiprofen, gentamicin, glaucoma medications (prostaglandins, carbonic anhydrase inhibitors, epinephrine or alpha agonists, beta blockers) locker), gramicidin, homatropine, hydrocortisone, hyoscine, ketorolac, ibuprofen, ketotifen, latanaprost, levobunolol, levocabastine, levofloxacin, loteprednol, medrysone, methazolamide, metipranolol, naphazoline, natamycin, nedocromil, neomycin, neuroprotective agents, nonsteroidal anti-inflammatory agents, nepafanec, norfloxacin, ofloxacin, Lopatadine, oxymetazoline, pemirolast, pheniramine, phenylephrine, pilocarpine, povidone, prednisolone, proparacaine, scopolamine, tetracaine, steroids, sulfacetamide, tetrahydrozoline, hypertonic tears, timolal, tobramycin, travaprost, trifluridine, trimethiprim, tropicamide, unoprostone, and zinc may all have some value in combination formulations. Prodrugs and related compounds, as well as any novel active pharmaceutical ingredients, can be combined with the block and graft copolymers described herein to reduce ADC toxicity or better manage adverse events associated with ADCs.

[0126] Based on the mechanism and nonspecific protection and inactivation approach, ADCs bearing tubulin inhibitors or epithelial cytotoxic agents are mitigated with this copolymer-based approach. The following ADCs are claimed in the present invention, but this list is by no means limiting. The copolymers discussed herein can be formulated together with the ADC itself or separately. The toxic payload can be a maytansinoid or auristatin, but can also be another type of general or other tubulin inhibitor. Tubulin inhibitors and tubulin polymerization inhibitors have particular utility in this embodiment. Tubulin inhibitors may include, but are not limited to, paclitaxel, epothilone, docetaxel, discodermolide, colchicine, combrestatin, 2-methoxyestradiol, methoxybenzenesulfonamide (E7010), vinblastine, vincristine, vinorelbine, vinfluin, dolastatin, halichondrin, hemiasterlin, cryptophycin 52, paclitaxel, vinca alkaloids, colchicine, etc. They may be tubulin-disrupting agents or may act by other cytotoxic mechanisms. DNA synthesis inhibitors, such as cytarabine, are also included. Other cancer drugs that can serve as payloads include monomethyl auristatin E (MMAE), DM1 (mertansine), T-DM1, maytansinoids, auristatins, DUO duostatin 5 and other duostatins, AF-HPA (auristatin F-hydroxypropylamide), PBD (pyrrolobenzodiazepine), MMAF (monomethyl auristatin F), Calich, sodium nitrate, calicheamicin, DM4 (ravtansine), SN-38, irinotecan metabolite, PF063801 01, Dxd, DNA topoisomerase I inhibitor, DOX, doxorubicin, PF063801 01, mitoxantrone, etoposide, tesirin, PBD dimer, pyrrolobenzodiazepine, and SG3199. Tubulin filament-targeting toxins, DNA-targeting toxins, RNA-targeting toxins, nanocarriers, protein toxins, and enzymes are also contemplated.

[0127] Various linkers in ADC drugs are claimed, and embodiments include those known in 2020 and those developed in the future when used to protect against or treat toxicity using copolymers in models or clinical settings. Embodiments include combinations with the following ADCs: Gemtuzumab ozogamicin, brentuximab vedotin, trastuzumab emtansine, inotuzumab ozogamicin, Polatuzumab vedotin-piiq, enfortumab vedotin, trastuzumab deruxtecan, IMGN242 (huC242-DM4), CanAg / DM4 / SPDB, IMGN242 (huC242-DM4), CanAg / DM4 / SPDB, trastuzumab emtansine, (T-DM1), SAR3419 (huB4-DM4), SGN-CD19A, CD19 / MMAF(auristatin) / mc AVE9633, Verantomab, mafodotin, CD33 / DM4 / SPDB, CD70 / MMAF (auristatin SGN-75 CD70 positive CD70 / MMAF (auristatin) / mc, SAR566658 CA6+, DS6 / DM4 / SPDB, CD33 / calicheamicin / Hydrazine, ephrin type A receptor 2 (EphA2) / mcMMAF (auristatin) / mc, lorvotuzumab, mertansine, D56 / DM1 / SPP, CD138 / DM4 / SPDB, FRa / DM4 / SPDB, AGS-16M8FMMAF, AGS-16C3FMMAF, ENPP3 / MMAF (auristatin), and the following, but not limited to: [Table 1-1]

[0128] [Table 1-2]

[0129] [Table 1-3]

[0130] [Table 1-4]

[0131] [Table 1-5]

[0132] [Table 1-6]

[0133] Novel, unpublished ADCs with potential for corneal toxicity can be treated with the methods of the present invention.

