WO2022084931A1 - Spray formulation for nasal/oral use with activity against respiratory coronaviruses and in particular the sars-cov-2 virus - Google Patents

Spray formulation for nasal/oral use with activity against respiratory coronaviruses and in particular the sars-cov-2 virus Download PDF

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WO2022084931A1
WO2022084931A1 PCT/IB2021/059753 IB2021059753W WO2022084931A1 WO 2022084931 A1 WO2022084931 A1 WO 2022084931A1 IB 2021059753 W IB2021059753 W IB 2021059753W WO 2022084931 A1 WO2022084931 A1 WO 2022084931A1
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nasal
oral use
spray formulation
ace2
formulation
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PCT/IB2021/059753
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French (fr)
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Antonio Bianchi
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Antonio Bianchi
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Priority claimed from IT102020000025207A external-priority patent/IT202000025207A1/en
Priority claimed from IT102021000016037A external-priority patent/IT202100016037A1/en
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Publication of WO2022084931A1 publication Critical patent/WO2022084931A1/en

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    • 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
    • A61K9/006—Oral mucosa, e.g. mucoadhesive forms, sublingual droplets; Buccal patches or films; Buccal sprays
    • 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
    • A61K31/13—Amines
    • A61K31/14—Quaternary ammonium compounds, e.g. edrophonium, choline
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A61K31/353—3,4-Dihydrobenzopyrans, e.g. chroman, catechin
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4425—Pyridinium derivatives, e.g. pralidoxime, pyridostigmine
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • 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
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
    • 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
    • A61K31/7135—Compounds containing heavy metals
    • A61K31/714—Cobalamins, e.g. cyanocobalamin, i.e. vitamin B12
    • 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
    • A61K31/717—Celluloses
    • 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
    • A61K31/726—Glycosaminoglycans, i.e. mucopolysaccharides
    • A61K31/728—Hyaluronic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/14—Alkali metal chlorides; Alkaline earth metal chlorides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • 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
    • A61K47/02—Inorganic compounds
    • 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
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A61K47/186—Quaternary ammonium compounds, e.g. benzalkonium chloride or cetrimide
    • 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
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • 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
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • A61K47/38—Cellulose; Derivatives thereof
    • 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
    • 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

Definitions

  • This invention refers to a spray formulation for nasal/oral use , with activity against respiratory Coronaviruses and, in particular, a solution to be sprayed into the nostrils (nasal spray : Zyvex Hypertonic Nasal Spray ) and into the throat (oral spray : Zyvex Advanced Oral Spray ) for the purpose of inhibiting or slowing down the activity of respiratory Coronaviruses .
  • nasal spray Zyvex Hypertonic Nasal Spray
  • oral spray Zyvex Advanced Oral Spray
  • Coronaviruses are viruses characterised by a morphology like a "crown" which can be seen under an electronic microscope . From a taxonomic point of view, they can be divided into three types :
  • - group 1 which includes viruses which affect cats , pigs and two human viruses (NL63 and 229E ) which cause respiratory infections ;
  • - group 2 which includes viruses of mice , pigs , sheep and five human viruses ;
  • the five human viruses of the group 2 are the OC43 virus , the HKU1 Virus , the SARS-CoV virus ( from Severe Acute Respiratory Syndrome which first appeared in 2002 in the Chinese province of Guangdong) (Weis s S . R . and Navas-Martin S . 2005 ) 1 , the MERS-CoV virus ( from Middle East Respiratory Syndrome which first appeared in 2012 in Saudi Arabia ) and the SARS-CoV-2 or COVID-19 virus (which appeared between the end of 2019 and the beginning of 2020 in the city of Wuhan, in China) .
  • the characteristics of the human Coronaviruses shown above can be summarised by stating that the NL63 and 229E viruses of group 1 and viruses OC43 and HKU1 of group 2 cause superficial infections of the upper respiratory tract , while the SARS-CoV, MERS-CoV and SARS-CoV-2 viruses cause severe infections of the lower respiratory tract.
  • the human coronaviruses enter the cells of the human organism through distinct receptors: the HCoV 229E virus bonds to aminopeptidase N, the HCoV OC43 and HCoV HKU1 viruses fundamentally use sugars to enter the human cells (portions containing Neu5, 9Ac2) ; while the HCoV NL-63 virus and the three viruses, SARS-CoV, MERS-CoV and SARS-CoV-2 viruses use the enzyme ACE2 (which enzyme converts angiotensin 2) .
  • the enzyme ACE 2 is present in numerous cells of the human organism (Wang Q. , Zhang Y., Wu L. et al. 2020) 2 .
  • SARS- CoV-2 and SARS-CoV Coronaviruses of binding to the ACE2 receptors and entering the human organism's cells depends, to a lesser extent, on the serine protease TMPRSS2 (Hoffmann M. , Kleine-Weber H., Schroeder S., Kruger N., Herrier T. et al. 2020) 3 , a potential target for the protease inhibitors.
