Classifications

• • 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
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
  • A61K38/46—Hydrolases (3)
  • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
  • A61K38/482—Serine endopeptidases (3.4.21)
  • A61K38/4826—Trypsin (3.4.21.4) Chymotrypsin (3.4.21.1)
• • 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
  • 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
  • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
• • 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
  • 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
• • 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
  • A61K9/08—Solutions
• • 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
  • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
  • C12Y304/21—Serine endopeptidases (3.4.21)
  • C12Y304/21004—Trypsin (3.4.21.4)

Definitions

• the present invention relates to a stable peptidase formulation for the treatment and prevention of microbial infections in the upper respiratory tract.
• Upper respiratory tract infections are caused by an acute infection, which involves the upper respiratory tract, including the nose, sinuses, pharynx, or larynx. Upper respiratory tract infection is one of the most common infectious illnesses in the general population, resulting in common cold and influenza, which are major contributors to unwanted absence from work and school. The vast majority of upper respiratory infections are caused by viruses. Over 200 different viruses have been isolated in patients with upper respiratory tract infections such as rhinoviruses, influenza viruses, Respiratory Syncytial Viruses (RSV), coronaviruses, parainfluenza viruses, adenoviruses, enteroviruses, metapneumo-viruses and unknown viruses (Heikkinen et al, 2003).
• RSV Respiratory Syncytial Viruses
• coronaviruses coronaviruses
• parainfluenza viruses adenoviruses
• enteroviruses enteroviruses
• metapneumo-viruses metapneumo-virus
• nasal swabs have yielded higher viral loads than throat swab implicating the nasal epithelium as a portal for initial infection and transmission (Sungnak et al, 2020). Given that nasal carriage is likely to be a key feature of transmission of SARS-CoV-2, drugs/vaccines administered intranasally could be highly effective in limiting spread.
• Viral infections varies from being relatively mild short-term infections, such as common cold or more severe viral infections such as influenza which spreads worldwide in seasonal epidemics, to aggressive life-threatening infections. In addition to the seasonal epidemics there have been several pandemics, the most devastating being the Spanish Flu of 1918, which caused 20-40 million deaths globally. Nearly half of the influenza-related deaths during the pandemic occurred among young, healthy adults for reasons that remain obscure but are suspected to be related to secondary bacterial infections (Mallia and Johnston, 2007). Bacterial superinfection in viral respiratory disease is a clinically well documented and physical damage to respiratory cells as a result of viral infection may lead to opportunistic adherence of bacteria. Given the magnitude of the cold flu and influenza health problems, in addition to new emerging viral infections that are difficult to treat such as MERS (Middle East respiratory syndrome) and COVID-19, the need for novel therapeutics against viral infections is evident.
• MERS Middle East respiratory syndrome
• Vaccination is currently the primary focus to prevent spread of influenza virus but due to mutations new vaccines must be developed each year. Development of antiviral drugs against common cold are difficult because of the diversity of viruses responsible for this disease. Such therapeutics should have broad-spectrum activity in order to be suited as an antiviral measure against diverse range of viruses.
• Other antiviral chemotherapies e.g., ICAM-1 blocker, capsid binding agents, and protease inhibitors have similarly failed to show a promising risk to benefit ratio (Allan et al, 2014).
• anti-viral compositions with broad spectrum activity (i.e. effective against various enveloped and nonenveloped viruses, RNA and DNA based genome, and independent on different viral proteins (mutated or not)), that reduce and/or prevents virus from binding to cells.
• Such compositions are ideally based on non-toxic compounds.
• anti-viral compositions should also be effective against secondary infections in the upper respiratory tract caused by bacteria and fungi, and target microbial/bacterial adhesion that do not induce resistance and furthermore has no or low impact on patients’ natural microbiota.
• Said formulation is a stable peptidase formulation for prevention and treatment of viral infections in the upper respiratory tract.
• the formulation can be administrated to the upper respiratory tract by nasal administration, thereby reaching the nasopharynx as well as by oral administration to reach the pharynx.
• This disclosure describes a product that functions as a complement to vaccines and systemic antivirals. It forms a thin protective broad-spectrum antiviral gel in the nasal cavity and/or throat (the pharynx) to prevent uptake of virus locally in upper respiratory tract or reduce propagation of viral infections. Virus is trapped and bound to the gel and then deactivated by a broad spectrum peptidase.
• the formulation constitutes an effective barrier, and exhibits low-to-no stinging when applied in the nasal cavity.
• Said formulation is capable of reducing SARS-CoV-2 virus by >99.9% in vitro.
• the present invention is also active against bacteria and fungi by targeting the same virulence factor as for viruses namely the binding interaction between microbials and cells. Said formulation does not induce microbial resistance and is therefore a good alternative to antibiotics. Thus, the formulation described herein carry high potential for clinical use and provides a solution to the challenge of outphasing the use of antibiotics.
• the disclosure relates to a stable composition
• a stable composition comprising: i. peptidase; ii. sugar alcohol;
• the disclosure relates to a composition
• a composition comprising: i. peptidase; ii. glycerol; iii. xylitol; iv. hyaluronic acid; and v. buffer.
• the disclosure relates to said composition for use in medicine.
• the disclosure relates to a composition
• a composition comprising or consisting essentially of: i. trypsin; ii. glycerol; iii. xylitol; iv. hyaluronic acid; v. CaCh; and vi. buffer.
• a composition as disclosed herein for use in the treatment of conditions selected from the group consisting of microbial infections, dermatological conditions, and oral conditions, in and/or on a mammal.
• compositions as disclosed herein for use in the treatment and/or prevention of upper respiratory tract infections, said composition being formulated for nasal and/or oral administration.
• the disclosure provides for a method for prevention and/or reduction of microbial infections, wherein the method comprises administration of the composition disclosed herein.
• the disclosure relates to a method for increasing microbial diversity, wherein the method comprises administration of the composition disclosed herein.
• the disclosure relates to a method of manufacturing the composition disclosed herein, said method comprising mixing: i. glycerol; ii. xylitol; iii. buffer; iv. hyaluronic acid; and v. peptidase.
• Carrageenan concentration of >0.2 mg/ml impacts activity.
• Figure 3 Enzyme stability in various formulations. Addition of >10% xylitol to 25-30 % glycerol was required in order to achieve high stability.
• CX is a commercially available, trypsin-containing mouth spray.
• microbial infection refers to an infection with a microorganism, such as a fungus, bacteria or virus.
• viral infection refers to any stage of a viral infection, including incubation phase, latent or dormant phase, acute phase, and development and maintenance of immunity towards a virus.
• upper respiratory tract the mouth, nose, sinus, middle ear, throat, pharynx, larynx, and trachea are included.
• preventing viral infection and “treating viral infection” means to inhibit the replication of the particular virus, to inhibit viral transmission, or to prevent the virus from establishing itself in its host, and to ameliorate or alleviate the symptoms of the disease caused by the viral infection, e.g. the sore throat, blocked and/or runny nose, cough and/or elevated temperature.
• the treatment is considered therapeutic if there is a reduction in viral load, and/or a decrease in morbidity and/or mortality.
• peptidase as used herein includes proteases, proteinases and proteolytic enzymes, functional homologues or functionalised derivatives thereof, capable of cleaving a polypeptide of any length, short or long, at a peptide bond, for example by hydrolysing the peptide bond.
• the substrate of a peptidase is thus a polypeptide.
• sugar alcohol refers to a sugar derivative, monosaccharides, disaccharides, oligosaccharides, whose carbonyl group (aldehyde or ketone, reducing sugar) has been reduced to a primary or secondary hydroxyl group.
• Non-limiting examples of sugar alcohols include: ethylenglycol (2-carbon), glycerol (3-carbon), erythritol (4-carbon), threitol (4-carbon), arabitol (5-carbon), xylitol (5-carbon), ribitol (5- carbon), mannitol (6-carbon), sorbitol (6-carbon), dulcitol (6-carbon), iditol (6-carbon), isomalt (12-carbon), maltitol (12-carbon), lactitol (12-carbon), polyglycitol and other relevant polyol derivatives.
