WO2021236626A1 - Technologies antivirales mucorétentives - Google Patents

Technologies antivirales mucorétentives Download PDF

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Publication number
WO2021236626A1
WO2021236626A1 PCT/US2021/032945 US2021032945W WO2021236626A1 WO 2021236626 A1 WO2021236626 A1 WO 2021236626A1 US 2021032945 W US2021032945 W US 2021032945W WO 2021236626 A1 WO2021236626 A1 WO 2021236626A1
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virus
cyclodextrin
viral
agent
subject
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PCT/US2021/032945
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English (en)
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Michael J. Caplan
Howard B. Sosin
H. Kim Bottomly
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N-Fold Llc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/716—Glucans
    • A61K31/724—Cyclodextrins

Definitions

  • the present disclosure provides various insights relating to cyclodextrins and inhibition of viral infection.
  • the present disclosure provides an insight that retention of a cyclodextrin agent at mucosal surface(s) may provide an "antiviral shield" that can reduce viral infectivity and/or reduce host susceptibility to viral infection.
  • technologies provided by the present disclosure may reduce infective burden associated with an exposure to a virus.
  • the present disclosure proposes that provided treatment may reduce the infectivity of the virus and/or otherwise attenuate its capacity to initiate a severe infection, without materially impeding availability of the virus to the immune system.
  • the present disclosure therefore teaches that described treatment(s) may offer a particular advantage of attenuating virus sufficiently to reduce or remove morbidity while preserving immune reactivity, so that exposure to the virus could achieve immunity without material morbidity.
  • the present disclosure proposes that provided technologies may effectively convert an incidence of an exposure to a relevant virus from a health risk to an attenuated live virus vaccination event.
  • provided technologies may involve administration prior to development of symptom(s) (and/or other hallmarks - such as detection of viral component(s) [ e.g genetic material] and/or of anti-viral antibodies in a biological sample of a relevant subject) of infection and/or prior to known exposure to a relevant virus.
  • provided technologies may involve administration during and/or after expected, suspected or known exposure and/or development of one or more symptoms or other hallmarks of infection.
  • provided technologies may involve administration of one or more doses by a particular route; in some embodiments, provided technologies may involve administration by a plurality of different routes.
  • the present disclosure identifies the source of a problem with prior approaches to using cyclodextrins in treatment and/or prophylaxis of viral infection, in particularly appreciating that they do not contemplate retention of cyclodextrin agents at relevant site(s) (e.g., mucosal surfaces).
  • mucoretention of cyclodextrin agent(s) as described herein may provide one or more surprising advantages including for example, reduced viral infectivity, reduced viral transmissibility, and/or potential for live vaccination via virus attenuation.
  • the present disclosure provides a method comprising administering a cyclodextrin agent to one or more mucosal surfaces in a subject susceptible to or suffering from a viral infection according to a regimen demonstrated to achieve sufficient retention of the cyclodextrin agent at mucosal surfaces that incidence or severity of infection with an enveloped virus is reduced.
  • a subject susceptible to or suffering from a viral infection does not display symptoms of infection with the virus.
  • viral load has not been detected in the subject.
  • viral nucleic acid has not been detected in the subject.
  • anti-viral antibodies have not been detected in the subject.
  • the subject is not known to have been exposed to the virus.
  • the subject is suspected to have been exposed to the virus. In some embodiments, the subject is known to have been exposed to the virus. In some embodiments, the subject does not develop symptoms of infection with the virus after subsequent exposure to the virus. In some embodiments, the subject does not develop serious illness after subsequent exposure to the virus. In some embodiments, serious illness involves hospitalization. In some embodiments, the subject develops anti-viral antibodies after exposure to the virus.
  • methods of the present disclosure further comprise after a step of administering (i.e., a “first administering”), performing at least one further administering., administering, optionally after assessing.
  • the step of administering comprises self-administering.
  • the step of administering comprises aerosol administration.
  • the step of administering comprises administering a nasal spray.
  • the step of administering comprises ingesting.
  • the step of administering comprises aerosol administration of at least one dose and ingestion of at least one dose.
  • the present disclosure provides a method of characterizing a cyclodextrin agent or a formulation there of by assessing one or more of: mucus binding, accumulation, and/or retention; ability to strip cholesterol from a cell membrane; ability to strop cholesterol from a viral envelope; ability to reduce viral infectivity; ability to reduce viral transmission; ability to reduce severity of viral disease; and/or extent of host immune response upon exposure to virus after treatment with the cyclodextrin agent or formulation thereof.
  • the present disclosure provides a combination comprising: a cyclodextrin agent; and a retentive material.
  • the present disclosure provides a method comprising steps of: administering a retentive material to mucosal surfaces in a subject at risk of or having experienced viral exposure; and subsequently administering a cyclodextrin agent to the mucosal surfaces.
  • affinity is a measure of the tightness with which two or more binding partners associate with one another. Those skilled in the art are aware of a variety of assays that can be used to assess affinity, and will furthermore be aware of appropriate controls for such assays. In some embodiments, affinity is assessed in a quantitative assay. In some embodiments, affinity is assessed over a plurality of concentrations (e.g., of one binding partner at a time). In some embodiments, affinity is assessed in the presence of one or more potential competitor entities (e.g., that might be present in a relevant - e.g., physiological - setting).
  • affinity is assessed relative to a reference (e.g., that has a known affinity above a particular threshold [a “positive control” reference] or that has a known affinity below a particular threshold [ a “negative control” reference”].
  • affinity may be assessed relative to a contemporaneous reference; in some embodiments, affinity may be assessed relative to a historical reference. Typically, when affinity is assessed relative to a reference, it is assessed under comparable conditions.
  • Analog refers to a substance that shares one or more particular structural features, elements, components, or moieties with a reference substance. Typically, an “analog” shows significant structural similarity with the reference substance, for example sharing a core or consensus structure, but also differs in certain discrete ways. In some embodiments, an analog is a substance that can be generated from the reference substance, e.g., by chemical manipulation of the reference substance.
  • Antiviral agent refers to a class of medication used specifically for treating viral infections by inhibiting, deactivating, or destroying virus particles.
  • an antiviral agent may be or comprise an agent of any chemical class or type (e.g., a carbohydrate, cell, lipid, metal, nucleic acid, polypeptide [e.g., an antibody], and/or small molecule).
  • an antiviral agent is or comprises an antibody or antibody mimic.
  • an antiviral agent is or comprises a nucleic acid agent (e.g., an antisense oligonucleotide, a siRNA, a shRNA, etc, or a viral vector or mRNA encoding a viral antigen that provokes a neutralizing immune response in the recipient) or mimic thereof.
  • an antiviral agent is or comprises a small molecule.
  • an antiviral agent is or comprises a naturally-occurring compound (e.g., small molecule).
  • an antiviral agent has a chemical structure that is generated and/or modified by the hand of man.
  • an antiviral agent is or comprises an antibody (e.g in serum or recombinantly produced) from a subject who has recovered from infection by the virus.
  • Two events or entities are “associated” with one another, as that term is used herein, if the presence, level, degree, type and/or form of one is correlated with that of the other.
  • a particular entity e.g., polypeptide, genetic signature, metabolite, microbe, etc
  • two or more entities are physically “associated” with one another if they interact, directly or indirectly, so that they are and/or remain in physical proximity with one another.
  • two or more entities that are physically associated with one another are covalently linked to one another; in some embodiments, two or more entities that are physically associated with one another are not covalently linked to one another but are non-covalently associated, for example by means of hydrogen bonds, van der Waals interaction, hydrophobic interactions, magnetism, and combinations thereof.