  • TMPRSS2 serine protease TMPRSS2
  • the expression of the ACE 2 enzyme is greater in the apical cells of the secretory, ciliated and suprabasal type (Kuhnemund M. , Lako M., Lee H., and Leroy S. et al. 2020) 4 .
  • the expression of the genes for ACE2 and TMPRSS2 is absent in the olfactory sensory neurons while it is present in the sustentacular cells and in the horizontal basal cells at a concentration like that of the respiratory epithelium (while the microvillar cells and the Bowman' s gland' s cells exhibit an expression of the TMPRSS2 genes which is the same as or greater than that of the respiratory epithelium, but with a lesser frequency of the expression of the ACE2 genes) .
  • the HCoV NL-63 virus and the three SARS-CoV, MERS-CoV and SARS-CoV-2 viruses can therefore penetrate the central nervous system not directly through the olfactory sensory neurons but through the support cells, the stem cells that are part of the olfactory epithelium (Brann D.H., Tsukahara T., Weinreb C., Lipovsek M. et al. 2020) 6 and probably the vascular pericyte cells accompanying the olfactory nerve.
  • experiments conducted with transgenic rats for the expression of the human ACE2 enzyme have shown how intranasal inoculation of the SARSCoV virus causes evident neural death in the absence of any sign of encephalitis.
  • the pattern of neuronal distribution of the viral antigen strongly suggested a penetration by the olfactory route, as said viral antigen is distributed in the olfactory bulb and in such regions as the pyriform and infralimbic cortex, the basal nuclei (the regions of the ventral pallidum and of the lateral preoptic area) and the mesencephalic nuclei (the region of the dorsal raphe) , all of which are regions with primary or secondary connections with the olfactory bulb (Netland J., Meyerhol D.K., Morre S., Cassell M. and Perlman S. 2008) 7 .
  • the problem faced by this invention is that of offering a spray formulation for nasal/oral use that inhibits or at least reduces the viral activity inside the nasal cavity before the viruses can penetrate the organism through the central nervous system or acces s the respiratory system in depth .
  • said formulation must act very quickly ( 30 seconds -1 minute ) .
  • a first ob ject of the invention is to of fer a spray formulation for nasal/oral use that can hinder Coronavirus bonding with the respective receptors , to delay acces s by said Coronaviruses into the cells of the nasal and throat epithelium . Due to the multiple reactions involved in the entrance of the virus into the epithelial cells , the formulation must neces sarily have a multitarget way of action .
  • a second obj ect of the invention is to of fer a spray formulation, of the type shown above , that exhibit a sufficiently high virucidal activity against the Coronaviruses to prevent their penetration into the cells of the organism, but without having a harmful cytotoxic activity on the cells of the nasal and throat epithelium .
  • Antiseptic substances have always played a key role in limiting or eradicating the harmful presence of pathogenic viruses in several environments .
  • these substances cannot be used on the mucosa or, when they are used, they must be used at very low concentrations .
  • Ammonium salt s form a special category of virucidal substances that can be used at low concentrations on the mucosa .
  • benzalkonium chloride in nasal sprays and swabs is also known, as a preservative, in concentrations lower than 0 . 02% , while for cetylpyridinium chloride the maximum concentration for solutions that are sprayed into the throat must not be greater than 0 . 05% as , above this concentration there is the risk of toxic reactions at pulmonary level (Kanno 2020 ) 9 , when inhaled .
  • concentrations of benzalkonium chloride Kampf G, Todt D, Pfaender S and Steinmann E, 2020
  • the inventor devised using a pair of active substances which have a particular synergistic effect .
  • the formulation consist s of the combination of a component forming a barrier matrix, as sociated with substances of a flavonoid type showing a specific antiviral action on the ACE2 and TMPRSS2 receptors .
  • the substances that form a barrier matrix, in order to carry out their action, must preferably have a bioadhesive activity and can be helped by pH-adjusting agents or by common antiseptic substances .
  • Substances that modulate the immune system and stimulate the topical defences of the oral mucosa and of the respiratory system for example Vitamin B12 and folic acid, can also contribute to the ef fectivenes s of the formulation, hindering the viral replication .
  • ( 1 ) causes the "entrapment" of the viral particles in the barrier matrix
  • ( 2 ) inhibits the virus bonding both with the ACE2 receptor and with the TMPRSS2 receptor, thus preventing or at least delaying the entrance of the Coronavirus into the cells of the nasal or throat epithelium;
  • two quaternary ammonium salts can preferably be used, such as benzalkonium chloride for the nasal solution and cetylpyridinium chloride for the oral solution.
  • the formulation contains a concentration of between 0.02% and 2% of substances that form the barrier matrix, between 0.03% and 3% of flavonoid substances provided with a specific antiviral action on the ACE2 and TMPRSS2 receptors, between 0.1% and 1% of pH-adjusting agents, and between 0.01% and 0.5% of antiseptic substances.
  • the formulation contains between 0.1% and 1% of the substances that form the barrier matrix, between 0.05% and 1% of the flavonoid substances provided with a specific antiviral action on the ACE 2 and TMPRSS2 receptors, between 0.1% and 0.3% of pH-adjusting agents and between 0.02% and 0.5% of antiseptic substances.
