WO2008105934A2 - Inactivation of toxic agents and pathogens using chitosan - Google Patents

Inactivation of toxic agents and pathogens using chitosan Download PDF

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WO2008105934A2
WO2008105934A2 PCT/US2007/079246 US2007079246W WO2008105934A2 WO 2008105934 A2 WO2008105934 A2 WO 2008105934A2 US 2007079246 W US2007079246 W US 2007079246W WO 2008105934 A2 WO2008105934 A2 WO 2008105934A2
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virus
filter
chitosan
human
hhv
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PCT/US2007/079246
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WO2008105934A3 (en
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Kenton W. Gregory
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Oregon Biomedical Engineering Institute, Inc.
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    • C12N7/00Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01DSEPARATION
    • B01D39/00Filtering material for liquid or gaseous fluids
    • B01D39/10Filter screens essentially made of metal
    • B01D39/12Filter screens essentially made of metal of wire gauze; of knitted wire; of expanded metal
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01DSEPARATION
    • B01D39/00Filtering material for liquid or gaseous fluids
    • B01D39/14Other self-supporting filtering material ; Other filtering material
    • B01D39/16Other self-supporting filtering material ; Other filtering material of organic material, e.g. synthetic fibres
    • B01D39/1607Other self-supporting filtering material ; Other filtering material of organic material, e.g. synthetic fibres the material being fibrous
    • B01D39/1615Other self-supporting filtering material ; Other filtering material of organic material, e.g. synthetic fibres the material being fibrous of natural origin
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01DSEPARATION
    • B01D39/00Filtering material for liquid or gaseous fluids
    • B01D39/14Other self-supporting filtering material ; Other filtering material
    • B01D39/16Other self-supporting filtering material ; Other filtering material of organic material, e.g. synthetic fibres
    • B01D39/1607Other self-supporting filtering material ; Other filtering material of organic material, e.g. synthetic fibres the material being fibrous
    • B01D39/1623Other self-supporting filtering material ; Other filtering material of organic material, e.g. synthetic fibres the material being fibrous of synthetic origin
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01DSEPARATION
    • B01D39/00Filtering material for liquid or gaseous fluids
    • B01D39/14Other self-supporting filtering material ; Other filtering material
    • B01D39/16Other self-supporting filtering material ; Other filtering material of organic material, e.g. synthetic fibres
    • B01D39/1638Other self-supporting filtering material ; Other filtering material of organic material, e.g. synthetic fibres the material being particulate
    • B01D39/1646Other self-supporting filtering material ; Other filtering material of organic material, e.g. synthetic fibres the material being particulate of natural origin, e.g. cork or peat
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    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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    • B01D2239/00Aspects relating to filtering material for liquid or gaseous fluids
    • B01D2239/02Types of fibres, filaments or particles, self-supporting or supported materials
    • B01D2239/0216Bicomponent or multicomponent fibres
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01DSEPARATION
    • B01D2239/00Aspects relating to filtering material for liquid or gaseous fluids
    • B01D2239/04Additives and treatments of the filtering material
    • B01D2239/0442Antimicrobial, antibacterial, antifungal additives
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01DSEPARATION
    • B01D2239/00Aspects relating to filtering material for liquid or gaseous fluids
    • B01D2239/04Additives and treatments of the filtering material
    • B01D2239/0464Impregnants
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01DSEPARATION
    • B01D2239/00Aspects relating to filtering material for liquid or gaseous fluids
    • B01D2239/04Additives and treatments of the filtering material
    • B01D2239/0471Surface coating material
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01DSEPARATION
    • B01D2239/00Aspects relating to filtering material for liquid or gaseous fluids
    • B01D2239/06Filter cloth, e.g. knitted, woven non-woven; self-supported material
    • B01D2239/065More than one layer present in the filtering material
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01DSEPARATION
    • B01D2251/00Reactants
    • B01D2251/90Chelants
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01DSEPARATION
    • B01D2251/00Reactants
    • B01D2251/95Specific microorganisms
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01DSEPARATION
    • B01D2253/00Adsorbents used in seperation treatment of gases and vapours
    • B01D2253/20Organic adsorbents
    • B01D2253/202Polymeric adsorbents
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01DSEPARATION
    • B01D2257/00Components to be removed
    • B01D2257/60Heavy metals or heavy metal compounds
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01DSEPARATION
    • B01D2257/00Components to be removed
    • B01D2257/91Bacteria; Microorganisms
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01DSEPARATION
    • B01D2259/00Type of treatment
    • B01D2259/45Gas separation or purification devices adapted for specific applications
    • B01D2259/4541Gas separation or purification devices adapted for specific applications for portable use, e.g. gas masks
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01DSEPARATION
    • B01D2259/00Type of treatment
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    • B01D2259/4583Gas separation or purification devices adapted for specific applications for removing chemical, biological and nuclear warfare agents
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    • C12N2740/00Reverse transcribing RNA viruses
    • C12N2740/00011Details
    • C12N2740/10011Retroviridae
    • C12N2740/16011Human Immunodeficiency Virus, HIV
    • C12N2740/16061Methods of inactivation or attenuation
    • C12N2740/16063Methods of inactivation or attenuation by chemical treatment

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  • Filtering Materials (AREA)

Abstract

Embodiments of the present invention provide methods and devices for filtering and/or inactivating toxic agents. More specifically, embodiments of the present invention provide methods and devices for filtering and/or inactivating toxic agents using chitosan filters.