  • Biocompatible refers to materials that do not cause significant harm to living tissue when placed in contact with such tissue, e.g., in vivo. In certain embodiments, materials are “biocompatible” if they are not toxic to cells. In certain embodiments, materials are “biocompatible” if their addition to cells in vitro results in less than or equal to 20% cell death, and/or their administration in vivo does not induce significant inflammation or other such adverse effects.
  • biological sample typically refers to a sample obtained or derived from a biological source (e.g., a tissue or organism or cell culture) of interest, as described herein.
  • a source of interest comprises an organism, such as an animal or human.
  • a biological sample is or comprises biological tissue or fluid.
  • a biological sample may be or comprise bone marrow; blood; blood cells; ascites; tissue or fine needle biopsy samples; cell-containing body fluids; free floating nucleic acids; sputum; saliva; urine; cerebrospinal fluid, peritoneal fluid; pleural fluid; feces; lymph; gynecological fluids; skin swabs; vaginal swabs; oral swabs; nasal swabs; washings or lavages such as a ductal lavages or broncheoalveolar lavages; aspirates; scrapings; bone marrow specimens; tissue biopsy specimens; surgical specimens; feces, other body fluids, secretions, and/or excretions; and/or cells therefrom, etc.
  • a biological sample is or comprises cells obtained from an individual.
  • obtained cells are or include cells from an individual from whom the sample is obtained.
  • a sample is a “primary sample” obtained directly from a source of interest by any appropriate means.
  • a primary biological sample is obtained by methods selected from the group consisting of biopsy (e.g., fine needle aspiration or tissue biopsy), surgery, collection of body fluid (e.g., blood, lymph, feces etc.), etc.
  • sample refers to a preparation that is obtained by processing (e.g., by removing one or more components of and/or by adding one or more agents to) a primary sample. For example, filtering using a semi-permeable membrane.
  • Such a “processed sample” may comprise, for example nucleic acids or proteins extracted from a sample or obtained by subjecting a primary sample to techniques such as amplification or reverse transcription of mRNA, isolation and/or purification of certain components, etc.
  • Comparable refers to two or more agents, entities, situations, sets of conditions, etc., that may not be identical to one another but that are sufficiently similar to permit comparison there between so that one skilled in the art will appreciate that conclusions may reasonably be drawn based on differences or similarities observed.
  • comparable sets of conditions, circumstances, individuals, or populations are characterized by a plurality of substantially identical features and one or a small number of varied features.
  • Dosage form or unit dosage form may be used to refer to a physically discrete unit of an active agent (e.g, a therapeutic or diagnostic agent) for administration to a subject. Typically, each such unit contains a predetermined quantity of active agent.
  • an active agent e.g, a therapeutic or diagnostic agent
  • such quantity is a unit dosage amount (or a whole fraction thereof) appropriate for administration in accordance with a dosing regimen that has been determined to correlate with a desired or beneficial outcome when administered to a relevant population (i.e., with a therapeutic dosing regimen).
  • a dosing regimen that has been determined to correlate with a desired or beneficial outcome when administered to a relevant population (i.e., with a therapeutic dosing regimen).
  • the total amount of a therapeutic composition or agent administered to a particular subject is determined by one or more attending physicians and may involve administration of multiple dosage forms.
  • dosing regimen may be used to refer to a set of unit doses (typically more than one) that are administered individually to a subject, typically separated by periods of time.
  • a given therapeutic agent has a recommended dosing regimen, which may involve one or more doses.
  • a dosing regimen comprises a plurality of doses each of which is separated in time from other doses.
  • individual doses are separated from one another by a time period of the same length; in some embodiments, a dosing regimen comprises a plurality of doses and at least two different time periods separating individual doses.
  • all doses within a dosing regimen are of the same unit dose amount.
  • a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount different from the first dose amount.
  • a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount same as the first dose amount.
  • a dosing regimen is correlated with a desired or beneficial outcome when administered across a relevant population (i.e., is a therapeutic dosing regimen).
  • Gel refers to viscoelastic materials whose rheological properties distinguish them from solutions, solids, etc.
  • a composition is considered to be a gel if its storage modulus (G') is larger than its modulus (G").
  • a composition is considered to be a gel if there are chemical or physical cross-linked networks in solution, which is distinguished from entangled molecules in viscous solution.
  • Susceptible to An individual who is “susceptible to” a disease, disorder, and/or condition is one who has a higher risk of developing the disease, disorder, and/or condition than does a member of the general public. In some embodiments, an individual who is susceptible to a disease, disorder and/or condition may not have been diagnosed with the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition may exhibit symptoms of the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition may not exhibit symptoms of the disease, disorder, and/or condition.
  • an individual who is susceptible to a disease, disorder, and/or condition will develop the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition will not develop the disease, disorder, and/or condition.
  • treatment refers to administration of a therapy that partially or completely alleviates, ameliorates, relives, inhibits, delays onset of, reduces severity of, and/or reduces incidence of one or more symptoms, features, and/or causes of a particular disease, disorder, and/or condition.
  • such treatment may be of a subject who does not exhibit signs of the relevant disease, disorder and/or condition and/or of a subject who exhibits only early signs of the disease, disorder, and/or condition.
  • such treatment may be of a subject who exhibits one or more established signs of the relevant disease, disorder and/or condition.
  • treatment may be of a subject who has been diagnosed as suffering from the relevant disease, disorder, and/or condition. In some embodiments, treatment may be of a subject known to have one or more susceptibility factors that are statistically correlated with increased risk of development of the relevant disease, disorder, and/or condition. Thus, in some embodiments, treatment may be prophylactic; in some embodiments, treatment may be therapeutic.
  • the present disclosure provides technologies for delivering cyclodextrin agent(s) to, and retaining them at, mucosal surfaces - e.g., the mucus layer and/or glycocalyx that sits atop the outward-facing surfaces of the epithelial cells of the nose, airway, lung, gut [2] and cornea [43]
  • the present disclosure teaches that such retention provides unexpected benefits including, for example, potential ability to deplete cholesterol from cell membranes at high risk sites of viral entry for extended periods of time and/or to deplete cholesterol from viral envelopes so as to effectively attenuate such viruses, reducing infectivity and potentially providing live vaccination.
  • the present disclosure proposes that achieving high local concentration of cyclodextrin agent at mucosal surface(s) may serve as a chemical shield that diminishes the infectious potential of relevant virus particles (specifically including coronaviruses, including SARS-CoV-2) as they traverse this cyclodextrin-agent-enriched barrier.
  • the retained cyclodextrin agent can reduce the concentration of cholesterol in adjacent target cell apical plasma membrane(s).
  • the retained cyclodextrin agent can strip cholesterol out of the membrane of an invading virus during its transit.
  • a cyclodextrin agent may be administered with sufficient frequency and/or at sufficient concentration and/or in an appropriate formulation so that a protective “layer” or “barrier” is formed over part or all of a relevant mucosal surface.
  • viral load e.g., early in infection
  • multiplicity of infection can be a determinant of disease severity and/or virus transmissibility.
  • provided technologies may reduce transmissibility of virus from one exposed person to another, for example, by attenuating virus to the extent that it cannot effectively survive transport to another subject and/or cannot establish infection even if transmitted.
  • cyclodextrin retention technologies provided herein may attenuate viruses without necessarily destroying them; in some such embodiments, sufficient exposure to the exposed subject’s immune system may occur to provide partial or total immunity to the relevant virus and/or to one or more related viruses.
  • Cholesterol has been determined to participate in, and in some cases to be required for, entry of certain viruses into mammalian cells. Specifically, viral receptors are often found in cholesterol-containing “rafts” in the plasma membranes of target cells (see, for example, Chazal & Gerber Microbiol &Molec Biol Rev 67:226, 2003).