[0134] Minimizing cytarabine corneal toxicity is also claimed because there is incorporation of molecules with a positive charge on the nitrogen, and PLL-g-PEG and other cationic graft copolymers can minimize this charged interaction by inactivating the negative charges on the surface cells.

[0135] Based on their physical chemistry, copolymers are effective in the embodiments herein because they reach the drug (pharmaceutical), the solution applied to the cells at risk, and the cells themselves, creating a dynamic, protective environment. When applied as droplets, suspensions, solutions, controlled delivery systems, or powders (possibly lyophilized), the graft copolymers are capable of migrating from the surface tissue to the ADC or other pharmaceutical in solution, tears, or extracellular fluids through physicochemical on-off binding involving electrostatic or hydrophobic interactions.

[0136] The experiments described herein predict that the formulations of the present invention will also be useful for preventing viral infection or ADC uptake or interaction with target cells, including, but not limited to, skin, mucous membranes (ocular, nasal, oral), and hair. Thus, these formulations may be applied in accordance with the present invention to epithelial tissues of the eye, respiratory tract, and gastrointestinal tract, mucous membranes, exposed wound surfaces, corneal and conjunctival surfaces, skin, and surgical and traumatic wounds and ulcers. These formulations may serve to protect the skin and other organs from viral infection and unwanted ADC interactions. Benefits may include reduced infection rates, reduced severity of infection, and reduced corneal epithelial toxicity. The copolymers can interact with both infectious agents or pharmaceutical agents and the surface of at-risk cells, inactivating any interactions and providing a protective effect.

[0137] For these human applications, the formulations may be in the form of lotions, gels, liquids, sprays, rinses, dissolvable wafers, or glycerin bars, to which water can be added to solubilize the graft or block copolymers and make them easier to apply. The formulations can be provided as individual or single-use products, or in industrial and / or multi-use dispenser volumes. In addition to the graft or block copolymer, such formulations can include any and all typical binders, excipients, and ingredients found in cosmetic sprays, lotions, soaps, shampoos, cleansers, and oral, nasal, and eye care products.

[0138] The formulations can also be used in accordance with the present invention in animals, including domestic pets, to reduce viral infections or ADC toxicity.

[0139] Other uses for these formulations will become apparent to those of skill in the art upon reading this disclosure, and such uses are encompassed by the present invention.

[0140] Sustained release formulations may prove particularly beneficial and are encompassed herein.

[0141] PLL-g-PEG is an example of a copolymer with bioadhesive and inactive moieties. PLL-g-PEG utilizes electrostatic bioadhesion due to the cationic backbone. The hydrophobic and anionic moieties utilize alternative bioadhesive interactions (hydrophobic, anionic). Formulations of the copolymer are safe for human use.

[0142] The following non-limiting examples are provided to further illustrate the present invention. Percentages are wt / wt %.

[0143] It has been reported that drugs can be secreted into tears. Lee, Brian A., et al. "Clinical and Histological Characterization of Toxic Keratopathy From Depatuxizumab Mafodotin (ABT-414), an Antibody-Drug Conjugate: [RETRACTED]." Cornea (2018). Note: Microcystic Epithelial Keratopathy (MEK). Regarding MEK: "Steroids are not an appropriate treatment." And: "...Confocal microscopy revealed multiple large, round, hyperreflective lesions throughout the epithelium that appeared to correlate with MEK seen clinically. Histologically, small cysts appeared to correlate with apoptotic cells enmeshed throughout the epithelium. In addition to the increased apoptotic cells seen in the histological specimens, immunohistochemistry revealed IgG-positive intracytoplasmic granules in the basal epithelium. Because the antibody component of ABT-414, depatuxizumab, is a monoclonal IgG1, it was suggested that ABT-414 itself was deposited within the basal epithelium. The direct presence of ABT-414 in the epithelium likely explains the increased apoptosis seen histologically."