Description

INACTIVATION OF TOXIC AGENTS AND PATHOGENS USING CHITOSAN (Attorney Docket No.: OBEI-001 PC)
Related Applications
[0001] This application claims the benefit of U.S. Provisional Application Serial
No. 60/826,684, filed on September 22, 2006, the contents of which are incorporated herein by reference in its entirety.
Technical Field
[0002] Embodiments of the present invention relate to methods and devices for filtering and/or inactivating toxic agents and pathogens.
Background
[0003] Weaponized agents, including chemical warfare agents, biological or biologic warfare agents, nuclear agents and radiological agents, remain a potentially devastating threat to deployed soldiers and civilians. Exposure to such toxic agents can markedly reduce military force effectiveness and can consume limited resources in the field.
[0004] More specifically, military personnel and civilians are exposed to a wide variety of pathogens, such as anthrax, botulism, smallpox hemorrhagic fever viruses, throughout the world, and are frequently exposed to a variety of highly infective illnesses transmittable through the air through simple touching, blood or bodily secretions. For example, the exposure of blood from HIV infected soldiers, foreign nationals or other patients encountered during military operations is particularly dangerous to individuals addressing bleeding or wounds with secretions that could be extremely infective. Similarly, herpes simplex types I and Il infect well over 20 million Americans and many US Service personnel.
[0005] With respect to viral pathogens, prevention is critically important.
Viruses' inherent ability to mutate into resistant strains and the protracted path for drug discovery equates into a lack of highly effective anti-viral treatments. Vaccines are an important but limited aspect of prevention due to constant viral mutations and resistance development.
[0006] Further, transmission of new lethal strains of avian flu is being reported in many countries, raising the potential of a viral pandemic that could threaten the lives of millions of persons and disable the health infrastructure of countries around the world, as demonstrated by the Severe Acute Respiratory Syndrome (SARS) outbreaks. Prevention of transmission of airborne infective influenza and other viruses may be the single most important defense against future pandemics. [0007] Several million Americans contract recurrent varicella zoster or shingles every year. This disease results in significant pain and disability - many times with chronic and permanent residual. Although, the use of vaccines has recently been reported to decrease recurrent varicella zoster outbreaks, the effectiveness of such treatments is only 50% and limited to younger patients. [0008] When a person is in an area where exposure to toxic agents in the air is a possibility, the best protection often is to wear a protective face mask or filter mask. Military personnel fighting in a war zone where chemical and biological agents may be present often wear such respiratory protective face masks along with protective clothing. Emergency workers such as police and fire fighters who may enter areas containing toxic substances in the air also wear such protective face masks for personal protection purposes.
[0009] A filter mask may provide protection to the wearer from harmful airborne substances by limiting the course of air so that it must flow through a filter. Such masks range from cheaper, single-use, disposable types to reusable models with replaceable cartridges. Because of their simple, passive design they are commonly used for filtration of tiny suspended solid or liquid particles and often referred to as particulate masks. For this purpose, they usually employ a dense, fine natural or synthetic fiber mesh. To aid filtration, the mesh is sometimes coated with substances that enhance the mask's effectiveness. For example, masks or respirator cartridges including nano-silver particles or iodine demonstrate antimicrobial properties. However, each of these materials have significant drawbacks. The antimicrobial properties of nano-silver coatings are comparatively weak and iodine itself is a highly toxic substance. Inhalation of iodine vapor may result in severe pulmonary irritation leading to pulmonary edema. [0010] The FDA recently acknowledged that chitosan, a linear polysaccharide produced from chitin (a structural element in the exoskeleton of crustaceans) has bactericidal activity. It is theorized that chitosan may have a similar ability to inactivate viruses. These findings indicate the strong potential that chitosan can inactivate bacteria and viruses in a variety of applications. Thus, the trapping and filtering of pathogens with a chitosan filter may decrease spread of infectious disease.
