A method for minimizing adverse effects mediated by external influences on cells, tissues, organ systems, and living organisms by utilizing the bioadhesion and steric interactions of copolymers having at least two sites.

Abstract

This invention provides a method for minimizing adverse effects mediated by external influences on cells, tissues, organ systems, and living organisms. [Solution] A method is provided for treating with an effective amount of a graft or block copolymer having cationic, hydrophobic, or anionic sites and hydrophilic inactivation sites. The copolymer is PLL-g-PEG. The copolymer exhibits bioadhesion through electrostatic and hydrophobic interactions, as well as inactivation by the hydrophilic sites. The copolymer is useful for reducing the viral infection rate of 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 tolerable. Usefulness and benefits can be obtained by treating epithelial cells and surfaces, including precursor or stem cell corneal epithelial cells, with the copolymer.

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 addresses significant problems currently faced in the biomedical field by using bioadhesive and inactivated copolymers (including 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 toxicities associated with antibody-drug conjugates (ADCs) and their toxic payloads. "ADC" is frequently used herein to represent antibody-drug conjugate therapeutics. An ADC is a complex molecule composed of an antibody conjugated to a biologically active cytotoxic (anticancer) payload or agent. Antibody-drug conjugates can be a type of bioconjugate and immunoconjugate. "ADC" can mean the host of various antibody-drug conjugates herein. An ADC combines the targeting ability inherent in monoclonal antibodies with the ability of cytotoxic agents to fight cancer. ADCs are often designed to be able to distinguish between healthy cells and tissues and diseased cells and tissues.

[0003] The viral infection that is particularly notable here is the infectivity of SARS-CoV-2. However, an important advance is that this effect is also effective against novel viruses, such as being able to suppress the infectivity of novel viruses to cells, tissues, or organisms where the host immune system is not yet developed or target antibodies and antiviral therapies are not yet available, through steric and electrostatic interactions. That is, this effect is broad, effective, and non-specific. Novel viruses are suitable for treatment by this approach.

[0004] Methods for mitigating the toxicity of ADCs are particularly related to the off-target entry of ADCs into non-neogeneic cells and adverse events associated with the inhibition of cellular processes mediated by the ADC payload. Neogeneic 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, leukocyte cancer, urethral cancer, and mesenteric lymphadenitis. Any or all of the cancers named herein and / or not named herein may be combined with claims relating to specific inventions of ADCs targeting such cancer cells or tissues.

[0005] Specifically, macropinocytosis-mediated toxicity is inhibited, reduced, and limited by steric and electrostatic interference acting at the molecular-cell-tissue (integrated or isolated) level, where the surface of a cell or tissue interacts with its local microenvironment. Cells include, but are not limited to, marginal stem cells, transient amplified cells, transient amplified cell daughter cells, basal epithelial cells, alar cells, and corneal epithelial cells and differentiated corneal epithelial cells.

[0006] The primary underlying polymer structure that is most useful (but not the only useful embodiment) in both settings is the cationic graft copolymer. This basic structure comprises a cationic backbone and grafted hydrophilic side chains. A typical example of such polymers is poly(L)lysine grafted (poly)ethylene glycol. Other molecular structures also achieve the interactions necessary to confer benefits. Other polymers may utilize charge, hydrophobic and deactivating sites to achieve these effects, and these polymers are the subject of this specification. Therapeutic methods are carried out by applying the aforementioned polymers (whether in a dissolved state or not) to cells, tissues, organs, living organisms, or mammals in amounts and for durations effective in achieving the intended beneficial effects.

[0007] Therefore, there is a need to reduce the severity and risk associated with novel viral diseases that spread to human populations and affect 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 in vitro coating of non-biological surfaces, coating of medical devices, and in the treatment of dry eye. An 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 chain has multiple positive charges and spontaneously adsorbs to negatively charged surfaces, while PEG is a hydrophilic polymer that functions as an unbound domain. The PEG sites inactivate surfaces, while the PLL sites adhere to charged components of cell membranes, antibodies, viruses, or viral proteins through electrostatic interactions. PLL-g-PEG is used in in vitro surface inactivation, experimental coating of medical devices, and as eye drops for dry eye lubrication and tear film stabilization. Far exceeding the treatment of dry eye, this invention represents the first identified approach to directly mitigate ADC corneal toxicity induced by 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 studied, researched, or put into practical use prior to this invention. Similarly, to the applicant's knowledge, no other effective polymer molecules have been studied or put into practical use in these respects prior to this invention. Multiple configurations of cationic graft copolymers effective in inhibiting viral infectivity and / or ADC-associated toxicity exist. This specification examines and addresses these alternative molecular approaches.

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

[0012] One aspect of the present invention relates to a method for preventing or reducing drug toxicity associated with ADCs to off-target cells and tissues. In particular, the present invention relates to a method for reducing the severity of corneal epithelial cytotoxicity in the setting of ADC therapy. Charged graft copolymers are safe and effective when applied to cells and tissues at risk to prevent or reduce corneal toxicity associated with ADCs. Exposure of human corneal epithelial cells, such as basal epithelial cells and their precursors, alar cells, and surface epithelial cells, to the cytotoxic payload carried on the ADC is reduced by the use of cationic graft copolymers for treatment. Exposure of epithelial cells to an effective amount of graft copolymer in a soluble state is effective in reducing the severity of corneal epithelial toxicity. The reduction in severity can be expressed on laboratory findings as a decrease in microcystic epithelial keratopathy, a decrease in the severity of punctate surface staining, a decrease in the incidence of epithelial abnormalities, a decrease in adverse ocular 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 present invention disclosed herein is a method for reducing viral infectivity by treating a tissue involved in transfection with an effective amount of a graft or block copolymer having cationic, hydrophobic, or anionic sites and hydrophilic inactivation sites. In one embodiment of the method, the copolymer is PLL-g-PEG. In another embodiment of the method, the graft copolymer of the formulation comprises a cationic backbone and water-soluble and nonionic side chains. In another embodiment of the method, the block copolymer of the formulation comprises at least one cationic block and at least one water-soluble and nonionic block. In another embodiment of the method, the block copolymer of the formulation comprises at least one hydrophobic block and at least one water-soluble and anionic, cationic, or nonionic block. In any of the above embodiments, the biological surface to which the copolymer formulation is administered is a mucous membrane selected from the ocular mucosa, oral mucosa, nasal mucosa and respiratory tract mucosa, respiratory tract epithelium, urinary tract mucosa, and gastrointestinal mucosa of the subject. In any of the above embodiments, the biological surface to which the copolymer formulation is administered is the surface of the eye. In any of the above embodiments, the viral infection is selected from coronaviruses, influenza viruses, Ebola viruses, and novel viruses transmitted by mucosal exposure, for example, the virus is SARS-CoV-2, but is not limited thereto. In any of the above embodiments, the graft copolymer or block copolymer of the formulation accounts for 0.001 to 40% of the formulation. In any of the above embodiments, the graft copolymer or block copolymer of the formulation accounts for 0.1 to 10% of the formulation. In any of the above embodiments, the inactivating effect is based on the interference of the SARS-CoV-2 spike protein and ACE2 receptors on cells at risk. 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 applying an effective amount of a copolymer having electrostatic and steric mediating properties applicable to cells affected by the toxicity in an off-target uptake pathway that damages non-nascent cells. In one embodiment of this aspect, the copolymer is selected from cationic graft copolymers, cationic block copolymers, hydrophobic graft copolymers, hydrophobic block copolymers, anionic graft copolymers, and anionic block copolymers. In one embodiment of this aspect, the copolymer is formulated in one or more approaches from powder, solution, suspension, topical, intravenous, oral, 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 in the case of 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 this application above.

[0015] In one ophthalmic aspect of the present invention, beneficial effects can be observed with topical treatment of the eye using ophthalmic drug products and / or commercially available formulations containing the copolymer described herein, at frequencies of once daily (one drop per eye per day), twice daily (two drops per eye per day), three times daily (three drops per eye per day), four times daily (four drops per eye per day), or up to every hour or more. Dosage may be as infrequent as once daily or less per affected eye. Dosage may be as needed or on a case-by-case basis. The range of necessary dosing frequencies may depend on the patient and the ADC.

[0016] In another aspect of the present invention, a method for reducing corneal epithelial toxicity associated with ADCs is disclosed herein, 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 into the subconjunctival space.

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

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

[0019] In another aspect of the present invention, a method for reducing the rate and severity of ocular adverse events associated with ADC use is disclosed herein, by delivering a copolymer having bioadhesive and inactivating sites, including a graft or block copolymer having cationic, hydrophobic, or anionic sites and hydrophilic inactivating sites, to the eye before initiating systemic ADC therapy.

[0020] Adverse eye events include, but are not limited to, corneal epithelial cell death, punctate superficial keratopathy or keratitis, corneal scarring, corneal infection, microcystic keratopathy, corneal cell damage, corneal cell apoptosis, eye irritation, foreign body sensation in the eye, blurred vision, eye pain, difficulty with visual tasks, photophobia, keratopathy, and corneal epitheliopathy.

[0021] In another aspect of the present invention, a method is disclosed herein for improving signs and symptoms of adverse ocular events associated with ADC use by delivering a copolymer having bioadhesive and inactivating sites, comprising a graft or block copolymer having cationic, hydrophobic, or anionic sites and hydrophilic inactivating sites, to the eye after initiation of systemic ADC therapy. Signs and symptoms include, but are not limited to, symptoms related to visual acuity, blurred vision, irritation, redness, and ophthalmic findings. Visual acuity is improved in eyes treated with the copolymer formulation.

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

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

[0024] In another aspect of the present invention, a method of reducing the uptake of an ADC by corneal epithelial cells (in any of culture, experimental models or in vivo) is disclosed herein by exposing the corneal epithelial cells to a copolymer having a bioadhesive and inactivating site, the copolymer comprising a graft or block copolymer having either a cationic, hydrophobic or anionic moiety and a hydrophilic inactivating moiety, prior to exposure to the ADC.

[0025] In another aspect of the present invention, a method of reducing the uptake of an ADC by corneal epithelial cells is disclosed herein by exposing the corneal epithelial cells to a copolymer having a bioadhesive and inactivating site, the copolymer comprising a graft or block copolymer having either a cationic, hydrophobic or anionic moiety and a hydrophilic inactivating moiety, after exposure to the ADC.

[0026] In another aspect of the present invention, a method of reducing the uptake of an ADC by corneal epithelial cells by macropinocytosis (or more generally pinocytosis) is disclosed herein by exposing corneal epithelial cells to a copolymer having a bioadhesive and inactivating site, the copolymer comprising a graft or block copolymer having either a cationic, hydrophobic or anionic moiety and a hydrophilic inactivating moiety, either before or after exposure to the ADC, wherein a formulation having an effective percentage based on weight / weight calculation of the copolymer is used.

[0027] In another aspect of the present invention, a method of reducing ocular adverse events associated with an ADC is disclosed herein by treating a patient with a copolymer having a bioadhesive and inactivating site, the copolymer comprising a graft or block copolymer having either a cationic, hydrophobic or anionic moiety and a hydrophilic inactivating moiety, wherein an effective amount is used. The treatment is topical in some embodiments.

[0028] In another aspect of the present invention, a method is disclosed herein for minimizing the deleterious effects resulting from exposure of cells to factors including an ADC having a cytotoxic payload that can cause SARS-Cov-2, novel viruses, viruses in an epidemic state, human pathological conditions and afflictions, using a copolymer that exhibits electrostatic and steric interactions at the cellular level and includes a graft or block copolymer having a cationic, hydrophobic or anionic moiety and a hydrophilic deactivating moiety.

[0029] In another aspect of the present invention, a method is disclosed herein for reducing ocular toxicity due to systemic exposure of humans 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 a cationic, hydrophobic or anionic moiety and a hydrophilic deactivating moiety. In one embodiment of this aspect, the cationic graft copolymer is PLL-g-PEG. In one embodiment of this aspect, the treatment is performed with an eye drop 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 in the range of 0.01 wt% to 5 wt% in the eye drop formulation. In one embodiment of this aspect, a low-toxicity preservative is selected from sodium perborate, stabilized oxychloro complexes, disappearing preservatives, hydrogen peroxide-based preservatives.