[0144] Experimental method reference: Zhao, Hui, et al. “Modulation of Macropinocytosis-Mediated Internalization Decreases Ocular Toxicity of Antibody-Drug Conjugates.” Cancer research 78.8 (2018): 2115-2126. Study Design for Cell Culture: Cell Lines and Reagents. All cells were maintained according to the vendor's protocol. Human primary corneal epithelial cells (HCECs) from Life Technologies (Cat. No. C018-5C) were cultured in Keratinocyte SFM (Cat. No. 17005-42). HCECs from ATCC (Cat. No. PCS-700-010) were cultured in Corneal Epithelial Cell Basal Medium (Cat. No. PCS-700-030) supplemented with a Corneal Epithelial Cell Growth Kit (Cat. No. PCS-700-040). Human umbilical vein endothelial cells (HUVECs, Cat. No. C-003-5C) and human dermal fibroblasts, adult (HDFa, Cat. No. C-013-5C) were obtained from Life Technologies. HUVECs were cultured in Medium supplemented with Low Serum Growth Supplement (LSGS, Cat. No. S-003-10). Human hematopoietic stem cells (HSCs) were grown in 200 mL of medium supplemented with LSGS. HDFa cells were grown in 10 mL of medium supplemented with LSGS. KU812 cells were obtained from ATCC (catalog number CRL-2099) and grown in RPMI 1640 + 10% FBS as previously described. Cell lines were passaged in our laboratory less than 6 months after resuscitation. Human cell lines were confirmed to be mycoplasma-negative using short tandem repeat profiling (Promega). Reagents for human hematopoietic stem cells (HSCs) and their differentiation into megakaryocytes have been previously described. T-DM1 (Kadcyla; Genentech / Roche) was purchased from Myoderm. Macropinocytic HSCs (10 cells / well) were grown overnight in 24-well plates and incubated with 1 mg / mL dextran-FITC (10,000 MW, Life Technologies) for 3 hours at 37°C or 4°C as a control.Cells were detached with trypsin and neutralized with neutralizing solution (Life Technologies, catalog no. R002100). Then, cells were washed three times with FACS staining buffer (FBS, BD Pharmingen, catalog no. 554656) and analyzed using an Attune acoustic focusing cytometer (Life Technologies). To examine the effects of 5-(N-ethyl-N-isopropyl) amiloride (EIPA), the indicated amount of EIPA was added to the cell culture 30 minutes before the addition of dextran-FITC. Mean fluorescence intensity ratios (MFIRs) were calculated by normalizing MFI values ​​at 37°C to those at 4°C. 100 mL of proliferation assay HCECs (500 cells / well) and HUVECs (2,000 cells / well) were seeded in collagen-coated 96-well plates (Corning, catalog no. 354650), and 100 mL of HDFa (2,000 cells / well) and KU812 (2,500 cells / well) were grown in 96-well tissue culture plates (Corning assay plates, catalog no. 3903). After 6 days of treatment with ADCs, the viability of treated cells relative to controls was measured using the CellTiter-Glo (CTG) luminescent assay kit (Promega, catalog no. G7572). CTG values ​​were normalized to mock-treated cells on day 6 (% maximum proliferation), and IC50 values ​​were obtained using a sigmoidal dose-response (variable slope) model using GraphPad Prism 6. Assay plates contained technical triplicates for each drug concentration, and the presented data are the average of at least two independent measurements. ANS assay ADC (2 mg / mL) was prepared in PBS and serially diluted 1:2 in a black-walled 96-well plate. An equal volume of 1,8-ANS (1-anilinonaphthalene-8-sulfonic acid, Thermo Fisher Scientific, catalog number A47) was added and incubated at room temperature for 30 minutes. Fluorescence signals were measured (e.g., 390 nm / em. 470 nm). The slope obtained by linear regression analysis was used as the hydrophobicity index. Cells were seeded (0.75 × 10 cells per well) in 8-well chamber slides for confocal microscopy. 5Cells were cultured for 48 hours before treatment and subsequent immunostaining. Next, cells were incubated with AGS-16C3F with or without co-incubation of 0.5 mg / mL Dextran-Texas Red (Molecular Probes; D18653) for 4 hours at 37°C. Inhibition of macropinocytosis was assessed by treating cells with EIPA for 30 minutes before AGS-16C3F / Dextran-Texas Red incubation. After the incubation period, unbound antibody was washed away with PBS, and cells were fixed with 4% paraformaldehyde for 20 minutes at room temperature. Cells were then permeabilized with PBS + 0.1% Triton-X-100 for 15 minutes, and nonspecific labeling was blocked with PBS + 10% normal goat serum. Internalized cytoplasmic AGS-16C3F bound to the cell surface was visualized by incubating cells with Alexa Fluor 488-conjugated goat anti-human IgG (Thermo Fisher Scientific, Catalog No. A-11013). Nuclei were visualized with TO-PRO-3 Iodide (Thermo Fisher Scientific, Catalog No. T3605), and coverslips were mounted for imaging using ProLong Gold Antifade reagent (Thermo Fisher Scientific, Catalog No. P36934). High-resolution laser confocal image sections were acquired using a Leica TCS SP5 II (63x oil immersion objective; NA 1 / 4 1.4) and sequentially scanned to minimize fluorophore crosstalk and false-positive colocalization. " Furthermore, rabbit techniques were also cited from Zhou et al.: "Animal Experiments and Welfare. In vivo xenograft tumor models and pharmacodynamic studies were performed as previously described. All experimental protocols were approved by Agensys' Institutional Animal Care and Use Committee. All procedures in toxicity studies complied with the Animal Welfare Act and regulations (9 CFR 3). Male Dutch Belted [Haz:(DB)SPF] rabbits weighing approximately 1.5-2.0 kg were used for ocular tolerability studies. Rabbits were allowed to acclimate for at least 6 days before the first dose.All animals were housed in individual hanging stainless steel cages, provided with food and water, and maintained under environmental conditions that complied with all animal welfare guidelines. Study drugs were administered intravenously to groups of three to four rabbits via the marginal ear vein, followed by saline flushes once weekly for up to six times (days 1, 8, 15, and 22). General health was assessed by weekly weight measurement and cageside observation. Ocular tolerability was assessed by external examination of the adnexa and anterior segment of each eye using a slit-lamp biomicroscope. Additionally, fundi were examined with an indirect ophthalmoscope after dilation with mydriatic agents. Corneal fluorescein staining was also performed to assess corneal damage. Fluorescein solution (approximately 1 mg / mL) was applied to the cornea with a cotton swab. At necropsy, eyes and other selected tissues were placed in fixative according to established protocols for IHC. All rabbit experiments were performed by Covance Laboratories. ” Standard statistical approaches are used to calculate the results.