[0011] Depleted uranium, or U-238 ("DU"), is a toxic, heavy metal byproduct of uranium enrichment that gives the world uranium suitable for use in nuclear weapons and reactor fuel. It is also used in munitions, ballast for airplanes, tank armor and other products. Researchers found that even though the alpha radiation from depleted uranium is relatively low, internalized DU as a metal can induce DNA damage and carcinogenic lesions in the cells that make up bones in the human body. The material in armor-piercing munitions ignites and burns on impact at temperatures of several thousand degrees Celsius. While burning, tiny particles, or dust, of uranium oxide aerosol are created. Wind can carry these considerable distances.
[0012] It is also theorized that chitosan may be an effective chelator of heavy metal ions, including uranium. Thus, absorption of uranium on a chitosan filter may be an effective way to protect military personnel and civilians from exposure to uranium dust contamination.
Summary
[0013] It has been found that chitosan is useful for methods and devices for filtering and/or inactivating viruses. The ability of chitosan to interact with and inactivate viruses is unexpected.
[0014] In accordance with embodiments of the present invention, chitosan and derivatives thereof may be included in an air purification device, thereby preventing viruses from being breathed into the nose and mouth of a person. In other embodiments, the chitosan or derivatives thereof may be included in a hand sanitizer thereby inactivating viruses from the hands or included in paper or cloth wipes for cleaning virus-contaminated surfaces, thereby contacting and inactivating the viruses. In another embodiment, gloves or masks may comprise chitosan or a derivative thereof to inactivate viruses.
[0015] In other embodiments, chitosan may be suspended in lotions or skin creams that are applied to skin, particularly hands and face, or internally within the vagina, for interacting with and thereby inactivating the transfer of viruses from one person to another, or to prevent a person from transferring the virus from external skin to internal cells.
[0016] In still another embodiment, the chitosan hereof may be ingested for internal interaction and inactivation of viruses within the gastrointestinal tract that have been or are about to be ingested. When wastes are expelled, viruses are retained on the chitosan and prevented from causing secondary infections. [0017] In another embodiment, chitosan may be vaginally inserted for interaction and inactivation of HIV or other sexually-transmitted viruses, in the same manner as a spermicidal foam or body heat-dissolving spermicidal cartridge. [0018] In still another embodiment, chitosan may be held in a vessel for filtering contact with blood, e.g., a secondary dialysis filter, or for filtering viruses from water in a virus-removing water purification step.
[0019] In another embodiment, chitosan may be used as, or form a portion of, a HVAC filtration media to prevent virus-contaminated air from passing between rooms; for example, between rooms in a hospital.
[0020] In another embodiment, chitosan may be used as a nasal lubricant by spraying a suspension of the chitosan or derivatives thereof in a carrier (water and/or organic solvent) into the nasal passages to coat nasal cells. In this manner, viruses entering the nose will interact with the chitosan and thereby will be inactivated to prevent infection.
[0021] In still another embodiment, a condom is coated with chitosan; in a cosmetically acceptable carrier, e.g., water and/or solvent. In the event of condom failure, the chitosan with and inactivates viruses before a sexual partner is infected. [0022] In another embodiment, a suspension of chitosan in a cosmetically acceptable carrier is packaged in a portable container, e.g., a tube or bottle, for use on the hands to periodically inactivate viruses held on a person's skin.
Brief Description of the Drawings
[0023] Embodiments of the present invention will be readily understood by the following detailed description in conjunction with the accompanying figures. [0024] FIG. 1 demonstrates that chitosan inhibits HIV-1 LAV replication in a standard virus growth assay in 174xCEM indicator cells. [0025] FIG. 2 shows that HIV is inactivated on 2% Chitosan/2% HAc filter [0026] Fig. 3 shows that HIV is inactivated on 0.5% Chitosan/0.25% HAc filter
Detailed Description of Embodiments of the Invention
[0027] In the following detailed description, reference is made to embodiments in which the invention may be practiced. It is to be understood that other embodiments may be utilized and structural or logical changes may be made without departing from the scope of the present invention. Therefore, the following detailed description is not to be taken in a limiting sense, and the scope of embodiments in accordance with the present invention is defined by the appended claims and their equivalents.
