WO2023081198A1 - Méthodes et compositions comprenant des cristaux d'acide protocatéchuique pour le traitement d'états provoqués par un virus enveloppé - Google Patents

Méthodes et compositions comprenant des cristaux d'acide protocatéchuique pour le traitement d'états provoqués par un virus enveloppé Download PDF

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WO2023081198A1
WO2023081198A1 PCT/US2022/048688 US2022048688W WO2023081198A1 WO 2023081198 A1 WO2023081198 A1 WO 2023081198A1 US 2022048688 W US2022048688 W US 2022048688W WO 2023081198 A1 WO2023081198 A1 WO 2023081198A1
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virus
pca
encephalitis
mammal
human
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PCT/US2022/048688
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Lanny Leo Johnson
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Lanny Leo Johnson
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Priority claimed from US17/453,432 external-priority patent/US20220054436A1/en
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Publication of WO2023081198A1 publication Critical patent/WO2023081198A1/fr

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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
    • A61P31/14—Antivirals for RNA viruses
    • 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
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/192—Carboxylic acids, e.g. valproic acid having aromatic groups, e.g. sulindac, 2-aryl-propionic acids, ethacrynic acid 
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0043—Nose
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
    • A61K9/0056—Mouth soluble or dispersible forms; Suckable, eatable, chewable coherent forms; Forms rapidly disintegrating in the mouth; Lozenges; Lollipops; Bite capsules; Baked products; Baits or other oral forms for animals

Definitions

  • This disclosure is directed to the use of protocatechuic crystals to destroy enveloped viruses in mammals.
  • Crystals can provide a therapeutic application by their physical nature. They have known cytotoxic properties which can achieve a therapeutic result. It is known that the physical properties of crystals can have an antimicrobial property independent or in conjunction with biochemical properties. Their many sharp edges have the potential to physically disrupt a microbe's integrity.
  • Enveloped viruses are particularly physically vulnerable. For example, the covering of the coronavirus, an enveloped virus, is surrounded by many projections like a crown. The projections are called prongs or spikes. These spikes are the virulent contact agent with the host cell. They penetrate the human cell, and the infection is then propagated. The spikes and especially the underlying thin lipid wall coating of the viral body are vulnerable to physical disruption. Physical disruption is one potential method of stopping the encoated virus cellular invasion and the clinical disease. Crystals that have a physical structure that is irregular, rough, and sharp have the potential to physically disrupt a microbes' spikes and covering.
  • a method of treating a disease or condition caused by an enveloped virus in a mammal including administering to the mammal a composition comprising protocatechuic acid crystals and disrupting the viral envelope of the enveloped virus.
  • the mammal may be a human.
  • the composition may include a pharmaceutically acceptable carrier.
  • the enveloped virus may be a coronavirus.
  • the enveloped virus may be a virus of the family Orthomyxoviridae.
  • the common denominator of an encoated virus, independent of its causation of differing clinical manifestations or diseases, is the physical vulnerability of the coating or envelope. PCA's therapeutic mode of action is to initially disrupt this inherent weakness and thus is effective against any and all encoated or enveloped viruses.
  • PCA has a multi-step process of inactivation.
  • the initial disruption is physical based upon the sharp protrusions of the PCA crystal.
  • the inactivation continues in that PCA has a low pH that further destroys the exposed RNA and or DNA.
  • PCA has anti protease and blocking properties to further limit any surviving encoated virus entry into a cell.
  • Fig. 1 shows crystals of PCA upon drying on a surface.
  • Fig. 2 shows a photomicrograph of crystals of PCA in a water droplet.
  • Fig. 3 shows a photomicrograph of crystals of PCA dissolved in ethanol.
  • Viruses are believed to be pieces of nucleic acids from various sources. Viruses are acellular, parasitic entities that are not considered to be alive. They have no plasma membrane, internal organelles, or metabolic processes, and they do not divide. They infect a host cell and use the host's replication processes to reproduce. Viruses infect all forms of organisms including bacteria, archaea, fungi, plants, and animals. Therefore, virus replication is entirely dependent on the host cells. [0015] Viruses are diverse. They vary in structure and target hosts. The structure of all viruses includes a protein shell called a capsid. Enveloped viruses have an additional layer that covers the capsid. An encapsulated virus thus has an envelope that is the outermost layer. This membrane is composed of lipids and proteins. Bumps, knobs, spikes, etc., structures may be present on the envelope.