  • the viral capsid binds to its receptor in the plasma membrane, and the virus enters the cell by invagination. Involvement of cholesterol in this process has been documented for various such viruses, including certain Papovaviridae viruses (e.g ., simian virus 40 (SV40); [56-60]), Picornaviridae viruses, and particularly certain enteroviruses(e.g., enterovirus 70; echoviruses such as EV1, EV11; coxackieviruses such as coxackie B viruses, coxackievirus A21; , etc.; [61-62], and Reoviridae viruses (e.g., certain rotaviruses [63-64]).
  • Papovaviridae viruses e.g ., simian virus 40 (SV40); [56-60]
  • enteroviruses e.g., enterovirus 70
  • echoviruses such as EV1, EV11
  • coxackieviruses such as co
  • viral and target cell membranes fuse, and in some cases form a fusion pore that promotes penetration of the viral nucleocapsid into the cell.
  • herpes viruses e.g., herpes simplex virus 1, herpes simplex virus 2, varicella-zoster virus
  • influenza viruses measles virus, etc.
  • Teachings of the present disclosure are relevant to infection by non- enveloped viruses that rely on cholesterol to achieve or establish infection to the extent that such teachings relate to depletion of cholesterol from target cell membranes.
  • teachings of the present disclosure relating to depletion of cholesterol from target cell membranes are also relevant to infection by enveloped viruses.
  • the present disclosure teaches that practice of provided technologies with respect to such enveloped viruses provides additional benefits.
  • depletion of cholesterol from viral envelopes can attenuate viral infectivity and/or transmissibility.
  • this approach to attenuation may preserve sufficient viral exposure to the exposed subject’s immune system to achieve partial or total immunity to the relevant virus and/or related virus (es).
  • Coronaviruses are a family of enveloped RNA viruses. Most coronaviruses that infect humans cause mild to moderate upper respiratory tract infections; many coronaviruses (e.g., types 229E, NL63, OC43, and HKU1) are described as having symptoms of the “common cold”. Certain coronaviruses cause very serious disease, and even death.
  • SARS coronavirus SARS coronavirus (SARS-CoV)
  • SARS-CoV severe acute respiratory syndrome
  • MERS coronavirus MERS coronavirus
  • SARS-CoV-2 first identified in humans in December 2019, causes coronavirus disease 2019 (COVID-19), and was declared a global pandemic by the World Health Organization on March 11, 2020.
  • cholesterol appears to be required for the effective binding of the viral S protein to the host cell receptor ACE2 [14, 15]
  • cholesterol may play an important role in aspects of the budding of the mature infectious virus.
  • the present disclosure proposes that reducing cholesterol concentrations in the membrane of the virus and/or in the plasma membranes of target cells might substantially reduce the susceptibility of these cells to be invaded by coronavirus, and/or to produce large quantities of infectious virus particles if they are successfully invaded.
  • the present disclosure appreciates that a large number of in vitro studies have demonstrated that cholesterol depletion of the plasma membranes of viruses and of cultured cells using methyl -b-cyclodextrin (Mbq ⁇ ) substantially reduces the extent of coronaviral infection [3-18] in cultured cells and in excised tissue samples.
  • Mbq ⁇ methyl -b-cyclodextrin
  • the present disclosure identifies the source of certain problems with merely delivering a cyclodextrin to a relevant site, including, for example, that it may be difficult or impossible to coordinate timing of administration with that of exposure sufficiently to achieve benefit.
  • the present disclosure provides solutions to this problem, including by providing certain technologies for retaining cyclodextrin agent(s) at mucosal surface(s).
  • the present disclosure further provides an insight that retention of cyclodextrin agent(s) at mucosal surface(s) may provide additional, and previously unexpected, advantages(s) including, for example, attenuating viral infectivity while permitting immune system exposure, thus potentially achieving effective immunization while reducing or preventing significant morbidity.
  • SARS-CoV-2 is thought to enter the body through the mouth, nose and eyes, and it initially infects the epithelial cells of the nose, airway, gut and cornea [1].
  • the SARS-CoV-2 virus In order to gain access to the ACE2 protein, which serves as its receptor on the lumen-facing apical plasma membranes of these epithelial cells, the SARS-CoV-2 virus must first penetrate the layer of mucus and the glycocalyx that covers the luminal surfaces of the nasal, airway and gut epithelia [2]
  • Technologies provided by the present disclosure administer and retain cyclodextrin agent(s) at relevant mucosal surface(s) (e.g in one or more of the mouth, nose, eyes, and/or gut), and are thus useful for prophylaxis and/or treatment of coronavirus infections, including with SARS-CoV-2.
  • Herpes viruses have a DNA genome that is encapsulated with a capsid surrounded by an amorphous protein coat within its envelope.
  • Cholesterol has been demonstrated to be required for cellular entry by at least
  • HSV1 [75-77] and varicella-zoster virus [78] Furthermore, reports have shown that cholesterol is required for various events in the HSV-1 life cycle beyond initial entry and transport of viral capsids to the nucleus [79]
  • herpes viruses can establish latent infections. Transmission of herpes simplex viruses typically requires close contact between a person who is shedding virus and a susceptible host; by contrast, varicella-zoster virus is usually transmitted by airborne routes. Infection is usually established within 4-6 days of exposure.
  • the present disclosure provides technologies for prophylaxis and/or treatment of herpes virus infection, for example by administration and retention of a cyclodextrin agent as described herein on mucosal (and/or skin) surfaces susceptible to exposure or suspected of having been exposed.
  • Influenza viruses are enveloped RNA viruses that cause outbreaks of illness, resulting in millions of infections, hundreds of thousands of hospitalizations, and tens of thousands of deaths each year. Moreover, influenza viruses have been responsible for pandemics in each of 1918, 1957, 1968, and 2009.
  • Technologies provided by the present disclosure administer and retain cyclodextrin agent(s) at relevant mucosal surface(s) (e.g., in one or more of the mouth, nose, eyes, and/or gut), and are thus useful for prophylaxis and/or treatment of influenza infections, including both seasonal and pandemic influenza infections.
  • Cyclodextrins are cyclic oligosaccharides that comprise a(l,4)-linked glucopyranose subunits. [19], which typically formhexamers (e.g., in a-cyclodextrins), heptamers (e.g., in b -cyclodextrins), or octamers (e.g., in g-cyclodextrins). Cyclodextrin structure creates a hydrophobic cavity whose size and characteristics (e.g., affinity for particular compounds) is influenced by, for example, the number of glucopyranose subunits in the ring structure and/or the degree of polymerization. [53]
  • Cyclodextrins are typically water soluble, and impact of moieties such as methyl groups etc on such water solubility has been established.
  • certain methylated cyclodextrins are known to have greater water solubility (e.g., as compared with their corresponding unmethylated analogs).
  • Certain cyclodextrins are known to bind cholesterol, and to be able to strip it out of, for example, cell and/or viral membranes
  • Methyl -b-cyclodextrin. in particular, has a central cavity that forms a 2: 1 complex with cholesterol.
  • Cyclodextrins are easy and inexpensive to produce, including in industrial quantities, for example through enzymatic modification of starch [21]
  • Cyclodextrins are generally considered to be non-toxic. They are in very wide use as FDA-approved food additives [22] They have also been administered orally to humans as a means for lowering plasma lipid levels by preventing intestinal cholesterol absorption [20] Moreover, as a consequence of their capacity to bind to small hydrophobic molecules like cholesterol, cyclodextrins have been extensively developed as carriers for the delivery of a variety of pharmaceuticals [19, 21, 23-28] Consequently, they are ingested or applied in a very large number of pharmaceutical and hygiene products [29], including oral rinses, eye drops and nasal sprays.
