Classifications

• • 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
  • A61K31/722—Chitin, chitosan
• • 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
  • A61K9/007—Pulmonary tract; Aromatherapy
  • A61K9/0073—Sprays or powders for inhalation; Aerolised or nebulised preparations generated by other means than thermal energy
• • 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
  • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
  • A61P31/14—Antivirals for RNA viruses
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • 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

Definitions

• the present invention relates to amphiphilic carbohydrate compounds and their use as antivirals.
• Viral binding to respiratory epithelial cell-surface receptors is a critical step in the infection of host cells and the COVID-19 infectious agent (SARS-CoV-2) gains entry to the cell via the viral spike protein S1 receptor binding domain (RBD) interacting with the angiotensin converting enzyme-2 (ACE-2) receptor (Tai, W. et al. Characterization of the receptor-binding domain (RBD) of 2019 novel coronavirus: implication for development of RBD protein as a viral attachment inhibitor and vaccine. Cellular & Molecular Immunology, doi:10.1038/s41423-020-0400-4 (2020) and Yan, R. et al. Structural basis for the recognition of the SARS-CoV-2 by full-length human ACE2.
• the ACE-2 receptor is highly expressed in nasal epithelial cells (Sungnak, W. et al. SARS-CoV-2 entry factors are highly expressed in nasal epithelial cells together with innate immune genes. Nat. Med., doi:10.1038/s41591 -020-0868-6 (2020)), making a nasal spray that inhibits viral entry into nasal epithelial cells an attractive prophylactic for use in the control of epidemics and pandemics.
• Viral receptor binding and internalisation occurs via an initial binding of the spike protein S1 binding domain to the ACE-2 receptor followed by a conformational change which results in membrane fusion of the viral particle with the epithelial cells and ultimately viral entry.
• Polymers such as sulphated glycopolymers have been shown to inhibit the viral binding of human papillomavirus to cell surface receptors (Soria-Martinez, L. et al. Prophylactic Antiviral Activity of Sulfated Glycomimetic Oligomers and Polymers. Journal of the American Chemical Society 142, 5252-5265, doi:10.1021/jacs.9b13484 (2020)).
• Sulphated chitosan compounds such as N-carboxymethylchitosan-N,O-sulfate were found to inhibit the synthesis of virus-specific proteins and the replication of HIV-1 in cultured T-cells as well as the replication of the Rausher murine leukemia virus in cultured mouse fibroblasts (Chirkov, S.
• quaternary ammonium compounds are viricidal due to mechanisms involving viral cell lysis and DNA binding (Gerba, C. P. Quaternary ammonium biocides: efficacy in application. Appl Environ Microbiol 81 , 464-469, doi:10.1128/AEM.02633-14 (2015)).
• WO2013/172725 discloses that N-(2- hydroxypropyl)-3-trimethylammonium chitosan chloride (HTCC), a chitosan QAC with a molecular weight of 50 - 190 kDa (based on viscosity), and a level of quaternary ammonium groups ranging from 57% to 77%, inhibits coronavirus infections (e.g. HCOV-NL63) in vitro by a mechanism that involves an inhibition of viral entry into the cell (Milewska, A. et al. HTCC: Broad Range Inhibitor of Coronavirus Entry. PLoS One 11 , e0156552, doi:10.1371/journal.
• HTCC inhibits the entry of SARS- COV-2 into cells (Milewska, A. et al. HTCC as a highly effective polymeric inhibitor of SARS-CoV-2 and MERS-CoV. bioRxiv https://doi.org/10.1101/2020.03.29.014183 (2020)).
• the HTCC disclosed has a relatively high molecular weight (50 - 190 kDa) and a high level of quaternary ammonium group substitution (57 - 77mole%).
• the quaternary ammonium group on HTCC is not the only factor essential for activity, as the following compounds were earlier found in Milewska, A. et al. Novel polymeric inhibitors of HCoV-NL63. Antiviral Res 97, 112-121 , doi: 10.1016/j.antiviral.2O12.11.006 (2013), to be non-active in inhibiting coronavirus entry into cells despite the presence of a quaternary ammonium group: O-(2-hydroxypropyl)-3-trimethylammonium poly(vinyl alcohol) chloride (HTPVA), N-(2-hydroxypropyl)-3-trimethylammonium dextran chloride, hydroxypropylcellulose-graff-poly(N-acrylamidopropyl-N,N,N- trimethylammonium chloride) (HPC-APTMAC), N-(3-ethylammonium) poly(allylamine) chloride, poly(methacryloyl aminopropyltrimethylammonium chlor
• oligochitosans without the quaternary ammonium group were inactive in inhibiting coronavirus entry into cells and HTCC was inactive in the viral inhibition of a number of other viruses (e.g. human herpes virus 1 , influenza A, adenoviruses and enteroviruses).
• chitosans of molecular weight 5 - 17 kDa have been shown to be more effective antiviral agents against tobacco mosaic virus than chitosans with a molecular weight of 130 kDa (Davydova, V. N. et al. [Chitosan antiviral activity: dependence on structure and depolymerization method], Prikl Biokhim Mikrobiol 47, 113-118 (2011 )).