• polyol refers to a compound comprising multiple alcohol groups, such as propylene glycol. As used herein, polyol does not necessarily have one hydroxy group on each carbon atom.
• buffer refers to an aqueous solution containing an acid-base mixture with the purpose of stabilizing pH.
• hyaluronic acid refers to a polymer comprising repeat disaccharide subunits of hyaluronan.
• the term also includes a polymer in which the disaccharide subunits may be derivatized at one or more positions of the D-glucuronic acid and/or the D-N-acetylglucosamine repeat subunit.
• hyaluronic acid encompasses hyaluronic acid, cross-linked forms, derivatized forms of hyaluronic acid, and pharmacologically acceptable salts thereof.
• pharmaceutically acceptable generally refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues, organs, and/or bodily fluids of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit/risk ratio.
• flavouring agent refers to any substance, natural or unnatural, that is capable of imparting a detectable flavour impact, especially at a concentration below 4 wt.%, more preferably below 2 wt.%.
• carrageenan refers to a family of linear sulfated polysaccharides which are usually extracted from red seaweeds. All carrageenans are polysaccharides made up of repeating galactose units and 3,6 anhydrogalactose, both sulfated and non-sulfated. Various types of carrageenans exist. Among them the main types are kappa, lambda and iota. Carrageenan are typically used for viscosity control or for achieving bioadhesion.
• bioadhesion refers to the ability of certain materials such as polymers, to adhere to biological or body tissue.
• mucoadhesion refers to the ability of certain materials to adhere, through the formation of chemical and/or physical bonds, to mucosa or mucosal tissue (as e.g. the nasal mucosa).
• viscosity refers to a fluid's resistance to flow.
• the unit of viscosity is Pascal-second (Pa s or Pas).
• oral refers to the introduction of a pharmaceutical composition into a subject by way of the oral cavity.
• the pharmaceutical composition is a liquid composition.
• nasal and “by nasal administration” as used herein refer to the introduction of a pharmaceutical composition into a subject by way of the nasal cavity.
• the pharmaceutical composition is a liquid composition.
• the term “tolerable” as used herein refers to the irritation, stinging sensation and secretory stimulation observed e.g. when the composition is used for nasal administration. Tolerability is rated from unacceptable to comfortable herein as assessed by the test subject.
• divalent cation refers to a positively charged ion of any metal from the periodic table having a valence of 2. Where an amount of divalent cation, in an aqueous solution, for example, is given in a percentage concentration, such as % w/w the concentration of divalent cation is based on the weight of the cation in relation to the total weight of the composition of which the cation forms part.
• w/w means a “weight/weight”.
• % w/w is synonymous to “wt%” or “weight-percent”.
• 10 g of a composition comprising 50 % w/w of A comprises 5 g A.
• phrases such as “a composition comprising X to Y % of A” are taken to mean a composition comprising A in the range of X to Y %, both thresholds included. That is, the composition does not comprise less than X % of A, and the composition does not comprise more than Y % of A, provided X is smaller than Y.
• the respiratory tract refers to the pathway that carries air to the lungs, and can be divided into upper and lower parts.
• the upper respiratory tract includes the nose, sinuses, throat (pharynx) and voice box (larynx).
• Mechanical barriers play a significant role in anti-viral defence of the upper respiratory tract.
• the tract is lined with a mucociliary blanket consisting of ciliated cells, mucous-secreting goblet cells, and sub-epithelial mucous-secreting glands. Foreign particles deposited in the nasal cavity or upper respiratory tract are trapped in mucus, carried to the back of the throat, and swallowed.
• Microbial adhesion where microbial surface proteins recognize cell surface protein, is the most common mode of establishing an infection in the host (Meena et al, 2020).
• This invention seeks to strengthen the natural mechanical barrier towards microbial infection. Blocking or hindering migration of virus to reach specific host cell receptors is an attractive way to provide a safe and efficacious protection against virus.
• the mucosa of the nasal cavity is more sensitive to hyperosmotic solutions than the oropharynx.
• Local irritation and nasal burning/pain has been reported after nasal administration of e.g. Nasalide® nasal spray, where up to 45% of patients noted nasal burning (Trangsrud et al, 2002).
• the present invention takes both the rapid clearance and the sensitivity of the nasal cavity into account.
• peptidase compositions for prevention of microbial infections are employed.
• the term "peptidase” as used herein includes proteases, proteinases and proteolytic enzymes and functional homologues or functionalized derivatives thereof, capable of catalyzing proteolysis in vivo, in the mammalian (e.g. human) body, thereby cleaving polypeptides of any length, short or long, at a peptide bond, for example by hydrolyzing the peptide bond.
• the substrate can be a naturally occurring polypeptide or a synthetic polypeptide.
• the peptidase may be either specific, being capable of hydrolysing only select peptide bonds, or may be non-specific, being capable of hydrolysing many different peptide bonds.
• Viruses and bacteria may be present in the nasopharynx without causing any respiratory symptoms.
• the upper respiratory tract hosts a vast range of commensals and potential pathogenic bacteria, which form a complex microbial community. This community is assumed to be constantly subject to synergistic and competitive interspecies interactions. Disturbances in the equilibrium, for instance due to the acquisition of new bacteria or viruses, may lead to overgrowth and invasion.
• Peptidases are very suitable for reduction or inhibition of unwanted, microbial adhesion such as viral adhesion, and furthermore peptidases are non-toxic, well-tolerated, and do not induce resistance.
• Compositions comprising peptidases can be used as therapeutic agents, as an alternative to antibiotics in treatments against microbial infections without interfering with the desired, healthy microbiota.
• the present disclosure relates to a stable peptidase composition for prevention and/or treatment of microbial infections.
• Said composition provides a means to strengthen the intrinsic barrier to increase protection against viruses.
• the disclosed composition comprises a peptidase, which can hydrolyse the microbial surface proteins thereby preventing microbial adhesion and the establishment of microbial infections.
• the composition comprises a mixture of sugar alcohols or polyols, capable of providing a stable composition, tolerable for nasal administration.
• composition of the present disclosure is more efficient against viruses and has improved shelf-life and activity compared to other known peptidase formulations (e.g. commercially available peptidase formulation). Furthermore the composition is suitable for nasal administration to humans and other mammals (as concluded from disclosed Example 4), which is a significant improvement, compared to other known peptidase formulations, which only are tolerable for oral administration.
• compositions comprising i. 0.005 to 1.0 % w/w peptidase; ii. 1.0 to 70 % w/w sugar alcohol.
• the composition comprises: i. 0.005 to 1.0 % w/w peptidase; ii. 20 to 70 % w/w glycerol; and iii. 1.0 to 65 % w/w xylitol.
• the composition comprises: i. 0.005 to 1.0 % w/w peptidase; ii. 20 to 70 % w/w glycerol; and iii. 1.0 to 65 % w/w xylitol, wherein the combination of components of said composition does not exceed 100%.
• the composition comprises 0.005 to 1.0 % w/w peptidase, such as 0.01 to 0.075 % w/w peptidase, such as 0.015 to 0.055 % w/w peptidase.
• said peptidase is selected from the group consisting of serine proteases (such as trypsins and chymotrypsins), threonine proteases, cysteine proteases, aspartate proteases, glutamic acid proteases and metalloproteases, functional homologues thereof, and functionalised derivatives thereof.
• said peptidase is a single type of peptidase, or a mixture of different types of peptidases.
• the combination of components of said composition does not exceed 100%.
• Peptidase activity can be determined by measuring the product formed during a defined time per given conditions per amount of peptidase.
• the specific activity of a peptidase is the enzymatic activity per protein concentration.
• the total peptidase activity will be determined by specific activity and purity.