[0030] In another aspect of the present invention, a method for reducing the discontinuation and dose reduction of ADCs in the treatment of human malignancies is disclosed herein, by applying an effective dose of a PLL-g-PEG eye drop formulation to the eye in a patient at risk to reduce corneal adverse events and alleviate concerns about ocular safety. In another embodiment of the present invention, ocular toxicity can be mitigated by topical treatment with a copolymer containing a graft or block copolymer having cationic, hydrophobic, or anionic sites and hydrophilic inactivation sites, if the toxicity is due to secondary effects of cytotoxicity on tear secretion and corneal epithelium. Another aspect is to utilize the method herein to reduce the risk of adverse outcomes or adverse courses due to corneal drug-related toxicity, selected from the effects of reducing the incidence of epithelial erosion, corneal ulcers, corneal epitheliopathy, punctate epitheliopathy, superficial corneal changes, inflammation or other changes of the corneal strom, and secondary bacterial infections.

[0031] In this specification, “to suffer an adverse effect” may mean, but is not limited to, a change from normal physiology and the function of cells, tissues, organs, or living organisms, including, but is not limited to, a decrease in cell viability, initiation of apoptosis, a decrease in the proliferation capacity of normally dividing cells, a decrease or improperity of cell adhesion and migration, changes in adhesion and junctions, including tight junctions, an inflammatory response to cells or tissues, changes in cell metabolism and catabolism, damage to cell bystanders, changes causing pain or discomfort, or visual changes, or other functional changes of organs, tissues, or living organisms, changes causing irregularities in the tear film, changes resulting in the uptake of fluorescein dye or other biopigments used for epithelial cell evaluation (lysamine green, rose bengal), eye damage, and changes affecting the health of normal cells. Adverse events include, but are not limited to, medical problems, cellular health problems, tissue health problems, organ health problems, or living organism health problems that occur during treatment with a drug, medicine, or other therapy. In this specification, drugs and medicines may be used interchangeably.

[0032] In this specification, the inactivation site can be a hydrophilic site, and is typically considered to be a hydrophilic site, but in alternative sites, inactivation is also possible in properties where hydrophilicity is not dominant.

[0033] In another embodiment of the present invention disclosed herein, the antibody is designed for ADCs with corneal toxicity, and the antibody further comprises an inactivation site, which is delivered to the eye in an amount effective to reduce ocular adverse events, such that the antibody having the inactivation site specifically and directly interacts with molecular components of the ADC that interfere with the activity of the ADC and / or the 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 resulting from the adverse effects of a drug, wherein the inhibition is mediated by a nonspecific (electrostatic or hydrophobic) interaction between a copolymer described herein, which includes a graft or block copolymer having cationic, hydrophobic, or anionic sites and hydrophilic inactivation sites, and a cytotoxic drug, thereby reducing the adverse effects of the drug having corneal toxicity through the effect of the inactivation sites. In some embodiments, the drug causing the corneal adverse events is an ADC, and in other embodiments, the drug or pharmaceutical is a low molecular weight. The pharmaceutical may be a biological substance, an antibody, and an antibody-drug conjugate, a high molecular weight or a low molecular weight. A low molecular weight drug is any organic compound that affects a biological process with a relatively low molecular weight of 900 daltons or less. A high molecular weight drug is an organic compound that affects a biological process with a relatively low molecular weight of 900 daltons or more. The high molecular weight may be an organic compound, a PEGylated compound, a protein, a biological substance, or a peptide. The peptide may be a low molecular weight or a high molecular weight depending on the molecular weight of the molecule. In the context of pharmaceuticals as defined herein, a biological substance or biological agent is a product produced from or containing components of a living organism.

[0035] In some embodiments, the agent is selected from the group of cationic amphiphilic agents, amiodarone, aminoquilon, chloroquine, hydroxychloroquine (plaquenil), amodiaquine, mepacrine, tafenoquine, thorazine, tamoxifen, NSAIDs, ibuprofen, indomethacin, naproxen, benoquine, atovaquone, suramin, tyrolone, perhexylline maleate, gentamicin, tobramycin, clarithromycin, ciprofloxacin, clofazimine, gold salts, vandetanib, and osimertinib, and the small molecule can be selected from kinase inhibitors, erlotinib, and cytarabine, but is not limited to these. It can reduce or prevent keratopathy pylorus, corneal deposition, epitheliopathy, keratopathy pylorus, corneal lines, Hudson-Steely lines, crystalline deposition, interstitial inflammation, and corneal opacity. For pharmaceutical agents associated with marginal stem cell dysfunction toxicity, it can prevent marginal stem cell dysfunction. It can reduce or prevent pannus, corneal neovascularization, and corneal conjunctival epithelialization. Although not limited to a specific method, copolymers inactivate cytotoxic agents found in tears and / or reaching marginal stem cells from the marginal circulation.

[0036] In another embodiment, this method reduces adverse events associated with the corneal toxicity of any drug by treating cells at risk with a copolymer or a solution applied to those cells.

[0037] In another aspect, this method reduces adverse events associated with the use of antibody-drug conjugates by applying an effective amount of copolymer to a solution that exposes non-nascent cells to damage, or, if such off-target pathways do not exist and toxicity is more direct, to target uptake, similarity of the cell receptor to the ADC binding site, or warhead entry into cells at risk.

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

[0039] The copolymers of the present invention have bioadhesive sites (cationic, anionic, hydrophobic) and inactivating sites (hydrophilic, sometimes chemically inactive, where "chemically inactive" means not having the ability to react significantly with cells or proteins covalently, electrostatically, or hydrophobically), thereby reducing the interaction between viruses and ADCs and cells damaged by infection or toxicity. Each site can have various MW sizes, and combinations of polymers can be utilized. Formulations having multiple copolymer structures can be used. The polydispersity between each polymer is known and expected during manufacturing, and this does not reduce the effectiveness.

[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 polyfunctional copolymers, which are incorporated herein by reference. U.S. Patent Application Publication 2004 / 0181172 was also considered, which discusses the collection of tears for tear analysis.

[0041] The method step of the present invention includes applying a copolymer having bioadhesive and inactivation sites to cells and tissues at risk.

[0042] A preferred embodiment of the present invention involves applying PLL-g-PEG to at-risk cells and tissues, preferably before exposure, but its use after exposure provides benefits in reducing the severity of adverse findings (reducing secondary epithelial cell damage or repeated exposure or infection or reinfection) and the degree of adverse events (minimizing continued uptake by ADCs).

[0043] While not limited to the specific mechanism of ADCs, on-target toxicity includes antibody / receptor-mediated uptake by epithelial cells that can lead to adverse events, and this approach can also reduce this on-target toxicity (particularly in local environments). Embodiments of the present invention also address this approach to improve or mitigate the toxicity of ADCs. For example, if corneal epithelial cells express cell surface receptors or protein targets that initiate cellular uptake, the copolymer reduces on-target toxicity by locally inactivating the ADC.

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

[0045] In some embodiments, copolymers are supplied as pharmaceutical compositions in eye drop bottles, preservative-free multi-dose bottles, standard three-piece bottles, and unit dozer / blow-fill seal containers. The filling volume of eye drop bottles is typically 1 ml to 30 ml. Examples of unit dozer filling include blow-fill seal containers of various sizes with filling volumes ranging from 0.1 ml to 1 ml (0.5 ml, 0.3 ml, 0.4 ml, 0.7 ml). Pharmaceutical ingredients can be supplied to consumer goods through aerosols, sprays, mist generators, mechanical or electronic spray bottles, pump spray bottles, mouthwashes, drinking solutions, powders, diluents, and other commercially available systems. Kits containing eye drops, nasal sprays, and mouthwashes can be marketed. Supplies of ADC-containing eye drops for the care of tumors and other diseases can be included in the kits.

[0046] One advantage is that there is currently no way to reduce the risk of infection by applying protective materials to mucous membranes, and this is necessary in stockpiling facilities for viral diseases.

[0047] One advantage is that, aside from supportive care such as warm compresses, bandaged contact lenses, and ophthalmic lubricants, there are no other treatments that reduce the risk of ADC corneal toxicity based on reducing ADC / epithelial cell interactions. [Brief explanation of the drawing]

[0048] [Figure 1] This is a projected image showing a SARS-CoV-2 virus particle in the presence of airway epithelial cells. 1. Airway epithelial cell nucleus. 2. Airway epithelial cell. 3. Cell villus. 4. ACE2 receptor. 5. SARS-CoV-2 virus particle RNA. 6. SARS-CoV-2 virus particle spike protein. 8. Virus binding to ACE2 receptor. 9. Virus entering the cell. 10. SARS-CoV-2 virus. [Figure 2]This is a projected image showing SARS-CoV-2 virus particles in the presence of airway epithelial cells and a cationic graft copolymer, and the interference conferred by the copolymer that prevents ACE2-related viral entry into cells. Although airway epithelial cells are shown in the figure, they can be replaced with other epithelial cells that can be infected with SARS-CoV-2. 1. Airway epithelial cell nucleus. 2. Airway epithelial cell. 3. Cell villus. 4. ACE2 receptor. 5. SARS-CoV-2 virus particle RNA. 6. SARS-CoV-2 virus particle spike protein. 7. Cationic graft copolymer. 11. Virus entry into cells is prevented due to the interference of the cationic graft copolymer (PLL-g-PEG in some embodiments). [Figure 3] This is a projected image showing an antibody-drug conjugate in the presence of corneal epithelial cells. 1. ADC. 2. Antibody component. 3. Toxic payload. 4. Corneal epithelial cells. 5. Microvilli of corneal epithelial cells. 6. Initiation of macropinocytosis. 7. Completion of macropinocytosis. 8. ADC involved in the macropinocytosis process, showing the ADC being captured from the extracellular fluid into corneal epithelial cells. 9. ADC with a toxic payload in epithelial cells and lysosomes, the payload being cleaved from the ADC and released into the cell, where it damages or kills the cell. [Figure 4] This is a projected image showing an antibody-drug conjugate in the presence of corneal epithelial cells and a cationic graft copolymer (PLL-g-PEG in one embodiment). Note that corneal epithelial cells may be transient amplified cells, wing cells, basal epithelial cells, or marginal epithelial stem cells. For simplification, surface epithelial cells are shown in the figure. 1. ADC. 2. Antibody component. 3. Toxicity payload. 4. Corneal epithelial cells. 5. Microvilli of corneal epithelial cells. 6. Initiation of macropinocytosis. 7. Completion of macropinocytosis. 8. PLL-g-PEG on the surface of corneal epithelial cells. 9. PLL-g-PEG in a lysed state. 10. PLL-g-PEG adhered to ADCs at several sites. 11. ADCs with a toxic payload are not present inside the epithelial cells. Cytotoxicity and cell death are prevented. [Figure 5]This is a projected diagram showing the benefits of cationic graft copolymer eye drop therapy in the presence of systemically administered ADCs. 1. Schematic diagram of the anterior surface of the eye. 2. Schematic diagram of the cornea. 3. Punctate superficial keratitis of the corneal surface. 4. Microcystic changes of the corneal epithelium observed in patients after 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 dissolved in the eye drops. 7. The corneal surface appears healthier in patients who have received ADC therapy and in patients treated with PLL-g-PEG eye drops. 8. Microcystic changes are less frequent in patients treated with PLL-g-PEG eye drops. 9. Punctate superficial keratitis is less frequent in patients treated with PLL-g-PEG eye drops, despite systemic ADC administration.

[0049] Detailed description of the invention Graft copolymers having positively charged and hydrophilic regions, or block copolymers having positively charged and hydrophilic regions, have now been shown to be effective in two important aspects relating to human health.

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

[0051] Secondly, these polymers are effective in reducing antibody-drug conjugate (ADC) toxicity to non-nascent cells. These polymers can interfere with the off-target cellular uptake of ADCs, which have a cytotoxic payload to cells that do not express the ADC's target receptor. They can also reduce uptake by locally treating tissues and inactivating ADCs through topical administration. (The copolymers can be present in the inner mucosal fluid or extracellular space to interfere with off-target ADC uptake). Specifically, with respect to off-target uptake, these cation grafts or cation block copolymers interfere with macropinocytosis of ADCs by human corneal epithelial cells, including basal epithelial cells, marginal stem cells, basal stem cells, alar cells, or surface epithelial cells. This reduction in exposure to the inner cell body, or cytoplasm including lysosomes, and, in some embodiments, to tubulin-forming components, provides benefits to cells and the organism.