[0028] Various operations may be described as multiple discrete steps in turn, in a manner that may be helpful in understanding embodiments of the present invention; however, the order of description should not be construed to imply that these operations are order dependent.
[0029] The description may use the phrases "in an embodiment," or "in embodiments," which may each refer to one or more of the same or different embodiments. Furthermore, the terms "comprising," "including," "having," and the like, as used with respect to embodiments of the present invention, are synonymous. [0030] In various embodiments of the invention, methods and devices for inactivating toxic agents is provided. In the following description, unless further particularized or otherwise noted, the term "toxic agents" or "toxic agent" is intended to refer to a broad class of poisonous substances, including but not limited to toxic gases and/or particulates, radioactive gases and/or particulates, weaponized agents, pathogens, and/or microbes. The term "pathogen" or "pathogens" is intended to refer to any bacterium, virus or other microorganism that can cause disease. The term "inactivate" or "inactivating" is intended to refer to rendering a toxic agent ineffective at causing disease or harmless to animals or humans. [0031] Although certain embodiments have been described herein for purposes of description of the preferred embodiment, it will be appreciated by those of ordinary skill in the art that a wide variety of alternate and/or equivalent embodiments or implementations calculated to achieve the same purposes may be substituted for the embodiments shown and described without departing from the scope of the present invention. Those with skill in the art will readily appreciate that embodiments in accordance with the present invention may be implemented in a very wide variety of ways. This application is intended to cover any adaptations or variations of the embodiments discussed herein. Therefore, it is manifestly intended that embodiments in accordance with the present invention be limited only by the claims and the equivalents thereof.
[0032] In various embodiments of the present invention, a virus may be inactivated by contacting the virus with chitosan or a derivative thereof. Exemplary viral families contemplated for inactivation by contact with chitosan as used herein include, but are not limited to herpesviridae, poxyiridae, adenoviridae, papovaviridae; parvoviridae, picornaviridae, togavihdae, flavivihdae, coronaviridae, calicivihdae, paramyxovihdae, rhabdovihdae, filoviridae, arenaviridae, orthomyxovihdae, bunyaviridae, retroviridae, hepadnavihdae, and combinations thereof. More specifically, viral species contemplated for inactivation by contact with chitosan as used herein include, but are not limited to virus herpes simplex type 1 (HHV-1 ), herpes simplex type 2 (HHV-2), varicella zoster virus (HHV-3), cytomegalovirus virus (HHV-5), human herpes virus type 6, 7, Epstein Barr virus (HHV-4), human herpes virus type 8, variola virus, molluscum contagiousum virus, human adenovirus, papillomavirus, BK virus, JC virus, human parvovirus (B 19), rhinovirus, hepatitis A virus, rubella virus, eastern equine encephalitis virus, human herpes virus type 8, yellow fever virus, dengue virus, west Nile virus, hepatitis C virus, human coronavirus, Norwalk virus, mumps virus, measles virus, respiratory syncitial virus (RSV), human parainfluenza virus 1 , rabies virus, ebola virus, lassa fever virus, influenza A, influenza B, influenza C, sin nombre virus, human immunodeficiency viruses, human T-cell leukemia viruses, hepatitis B virus, and combinations thereof. [0033] The term "chitosan," as described herein, generally refers to a deacetylated derivative of chitin. The term "chitosan" may include both ideal chitosan, which is a linear polysaccharide of β-(1 -4)-2-amino-2-deoxy-D- glucopyranose, as well as any chitin/chitosan co-polymer such as linear polysaccharides composed of randomly distributed β-(1-4)-linked D-glucosamine (deacetylated unit) and N-acetyl-D-glucosamine (acetylated unit). The term "chitosan" may also include one or more derivatives of chitosan, such as derivatives of chitosan functionalized with an amino acid. For example, the free amine on chitosan may be functionalized with arginine ("chitosan-arginine") or guanidine ("chitosan-guanidine"). Any one or any mixture of two or more of the above chitosan and/or chitosan derivatives may be capable of contacting, absorbing and/or binding a toxic agent, or combination of toxic agents, thereby inactivating the toxic agent(s). [0034] Various embodiments of the present invention include a filter comprising chitosan. The term "filter" or "filters" as used herein includes, but is not limited to, a filter as a surgical mask; a filter for a face mask, a filter for first responders giving mouth to mouth resuscitation; a filter for a respirator, a filter for bodily fluids (e.g., blood and blood transfusions) or a HVAC filter (e.g., for a ventilation system in a building or vehicle).