  • cyclodextrins ability to deplete cholesterol from lipid membranes has led to studies of its use to treat conditions associated with excess or inappropriate cholesterol accumulation.
  • HP CD 2-hydroxypropyl ⁇ -cyclodextrin
  • HP CD delivered either intravenously or intrathecally, is currently in advanced human trials designed to test its ability to deplete cholesterol from cells that have excessive levels of cholesterol in patients with the genetic disorder Niemann-Pick C disease [36-38]; both routes of delivery of HRbO ⁇ are well tolerated.
  • aerosolized b6 ⁇ has shown promise in preclinical animal testing for its ability to decrease the elevated levels of cholesterol that damage pulmonary surfactant in certain patients suffering from Acute Respiratory Distress Syndrome [39] and/or Cystic Fibrosis [40]
  • cyclodextrins constitute safe and aerosol-deliverable compounds with which to control the levels of cholesterol in the plasma membranes of airway epithelial cells, and furthermore that they can be safely applied to the surfaces of the oral, ocular, nasal and gut epithelia.
  • certain studies and/or proposals suggest the possibility of an anti-viral role for cyclodextrins; the present disclosure identifies the source of a problem(s) with such studies and proposals, provides solutions for such problem(s) and furthermore provides insights of unexpected benefits associated with such provided solutions.
  • the present disclosure provides technologies for administering cyclodextrin agent(s) to, and retaining them at, mucosal surfaces.
  • the present disclosure teaches that such retention provides unexpected benefits including, for example, potential ability to deplete cholesterol from cell membranes at high risk sites of viral entry for extended periods of time and/or to deplete cholesterol from viral envelopes so as to effectively attenuate such viruses, reducing infectivity and potentially providing live vaccination.
  • a cyclodextrin agent is or comprises an a- cyclodextrin, a b -cyclodextrin, and/or a g-cyclodextrin.
  • a cyclodextrin agent is or comprises a derivative of a natural cyclodextrin - i.e., comprises one or more modifying moieties (e.g., methyl groups). A variety of such derivatives are known, as are effective synthesis technologies, etc. [74]
  • a cyclodextrin agent is or comprises one or more of
  • the present disclosure provides cyclodextrin agents that include one or more mucoadhesive moieties.
  • a cyclodextrin agent e.g., an a-cyclodextrin, a b - cyclodextrin, and/or a g-cyclodextrin, with or without one or more modifying moieties such as methyl groups and/or mucoadhesive moieties, is or comprises a polymeric cyclodextrin.
  • a utilized cyclodextrin agent will be biocompatible. Certain cyclodextrins (e.g., unmodified cyclodextrin) can show toxicities. It may be desirable to utilize a cyclodextrin agent that is modified relative to a parent cyclodextrin (e.g., to a natural cyclodextrin) and displays improved biocompatibility/decreased toxicity relative to such parent cyclodextrin.
  • a cyclodextrin agent that is modified relative to a parent cyclodextrin (e.g., to a natural cyclodextrin) and displays longer half-life (e.g., less degradation) than such parent cyclodextrin under relevant conditions (e.g., under storage conditions and/or under conditions that are or mimic one or more features of a human mucosal surface or other relevant physiological location such as saliva).
  • such modified cyclodextrin may display reduced susceptibility to degradation (e.g., by amylase).
  • a cyclodextrin may be or comprise a methyl-b- or hydroxy propyl -b-cyclodextrin (e.g., may be a modified methyl-b- or hydroxypropyl b- cyclodextrin), as various methyl-b- and/or hydroxypropyl-b-cyclodextrins have been demonstrated to be particularly effective at binding cholesterol.
  • technologies provided herein utilize a cyclodextrin agent that itself is mucoadhesive (e.g., that includes a mucoadhesive moiety).
  • mucoadhesive moieties and entities are known in the art.
  • various saccharides e.g., alginate, chitosan, hydroxypropylmethylcellulose [HPMC]
  • other polymers e.g., carbomers, polyacrylic acid (PAA), poly(lactic-co- glycolic acid) (PLGA), polyethylene oxide (PEO), polypropylene glycol (PPG), and certain thiolated polymers (“thiomers”, e.g., as may have been thiolated, for example, by conjugation with one or more cysteine ethyl esters; in some particular embodiments, a thiomer may be or comprise a thiolated polyacrylic acid) have been demonstrated to have mucoadhesive properties, as have certain fatty agents like glycerin.
  • one or more such mucoadhesive entities or moieties may be combined and/or conjugated with a CD for use as
  • a mucoadhesive cyclodextrin may comprise (e.g., may be modified relative to a parent cyclodextrin) one or more amino groups and/or one or more sulfate groups (e.g., attached to the cyclodextrin backbone), which may impart increased mucosal binding, accumulation, and/or retention relative to the parent cyclodextrin.
  • a mucoadhesive cyclodextrin may be modified (relative to a parent cyclodextrin) with (i.e., covalently linked to) one or more entities (such as alginate, carbomer, chitosan and/or a chitosan derivative (e.g., quaternary amonium chitosan, thiolated chitosan, etc), glycerin, hydroxypropylmethylcellulose, polyacrylic acid (PAA), poly(lactic-co-glycolic acid) (PLGA), polyethylene oxide (PEO), polypropylene glycol (PPG) , thiolated polymer, etc) characterized by mucoadhesive properties, such that the modified cyclodextrin shows increased mucosal binding, accumulation, and/or retention relative to the parent cyclodextrin.
  • one or more entities such as alginate, carbomer, chitosan and/or a chito
  • provided technologies involve administration of a cyclodextrin agent in or with one or more retentive materials.
  • a cyclodextrin agent that may not itself display significant mucoadhesive properties and/or mucoretention may be combined with ( e.g ., in a single formulation or via combination administration) one or more other materials with which it associates to achieve mucoretention of the cyclodextrin agent as described herein.
  • Combination with a retentive material is not limited to cyclodextrin agents that may not themselves display significant mucoadhesive properties and/or mucoretention, however; in some embodiments, mucoadhesive cyclodextrin agents may be so combined.
  • a cyclodextrin agent e.g., a cyclodextrin or a modified cyclodextrin, which may be a mucoadhesive cyclodextrin
  • a retentive material is administered first, followed by administration of the cyclodextrin agent, which may then associate with (e.g., become entrapped or otherwise retained by) the retentive material.
  • a cyclodextrin agent may be combined with a mucoadhesive material such as alginate, carbomer, chitosan and/or a chitosan derivative (e.g., quaternary ammonium chitosan, thiolated chitosan, etc), glycerin, hydroxypropylmethylcellulose, polyacrylic acid (PAA), poly(lactic-co-glycolic acid) (PLGA), polyethylene oxide (PEO), polypropylene glycol (PPG) , and/or a thiolated polymer.
  • a mucoadhesive material such as alginate, carbomer, chitosan and/or a chitosan derivative (e.g., quaternary ammonium chitosan, thiolated chitosan, etc), glycerin, hydroxypropylmethylcellulose, polyacrylic acid (PAA), poly(lactic-co-glycolic acid) (
  • a material may be considered to be a “retentive material” by virtue of sufficient viscosity that, once applied, it remains reasonably associated with mucosal surface(s) even without specific mucosal adherence ( e.g relative to other surface(s)).
  • a retentive material may be or comprise a viscous liquid or a gel.
  • a retentive material (which may, for example, be or comprise a mucoadhesive material and/or a viscous material) may be considered an “artificial mucus” with which a cyclodextrin agent (e.g., a cyclodextrin or a modified cyclodextrin, which may be a mucoadhesive cyclodextrin) may be mixed or otherwise administered in combination.