[0052] While not bound by theory, the method of interference involves the graft or block copolymer inactivating the surface of the affected cell and / or ADC or virus, thereby preventing or reducing cellular uptake. Embodiments of cationic graft copolymers or other polymers interact with biological surfaces and / or the surface of viruses or ADCs.

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

[0054] definition "Off-target uptake" means that ADCs are taken up into cells by mechanisms that are different from, or completely independent of, the antibody-cell receptor interactions for which targeted therapy was designed.

[0055] A "novel virus" refers to something new to its host (human or other) or something that humans have never encountered before.

[0056] A host is an organism that normally inhabits a microorganism that causes bacteria, viruses, protozoa, or other diseases.

[0057] "Biological surface" means the surface of cells, tissues, and body organs, 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 with epithelial cells; the epithelial layers of the digestive tract and skin include membranes such as mucous membranes, including the oral mucosa, nasal mucosa, respiratory 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 the surfaces of viruses and ADCs to impart beneficial properties.

[0058] "Formulation" means a solution, suspension, powder, spray, wash, or eye drop administered to cells, tissues, organs, or mammals to be treated, containing the components necessary to enable the beneficial effects of the graft or block copolymer to occur. Formulations may or may not contain the pharmaceutically active ingredient. In this specification, graft copolymers may or may not be considered pharmaceutically active ingredients in the terminology of regulatory authorities. Eye drop formulations may have varying proportions (effective range) of copolymers, may have a pH of 3.9 to 9.9, may have a molar osmotic concentration of 150 to 400, and may have a viscosity of 1.0 to 15 cP. Viscosity may be even higher in some embodiments. Formulations are safe for subconjunctival injection.

[0059] "Microtubule disruption" is considered a particularly important cytotoxic effect in this specification. 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), meitansine, aurastatin, DM4 (ravutansine), and DM1 (meltansine). Other cytotoxic agents are listed elsewhere, some in combination with antibodies. All are included, and unknown cytotoxic agents are also included in various embodiments.

[0060] A "cytotoxic payload," sometimes also called a "warhead," is used to bind the antibody to the warhead. The linker is cleaved by intracellular and, possibly extracellular, enzymes. In particular, the linker is cleaved in lysosomes, releasing the warhead into the cell. Enzymes in extracellular fluid or tears may also cleave the linker, releasing the warhead. Thus, the warhead may enter the cell directly, or it may leak out of the cell, causing a bystander effect (damaging nearby cells that did not take up the ADC). Any and many (known and unknown) linkers are considered in embodiments of the present invention. The linker may be cell and therapeutic context specific.

[0061] As used herein, the term “pharmaceutical composition” refers to a composition formulated with an active agent together with one or more pharmaceutically acceptable carriers. This composition is suitable for administration to human or animal subjects. The active agent is present in a unit dose appropriate for administration in a therapeutic regime that exhibits a statistically significant probability of achieving a predetermined therapeutic effect when administered to the relevant population.

[0062] The copolymer formulations in the present invention include embodiments using many types of formulations, including gels, lotions, creams, ointments, sprays, wipes, and ointments. The formulations may or may not contain preservatives. The formulations may be monophasic or polyphasic. Although not limited thereto, phasic formulations have different amounts of different components to affect efficacy and duration of activity. Some formulations exhibit long-lasting activity, while others exhibit short-lasting activity. In general, a single application provides protection for a meaningful duration. As used herein, “meaningful duration” means 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 to 12 hours. In some embodiments, the duration is longer than 12 hours. Sustained-release formulations extend the duration of action. The duration of activity can be shorter or longer in relation to viral protection and reduction of ADC toxicity, as determined in development. Regarding methods for attenuating and mitigating ADC toxicity, administration can be carried out on a daily or hourly basis, depending on the formulation and / or ADC payload, the patient's condition and underlying circumstances, and the formulation itself. The use of these approaches in vitro (and in vivo) has practical value and may be shorter or longer in some in vitro models.

[0063] Animal data from other uses of small interfering RNA molecules have shown that PLL-g-PEG can be tolerated intravenously. PLL-g-PEG is sufficiently tolerable locally and is therefore of particular value here. "Adjustable" means that the cationic graft copolymer can be prepared in various ways to maintain its usefulness. For example, the length of the PLL chain, the graft ratio to the number of monomer bonds in the polymer, and the length of the hydrophilic side chain are adjustable. As an example, the PLL-g-PEG molecule used in the experimental demonstration or practical application of the present invention includes PLL(15,000-30,000 daltons)-graft(3.5 ratio)-PEG(5,000). Alternative molecular weights and graft ratios may have the same, higher, or slightly lower effect. In this specification, all variations of PLL-g-PEG are valid and referred to as PLL-g-PEG. Mimics are similarly adjustable. Because PEG is a hydrophilic molecule, it has been used for inactivating microscope slides. Polyethylene glycol has low toxicity and is used in a variety of products. This polymer is used as a lubricating coating for various surfaces in both aqueous and non-aqueous environments.

[0064] This section describes the properties of PLL-g-PEG. Polylysine (PLL) promotes adhesion to proteins and cell surfaces. PLLs may be greater than 30,000 daltons and greater than 60,000 daltons, or less than 15,000 daltons. A range of polydispersity is acceptable. Polydispersity values ​​of 0.3, 0.5, 0.8, 1, or 1.2 are considered acceptable (including endpoints), as are the range greater than 0.1–3. PLLs may also be reported as a single size (e.g., 20,000 daltons). Larger or smaller PLL molecules are also valid in this setting. The preferred configuration is an average PLL size of 10,000–40,000, but larger or smaller PLL sizes are also valid. Polydispersity values ​​may vary and may still be valid. The number of monomer bonds in the lysine chain (L-lysine, D-lysine, α- or ε-polylysine, but not limited to these) can be 50 to 200 in some embodiments, and can also be longer or shorter (fewer or more monomer bonds). The graft ratio is optimally 3.5 or 4 PLL:PEG, but a reasonable preferred range is 2 to 6. Above 6, 7, 8, 9, or 10 are acceptable in some embodiments. The upper limit is simply where tolerability decreases. While a decrease in effectiveness is expected below a graft ratio of 2, the lower limit at which optimal benefits are obtained from the mechanism by which the cationic PLL chain utilizes charge for bioadhesion is probably 1.1. Hydrophobic chains and other configurations may have different success rates and are also adjustable. The hydrophilic portion in this case is PEG, which can have different sizes or lengths. PEG 5,000 Dalton is a preferred embodiment, but PEG 2,000 is also effective. The effective range of PEG molecular weights for this particular molecule includes PEG 1000 to PEG 20000. A range and variable polydispersity are acceptable (0.1 to 2 or greater). The monomer number of ethylene glycol can be counted similarly as a substitute for the molecular weight reported by Dalton. Here, a reasonable range for the monomer number is 22 to 250. In some embodiments, the monomer number is 50 to 150.In some embodiments, this is greater than 250. These factors are adjustable, and the same or similar effects can be observed while maintaining PLL size, graft ratio, and PEG size. Optimal configurations are described but are not limited thereto. 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 graft ratio, skeleton size, and hydrophilic / inactivation site size.

[0065] Accordingly, the present invention provides a method for inhibiting, reducing, or preventing the infectivity of a virus (e.g., SARS-CoV-2) and reducing the severity of the disease course in a subject by superficially or topically administering a formulation comprising a graft copolymer having positively charged and hydrophilic parts, or a block copolymer having positively charged and hydrophilic parts, to the biological surface of the subject. Because charge dynamics are complex, methodologies for inactivation based on negative charge can also be identified, and these findings are also discussed and utilized herein. Formulations having the polymers claimed herein can be administered superficially or topically to the biological surface of a subject, including but not limited to the skin, mucous membranes, oral mucosa, nasal mucosa, and the surface of the eye, according to the method of the present invention. As used herein, “subject” means all animals, in particular, but not limited to humans and dogs, as well as mammals such as farm animals such as cattle, sheep, and pigs.

[0066] The graft copolymers used in the methods and formulations of the present invention are polymers having linear sections of repeating units called a “backbone,” typically with at least one side chain (called a “graft”) of repeating units of different chemicals branching off from a point along the backbone. In one embodiment, the graft copolymer comprises a cationic backbone and water-soluble and nonionic side chains. In another embodiment, the graft copolymer comprises a water-soluble and nonionic backbone and cationic side chains. In yet another embodiment, negative or anionic graft copolymers are utilized.

[0067] The block copolymer used in the method and formulation of the present invention is a polymer in which a linear section of the first section of a repeating unit is bonded at both ends to a linear section of a subsequent repeating unit that is not chemically similar to the first one.

[0068] Formulations for use in the method of the present invention include, but are not limited to, blocks or graft copolymers having one section of a skeleton, 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 surface including corneal and conjunctival epithelial cells, and marginal stem cells, marginal cells, marginal epithelial stem cells, basal stem cells, basal cells, early transient amplified cells, transient amplified cells and corneal epithelial cells in general, as well as sugar coatings and microvilli associated with those cells. In addition to bioadhesion by electrostatic force, any other, chemically distinct section, skeleton, graft or block is hydrophilic and, in one embodiment, induces inactivation and reduction of interaction between a virus and another ADC. The interaction is effectively reduced for beneficial effects on human health. In particular, it can reduce the transmission of viruses and contact diseases in those with insufficient host or herd immunity to prevent epidemics or pandemics. One embodiment is a method for reducing the infectivity of a novel virus in which humans have not acquired herd immunity, which helps to mitigate the severity of diseases, epidemics, and pandemics.

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

[0070] Human health is improved by reducing the overall toxicity of ADCs, particularly to the eyes and cornea. Specifically, corneal epithelial health is promoted. Patients can better tolerate ADCs for the treatment of malignant diseases or other conditions while maintaining healthier corneal epithelium, resulting in less significant vision / visual impairment and fewer ocular symptoms. The tissue-adherent sections of the graft or block copolymer in the formulations used in the methods of the present invention may be cationic, in which case the polymer adheres to the biological surface by electrostatic attraction. The interaction may be anionic, or hydrophobic using hydrophobic moieties in the combinations herein, and both hydrophobic and anionic interactions are promising.

[0071] Examples of cationic polymer sections of graft 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 their repeating units, as well as homo and copolymers of poly(aminoethyl methacrylate) containing N,N-dimethylaminoethyl methacrylate repeating units. Other cationic polymer sections that can be used include chitosan (a copolymer of glucosamine and N-acetylglucosamine, where 5-100% of the repeating units are glucosamine) and its synthetic derivatives. Examples of hydrophobic polymer sections of graft 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 nonionic if the tissue-adhering section is cationic, and may be anionic if the tissue-adhering section is nonionic (and hydrophobic).

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

[0073] Examples of nonionic hydrophilic polymer sections of formulations useful in the method of the present invention include, but are not limited to, polyethylene glycol (PEG), polyvinyl alcohol, and polyvinylpyrrolidinone. Examples of anionic hydrophilic polymer sections include homopolymers and copolymers containing, for example, acrylic acid, methacrylic acid, itaconic acid, maleic acid, styrene sulfonic acid, carboxymethylcellulose, carboxyethylcellulose, succinyl chitosan, and cellulose sulfate.

[0074] The terms "block" or "graft copolymer" are intended to represent the architecture of a polymer.

[0075] "Mimic polymers" refer to alternative chemical / molecular approaches to produce the same behavior and attributes as effective and tested polymers. For example, multiple mimics exist, such as PLL-g-PEG, which is effective. Their effects are primarily derived from polymer inactivation.

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

[0077] Inactivation can occur with ADCs, cytotoxic drugs, or viruses. Inactivation can also occur by making it more difficult for ADCs, drugs, or viruses to take up cells through interactions on the cellular surface.

[0078] A graft copolymer may have a cationic (or nonionic hydrophobic or anionic) skeleton composed of polymers selected from the above list and hydrophilic grafts, or a hydrophilic skeleton and cationic (or nonionic hydrophobic) grafts selected from the above list. In the case of a graft copolymer, the grafts may arise from all repeating units of the skeleton or may be intermittently spaced along the skeleton (at uniform or random intervals). For example, a polymer useful in formulations for use in the methods of the present invention is PLL-g-PEG, in which the skeleton is a cationic polymer poly(L-lysine) and the grafts are made of polyethylene glycol, which is a hydrophilic polymer. The PLL skeleton may be 3 to several thousand repeating units long, and the PEG grafts may be 1 to several thousand repeating units long. The PEG grafts can be attached to all PLL repeating units, to every other PLL repeating unit, to every two repeating units, or at less frequent intervals. In one embodiment, on average, there is one PEG graft for every two PLL repeating units. Similar properties can be applied to mimic polymers.