• a desired activity can be achieved by using a relatively impure peptidase at a relatively higher concentration, or it can be achieved using a relatively pure peptidase at a relatively lower concentration.
• the optimal concentration of peptidase to be used can be assessed by measuring the enzyme activity. Trypsin
• Said peptidase may be a protease such as trypsin, which predominantly cleaves peptide chains at the carboxyl side of the amino acids lysine and arginine.
• the peptidase is a protease.
• said protease is trypsin.
• the protease is a functional homologue or a functionalized derivative of trypsin having proteolytic activity.
• the composition comprises 0.005 to 1.0 % w/w protease, such as 0.005 to 1.0 % w/w protease. In a further embodiment of the disclosure, the composition comprises 0.015 to 0.055 % w/w protease. In a specific embodiment of the disclosure, the composition comprises essentially 0.015 to 0.055 % w/w protease, such as 0.015 to 0.055 % w/w protease.
• the composition comprises 0.005 to 1.0 % w/w trypsin, a functional homologue or a functionalized derivative thereof, such as 0.005 to 1.0 % w/w trypsin, a functional homologue or a functionalized derivative thereof.
• the composition comprises 0.015 to 0.055 % w/w trypsin, a functional homologue or a functionalized derivative thereof.
• the composition comprises essentially 0.015 to 0.055 % w/w trypsin, a functional homologue or a functionalized derivative thereof, such as 0.015 to 0.055 % w/w.
• composition of the present disclosure is a stable peptidase formulation.
• the peptidase may be stabilised by a salt.
• composition further comprises a salt. In another embodiment, said composition comprises a divalent cation.
• divalent cation refers to a positively charged ion of any metal from the periodic table having a valence of 2, such as calcium.
• the invention discloses a composition comprising trypsin, said composition further comprising a salt. In another embodiment, the invention discloses a composition comprising trypsin, said composition further comprising a salt with a divalent cation.
• the invention discloses a composition comprising a peptidase, said composition further comprising a salt with a divalent cation, such as 0.003 to 0.2 % w/w salt comprising a divalent cation, such as 0.003 to 0.15 % w/w, such as 0.003 to 0.1 % w/w salt comprising a divalent cation.
• a salt with a divalent cation such as 0.003 to 0.2 % w/w salt comprising a divalent cation, such as 0.003 to 0.15 % w/w, such as 0.003 to 0.1 % w/w salt comprising a divalent cation.
• the composition comprising trypsin, a functional homologue, or a functionalized derivative thereof further comprises 0.003 to 0.2 % w/w salt comprising a divalent cation, such as 0.003 to 0.15 % w/w, such as 0.003 to 0.1 % w/w salt comprising a divalent cation.
• the divalent cation is calcium (Ca 2+ ).
• the salt comprising the divalent cation is a pharmaceutically acceptable salt of calcium, including hydrates.
• the pharmaceutically acceptable salt of calcium is CaCh.
• the salt comprising a divalent cation is a pharmaceutically acceptable inorganic salt of calcium, including hydrates thereof, such as calcium chloride dihydrate.
• the salt comprising a divalent cation is a pharmaceutically acceptable organic salt of calcium, such as calcium acetate or calcium citrate.
• the composition comprises 0.003 to 0.2 % w/w calcium, such as 0.003 to 0.15 % w/w, such as 0.003 to 0.1 %.
• the composition further comprises calcium ions, such as 0.003 to 0.2 % w/w calcium ions, such as 0.003 to 0.15 % w/w calcium ions, such as 0.003 to 0.1 % w/w calcium ions.
• Calcium ions means Ca 2+ .
• the composition comprises 0.002 to 0.2 % w/w calcium chloride, such as 0.005 to 0.02 % w/w calcium chloride, such as 0.010 to 0.015 % w/w calcium chloride.
• the composition comprises 0.002 to 0.2 % w/w calcium chloride dihydrate, such as 0.005 to 0.02 % w/w calcium chloride dihydrate, such as 0.010 to 0.015 % w/w calcium chloride dihydrate.
• the composition comprises: i. 0.005 to 1.0 % w/w trypsin; ii. 1.0 to 70 % w/w sugar alcohol; and iii. 0.010 to 0.015 % w/w calcium dichloride dihydrate.
• composition of the present disclosure is a stable peptidase formulation. Deactivation of the peptidase improves its stability in the composition. One way to deactivate the peptidase is by reducing the water activity of the composition. Water activity of an aqueous solution can be reduced by presence of ions and/or other molecules.
• the present invention comprises salts, sugar alcohols, and/or polyols, which in part act to reduce the water activity in the composition. Reducing the water activity in turn deactivates the peptidase. Furthermore it is beneficial for the purpose of the present disclosure that said composition is hyperosmotic. Hyperosmotic compositions generally stimulate nasal secretion, which has a rinsing effect of the nasal mucosa cleaning the infected surface of all the contaminants, such as virus particles and free-floating bacteria, without any toxic effect on the cells or the cellular matrix.
• the composition comprises one or more of a diol, a triol, or a polyol, such as glycerol, propylene glycol, or a sugar alcohol.
• the composition comprises 35 to 70 % w/w sugar alcohol, such as 40 to 65 % w/w sugar alcohol, such as 45 to 60 % w/w sugar alcohol.
• the sugar alcohol may be glycerol, erythritol, xylitol, mannitol, arabitol, maltitol, lactitol, isomalt, sorbitol, hydrogenated starch hydrolysates or propylene glycol and any other suitable polyol or mixtures thereof providing a stable solution of the peptidase.
• the oral cavity tolerates high concentrations of glycerol well whereas high concentrations of glycerol has a painful, stinging effect on the thinner, more sensitive nasal mucosa.
• a composition comprising 50% glycerol cannot be sprayed into nasal cavity because it stings severely due to the high hypertonic effect of glycerol. 10-25% glycerol can be accepted at low volumes.
• the enzymes disclosed herein are not stable for long term storage, severely reducing their use in treatment and/or prevention of infections.
• the glycerol concentration in said formulation for nasal use needs to be diluted compared to formulations for oral use, while maintaining a stable composition.
• the disclosed composition comprising a mixture of the sugar alcohols glycerol and xylitol, provides a stable composition, said stable composition being tolerable for nasal administration.
• Xylitol is a sugar alcohol with properties similar to glycerol. Xylitol can have a protein stabilizing effect. Xylitol furthermore has a sweetness intensity equal to sucrose.
• the composition comprises 20 to 70 % w/w glycerol, such as 25 to 65 % w/w glycerol. In one specific embodiment, the composition comprises 27 to 57 % w/w glycerol.
• the composition comprises 1.0 to 65 % w/w xylitol, such as 3.0 to 50 % w/w xylitol, such as 4.5 to 30 % w/w xylitol.
• the composition comprises a combined amount of glycerol and xylitol of at least 40 % w/w, such as at least 45 % w/w, such as at least 50 % w/w.
• the composition of the present invention comprises: i. 0.005 to 1.0 % w/w peptidase; ii. 20 to 70 % w/w glycerol; and iii. 1.0 to 65 % w/w xylitol.
• i. 0.005 to 1.0 % w/w peptidase ii. 20 to 70 % w/w glycerol
• iii. 1.0 to 65 % w/w xylitol for the compliance of the patients taking a nasal spray, the taste and tolerance of the product is of particular importance.
• This invention presents a tolerable formulation with a sweet, pleasant taste.
• buffer is intended to mean an aqueous solution containing an acid-base mixture with the purpose of stabilising pH. pH is of importance for both the stability of the peptidase, the activity of the peptidase, and the tolerability of the composition when used for treatment of microbial infections in the oral and the nasal cavity.