  • the envelope protects the virus.
  • Envelopes are typically composed of a thin layer of phospholipid and protein material.
  • the envelope surface serves to identify and bind to receptor sites on the host cell membrane.
  • Enveloped viruses need both an intact capsid and the envelope to infect cells.
  • the envelope also helps avoid detection by the host immune system because it makes the virus appear as any other host cell.
  • the viral envelope fuses with the host's membrane, allowing the capsid and viral genome to enter and infect the host.
  • Enveloped viruses are adaptable and can quickly adapt to evade the immune system. Enveloped viruses can cause persistent infections and must transfer from host to host.
  • enveloped viruses include many that cause notorious diseases in humans, such as COVID-19, Influenza, Hepatitis B and C, and Hemorrhagic Fever (Ebola Virus Disease), DNA viruses, Herpesviruses, Poxviruses, Hepadnavirus, Asfarviridae, RNA viruses, Flaviviruses, Alphaviruses, Togaviruses, Coronaviruses, Hepatitis D, Orthomyxoviruses, Paramyxoviruses, Rhabdovirus, Bunyaviruses, Filoviruses, Retroviruses, and Retroviruses.
  • COVID-19 Influenza
  • Hepatitis B and C and Hemorrhagic Fever (Ebola Virus Disease)
  • DNA viruses Herpesviruses, Poxviruses, Hepadnavirus, Asfarviridae, RNA viruses, Flaviviruses, Alphaviruses, Togaviruses, Coronaviruses, Hepatitis D, Orthomy
  • envelope viruses are easier to sterilize than non-enveloped viruses.
  • Common disinfectants, including alcohol, will disrupt the oily envelope and its components destroying the ability for the virus to infect host cells.
  • Enveloped viruses have limited survival outside host environments and typically must transfer directly from host to host. This factor provides a means of mitigation by attacking the virus in transit whether in air or on hard surfaces. Examples of diseases caused by enveloped viruses include ones that cause notorious diseases in humans, such as COVID-19, Influenza, Hepatitis B and C, and Hemorrhagic Fever (Ebola Virus Disease).
  • viruses that can be treated by the present methods and compositions include the family of coronaviruses.
  • Coronaviruses are enveloped viruses of the subfamily Orthocoronavirinae in the family Coronaviridae.
  • 229E alpha coronavirus
  • NL63 alpha coronavirus
  • OC43 beta coronavirus
  • HKU1 beta coronavirus
  • SARS-CoV the virus that causes SARS
  • MERS-CoV the virus that causes Middle East Respiratory Syndrome, or MERS
  • SARS-CoV-2 the virus that causes Covid-19. See Coronavirus, Wikipedia, the free encyclopedia
  • viruses that can be treated by the present methods and compositions include the family of enveloped influenza viruses Orthomyxoviridae.
  • Alphainfluenzavirus influenza A virus
  • Betainfluenzavirus (influenza B virus) including Victoria, Yamagata, Deltainfluenzavirus (influenza D virus), Gammainfluenzavirus (influenza C virus), Isavirus, Thogotovirus including Batken virus, Bourbon virus, Jos virus, and Quaranjavirus.
  • enveloped viruses that can be treated by the compositions and methods disclosed herein include: gingiva stomatitis, vesicles and ulcers in the mouth, herpes genitalis, vesicles and ulcers on genitalia; herpes labialis, cold sores, fever blisters, vesicles and ulcers of lips, herpes gladiatorum, clusters of vesicles and ulcers on skin, encephalitis, keratoconjunctivitis, whitlow (felon) a purulent infection involving the pulp of the distal phalanx of a finger, Human herpesvirus 3 (varicella-zoster virus), chickenpox (varicella), epithelial cell infection resulting in an exanthem of macules, papules, pustules, vesicles and shallow ulcers shingles (zoster), peripheral nerve cell infection with an eruption in the overlying epidermis Human herpes
  • encephalitis Louis encephalitis virus (encephalitis), Dengue virus, dengue, Hepatitis G virus, acute hepatitis via blood transfusions, West Nile virus (fever, rash, hemorrhage and shock), Japanese B encephalitis virus, Japanese encephalitis, Murray Valley encephalitis virus, Murray Valley encephalitis, Central European tick-borne encephalitis virus (encephalitis), Far eastern tick-born encephalitis virus (encephalitis), Kyasanur forest virus, (encephalitis), Louping ill virus (encephalitis), Powassan virus, (encephalitis), Omsk hemorrhagic fever virus (hemorrhagic fever), Kumilinge virus (encephalitis), Absetarov anzalova hypr virus (encephalitis), llheus virus (encephalitis), Rocio encephalitis virus (encephalitis), Langat virus (encephalitis), Lymphocytic choriomeningitis virus, lymphocytic
  • the disclosure also provides methods and compositions that effectively deliver PCA compositions to an affected mammal including humans. This includes intravenous administration and oral administration including intra nasal, peritoneal, nebulizer, ventilator, and transcutaneous.