[0145] The entire contents of all references, publications and patent documents are incorporated herein by reference.

[0146] Example Results show a significant benefit for the primary endpoint and for several secondary analyses.

[0147] All animal experiments are conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals and Institutional Animal Care and Use Committee (IACUC) policies. Specific protocols are approved by appropriate review boards.

[0148] These experimental results, in whole or in part, demonstrate the utility of the present invention. When scientific data are inconsistent, as they sometimes are, variations in experimental conditions that adequately separate the important variables demonstrate physical benefits.

[0149] In all examples, target cell safety is maintained in the presence of the copolymer. Human and mammalian formulations are well tolerated.

[0150] Example 1: The presence of SARS-CoV-2 viral particles in cultured airway epithelial cells results in extremely high infection rates and subsequent death of these cells. A cationic graft copolymer is added to this system in solution at concentrations of 0.001, 0.01, 0.1, 1, 2, and 3% by weight for a period long enough to allow significant infection rates, e.g., several hours. Viral infectivity is reduced, and cell viability is improved in a dose-response manner. The negative control is simply a solution without any cationic graft copolymer. To demonstrate this beneficial effect, multiple experiments are performed. One involves cell viability measured by cytometry, and the other involves quantitative PCR to measure viral genome copy number. In one experiment, viable airway epithelial cells are counted in solution to obtain the number of viable airway epithelial cells / ml. PLL(20)-g[3.5]-PEG(5) is added in different amounts as a lyophilized powder, and the test concentrations are provided in various wells and cell cultures (standard growth and survival media). Add the same amount of SARS-CoV-2 virus particles (absolute virus particle count) per well. After 24 hours, count the cell viability. Preliminary experiments indicate the optimal number of virus particles to add. Empirical data suggest that 10,000 virus particles per ml are effective for infecting airway cells, resulting in the death of most cultured cells in the well after 24 hours. Solutions containing PLL-g-PEG demonstrated improved cell viability, as shown in Table 1 below.

[0151] [Table 2]

[0152] Another method demonstrating the same efficacy of cationic graft copolymer intervention in the setting of SARS-CoV-2 infection is performed using a similar design, except that the endpoint is viral genome RNA copy number. In this experiment, the same PLL-g-PEG concentration is used, but after exposure and a 2-hour incubation period, all virus and PLL-g-PEG media is washed off from the cells. After 24 hours, the solution is examined for viral RNA copy number. SARS-CoV-2 virus detection is performed using the One Step Prime Script RT-PCR kit on a Light Cycler 480 real-time PCR system with primers: forward primer: 5'-AGAAGATTGGTTAGATGATGATAGT-3'; reverse primer: 5'-TTCCATCTCTAATTGAGGTTGAACC-3'; and probe: 5'-FAM-TCCTCACTGCCGTCTTGTTG ACCA-BHQ1-3'. All experiments are performed in triplicate. Table 2 shows the results.

[0153] [Table 3]

[0154] These studies will be performed in enough well plates (triplicates) to demonstrate statistical significance. This experiment puts into practice the specific option of utilizing cationic graft copolymers to reduce SARS-CoV-2 viral infectivity in at-risk cells and, therefore, tissues and organisms.