[0035] The filter may comprise low-modular meshes and/or films and/or weaves of synthetic and naturally occurring polymers. For example, the filter may comprise cellulose polymers, polyethylene, polypropylene, metallocene polymers, polyurethanes, polyvinylchloride polymers, polyesters, polyamides or combinations thereof. In other embodiments, the filter may also include a yarn, such as a cotton yarn.
[0036] In embodiments, the filter may comprise meshes and/or films and/or weaves of chitosan ("chitosan material" or "chitosan materials"). For example, the filter can be made of chitosan or a chitosan material may be contained in or on a filter. The term "contained in or on" is intended to refer to, but not limited to, coating, interweaving, mixing, copolymehzing, grafting, coupling, layering, joining and/or binding chitosan onto or into or with a filter.
[0037] In such embodiments, the filter may include a polymer material interwoven with chitosan. In another embodiment, the filter may comprise a chitosan co-polymer. In yet another embodiment, the filter may comprise woven or spun chitosan fibers, such as chitosan microfibers. The spun chitosan fibers may be produced using electrospinning or electrospraying techniques. [0038] In an embodiment, individual fibers or strands of a filter may be coated with chitosan. In another embodiment of the present invention, a filter may include chitosan grafted with other biopolymers (e.g. carbohydrate macromolecules) or synthetic polymers (a "chitosan graft").
[0039] In an embodiment of the present invention, at least a portion of a surface of the filter may be coated with a chitosan layer. In such an embodiment, the chitosan layer may comprise chitosan, a chitosan co-polymer, a chitosan graft, chitosan beads, chitosan gels/hydrogels, chitosan films, woven or spun chitosan fibers or other chitosan biomaterials. [0040] In an embodiment of the present invention, a chitosan material may comprise a distinct layer of the filter. In such an embodiment, the chitosan material may be combined with one or more additional filter layers to form the air filter. For example, a chitosan layer may be sandwiched between additional filter layers. In various embodiments, the additional filter layers may comprise low-modular meshes and/or films and/or weaves of synthetic and naturally occurring polymers. [0041] In various embodiments of the present invention, the air filter may be capable of filtering toxic agents. Thus, a user may use the air filter as a barrier to the toxic agents. For such embodiments, the air filter may substantially absorb the toxic agents. In other embodiments, the air filter may substantially chelate the toxic agents. As used herein, the terms "substantial" or "substantially" generally refer to binding, chelating and/or inactivating one or more toxic agents effective to reduce exposure to these agents.
[0042] In various embodiments, the air filter may substantially inactivate toxic agents. Where the toxic agents include pathogens, the air filter may act as a virucidal, bactericidal or fungicidal agent. For example, the air filter may be capable of inactivating a bacterium such as Methicillin-resistant Staphylococcus aureus. [0043] In embodiments of the present invention, the air filter may be adapted to fit in an air passageway to direct air through the air filter. For example, an air passageway may be part of a ventilation system in a building or vehicle (e.g. an air duct). Thus, a filter system, including the air filter and air passageway, may be used to protect the inhabitants of various building, facilities, shelters and/or vehicles from toxic agents. In other embodiments, an air passageway may be part of a respirator or face mask. [0044] In various embodiments of the present invention, a filter system for filtering air inhaled by a user may comprise a face mask and a strap. In various embodiments, the face mask may be a cone mask, cup mask or a surgery mask (with or without a shield). In an embodiment, at least a portion of the face mask comprises an air filter such that toxic agents in the air inhaled by a user of the face mask may be trapped, inactivated or both by the air filter. In another embodiment, the air filter may be removable. An additional material layer may create a barrier layer between a user's skin and air filter to protect the user from irritation. [0045] Embodiments of the present invention include a respirator with a cartridge comprising one or more chitosan air filters. During operation, inhaled air is directed through the one or more chitosan air filters to remove toxic agents. In such embodiments, the cartridge may be removable.
Examples
[0046] Example 1 : Evaluation of the Anti-HIV Properties of Chitosan
[0047] Methods. HIV-susceptible indicator cell lines 174xCEM and HeLa
CD4 were expanded in tissue culture and cryopreserved in liquid nitrogen in multiple vials. Cells were originally obtained from the ATCC (Manassas, VA). Stocks of X4 /T cell-tropic HIV-1 isolate LAV and R5/macrophage-tropic HIV-1 isolate JR-FL were expanded in 174xCEM and aliquots of virus stored at -800C. Virus isolates were originally obtained from the NIH AIDS Research & Reference Reagent Program (Germantown, MD). HIV-1 stocks were titered by p24 ELISA and by infectious center assay on MAGI cells (ATCC). Ultrapure chitosan was obtained from Novamatrix (Oslo, Norway).