• buffers are Trizma, Bicine, Tricine, MOPS, MOPSO, MOBS, Tris, Hepes, HEPBS, MES, phosphate, carbonate, acetate, citrate, glycolate, lactate, borate, ACES, ADA, tartrate, AMP, AMPD, AMPSO, BES, CABS, cacodylate, CHES, DIPSO, EPPS, ethanolamine, glycine, HEPPSO, imidazole, imidazolelactic acid, PIPES, SSC, SSPE, POPSO, TAPS, TABS, TAPSO and TES.
• the buffer may be a mixture of two or more buffer systems.
• the buffer is any buffer above, including mixtures of two or more buffers.
• the composition comprises a buffer stabilizing the pH of the composition.
• the buffer may be selected from the group consisting of tris(hydroxymethyl)amino methane (Tris), 3-(/V-morpholino)propane sulfonic acid (MOPS), phosphate, or any other suitable buffer, such as pharmaceutically acceptable buffers.
• the composition of the present disclosure comprises 0.01 to 2.4 % w/w buffer, such as 0.07 to 0.3 % w/w buffer, such as 0.09 to 0.2 % w/w buffer.
• the composition comprises a concentration of buffer in the range 1 to 200 mM, such as 5 to 25 mM, such as 8 to 15 mM.
• the pH of the composition is adjusted to be suitable for use in respectively the nasal and the oral cavity.
• the nasal cavity has a pH range of 5.5-6.5.
• the pH of the throat has a range 7.2 to 8.5.
• the composition has a pH in the range 5.5 to 8.5, such as 6.0 to 7.0, such as 6.3 to 6.7.
• the composition has a pH in the range 6.2 to 7.0, such as 6.4 to
• the composition has a pH in the range 7.2 to 8.5, such as
• the pH of solutions can be assessed in a number of ways.
• pH can be assessed using a pH electrode or a pH indicator.
• the composition comprises: i. 0.005 to 1.0 % w/w peptidase; ii. 20 to 70 % w/w glycerol; iii. 1.0 to 65 % w/w xylitol; and iv. 0.01 to 1.2 % w/w buffer.
• the composition further comprises water, such as at the most 50 % w/w water, such as at most 40 % w/w, such as at most 30 % w/w.
• the composition may advantageously comprise ingredients giving mucoadhesive properties to the composition.
• the disclosed composition may comprise polymers to increase the residence time of the composition (e.g. gel).
• the polymer(s), relative amounts, and concentrations are selected to provide a film that is mucoadhesive. Mucoadhesive performance is typically observed for polymers possessing charged groups or nonionic functional groups capable of forming hydrogen bonds. Negatively charged polysaccharides such as hyaluronate salts of hyaluronic acid, and sulfated polysaccharides such as carrageenan are found to be efficient.
• the composition comprises polymers capable of increasing the residence time of the composition in mucosa.
• the composition comprises a polymer selected from the group consisting of hyaluronic acid and hyaluronic acid derivatives. Such function as a means to strengthen the gel, increase mucoadhesion and to reduce irritation potential in the nasal cavity.
• polymers are included to strengthen the composition by forming a gel, to reduce irritation potential in the nasal cavity.
• the composition comprises hyaluronic acid.
• the composition comprises hyaluronic acid, such as 0.0001 to 2.0 % w/w hyaluronic acid, such as 0.001 to 1.5 % w/w, such as 0.01 to 1.0 % w/w, such as 0.02 to 0.5 % w/w hyaluronic acid.
• hyaluronic acid such as 0.0001 to 2.0 % w/w hyaluronic acid, such as 0.001 to 1.5 % w/w, such as 0.01 to 1.0 % w/w, such as 0.02 to 0.5 % w/w hyaluronic acid.
• the composition comprises or consists essentially of: i. 0.005 to 1.0 % w/w peptidase; ii. 20 to 70 % w/w glycerol; iii. 1.0 to 65 % w/w xylitol; iv. 0.0001 to 1.5 % w/w hyaluronic acid; and v. 0.01 to 1.2 % w/w buffer.
• the composition comprises or consists essentially of: i. 0.005 to 1.0 % w/w trypsin; ii. 20 to 70 % w/w glycerol; iii. 1.0 to 65 % w/w xylitol; iv. 0.0001 to 1.5 % w/w hyaluronic acid; v. 0.002 to 0.2 % w/w CaCh; and vi. 0.01 to 1.2 % w/w buffer.
• the composition comprises or consists essentially of: i. 0.015 to 0.055 % w/w trypsin; ii. 27 to 57 % w/w glycerol; iii. 4.5 to 30 % w/w xylitol; iv. 0.0002 to 0.02 % w/w hyaluronic acid; v. 0.010 to 0.015 % w/w CaCh; and vi. 0.1 to 0.14 % w/w T ris buffer.
• the pH of said composition is between 5.5 to 8.5, such as 6.0 to 7.0, such as 6.3 to 6.7.
• the pH of said composition is between 6.2 to 7.0, such as 6.4 to 6.6.
• the pH of said composition is between 7.2 to 8.5, such as 7.4 to 7.6.
• the composition comprises water. In a specific embodiment of the disclosure, at least part of the water of the composition originates from the buffer.
• the presence of free water can activate and/or destabilize the peptidase.
• Water in the composition may be deactivated by other components, such as for instance glycerol and other alcohols describes herein.
• the composition comprises at most 50 % w/w water, such as at most 40 % w/w, such as at most 30 % w/w water.
• it is important that the water activity in the composition is low.
• the composition is the form of a spray, a lozenge, a pastille, a chewing gum, a gel, or a liquid.
• composition comprises carrageenan.
• Carrageenan is typically used for viscosity control or for achieving bioadhesion.
• the term “carrageenan” refers to a family of linear sulfated polysaccharides which are usually extracted from red seaweeds. Carrageenans are polysaccharides made up of repeating galactose units and 3,6 anhydrogalactose, both sulfated and non-sulfated.
• the composition comprises no more than 1 % w/w carrageenan, such as no more than 0.5 % w/w carrageenan, such as no more than 0.1 % w/w carrageenan.
• the smell and taste of the product is of particular importance.
• This invention presents a tolerable formulation with a sweet pleasant taste in part due to the presence of xylitol.
• the smell and taste may be further improved, if the composition further comprises a flavouring agent.
• flavouring agent refers to any substance, natural or unnatural, that is capable of imparting a detectable flavour impact, especially at a concentration below 4 % w/w, more preferably below 2 % w/w.
• suitable flavours or flavouring agents include, but are not limited to, eucalyptus, mint, such as peppermint and spearmint, menthol, chocolate, liquorice, citrus and other fruit flavours, gamma octalactone, vanillin, ethyl vanillin, breath freshener flavours, spice flavours such as cinnamon, methyl salicylate, linalool, bergamot oil, geranium oil, lemon oil and ginger oil.
• the composition comprises a flavouring agent.
• flavouring agent is natural or unnatural, e.g. synthetic.
• the natural or unnatural flavouring agent is spearmint or eucalyptus.
• the composition comprises 0.01 to 0.4 % w/w flavouring agent, such as 0.02 to 0.1 % w/w flavouring agent, such as 0.035 to 0.055 % w/w flavouring agent compound.
• the composition comprises or consists essentially of: i. 0.02 to 0.05 % w/w peptidase; ii. 29 to 56 % w/w glycerol; iii. 4 to 25 % w/w xylitol; iv. 0.0002 to 0.0004 % w/w hyaluronic acid; v. 0.1 to 0.2 % w/w buffer; vi. 0 to 0.1 % w/w carrageenan; and vii. 0 to 0.05 % w/w flavouring agent.
• the disclosure of the present invention is a stable composition for treatment of microbial infections, in particular viral infections in the upper respiratory tract.
• Virus replication takes place in the upper respiratory tract.
• oral sprays reach the oropharynx while the nasal cavity including the nasopharynx is not reached with the oral administration route.
• a sprayable composition which can be administered through the nose to reach the nasal cavity and the nasopharynx, using a nasal spray.
• the composition preferably has a viscosity which is at most 0.015 Pa s.