  • the substances that form the barrier matrix are chosen from hyaluronic acid, hydroxyethyl cellulose and hydroxypropyl methylcellulose .
  • the substances which inhibit the bond of the spike protein with the ACE2 receptor and with the TPRMSS2 receptor are selected from flavonoids such as quercetin, hesperidin, baicalin, scutellarin, luteolin, myricetin and epigallocatechin .
  • the barrier matrix is kept at a low pH, i . e . , an acid pH, thanks to the addition of pH-adjusting agents such as citric acid, sodium citrate, lactic acid, and ascorbic acid .
  • the pH is kept between 3 and 5 , preferably between 3 . 5 and 4 . 5 .
  • the barrier matrix can preferably include humectant agent s such as glycerol, propylene glycol and polyethylene glycols .
  • humectant agent s such as glycerol, propylene glycol and polyethylene glycols .
  • Glycerol is the humectant agent of choice which prevent s the dryness of the biofilm when it contacts the nasal and throat mucosa .
  • the saline solution in which the components of the formulation are dis solved can be a traditional isotonic solution ( 0 . 9% NaCl ) or, preferably, a hypertonic solution ( from 1 . 2% to 3% of NaCl ) .
  • a traditional isotonic solution 0 . 9% NaCl
  • a hypertonic solution from 1 . 2% to 3% of NaCl
  • the formulation can be as sociated with substances such as Vitamin B12 ( 0 . 0025%-0 . 50% ) and folic acid ( 0 . 00125%-0 . 25% ) .
  • the spray formulation of this invention preferably includes hyaluronic acid, hydroxyethyl cellulose and hydroxypropyl methylcellulose as substances forming the barrier matrix, quercetin as inhibitor of bonding formation between virus and ACES2 and TPRMSS2 receptors , citric acid and sodium citrate as pH-adjusting agents , glycerol as humectant agent and benzalkonium chloride and cetylpyridinium chloride as antiseptic substances , sodium chloride that can be added as an osmotic agent, with the function of contributing to greater stability of the epithelial membranes and Vitamin B12 and folic acid as stimulants of the topical defences of the oral mucosa and of the upper respiratory tract.
  • Example 1 The substance that forms the barrier matrix in Example 1 has been modified by using an equivalent quantity of hydroxypropyl methylcellulose instead of hyaluronic acid.
  • Vitamin B12 0.0025%
  • the same substances which form the barrier matrix in this invention have been previously tested in a similar in vitro test and can inhibit under the same conditions of pH the growth of the Coronavirus only after 3-4 hours (Brambilla L, 2015 ) 13 .
  • Quercetin has shown that it can block the entry of the SARS-Coronavirus virus into the Vero E6 cells by 50% (Yi L, Zi K, Yuan K, Qu X, Chen J, Wang G et al . 2004 ) 14 .
  • the mechanism of action determined by the presence of the barrier layer allows obtaining an effectiveness of 99 . 99% in inhibiting the penetration of the virus in the Vero E 6 cells .
  • the formulation described here thanks to the special composition of the acid barrier matrix allows reducing the concentration of benzalkonium chloride to 0 . 02% and of cetylpyridinium chloride to 0 . 05% but obtaining a particularly high reduction of the proliferation of the SARS-CoV-2 virus of 4 logic after only 30 seconds .
  • the formulation of the invention therefore allows to attain a clear improvement with respect to the action of the individual component s thereof , in terms of a greater activity ( reduction of 4 logic of the viral load at lower and therefore safer concentrations compared to those previously known) and much faster times of action ( 30 seconds ) compared to those known for the individual components .

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Abstract

A spray formulation for nasal/oral use, for the protection against Coronavirus infections wherein the ACE2 receptor and/or the TMPRSS2 receptor is used by Coronavirus for penetrating the cells of the nasal and throat mucosa, consisting of a saline solution comprising, as active substances, at least one substance forming a barrier matrix and at least one substance provided with a specific antiviral action on the ACE2 and TMPRSS2 receptors.

Description

SPRAY FORMULATION FOR NASAL/ORAL USE WITH ACTIVITY AGAINST RESPIRATORY CORONAVIRUSES AND IN PARTICULAR THE SARS-COV-2 VIRUS ★ ★ ★ ★ ★
FIELD OF THE INVENTION
This invention refers to a spray formulation for nasal/oral use , with activity against respiratory Coronaviruses and, in particular, a solution to be sprayed into the nostrils (nasal spray : Zyvex Hypertonic Nasal Spray ) and into the throat (oral spray : Zyvex Advanced Oral Spray ) for the purpose of inhibiting or slowing down the activity of respiratory Coronaviruses . STATE OF THE PRIOR ART
Coronaviruses are viruses characterised by a morphology like a "crown" which can be seen under an electronic microscope . From a taxonomic point of view, they can be divided into three types :
- group 1 , which includes viruses which affect cats , pigs and two human viruses (NL63 and 229E ) which cause respiratory infections ;
- group 2 , which includes viruses of mice , pigs , sheep and five human viruses ; and
- group 3 , which essentially concerns only aviary viruses .