• Carrageenans anionic sulphated carbohydrates have been shown to reduce the duration of disease by 3 days, reduce the number of relapses over a 21 day period by three-fold in influenza and common cold patients, and prevent influenza A viral infections in mice, by preventing viral interaction with relevant cell surface receptors, as disclosed by Koenighofer et al. (“Carrageenan nasal spray in virus confirmed common cold: individual patient data analysis of two randomized controlled trials. Multidisciplinary Resp Med 9, 57, 2014) and Leibbrandt et a., (“lota-carrageenan is a potent inhibitor of influenza A virus infection”, PLoS One 5, e14320, 2010).
• the current invention is aimed at inhibiting viral infections and/ or limiting their severity and is focused on the use of polymers as viral inhibition agents and their use for the treatment or prophylaxis of viral infections.
• an amphiphilic carbohydrate compound of molecular weight less than 50kDa of formula (I) for use in the prevention or treatment of a viral infection wherein: the level of unit A is from 0% to 26 mole%; the level of unit D is from 1 % to 95.5 mole%; the level of unit H is from 1 % to 95.5 mole%; the level of unit Q is from 3% to 40 mole%; the level of unit T is from 1 % to 94.5 mole%;
• Ri, R2, R3, R4 and R10 are independently hydrogen or any linear, branched or cyclo form of an alkyl, alkenyl, alkynyl, aryl, acyl group, a sugar substituent selected from glucose, galactose, fructose, and muramic acid, or oligo polyoxa C1-C3 alkylene units, optionally substituted with amine, amide or alcohol; wherein at least one of R1, R 2 , R3, R4 and Rio is not hydrogen;
• Rs is a hydrophobic, substituted or unsubstituted, linear, branched or cyclo form of a C4-30 alkyl, C4-30 alkenyl, C4-30 alkynyl, C4-30 aryl, C4-30 amide, C4-30 alcohol or C3-30 acyl group;
• Re, R7, and Rs are independently any linear, branched, or cyclo forms of any alkyl, alkenyl, alkynyl, aryl or acyl group;
• R9 may be present or absent and, when present, is a substituted or unsubstituted alkyl group, a substituted or unsubstituted amine group or a substituted or unsubstituted amide group;
• R11 is a substituted or unsubstituted alkyl group, a substituted or unsubstituted ether group or a substituted or unsubstituted alkene group or hydrogen;
• R12 is a substituted or unsubstituted alkyl group, a substituted or unsubstituted ether group or a substituted or unsubstituted alkene group
• 13 is a substituted or unsubstituted alkyl group, a substituted or unsubstituted ether group or a substituted or unsubstituted alkene group or hydrogen; or a salt thereof.
• a pharmaceutical composition for use in the prevention or treatment of a viral infection comprising one or more pharmaceutically acceptable excipients, and an amphiphilic carbohydrate compound having a molecular weight of less than 50kDa and is represented by the general formula:
• a method of prevention or treatment of a viral infection wherein a compound or a composition according to the first or second aspect of the invention is administered to the patient, or healthy individual for the purposes of viral prophylaxis.
• the presently claimed compounds possess significant advantages for use in viral inhibition and specifically the clinical prevention of viral infections.
• the compounds are mucoadhesive, chemically stable for at least 18 months and in the case of GCPQ has been subjected to a Good Laboratory Practice toxicology screen, with a no observed adverse effect level defined (Siew, A. et al. Enhanced oral absorption of hydrophobic and hydrophilic drugs using quaternary ammonium palmitoyl glycol chitosan nanoparticles. Molecular Pharmaceutics 9, 14-28, doi: 10.1021 /mp200469a (2012) and Chooi, K. W. et al.
• GCPQ quaternary ammonium palmitoyl glycol chitosan
• Nanoparticulate peptide delivery exclusively to the brain produces tolerance free analgesia. J Control Release 270, 135- 144, doi:10.1016/j.jconrel.2017.11 .041 (2017)). These advantages mean that GCPQ may be given as a nasal spray or by other means for the prevention and treatment of specific viral infections.
• US8470371 discloses GCPQ and specifies its use for drug delivery and not as a virus inhibitor.
• FIG. 1 Cytotoxicity of GCPQs in vitro. Cell viability was assessed using an XTT assay on Vera E6 cells (A) and A549/ACE2 cells (B). Relative viability of cells (percentage of the untreated control) is shown on y-axis. All assays were performed in triplicate, and average values with standard errors are presented. The letters a to d refer to the GCPQs shown in Table 2.
• Figure 2 Antiviral activity of GCPQs against SARS-CoV-2. Virus replication was evaluated using RT-qPCR. The data are presented as a number of RNA copies per ml of the original sample (top) or as Log Removal Value (LRV) (bottom) compared to untreated samples. The assay was performed in triplicate, and average values with standard errors are presented.
• LRV Log Removal Value
• FIG. 3 Replication of SARS-CoV-2 in fully differentiated tissue cultures of the human respiratory epithelium (HAE cultures) in the presence or absence of GCPQ. Virus replication was evaluated using RT-qPCR. The data are presented as a number of viral copies per ml. The assay was performed in triplicate, and median values with range are presented.
• Figure 4 Sagittal SPECT/CT images of radiolabelled GCPQ (10 mg/ kg) at 30 min, 2h 30 min and 24 hours after nasal administration to male mice (a-c), the nasal delivery device (Naltos device) that may be used to deliver the prophylactic GCPQ powder, permission from Alchemy Pharmatech Ltd.
• amphiphilic carbohydrate compound used in this invention is a chitosan derivative. With reference to the formulae in this invention, all percentages refer to mole%. In formula I, it is understood that A + D + H + Q + T will be equal to 100%. It should also be understood that A, D, H, Q and T may form any arrangement in the amphiphilic carbohydrate compound. The arrangement may therefore be entirely random or as a block copolymer form such as ADHQTADHQT etc.