• administration to the upper respiratory tract is required.
• composition formulated as a spray, lozenge, pastille, chewing gum, gel, or liquid.
• the composition is a sprayable composition.
• the composition is in the form of a spray.
• the composition is a nasal spray or a throat spray.
• the viscosity of the composition must allow for the composition to be sprayable.
• the composition has a viscosity which is at most 0.015 Pa s. This is supported by the findings of Example 6 disclosed herein.
• the nasal spray targets the nasal cavity, the primary site for upper respiratory tract infections.
• the disclosure of the present invention is a stable composition for treatment of microbial infections, in particular viral infections in the upper respiratory tract.
• the composition of the present disclosure can complement vaccines and systemic antivirals. It forms a thin protective broad-spectrum antiviral gel in nasal cavity and throat to prevent or reduce propagation of viral infections. Virus is trapped and bound to the gel and then deactivated by a broad spectrum peptidase. In comparison with antiviral drugs that works systemically and within cells to inhibit replication systems, the disclosed composition reduces and/or prevents up-take of virus locally in upper respiratory tract. These mechanisms of action complement each other to achieve more efficient treatment and/prevent of infections.
• the present invention is also active against bacteria and fungi by targeting the same virulence factor as for viruses namely the proteins responsible for the adhesion between the microbials and the cells.
• the virus infection is selected form the group consisting of common cold, pneumonia, bronchitis, severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), sinusitis, otitis and pharyngitis.
• SARS severe acute respiratory syndrome
• MERS Middle East respiratory syndrome
• sinusitis otitis and pharyngitis.
• One embodiment provides a method for treatment and/or prevention of virus infection in a mammal comprising administering to the subject a therapeutically-effective amount of a polypeptide having peptidase activity.
• Influenza is a more severe viral infection, which spreads worldwide in seasonal epidemics infecting up to 20% of the population which depending on the circulating virus, can cause substantial mortality.
• composition of the disclosure is for use in medicine.
• the disclosure relates to a composition for use in treating and/or preventing of microbial infections.
• the disclosure relates to a composition for use in the treating and/or preventing of a viral infection, a bacterial infection, a fungal infection, and/or a yeast infection.
• the disclosure relates to a composition for use in treating and/or preventing a viral infection.
• the disclosure relates to a composition for use in treating and/or preventing a viral infection from a virus selected from the group consisting of rhinovirus, influenza virus such as influenza A virus, Respiratory Syncytial Virus (RSV), coronavirus, parainfluenza virus, adenovirus, enterovirus, metapneumovirus, and other infectious viruses.
• a virus selected from the group consisting of rhinovirus, influenza virus such as influenza A virus, Respiratory Syncytial Virus (RSV), coronavirus, parainfluenza virus, adenovirus, enterovirus, metapneumovirus, and other infectious viruses.
• influenza virus such as influenza A virus, Respiratory Syncytial Virus (RSV), coronavirus, parainfluenza virus, adenovirus, enterovirus, metapneumovirus, and other infectious viruses.
• RSV Respiratory Syncytial Virus
• the disclosure relates to a composition, for use in treating and/or preventing infectious diseases such as common cold, flu, rhinitis, sinusitis, bronchitis, Severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), coronavirus disease 2019 (COVID-19), Pneumonia, Viral Meningitis, Herpangina, herpesvirus, papillomavirus or any other disease caused by viral infections.
• infectious diseases such as common cold, flu, rhinitis, sinusitis, bronchitis, Severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), coronavirus disease 2019 (COVID-19), Pneumonia, Viral Meningitis, Herpangina, herpesvirus, papillomavirus or any other disease caused by viral infections.
• the disclosure relates to a composition, for use in treating and/or preventing an upper respiratory tract viral infection.
• the disclosure relates to a composition, for use in treating and/or preventing an upper respiratory tract viral infection resulting in common cold, flu, rhinitis, sinusitis, COVID-19, or other diseases caused by upper respiratory tract viral infections.
• the composition as disclosed herein is for use in the prevention and/or treatment of symptoms related to viral infection, such as sore throat, malaise, rhinorrhea, nasal congestion, headache, cough, sneezing, and/or fever.
• symptoms related to viral infection such as sore throat, malaise, rhinorrhea, nasal congestion, headache, cough, sneezing, and/or fever.
• composition as disclosed herein is for use in the prevention and/or treatment of a disease, disorder or condition selected from the group consisting of microbial infections and/or oral conditions.
• composition as disclosed herein is for use in the prevention and/or treatment of microbial infection selected from the group consisting of viral infections, bacterial infections, fungal infections, and yeast infections.
• composition as disclosed herein is for use in the prevention and/or treatment of a microbial infection and/or oral condition such as a gum disease in and/or on a mammal.
• the composition as disclosed herein is for use in the prevention and/or treatment of a microbial infection and/or oral condition, such as gingivitis in and/or on a mammal.
• the disclosure provides a composition comprising i. peptidase; ii. glycerol; iii. xylitol; iv. hyaluronic acid; and v. buffer for use in medicine.
• the peptidase of said composition is trypsin.
• the composition comprising trypsin further comprises a divalent cation such as calcium (Ca 2+ ).
• compositions comprising i. peptidase; ii. glycerol; iii. xylitol; iv. hyaluronic acid; and v. buffer for use in the prevention and/or treatment of a disease, disorder or condition selected from the group consisting of microbial infections and/or oral conditions
• the microbial infection and/or oral condition is gingivitis or a gum disease in and/or on a mammal.
• compositions comprising: i. 0.005 to 1.0 % w/w peptidase; ii. 20 to 70 % w/w glycerol; iii. 1.0 to 65 % w/w xylitol; iv. 0.0001 to 1.5 % w/w hyaluronic acid; and v. 0.01 to 1.2 % w/w buffer for use in the prevention and/or treatment of a disease, disorder or condition selected from the group consisting of microbial infections, and oral conditions.
• compositions comprising: i. 0.015 to 0.055 % w/w trypsin; ii. 27 to 57 % w/w glycerol; HI. 4.5 to 30 % w/w xylitol; iv. 0.0002 to 0.02 % w/w hyaluronic acid; v. 0.010 to 0.015 % w/w CaCh; and vi. 0.1 to 0.14 % w/w buffer for use in the prevention and/or treatment of a disease, disorder or condition selected from the group consisting of microbial infections, and oral conditions.
• compositions as disclosed herein for use in preventing and/or treating a microbial infection selected from the group consisting of viral infections, bacterial infections, fungal infections, and yeast infections.
• compositions as disclosed herein for use in preventing and/or treating a microbial infection, which is a viral infection.
• the composition is for use in the treatment of an upper respiratory tract viral infection.
• the microbial infection is an upper respiratory tract viral infections.
• One embodiment provides for a composition as disclosed herein for use in in preventing and/or treating an upper respiratory tract infection resulting in common cold, flu, rhinitis, sinusitis, COVID-19, or another disease caused by upper respiratory tract infections.
• compositions as disclosed herein for use in in preventing and/or treating a viral infection from a virus selected from the group consisting of Rhinovirus, Influenza virus, Respiratory Syncytial Virus (RSV), Coronavirus, Parainfluenza virus, Adenovirus, Enterovirus, Metapneumovirus, and other infectious viruses.
• a virus selected from the group consisting of Rhinovirus, Influenza virus, Respiratory Syncytial Virus (RSV), Coronavirus, Parainfluenza virus, Adenovirus, Enterovirus, Metapneumovirus, and other infectious viruses.