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

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

[0081] Examples of water-soluble and anionic copolymer blocks or skeletons include, but are not limited to, polyacrylic acid (PAA), polymethacrylic acid, sodium polystyrene sulfonate, carboxylated celluloses such as carboxymethylcellulose (CMC), polyitaconic acid, polymaleic acid, polyacrylamide propanesulfonic acid, anionic natural rubber, 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 halide, polyaminostyrene, chitosan, polyethyleneimine, polyallylamine, polyetheramine, polyvinylpyridine, positively charged functional polysaccharides, polyamino acids, and, for example, but not limited to these, 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- Polymers 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 oxides, polytetramethylene oxide, polyethylene, polypropylene, polystyrene, polyacrylates such as polymethyl acrylate (PMA), polymethacrylates such as polymethyl methacrylate (PMMA), polysulfones, polyether ether ketones (PEEKs), polyphosphatidines, polycarbonates, polyacetals, and polysiloxanes.

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

[0085] As will be understood by those skilled in the art upon reading this disclosure, grafts and blocks, as well as triblocks and dendrimers, are intended, and these configurations can also be used as embodiments of the present invention.

[0086] One exemplary block copolymer having a triblock configuration is PLURONIC® F127, also known as Poloxamer 407, which comprises a polyethylene oxide hydrophilic block ("PEO"), a polypropylene oxide hydrophobic block ("PPO"), and another PEO block. Other block copolymers for use in the present invention may consist of only one hydrophilic block and one hydrophobic block, or several alternating blocks, for example, a PPO-PEO-PPO block copolymer (PLURONIC®, ethylene oxide and propylene oxide-based block copolymer, BASF, Floram 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, and 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 PLURONIC P 65, PLURONIC P 84, and PLURONIC P 85 are examples, but are not limited to these. Where appropriate, block copolymers of all particle sizes are included; for example, PLURONIC F 127 and PLURONIC F 87 are available as prill and microprill products. For example, nonionic surfactants containing hydrophobic segments and PEO blocks are considered block copolymers here.

[0087] Additional exemplary block or graft copolymers that can be used in the present invention are disclosed in U.S. Patent No. 5,578,442 and U.S. Patent No. 5,834,556, the teachings of which are incorporated herein by reference in their entirety.

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

[0089] Copolymer combinations are considered and included herein, but the proportions of the formulations are applicable to different components.

[0090] In the human examples described herein, when the formulations are delivered as a solution or suspension, the amount of copolymer is approximately 0.1% to 5%. Furthermore, 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 can be incorporated into pharmaceutical formulations and coatings for use in the present invention include, but are not limited to, PLURONIC gelling agents such as F127, F108, and the aforementioned additional PLURONIC agents. Furthermore, in one embodiment, these components are used in amounts less than or equal to the amount required for gelling activity.

[0092] Other components (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 the oral cavity, topically, and on mucous membranes, skin 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, viscoelastic substances, and hyaluronic acid, as well as combinations thereof. Additional components include those commonly found in mouthwashes, nasal sprays, shampoos, soaps, and conditioners. Such components may be included in the formulations in various proportions ranging from less than 0.1% to 99% by weight, more preferably less than 1% to 10% by weight. Other components that may be included in these pharmaceutical formulations include, but are not limited to, preservatives such as polyxetonium, polyquaternium-42, polyquaternium-1, polyquat, alkylhydroxybenzoate preservatives, parabens, hydrogen peroxide, benzalkonium chloride, cetylpyridimine chloride, cetalkonium chloride, sodium perborate, Purite, eluting preservatives, polyhexamethylene biguanide (PHMB), chlorobutanol, benzododecinium bromide, "ionic buffer systems," povidone, silver, silver sulfate, betadine, and other disinfectants, as well as trademarked and untrademarked preservatives. PLL-g-PEG may also act as a preservative. Antibiotics and antiviral agents (whether low molecular weight or biological agents) may also be included in the formulations. In some embodiments, preservative-free formulations are preferred.

[0093] Furthermore, in some embodiments, the formulations and coatings may contain one or more additional pharmaceutically active ingredients. Examples include, but are not limited to, anesthetics, antibiotics, antivirals, anti-inflammatory agents, intraocular pressure lowering agents, artificial tears, lubricants, dilators, immunosuppressants, anti-angiogenic agents, monoclonal antibodies, proteins, peptides, neuroprotective agents, small molecules, and antibodies. In some embodiments, the formulations are delivered before the use of personal protective equipment.

[0094] Some example, but not limited to, additional drugs that may be included in these formulations include antiviral agents, antiretroviral agents, 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, and others used for HIV inhibition. Lipla (fixed-dose drug), paravir, baloxavir marboxil (Xofluza), bictarvy, boceprevir (Victrelis), cidofovir, cobicistat (Tyvost), combivir (fixed-dose drug), daclatasvir (Daclinza), darunavir, derevirdin, descovy, didanosine, docosanol, dolutegravir, doravirine (Pifeltro), ecoliva, edoxudine, efavirenz, elvitegravir, emtricitabine, enfuvirtide, entecavir, etravirine (Intelence), famciclovir, fixed-dose combination drug (anti- Retroviral agents), fomivirsen, fosanprenavir, foscarnet, phosphonet, fusion inhibitors, ganciclovir (cytoben), ibasitabine, ivalizumab (trogarzo), idoxuridine, imiquimod, immunovir, indinavir, inosine, integrase inhibitors, type I interferon, type II interferon, type III interferon, interferon, lamivudine, letermovir (prevymis), lopinavir, roviride, maraviloc, methisazone, moloxidine, nelfinavir, nevirapine, nexavi Lu, nitazoxanide, Norvir, nucleoside analogs, oseltamivir (Tamiflu), pegylated interferon alfa-2a, pegylated interferon alfa-2b, penciclovir, peramivir (rapivab), preconalil, podophyllotoxin, protease inhibitors (pharmacology), pyramidine, raltegravir, remdesivir, reverse transcriptase inhibitors, ribavirin, rilpivirine (Edurant), rimantadine, ritonavir, saquinavir, simeprevir (Olysio), sofosbuvir, stabuzin, synergistic enhancers (antiretroviral agents), telaprevir,Terbivudine (Taizeca), tenofovir alafenamide, tenofovir disoproxil, tenofovir, tipranavir, trifluridine, trizivir, tromantadine, tolvada, valacyclovir (Valtrex), valganciclovir, bicriviroc, vidarabine, viramidine, zalcitabine, zanamivir (Relenza), zidovudine. The formulations may also include hydroxychloroquine, chloroquine, and azithromycin. Potential formulations include antibody products having poly or monoclonal properties, antibodies, proteins, and biomolecules selected from those that bind to antigens. Antibody fragments, trap molecules, and other antibodies, cell receptors, and protein-binding biomolecules related to ADCs and the conditions addressed in this application may be known, under development, undeveloped, or in the idea stage. Importantly, the claimed copolymers may be combined with other effective agents in the management of the disease being addressed, including synergistic therapies, to improve performance or efficacy. Biomolecules are produced by living organisms. This includes synthesized antibodies, recombinant proteins, and other high and low molecular weight molecules, some of which have new nomenclature developed through novel technologies. ACE2 receptor blockers and steroids (such as glucocorticoids, androgens, and estrogens) are also potential formulation components and are considered within various embodiments of this invention.

[0095] The pH of the formulation of the present invention is within a physiological range depending on the administration site and the site of the biological surface or membrane to be modified. Typically, the pH is 3 or higher, for example, 5.6 or higher and 9 or lower.

[0096] The formulations may include novel or established dry eye or corneal therapeutic agents, including, but are not limited to, cyclosporine, refettigrast, LFA-antagonists, steroids, e.g., loteprednol, dexamethasone, flucinolone, difluprednate, aldehyde scavengers, fonaldepar, varenicline, bisomitin, kinases, silk-derived proteins, voclosporine, omega-3 fatty acids, and any easing or lubricant of OTC monographs fully referenced herein. Importantly, some embodiments of the formulations have very low viscosity (lower than most artificial tear products on the market). The advantages of this are that vision is not blurred upon instillation, the spray functions easily through a small diameter nozzle, and the mouthwash rinses completely and easily and exhibit good tolerability. Electrospray and other microelectronic and mechanical delivery systems, high-precision multi-jet systems and other approaches to microdose release are, among other possible delivery systems. The copolymers exist as follows: The residual bioadhesive polymers, naturally, 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 this invention demonstrate the value of graft or block copolymers as a mechanism of action.

[0097] As described in the in vitro experiment of Example 1, this approach is effective in reducing the infectivity of the virus. Other examples have shown a reduction in ADC entry into epithelial cells and a reduction in 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 showed good tolerability. For example, in several initial human exposures, no irritation or discomfort was reported in any of the subjects. Moreover, there were no reports of blurred vision with single or repeated instillation of 50 microliters or less. Therefore, one embodiment of the present invention is a product having lower viscosity.

[0099] However, as will be understood by those skilled in the art by reading this disclosure, alternative ophthalmic delivery methods may be used, including, but are not limited to, intraocular, periocular, conjunctival, subconjunctival, transconjunctival, periocular, retroocular, sub-Tenon's capsule, transscleral, topical gel, topical dispersion, intraorbital, intrascleral, intravitreous, subretinal, transretinal, choroidal, uveal, intracavitary, transcorneal, intracorneal, intralenical (including phakic and pseudolentic) and intraoptic nerve or adjacent delivery methods. The present invention can be used in conjunction with other delayed-release formulations of polymers for extending drug delivery. The present invention can be used in conjunction with depot formulations. The present invention can be administered intravenously to treat other ADC toxicity such as thrombocytopenia.

[0100] A key aspect of this invention, for use in ADC toxicity and viral infection, is the existence of in vitro and in vivo opportunities with commercial and product development value. For example, these copolymers with bioadhesive and inactivation sites have been shown to be effective under these conditions both in vitro and in humans, making their use in development valuable. Cells can be treated with copolymer formulations, and then ADCs can be added. Tissue cultures, including corneal epithelial models, can also be used. Approaches to improve uptake by off-target or macropinocytosis are known, so those with low uptake in the presence of copolymers can be selected for development. Models can include epithelial cell lines, harvested epithelial cells, megakaryocytes, other cells, and human umbilical vein endothelial cells. Cell types with toxicity risks can be used in several laboratory development procedures. Similarly, antiviral effects in therapeutic and prophylactic measures can be evaluated in laboratory / development settings of viral and copolymer exposure, and synergistic and optimal molecules and formulations can be selected for human development. Therefore, the claims for the broad treatment of cells are appropriate and useful inventions for advancing therapeutics for human health, which are the subject of this specification.

[0101] COVID-19, an infection caused by SARS-CoV-2, is a devastating disease for many patients, with a mortality rate of up to 3% and a high number of infected individuals requiring hospitalization or ICU treatment. Millions of people have been infected, and the mortality rate continues to rise by hundreds of thousands. More than 3 million people have been infected worldwide, and more than 200,000 have died within the last six months. Elderly patients and those with pre-existing conditions are at higher risk of morbidity and mortality. High viral load exposure also poses a significant risk to morbidity. SARS-CoV-2 has caused a global pandemic, and better alternative therapies are needed. There is also the possibility of a new pandemic. The SARS-CoV-2 spike protein can be inactivated using graft copolymer and block copolymer approaches, but these approaches are not specific to a single virus strain.

[0102] "Viral infectivity" refers to the exposure of host cells at risk to pathogenic viruses. Utilizing protein receptors and other uptake mechanisms, viral particles can enter cells, release their RNA or DNA, and hijack the cell's protein synthesis mechanisms, nucleotide mechanisms, or other metabolic processes to produce more viral particles, which can then be released to infect other cells or other organisms. While not limited to specific mechanisms, formulations containing the above-mentioned graft copolymers (which are cationic, hydrophobic, anionic, and have hydrophilic side chains), such as PLL-g-PEG, adhere to the biological surface 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 sites, such as those in PLL, contribute to bioadhesion. Hydrophilic sites (e.g., PEG) prevent and / or reduce interaction with target cells. When applied as droplets, spray mist, or rinse, graft copolymers can migrate from surface tissues to viral particles via physicochemical on-off coupling associated with electrostatic interactions. Inactivation sites can be nonionic, nonionic and inert, as well as hydrophilic. Graft copolymers also protect at-risk cells and, in some cases, directly interfere with receptor proteins (e.g., ACE2) to enhance their activity. Combined exposure to viral particles and at-risk cells significantly reduces their interactions and viral infectivity. Reduced interactions are beneficial to cells, tissues, and organisms, including humans, by reducing exposure to pathogens or toxins, thereby lowering morbidity and mortality associated with these agents. For example, lower viral loads lead to reduced severity of subsequent infections and increased opportunities 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 would be an important additional approach to safely protect subjects.