  • a cyclodextrin agent e.g., a cyclodextrin or a modified cyclodextrin, which may be a mucoadhesive cyclodextrin
  • a retentive material may reduce incidence or severity of viral infection.
  • the administration of a retentive material as described herein may itself provide some protective effect, if only, for example, by providing some barrier or coating that may slow or otherwise inhibit viral exposure to relevant receptors.
  • a retentive material is administered without a cyclodextrin agent (e.g. before, after, or concurrently with one or more administrations of a cyclodextrin agent and/or independent of any administration of cyclodextrin agent).
  • a retentive material is administered repeatedly without a cyclodextrin agent.
  • a subject receives, one or more doses of a retentive material prior to administration of a cyclodextrin agent.
  • a retentive material is administered in any manner herein described for cyclodextrin agents.
  • a cyclodextrin agent may be combined with a hydrogel or other material, such as those established for use in nasal sprays.
  • nasal sprays are known in the art including, for example, Ayr Saline Nasal Gel (produced BF Ascher & Co., Inc), which includes water, glycerin, methyl gluceth-10, propylene glycol, PEG/PPG-18/18 dimethicone, aloe barbadensis leaf juice (aloe vera gel), carbomer, sodium chloride, xanthan gum, glyceryl acrylate/acrylic acid copolymer, potassium sorbate, sodium hydroxide, diazolidinyl urea, glycine soja (soybean) oil, geranium maculatum oil, tocopheryl acetate, citric acid, sodium benzoate, sodium sulfite, and coloring; other nasal gel products, such as NasoGelTM (produced by
  • cyclodextrin agents as described herein will be formulated for delivery to particular mucosal surface(s) of interest, for example in nasal passages, oral cavity, lungs, and/or gastrointestinal tract ( e.g ., in the gut).
  • cyclodextrin agent(s) will be formulated for aerosol administration (e.g., via the nose and/or the mouth).
  • cyclodextrin agent(s) will be formulated for ingestion.
  • ingestible formulations will comprise an enteric coating or other technology to protect the cyclodextrin agent(s) from exposure to gastric acid and to permit their release in the small intestine.
  • enteric coatings which may be applied, for example, to a tablet, capsule, lozenge, or other ingestible dosage form.
  • Certain such enteric coatings are prepared, for example, from fatty acids, plant fibers, waxes, and/or combinations thereof.
  • cyclodextrin agent(s) will be formulated for administration to the eye (e.g., in eye drops).
  • a provided formulation may be or comprise beads, particles (e.g., nanoparticles), etc.; in some such embodiments, beads or particles may be or comprise a polymer or a polymeric material.
  • a provided formulation may be or comprise a gel, a liquid, and/or a solid.
  • a provided formulation may be or comprise a powder.
  • a provided formulation may be or comprise a dispersion, an emulsion, or a suspension.
  • an aerosol formulation may be or comprise a spray (e.g., a nasal spray).
  • a provided formulation may comprise or consist of a cyclodextrin agent (e.g., a cyclodextrin or a modified cyclodextrin, which may be a mucoadhesive cyclodextrin) combined with a carrier formulation otherwise available in an approved or over-the counter product.
  • a cyclodextrin agent e.g., a cyclodextrin or a modified cyclodextrin, which may be a mucoadhesive cyclodextrin
  • a carrier formulation otherwise available in an approved or over-the counter product.
  • nasal spray products are available, some of which were developed and/or are marketed to include a particular pharmaceutically active agent.
  • a cyclodextrin agent e.g., a cyclodextrin or a modified cyclodextrin, which may be a mucoadhesive cyclodextrin
  • a cyclodextrin agent is added to or otherwise administered together with such nasal spray product (optionally without the pharmaceutically active agent with which it was developed and/or is otherwise marketed).
  • a cyclodextrin agent e.g., a cyclodextrin or modified cyclodextrin, which may be a mucoadhesive cyclodextrin
  • a retentive material e.g., a cyclodextrin or modified cyclodextrin, which may be a mucoadhesive cyclodextrin
  • a provided formulation i.e., that is or comprises a cyclodextrin agent and/or a retentive material
  • such characterization may be assessed relative to an appropriate reference (e.g., to a control that lacks any cyclodextrin or that is or comprises a natural cyclodextrin that is not mucoadhesive and/or that is not mixed or otherwise combined with a retentive material or otherwise formulated for mucoretentive) and/or to one or more alternative cyclodextrin agent(s) and/or formulation(s).
  • an appropriate reference e.g., to a control that lacks any cyclodextrin or that is or comprises a natural cyclodextrin that is not mucoadhesive and/or that is not mixed or otherwise combined with a retentive material or otherwise formulated for mucoretentive
  • alternative cyclodextrin agent(s) and/or formulation(s) e.g., to a control that lacks any cyclodextrin or that is or comprises a natural cyclodextrin that is not mucoadhe
  • a cyclodextrin agent and/or formulation may be characterized, for example, for one or more of: ability to bind to, accumulate in, and/or be retained in a relevant mucosal surface(s) (e.g., in a mucus or gly cocalyx layer); in some embodiments, such assessment may be made in vitro (e.g., using cultured cells such as epithelial cells and/or other cells; preferably with respect to human cells). In some embodiments, such assessment may be made in vivo (e.g., in a model organism or a human)
  • a cyclodextrin agent and/or formulation may be characterized, for example for its ability to deplete cholesterol from a cell membrane (e.g., a target cell membrane, such as a membrane of a human cell such as a human epithelial cell) and/or from an envelope of an enveloped virus); in some embodiments, such assessment may be made in vitro (e.g., using cultured cells such as epithelial cells and/or other cells; preferably with respect to human cells). In some embodiments, such assessment may be made in vivo (e.g., in a model organism or a human).
  • a cyclodextrin agent and/or formulation may be characterized, for example, for its ability to impact viral infectivity and/or transmissibility and/or for its impact on infection severity.
  • a cyclodextrin agent and/or formulation may be assessed for its ability to reduce viral load in a subject after exposure to virus under particular conditions (e.g., level of exposure, time of exposure, and/or route of exposure, etc )
  • a cyclodextrin agent and/or formulation may be characterized, for example, for its ability to attenuate a virus (e.g., an enveloped virus) and/or for its ability to permit exposure to the immune system of a host exposed to the virus.
  • immune system exposure may be assessed by detection of antibodies or other indicators of antibody response.
  • reduced infectivity and/or reduced severity of disease combined with detection of antibody response may indicate that provided cyclodextrin agents and/or formulations have provided a “viral shield” that effectively converted a viral exposure event to a live immunization event.
  • one or more detected antibodies may be characterized to assess its neutralization characteristics, e.g., by assessing binding to a known neutralizing target and/or to inhibit one or more key steps of viral life cycle (e.g., infection, replication, expression, encapsulation, release).
  • neutralization characteristic(s) may be assessed by assessing ability to protect a separate organism from infection and/or to reduce severity of disease, by administration of the antibody before, during, and/or after exposure to virus.
  • a cyclodextrin agent or formulation as described herein may be assessed using one or more assays as described herein (see Examples).
  • provided cyclodextrin agent(s) and/or formulation(s) may be administered to individuals with no known exposure to a relevant virus, expected to be at risk of exposure to such virus, and/or having had known exposure to virus.
  • provided cyclodextrin agent(s) and/or formulation(s) may be administered to individuals who do not display any known symptoms or features of infection with a relevant virus. In some embodiments, provided cyclodextrin agent(s) and/or formulation(s) may be administered to individuals who display one or more, but not all known symptoms and/or features of infection with a relevant virus. In some embodiments, provided cyclodextrin agent(s) and/or formulation(s) may be administered to individuals who have not been diagnosed as having been infected with the relevant virus. In some embodiments, provided cyclodextrin agent(s) and/or formulation(s) may be administered to individuals who have been diagnosed with the relevant virus.