• viruses which are capable of infecting and/or entering a cell
• viruses such as Corynebacterium diphtheria, Lassa fever virus (Arenavirus), Astrovirus, Hantavirus, Rift Valley Fever virus (Phlebovirus), Calicivirus, Ebola virus, Marburg Virus, Japanese encephalitis virus, Dengue virus, Yellow fever virus, Hepatitis C virus, Hepatitis G virus, Hepatitis B virus, Hepatitis D virus, Herpes simplex virus, Herpes simplex virus), Cytomegalovirus, Epstein Barr virus, Varicella Zoster Virus, Human Herpesvirus, Human Herpesvirus, Rubella virus, Mumps virus, Morbillivirus, Measles virus, Papillomaviruses, JC virus (Polyomavirus), BK virus (Polyomavirus), Parvovirus, Coxsackie virus (A and B), Hepatitis A virus, Polioviruses,
• viruses such as Cor
• compositions as disclosed herein for use in in preventing and/or treating a viral infection causing infectious diseases such as common cold, flu, rhinitis, sinusitis, bronchitis, Severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), coronavirus disease 2019 (COVID-19), Pneumonia, Viral Meningitis, Herpangina, herpesvirus, papillomavirus or any other disease caused by viral infections.
• infectious diseases such as common cold, flu, rhinitis, sinusitis, bronchitis, Severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), coronavirus disease 2019 (COVID-19), Pneumonia, Viral Meningitis, Herpangina, herpesvirus, papillomavirus or any other disease caused by viral infections.
• compositions as disclosed herein for use in the in preventing and/or treating symptoms related to viral infection, such as sore throat, malaise, rhinorrhea, nasal congestion, headache, cough, sneezing, and/or fever.
• compositions as disclosed herein for use in the manufacture of a medicament for in preventing and/or treating a disease, disorder or condition selected from the group consisting of microbial infections, dermatological conditions and oral conditions.
• composition is for oral use. In another embodiment, the composition is for nasal use.
• the composition for nasal use has a pH in the range 6.2 to 7.0, such as 6.4 to 6.6. In another embodiment, the composition for oral use has a pH in the range 7.2 to 8.5, such as 7.4 to 7.6.
• One embodiment provides for a method of treating or preventing of a disease, disorder or condition selected from the group consisting of microbial infections, dermatological conditions, and oral conditions, said method comprising administering the composition disclosed herein to an individual in need thereof.
• One embodiment provides for a method of treating or preventing of a disease, disorder or condition selected from the group consisting of microbial infections, dermatological conditions, and oral conditions, said method comprising administration of a composition comprising: i. peptidase; ii. glycerol; iii. xylitol; iv. hyaluronic acid; and v. buffer.
• the peptidase of said composition is trypsin.
• the composition comprising trypsin further comprises a divalent cation, such as calcium (Ca 2+ ).
• One embodiment provides for a method of preventing and/or reducing viral infections, said method comprising administering the composition disclosed herein to a subject in need thereof.
• One embodiment provides for a method of increasing microbial diversity, said method comprising administering a composition is disclosed herein to a subject in need thereof.
• One embodiment provides for a method of manufacturing the composition disclosed herein, said method comprising mixing; i. sugar alcohol ii. buffer; iii. hyaluronic acid; and iv. peptidase.
• One further embodiment provides for a method of manufacturing the composition disclosed herein, further comprising mixing; i. carrageenan; and/or ii. a flavouring agent.
• One embodiment provides for a method of increasing microbial diversity, said method comprising administering the composition disclosed herein.
• Example 1 Enzyme activity as function of dilution in Tris-HCI buffer
• the purpose of the experiments was to measure enzyme activity as a function of dilution in Tris-HCI buffer. Further, the carrageenan concentration on enzyme activity was evaluated.
• the purpose of the experiment was to measure enzyme stability in various nasal spray formulations.
• N01-N04 contained CaC/2 1 mM, trypsin 0.2 mg/ml, hyaluronic acid 2 pg/ml, Carrageenan 0.5 mg/ml.
• N05 contained CaCF 1 mM, trypsin 0.2 mg/ml, hyaluronic acid 2 pg/ml.
• the formulations were put into sealed polypropylene bottles.
• the stability of products from each formulation composition were tested by placing them at a stressed condition (40 ⁇ 2°C/100% RH) for up to 115 days.
• Enzyme activity was measured using same method as described in Example 1.
• the purpose of the experiment was to measure enzyme stability in various throat spray formulations.
• Table 2 Formulations used in stability studies of throat sprays.
• the purpose of the experiment was to evaluate tolerability, irritation/stinging sensation and experienced secretory stimulation of various nasal spray formulations by spraying the formulation into the nasal cavity.
• the nasal spray is intended to be hyperosmotic, thereby stimulating nasal secretion, which has a rinsing effect of the nasal mucosa.
• the glycerol concentration in the formulation for nasal use were diluted compared to formulations for oral use.
• ⁇ Formulation 1-4 and 6 contained glycerol, xylitol, water. **Formulation 5 contained glycerol, xylitol, Tris-HCI, Hyaluronic acid, CaCF and trypsin (concentrations equivalent to N03, Example 2). *** CX is a commercially available trypsin-comprising mouth spray.
• Tolerability was determined immediately after spraying using a 4 graded scale: 0; unacceptable, 1; uncomfortable, 2; acceptable, 3; comfortable.
• Secretory stimulation was determined after 2 minutes using a 3 graded scale: 0; no secretion, 1 ; secretion, 2; runny nose.
• Spraying in the nose was repeated 2 times and tolerability and secretory stimulation was noted after each spray.
• Example 5 Virucidal efficacy of throat spray and nasal spray formulations
• the purpose of the experiment was to determine virucidal efficacy of a throat spray formulation (for oral use) and a nasal spray formulation against SARS-CoV-2 virus.
• a nasal spray targets the nasal cavity, which is the primary site for viral upper respiratory tract infections.
• This study was designed to measure the virucidal effectiveness of the disclosed formulations, and thereby determine the potential of the formulations for inactivation of the target virus - SARS-CoV-2 - in suspension.
• E1052 Severe Acute Respiratory Syndrome-Related Coronavirus 2 (SARS-CoV-2, COVID-19 virus), Strain: USA-WA1/2020, Source: BEI resources, NR-52281, was used.
• the virus stock was stored at an ultra-low temperature prior to use, and thawed on the day of the test.
• the challenge virus stock contained 5.0% serum.
• the composition of the tested formulations were prepared as described in Table 5.
• the test formulations were preequilibrated to the test temperature prior to the study. The test solutions were evaluated at one contact time at one replicate.
• Selected dilutions were inoculated onto the host cells to assay for the quantity of infectious virus units.
• Virus recovery control a virus recovery control
• neutralizer effectiveness/viral interference control a cytotoxicity control
• media negative control a virus stock titer control
• virus recovery control a virus recovery control
• neutralizer effectiveness/viral interference control was performed in order to determine if residual active ingredients were present after neutralization and if the neutralized test substance interfered with virus infectivity. All controls were performed at the same time as the test, incubated under the same conditions and assayed in the same manner as the test.
• CPE viral-induced cytopathic effect
• the 50% tissue culture infective dose per mL was determined using the method of Spearman-Karber (Karber G. et al, 1931) or other appropriate methods such as Reed and Muench (Reed and Muench, 1938).
• LRF Log10 Reduction Factor
• Log10 Reduction Factor Virus Recovery Control (Log10 TCID50) - Test (Log 10 TCID50) where:
• TS01 and TS02 The formulation of TS01 and TS02 is presented in Table 5. Only the nasal spray contains carrageenan. The virucidal effects of throat spray (TS01) and nasal spray (TS02) are presented in 6.
• the purpose of this experiment was to measure how viscosity changed upon adding carrageenan.
• a throat spray solution TS03 (TS01 + 0.05 wt % carrageenan) was also evaluated.
• the rheological characteristics of the formulations were examined at high shear rates using continuous shear techniques and were performed with a controlled stress Malvern Rheometer (Malvern Instruments) using plate geometry radius 20 mm.
• the frequency sweep method was performed between 0.1 Hz and 10 Hz, with a shear strain of 0.8%, at 25 °C, while the table of shear rate method was performed by increasing the shear rate from 0.1 s -1 to 100 s -1 , at 25 °C.