[0103] Similar formulations have already been used in the eyes 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 that this formulation is safe for use in humans and animals.

[0104] These formulations can also be widely used, in accordance with the present invention, to reduce the transmission of SARS-CoV-2 and novel viruses via nasal and inhalation applications in environments including, but not limited to, hospitals, emergency departments, intensive care units, aircraft, kindergartens and schools, homes of infected individuals, and nursing homes and chronic care facilities. Healthcare workers and first responders may also benefit. A “novel” or “new” virus means a virus that has mutations or general characteristics that indicate that humans, generally the majority of the population, are not resistant through prior exposure or vaccination. “Transfection” means the way in 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 an infectious disease in a community at a particular time. The present invention relates in particular 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 a contact-borne infectious disease within a population, which occurs when a sufficiently high percentage of individuals have immunity to the disease due to antibody production resulting from vaccination or prior exposure to the virus. Herd immunity requires resistance to a considerable number of viruses. The formulation has significant utility in children and adults with weakened constitutions, such as immunodeficiency, chronic diseases, and other chronic health conditions that make the host more susceptible to everyday illnesses, such as cystic fibrosis.

[0105] Microcystic epithelial lesions of the corneal epithelium are a currently identified problem known to be associated with and / or caused by ADC therapy, and have recently become clinically significant as ADCs with corneal epithelial toxicity have become commercially available after many years of development. ADCs are being developed as effective treatments for many forms of tumor diseases. These are costly and resource-intensive development programs, and the need for limitations on use due to ocular toxicity is significant. Human toxicity may not manifest until clinical trials. It is important that patients can maintain ADC therapy toward the optimal tumor (or other indication) disease outcome (survival or progression-free response, beneficial effect), and corneal epithelial toxicity is an adverse event that may limit treatment or cause patients to discontinue ADC-related treatment. Serious ophthalmic and other types of adverse events associated with ADCs are a problem for patients and treating physicians. The payload or warhead, once inside the cell, is thought to cause 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] While not bound by any specific theory or mechanism of toxicity, the discovery of beneficial effects is demonstrated in vitro and in vivo as a way to explain the presumed usefulness of the present invention.

[0107] Based on clinically observed toxicity, ADCs can be exposed to the eye through one of two methods. First, ADCs can reach daughter cells of marginal stem cells, and ultimately basal stem cells and basal epithelial cells, through release into the extracellular space from the marginal circulation (including, but not limited to, the palisades of Vogt, which have a distinct vascular system with thin, almost invisible hairpin loops in which arterial and venous components are oriented radially), subsequent nonspecific uptake by basal cells, and their migration to the cornea.

[0108] Macropinocytosis is described as the mechanism by which ADCs enter cells, such as corneal cells, that are affected by toxicity, because the antibody portion of ADCs typically lacks the specific receptors that they use to enter the cell. Therefore, the entry of ADCs into cells can be considered nonspecific and off-target. Embodiments of the present invention described herein improve or reduce corneal cytotoxicity induced by off-target ADCs, such as marginal stem cells, daughter cells, transient amplification cells, alar cells, basal cells, corneal epithelial cells, and terminal epithelial differentiated cells.

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

[0110] Because superficial corneal epithelial cells do not receive a blood supply, they must obtain fluids and nutrients through some means independent of direct blood supply. Epithelial cells utilize macropinocytosis as a method of nutrient acquisition. The cell's plasma membrane contains a combination of sphingoglycolipids, cholesterol, and protein receptors, which are organized into microdomains of glycolipid proteins called lipid rafts. Internalization of lipid rafts is a process recorded in corneal epithelial cells regarding the internalization of extracellular material. Macropinocytosis is a type of endocytosis in which extracellular material is captured by the cell (macropinocytosis is an endocytosis that takes extracellular material into the cell). (Macropinocytosis is a means by which eukaryotic cells take in extracellular fluids and dissolved molecules. In embodiments herein, the term pinocytosis may also be used. Pinocytosis is the take-in of fluids into a cell by the budding of vesicles from the cell membrane. Micropinocytosis is considered herein and used as a term in embodiments, and may be used in any particular embodiment in which either macropinocytosis or pinocytosis is used collectively. Micropinocytosis is the take-in of macromolecules or other chemicals into a cell by membrane invasion and the entrapment of relatively small vesicles.) Toxins and pathogens invade many types of cells by endocytosis and macropinocytosis. ADCs can enter cells by macropinocytosis.

[0111] Macropinocytosis occurs in many cell types. Macropinocytosis has been found to be one way in which ADCs enter corneal epithelial cells. While this method of taking up extracellular components into cells is natural, inhibiting it in certain environments can be beneficial. Macropinocytosis is thought to be the way in which surface epithelial cells take up ADCs. It is the least specific pathway and is directed by actin-driven membrane overhangs that form large endocytic vesicles known as macropinosomes. Ultimately, these vesicles fuse with lysosomes. Once ADCs enter lysosomes, the payload, particularly the maytansinoids and auristatins in the setting of ADV tox, are toxic. However, tubulin inhibitors can generally cause corneal toxicity. Generally, the most adverse effects from toxic payloads, or tubulin inhibitors, occur when cells are exposed to either dividing (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 a linker by intracellular enzymes. This is how ADCs deliver toxins to target (newly formed) cells, but other cells are also affected, and ADCs can enter even if they do not express receptors / proteins that interact with ADCs. Pinocytosis is one way ADCs enter cells. Payloads other than tubulin inhibitors can also exhibit corneal epithelial toxicity, and this specification examines these alternative cytotoxic payloads. Once the payload is cleaved from the ADC, it actively exerts its chemical functions, such as inhibiting the polymerization of tubulin into microtubules necessary for cell division and, in some cases, cell migration. The presence of a tubulin inhibitor in dividing cells can cause cell death (followed by apoptosis and pycnosis). In ADC toxicity, apoptosis and pycnosis have been observed in corneal epithelial cells. It is presumed that these pycnosed cells are the cause of microcystic keratopathy, a laboratory finding. Dead or dysfunctional cells are observed in the epithelial layer on slit-lamp examination. That degree can be quantified.

[0113] Once an ADC is cleaved in a cell that has taken it up, cytotoxic molecules can be released from that cell, potentially causing a bystander effect. Therefore, reducing uptake and cleavage into cells may be an intervention with excessive relative reduction (bystander death can protect more cells than actually take up ADC in one cell). Bystander death means that once cleaved, the cytotoxic substance is released into the extracellular space and enters subsequent cells. Therefore, if taken up by more superficial cells, it can migrate within the corneal epithelium. It can also be released at the periphery after being taken up by stem cells, released to other nearby stem cells, and diffuse inward to basal stem cells. Such diffusion of cytotoxic drugs can be reduced by the method of the present invention. This approach not only reduces uptake and cleavage but also allows for the restriction of local cytotoxic migration and reuptake by polymers. Regardless of the mechanism, the effect is practical and valuable.

[0114] In humans, toxicity is sometimes limited to the epithelial layer (excluding the interstitium and endothelium), or potentially limited to the interstitium and endothelium. (Note that the interstitium or endothelium may be involved secondarily or in some ADC designs.) Normal replenishment of surface epithelial cells is impaired, leading to abnormalities in the surface epithelium, which may present with punctate staining, corneal epithelial defects, and abnormal refractive surfaces. Visual acuity is often negatively affected. Patients may experience symptoms such as blurred vision, dry eyes, corneal foreign body sensation, ocular discomfort, and ocular irritation. Corneal infection, interstitial keratitis, and ulcerative keratitis have been reported with ADC therapy. These are all serious adverse events, and it is not uncommon for these toxicities to necessitate discontinuation or postponement of administration. Histopathological examination of the eye in the case of cytarabine (similar to cases encountered with some ADCs) reveals severe degeneration of rapidly dividing basal epithelial cells, leading to the formation of parathyroid cysts.

[0115] Eye irritation is a toxic or adverse event associated with ADCs, but it is not the only symptom. Corneal toxicity from ADCs can lead to or may lead to corneal punctate superficial epitheliopathy, corneal erosion and epithelial defects, corneal ulcers, corneal infections, and corneal perforations. Foreign body sensation and decreased vision may occur. To save lives, drug discontinuation, postponement, and dose reduction may be necessary. Patients need vision for driving and reading. Vision is a crucial factor affecting quality of life. An advantage of this invention is that it allows patients to maintain a more regular dosing schedule and minimize eye symptoms. Patients will be able to achieve better outcomes with fewer serious adverse events without interrupting ADCs.

[0116] Corneal toxicity associated with ADCs has no therapeutic mechanism, as described herein. The limit of intervention is symptomatic management. Commercially available lubricating eye drops, punctal occlusion, and bandage contact lenses can help with symptoms.

[0117] In one embodiment, the method involves reducing adverse events associated with the use of antibody-drug conjugates by applying an effective amount of a copolymer having electrostatic and steric mediating properties applicable to cells affected by the toxicity, in setting up off-target uptake pathways (but not necessarily limited to off-target uptake pathways) that damage non-neogeneic cells.

[0118] While not bound by any particular theory, the usefulness of the present invention can be manifested through the interference, mitigation, and inhibition described herein.

[0119] Another relevant approach to managing locally occurring ADC toxicity due to off-target uptake is to use antibodies against the ADCs themselves. Antibodies formulated for specific ADCs would be designed to have inactivation sites, such as PEGylation, which thereby reduce the ADC's ability to enter cells via macropinocytosis. The ADCs would then be delivered locally, for example, to inhibit uptake by pinocytosis. Antibodies may have PEGylation or other inactivation sites as described herein, by modifying the antibody against the ADC by conjugating it to a hydrophilic polymer, for use in cell culture, laboratory, and in vivo human use. Laboratory use is important because, in embodiments where the copolymer or antibody is shown to reduce ADC-associated toxicity or other drug corneal toxicity, clinical development can be pursued by reducing the risk of ocular adverse events that complicate treatment.

[0120] Cationic graft copolymers interfere with the uptake of ADCs into cells (corneal epithelial cells). This unique methodological approach improves the health of the corneal epithelium, resulting in improved laboratory and symptomatic findings and reduced eye risks. Reducing cell death (e.g., toxicity) associated with ADCs offers numerous advantages. Intravenous administration and other options exist for reducing toxicity, and therefore the scope of the claims is broad. Megakaryocytes can be used for macropinocytosis for the internalization of ADCs, and therefore, options for reducing this known adverse event associated with ADCs are addressed in this invention.

[0121] Epithelial cells have a negative charge on their surface (3.6 × 10 -4It is known that ) have negative charges. Therefore, cationic graft and block copolymers inactivate these negative charges that are thought to be involved in ADC uptake. There is evidence that positively charging ADCs increases their toxicity and negatively charging them reduces uptake. A key point of the present invention is that we have found that inactivation by electrostatic (or, in some embodiments, hydrophobic) interactions is beneficial. By reducing ADC interactions on the cell surface, ADC entry is reduced in one embodiment. By protecting the cell as a whole, the toxicity of ADCs is also reduced. Other (non-epithelial) cells also have charged regions (and hydrophobic regions) that allow interference with the reduction of these interactions. Copolymers can also act on major protein components on cells and ADCs to restrict uptake.