[0155] Example 2: A clinical study will be conducted to demonstrate the clinical benefit of this invention. Humans at risk for SARS-CoV-2 virus infection (COVID19) will be treated with topical formulations of PLL-g-PEG. These formulations are liquid-based. Individuals at risk for SARS-CoV-2 virus infection due to high-risk exposure in the workplace will be enrolled in the study. 1,000 patients will be enrolled in a 1:1:1 randomization scheme. The study is double-blind. Dose A will be PLL-g-PEG as follows: 0.1% eye drops, 0.1% nasal spray, and 0.1% mouthwash; Dose B will be 0.5% eye drops, 0.5% nasal spray, and 0.5% mouthwash. Cohort C will receive saline only in the eye drops, nasal spray, and mouthwash. Participants were equally stratified by gender, age (over 60 years, under 60 years), and whether they were frontline emergency medical personnel in a large-scale hospital setting for acute care of COVID-19, or first responders to police and emergency medical technician calls. All participants tested negative for SARS-CoV-2 at enrollment and were provided with the solution to use. Instructions were to apply to the ocular, nasal, and oral mucosa immediately before entering the risk environment. Application should be repeated every four hours as needed based on exposure risk. PLL-g-PEG formulation or saline, along with standard precautions, were used. The study period was two weeks for treatment or control. Four-week endpoints included: A) the number of infected individuals in treatment or control groups after initiation of treatment, and B) the severity of the disease, graded as 1 (resolved without hospitalization), 2 (required hospitalization), and 3 (required ICU or death). In a secondary experiment, swab tests demonstrated statistically equivalent amounts of viral particles on patients' personal protective equipment and inanimate surfaces, such as counters and bedside tables in their rooms. Despite the personal safety precautions taken by at-risk individuals, mucosal exposure remains significant. The use of cationic graft copolymer formulations prevents infection in at-risk individuals, indicating a reduction in infection rates and disease severity.

[0156] The results are shown in Table 3.

[0157] [Table 4]

[0158] Note that the in vivo and in vitro experiments utilize the text and design of Zhou et al.

[0159] Example 3: ADC in vitro The ADC is AGS-16C3F, an antibody-drug conjugate against ectonucleotide pyrophosphatase / phosphodiesterase 3 (ENPP3) containing a mcMMAF linker-payload for the treatment of metastatic renal cell carcinoma. AGS-16C3F or rituxumab-mcMMAF can be used as the investigational drug (ADC that binds to CD20), or other ADCs can be used.

[0160] Other ADCs have been reported to cause ocular toxicity in patients, but the mechanisms are still being elucidated. The present invention is an approach based on experiments and new ideas regarding this toxicity.

[0161] Human primary corneal epithelial cells were cultured in Keratinocyte SFM, and ATCC HCEC cells were cultured in Corneal Epithelial Cell Basal Medium supplemented with a Corneal Epithelial Cell Growth Kit. Human cell lines were confirmed to be mycoplasma-negative using short tandem repeat profiling. Cells were seeded into multiple 8-well chamber slides and cultured for 48 hours before treatment and subsequent immunostaining. Cells were treated with a negative control (no copolymer) and also with just PLL and PEG (not grafted together). Other cell cultures are exposed to PLL-g-PEG (PLL(20-g[3.5]-PEG(5)) as the primary example. Other PLL-g-PEGs are tested as well. The concentrations of PLL-g-PEG are 0.001 wt%, 0.01 wt%, 0.1 wt%, 0.3 wt%, 0.5 wt%, and 1 wt%. Keratocytes can retain viability after treatment with PLL-g-PEG, but in an in vitro experimental setting, some keratocytes may die during the experiment. Next, the keratocytes are incubated with AGS-16C3F (or rituxumab-mcMMAF or another ADC) with or without co-incubation of 0.5 mg / mL Dextran-Texas Red at 37 °C for 4 h. Inhibition of macropinocytosis as a control is measured using AGS-16C3F (or another ADC) / Dextran-Texas Red. Cell surface binding and internalized AGS-16C3F (or other ADCs) are visualized by incubating cells with Alexa Fluor 488-conjugated goat anti-human IgG. Nuclei are visualized with TO-PRO-3 Iodide, and coverslips are mounted using ProLong Gold Antifade reagent for imaging.High-resolution laser confocal image sections were acquired using a Leica TCS SP5 II (63x oil immersion objective; NA 1 / 4 1.4), which were scanned sequentially to minimize fluorophore crosstalk and false-positive colocalization.

[0162] Prepare ADC (2 mg / mL or other concentrations) in PBS and perform 1:2 serial dilutions in a black-walled 96-well plate (21, 23). Add an equal volume of 1,8-ANS and incubate at room temperature for 30 minutes. Measure the fluorescent signal.

[0163] result: Upon microscopic examination, numerous, poorly stained ADCs penetrated into the copolymer-treated epithelial cells. A dose response was shown.

[0164] We use models and designs, but we do not, do not, or have ever obtained ideas or intellectual property rights from Zhou or any other authors of the cited documents.

[0165] See Table 4.

[0166] [Table 5]

[0167] Therefore, PLL-g-PEG, and thus mimetics, are effective interventions to reduce corneal toxicity associated with ADCs.

[0168] Megakaryocyte experiments and the use of mimics are also effective.