The five human viruses of the group 2 are the OC43 virus , the HKU1 Virus , the SARS-CoV virus ( from Severe Acute Respiratory Syndrome which first appeared in 2002 in the Chinese province of Guangdong) (Weis s S . R . and Navas-Martin S . 2005 ) 1, the MERS-CoV virus ( from Middle East Respiratory Syndrome which first appeared in 2012 in Saudi Arabia ) and the SARS-CoV-2 or COVID-19 virus (which appeared between the end of 2019 and the beginning of 2020 in the city of Wuhan, in China) .
The characteristics of the human Coronaviruses shown above can be summarised by stating that the NL63 and 229E viruses of group 1 and viruses OC43 and HKU1 of group 2 cause superficial infections of the upper respiratory tract , while the SARS-CoV, MERS-CoV and SARS-CoV-2 viruses cause severe infections of the lower respiratory tract.
The human coronaviruses enter the cells of the human organism through distinct receptors: the HCoV 229E virus bonds to aminopeptidase N, the HCoV OC43 and HCoV HKU1 viruses fundamentally use sugars to enter the human cells (portions containing Neu5, 9Ac2) ; while the HCoV NL-63 virus and the three viruses, SARS-CoV, MERS-CoV and SARS-CoV-2 viruses use the enzyme ACE2 (which enzyme converts angiotensin 2) . The enzyme ACE 2 is present in numerous cells of the human organism (Wang Q. , Zhang Y., Wu L. et al. 2020) 2. The possibility of the SARS- CoV-2 and SARS-CoV Coronaviruses of binding to the ACE2 receptors and entering the human organism's cells depends, to a lesser extent, on the serine protease TMPRSS2 (Hoffmann M. , Kleine-Weber H., Schroeder S., Kruger N., Herrier T. et al. 2020) 3, a potential target for the protease inhibitors. At the nasal level, the expression of the ACE 2 enzyme is greater in the apical cells of the secretory, ciliated and suprabasal type (Kuhnemund M. , Lako M., Lee H., and Leroy S. et al. 2020) 4.
It is already well known that numerous viruses, once they have entered the human organism can affect the central nervous system. The entrance of viruses into the brain can take place either through hematic route or through retrograde neuronal routes. In the latter case, the virus uses the axonal transport machinery to obtain access to the central nervous system. For a respiratory virus, this last case implies the possibility of infecting the receptors and the olfactory bulb and this possibility has been shown in coronaviruses, in studies on mice after nasal inoculation, both for the HCoV-OC43 virus and for the virus of SARS-CoV (Desforges M., Le Copuanec A., Debeau P., Bourgouin A., Lajoie L., Dube M. and Talbot P.J. 2019)5.
The expression of the genes for ACE2 and TMPRSS2 is absent in the olfactory sensory neurons while it is present in the sustentacular cells and in the horizontal basal cells at a concentration like that of the respiratory epithelium (while the microvillar cells and the Bowman' s gland' s cells exhibit an expression of the TMPRSS2 genes which is the same as or greater than that of the respiratory epithelium, but with a lesser frequency of the expression of the ACE2 genes) . The HCoV NL-63 virus and the three SARS-CoV, MERS-CoV and SARS-CoV-2 viruses can therefore penetrate the central nervous system not directly through the olfactory sensory neurons but through the support cells, the stem cells that are part of the olfactory epithelium (Brann D.H., Tsukahara T., Weinreb C., Lipovsek M. et al. 2020) 6 and probably the vascular pericyte cells accompanying the olfactory nerve. Moreover, experiments conducted with transgenic rats for the expression of the human ACE2 enzyme have shown how intranasal inoculation of the SARSCoV virus causes evident neural death in the absence of any sign of encephalitis. The pattern of neuronal distribution of the viral antigen strongly suggested a penetration by the olfactory route, as said viral antigen is distributed in the olfactory bulb and in such regions as the pyriform and infralimbic cortex, the basal nuclei (the regions of the ventral pallidum and of the lateral preoptic area) and the mesencephalic nuclei (the region of the dorsal raphe) , all of which are regions with primary or secondary connections with the olfactory bulb (Netland J., Meyerhol D.K., Morre S., Cassell M. and Perlman S. 2008) 7. Furthermore, in a recent study the expression of ACE2 in the olfactory epithelium of the nasal cavities was found greater than that in the respiratory epithelium (Chen M., Shen W. , Rowan N.R., Kulaga H., Hille A., Ramanathan M. and Lane A.P. 2020) 8; this circumstance allows to postulate that the main route of penetration of the Coronavirus in the organism is represented by the olfactory epithelium and by the central nervous system and that the (often sudden) severity of certain respiratory syndromes can be ascribed to an infection of the encephalic trunk (which includes the nucleus of the solitary tract and the ambiguous nucleus) .
In the above-described medical picture, the problem faced by this invention is that of offering a spray formulation for nasal/oral use that inhibits or at least reduces the viral activity inside the nasal cavity before the viruses can penetrate the organism through the central nervous system or acces s the respiratory system in depth . To perform an action of this type , said formulation must act very quickly ( 30 seconds -1 minute ) .