[0122] It is also important that there are multiple methods for delivering cationic graft copolymers (or other effective blocks or graft copolymers) to target tissues. PLL-g-PEG has been found to be safe for intravenous injection in mammals. Ophthalmic administration and topical exposure have been found to be safe. This polymer can reach target marginal cells by intravenous injection. Subconjunctival delivery can also be a valuable approach to improve toxicity because this approach provides both a reservoir and access to daughter cells, including marginal stem cells and basal epithelial cells. The conjunctiva also exhibits some permeability to macromolecules. Because the intercellular spaces of the conjunctival epithelium are wider than those of the cornea, it exhibits higher permeability to larger molecules. Therefore, locally delivered PLL-g-PEG can reach stem cells (progenitor cells of corneal surface epithelial cells) and reduce toxicity by interfering with ADC corneal toxicity. Topical delivery to the cornea (or eye) can interfere with any ADCs that penetrate the surface cornea or conjunctival epithelium. Therefore, the release of the payload and its subsequent migration to basal cells through corneal layers 5 or 6 are reduced. Larger molecules can, naturally, penetrate the superficial epithelium. Thus, PLL-g-PEG, or other specified and claimed graft or block copolymers, can protect basal epithelial cells located deeper within the epithelial layers. These cells reach this space. Similarly, ADCs can be exposed to deeper corneal epithelial cells. Therefore, copolymers can interfere with ADCs to block their interaction with the cell capture process, and can also interfere at the corneal cell surface to prevent ADC entry into cells. These effects provide an effective approach to reduce corneal toxicity associated with ADCs. In the case of corneal cells, basal cells and other potentially adverse cells are protected, resulting in less toxicity to some extent, and fewer signs and symptoms of ADC corneal toxicity exhibited by patients. This option has significant implications and applies to other approaches to minimizing off-target ADC toxicity. Since corneal cells typically do not exhibit the receptors targeted by ADCs, toxicity is off-target.In some embodiments, these copolymers may be beneficial for topical application if there are components for on-target uptake based on the presence of receptors on corneal cells.

[0123] Delivery may be localized, localized, or systemic. Many formulations exist. Copolymers (PLL-g-PEG) can reduce corneal tissue exposure to ADCs and reduce uptake. Interference is steric and charge-based. Uptake is reduced by inactivating ADCs and the cell surface. Charged or hydrophobic active sites adhere to cells or ADCs. The hydrophilic component of the polymer reduces ADC-corneal interaction and thus reduces uptake. The binding of the copolymer to the conjunctiva or cornea can also function as a reservoir for interference and inactivation of ADCs as they move from the tear film to the tear drainage system. The selected copolymer is non-toxic. The concentration of eye drops may range from 0.01 to 10% by weight, but more commonly from 0.5 to 3% by weight. Subconjunctival formulations may have 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 offer safety at a given time, and it may be beneficial to include other agents or polymer components in the eye drops to mitigate ADC toxicity or provide viral protection. Subconjunctival delivery may require fewer doses. Topical delivery is suitable for long-term use. In many embodiments, preservatives are avoided to help protect the corneal surface and avoid damaging epithelial cells. Exemplary additional pharmaceutically active ingredients for ophthalmic use include, but are not limited to, lubricants, mitigants, and sterile water, as well as other standard excipients. In some embodiments, including novel protective agents in which synergistic effects with copolymers may exist, it may be beneficial to combine them with other active agents.Also, antibiotics (fluoroquinolones, vancomycin, cephalosporins, gentamicin, erythromycin, azithromycin, sulfonamides, bacitracin, gatifloxacin, levofloxacin, moxifloxacin, ofoxacin), acetazolamide, antazoline, aspirin, atropine, azelastine, bacitracin, betaxolol, bimatoprost, plant-derived drugs, such as zeaxanthin lutein, etc. Copinbrimonodine, brinzolamide, carbachol, carteolol, ciprofloxacin, ofloxacin, cromarin, cyclosporine, dapiprazole, dexamethasone, diclofenac, dipivifren, dorzolamide, epinephrine, erythromycin, fluoromethalone, flurbiprofen, gentamicin, glaucoma treatment drugs (prostaglandins, carbonic anhydrase inhibitors, epinephrine or α-agonists, β-br Rocker), gramicidin, homatropin, hydrocortisone, hyostine, ketorolac, ibuprofen, ketotifen, latanaprost, levobunolol, levocabastine, levofloxin, loteprednol, medrison, metazolamide, metipranolol, naphazoline, natamycin, nedocromil, neomycin, neuroprotective agents, nonsteroidal anti-inflammatory drugs, nepafanec, norfloxacin, ofloxacin, o Lopatadine, oxymetazoline, pemirolast, pheniramine, phenylephrine, pilocarpine, povidone, prednisolone, propalacaine, scopolamine, tetracaine, steroids, sulfacetamide, tetrahydrozoline, hypertonic tears, timoral, 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 pharmacoactive ingredients, can be used in combination with the block and graft copolymers described herein to reduce ADC toxicity or to better manage adverse events associated with ADCs.

[0126] Based on the mechanism and non-specific protective and inactivation approaches, ADCs having tubulin inhibitors or epithelial cytotoxic agents are mitigated by this copolymer-based approach. The following ADCs are claimed in this invention, but this list is not limiting. The copolymers discussed herein may be formulated together with the ADC itself or separately. The toxic payload may be a meitansinoid or auristatin, but may be another type in general or another tubulin inhibitor. Tubulin inhibitors and tubulin polymerization inhibitors have particular utility in this embodiment. Tubulin inhibitors may include, but are not limited to, paclitaxel, epothilon, docetaxel, discodermolide, colchicine, combrestatin, 2-methoxyestradiol, methoxybenzenesulfonamide (E7010), vinblastine, vincristine, vinorelbine, vinfluin, dorastatin, halichondrin, hemiastalin, cryptophycin 52, paclitaxel sites, vinca alkaloid sites, colchicine sites, etc. They may also be tubulin disruptors or act through other cytotoxic mechanisms. DNA synthesis inhibitors such as cytarabine are also included. Other anticancer agents that can function as payloads include monomethyl auristatin E (MMAE), DM1 (meltansine), T-DM1, meitansinoids, auristatin, DUO duostatin 5 and other duostatins, AF-HPA (aulistatin F-hydroxypropylamide), PBD (pyrrolobenzodiazepine), MMAF (monomethyl auristatin F), Calich, sodium nitrate, calicheamicin, DM4 (labtansine), SN-38, irinotecan metabolites, PF063801 01, Dxd, DNA topoisomerase I inhibitors, DOX, doxorubicin, PF063801 01, mitoxantrone, etoposide, tecilin, PBD dimer, pyrrolobenzodiazepine, and SG3199. Toxins targeting tubulin filaments, toxins targeting DNA, toxins targeting RNA, nanocarriers, protein toxins, and enzymes are also considered.

[0127] Various linkers in ADC agents have been claimed, and when used in model or clinical copolymer applications for protection from toxicity or treatment, embodiments include those known in 2020 as well as those to be developed in the future. Embodiments also include combinations with the ADCs described below: 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, Bellantomab fodotin, CD33 / DM4 / SPDB, CD70 / MMAF (Auristatin) SGN-75 CD70 positive, CD70 / MMAF (Auristatin) / mc, SAR566658 CA6+, DS6 / DM4 / SPDB, CD33 / Calichaemycin / Hydrazine, Ephrin type A receptor 2 (EphA2) / mcMMAF (Auristatin) / mc, lorbotuzumab, meltansine, D56 / DM1 / SPP, CD138 / DM4 / SPDB, FRa / DM4 / SPDB, AGS-16M8FMMAF, AGS-16C3FMMAF, ENPP3 / MMAF (Auristatin), and, but not limited to, the following: [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 corneal toxicity can be treated by the method of the present invention.

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

[0135] Based on physicochemistry, copolymers are effective in the embodiments described herein because they reach drugs (pharmaceuticals), solutions applied to cells at risk, and the cells themselves to create a dynamic protective environment. When applied as droplets, suspensions, solutions, controlled delivery systems, or powders (sometimes lyophilized), graft copolymers can migrate from surface tissues to ADCs or other pharmaceuticals in solutions, tears, or extracellular fluid through physicochemical on-off coupling associated with electrostatic or hydrophobic interactions.

[0136] From the experiments described herein, it is expected that the formulations of the present invention will also be useful in preventing viral infection or ADC uptake or interaction with target cells, including but not limited to the skin, mucous membranes (eyes, nose, oral cavity) and hair. Therefore, these formulations can also be applied according to the present invention to epithelial tissues of the eyes, respiratory tract and gastrointestinal tract, mucous membranes, exposed wound surfaces, corneal and conjunctival surfaces, skin, and surgical and traumatic wounds and ulcers. These formulations can play a role in protecting 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. Copolymers can interact with both infectious agents or pharmaceuticals and the surface of cells at risk to inactivate any interaction and provide a protective effect.

[0137] For these human applications, formulations may be in the form of lotions, gels, liquids, sprays, rinses, soluble wafers, or glycerin bars, to which water can be added to solubilize the graft copolymer or block copolymer for easier application. Formulations may be supplied as individual or single-use products, or in volumes for industrial and / or multi-use dispensers. In addition to graft or block copolymers, such formulations may contain 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 formulation can also be used in accordance with the present invention for animals, including household pets, to reduce viral infection or ADC toxicity.

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

[0140] Sustained-release formulations have been demonstrated to be particularly beneficial and are included herein.

[0141] PLL-g-PEG is an example of a copolymer possessing bioadhesive and inactivation sites. PLL-g-PEG utilizes electrostatic bioadhesion through its cationic backbone. Hydrophobic and anionic sites utilize alternative bioadhesive interactions (hydrophobic and anionic). Formulations of this copolymer are safe for use in humans.

[0142] The following non-limiting embodiments are provided to further illustrate the present invention. Percentages are expressed as weight / weight %.

[0143] It has been noted that the drug may be secreted into the tear fluid. From 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, highly reflective lesions throughout the epithelium that appear to correlate with clinically observed MEK. Histologically, the microcysts appeared to correlate with apoptotic cells involved throughout the epithelium. In addition to the increase in apoptotic cells observed in the histological specimen, immunohistochemically, IgG-positive intracytoplasmic granules were observed in the basal epithelium. Since depatuxizumab, the antibody component of ABT-414, is monoclonal IgG1, it was suggested that ABT-414 itself was deposited in the basal epithelium. The direct presence of ABT-414 in the epithelium likely explains the histologically observed increase in apoptosis."