• A is in the range 0% to 26 mole%.
• unit A may be absent.
• the amphiphilic carbohydrate compound shows a degree of acetylation.
• A is in the range 0.5% to 20 mole%, more preferably in the range 0.5% to 15 mole%, even more preferably in the range 0.5% to 10 mole%, even more preferably in the range 0.5% to 5 mole% or 0.5 to 4 mole% or 0.5 to 3 mole%.
• A is in the range 2 to 20 mole%, preferably 2 to 15 mole%, more preferably in the range 2 to 10 mole%, even more preferably in the range 2 to 5 mole% or 2 to 4 mole%.
• A is in the range 1 to 20 mole%, preferably 1 to 15 mole%, more preferably in the range 1 to 10 mole%, even more preferably in the range 1 to 5 mole% or 2 to 5 mole%.
• D is in the range 1 to 95.5 mole%. In a preferred embodiment of the invention, D is in the range 2% to 94.5 mole%, preferably in the range 10% to 94.5 mole%, more preferably in the range 10% to 90 mole%, typically in the range 20 to 80 mole% or 50% to 75 mole%, more preferably in the range 55% to 75 mole%, even more preferably in the range 65% to 75 mole%.
• T is in the range 1 to 94.5 mole%. In a preferred embodiment of the invention, T is in the range 2% to 94.5 mole%, preferably in the range 2% to 90 mole%, more preferably in the range 5% to 80 mole%. In a further preferred embodiment, T is in the range 5% to 70 mole%, for instance 5% to 60 mole% or 5% to 50 mole%. In an alternative embodiment, T is in the range 10% to 30 mole%, more preferably in the range 10 to 20 mole% or 20% to 30 mole%.
• the level of the quaternary ammonium unit, unit Q is no more than 40mole%.
• Q is in the range 3% to 40mole%.
• Q is in the range 3% to 30 mole%. It is preferably present in the range 5% to 30 mole%, for instance 5% to 20 mole%, 5% to 15 mole% or 5 to 10 mole%, or for instance 5 to 20 mole%, 10 to 20 mole% or 15 to 20 mole%.
• H is in the range 1 to 95.5mole%. In a preferred embodiment of the invention, H is in the range 1 % to 40 mole%, 1 % to 30mole% or 1 % to 20 mole%, more preferably in the range 1 % to 10 mole%, even more preferably in the range 1 % to 5 mole%. In some embodiments, H is in the range 0.5% to 20 mole% or 1 % to 20 mole%, for instance, 1 to 10 mole% or 1 to 5 mole%.
• the amphiphilic carbohydrate may be in the form of a salt.
• the salt can comprise a chloride, iodide, acetate or glucuronide salt.
• the molecular weight of the amphiphilic carbohydrate compound has a molecular weight of less than 50kDa. It is preferably in the range 1-40kDa or 1 -30kDa, for instance 5- 30kDa or 10-30kDa. Molecular weight is preferably measured using Gel-permeation chromatography - multi-angle light scattering (GPC-MALLS). The molecular weight will be the mean average measurement for all the polymer chains present in a sample.
• GPC-MALLS Gel-permeation chromatography - multi-angle light scattering
• amphiphilic carbohydrate compound is capable of self-assembly into nanoparticles in aqueous media.
• Ri, R2, R3, R4 and R10 are independently hydrogen or any linear, branched or cyclo form of an alkyl, alkenyl, alkynyl, aryl, acyl group, a sugar substituent selected from glucose, galactose, fructose, and muramic acid, or oligo polyoxa C1-C3 alkylene units, optionally substituted with amine, amide or alcohol.
• these groups are independently selected from hydrogen, a substituted or unsubstituted alkyl group, a substituted or unsubstituted ether group, or a substituted or unsubstituted alkene group.
• R1, R2, R3, R4 and R10 may be C1-C4 linear alkyl groups.
• R1, R2, R3, R4 and R10 may be C1-C4 linear glycol-based groups.
• R1, R2, R3, R4 and R10 are any of the following sugar substituents: glucose, galactose, fructose, and muramic acid.
• Ri, R2, R3, R4 and R10 may be oligo polyoxa C1-C3 alkylene units such as ethylene glycol oligomers. All of R1, R2, R3, R4 and R10 may be CH2CH2OH.
• R1, R2, R3, R4 and R10 may also be hydrophilic.
• R5 is a hydrophobic, substituted or unsubstituted, linear, branched or cyclo form of a C4-30 alkyl, C4-30 alkenyl, C4-3oalkynyl, C4-3oaryl, amine, C4-30 amide, C4-30 alcohol or C3-30 acyl group.
• the group R5 is preferably selected from a substituted or unsubstituted group which is an acyl group such as a Cs-so acyl group, an alkyl group such as a C4-30 alkyl group, an alkenyl group such as a C4-30 alkenyl group, an alkynyl group such as a C4-30 alkynyl group, an aryl group such as a C 5 -2oaryl group, a multicyclic hydrophobic group with more than one C4-C8 ring structure such as a sterol (e.g.
• a multicyclic hydrophobic group with more than one C4-C8 heteroatom ring structure a polyoxa C1-C4 alkylene group such as polyoxa butylene polymer, or a hydrophobic polymeric substituent such as a poly (lactic acid) group, a poly(lactide-co- glycolide) group or a poly(glycolic acid) group.