  • Exemplary symptoms of coronavirus infection, and particularly of COVID19 may include, for example, chills, cough, difficulty breathing or shortness of breath, fever, loss of taste or smell, muscle pain, sore throat, particularly if combined with one or more of bluish lips or face, confusion, inability to wake or stay awake, and/or pain or pressure in the chest.
  • Exemplary symptoms of herpes virus infection particularly include rashes.
  • Herpes zoster infection is characterized by itchy rashes (chicken pox) in children and painful rashes (shingles) in adults.
  • Infection with herpes simplex virus (1 and 2) is typically characterized by recurring sores, such as cold sores on the mouth and/or sores in the genital area.
  • Exemplary symptoms of influenza virus infection may include, for example, cough, fatigue, fever, runny nose, sore throat, vomiting (especially in children), particularly if combined with confusion, dizziness, difficulty breathing or shortness of breath, inability to wake or stay awake, lack of urination, pain or pressure in the chest, seizures, and/or severe weakness or unsteadiness.
  • provided cyclodextrin agent(s) and/or formulation(s) may be administered to individuals who display one or more features of acute respiratory distress syndrome (ARDS), pneumonia, and/or other respiratory damage or distress.
  • ARDS acute respiratory distress syndrome
  • viral infection may be determined by detection of one or more viral components, and particularly of viral nucleic acid), in a biological sample (e.g ., a sample of blood, feces, mucus, saliva, sputum, tears, urine, etc) from the relevant individual.
  • a biological sample e.g ., a sample of blood, feces, mucus, saliva, sputum, tears, urine, etc.
  • viral infection may be determined by detection of one or more features of an immune response to a virus (e.g., of one or more antibodies to a viral component).
  • provided cyclodextrin agent(s) and/or formulation(s) may be administered to individuals who have received or are receiving other therapy to treat the viral infection or one or more symptoms of it.
  • provided cyclodextrin agent(s) and/or formulation(s) may be administered to individuals who have received one or more cough suppressants, fever reducers, and/or pain relievers.
  • provided cyclodextrin agent(s) and/or formulation(s) may be administered to individuals who have received and/or are receiving antiviral therapy (e.g., with one or more antibody, cellular, nucleic acid, or small molecule therapeutics that specifically targets the virus and/or cells infected with it) and/or anti-inflammatory therapy.
  • provided cyclodextrin agent(s) and/or formulation(s) may be administered to individuals who are or have been one a ventitator [0105] In some embodiments, provided cyclodextrin agent(s) and/or formulation(s) may be administered to individuals who have received a vaccine for the relevant virus, or for other member(s) or strain(s) of the relevant viral clade, class, or family.
  • the present disclosure provides both therapeutic and prophylactic uses for provided retentive cyclodextrin technologies.
  • provided technologies involve administration of cyclodextrin agent(s) and/or formulation(s) to mucosal surface(s) in nasal passages, oral cavity, lungs, eyes, and/or gastrointestinal tract ( e.g ., the gut).
  • provided technologies involve administration of an aerosol formulation (e.g., via the nose and/or the mouth).
  • provided technologies involve administration of an ingestible formulation (e.g., a liquid, capsule or tablet, etc).
  • provided technologies involve administration of eye drops.
  • provided technologies involve administration by two or more distinct routes (e.g., aerosol and ingestion).
  • provided technologies involve administration of one or more doses of cyclodextrin agent(s) and/or formulation(s) prior to development of symptom(s) (or other hallmark(s) of infection, such as detectable viral load and/or anti -viral antibodies in one or more biological samples) and/or prior to a known or suspected incidence of exposure (e.g., prior to leaving a protected or known safe space and/or prior to entering a space where known or expected risk of exposure is elevated); in some embodiments, multiple doses may be administered, via one or more routes, prior to development of symptom(s) (and/or other hallmark(s)) and/or prior to a known or suspected incidence of exposure.
  • provided technologies involve administration of one or more doses of cyclodextrin agent(s) and/or formulation(s) prior to development of symptom(s) (and/or other hallmark(s) of viral infection) but after to a known or suspected incidence of exposure (e.g., after leaving a protected or known safe space and/or after entering a space where known or expected risk of exposure is elevated); in some embodiments, multiple doses may be administered, via one or more routes, prior to development of symptom(s) (and/or other hallmark(s)) but after the known or suspected incidence of exposure.
  • provided technologies involve administration of one or more doses cyclodextrin agent(s) and/or formulation(s) after development of one or more symptoms (and/or other hallmark(s)) of viral infection.
  • multiple doses may be administered, via one or more routes, after development of symptom(s)
  • multiple doses may be administered, via multiple different routes, after development of symptom(s) (and/or hallmark(s).
  • at least one ingestible dose is administered after development of symptom(s) (and/or hallmark(s)).
  • at least one aerosol dose is administered after development of symptom(s) (and/or hallmark(s)).
  • each of at least one ingestible dose and at least one aerosol dose is administered after development of symptom(s) (and/or hallmark(s)).
  • timing, route(s), and/or frequency of administration may be adjusted as desired (e.g ., depending upon the particular virus(es) and/or susceptibility(ies) of the relevant subject. For example, in some cases, regular (e.g., seasonal, monthly, weekly, daily, twice a day, thrice a day, every 6 hours, every 4 hours, every 3 hours, every 2 hours, every hour) dosing may be desired, for example year-round and/or during a particular season (e.g., winter/cold and flu season). Alternatively or additionally, in some cases, intermittent and/or situational (e.g., relative to a planned, expected, or received exposure) administration may be desirable. In some embodiments, administration is 2 or more times per day. In some embodiments, administration is twice a day. In some embodiments, administration is twice a day for 1, 2,
  • administration is twice a day for 1, 2, 3, 4, or 5 day(s).
  • administration is twice a day for 1, 2, 3, 4, or 5 day(s) followed by administration once a day thereafter.
  • administration is episodic, for example occurring multiple times shortly (e.g., within minutes, hours, 1 ⁇ 2 day, one day, or a few days) before an anticipated exposure or risk thereof and, optionally continuing during and/or after an expected or suspected exposure.
  • provided cyclodextrin agent(s) and/or formulation(s) may be self-administered.
  • an administered dose may provide benefit for a period of time that lasts at least 10 minutes, 20 minutes, 30 minutes, 45 minutes, 60 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 30 hours, 36 hours, 42 hours, 48 hours, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 2 months, 3 months, 4 months, 5 months, 6 months or more.
  • provided technologies involve administration of individual doses or regimens (where a regimen may comprise a plurality of doses that together have been determined to achieve a particular effect) separated by at least a designated period of time which may be, for example, one or more hours, one or more days, one or more weeks, one or more months, or even as much as a year.
  • Example 1 Characterizing Binding of Cyclodextrin Agent(s) and/or Formulation(s) Thereof to Mucus and/or Gly cocalyx Layer of Epithelial Tissue Explants
  • Bodipy-labeled CD agent formulations will be suspended in phosphate buffered saline solution and will be applied at varying concentrations and for varying durations to the mucosal surface of the epithelial explants and cell cultures. Following these incubations, the tissue samples will be rinsed with phosphate-buffered saline to remove unbound CD.
  • Bodipy-cholesterol delivered in association with bovine serum albumin [5] to assess to what degree the CD formulations enhance Bodipy-cholesterol association with the mucosal surfaces of the epithelial cells.