[0048] It was determined that a 5mg/ml stock solution of chitosan could be kept in solution with a 1.2% (v/v) glacial acetic acid vehicle. A dilution series of chitosan stock was tested on adherent (Hela-CD4) and non-adherent (174xCEM) indicator cells to determine the cut-off at which chitosan/vehicle exhibited nonspecific cellular toxicity. A cut-off of 200ug/ml (0.05% vehicle) was established. [0049] CEMxI 74 cells were exposed to HIV-1 LAV at an MOI of 0.2 for 4 hours to allow virus binding and infection of approximately 10-20% of cells. Cells were rinsed to remove viral inoculum and cultured in the presence of Chitosan solutions (range of 10-200 ug/ml) for 4 days to allow the effect of chitosan on the spread of HIV through the culture to be assessed. Controls included cells cultured in the presence of medium alone (positive control for HIV replication) and cells cultured in the presence of AZT, an inhibitor of HIV reverse transcriptase (positive control for HIV inhibition). To control for any effect of the acetic acid carrier on HIV replication, cultures exposed to the equivalent concentration of acetic acid for each chitosan dose were included. On day 4 post-infection, the culture supernatants were harvested and tested for their levels of p24 Gag (an HIV structural protein present in HIV virions) by ELISA assay as a measure of HIV growth. Three independent experiments were performed.
[0050] Results. At the lowest concentration of chitosan tested (10ug/ml) inhibition of HIV replication was seen in only 1 of 3 experiments. At the highest concentration of chitosan tested (200ug/ml), the equivalent carrier control (0.05% acetic acid) affected HIV replication in 2 of 3 experiments. Thus, neither the lowest or highest doses of chitosan tested yielded reproducible or chitosan-specific results. However, chitosan at doses of 25, 50 and 100ug/ml had a significant and reproducible inhibitory effect on HIV replication in 174xCEM cells when compared to replication in untreated cells (Figure 1 ) or cells exposed to the equivalent dose of acetic acid carrier (Figure 1 ). [0051] As shown in Figure 1., chitosan inhibits HIV-1 LAV replication in a standard virus growth assay in 174xCEM indicator cells. HIV replication in the presence of Chitosan solution is expressed as a percentage of HIV replication in the presence of medium alone, which is adjusted to 100% (Figure 1 , A). HIV replication in the presence of Chitosan solution is expressed as a percentage of HIV replication in the presence of the equivalent concentration of acetic acid carrier, which is adjusted to 100% (Figure 1., B). Each bar represents the average ±SD of 3 independent experiments. Exposure of cells to a known HIV inhibitor (AZT) allowed a p24 output approximately 10% of that seen in the presence of medium alone. [0052] Summary. Chitosan solutions greater than 10 ug/ml but less than 200 ug/ml selectively and reproducibly inhibit HIV-1 replication and spread in typical HIV assay. The mode of inhibition is as yet unknown, but could include direct inactivation/destabilization of virus particles, inhibition of HIV-receptor binding and entry, or inhibition of intracellular events in the HIV life cycle that culminate in reduced egress of infectious virus. In addition, chitosan could contribute to HIV inhibition via its known ability to enhance cellular innate immunity. [0053] Example 2: Evaluation of the Anti-HIV Properties of Chitosan
Filters
[0054] Methods. Standard surgical masks were cut along seams, such that all three layers were separated. The inside layer (the layer that would contact the person's face) was cut into 2"x4" strips and put into sealable plastic bag. Similarly the Middle layer (white middle layer of the mask) was cut into 2"x4" strips and put in same sealable plastic bag. Same cut and packaging was performed for the outside (green layer). [0055] The following solutions were made for all coatings and controls: 2% food grade chitosan (Primex) 2% glacial acetic acid (HAc) and 0.5% food grade chitosan in 0.25% HAc, and all solvent control solutions.