• the R5 group may be linear, branched or cyclo groups.
• a particularly preferred class of R5 substituents are linked to the chitosan monomer unit via an amide group (including the pendant NH in the formula), for example as represented by the formula CH3(CH2) n CO-, where n is between 2 and 28.
• amide groups are produced by the coupling of carboxylic acids to the amine group of chitosan.
• Re, 7, and Rs are independently any linear, branched, or cyclo forms of any alkyl, alkenyl, alkynyl, aryl or acyl group.
• Re, 7 and Rs are preferably independently selected from a substituted or unsubstituted alkyl group such as a C1.10 alkyl group.
• Re, R7 and/ or Rs may be linear or branched.
• Re, R7 and Rs are independently selected from methyl, ethyl or propyl groups and are preferably methyl groups.
• Re, 7 and Rs form a quaternary ammonium group which is hydrophilic. Hydrophilic groups are groups which are well hydrated by water and associate on a molecular level with water.
• the Rg group may be present or absent in the general formula.
• Rg may be present or absent and, when present, is a substituted or unsubstituted alkyl group, a substituted or unsubstituted amine group or a substituted or unsubstituted amide group.
• Rg is not a 2-hydroxypropyl group. When absent, it provides a quaternary ammonium functional group that is directly linked to the monomer unit of the chitosan backbone.
• the Rg group When the Rg group is present it may be a unsubstituted or substituted alkyl group (e.g.
• Ci.io alkyl group but generally not a 2-hydroxypropyl group -CH2-CH(OH)-CH2-) for example as represented by -(CH2) n - wherein n is preferably 1 to 4.
• RgN+ReRzRa substituent is provided by coupling betaine (-OOC-CH2-N + -(CH3)3) to the amine of the D unit providing an amide group such as in: -NH-CO-CH2-N+R6R7R8.
• R11 is a substituted or unsubstituted alkyl group, a substituted or unsubstituted ether group or a substituted or unsubstituted alkene group or hydrogen.
• Rn is selected from hydrogen and a substituted or unsubstituted alkyl group such as a C1.10 alkyl group.
• Rn may be linear or branched.
• Rn is selected from methyl, ethyl or propyl groups and is preferably a methyl group.
• it is an OH- substituted alkyl group, preferably of formula CH2CH2OH.
• R12 is a substituted or unsubstituted alkyl group, a substituted or unsubstituted ether group or a substituted or unsubstituted alkene group.
• R12 is selected from substituted or unsubstituted alkyl group such as a C1-10 alkyl group.
• R12 may be linear or branched.
• R12 is selected from methyl, ethyl or propyl groups. Alternatively, it is an OH-substituted alkyl group, preferably of formula CH2CH2OH.
• R12 is a C1-10 alkyl group.
• R12 may be linear or branched.
• R12 is selected from methyl, ethyl or propyl groups and is preferably a methyl group.
• R13 is a substituted or unsubstituted alkyl group, a substituted or unsubstituted ether group or a substituted or unsubstituted alkene group or hydrogen.
• R13 is selected from hydrogen and a substituted or unsubstituted alkyl group such as a C1.10 alkyl group.
• R ⁇ may be linear or branched.
• R13 is selected from methyl, ethyl or propyl groups, most preferably a methyl group. Alternatively, it is an OH- substituted alkyl group, preferably of formula CH2CH2OH.
• Most preferably R13 is hydrogen.
• the total number of monomer units of A+D+H+Q+T may be about 5 to 100.
• the total number of monomer units of A+D+H+Q+T may be less than about 220.
• the amphiphilic carbohydrate compound is a form of N-palmitoyl,N-monomethyl,N,N-dimethyl,N,N,N-trimethyl-6-O-glycolchitosan (GCPQ).
• GCPQ N-palmitoyl,N-monomethyl,N,N-dimethyl,N,N,N-trimethyl-6-O-glycolchitosan
• substituents described herein may be either unsubstituted or substituted with one or more additional substituents as is well known to those skilled in the art.
• substituents include halo; hydroxyl; ether (e.g., Ci.
• acyl e.g. C1-7 alkylacyl, C5-20 arylacyl
• acylhalide carboxy; ester; acyloxy; amido; acylamido; thioamido; tetrazolyl; amino; nitro; nitroso; azido; cyano; isocyano; cyanato; isocyanato; thiocyano; isothiocyano; sulfhydryl; thioether (e.g., Ci.
• alkylthio sulphonic acid; sulfonate; sulphone; sulfonyloxy; sulfinyloxy; sulfamino; sulfonamino; sulfinamino; sulfamyl; sulfonamide; C1.7 alkyl (including, e.g., unsubstituted C1.7 alkyl, C haloalkyl, C1.7 hydroxyalkyl, C1.7 carboxyalkyl, C1.7 aminoalkyl, Cs-2o aryl- C1.7 alkyl); C3-20 heterocyclyl; and C5-20 aryl (including, e.g., C5-20 carboaryl, C5-20 heteroaryl, C1.7 alkyl-C5.20 aryl and C5-20 haloaryl) groups.
• C1.7 alkyl including, e.g., unsubstituted C1.7 alkyl, C haloalkyl, C1.7
• ring structure as used herein, pertains to a closed ring of from 3 to 10 covalently linked atoms, yet more preferably 3 to 8 covalently linked atoms, yet more preferably 5 to 6 covalently linked atoms.