  • the present invention also provides compositions and methods for use in the treatment of diseases caused by enveloped viruses to localized affected areas of a mammal as well. This would include the skin, lungs, and nasal cavities.
  • the present disclosure also provides compositions and methods for use in the treatment of a variety of symptoms related to enveloped viruses.
  • the ready absorption places the PCA in solution in the plasma.
  • PCA in solution in the plasma.
  • cells, tissues and organs which will kill enveloped viruses on contact. This will last throughout the known presence of PCA in the mammalian body for several days. Additional interval doses over time may give a continued effect.
  • the PCA known metabolism is that it is subsequently found in urine and feces intact and as subsequent metabolites.
  • the ready absorption places the PCA in solution in the plasma which will kill enveloped viruses on contact. This will last throughout the known presence of PCA in the mammalian body for several days.
  • the PCA known metabolism is that it is subsequently found in urine and feces intact and as subsequent metabolites. Additional interval doses over time may give a continued effect.
  • the administration places the PCA in solution in the plasma which will kill enveloped viruses on contact. This will last throughout the known presence of PCA in the mammalian body for several days.
  • the PCA known metabolism is that it is subsequently found in urine and feces intact and as subsequent metabolites. Additional interval doses over time may give a continued effect.
  • Also disclosed are methods of treatment of the pathological condition caused by enveloped viruses comprising the intravenous and intraperitoneal route.
  • the present disclosure provides multiple routes of therapeutic delivery of PCA.
  • Normal size crystals e.g., approximately 177 pm
  • powder may be delivered by the oral route including liquid, or capsule.
  • Smaller sized crystals and particles may also be used for intravenous, intraperitoneal and aerosol delivery to a patient.
  • the therapeutic compositions and compounds may be administered, for example, orally and topically.
  • the therapeutic compositions and compounds may also be administered by various conventional routes including, for example, oral, topical, buccal, injection, pulmonary, intravenous, inhalant, subcutaneous, sublingual, and/or transdermal.
  • the pharmaceutical composition can comprise: PCA and a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier can be selected from, but not limited to, any carrier, diluent, or excipient compatible with the other ingredients of the composition.
  • the pharmaceutical composition can comprise PCA and an acceptable delivery carrier.
  • the delivery can be formulated and administered as known in the art, e.g., for topical, oral, buccal, including lozenges, injection, intravenous, inhalant, subcutaneous, sublingual and/or transdermal.
  • said topical delivery carrier may be formulated and administered to any surface or cavity of the body.
  • the acceptable delivery can be selected from any dermal or transdermal carrier compatible with the other ingredients of the composition.
  • the acceptable delivery carrier is a biodegradable microsphere or a slow release bioabsorbable material.
  • the acceptable delivery carrier can be selected from 50/50 D, L lactide/glycol ide or 85/15 D, L lactide/glycolide, both of which are amorphous physically and, therefore, are non- reactive when used as a carrier in a composition that is delivered in or to the body.
  • the pharmaceutical composition comprising PCA may be formulated for an aerosol spray.
  • the aerosol spray may include PCA, a liquid vehicle, and a stabilizer.
  • the liquid vehicle may include water, or an alcohol and the stabilizer may include an oil.
  • the oil is an essential oil.
  • the essential oil may be lemon oil.
  • the aerosol spray compositions may comprise principally protocatechuic acid, liquid vehicle, and stabilizer as the main ingredients. In preferred embodiments, the aerosol spray compositions may comprise only protocatechuic acid, a liquid vehicle, and a stabilizer.
  • An oral dose for humans could be a minimum of 500-1000 milligrams per day.
  • the dose for administration may be interdependent on the other factors.
  • the dose for aerosol spray, ventilation and/or nebulizer may be a solution. It could be as low as, for example, 25 pM in water. It could be 1 to 30% by weight depending upon a vehicle's solubility properties.