[0144] Source of experimental methodology: Zhao, Hui, et al. “Modulation of Macropinocytosis-Mediated Internalization Decreases Ocular Toxicity of Antibody-Drug Conjugates.” Cancer research 78.8 (2018): 2115-2126. Cell Culture Test Design: "Cell lines and reagents. All cells were maintained according to the vendor's protocol. Human primary corneal epithelial cells (HCEC) (catalog no. C018-5C) from Life Technologies were cultured in keratinocyte SFM (catalog no. 17005-42), and HCEC cells from ATCC (catalog no. PCS-700-010) were cultured in corneal epithelial cell basal medium (catalog no. PCS-700-030) supplemented with a corneal epithelial cell growth kit (catalog no. PCS-700-040). Human umbilical vein endothelial cells (HUVEC, catalog no. C-003-5C) and human dermal fibroblasts, adult (HDFa, catalog no. C-013-5C) were obtained from Life Technologies. HUVEC was cultured in Medium medium supplemented with low serum growth supplement (LSGS, catalog no. S-003-10)." Cells were grown in 200°C. HDFa cells were grown in Medium 10°C supplemented with LSGS. KU812 cells were obtained from ATCC (catalog number CRL-2099) and grown in RPMI1640 + 10% FBS as described above. Cell lines were passaged in our laboratory less than 6 months after resuscitation. Human cell lines were identified using short tandem repeat profiling (Promega) and confirmed to be mycoplasma-negative. Reagents for the differentiation of human hematopoietic stem cells (HSCs) and megakaryocytes have been previously reported. T-DM1 (Kadcyla; Genentech / Roche) was purchased (Myoderm). Macropinocytotic HSCs (10⁵ cells / well) were grown overnight in 24-well plates and incubated with 1 mg / mL dextran-FITC (10,000 MW, Life Technologies) as a control at 37°C or 4°C for 3 hours.Cells were detached with trypsin and neutralized with a neutralizing solution (Life Technologies, catalog number R002100). The cells were then washed three times with FACS staining buffer (FBS, BD Pharmingen, catalog number 554656) and analyzed using an Attune acoustic focusing cytometer (Life Technologies). To investigate the effect of 5-(N-ethyl-N-isopropyl)amylolide (EIPA), the indicated amount of EIPA was added to the cell culture 30 minutes before dextran-FITC addition. The mean fluorescence intensity ratio (MFIR) was calculated by normalizing the MFI value at 37°C to the value at 4°C. 100 mL of proliferation assay HCEC (500 cells / well) and HUVEC (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 plate, catalog no. 3903). After treatment with ADC for 6 days, the viability of treated cells relative to the control was measured using the CellTiter-Glo (CTG) luminescence 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 GraphPad Prism 6 with a sigmoid dose-response (variable slope). Assay plates contained technical triplicates for each drug concentration, and the presented data are the mean 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. Equal volumes of 1,8-ANS (1-anilinonaphthalene-8-sulfonic acid, Thermo Fisher Scientific, catalog number A47) were 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 defined as the hydrophobicity index. Cells were seeded on 8-well chamber slides using a confocal microscope (0.75 × 10⁶ cells per well). 5Cells were cultured for 48 hours before treatment and subsequent immunostaining. Next, cells were incubated with AGS-16C3F for 4 hours at 37C with or without co-incubation with 0.5 mg / mL Dextran-Texas Red (Molecular Probes; D18653). Inhibition of macropinocytosis was evaluated by treating cells with EIPA for 30 minutes before incubation with AGS-16C3F / Dextran-Texas Red. After the incubation period, unbound antibodies were washed off with PBS, and cells were fixed with 4% paraformaldehyde at room temperature for 20 minutes. Subsequently, cells were permeabilized with PBS + 0.1% Triton-X-100 for 15 minutes, and nonspecific labeling was blocked with PBS + 10% normal goat serum. Cytoplasmic AGS-16C3F bound to the cell surface and internalized was visualized by incubating cells with Alexa Fluor 488-labeled goat anti-human IgG (Thermo Fisher Scientific, catalog number A-11013). The nuclei were visualized with TO-PRO-3 Iodide (Thermo Fisher Scientific, catalog number T3605), and coverslips were attached using ProLong Gold Antifade reagent (Thermo Fisher Scientific, catalog number P36934) for imaging. High-resolution laser confocal image sections were acquired using a Leica TCS SP5 II (63x oil immersion objective lens; NA 1 / 4 1.4), and these were sequentially scanned to minimize phosphor crosstalk and false-positive colocalization. Furthermore, the techniques for rabbits were also cited from Zhou et al.: "Animal experiments and welfare: In vivo xenograft tumor models and pharmacodynamic studies were conducted as described above. All experimental protocols were approved by the Agensys Facility Animal Management and Use Committee. All procedures in toxicity testing complied with the Animal Welfare Act and Regulations (9 CFR 3). Male Dutch Belted rabbits [Haz:(DB)SPF] weighing approximately 1.5–2.0 kg were used for ocular tolerability testing. The rabbits were acclimatized for at least 6 days before the first dose."All animals were housed in individual suspended stainless steel cages, provided with feed and water, and maintained under environmental conditions compliant with all animal welfare guidelines. The test drug was administered intravenously to groups of 3-4 rabbits via the ear margin vein, followed by weekly saline flushing for up to 6 times (days 1, 8, 15, and 22). Overall health was assessed by weekly weight measurements and cage-side observations. Ocular tolerance was assessed by visual examination, i.e., examination of the adnexa and anterior segment of each eye using a slit-lamp biomicroscope. Furthermore, the fundus was examined with an indirect ophthalmoscope after dilation with a pupil-dilating agent. Corneal fluorescein staining was also performed to examine corneal damage. Fluorescein solution (approximately 1 mg / mL) was applied to the cornea with a cotton swab. At necropsy, the eyes and other selected tissues were placed in a fixative according to established procedures for IHC. All rabbit experiments were performed by Covance Laboratories. The results will be calculated using standard statistical approaches.

[0145] All references, publications, and patent documents are incorporated herein by reference in their entirety.

[0146] Examples The results demonstrate 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 the guidelines of the Institutional Animal Care and Use Committee (IACUC). Specific protocols are approved by the appropriate review boards.

[0148] These experimental results, in whole or in part, demonstrate the usefulness of the present invention. If, as is sometimes the case with scientific data, there are inconsistencies in the data, the physical benefits can be demonstrated by variations in experimental conditions that appropriately separate the important variables.

[0149] In all examples, the safety of target cells was maintained in the presence of the copolymer. The formulations for human and mammalian use are well tolerable.

[0150] Example 1: The presence of airway epithelial cells in culture and SARS-CoV-2 virus particles results in a very high infection rate and subsequent death of these cells. Cationic graft copolymers are added to this system in soluble form at concentrations of 0.001, 0.01, 0.1, 1, 2, and 3% by weight for a duration sufficient to allow a significant infection rate, e.g., several hours. The infectivity of the virus decreases, and cell viability improves on a dose-response basis. The negative control is simply a solution containing no cationic graft copolymer. To demonstrate this beneficial effect, several experiments are performed. One involves cell viability measured by cytometry, and another involves quantitative PCR to measure the copy number of the viral genome. 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 lyophilized powder to provide test concentrations in various wells and cell cultures (standard growth and viability media). The same amount of SARS-CoV-2 virus particles were added to each well, based on the absolute number of virus particles per well. After 24 hours, cell viability was counted. Prior experiments indicated the optimal number of virus particles to add. Empirical data suggest that 10,000 virus particles per ml are effective in infecting airway cells, and that after 24 hours, most of the cultured cells in the wells are killed. Solutions containing PLL-g-PEG showed 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 a SARS-CoV-2 infection setting is performed using a similar design, except the endpoint is the RNA copy number of the viral genome. In this experiment, the same PLL-g-PEG concentration is used, but all viral and PLL-g-PEG media are washed off the cells after exposure and a 2-hour incubation period. After 24 hours, the solution is examined for viral RNA copy number. SARS-CoV-2 virus detection is performed using a One Step Prime Script RT-PCR kit on a Light Cycler 480 real-time PCR system with primers. The following sequences are used: forward primer: 5'-AGAAGATTGGTTAGATGATGATAGT-3'; reverse primer: 5'-TTCCATCTCTAATTGAGGTTGAACC-3'; and probe: 5'-FAM-TCCTCACTGCCGTCTTGTTG ACCA-BHQ1-3'. All experiments are performed in triple replicates. These results are shown in Table 2.

[0153] [Table 3]

[0154] These studies will be conducted in well plates (triple replicates) sufficient to demonstrate statistical significance. This experiment puts into practice a specific option of using cationic graft copolymers to reduce the infectivity of SARS-CoV-2 virus to at-risk cells, and consequently to tissues and organisms.

[0155] Example 2: A clinical study will be conducted to demonstrate the clinical benefit of the present invention. Individuals at risk of SARS-CoV-2 virus infection (COVID-19) will be treated with topical formulations of PLL-g-PEG. These formulations are liquid-based. Individuals at risk of SARS-CoV-2 virus infection based on high-risk exposure in the workplace will be enrolled in the study. 1000 patients will be enrolled in a 1:1:1 randomization scheme. This study is double-blind. Dosage A consists of PLL-g-PEG in the following forms: 0.1% eye drops, 0.1% nasal spray, and 0.1% mouthwash; Dosage B consists of 0.5% eye drops, 0.5% nasal spray, and 0.5% mouthwash. Cohort C will receive only saline in the form of eye drops, nasal spray, and mouthwash. Participants were equally stratified by sex, age (60 years or older, under 60 years), and whether they were first responders to medical calls or first responders of emergency medical personnel, police officers, or emergency medical technicians in large hospital settings for the treatment of acute COVID-19. At enrollment, all participants were SARS-CoV-2 negative and provided with the solution to be used. Instructions were to apply the solution to the eyes, nose, and oral mucosa immediately before entering the risk environment. Application should be repeated every 4 hours as needed, based on the risk of exposure. Standard precautions should be used in addition to the PLL-g-PEG preparation or saline. The study period was 2 weeks for treatment or control. The outcomes after 4 weeks were A) the number of infected subjects in the treatment or control group after the start of treatment, and B) the severity, graded as 1-resolved without hospitalization, 2-required hospitalization, and 3-required ICU or death. As a secondary experiment, swab testing demonstrated that statistically equivalent amounts of viral particles adhered to patients' personal protective equipment and to inanimate surfaces such as room counters and bedside tables. Individuals at risk of infection practice personal safety measures, but despite these precautions, mucosal exposure in these individuals remains significant. The use of cationic graft copolymer formulations prevents infection in individuals at risk, resulting in reduced infection rates and disease severity.

[0156] The results are shown in Table 3.

[0157] [Table 4]

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

[0159] Example 3: In vitro ADC The ADC is AGS-16C3F. AGS-16C3F is an antibody-drug conjugate against ectonucleotide pyrophosphatase / phosphodiesterase 3 (ENPP3) containing an 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 under investigation. This invention is an experimental and novel approach to this toxicity.

[0161] Human primary corneal epithelial cells are cultured in keratinocyte SFM, and HCEC cells of ATCC are cultured in corneal epithelial cell basal medium supplemented with a corneal epithelial cell growth kit. Human cell lines are identified using short tandem repeat profiling to confirm mycoplasma negativity. Cells are seeded on multiple 8-well chamber slides and cultured for 48 hours before treatment and subsequent immunostaining. Cells are treated with a negative control (without copolymer) and also with PLL and PEG (not grafted together). Other cell cultures are exposed to PLL-g-PEG (PLL(20-g[3.5]-PEG(5)) as a primary example. Other PLL-g-PEGs are tested similarly. 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. Corneal stromal cells can retain viability after treatment with PLL-g-PEG, although some corneal stromal cells may die during the experiment in an in vitro experimental setting. Next, corneal stromal cells are incubated with AGS-16C3F (or rituxumab-mcMMAF or another ADC) for 4 hours with or without co-incubation of 0.5 mg / mL Dextran-Texas Red. Inhibition of macropinocytosis as a control is tested with AGS-16C3F (or another ADC) / Dextran-Texas Cells are evaluated by treating them with EIPA for 30 minutes before incubation with Red. After incubation, unbound antibodies and copolymers are washed off with PBS, and cells are fixed with 4% paraformaldehyde at room temperature for 20 minutes. Subsequently, cells are permeabilized with PBS + 0.1% Triton-X-100 for 15 minutes, and nonspecific labeling is blocked with PBS + 10% normal goat serum. AGS-16C3F (or other ADCs) bound to and internalized on the cell surface is visualized by incubating cells with Alexa Fluor 488-labeled goat anti-human IgG. The nuclei are visualized with TO-PRO-3 iodide, and coverslips are attached using ProLong Gold Antifade reagent for imaging.High-resolution laser confocal image sections are acquired using a Leica TCS SP5 II (63x oil immersion objective lens; NA 1 / 4 1.4), and these are sequentially scanned to minimize phosphor crosstalk and false positive colocalization.

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

[0163] result: Microscopic examination revealed that numerous ADCs, which were not heavily stained, were found to have penetrated copolymerized epithelial cells. A dose-response relationship has been demonstrated.

[0164] While models and designs are used, no ideas or intellectual property rights were, are not, or have never been obtained from Zhou or any other authors of the cited references.

[0165] See Table 4.

[0166] [Table 5]

[0167] Therefore, PLL-g-PEG, and by extension its imitations, is an effective intervention to reduce corneal toxicity associated with ADCs.

[0168] Experiments with megakaryocytes and the use of imitations are also effective.

[0169] Experiments will be conducted using ADCs, tubulin-destroying agents, and other cytotoxicities, but the effectiveness is not limited to a single type of ADC. In fact, it is possible to mitigate all ADCs with corneal epithelial toxicity using this approach. This approach is an ADC-independent method for reducing the corneal toxicity of this class effect.