In the context of this problem, a first ob ject of the invention is to of fer a spray formulation for nasal/oral use that can hinder Coronavirus bonding with the respective receptors , to delay acces s by said Coronaviruses into the cells of the nasal and throat epithelium . Due to the multiple reactions involved in the entrance of the virus into the epithelial cells , the formulation must neces sarily have a multitarget way of action .
A second obj ect of the invention is to of fer a spray formulation, of the type shown above , that exhibit a sufficiently high virucidal activity against the Coronaviruses to prevent their penetration into the cells of the organism, but without having a harmful cytotoxic activity on the cells of the nasal and throat epithelium .
Antiseptic substances have always played a key role in limiting or eradicating the harmful presence of pathogenic viruses in several environments . Unfortunately, these substances cannot be used on the mucosa or, when they are used, they must be used at very low concentrations . Ammonium salt s form a special category of virucidal substances that can be used at low concentrations on the mucosa .
The use of benzalkonium chloride in nasal sprays and swabs is also known, as a preservative, in concentrations lower than 0 . 02% , while for cetylpyridinium chloride the maximum concentration for solutions that are sprayed into the throat must not be greater than 0 . 05% as , above this concentration there is the risk of toxic reactions at pulmonary level (Kanno 2020 ) 9, when inhaled . Unfortunately, according to the data of scientific trials , the concentrations of benzalkonium chloride (Kampf G, Todt D, Pfaender S and Steinmann E, 2020 ) 10 active against the Coronavirus , to obtain a reduction of 3 Logic ( i . e . , -3 on a decimal logarithmic scale ) after one minute are at least of 0 . 05% whereas those of cetylpyridinium chloride must be of at least 0 . 07 % ( Green A, Roberts G, Tobery T, Vincent C, Barili M, Jones C 2020 ) 11 .
SUMMARY OF THE INVENTION
This problem is solved, and these ob ject s are achieved through a spray formulation for nasal/oral use having the features defined in claim 1 . Other preferred features of the afore-mentioned spray formulation are defined in the secondary claims . BRIEF DESCRIPTION OF THE DRAWINGS
Further features and advantages of the spray formulation for nasal/oral use according to this invention will however be clearer from the following detailed description of a preferred embodiment of the same, provided merely as a not limiting example . DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
According to this invention, to solve the problem shown above with a formulation that can be easily provided, with a low cost and certain effectiveness , the inventor devised using a pair of active substances which have a particular synergistic effect . The formulation consist s of the combination of a component forming a barrier matrix, as sociated with substances of a flavonoid type showing a specific antiviral action on the ACE2 and TMPRSS2 receptors . The substances that form a barrier matrix, in order to carry out their action, must preferably have a bioadhesive activity and can be helped by pH-adjusting agents or by common antiseptic substances . Substances that modulate the immune system and stimulate the topical defences of the oral mucosa and of the respiratory system, for example Vitamin B12 and folic acid, can also contribute to the ef fectivenes s of the formulation, hindering the viral replication .
The formulation of the invention, as so conceived,
( 1 ) causes the "entrapment" of the viral particles in the barrier matrix;
( 2 ) inhibits the virus bonding both with the ACE2 receptor and with the TMPRSS2 receptor, thus preventing or at least delaying the entrance of the Coronavirus into the cells of the nasal or throat epithelium; and
(3) forms a bioadhesive film on the mucosa of the nose and throat epithelium.
Precisely the fact that for a prolonged period of time the viruses are compelled to remain in an extra-cellular position, where they naturally cannot survive, fosters their rapid inactivation, which inactivation can be further boosted by the presence in the formulation of common antiseptic substances, even when they are used in so low concentrations as not to exhibit any adverse effects. To this end, in the formulation two quaternary ammonium salts can preferably be used, such as benzalkonium chloride for the nasal solution and cetylpyridinium chloride for the oral solution.
When the formulation of the invention is then further supplemented by the additional components described above, it can also
(4) keep a low pH, i.e., an acid pH, which hinders the short-term survival of the virus before it penetrates the cells; and
(5) hinder viral replication through the stimulus of the topical defences of the respiratory and oral mucosa with an action that helps or replaces the barrier action.
Preferably, the formulation contains a concentration of between 0.02% and 2% of substances that form the barrier matrix, between 0.03% and 3% of flavonoid substances provided with a specific antiviral action on the ACE2 and TMPRSS2 receptors, between 0.1% and 1% of pH-adjusting agents, and between 0.01% and 0.5% of antiseptic substances.
More preferably, the formulation contains between 0.1% and 1% of the substances that form the barrier matrix, between 0.05% and 1% of the flavonoid substances provided with a specific antiviral action on the ACE 2 and TMPRSS2 receptors, between 0.1% and 0.3% of pH-adjusting agents and between 0.02% and 0.5% of antiseptic substances. The substances that form the barrier matrix are chosen from hyaluronic acid, hydroxyethyl cellulose and hydroxypropyl methylcellulose . The substances which inhibit the bond of the spike protein with the ACE2 receptor and with the TPRMSS2 receptor are selected from flavonoids such as quercetin, hesperidin, baicalin, scutellarin, luteolin, myricetin and epigallocatechin .