  • the resultant dose concentration becomes 3%. If a certain concentration is delivered intravenously the variables include speed of flow and duration.
  • One example dosage could be 50 mg/kg, IV, every 6 hours for 4 days.
  • water may be the vehicle for treatment of mucous membranes of the nose, mouth, pharynx, respiratory tree, and lungs.
  • the compositions may be sprayed, misted, or fogged onto mammalian and human skin, the lungs and/or nasal cavities, etc., to kill and protect from enveloped viruses.
  • Electrostatic means of spraying including sanitizing or disinfection are also included.
  • the PCA has at least some liquid or moisture present.
  • the PCA may be dissolved in water, alcohol, saline, including saline 0.9% salt, or some other liquid, including droplets. Once dissolved in a liquid, the PCA may become crystalline in form and may then physically disrupt a virus and become virucidal. Nevertheless, dry PCA powder in amorphous or crystalline form is also included in this disclosure.
  • Drugs that change the pH at the surface of a cell membrane inhibit the fusion of the virus to the cell membrane. It can also inhibit nucleic acid replication, glycosylation of viral proteins, virus assembly, new virus particle transport, virus release, and other processes to achieve its antiviral effects.
  • PCA has an acid pH of 5.4 which is disruptive to viral coating and damaging to now exposed contents of RNA or DNA.
  • COVID-19 main protease (Mpro) is the key enzyme of coronavirus which plays a crucial role in virus replication and transcription, which can be targeted to retard the growth of virus inside the host.
  • Mpro main protease
  • pplab responsible for generating viral genome
  • Mpro can act as potential target for structure-based drug discovery as this enzyme not only involved in autocatalytic cleavage of itself and key viral enzymes, as well as lacks any close homologues among human hosts. Targeting this enzyme using suitable protease small molecule inhibitor holds immense potential to curb virus replication and transcription which are critical steps in virus life cycle.
  • PCA has anti-protease properties.
  • Fig. 1 shows crystals of PCA upon drying on a surface.
  • Fig. 2 shows a photomicrograph of crystals of PCA in a water droplet.
  • Fig. 3 shows a photomicrograph of crystals of PCA dissolved in ethanol. The figures show that PCA exists as a crystal upon drying and in solution.
  • PCA Protocatechuic acid
  • IUPAC 3,4 dihydroxybenzoic acid
  • PCA is the primary metabolite of cyanidin-3-glucoside.
  • PCA is common in the human diet in many vegetables and fruits.
  • the human bowel bacteria manufacture small amounts daily.
  • PCA upon ingestion perfuses all the cells and tissues of the human body in matter of a few minutes. The entire metabolism is known with duration of eight hours prior to excretion in the urine and feces.
  • PCA is safe for human consumption.
  • PCA has an existing FDA G.R.A.S. designation as Generally Recognized As Safe as a flavoring substance. Its FEMA number is 4430.
  • PCA is non-toxic. There are no known allergy or mutagenic effects.
  • PCA is a powerful antioxidant; 10 times more powerful than vitamin E. Antioxidants are fundamental to health.
  • PCA is a powerful antiinflammatory reagent. Inflammation is known to be the common denominator of all disease.
  • PCA enhanced the genetic expression in vitro studies of local growth factors in human and rabbit synovium, rodent skin and human osteoblasts and mesenchymal stem cells to produce bone. There are known to be many, and varied, health benefits of protocatechuic acid.
  • a 'pharmaceutically acceptable carrier' is as described above and is generally any substrate used in the process of drug delivery which serves to improve the selectivity, effectiveness, and/or safety of drug administration. See Drug carrier, Wikipedia, the free encyclopedia, last edited 16 October 2021, herein incorporated by reference.
  • mammal' as used herein are a group of vertebrate animals constituting the class Mammalia.
  • mammal refers to primates and most preferably humans. See Mammal, Wikipedia, the free encyclopedia, last edited 18 October 2021, herein incorporated by reference.
  • the term 'administration' refers to the administration of a drug to a mammal or human and can be oral; injection into a vein (intravenously, IV), into a muscle (intramuscularly, IM), into the space around the spinalcord (intrathecally), or beneath the skin (subcutaneously), placed under the tongue (sublingually) or between the gums and cheek (buccally), inserted in the rectum (rectally) or vagina (vaginally), placed in the eye (by the ocular route) or the ear (by the otic route), sprayed into the nose and absorbed through the nasal membranes (nasally), breathed into the lungs, usually through the mouth (by inhalation) or mouth and nose (by nebulization), applied to the skin (cutaneously) for a local (topical) or body wide (systemic) effect, delivered through the skin by a patch (transdermally) for a systemic effect. See Drug Administration, Merck Manual, Consumer version, Last full review/re
  • a viral envelope refers to the outermost layer of an encapsulated or encoated virus. Bumps, knobs, spikes, etc., structures may be present on the envelope. The envelope protects the virus.