  • CD agent formulations assessed in accordance with this Example and particularly those that manifest the most extensive and/or enduring mucosal binding, can be further assessed, for example using one or more other assays as described herein.
  • mucoadhesive CD agent formulations which bind to, are retained at, and/or accumulate (i.e., which achieve accumulation and/or retention of CD) in mucus and/or gly cocalyx layers at mucosal surfaces of nasal, airway and/or intestinal epithelial cells can remove cholesterol from apical plasma membrane domains of the epithelial cells themselves and/or from any enveloped viral particles that traverse these mucus and glycocalyx layers.
  • the present Example describes use of a well-characterized fluorescent cholesterol-binding molecular probe, GFP-D4 [6] to assess such cholesterol depletion.
  • This GFP-D4 molecular probe constitutes a fusion between GFP and the cholesterol-binding D4 fragment of the pore forming toxin perfringolysin-O.
  • a construct encoding this fusion can be expressed in bacteria; a His-tag (or other appropriate tag) can be included in the fusion protein, which can facilitate its purification. Fusions that include a His-tag, for example, can be readily purified by nickel affinity chromatography. The cell and/or tissue preparations will be incubated with unmodified MbOO and HPpCD. or with CD agent formulation(s) as described herein.
  • control samples will be incubated with saline alone. Following incubation for two hours at 37oC, the cell and/or tissue samples will be rinsed with phosphate buffered saline and then incubated for two additional hours with GFP-D4. The samples will be rinsed again with phosphate buffered saline and then examined by confocal fluorescence microscopy. All of the resultant images will be analyzed and the signal intensity per unit area will be determined using ImageJ software.
  • Control incubations will be performed with GFP that is not conjugated to D4 to establish the baseline signal that is attributable to non-specific binding of the fluorescent probe to the epithelial cell mucosal surfaces.
  • the present disclosure teaches that cells and/or tissues exposed to CD agent formulations that are mucoadhesive will have less cholesterol in their apical plasma membranes and thus will exhibit less GFP-D4 labeling than will cells treated with unmodified CDs or with saline alone. The magnitude of this difference will provide a quantitative assessment of the degree to which mucoadhesive CD agent formulation(s) extract cholesterol from mucosal epithelial cell apical plasma membranes and thus by extension reduce the likelihood of their infection by SARS-CoV-2.
  • Example 3 Characterizing Degree of Cholesterol Stripping from Membrane of Enveloped Virus(es)
  • mucus and/or glycocalyx layer(s) associated with mucosal surfaces of nasal, airway and/or intestinal epithelial cells can strip cholesterol out of membranes of enveloped viruses as they diffuse through such layer(s) en route to infecting underlying cells.
  • the present Example describes use of a well characterized in vitro system and a readily-available and easy to work with cholesterol-containing enveloped virus to assess how envelope cholesterol content may impact diffusion of viral particles through CD- loaded intestinal mucus.
  • Mucus will be collected from porcine intestine according to standard methods
  • This mucus will be cleaned, for example by mixing it with gentle stirring with 5 ml/g of 100 mM NaCl, after which the mucus will be recovered by low speed centrifugation and the supernatant will be discarded.
  • the fluorescent GFP-D4 cholesterol-binding probe will be added for a one hour incubation at 4°C. Unbound GFP-D4 will then be removed in the supernatant following centrifugation and the mucus will be examined by confocal fluorescence microscopy. All of the resultant images will be analyzed and the signal intensity per unit area will be determined using ImageJ software.
  • Control incubations will be performed with GFP that is not conjugated to D4 to establish the baseline signal that is atributable to non-specific binding of the fluorescent probe to the viral membranes or to the mucus.
  • mucus that had not received virus during the second incubation will provide a measurement of the baseline signal attributable to the non-specific sticking of the GFP-D4 probe to the mucus. Once these baselines are established and subtracted, the remaining fluorescent signal detected in the mucus samples will be attributed to the binding of the GFP-D4 probe to the cholesterol-rich membranes of the lentivirus [8]
  • Example 4 Characterizing Impact of CD Agent Formulations on Ability of a Cholesterol- Dependent Virus to Infect Nasal. Airway and/or Intestinal Epithelial Cells
  • CD agent(s) in mucus and/or glycocalyx layers at mucosal surface(s) of epithelial cells as described herein can have a surprising benefit of creating an “anti-viral” shield that will strip cholesterol from the membrane of an enveloped virus during its passage through this layer.
  • the present disclosure provides a further insight that cholesterol-dependent enveloped virus subjected to this cholesterol depletion will be atenuated and thus will manifest reduced capacity to infect underlying cells.
  • the present Example describes use of two viral infection reporter systems to characterize CD agent(s) and/or formulation(s) as described herein with respect to such viral attenuation.
  • VSV G pseudotyped lentivirus is extraordinarly dependent upon cholesterol in its own membrane as well as in that of the target cell in order to be maximally infective
  • SARS-CoV-2 pseudotyped lentivirus has become commercially available (www.integralmolecular.com/covidl9/). Since the binding and fusion of the SARS-CoV-2 pseudotyped lentivirus are dependent upon S spike glycoprotein of the SARS-CoV-2, it is likely that this pseudotyped lentivirus shares the cholesterol requirement of the parent SARS-CoV-2 coronavirus.
  • Both of these lenti viral constructs drive the expression of GFP and thus can serve as quantitative reporters of viral infection.
  • These lentivirus reporter probes will be used to assess effects of accumulating and/or retaining CD (e.g., via a CD agent formulation as described herein) in mucus and/or glycocalyx layers as described herein on susceptibility of nasal, airway and intestinal epithelial cells to infection. The extent of infection will be measured by assessing the magnitudes of the GFP fluorescence signals in the target cells.
  • Nasal, airway and intestinal epithelial explants and cultures will be prepared and maintained according to established technologies [1-3] These cell and tissue preparations will be incubated with unmodified M CD, HRbO ⁇ . and/or with CD agent formulation(s) as described herein (which may, for example, be or comprise mucoadhesive variant(s) of M CD and/or HRbO ⁇ ) in saline. In addition, control samples will be incubated with saline alone.
  • the epithelial cell samples will be rinsed with phosphate buffered saline and then incubated for one hour with a range of titers of the VSV G pseudotyped lentivirus, with a range of titers of the SARS-CoV-2 pseudotyped lentivirus, or with saline alone. After rinsing to remove free virus, the cell and tissue preparations will be incubated for twenty-four hours at 37°C. At the end of this incubation the cells will examined by confocal fluorescence microscopy. All of the resultant images will be analyzed and the signal intensity per unit area will be determined using ImageJ software.
  • the level of fluorescence detected in the cell and tissue samples treated with the maximal doses of the most effective CD agent formulation(s) will produce fluorescent signals that are not distinguishable from the level of background fluorescence that is observed in cell and tissue samples that did not receive virus. Observation of such behavior will inducate that the accumulation and/or retention of CD (e.g., of the relevant CD agent) in mucus endows an epithelial mucus and/or glycocalyx layer with a capacity to attenuate cholesterol-dependent enveloped virus particles and to reduce or completely abrogate capacity of such viral particles to infect the underlying epithelial cells.
  • CD e.g., of the relevant CD agent
  • this assay will assess relative effectiveness of CD agent preparation(s) as described herein in attenuating invading cholesterol-dependent and SARS-CoV-2 S protein dependent viruses, including for example in reducing their capacity to productively infect their target cells.
  • the present Example describes assessment of the efficacy of CD agent formulation(s) as described here, and particularly of their accumulation and/or retention in mucus and/or glycocalyx layer(s) to attenuate the infection of human airway epithelial cells by the SARS-CoV-2 virus, derived from a human patient isolate.