• the shear stress was measured by this method and the apparent viscosity was calculated by dividing the shear stress by the shear rate.
• Example 7 Virucidal efficacy of throat spray against Influenza A virus
• the purpose of the experiment was to determine virucidal efficacy of a throat spray formulation against Influenza A virus strain H3N2.
• Example 5 This study had the same experimental set up as thoroughly described in Example 5. This study also followed the ASTM International test method designated E1052 “Standard Test Method to Assess the Activity of Microbicides against Viruses in Suspension”. The following specific changes to the experimental set up in Example 5 were made.
• Virus tested Influenza A Virus strain, H3N2, Charles River Laboratories.
• Host cell line MDCK cells, ATCC CCL-34.
• Dilution medium Minimum Essential Medium (MEM) + 1.0 pg/ml Trypsin.
• Neutralizer MEM + 1% Fetal Bovine Serum (FBS). Incubation time 6 days.
• the throat spray solution Table 5, was tested for its ability to inactivate Influenza A Virus (H3N2), exposed to the TS01 throat spray solution in suspension for 30 minutes at 36°C.
• the viral stock titer control for the assay confirmed that the appropriate titer was used in the experiment and sufficient amount of virus was recovered for the virus recovery control. Cytotoxicity was not detected at any dilution. All controls met the criteria for a valid test.
• the viral reductions for the TS01 solution against Influenza A Virus (H3N2) was a log reduction of 1.25 corresponding to 94.4% reduction.
• the TS01 solution is also effective against Influenza virus in vitro.
• CPE cytopathic effect
• HEp-2 cells were seeded in clear 96-well plates at 1.30E+04 cells per well. T01 and T02 were diluted 1 :10 in PBS and added in triplicate to HEp-2 cells. Cells were incubated for 1-hour at 37°C . Approximately 200 PFU/well of RSV A2 (0.02 MOI) was then added to the TA/cells wells. Virus only and cells only were also included for controls and calculation, as well as an internal assay control was also included for assay validation. HEp-2 cells were incubated for 3 days at 37°C. On day 3 post infection cells were immuno-stained and optical density was read and the percentage of virus inhibition was determined.
• T01 and T02 were diluted 1:10 in PBS for 3-days incubation with HEp-2 cells seeded in black 96-well plates; cells only and medium only wells were added. After 3 days cells were lysed for evaluation of the ATP content using Promega’s Cell Titer Gio kit. The luciferase luminescence in relative light units (RLU) was read and the percentage of cytotoxicity was determined
• T01 and T02 demonstrated inhibition against RSV but both solutions also demonstrated high toxicity against HEp-2 cells, Table 7. It is well known that high glycerol concentration is cytotoxic for human cells in general due to its high osmolality and therefore further dilutions could be performed to verify inhibition, at least for the T01 formulation.
• VSV Vesicular Stomatitis virus glycoprotein
• S SARS-CoV-2 B.1.1.529 (Omicron) spike (S) protein
• T01 and T02 were incubated together with cells and then virus was added to infect cells.
• virus and T01 or T02 were pre-incubated and then added to cells.
• Vero cells were seeded in black 96-well plates at 5.00E+04 cells per well. T01 and T02 were diluted 1 :1 and 1:10 in PBS and added in triplicate to Vero cells. Cells were incubated for 1-hour at 37°C. Approximately 10,000 RLU of rVSV-SARS- CoV-2 B.1.1.529 Omicron was then added to the TA/cells wells. Virus only and cells only were also included for controls and calculation, as well as an internal assay control was also included for assay validation. Vero cells were incubated for 24-hours at 37°C. Firefly Luciferase activity was detected using the Bright-GloTM Assay System kit (Promega) and the percentage of virus inhibition was determined.
• T01 and T02 were diluted 1 :1 and 1:10 in PBS for 24-hours incubation with Vero cells seeded in 96-well black plates; cells only and medium only wells were added. After 24- hours, cells were lysed for evaluation of the ATP content using Promega’s Cell Titer Gio kit. The luciferase luminescence in relative light units (RLU) was read and the percentage of cytotoxicity was determined.
• RLU relative light units
• Example 10 Stability of a nasal and throat spray formulation
• the purpose of the experiment was to measure enzyme stability in TS01 and TS02.
• TS01 , TS02, TS02 without xylitol and a commercially available, trypsin-containing mouth spray (CX) were stored in sealed glass bottles.
• the stability of trypsin in each formulation composition were tested by placing them under stressed conditions (40 ⁇ 2°C/100% RH) for up to 97 days. Enzyme activity was measured using same method as described in Example 1
• TS01 expressed the highest stability. Both TS01 and TS02 is more stable than CX. TS02 without xylitol is not stable
• a composition comprising: i. 0.005 to 1.0 % w/w peptidase; and ii. 1.0 to 70 % w/w sugar alcohol.
• composition according to item 1 comprising: i. 0.005 to 1.0 % w/w peptidase; ii. 20 to 70 % w/w glycerol; and iii. 1.0 to 65 % w/w xylitol. 3. The composition according to any one of the preceding items, wherein the composition comprises 0.005 to 1.0 % w/w peptidase, such as 0.01 to 0.075 % w/w peptidase, such as 0.015 to 0.055 % w/w peptidase.
• composition according to any one of the preceding items wherein the peptidase is selected from the group consisting of serine proteases, threonine proteases, cysteine proteases, aspartate proteases, glutamic acid proteases and metalloproteases, functional homologues thereof, or functionalised derivatives thereof.
• composition according to any one of the preceding items, wherein the peptidase is trypsin, a functional homologue, or a functionalized derivative thereof.
• composition according to any one of the preceding items wherein the composition comprises 0.005 to 1.0 % w/w trypsin, such as 0.01 to 0.075 % w/w trypsin, such as 0.015 to 0.055 % w/w trypsin.
• composition according to any one of the preceding items comprising: i. 0.005 to 1.0 % w/w trypsin; ii. 20 to 70 % w/w glycerol; and iii. 1.0 to 65 % w/w xylitol.
• composition according to any one of the preceding items, wherein the composition comprises 0.005 to 1.0 % w/w trypsin, such as 0.01 to 0.075 % w/w trypsin, such as 0.015 to 0.055 % w/w trypsin.
• composition according to any one of the preceding items, wherein the composition further comprises 0.003 to 0.2 % w/w salt comprising a divalent cation, such as 0.003 to 0.15 % w/w, such as 0.003 to 0.1 % w/w salt comprising a divalent cation. 11.
• composition according to any one of the preceding items, wherein the peptidase is trypsin, a functional homologue thereof, or a functionalized derivative thereof and wherein the composition further comprises 0.003 to 0.2 % w/w salt comprising a divalent cation, such as 0.003 to 0.15 % w/w, such as 0.003 to 0.1 % w/w salt comprising a divalent cation.
• composition according to any one of the preceding items, wherein the composition comprises 35 to 70 % w/w sugar alcohol, such as 40 to 65 % w/w sugar alcohol, such as 45 to 60 % w/w sugar alcohol.
• sugar alcohol is glycerol, erythritol, xylitol, mannitol, arabitol, maltitol, lactitol, isomalt, sorbitol, hydrogenated starch hydrolysates or propylene glycol, or other relevant polyols.
• composition according to any one of the preceding items wherein the composition comprises 20 to 70 % w/w glycerol, such as 25 to 65 % w/w glycerol, such as 27 to 57 % w/w glycerol.
• composition according to any one of the preceding items wherein the composition comprises 1.0 to 65 % w/w xylitol, such as 3.0 to 50 % w/w xylitol, such as 4.5 to 30 % w/w xylitol.
• composition according to any one of the preceding items, wherein the composition further comprises a buffer.