[0169] Although experiments will be conducted with ADCs, tubulin-disrupting agents, and other cytotoxic agents, this effectiveness is not limited to a single ADC. In fact, all ADCs with corneal epithelial toxicity can be mitigated with this approach. This approach is ADC-independent and provides a method for reducing the corneal toxicity of this class effect.

[0170] PLL-g-PEG can also be added slightly after the ADC is added to the solution, so in some embodiments, the benefits remain the same if the ADC exposure precedes the PLL-g-PEG exposure.

[0171] Example 4: ADC in vivo Corneal toxicity associated with ADCs is reduced in in vivo models. Although animal models for ADC toxicity are imperfect (the cynomolgus monkey model is not optimal), rabbits are commonly used to test potential drug-mediated ocular toxicity and are chosen to investigate the ocular toxicity of ADCs. AGS-16C3F and other ADCs are useful for these experiments. In vivo animal toxicity, while very common in humans, is somewhat variable as an animal model. Rabbits exhibit variable toxicity with various ADCs. For this experiment, multiple ocular experiments testing AGS-16C3F (an antibody-drug conjugate against ectonucleotide pyrophosphatase / phosphodiesterase 3 (ENPP3) containing a mcMMAF linker-payload for the treatment of metastatic renal cell carcinoma) and other ADCs with tubulin inhibitory payloads will be conducted in rabbits. Five rabbits will be dosed per group. There is a non-ADC-treated control group that showed no toxicity at doses of 10 and 15 mg / kg of anuAGS-16C3F. Rabbits treated with 15 mg / kg exhibited reversible conjunctival hyperemia, pericorneal haze, corneal edema, and ciliary body flushing. Similarly, once-weekly administration of 10 mg / kg resulted in ocular toxicity. Compared to no exposure to PLL-g-PEG via topical treatment at the start of ADC administration, this study demonstrated reduced ocular toxicity in rabbits treated with 0.5%, 1%, and 2% PLL-g-PEG topical eye drops three times daily. The 0.5%, 1%, and 2% PLL-g-PEG dose groups, respectively, showed average benefit of >5%, >7.5%, and >10%. Daily administration was performed with PLL-g-PEG eye drops and control. ADC injections were administered weekly. PLL-g-PEG also showed beneficial effects one week after ADC administration, but the benefit decreased by approximately 20% in each group by the end of the study. Animals treated with PLL-g-PEG experienced less ADC-associated toxicity compared with controls (artificial tears only). Both formulations contained no preservatives. Preservative-containing formulations were effective but could have the undesirable effect of causing or worsening corneal epitheliopathy. However, rabbit models have shown beneficial effects even with the use of preservative-containing solutions.

[0172] Subconjunctival formulations also provide protection when delivered subconjunctivally once weekly.

[0173] Although intravenous formulations are safe, tolerable, and effective, local delivery is more effective.

[0174] Results will be recorded by laboratory findings (including staining) and histopathology (reduction of infiltration and tissue damage) on days 21 and 42.

[0175] The use of PLL-g-PEG solution improves recovery time in treated animals.

[0176] Further experiments with different polymer configurations are also valid.

[0177] It should be noted that while the findings in rabbits do not identically replicate those in humans with pycnotic keratosis, this model demonstrates anterior corneal abnormalities both clinically and histopathologically. We selected sample ADCs and attempted to replicate the results with other ADCs, including tubulin inhibitors.

[0178] See Table 5 [Table 6]

[0179] Example 5: ADC in humans This study will be conducted in patients receiving ADCs for oncological conditions, using ADCs with known corneal toxicity. The commercially available ADCs will be utilized in the clinical treatment setting of approved ADCs, as well as in patients receiving unapproved ADCs under clinical investigation as part of the regulatory approval process. Therefore, this randomized controlled trial will include patients receiving multiple different ADCs. Stratification will be based on the ADC and existing ocular discomfort.

[0180] Empirically, a total of 25 subjects per group is deemed sufficient to detect differences in ophthalmologic findings, so 75 patients will be enrolled.

[0181] All patients will be enrolled at the start of treatment. Patients will be randomized to receive preservative-free artificial tears only, 1% PLL-g-PEG eye drops (preservative-free), or 2% PLL-g-PEG eye drops (preservative-free). All patients will be instructed to administer the eye drops in both eyes four times daily, starting one day before ADC administration in cycle 1. Ophthalmologic examinations will be performed at baseline and at each cycle through cycle 4. Each cycle is 21 days long. After cycle 4, patients randomized to a treatment group may continue receiving PLL-g-PEG. Patients randomized to artificial tears may cross over to PLL-g-PEG eye drops.

[0182] The severity of corneal microcystic epitheliopathy or superficial punctate keratopathy or keratopathy is assessed using objective analysis to estimate the density and area of ​​epitheliopathy and / or assessment of epithelial damage by staining with vital dyes such as fluorescein. A score is calculated by multiplying the percentage of corneal damage by a correction factor (1-3) for overall clinical severity. The endpoint of the study is the worst score achieved during the study period.