  • EpiAirway human airway epithelial cell cultures such as are available from MatTek
  • qPCR quantitative PCR
  • MatTek EpiAirway human airway epithelial cell cultures will be treated for one hour with saline or with varying quantities of one or more CD agent formulation(s) as described herein (e.g., those formulation(s) that may have exhibited the particularly promising propert(ies) in one or more other assessment(s) as described herein, such as in one or more of the other Examples presented herein).
  • CD agent formulation(s) as described herein (e.g., those formulation(s) that may have exhibited the particularly promising propert(ies) in one or more other assessment(s) as described herein, such as in one or more of the other Examples presented herein).
  • Nonadherent CD agent will be removed by rinsing with saline, after which varying titers of SARS-CoV-2 or saline alone will be added to the medium, bathing the apical surfaces of the cells, and will be allowed to incubate for one hour at 37°C, according to the standard method [9] Unbound virus will be removed by rinsing with saline, after which the cells will be allowed to incubate for twenty- four hours at 37°C. The cells will then be harvested and RNA will be prepared for qPCR analysis of viral load.
  • the present disclosure teaches that accumulation and/or retention of CD (e.g., via a CD agent formulation as described herein) in mucus and/or glycocalyx layer(s) will produce a dose-dependent reduction in infectivity of SARS-CoV-2.
  • CD agent formulation(s) e.g., via a CD agent formulation as described herein
  • viral titers e.g., the extent to which each CD agent formulation reduces SARS-CoV-2 infectivity can be both determined precisely and compared with that of other CD agent formulations and/or of relevant controls (e.g., no CD agent, standard CD that has not been modified and/or formulated as described herein for mucoadhesion, etc).
  • the present Example describes characterization of whether a CD agent formulation as described herein is effective in attenuating or preventing infection by SARS- CoV-2 in a relevant animal model, using the well characterized susceptibility of the golden Syrian hamster to infection with SARS viruses, including SARS-CoV-1 [10] and SARS- CoV-2 [11], when exposed via intranasal challenge.
  • Intranasal challenge of the golden Syrian hamster with SARS-CoV-2 leads to an infection whose severity is dose dependent and is characterized by weight loss, respiratory distress, histopathological changes in the lung, nose and trachea, and high levels of cytokine production. In addition, high levels of virus accumulate in the lung, nose, trachea and intestine [11]
  • the SARS-CoV-2 infection is not lethal in the golden Syrian hamster and resolves within fourteen days. Recovered animals produce anti-viral antibodies as part of an immune response that would likely protect them from subsequent infection.
  • Co- housed naive cage-mates of infected animals who have not themselves received intranasal challenge acquire the infection via passive spread of the virus. The disease course in these passively infected animals is very similar to that which characterizes the animals that received nasal challenge with the exception that the weight loss is much less pronounced. Passively infected animals also develop protective immunity.
  • CD agent formulations will be utilized in a synthetic mucus vehicle solution that contains Na hyaluronate and glycerol to recapitulate the compositions of widely available and well-tolerated over-the-counter nasal sprays.
  • CD agent in this synthetic mucus solution will be administered to animals via spraying with an intranasal pipette.
  • Both the +NVC and the -NVC animals will be pretreated with CD agent formulation one hour before the +NVC cohort receives the intranasal viral challenge. Both the +NVC and the -NVC animals will receive additional single daily doses of the CD agent formulation for each of the remaining days of the experiment. Group 2. The -NVC animals will be pretreated with CD agent formulation one hour before the +NVC cohort receives the intranasal viral challenge. The +NVC animals will not receive any pretreatment with CD agent formulation. The -NVC animals will receive additional single daily doses of the CD agent formulation for each of the remaining days.
  • the +NVC animals will be pretreated with CD agent formulation one hour before they receive their intranasal viral challenge.
  • the -NVC animals will not receive any pretreatment with CD agent formulation.
  • the +NVC animals will receive additional single daily doses of the CD agent formulation for each of the remaining days.
  • Both the +NVC and the -NVC animals will be pretreated with synthetic mucus solution one hour before the +NVC cohort receives the intranasal viral challenge. Both the +NVC and the -NVC animals will receive additional single daily doses of the synthetic mucus for each of the remaining days.
  • the histopathological appearance of the lung will be evaluated and the level of viral N protein expression will be assessed by immunofluorescence analysis of tissue sections prepared from the nasal turbinate, trachea, lung, and intestine.
  • the levels of cytokine production (including interferon-g, and interleukin-6) will be determined by performing qPCR on RNA prepared from the lungs [11]
  • anti-viral antibodies may indicate a level of immunity, independent of severity of disease.
  • detected antibodies may be assessed for neutralizing activity (e.g for ability to protect another organism from infection and/or to reduce severity of an infection).
  • the present disclosure teaches that accumulation and/or retention of CD (e.g., via a CD agent formulation as described herein) can reduce severity of infection. That is, animals treated with a CD agent formulation as described herein will experience a substantially milder disease course than animals that received the synthetic mucus vehicle alone or no treatment at all. The present disclosure also teaches that +NVC animals that received treatment with a CD agent formulation as described herein will produce milder infections in their untreated -NVC naive cage mates.
  • the present disclosure teaches that, for at least some CD agent formulations and/or recipients, treatment-induced reduced severity of the disease course will not suppress the mounting of an exuberant immune response in directly challenged and/or naive co-housed animals, since the action of the accumulated and/or retained CD (e.g., CD agent) should result in the production of an attenuated virus whose infectivity is reduced but whose immunogenicity remains intact.
  • CD agent e.g., CD agent
  • CD agent formulation(s) as described herein can serve as effective prophylactic “anti-viral shields” that have the potential to protect uninfected individuals from the potentially devastating consequences of environmental exposure to the virus.
  • anti-viral shields Such a result would constitute an extremely important and unexpected result that has the potential to dramatically reduce the world-wide public health and economic consequences of the current COVID-19 pandemic.
  • Extracellular simian virus 40 transmits a signal that promotes virus enclosure within caveolae. Exp. Cell Res. 246: 83-90.
  • Lipid rafts and HIV pathogenesis host membrane cholesterol is required for infection by HIV type

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Abstract

La présente invention concerne des technologies pour accumuler et/ou retenir des agents de cyclodextrine au niveau de surfaces mucosales, avec des effets surprenants et bénéfiques dans la prophylaxie et/ou le traitement d'infections virales.
PCT/US2021/032945 2020-05-18 2021-05-18 Technologies antivirales mucorétentives WO2021236626A1 (fr)

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Cited By (1)

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WO2023180567A1 (fr) * 2022-03-24 2023-09-28 Fundacion Privada Institut De Recerca De La Sida-Caixa Cyclodextrines destinées à être utilisées dans une thérapie contre une infection à coronavirus

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WO2004009029A2 (fr) * 2002-07-22 2004-01-29 La Jolla Biosciences Compositions et procedes de traitement et de prevention d'infections
WO2013166503A1 (fr) * 2012-05-04 2013-11-07 The Regents Of The University Of California Thérapie antivirale large par des oxystérols de modification de membrane

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WO2004009029A2 (fr) * 2002-07-22 2004-01-29 La Jolla Biosciences Compositions et procedes de traitement et de prevention d'infections
WO2013166503A1 (fr) * 2012-05-04 2013-11-07 The Regents Of The University Of California Thérapie antivirale large par des oxystérols de modification de membrane

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WO2023180567A1 (fr) * 2022-03-24 2023-09-28 Fundacion Privada Institut De Recerca De La Sida-Caixa Cyclodextrines destinées à être utilisées dans une thérapie contre une infection à coronavirus

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