• a ring may be an alicyclic ring, or aromatic ring.
• alicyclic ring as used herein, pertains to a ring which is not an aromatic ring.
• carrier ring refers to a ring wherein all of the ring atoms are carbon atoms.
• Carboaromatic ring as used herein, pertains to an aromatic ring wherein all of the ring atoms are carbon atoms.
• heterocyclic ring refers to a ring wherein at least one of the ring atoms is a multivalent ring heteroatom, for example, nitrogen, phosphorus, silicon, oxygen or sulphur, though more commonly nitrogen, oxygen, or sulphur.
• the heterocyclic ring has from 1 to 4 heteroatoms.
• the above rings may be part of a “multicyclic group”.
• the amphiphilic carbohydrate compound is N-palmitoyl-N-monomethyl-N,N- dimethyl-N,N,N-trimethyl-6-O-glycolchitosan, otherwise known as quaternary ammonium palmitoyl glycol chitosan (GCPQ).
• GCPQ quaternary ammonium palmitoyl glycol chitosan
• Re, R? and Rs are methyl
• Ri, R2, R3, R4 and R10 are -CH2CH2OH
• R9 is absent
• R13 is hydrogen.
• Rn and R12 are either hydrogen or methyl and both may not be hydrogen.
• the palmitoylation level (corresponding to group H) is preferably between 5-50% per monomer, for instance, 10-20% per monomer.
• the quaternisation level (Q) is preferably between 3-40% per monomer, preferably 10-30% per monomer.
• the molecular weight of the GCPQ is typically in the range 1 -40kDa or 1 -30kDa, for instance 5-30kDa or 10-30kDa.
• amphiphilic carbohydrate compound is capable of self-assembling into particles in aqueous media without the presence of other agents such as tripolyphosphate. Generally, micelles are formed.
• the amphiphilic carbohydrate may form particulate aggregates. These may be formed by the aggregation of individual amphiphile molecules and have a mean particle size of between 10 nm and 20 pm.
• the mean particle size can readily be determined microscopically or by using photon correlation spectroscopy and is conveniently determined in aqueous dispersions prior to filtration.
• the polymeric micellar aggregates have a minimum mean particle size of at least 10 nm, and more preferably at least 30 nm, and a maximum mean particle size which is preferably 10pm or less.
• the amphiphilic carbohydrate compound may be used alone or formulated with one or more drugs.
• the drug may be a hydrophobic drug.
• the amphiphilic carbohydrate compound is capable of self-assembly into nanoparticles in aqueous media.
• Pharmaceutical compositions of the present invention may comprise nanodispersions of the nanoparticles of the amphiphilic carbohydrate compound.
• amphiphilic carbohydrate compound of the invention is useful against viral infections perse. Therefore, it may be used alone in the treatment or prevention of a viral infection. Thus the amphiphilic carbohydrate compound is itself an active agent. In a pharmaceutical composition of the invention, it may be the only active agent/ingredient, i.e. there may be no further drugs present. For instance, a pharmaceutical composition may consist essentially only of the amphiphilic carbohydrate compound and one or more pharmaceutically acceptable excipients.
• the amphiphilic carbohydrate compound forms nanoparticles which can be loaded with drug.
• the drug is typically encapsulated by the self-assembled positively charged amphiphilic polymers.
• a dispersion of carbohydrate and drug may be formed which is clear or translucent.
• the amphiphilic compound is mixed with drug and a dispersion is prepared by vortexing and probe sonicating the mixture or by high-pressure homogenisation of the mixture.
• the drug is a hydrophobic drug.
• a hydrophobic drug is one which is poorly soluble in aqueous media, such as water.
• poorly soluble drugs is meant where one gram of a drug requires more than 10,000 ml of solvent (water) to be solubilised. Alternatively, this means a drug which has a solubility of less than O.l mgmL' 1 in water.
• the hydrophobic drug is typically encapsulated by the amphiphilic carbohydrate compound.
• the hydrophobic drug may be an analgesic, antibiotic, anticoagulant, antidepressant, anticarcinogen, anticarcinoma, anti-inflammatory, antihistamine, antiemetic, anxiolytic, anticonvulsive, antipsychotic, antipyretic, antiviral, antidiabetic, sedative, antihypertensive or a cardiovascular drug.
• the hydrophobic drug may act as a diuretic or antidiuretic, chronotrope, inotrope, decongestant, bronchodilator, anticholinergic, antithrombotic, antimicrobial or antifungal.
• a drug When a drug is present, it may be an anti-viral drug, which further potentiates the action of the amphipilic carbohydrate compound. Typically, the drug is effective against respiratory viruses.
• the amphiphilic carbohydrate compound of the invention is effective as an anti-viral agent.
• the virus may be for instance selected from coronaviruses, influenza and ebola, herpes viruses, adenoviruses and enteroviruses.
• the compound may be effective against human herpes virus 1 or influenza A.
• the amphiphilic carbohydrate compound is used in the prevention and/or treatment of infections caused by alphacoronaviruses or betacoronaviruses.
• the coronavirus may be severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), otherwise known as Covid-19.
• amphiphilic carbohydrate compound of the invention may be effective via inhibition of viral entry into cells.
• Polymers of the invention may be useful in the treatment and prevention of respiratory infections manifested by insufficiency and impairment of respiratory functions in humans or animals, for instance in the treatment and prevention of infections of the upper respiratory tract, lower respiratory tract infections, croup in children, gastrointestinal infections, infections of the nervous system, and Kawasaki disease.