[0183] Visual acuity is measured using logMAR.

[0184] The primary analysis will use the worst eye for each patient.

[0185] Also, observe both eyes independently.

[0186] Adverse events will be recorded.

[0187] The crossover group will be evaluated for symptom improvement.

[0188] The results are as follows: [Table 7]

[0189] Adverse events of blurred vision and eye irritation were most severe in the control group and less severe in the low and high dose groups.

[0190] The rate of drug withdrawals and dose delays in each group was highest in the control group and lower in the low and high dose groups.

[0191] PLL-g-PEG-treated eyes had less superficial corneal punctate keratitis. Visual acuity was, on average, better in eyes and patients treated with PLL-g-PEG (or copolymer).

[0192] This experiment exemplarily demonstrates the clinical value of this intervention with a PLL-g-PEG solution applied topically to the eye to reduce the adverse effects of ADC on the cornea.

[0193] Concurrent intravenous delivery of 1% PLL-g-PEG solution per dosing cycle resulted in less thrombocytopenia in treated subjects, thereby demonstrating a broader effect of reducing ADC toxicity to systems extending beyond the corneal epithelium, thus supporting the broader patent claim.

[0194] These experiments are successfully repeated with other copolymers.

[0195] Example 7 Various novel ADCs will be preclinically evaluated for uptake into human corneal epithelial cells in the presence of PLL-g-PEG formulations, and those specific ADCs that show the greatest benefit (greatest reduction in uptake) in the presence of PLL-g-PEG can be advanced to the clinic. One reason for their selection is that there are now known and effective clinical methods for treating, mediating, mitigating, reducing, and preventing ADC toxicity in that cell type by co-treatment with the claimed copolymers discussed herein.