[0056] For each filter layer, or solution the following protocol was applied in a class 1000 clean room. 30.Og of solution was poured into a 4 inch X 4 inch aluminum mold for coating procedures. The filter layers were then submerged into solution, such that the entire 2 inch X 4 inch strip was submerged. Using a rubber brayer, the sample was compressed and rolled over back and forth while in solution. This was the top-side of the filter. The opposite side of the sample was coated in a similar way, by flipping the sample in the solution and compress rolling. To remove excess solution, the sample was removed from solution and a squeegee was used to draw the solution off the samples. Each sample was put (Top-side up) in a 2 inch X 4 inch aluminum mold for freeze drying.
[0057] All samples including were freezed dried to remove the solvent. Next, each set of filters (one concentration all three layers) was packaged into a foil pouch, vacuum sealed and gamma sterilized.
[0058] Filters were cut into 2mm squares and placed in the bottom of 12-well trays. HIV (3OuI drop) was placed on filter and allowed to adsorb into the material. As a control, 3OuI of HIV was placed into empty well for the same time period. Each filter was subsequently rinsed in medium and placed back into its well - to test if the rinsed filter remains infectious or if the virus can be rinsed out. The rinse medium was then placed into wells to test for presence of infectious eluted virus. Each of these conditions were performed in duplicate. Next, an HIV-susceptible T cell line was added to each well in culture medium. The medium was harvested at day 4, post infection for p24 testing. [0059] Summary of Test Samples
3-layer 0.5% Chitosan filters:
0.5% Primex/0.25% HAc Control 0.25% HAc
3-layer 2% Chitosan filters:
2% Phmex/2% HAc Control 2% HAc
[0060] Results. As shown in Figures 2 and 3, chitosan filters are anti-viral.
Adsorbed HIV loses most of its infectivity. Virus either cannot be eluted or eluted virus loses most of its infectivity. Testing of control filters demonstrates that at least some of the anti-viral effect is due to either physical trapping in filter or in the HAc vehicle.

Claims

ClaimsWhat is claimed is:
1. A method of inactivating a virus comprising contacting the virus with chitosan or derivative thereof.
2. The method according to claim 1 , wherein the chitosan or derivative thereof is contained in or on a filter.
3. The method according to claim 1 , wherein the virus is trapped in a filter.
4. The method according to claim 3, wherein the filter is an air filter.
5. The method according to claim 1 , wherein the virus is capable of infecting a human or an animal.
6. The method according to claim 5, wherein the virus is virus herpes simplex type 1 (HHV-1 ), herpes simplex type 2 (HHV-2), varicella zoster virus (HHV-3), cytomegalovirus virus (HHV-5), human herpes virus type 6, 7, Epstein Barr virus (HHV-4), human herpes virus type 8, variola virus, molluscum contagiousum virus, human adenovirus, papillomavirus, BK virus, JC virus, human parvovirus (B 19), rhinovirus, hepatitis A virus, rubella virus, eastern equine encephalitis virus, human herpes virus type 8, yellow fever virus, dengue virus, west Nile virus, hepatitis C virus, human coronavirus, Norwalk virus, mumps virus, measles virus, respiratory syncitial virus (RSV), human parainfluenza virus 1 , rabies virus, ebola virus, lassa fever virus, influenza A, influenza B, influenza C, sin nombre virus, human immunodeficiency viruses, human T-cell leukemia viruses or hepatitis B virus.
7. The method according to claim 3, wherein at least a portion of the filter is coated with chitosan.
8. The method according to claim 3, wherein at least a portion of the filter is impregnated with chitosan.
9. The method according to claim 3, wherein the filter is capable of substantially inactivating the virus.
10. The method according to claim 3, wherein the filter is adapted to fit in an air passageway.
11. The method according to claim 10, wherein the filter is part of a face mask.
12. A filter for removing one or more toxic agents from a volume of air, comprising chitosan or a derivative thereof.
13. The filter of claim 12, wherein at least a portion of the air filter is coated with chitosan.
14. The filter of claim 12, wherein at least a portion of the air filter is impregnated with chitosan.
15. The filter of claim 12, wherein the air filter is capable of substantially inactivating the one or more toxic agents.
16. The filter of claim 12, wherein the one or more toxic agents include one or more pathogens, heavy metals, or radioactive agents.