The barrier matrix is kept at a low pH, i . e . , an acid pH, thanks to the addition of pH-adjusting agents such as citric acid, sodium citrate, lactic acid, and ascorbic acid . The pH is kept between 3 and 5 , preferably between 3 . 5 and 4 . 5 .
The barrier matrix can preferably include humectant agent s such as glycerol, propylene glycol and polyethylene glycols . Glycerol is the humectant agent of choice which prevent s the dryness of the biofilm when it contacts the nasal and throat mucosa .
The saline solution in which the components of the formulation are dis solved can be a traditional isotonic solution ( 0 . 9% NaCl ) or, preferably, a hypertonic solution ( from 1 . 2% to 3% of NaCl ) . In this last case , in fact , the use of the formulation causes a depolarisation of the cellular membranes of the nasal mucosa, contributing to render the cells more impervious to penetration by the Coronavirus .
Lastly, to obtain a slowdown of the viral replication, thanks to the stimulus of the topical defences of the respiratory mucosa, the formulation can be as sociated with substances such as Vitamin B12 ( 0 . 0025%-0 . 50% ) and folic acid ( 0 . 00125%-0 . 25% ) .
The spray formulation of this invention preferably includes hyaluronic acid, hydroxyethyl cellulose and hydroxypropyl methylcellulose as substances forming the barrier matrix, quercetin as inhibitor of bonding formation between virus and ACES2 and TPRMSS2 receptors , citric acid and sodium citrate as pH-adjusting agents , glycerol as humectant agent and benzalkonium chloride and cetylpyridinium chloride as antiseptic substances , sodium chloride that can be added as an osmotic agent, with the function of contributing to greater stability of the epithelial membranes and Vitamin B12 and folic acid as stimulants of the topical defences of the oral mucosa and of the upper respiratory tract.
Formulation Examples
Example 1
Nasal spray - Zyvex Hypertonic Nasal Spray
Hyaluronic acid 0.1%
Sodium chloride 1.5%
Benzalkonium chloride 0.02%
Sodium citrate 0.1%
Citric acid 0.1%
Glycerol 0.5%
Quercetin 0.1%
Xylitol 10.0%
Water as required to reach 100.0%
Example 2
Nasal spray - Zyvex Hypertonic Nasal Spray
The substance that forms the barrier matrix in Example 1 has been modified by using an equivalent quantity of hydroxypropyl methylcellulose instead of hyaluronic acid.
Hydroxypropyl methylcellulose 0.3%
Sodium chloride 1.5%
Benzalkonium chloride 0.02%
Sodium citrate 0.1%
Citric acid 0.1%
Glycerol 0.5%
Quercetin 0.1%
Xylitol 10.0%
Water as required to reach 100.0% Example 3
Oral spray - Zyvex Advanced Oral Spray
Hydroxyethyl cellulose 0.2%
Cetylpyridinium chloride 0.05%
Xylitol 5.0%
Glycerol 5.0%
Quercetin 0.05%
Saccharose 5.0%
Eucalyptol 0.1%
Colouring 0.0025%
Aroma 0.3975%
Water as required to reach 100.0%
Example 4
Nasal and oral spray - Zyvex Hypertonic Nasal Spray and Zyvex
Advanced Oral Spray
Hydroxypropyl methylcellulose 0.3%
Sodium chloride 1.5%
Cetylpyridinium chloride 0.05%
Sodium citrate 0.1%
Glycerol 0.5%
Quercetin 0.1%
Xylitol 10.0%
Water as required to reach 100.0%
Example 5
Nasal and oral spray - Zyvex Hypertonic Nasal Spray and Zyvex
Advanced Oral Spray, with stimulation of the immune defences
Hydroxypropyl methylcellulose 0.3%
Sodium chloride 1.5%
Cetylpyridinium chloride 0.05%
Sodium citrate 0.1%
Citric acid 0.1%
Glycerol 0.5%
Quercetin 0.05%
Xylitol 10.0%
Vitamin B12 0.0025%
Folic acid 0.00125%
Water as required to reach 100.0%
The results in inhibiting the viral growth on in vitro tests in relation to the SARS-CoV-2 virus on a culture medium of Vero E6 cells, according to the standard protocol EN14476 : 2013/FprAl : 2015, has shown a marked and rapid inhibition of the viral growth after only 30 seconds from application of the formulation of this invention, according to any one of the Examples shown above, on the cell culture defined above, as shown in Table 1 below.