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

[0171] Example 4: In vivo ADC Corneal toxicity associated with ADCs is reduced in in vivo models. While animal models for ADC toxicity are imperfect (cynomolgus monkey models are not optimal), rabbits are commonly used to test potential drug-mediated ocular toxicity and are selected to investigate the ocular toxicity of ADCs. AGS-16C3F and other ADCs are useful in these experiments. In vivo animal toxicity is very common in humans but somewhat variable in animals as a model. Rabbits exhibit varying toxicity to various ADCs. For this experiment, multiple eye experiments will be conducted in rabbits testing AGS-16C3F (AGS-16C3F is an antibody-drug conjugate against ectonucleotide pyrophosphatase / phosphodiesterase 3 (ENPP3) containing an mcMMAF linker-payload for the treatment of metastatic renal cell carcinoma) and other ADCs with a tubulin inhibitor payload. Five rabbits will be administered to each group. There are non-ADC-administered control groups that do not show toxicity with doses of 10 and 15 mg / kg of anuAGS-16C3F. Rabbits administered 15 mg / kg exhibit reversible conjunctival hyperemia, pericorneal haze, corneal edema, and ciliary body flush. Similarly, ocular toxicity occurs even with a once-weekly dose of 10 mg / kg. This experiment shows reduced ocular toxicity in rabbits administered 0.5%, 1%, and 2% PLL-g-PEG topical eye drops three times daily compared to rabbits without exposure to PLL-g-PEG from topical treatment at the start of ADC administration. The average benefit was better by more than 5%, 7.5%, and 10% in the 0.5%, 1%, and 2% PLL-g-PEG dose groups, respectively. Administration is performed daily with PLL-g-PEG eye drops and controls. ADC infusion is performed once a week. Even with PLL-g-PEG administration, beneficial effects were observed starting one week after ADC administration, but the benefits decreased by approximately 20% in each group by the end of the experiment. Animals administered PLL-g-PEG showed less toxicity associated with ADC compared to the control group (artificial tears only). All formulations were preservative-free. While formulations with preservatives are effective, they have the undesirable effect of causing or worsening corneal epitheliopathy. However, in rabbit models, the use of preservative solutions has been shown to be beneficial.

[0172] Subconjunctival preparations also provide protection if delivered subconjunctivally once a week.

[0173] Intravenous formulations are safe, tolerable, and effective, but local delivery is more effective.

[0174] The results are recorded by laboratory findings (including staining) and histopathological examination on days 21 and 42 (reduction of infiltration and tissue damage).

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

[0176] Other experiments using different polymer compositions would also be beneficial.

[0177] While the findings in rabbits do not identically reproduce those of human pycnoses, it should be noted that this model clinically and histopathologically demonstrates abnormalities in the anterior cornea. Select a sample ADC and repeat the results using other ADCs containing tubulin inhibitors.

[0178] See Table 5 [Table 6]

[0179] Example 5: ADC in humans This study will be conducted in patients prescribed ADCs for neoplastic diseases, using ADCs known to have corneal toxicity. Commercially available ADCs will be used in the clinical 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 involve patients receiving multiple different ADCs. Stratification will be performed based on the ADC and pre-existing eye discomfort.

[0180] Based on empirical observations, a total of 25 subjects from each group is deemed sufficient to detect differences in ophthalmic findings; therefore, 75 patients will be enrolled.

[0181] All patients will be enrolled at the start of treatment. Patients will be randomly assigned to receive either 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 instill the eye drops four times daily in both eyes, starting one day before ADC administration in Cycle 1. Ophthalmic examinations will be performed at baseline and in each cycle up to the fourth cycle. One cycle is 21 days long. From the fourth cycle onward, patients assigned to the treatment group may continue with PLL-g-PEG. Patients randomly assigned to artificial tears may cross over to PLL-g-PEG eye drops.

[0182] The severity of corneal microcystic epithelial disease, punctate superficial keratitis, or keratopathy is assessed using objective analysis to estimate the density and extent of epithelial lesions, and / or evaluation of epithelial damage by staining with a biological dye such as fluorescein. A score is used, obtained by multiplying the corneal damage rate by an overall clinical severity correction factor (1-3). The evaluation criterion for the study is the worst score during the study period.

[0183] Visual acuity is measured using logMAR.

[0184] In the primary analysis, the worst eye in each patient is used.

[0185] Furthermore, observe each eye independently.

[0186] Record any adverse events.

[0187] In the crossover group, we will evaluate the improvement in symptoms.

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

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

[0190] Drug discontinuation and administration delays were most frequent in the control group and least frequent in the low-dose and high-dose groups.

[0191] Eyes treated with PLL-g-PEG show fewer cases of punctate superficial keratitis. Visual acuity is, on average, better in eyes and patients treated with PLL-g-PEG (or copolymer).

[0192] This experiment exemplifies the clinical value of this intervention using a PLL-g-PEG solution applied topically to the eye to reduce the adverse effects of ADCs on the cornea.

[0193] Simultaneous intravenous delivery of a 1% PLL-g-PEG solution with each administration cycle demonstrates less thrombocytopenia in treated subjects, thereby demonstrating a broader effect in reducing ADC toxicity to the system extending beyond the corneal epithelium, and thus supporting broader patent claims.

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

[0195] Example 7. Preclinically evaluating various novel ADCs for uptake into human corneal epithelial cells in the presence of PLL-g-PEG formulations allows us to advance these specific ADCs, which show the greatest benefit (most significant reduction in uptake) in the presence of PLL-g-PEG, to clinical use. One reason for this selection is the existence of known and effective clinical methods for treating, mediating, mitigating, reducing, and preventing ADC toxicity in their cell types through co-treatment with the claimed copolymers discussed herein.

[0196] Three variations of ADCs targeting cell receptors found in neoplastic diseases with unmet 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% by weight) are used in similar in vitro tests of ADC uptake into human epithelial cells. The potential toxicity of the ADCs is re-evaluated. PLL-g-PEG at concentrations of 0.01% or higher (not necessarily excluding the endpoints) was found to significantly reduce the uptake of one of the three ADCs in vitro, by a rate that may vary depending on the ADC, but remains effective. This effect has been confirmed in an in vivo rabbit model. Thus, because methods for minimizing uptake by topical application of PLL-g-PEG are known, this molecule can enter clinical practice faster than other molecules. Other bioadhesive / inactivating copolymers are also used in ADC development, similar to the experiments described above.

Claims

1. A method for reducing viral infectivity by treating tissue involved in transfection with an effective amount of a graft or block copolymer having cationic, hydrophobic, or anionic sites and hydrophilic inactivation sites.

2. The method according to claim 1, wherein the copolymer is PLL-g-PEG.

3. The method according to claim 1, wherein the graft copolymer of the formulation comprises a cationic backbone and water-soluble and nonionic side chains.

4. The method according to claim 1, wherein the block copolymer of the formulation comprises at least one cationic block and at least one water-soluble and nonionic block.

5. The method according to claim 1, wherein the block copolymer of the formulation comprises at least one hydrophobic block and at least one block that is water-soluble and anionic, cationic, or nonionic.

6. The method according to any one of claims 1 to 5, wherein the biological surface to which the copolymer formulation is administered is a mucous membrane selected from the ocular mucosa, oral mucosa, nasal mucosa and respiratory tract mucosa, respiratory tract epithelium, urinary tract mucosa, and gastrointestinal mucosa of the subject.

7. The method according to claim 6, wherein the biological surface to which the copolymer formulation is administered is the surface of the eye.

8. The method according to any one of claims 1 to 5, wherein the viral infection is selected from coronavirus, influenza virus, Ebola virus and novel viruses transmitted by mucosal exposure.

9. The method according to claim 8, wherein the virus is SARS-CoV-2.

10. The method according to any one of claims 1 to 9, wherein the graft copolymer or block copolymer of the formulation constitutes 0.001 to 40% of the formulation.

11. The method according to any one of claims 1 to 9, wherein the graft copolymer or block copolymer of the formulation constitutes 0.1 to 10% of the formulation.

12. The method according to claim 8, wherein the inactivation effect is based on the interference of the SARS-Cov-2 spike protein and the ACE2 receptor on cells at risk.

13. The therapeutic effect is general steric inhibition, as described in claim 1.

14. A method for reducing adverse events associated with the use of antibody-drug conjugates that damage non-neogeneic cells (which exhibit adverse effects) by applying an effective amount of a copolymer having electrostatic and steric mediating properties to cells and tissues (which are adversely affected by the use of antibody-drug conjugates).

15. The method according to claim 14, wherein the copolymer is selected from cationic graft copolymers, cationic block copolymers, hydrophobic graft copolymers, hydrophobic block copolymers, anionic graft copolymers, and anionic block copolymers.

16. The method according to claim 14 or 15, wherein the copolymer is formulated by one or more approaches from among a powder, a solution, a suspension, a topical agent, an intravenous agent, an oral agent, an oral rinse, a nasal spray, and an eye drop.

17. The method according to claim 14 or 15, wherein the proportion of the copolymer solution is at least 0.01% by weight.

18. The method according to claim 14 or 15, wherein the proportion of the copolymer solution is up to 40% by weight in the case of a solution and a suspension.

19. The method according to claim 14, wherein the copolymer is PLL-g-PEG.

20. The method according to claim 14, wherein the copolymer is selected from the list of combinations described in the present application.

21. 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.

22. The method according to claim 21, wherein the copolymer is applied to corneal epithelial cells and conjunctival epithelial cells.

23. The method according to claim 22, wherein the copolymer is PLL-g-PEG.

24. The method according to claim 14, wherein the preparation is a solution for delivery into the subconjunctival space.

25. A method for reducing adverse events associated with the use of antibody-drug conjugates in humans, comprising applying an effective amount of a copolymer having electrostatic and steric mediating properties to cells involved in the adverse events.

26. The method according to claim 25, wherein the copolymer is selected from cationic graft copolymers, cationic block copolymers, hydrophobic graft copolymers, hydrophobic block copolymers, anionic graft copolymers, and anionic block copolymers.

27. The method according to claim 26, wherein the copolymer is selected from the polymers of the present disclosure listed above.

28. A method for reducing microcyst-like epithelial toxicity associated with cytotoxicity cleaved from ADCs by applying an effective amount of a copolymer having bioadhesive and inactivating properties to corneal epithelial cells.

29. The method according to claim 28, wherein the copolymer is PLL-g-PEG.

30. A method for 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 before initiating systemic ADC therapy.

31. A method for improving signs and symptoms of adverse ocular events associated with ADC use by delivering a copolymer having bioadhesive and inactivation sites to the eye after initiation of systemic ADC therapy.

32. A method for reducing the uptake of ADCs into cells by exposing them to a copolymer having bioadhesive and inactivation sites, including marginal stem cells, transiently amplified cells, basal epithelial cells, alar cells, and corneal epithelial cells, before exposure to ADCs.

33. A method for reducing the uptake of ADC into cells by exposing them to a copolymer having bioadhesive and inactivation sites after exposure to ADC, wherein the cells are marginal stem cells, transiently amplified cells, basal epithelial cells, alar cells, and corneal epithelial cells.

34. A method for reducing macropinocytosis-mediated uptake of ADCs by cells by exposing marginal stem cells, marginal epithelial cells, marginal epithelial daughter cells, transient amplified cells, basal epithelial cells, alar cells, corneal epithelial cells, and differentiated corneal epithelial cells to a copolymer having bioadhesive and inactivation sites before or after exposure to ADCs, and using a formulation having an effective ratio based on weight / weight calculation of the copolymer.

35. A method for treating patients with a copolymer having bioadhesive and inactivation sites, thereby reducing adverse ocular events associated with ADCs by using an effective amount.

36. A method for minimizing adverse effects resulting from exposure of cells to factors selected from SARS-CoV-2, novel viruses, viruses in an epidemic state, human pathological conditions, and ADCs having cytotoxic payloads that can cause disease, using copolymers that exhibit electrostatic and steric interactions at the cellular level.

37. A method for reducing ocular toxicity caused by systemic exposure to an ADC containing a tubulin-destroying agent as a payload in humans by treating the eyes with an effective amount of a cationic graft copolymer formulation.

38. The method according to claim 37, wherein the cationic graft copolymer is PLL-g-PEG.

39. The method according to claim 38, wherein the treatment is performed using an eye drop formulation.

40. The method according to claim 39, wherein the preparation is free of preservatives.

41. The method according to claim 37, wherein the cationic graft copolymer is PLL-g-PEG in an eye drop formulation at a concentration in the range of 0.01% to 5% by weight.

42. A method to reduce the discontinuation and dose reduction of ADCs in the treatment of human malignancies by applying an effective dose of PLL-g-PEG eye drops to the eye in patients at risk, thereby reducing corneal adverse events and ocular safety concerns.

43. An antibody for ADCs with corneal toxicity, having an inactivation site, and delivered to the eye in an amount effective in reducing adverse ocular events.

44. A method for reducing corneal cytotoxicity caused by a pharmaceutical selected from the group consisting of ADCs, biological substances, low molecular weight substances, high molecular weight substances, and peptides, comprising topically applying a copolymer having bioadhesive and inactivating components to an eye at risk of adverse effects caused by the pharmaceutical.

Images