17. The filter of claim 12, wherein the one or more pathogens includes one or more viruses or bacteria.
18. The filter of claim 16, wherein the virus is virus herpes simplex type 1 (HHV-1 ), herpes simplex type 2 (HHV-2), varicella zoster virus (HHV-3), cytomegalovirus virus (HHV-5), human herpes virus type 6, 7, Epstein Barr virus (HHV-4), human herpes virus type 8, variola virus, molluscum contagiousum virus, human adenovirus, papillomavirus, BK virus, JC virus, human parvovirus (B 19), rhinovirus, hepatitis A virus, rubella virus, eastern equine encephalitis virus, human herpes virus type 8, yellow fever virus, dengue virus, west Nile virus, hepatitis C virus, human coronavirus, Norwalk virus, mumps virus, measles virus, respiratory syncitial virus (RSV), human parainfluenza virus 1 , rabies virus, ebola virus, lassa fever virus, influenza A, influenza B, influenza C, sin nombre virus, human immunodeficiency viruses, human T-cell leukemia viruses or hepatitis B virus.
19. The filter of claim 16, wherein the bacteria is Methicillin-resistant Staphylococcus aureus.
20. The filter of claim 12, wherein the air filter is capable of substantially chelating the one or more heavy metals.
21.The filter of claim 12, further comprising an air passageway, wherein the air filter is adapted to fit in the air passageway.
22. The filter of claim 12, further comprising a face mask coupled to the air filter.
23. The filter of claim 20, wherein at least a portion of the face mask comprises the air filter.
24.A method of removing one or more toxic agents from a volume of air, comprising: trapping the one or more toxic agents in an air filter, wherein the air filter comprises chitosan.
25. The method of claim 22, wherein at least a portion of the air filter is coated with chitosan.
26. The method of claim 22, wherein at least a portion of the air filter is impregnated with chitosan.
27. The method of claim 22, wherein at least a portion of the air filter is a chitosan material.
28. The method of claim 22, wherein said trapping the one or more toxic agents in an air filter comprises substantially inactivating the one or more toxic agents.
29. The method of claim 22, wherein said trapping the one or more toxic agents in an air filter comprises substantially chelating the one or more toxic agents.
30. The method of claim 22, wherein the one or more toxic agents include one or more pathogens, heavy metals, or radioactive agents.
31. The method of claim 28 wherein the one or more pathogens includes one or more bacteria or viruses.
32. The method of claim 30, wherein the virus is virus herpes simplex type 1 (HHV- 1 ), herpes simplex type 2 (HHV-2), varicella zoster virus (HHV-3), cytomegalovirus virus (HHV-5), human herpes virus type 6, 7, Epstein Barr virus (HHV-4), human herpes virus type 8, variola virus, molluscum contagiousum virus, human adenovirus, papillomavirus, BK virus, JC virus, human parvovirus (B 19), rhinovirus, hepatitis A virus, rubella virus, eastern equine encephalitis virus, human herpes virus type 8, yellow fever virus, dengue virus, west Nile virus, hepatitis C virus, human coronavirus, Norwalk virus, mumps virus, measles virus, respiratory syncitial virus (RSV), human parainfluenza virus 1 , rabies virus, ebola virus, lassa fever virus, influenza A, influenza B, influenza C, sin nombre virus, human immunodeficiency viruses, human T-cell leukemia viruses or hepatitis B virus.
33. The method of claim 30, wherein the bacteria is Methicillin-resistant Staphylococcus aureus.
34.The method of claim 22, wherein the air filter is adapted to fit in an air passageway.
35. The method of claim 22, wherein the air filter is coupled to a face mask.
36. The method of claim 31 , wherein at least a portion of the face mask comprises the air filter.
PCT/US2007/079246 2006-09-22 2007-09-21 Inactivation of toxic agents and pathogens using chitosan WO2008105934A2 (en)

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CN114767707A (en) * 2021-01-22 2022-07-22 艾美科健株式会社 Composition for preventing or treating coronavirus infection diseases comprising low molecular chitosan
KR20220106912A (en) * 2021-01-22 2022-08-01 아미코젠주식회사 Composition for preventing or treating coronavirus infection comprising low molecular weight chitosan
KR102444839B1 (en) * 2021-01-22 2022-09-19 아미코젠주식회사 Composition for preventing or treating coronavirus infection comprising low molecular weight chitosan
CN114767707B (en) * 2021-01-22 2024-05-03 艾美科健株式会社 Composition for preventing or treating coronavirus infection diseases comprising low molecular chitosan
WO2022161559A1 (en) * 2021-01-28 2022-08-04 Apodis Gmbh Filter element for filtering viruses and/or bacteria from a fluid
WO2022185326A1 (en) * 2021-03-03 2022-09-09 Amitava Mazumder Antiviral, anti sars-cov-2, anti h1n3, antibacterial and antimicrobial compositions and methods of preparation thereof

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