[0196] Three variations of ADCs targeting cellular receptors found in oncological diseases with unmet medical needs are evaluated in the laboratory. All show significant uptake by macropinocytosis into human corneal epithelial cells. Next, PLL-g-PEG solutions at various concentrations (ranging from 0.01 to 3 wt%) are used in similar in vitro tests of ADC uptake into human epithelial cells. Potential toxicity of the ADCs is reassessed. Concentrations of PLL-g-PEG above 0.01% (not necessarily including the endpoints) were found to significantly reduce the in vitro uptake of one of the three ADCs, at a rate that can vary based on the ADC, yet remain effective. This effect is confirmed in an in vivo rabbit model. Thus, because methods for topical application of PLL-g-PEG are known to minimize uptake, this molecule may advance to the clinic more quickly than other molecules. Other bioadhesive / inactivating copolymers may also be used in ADC development, similar to the experiments described above. Preferred embodiments of the present invention are described below. Embodiment 1: A method of reducing adverse events associated with the use of antibody drug conjugates that damage non-neoplastic cells (which exhibit the adverse effects) by applying to cells and tissues (which are adversely affected by the use of the antibody drug conjugate) an effective amount of a copolymer having electrostatic and steric mediation properties. Aspect 2: The method of aspect 1, wherein the copolymer is selected from a cationic graft copolymer, a cationic block copolymer, a hydrophobic graft copolymer, a hydrophobic block copolymer, an anionic graft copolymer, and an anionic block copolymer. Embodiment 3: The method of embodiment 1, wherein the copolymer is PLL-g-PEG. Aspect 4: The method of aspect 1, wherein the copolymer is selected from the list of combinations described in the present application supra. Aspect 5: A method of reducing corneal epithelial toxicity associated with ADCs by administering a copolymer having bioadhesive and inactivating components to cells at risk for off-target drug uptake. Embodiment 6: The method of embodiment 5, wherein the copolymer is applied to corneal epithelial cells and conjunctival epithelial cells. Embodiment 7: The method of embodiment 6, wherein the copolymer is PLL-g-PEG. Aspect 8: The method of aspect 5, wherein the agent is a solution for delivery to the subconjunctival space. Embodiment 9: A method for reducing an adverse event associated with the use of an antibody drug conjugate in a human, comprising applying an effective amount of a copolymer having electrostatic and steric mediation properties to a cell involved in said adverse event. Embodiment 10: The method of embodiment 9, wherein the copolymer is selected from a cationic graft copolymer, a cationic block copolymer, a hydrophobic graft copolymer, a hydrophobic block copolymer, an anionic graft copolymer, and an anionic block copolymer. Embodiment 11: The method of embodiment 10, wherein the copolymer is selected from the polymers in this disclosure listed above. Aspect 12: A method of reducing microcystoid epithelial toxicity associated with cytotoxins cleaved from ADCs by applying to corneal epithelial cells an effective amount of a copolymer having bioadhesive and inactivating properties. Embodiment 13: The method of embodiment 12, wherein said copolymer is PLL-g-PEG. Embodiment 14: A method of reducing the rate and severity of ocular adverse events associated with ADC use by delivering a copolymer having bioadhesive and inactivation sites to the eye prior to the initiation of systemic ADC therapy. Embodiment 15: A method of ameliorating signs and symptoms of ocular adverse events associated with ADC use by delivering a copolymer having bioadhesive and inactivation moieties to the eye after initiation of systemic ADC therapy. Embodiment 16: A method of reducing uptake of an ADC into limbal stem cells, transiently amplifying cells, basal epithelial cells, wing cells, and corneal epithelial cells by exposing the cells to a copolymer having bioadhesive and inactivation moieties prior to exposure to the ADC. Embodiment 17: A method of reducing uptake of an ADC into limbal stem cells, transient amplifying cells, basal epithelial cells, wing cells, and corneal epithelial cells by exposing the cells to a copolymer having bioadhesive and inactivation moieties after exposure to the ADC. Embodiment 18: A method of reducing macropinocytotic uptake of an ADC into any of limbal stem cells, limbal epithelial cells, limbal epithelial daughter cells, transient amplifying cells, basal epithelial cells, wing cells, corneal epithelial cells, and differentiated corneal epithelial cells by exposing said cells to a copolymer having bioadhesive and inactivation moieties before or after exposure to an ADC, wherein the copolymer has an effective weight / weight ratio, and Embodiment 19: A method of treating a patient with a copolymer having bioadhesive and inactivation sites, wherein an effective amount is used to reduce ocular adverse events associated with an ADC. Embodiment 20: A method of using copolymers that exhibit electrostatic and steric interactions at the cellular level to minimize adverse effects resulting from exposure of said cells to an agent selected from SARS-Cov-2, a novel virus, an epidemic virus, an ADC with a cytotoxic payload that may lead to human pathology and disease. Embodiment 21: A method of reducing ocular toxicity from systemic exposure in humans to an ADC having a tubulin disrupting agent as payload by treating the eye with an effective amount of a cationic graft copolymer formulation. Embodiment 22: The method of embodiment 21, wherein the cationic graft copolymer is PLL-g-PEG. Aspect 23: The method of aspect 22, wherein said treatment is administered by an eye drop formulation. Aspect 24. The method of Aspect 23, wherein the formulation is preservative-free. Aspect 25: The method of Aspect 21, wherein the cationic graft copolymer is PLL-g-PEG at a concentration ranging from 0.01% to 5% by weight in the ophthalmic formulation. Aspect 26: A method of reducing ADC withdrawal and dose reduction in the treatment of human malignancies by applying an effective amount of a PLL-g-PEG eye drop formulation to the eye to reduce corneal adverse events and ocular safety concerns in at-risk patients. Embodiment 27: An antibody directed to an ADC with corneal toxicity, the antibody having an inactivation site and delivered to the eye in an amount effective to reduce ocular adverse events. Aspect 28: A method for reducing corneal cell toxicity caused by a pharmaceutical agent selected from the group consisting of ADCs, biological agents, small molecules, polymers, and peptides, comprising topically applying a copolymer having bioadhesive and inactivating components to an eye at risk of adverse effects caused by said pharmaceutical agent.

Claims

1. Use of a copolymer having electrostatic and steric mediator properties in the manufacture of a formulation for reducing adverse events associated with the use of an antibody-drug conjugate that damages non-neoplastic cells, wherein the formulation is applied to cells or tissues that suffer adverse effects from the use of the antibody-drug conjugate in an effective amount of the copolymer, the copolymer being PLL-g-PEG, and the cells or tissues being corneal epithelial cells or corneal epithelial tissue.

2. 1. Use of a copolymer having electrostatic and steric mediating properties in the manufacture of a formulation for reducing adverse events associated with the use of an antibody-drug conjugate in a human, wherein the formulation is applied to cells or tissues involved in the adverse events in an effective amount of the copolymer, the copolymer being PLL-g-PEG, and the cells or tissues being corneal epithelial cells or corneal epithelial tissue.

3. 1. Use of a cationic graft copolymer in the manufacture of a formulation for reducing ocular toxicity resulting from systemic exposure in humans to an ADC having a tubulin disrupting agent as a payload, wherein the formulation is applied to cells or tissues involved in the ocular toxicity in an effective amount of the cationic graft copolymer, wherein the cationic graft copolymer is PLL-g-PEG, and the cells or tissues are corneal epithelial cells or corneal epithelial tissue.

4. The use according to claim 3, wherein the formulation is an eye drop formulation.

5. 4. The use according to claim 3, wherein the formulation is preservative-free.

6. The use according to claim 4, wherein the formulation comprises PLL-g-PEG at a concentration ranging from 0.01% to 5% by weight in the eye drop formulation.

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