• the drug, if present with the amphiphilic carbohydrate is preferably present at a concentration in the range 0.001 -10% w/v. When concentrations are expressed in % w/v, this means the amount of solid, in g, contained in 100mL or 100g of composition.
• the ratio of amphiphilic carbohydrate compound to drug (if present) is within the range of 1 :1 to 50:1 , more preferably 1 :1 to 20:1 .
• the ratio of amphiphilic carbohydrate compound to drug to pharmaceutically acceptable carrier may be about 1 -40mg:1 mg:1g, for instance 1 - 5 mg: 1 mg: 1g.
• the pharmaceutical composition may be in the form of any of the following: tablets, suppositories, liquid capsule, powder form, or a form suitable for pulmonary or nasal delivery.
• typically used carriers include sucrose, lactose, mannitol, maltitol, dextran, corn starch, typical lubricants such as magnesium stearate, preservatives such as paraben, sorbin, anti-oxidants such as ascorbic acid, a- tocopheral, cysteine, disintegrators or binders.
• effective diluents include lactose and dry corn starch.
• a liquid for oral use includes syrup, suspension, solution and emulsion, which may contain a typical inert diluent used in this field, such as water, in addition, sweeteners or flavours may be contained.
• Suppositories may be prepared by admixing the compounds of the present invention with a suitable non-irritative excipient such as those that are solid at normal temperature but become liquid at the temperature in the intestine and melt in the rectum to release the active ingredient, such as cocoa butter and polyethylene glycols.
• a suitable non-irritative excipient such as those that are solid at normal temperature but become liquid at the temperature in the intestine and melt in the rectum to release the active ingredient, such as cocoa butter and polyethylene glycols.
• Additional ingredients that may be included in the formulation include tonicity enhancers, preservatives, solubilisers, non-toxic excipients, demulcents, sequestering agents, pH adjusting agents, co-solvents and viscosity building agents.
• Tonicity is adjusted if needed typically by tonicity enhancing agents.
• Such agents may, for example be of ionic and/or non-ionic type.
• ionic tonicity enhancers are, but are not limited to, alkali metal or earth metal halides, such as, for example, CaCI2, KBr, KOI, LiCI, Nal, NaBr or NaCI, Na2SO4 or boric acid.
• Non-ionic tonicity enhancing agents are, for example, urea, glycerol, sorbitol, mannitol, propylene glycol, or dextrose. These agents may also serve as the active agents in certain embodiments.
• buffers may be especially useful.
• the pH of the present solutions should be maintained within the range of 5.5 to 8.5,
• the challenge is that for ionic complexation to occur may require a more acidic or alkaline pH.
• Suitable buffers may be added, such as, but not limited to, boric acid, sodium borate, potassium citrate, citric acid, sodium bicarbonate, TRIS, and various mixed phosphate buffers (including combinations of Na2HPO4, NaH2PO4 and KH2PO4) and mixtures thereof.
• buffers will be used in amounts ranging from about 0.05 to 2.5 percent by weight
• the pharmaceutical composition may be formulated for administration by any route, for instance, oropharyngeal, oral, parenteral, nasal, by inhalation, topical ocular or topical.
• the drug is preferably delivered to the human or animal body by intranasal or oropharyngeal delivery.
• the formulation may be a powder or liquid dispersion.
• Formulation of the pharmaceutical composition into a nasal and oropharyngeal spray for intranasal or oropharyngeal delivery is particularly preferred.
• the delivery of the nasal and/or oropharyngeal spray may be accomplished by a spay device such as a NaltosTM nasal delivery device (Alchemy Pharmatech).
• the dose can be determined on age, body weight, administration time, administration method, combination of drugs, the severity of the clinical condition orthe actual condition for which a patient is undergoing therapy and other factors. While the daily doses may vary depending on the conditions and body weight of patients, the species or active ingredient, and administration route, in the case of oral use, the daily doses may be about 0.1 mg - 2 g/person/day, or from 0.5-1000 mg/person/day or from 5 - 500 mg/person/day or from 10 - 250 mg/person/day or from 25 - 200 mg/person/day.
• the current invention is aimed at inhibiting viral infections and/ or limiting their severity and is focused on the use of polymers as viral inhibition agents and their use for the treatment or prophylaxis of viral infections.
• compositions of the present invention may reduce viral replication such that the titre of virus measured post treatment as compared to pre-treatment is at least twofold or threefold or fourfold or fivefold or tenfold or one hundredfold less.
• the compositions of the present invention may reduce viral replication such that the titre of virus measured post treatment as compared to pretreatment is between 10 to 90% or 10 to 75% or 10 to 50% or 10 to 25% that of the pretreatment titre.
• DMEM - Dulbecco's Modified Eagle Medium (Thermo Fisher Scientific, Tru) supplemented with 2-3% heat-inactivated Foetal Bovine Serum (FBS) (Thermo Fisher Scientific, Tru), penicillin (100 U/mL) (Thermo Fisher Scientific, Tru) and streptomycin (100 pg/mL) (Thermo Fisher Scientific, Poland), and ciproflocaxin (5 pg/mL);
• FBS Foetal Bovine Serum
• penicillin 100 U/mL
• streptomycin 100 pg/mL
• ciproflocaxin 5 pg/mL
• Cultures MucilAirTM Human Airway Epithelial (HAE) cultures were used for the ex vivo analysis (Epithelix Sari, Switzerland). All cultured were carried out at 37°C under 5% CO2; e.