Table 1
Virucidal activity of the Zyvex Advanced Oral Spray and of the Zyvex Hypertonic Nasal Spray against SARS-CoV-2
Figure imgf000012_0001
1 Logic = 95% of inhibition
2 Logic = 99% of inhibition
3 Logic = 99 . 9% of inhibition
4 Logic = 99 . 99% of inhibition
An inhibition action of the viral growth at 99 . 99% after only 30 seconds is significant of a marked virucidal activity . This marked antiviral activity confirms the usefulnes s of this invention . When the individual components of the formulation that could have an antiviral activity against the SAARS-CoV-2 virus are considered, it can be observed that the Xylitol used in the same in vitro test described above showed a reduction of 2 . 5 logic only after 25 minutes (Cannon ML, Westover JB, Bleher R, Sanchez-Gonzalez MA, Ferrer G . 2020 ) 12 . The same substances which form the barrier matrix in this invention have been previously tested in a similar in vitro test and can inhibit under the same conditions of pH the growth of the Coronavirus only after 3-4 hours (Brambilla L, 2015 ) 13 . Quercetin has shown that it can block the entry of the SARS-Coronavirus virus into the Vero E6 cells by 50% (Yi L, Zi K, Yuan K, Qu X, Chen J, Wang G et al . 2004 ) 14 . In this invention, the mechanism of action determined by the presence of the barrier layer allows obtaining an effectiveness of 99 . 99% in inhibiting the penetration of the virus in the Vero E 6 cells .
Lastly, as far as the antiseptic substances made up of quaternary ammonium salts (benzalkonium chloride and cetylpyridinium chloride ) are concerned, they have been able to reduce the growth of Coronavirus by a 3 Logic after at least 1 minute but only at a concentration of 0 . 05% for the benzalkonium chloride (Kampf G, Todt D, Pfaender S and Steinmann E , 2020 ) 10 and of 0 . 07 % for cetylpyridinium chloride (Green A, Roberts G, Tobery T, Vincent C, Barili M, Jones C 2020 ) 11 . The formulation described here , thanks to the special composition of the acid barrier matrix allows reducing the concentration of benzalkonium chloride to 0 . 02% and of cetylpyridinium chloride to 0 . 05% but obtaining a particularly high reduction of the proliferation of the SARS-CoV-2 virus of 4 logic after only 30 seconds .
The formulation of the invention therefore allows to attain a clear improvement with respect to the action of the individual component s thereof , in terms of a greater activity ( reduction of 4 logic of the viral load at lower and therefore safer concentrations compared to those previously known) and much faster times of action ( 30 seconds ) compared to those known for the individual components .
It is intended however that the invention is not to be considered limited to the special formulations illustrated above , which are only exemplary embodiment s thereof , but that dif ferent variant s are pos sible, all within reach of the skilled man in the art , without departing from the scope of protection of the invention, which is solely defined by the following claims . BIBLIOGRAPHY
1 - Weiss S.R., Navas Martin S. Coronavirus Pathogenesis and the Emerging Pathogen Severe Acute Respiratory Syndrome Coronavirus - Microbiology and Molecular Biology Review, 2005, Vol 69 (4) , p 635-664.
2 - Wang Q, Zhang Y, Wu L, Niu S, Song C, Zhang Z, Lu G, Qiao C, Hu Y , Yuen KY, Wang Q, Zhou H, Yan and Qi J. Structural and Functional Basis of SARS-CoV-2 Entry by Using Human ACE2. Cell, 2020, 181, 894-904
3 - Hoffmann M., Kleine-Weber H., Schroeder S., Kruger N., Herrier T., Erichsen S., Schiergensen T.S., Herrier Wu N.H., Nitsche A., Muller M.A., Drosten C., and Pohlamnn S. - SARS-CoV- 2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor. - Cell, 2020, 181, 276- 280.
4 - Kuhnemund M., Lako M. , Lee H., Leroy S., Linnarson S., Lundberg J., Meyer K. , Miao Z., Misharin A.V., Nawijn M., Nikolic M.C., Noseda M. , Ordova-Monatnes, Oudit G.Y., Pe'er D., Powell J., Quake S., Ra jagopal J., Rao Tata P., Rawlins E. et al. - Nature medicine, 2020, 26, 681-687.
5 - Desforges M. , Le Coupanec A., Dubeau P., Bourgouin A., La oie L., Dube A., and Talbot P.J. - Human Coronavirus and Other Respiratory Viruses: Understimated Opportunistic Pathogens of Central Nervous System. - Viruses 2019, 12 (14) : 2-28.
6 - Brann D.H., Tsukahara T., Weinreb C., Lipovsek M. , Van der Berge K., Gong B., Chance R. , Macaulay I.C., Chou H.J., Fletcher R., Das D., Street K. , Roux de Bezieux H., Choi Y.G., Risso D., Dudoit S., Purdom E., Mill J.S., Hachem R.A., Matsunami H., Logan D.W., Goldstein B.J., Grubb M.S., Ngai J. and Datta S.R. - Non-neuronal expression of SARS-CoV-2 entry genes in the olfactory system suggest mechanism underlying Covid-19 associated anosmia - bioRvix,
Doi. org/10.1101/2020.03.25.009084.
7 - Netland J., Meyerholz D.K., Morre S., Cassell M. , and Perlman S. - Severe Acute Syndrome Coronavirus Infection Causes Neuronal Death in the Absence of Encephalitis in Mice Transgenic for Human ACE2 - Journal of Virology. 2008, 82 (15) , 7264-7275.