• composition according to any one of the preceding items wherein the composition comprises 0.01 to 2.4 % w/w buffer, such as 0.07 to 0.3 % w/w buffer, such as 0.09 to 0.2 % w/w buffer. 19. The composition according to any one of the preceding items, wherein the concentration of the buffer is 1 to 200 mM, such as 5 to 25 mM, such as 8 to 15 mM.
• Tris tris(hydroxymethyl)amino methane
• MOPS 3-(/V-morpholino)propanesulfonic acid
• phosphate phosphate
• composition according to any one of the preceding items, wherein the composition further comprises water.
• composition according to any one of the preceding items, wherein the composition comprises at most 50 % w/w water, such as at most 40 % w/w, such as at most 30 % w/w.
• composition according to any one of the preceding items, wherein the composition further comprises hyaluronic acid, such as 0.0001 to 2.0 % w/w hyaluronic acid, such as 0.0001 to 1.5 % w/w, such as 0.0001 to 1.0 % w/w, such as 0.001 to 1.5 % w/w, such as 0.01 to 1.0 % w/w, such as 0.02 to 0.5 % w/w, such as 0.0001 to 0.1 % w/w, such as 0.0001 to 0.01 %.
• hyaluronic acid such as 0.0001 to 2.0 % w/w hyaluronic acid, such as 0.0001 to 1.5 % w/w, such as 0.0001 to 1.0 % w/w, such as 0.001 to 1.5 % w/w, such as 0.01 to 1.0 % w/w, such as 0.02 to 0.5 % w/w, such as 0.0001 to 0.1 %
• composition according to any one of the preceding items, wherein the divalent cation is calcium.
• the salt comprising a divalent cation is a pharmaceutically acceptable salt of calcium, including hydrates thereof.
• composition according to any one of the preceding items wherein the composition further comprises 0.003 to 0.2 % w/w calcium salt, such as 0.003 to 0.15 % w/w calcium salt, such as 0.003 to 0.1 % w/w calcium salt.
• calcium ions such as 0.002 to 0.2 % w/w calcium ions, such as 0.003 to 0.15 % w/w calcium ions, such as 0.003 to 0.1 % w/w calcium ions.
• composition according to any one of the preceding items, wherein the salt comprising a divalent cation is CaCh.
• composition according to any one of the preceding items, wherein the salt comprising a divalent cation is a pharmaceutically acceptable inorganic salt of calcium, including hydrates thereof.
• composition according to any one of the preceding items wherein the composition comprises 0.003 to 0.2 % w/w divalent calcium, such as 0.003 to 0.15 % w/w, such as 0.003 to 0.1 %.
• composition according to any one of the preceding items wherein the composition comprises 0.002 to 0.2 % w/w calcium chloride, such as 0.005 to 0.02 % w/w calcium chloride, such as 0.010 to 0.015 % w/w calcium chloride.
• composition according to any one of the preceding items, wherein the composition comprises or consists essentially of: i. 0.005 to 1.0 % w/w trypsin; ii. 20 to 70 % w/w glycerol; iii. 1.0 to 65 % w/w xylitol; iv. 0.0001 to 1.5 % w/w hyaluronic acid; v. 0.002 to 0.2 % w/w CaCh; and vi. 0.01 to 1 .2 % w/w buffer.
• composition according to any one of the preceding items wherein the composition comprises or consists essentially of: i. 0.015 to 0.055 % w/w trypsin; ii. 27 to 57 % w/w glycerol; iii. 4.5 to 30 % w/w xylitol; iv. 0.0002 to 0.02 % w/w hyaluronic acid; v. 0.010 to 0.015 % w/w CaCh; and vi. 0.1 to 0.14 % w/w buffer.
• composition according to any one of the preceding items further comprising carrageenan.
• composition according to any one of the preceding items wherein the composition comprises no more than 1 % w/w carrageenan, such as no more than 0.5 % w/w carrageenan, such as no more than 0.1 % w/w carrageenan.
• composition according to any one of the preceding items further comprising a flavouring agent.
• flavouring agent is natural or unnatural.
• composition according to any one of the preceding items wherein the composition comprises 0.01 to 0.4 % w/w flavouring agent, such as 0.02 to 0.1 % w/w flavouring agent, such as 0.035 to 0.055 % w/w flavouring agent compound.
• composition according to any one of the preceding items, wherein the composition comprises or consists essentially of: i. 0.02 to 0.05 % w/w peptidase; ii. 29 to 56 % w/w glycerol; iii. 4 to 25 % w/w xylitol; iv. 0.0002 to 0.0004 % w/w hyaluronic acid; v. 0.01 to 0.02 % w/w CaCh; vi. 0.1 to 0.2 % w/w buffer; vii. 0 to 0.1 % w/w carrageenan; and viii. 0 to 0.05 % w/w flavouring agent.
• composition according to any one of the preceding items wherein the composition is delivered in the form of a spray, lozenge, pastille, chewing gum, gel or liquid.
• composition according to any one of the preceding items, wherein the composition is a sprayable composition.
• composition according to any one of the preceding items for use in medicine is a composition according to any one of the preceding items for use in medicine.
• composition according to any one of the preceding items for use in the prevention and/or treatment of a disease, disorder or condition selected from the group consisting of microbial infections, and oral conditions, such as gingivitis or gum disease in and/or on a mammal.
• microbial infection is selected from the group consisting of viral infections, bacterial infections, fungal infections, and yeast infections.
• composition for use according to any one of the preceding items wherein the viral infection is from a virus selected from the group consisting of rhinovirus, influenza virus such as influenza A virus, Respiratory Syncytial Virus (RSV), coronavirus, parainfluenza virus, adenovirus, enterovirus, metapneumovirus, and other infectious viruses.
• influenza virus such as influenza A virus, Respiratory Syncytial Virus (RSV), coronavirus, parainfluenza virus, adenovirus, enterovirus, metapneumovirus, and other infectious viruses.
• RSV Respiratory Syncytial Virus
• composition for use according to any one of the preceding items wherein the viral infection cause infectious diseases such as common cold, flu, rhinitis, sinusitis, bronchitis, Severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), coronavirus disease 2019 (COVID-19) such as the COVID-19 omicron variant, Pneumonia, Viral Meningitis, Herpangina, herpesvirus, papillomavirus or any other disease caused by viral infections.
• infectious diseases such as common cold, flu, rhinitis, sinusitis, bronchitis, Severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), coronavirus disease 2019 (COVID-19) such as the COVID-19 omicron variant, Pneumonia, Viral Meningitis, Herpangina, herpesvirus, papillomavirus or any other disease caused by viral infections.
• infectious diseases such as common cold, flu, rhinitis, sinusitis, bronchi
• symptoms related to viral infection such as sore throat, malaise, rhinorrhea, nasal congestion, headache, cough, sneezing, and/or fever.
• composition according to any one of the preceding items in the manufacture of a medicament for the treatment and prevention of a disease, disorder or condition selected from the group consisting of microbial infections, dermatological conditions and oral conditions.
• a method of treating and/or preventing of a disease, disorder or condition selected from the group consisting of microbial infections, dermatological conditions, and oral conditions comprising administration of the composition according to any one of the preceding items to an individual in need thereof.
• a method for prevention and/or reduction of viral infection comprising administration of a composition according to any one of the preceding items.
• a method for increasing microbial diversity comprising administration of a composition according to any one of the preceding items.
• composition according to any one of the preceding items comprising mixing; i. peptidase; and ii. sugar alcohol.
• a method of manufacturing the composition according to any one of the preceding items comprising mixing; i. trypsin; ii. glycerol; and iii. xylitol.
• a method of manufacturing the composition according to any one of the preceding items comprising mixing; i. glycerol; ii. xylitol; iii. buffer; iv. hyaluronic acid; and v. peptidase.
• the method according to any one of the preceding items further comprising mixing; i. carrageenan; and/or ii. a flavouring agent.

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• 2022
  • 2022-08-03 CN CN202280054111.3A patent/CN117794513A/zh active Pending
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