• mice Transgenic mice expressing human ACE2 protein under cytokeratin 18 promoter (Jackson laboratory, USA) f. XTT cell viability kit (Biological Industries, Israel) g. Viral DNA/RNA isolation kit (A&A Biotechnology, Tru) h. High-capacity cDNA reverse transcription kit (Thermo Fisher Scientific, Tru) i. Real-time qPCR kit (RT-HS-PCR mix probe, A&A Biotechnology, Poland) j. Real-time qPCR oligonucleotides are listed in Table 1.
• GCPQs used in the experiments are listed in Table 2.
• the compounds were resuspended in 1 x PBS to the final concentration of 5 mg/mL. All stocks were stored at 4°C.
• Mock controls (cell lysate without the virus) and medium (supplemented DMEM - please see above) controls were included. Each compound was present during and after the infection. The cells were then incubated for 2 hours at 37°C and 5% CO2. Afterward, the cells were washed three times with PBS, and each compound was re-applied into the cell monolayer. 100pL of the cell culture supernatants were subsequently collected from each designated well after two days of culture at 37°C and in a 5% CO2 environment. The experiments were carried out in triplicate.
• Virus replication inhibition in HAE was evaluated by infecting MucilAirTM with SARS-CoV-2 isolate 026V-03883 ⁇ in vitro) virus at 5000 TCID50/mL in the presence of GCPQa or PBS.
• GCPQa diluted in PBS was added to the apical side of the insert (200 g/ml or 500 pg/ml) and incubated at 37°C for 30 minutes before the infection. After the pre-incubation was completed, the compound was removed and fresh dilutions of the compound with the virus were added and incubated for 2 hours at 37°C.
• RNA samples were prepared with a High Capacity cDNA Reverse Transcription Kit according to the manufacturer’s instructions. Briefly, 5pL of isolated RNA was mixed with 2 x concentrated RT-PCR mix and incubated as follows: 10 min at 25°C, 2 hrs at 37°C, 5 min at 85°C. cDNA was stored at -20°C until use. Viral RNA yield was assessed using real-time PCR (7500 Fast Real-Time PCR; Life Technologies, Poland). cDNA was amplified in a reaction mixture containing 1 x qPCR Master Mix (A&A Biotechnology, Poland), in the presence of probe (100nM) and primers (450nM each), sequences provided in Table 1.
• the reaction was carried out according to the scheme: 2 min at 50°C and 10 min at 92°C, followed by 40 cycles of 15 s at 92°C and 1 min at 60°C.
• DNA standards were prepared, as described before (Milewska, A. et al. Replication of Severe Acute Respiratory Syndrome Coronavirus 2 in Human Respiratory Epithelium. J Virol 94, doi:10.1128/JVL00957-20 (2020)).
• the number of viral RNA copies/mL was calculated by comparing the value obtained for each well with that of serial dilutions of samples containing a known number of cDNA copies/mL (standards).
• the difference in viral yield was also analysed as the log removal value (LRV), showing the relative decrease in the amount of virus in cell culture media compared to the control.
• GCPQ-BH (20 mg) and 100 mg GCPQ were dissolved in methanol with stirring, then the methanol was removed under vacuum and Tris-HCL buffer (25 mM, pH 4.8, 1 .8 mL) was added to the dry film to produce a final concentration of 66.7 mg/mL.
• Tris-HCL buffer 25 mM, pH 4.8, 1 .8 mL
• This solution was then added to a tube containing the I 125 (1 mCi, 17 Ci/mg, 0.392 nmol, Perkin-Elmer, USA) and four iodination beads® (Thermo Scientific Pierce, UK). The reaction was incubated for 1.5 hours at room temperature, after which the reaction was terminated by separating the solution from beads.
• PD Spin Trap G-25 Columns (GE Healthcare Life Sciences, UK), that are prepared by vortexing and discarding of the eluting storage buffer by centrifuging (2800 rpm for 1 min), were used in order to remove the free iodine (with the free iodine removed through the addition of 50 pL of the reaction per column and centrifuging at 2,800 rpm for 2 min).
• the eluent was placed in Amicon ultra centrifugal filters (3 kDa, Millipore, USA) with 200 pL H2O, and was subject to repeated washes (through centrifuging at 10,000 rpm for 10 min), until the washed out water produced negligible counts.
• SPECT/CT scans of the mouse head at 30 min, 2h 30 min and 24 h after nasal administration were acquired (Figure 4) using a NanoSPECT/CT scanner (Mediso, Hungary).
• SPECT images were obtained over 30 minutes using a 4-head scanner with nine 1 .4 mm pinhole apertures in helical scan mode with a time per view of 60 seconds.
• CT images were subsequently acquired using a 45 kilo volt peak (kVp) X-ray source, 500 ms exposure time in 180 projections, a pitch of 0.5 with an acquisition time of 4:30 minutes.
• Body temperature was maintained by a warm air blower and the respiration and core body temperature was monitored throughout.
• CT images were reconstructed using Bioscan InVivoScope (Bioscan, USA) software in voxel size 124 x 124 x 124 pm, whereas SPECT images were reconstructed using HiSPECT (ScivisGmbH, Bioscan) in a 256 x 256 matrix. Images were fused and analysed using VivoQuant (Invicro, A Konica Minolta Company) software. 3D Regions of Interest (ROIs) were created for the uptake within the nares for each time point and the activity calculated as the percentage of the administered dose. Representative images are scaled the same (same min and max). After the final scan the mouse was sacrificed and the entire head of the mouse analysed using a curimeter (Capintech, Mirion Technologies, UK) for ex vivo validation of 125 l concentration.