8 - Chen M., Shen W. , Rowan N.R., Kulaga H., Hillel A., Ramanathan Jr M., and Lane A.P. - Elevated ACE2 expression in the olfactory neuroepithelium: implication for ansomia and upper respiratory. SARS-CoV-2 entry and replication - bioRxiv,
Doi. org/10.1101/2020.05.08.084996. 9 - Kanno S, Hirano S, Kato H, Fukuta M, Mukai T and Aoki Y., 2020 Benzalkonium chloride and cetylpiridinium chloride induce a poptosis in human lung epithelial cells and alter surface activity of pulmonary surfactant monolayesr. Chem . Biol. Interact .317 : 108962.
10 “ Kampf G, Todt D, Pfander S , Steinmann E. 2020. Persistence of coronavirus on inanimate surfaces and their inactivation with biocidal agents. Journal of Hospital Infection , 104:246-251.
11 - Green A. Roberts G, Tobery T, Vincent C, Barili M, Jones C. 2020. In vitro assessment of the virucidal activity of four mouthwashes containing Cetylpyridinium Chloride, ethanol, zinc and a mix of enzyme and proteins against a human coronavirus.
12 - Cannon ML, Westover JB, Bleher R, Sanchez-Gonzalez MA, and Ferrer G. (2020)- In Vitro Analysis of the Anti-Viral Potential of nasal Spray Constituents Against SARS-CoV-2
13 - Brambilla L, Evaluation of the Capability of the Product camuvir Barriera to Inhibit the Virus Infectivity on the Host Cells. Final report Eurofins S-2014.03249 SAMi .
14 - Yi L, Li Z, Yuan K, Qu X , Chen J, Wang G et al . Small molecules blocking the entry of severe acute respiratory syndrome coronavirus into host cells. J. Virol (2004) , 78:11334, doi: 10: 1128/ JVL .78.20.11334-11339

Claims

1 ) Spray formulation for nasal/oral use, for the protection against Coronavirus infections wherein the ACE2 receptor and/or the TMPRSS2 receptor is used by Coronavirus for penetrating the cells of the nasal and throat mucosa, characterised in that it consist s of a saline solution comprising, as active substances , at least one substance forming a barrier matrix and at least one substance provided with a specific antiviral action on the ACE2 and TMPRSS2 receptors .
2 ) Spray formulation for nasal/oral use as in claim 1 , wherein the aforesaid substance forming a barrier matrix is selected from hyaluronic acid, hydroxyethyl cellulose or hydroxypropyl methylcellulose or their combinations .
3 ) Spray formulation for nasal/oral use as in claim 1 , wherein the aforesaid substance provided with a specific antiviral action on the ACE2 and TMPRSS2 receptors is selected from flavonoids such as quercetin, hesperidin, baicalin, scutellarin, luteolin, myricetin and epigallocatechin or their combinations .
4 ) Spray formulation for nasal/oral use as in claim 1 , wherein the concentration of said substance forming a barrier matrix is comprised between 0 . 02% and 2% and the concentration of said substance provided with a specific antiviral action on the ACE2 and TMPRSS2 receptors is comprised between 0 . 03% and 3% .
5 ) Spray formulation for nasal/oral use as in claim 1 , wherein the concentration of said substance forming a barrier matrix is comprised between 0 . 1% and 1% and the concentration of said substance provided with a specific antiviral action on the ACE2 and TMPRSS2 receptors is comprised between 0 . 05% and 1% .
6 ) Spray formulation for nasal/oral use as in any one of the previous claims 1-5 , also comprising between 0 . 1% and 1% and preferably between 0 . 1% and 0 . 3% of pH-adjusting agent s , to adjust the value of the pH of the formulation in an acid field between 3 and 5 , preferably between 3 . 5 and 4 . 5 . 7) Spray formulation for nasal/oral use as in any one of the previous claims 1-5, also comprising between 0.01% and 0.5% and preferably between 0.02% and 0.5% of antiseptic substances.
8) Spray formulation for nasal/oral use as in claim 7, wherein said antiseptic substance is selected from benzalkonium chloride for the nasal solution and cetylpyridinium chloride for the oral solution.
9) Spray formulation for nasal/oral use as in any one of the previous claims 1-5, also comprising between 0.0025% and 0.50% of Vitamin B12 and between 0.00125% and 0.25% of folic acid.
10) Spray formulation for nasal/oral use as in any one of the previous claims, wherein said saline solution in an isotonic solution (NaCl 0.9%) .
11) Spray formulation for nasal/oral use as in any one of the previous claims, wherein said saline solution is a hypertonic solution (NaCl 1.2% to 3%) .
12) Use of a spray formulation for nasal/oral use as in any one of the previous claims to prevent spreading of Coronavirus in the respiratory tract and in the central nervous system.
PCT/IB2021/059753 2020-10-23 2021-10-22 Spray formulation for nasal/oral use with activity against respiratory coronaviruses and in particular the sars-cov-2 virus WO2022084931A1 (en)

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