  • Envelopes are typically composed of a thin layer of phospholipid and protein material.
  • the envelope surface serves to identify and bind to receptor sites on the host cell membrane. See Viral envelope, Wikipedia, the free encyclopedia, last edited 16 September 2021, herein incorporated by reference.
  • An encapsulated virus or encoated virus or enveloped virus is a virus which has a viral envelope.
  • Viral inactivation renders viruses unable to infect. Viral inactivation stops a virus from contaminating a particular product by rendering them non-infectious. Preferably this is done by chemically altering or physically disrupting a viral envelope. See Virus inactivation, Wikipedia, the free encyclopedia, last edited 22 October 2021, herein incorporated by reference.
  • Saliva testing was performed with PCA. This example replicated the practical clinical use of a lozenge to deliver the therapeutic crystals of PCA to the oral cavity and tongue's part of the pharynx and substantiated the lasting duration of PCA coating on those anatomical structure's irregular surfaces of the oral pharynx.
  • This example shows nasal hair Coating with Protocatechuic acid crystals.
  • the nasal hair has a natural filtering function. Therefore, a PCA coating can be applied with raw crystals, but also with a variety of vehicles; water, glycerin, propylene glycol and or mixtures with alcohols that put the powder into small crystals intimately attached to the hair.
  • Photomicrographs of alcohol solution of PCA on hair with polarized light show the intimacy of crystals on the hair. Photomicrographs show crystals covering the end of the cut hair.
  • Example 3 demonstrates the results studies utilizing a methodology replicating the clinical therapeutic environment whereby the PCA crystal engages the SARS CoV-2 virus in an aqueous environment. These studies demonstrate the effectiveness of Protocatechuic Acid (PCA) against SARS-CoV-2, the causative virus for COVID19.
  • PCA Protocatechuic Acid
  • the Test Article (TA) used for this study was Protocatechuic Acid (PCA).
  • PCA Protocatechuic Acid
  • the TA was received as an off-white powder.
  • the PCA solution was prepared to be 30% PCA w/v in Ethanol.
  • the PCA was prepared in 5g increments to pre-warmed 50-60 mL ethanol until dissolved for a total of 30g PCA in the solution. Additional ethanol was then added volumetrically to be equivalent to 100m L.
  • Test Substrates were a Plastic-type material sourced from a clear plastic laboratory bottle (Corning 431731 Octagonal bottle, 150mL), cloth (the top layer of a N95 mask [3M 8210]), and a Sponsor-provided wire mesh to serve as a substrate for the TA. All test substrates were cut to approximately l"x 1" in size. The test substrates were submerged into the PCA solution and dried horizontally to allow for even coating. After the substrate was thoroughly dried, the test substrate was re-submerged into the PCA solution for an additional coating.
  • the Test Virus used for this study was 2019 Novel Coronavirus, Isolate USA-WA1/2020 (SARS-CoV-2).
  • SARS-CoV-2 The virus was stored at approximately ⁇ -65°C prior to use.
  • the multiplicity of infection (MOI) was 0.01 TCID 50 /cell.
  • the Cell Culture used for the TCID 50 test was African Green Monkey Kidney Cells (Vero E6 cells) that were maintained in Dulbecco's Minimum Essential Medium with 10% fetal calf serum (DMEM-2). All growth media contained heat-inactivated fetal calf serum and antibiotics. [0064] The test design is shown below in Table 1. This test assesses the TA on a substrate in various conditions as shown in Table 1.
  • Test Substrate was coated with PCA as described above.
  • the test substrates were treated with PCA twice and allowed to fully dry overnight. In general, the time from the first coat to the next day's virus exposure was approximately 24 hours.
  • the treated Test Substrate plus TA was placed into a sterile 6 well cell culture plate and approximately 100 pL total of a >1 x 10 s TCID 5 o/mL SARS-CoV-2 virus was such that 50 pL of virus was layered on each side of the treated test substrates. This was the procedure used for the initial Day 1 experiment.