• a curimeter Capintech, Mirion Technologies, UK
• mice expressing the human ACE2 protein under the human cytokeratin 18 promoter were experimented for antiviral activity against SARS-CoV-2 (Munchen-1.2 2020/984).
• the mice were quarantined for at least 7 days prior to the experiment.
• GPCQa was administered once daily intranasally (20 mg/kg per day).
• the treatment control group received remdesivir intramuscularly (25 mg/kg per day).
• SARS-CoV-2 virus Unchen-1.2 2020/984; 5 pl to each nostril
• FIG. 5 shows the antiviral activity in the brains and from the respiratory tract swabs following nasal dosing of GCPQ and Remdesivir.
• GCPQs The antiviral activity of GCPQs was analysed on Vero E6 and A549/ACE2+ cells. Each analysis was performed in triplicate. The results of the experiment are presented in Figure 2. The analysis demonstrated that the presence of chitosans GCPQa and GCPQc at nontoxic concentrations significantly hamper SARS-CoV-2 replication in vitro. GCPQa and GCPQc showed anti-coronaviral activity at non-toxic concentrations. GCPQa showed the highest toxicity and anti-SARS-CoV-2 potential ( ⁇ 3 to 4 logs decrease in viral load at 10 pg/mL).
• the initial viral titre used in these experiments was significantly higher than contained in human infective influenza breath samples (3 X 10 5 pfu in this study vs influenza qPCR RNA copy numbers of 3.8 x 10 4 in a 30 minute fine aerosol breath sample and 1.2 x 104 in a 30 minutes coarse aerosol breath sample according to Yan et al. (Yan, J. et al. Infectious virus in exhaled breath of symptomatic seasonal influenza cases from a college community. Proceed Natl Acad Sci 115, 1081, doi:10.1073/pnas.1716561115 (2016).
• the assay according to Figure 5 showed an inhibition of SARS-CoV2 replication in the presence of GCPQ at non-toxic concentrations in both the respiratory tract swabs and brains relative to the control. Relative to the Remdisivir, the average number of RNA copies with GCPQ in the respiratory tract swabs was almost level, but overall gave slightly less inhibition. Further, in the brains assay, the average inhibition with GCPQ was greater than the Remdisivir, however the error bars were larger owing to a single result with high SARS-CoV-2 copies/ml.
• the high initial viral titre (comparatively higher than contained in a human infective droplet) in this model means that it is more difficult to prevent viral replication in tissues as GCPQ is not absorbed into these areas.
• the trend towards a reduction of the viral titres in the nasal and respiratory passages provide evidence that GCPQ is likely to limit viral transmission and indeed act as a prophylactic.
• a derivatised chitosan compound with a 6-O-glycol group (lacking in HTCC), a hydrophobic acyl group (lacking in both HTCC and HM-HTCC; the latter derivatised with N-dodecyl groups), a lower molecular weight than HTCC, a trimethyl quaternary ammonium group directly in place of the C2 amine group in chitosan, unlike HTCC (which has the hydroxypropyltrimethylammonium group attached to the C2 nitrogen), and a lower level of quaternary ammonium substitution ( ⁇ 40 mole%) than HTCC, is effectively able to inhibit viral entry into cells. Large structural differences between the prior art and the compounds of the present invention would not lead to the expectation that the compounds of the present invention would exhibit anti-viral activity.
• a low molecular weight clearly promotes activity against SARS-COV-2 in mammalian cells (Table 2 and Figures 2 - 3) and this is correlated with the ease with which this polymer may be incorporated into aqueous media.
• Underivatised glycol chitosan of molecular weights 40 - 100 kDa were not active (data not shown), demonstrating that quaternary ammonium and possibly palmitoyl groups are important determinants of activity.
• GCPQ possesses advantages for use in viral inhibition and specifically the clinical prevention of viral infections as GCPQ is mucoadhesive, has a long residence time in the nares ( Figure 4) and is chemically stable for at least 18 months.
• GCPQ also self assembles into nanoparticles and these nanoparticles may be clustered into microparticles for nasal delivery.
• GCPQ may therefore be used as a molecular mask nasal spray for the prevention of coronavirus infections.
• Reduction in brain levels of the virus (Figure 5) provide encouraging evidence that there is a possibility that the neurological symptoms experienced with SARS-COV-2 infections as reported in The Lancet Neurology (“Long COVID: understanding the neurological effects”, The Lancet Neurol., 20, 247, 2021 ) may indeed be reduced with the use of the anti-viral prophylactic.
• MMS019 After treatment with MMS019, there was a marked inhibition of viral replication in the mouse nasal passages, and decreased levels of the virus were also recorded for the brain tissue, indicating the limited systemic infection. No adverse effects were observed during the experiment.
• GCPQ As GCPQ’s activity may not be predicated specifically on the recognition of particular epitopes but appears to be based on electrostatic interactions between GCPQ and the virus, GCPQ may be applied to a wide variety of viral infections. These advantages mean that GCPQ polymer may be used as a nasal spray or by other means for the prevention and treatment of other specific viral infections, in addition to SARS-CoV-2.

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