  • the treated Test Substrate plus TA was placed into a sterile 6 well cell culture plate and the same amount of virus was layered onto both sides of the test substrate. However, an addition 50 pL of DMEM was added to each side to reduce the inactivation of the virus due to desiccation.
  • a glass coverslip was also added to help mitigate against evaporation.
  • the virus was contact with the Test substrates for approximately 10 minutes (Groups 1, 2, and 3, Control groups 7, 8, and 9), 60 minutes (Groups 4, 5, and 6, Control Groups 10, 11, and 12). Each substrate per time per test article was performed in duplicate.
  • a cell culture-only control was included to indicate that cells without any TA or virus remain healthy throughout the assay. Virus-only controls without substrate was added for each timepoint to verify that the assay was performing as expected.
  • the treated substrate was washed with 1 mL of cell culture media (DMEM-2) for approximately 5-10 minutes within the 6 well cell culture plate and the glass cover slip removed if necessary. This was the equivalent to a 10-fold dilution.
  • the plate was gently stirred via an orbital shaker to enhance the recovery of the virus.
  • the cell culture media (DMEM-2) used to wash the Test Substrate was serially diluted 10-fold and transferred into respective wells of a 96-well plate which contained a monolayer of African Green Monkey Kidney Cells (Vero E6 cells) for titration.
  • the TCID 50 assay was performed non-GLP according to IITRI Standard Operating Procedures for the assay.
  • the TCID 50 titers was calculated using the method of Reed-Meunch.
  • Test Articles, Test Substrates, and virus were prepared according to protocol and each preparation was noted in the study notebook for this study.
  • a Log difference is defined as the averaged TCID 50 Logio/mL from virus control on substrates - TCID 50 Logio/mL from replicate test group. Log difference indicates amount of reduction in infectious virus when comparing the virus control on substrate to the test group.
  • TCID50 For Day 2, after coating the Test Substrates with PCA as described above (Groups shown in Table 1 above), a TCID50 was performed at 10 minutes or 60 minutes after initial application of the virus. There was a modification to the procedures to see if the viral titers could be increased. To mitigate against evaporation during the incubation periods, these modifications included adding an additional 50 pl of DMEM on each side of the test substrate and a glass coverslip was placed on top of the test substrate.
  • a Log difference is defined as the averaged TCIDso Logio/mL from virus control on substrates - TCIDso Logio/mL from replicate test group. Log difference indicates amount of reduction in infectious virus when comparing the virus control on substrate to the test group.
  • a PCA coating on the three test substrates appeared to show some effectiveness in reducing infectious virus titers in the experimental condition shown in the protocol after the 10 minutes and 60 minutes post-exposure incubation when compared to the virus control on substrate. From both the Day 1 and the confirmatory runs, the log reduction varied between a 0.63 to a 2.38 log reduction.
  • the second laboratory test utilized test coupons made of solid stainless steel, plastic and K95 mask were coated in 30% w/v PCA in 70% ethanol. Each coupon was dipped in PCA, allowed to dry, dipped again, and allowed to dry with the opposite side of the coupon facing up. Once dry, 200 ul virus was added to each coupon and allowed to dry (45 minutes - lh drying time). Virus was recovered by adding 2 ml DMEM/F12 media and washing the coupon, without scraping so as not to dislodge PCA crystals. A yellow color change in the media was observed indicating acidification of the media upon addition to the PA-coated coupon. The recovered virus was added to empty 96 well plates and diluted 1:10 down the plate.
  • Table 6 The following are the results when the SARS CoV 2 virus was delivered in aqueous droplet on the PCA coated article immediately after drying. The virucidal effect was measured at 2 hours in this test.

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Abstract

Dans des modes de réalisation, une méthode de traitement d'une maladie ou d'un état provoqué par un virus enveloppé chez un mammifère, comprenant l'administration au mammifère d'une composition comprenant des cristaux d'acide protocatéchuique et la lyse de l'enveloppe virale du virus enveloppé. Le mammifère peut être un être humain. La composition peut comprendre un support pharmaceutiquement acceptable. Le virus enveloppé peut être un coronavirus. Le virus enveloppé peut être un virus de la famille des Orthomyxoviridae.
PCT/US2022/048688 2021-11-03 2022-11-02 Méthodes et compositions comprenant des cristaux d'acide protocatéchuique pour le traitement d'états provoqués par un virus enveloppé WO2023081198A1 (fr)

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US17/453,432 2021-11-03

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