WO2023046680A1 - Combination of cetylpyridinium and benzydamine with virucidal effect on sars-cov-2 - Google Patents

Abstract

The invention relates to a pharmaceutical dosage form comprising (i) a physiologically acceptable cetylpyridinium salt and (ii) benzydamine or a physiologically acceptable salt thereof for use in the prevention and/or treatment of a coronavirus infection. Preferably, the coronavirus is a severe acute respiratory syndrome-related coronavirus, more preferably a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

Description

Combination of cetylpyridinium and benzydamine with virucidal effect on SARS-CoV-2

[0001] Priority is claimed of European patent application no. 21198100.6 filed on September 21, 2021.

[0002] The invention relates to a pharmaceutical dosage form comprising (i) a physiologically acceptable cetylpyridinium salt and (ii) benzydamine or a physiologically acceptable salt thereof for use in the prevention and/or treatment of a coronavirus infection in early phases of infection. Preferably, the coronavirus is a severe acute respiratory syndrome-related coronavirus, more preferably a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0003] Respiratory tract infections are among the most common diseases that affect humans, with typical symptoms, such as rhinitis, cough, fever, and sore throat. Among the microbes, viruses are the most common causative agents of inflammation in the oral cavity and throat area. Bacterial respiratory tract infections are less common, and often develop after a viral infection. The most common viruses associated with respiratory tract infections are picomaviruses (human rhinoviruses (HRV)), coronaviruses (common human coronavirus (hCoV) OC43, 229E, NL63, HKU1), ortho- and paramyxoviruses (influenza viruses, parainfluenza viruses (PIV1-4)), and respiratory syncytial virus), respiratory adenoviruses and others (M. Morokutti-Kurz et al., Int J Gen Med. 2017, 10:53-60. LRA138592).

[0004] Many of the circulating human pathogenic viruses are enveloped with a lipid bilayer, and infect their target cells by inducing fusion of the viral envelope with the cell membrane. Many classes of enveloped viruses are human pathogens, including respiratory viruses such as influenza viruses (four genera in the Orthomyxovirus family: influenza A, B, C and D), the respiratory syncytial virus (RSV, Pneumoviridae family), coronaviruses (('oronaviridae family), and others (F.A. Rey et al. Cell, 2018, 172(6): 1319-1334).

[0005] Human coronaviruses are commonly circulating viruses, and they were considered the second most common viral agent responsible for 10-15% of all upper respiratory tract infections in humans (R. Eccles, Lancet Infect Dis, 2005, 5: 718-25). They replicate in the nasopharynx and generally cause mild, self-limited upper respiratory tract infections with short incubation periods, although lower tract respiratory infections and pneumonia have occasionally been described (S. Su et al., Trends Microbiol. 2016, 24(6):490-502).

[0006] Most respiratory tract infections are self-limiting and require no specific treatment. However, patients often use different self-medication therapies that can treat both the symptoms and the cause. [0007] Cetylpyridinium chloride, a quaternary ammonium compound, is an antiseptic with the actions and uses typical of cationic surface-active agents (surfactants). In addition to its emulsifying and detergent properties, quaternary ammonium compounds show bactericidal activity against gram-positive and, in higher concentrations, some gram-negative bacteria. They also have variable antifungal activity and are effective against some viruses (H. Hammacher, Selbstmedikation _a Mund- und Rachentherapeutika. Stuttgart: DAV 1990, 6/23-6/30, LRA077630; A. Kramer, Hagers Handbuch der Pharmazeutischen Praxis, 5. ed. 1990, Bd7, Springer, Berlin-Heidelberg). Their virucidal activities are not widely reported, although some reports against enveloped viruses were made in the literature relating to surface disinfection (J. Shirai et al, J Vet Med Sci 2000, 62(l):85-92). Cetylpyridinium chloride is usually used in medicated oral rinses, throat lozenges and sprays.

[0008] P.K. Mukherjee et al., BMC Infectious Diseases (2017) 17:74, 1-8 relates to a randomized, double-blind, placebo-controlled clinical trial to assess the safety and effectiveness of a dual-action oral topical formulation containing cetylpyridinium chloride against upper respiratory infections.

[0009] D.L. Popkin et al., Pathogens and Immunity, 2017;2(2):253-69 discloses that cetylpyridinium chloride exhibits potent, rapid activity against influenza viruses in vitro and in vivo.

[0010] WO 2005/046738 A2 relates to virucidal activities of cetylpyridinium chloride.

[0011] US 2011/0052510 Al relates to a tooth movement activating composition for use in orthodontics containing an antiseptic compound and an antibacterial agent.

[0012] CN 103 893 171 A relates to the technical field of medicines and in particular discloses an application of benzydamine hydrochloride in preparing a medicament for preventing or treating influenza virus infections.

[0013] V.V. Lebedeva et al. Epidemiology and Vaccine Prophylaxis. 2021; 20 (3): 83-89 reports about antiviral activity of benzydamine hydrochloride against SARS-CoV-2 in an in vitro model.

[0014] A fixed dose combination of cetylpyridinium chloride and benzydamine hydrochloride is commercially available under the tradenames Septolete® and Septabene®.

[0015] The fixed dose combination is provided in form of an oromucosal spray containing 1.5 mg/ml benzydamine hydrochloride and 5.0 mg/ml cetylpyridinium chloride in solution. One actuation contains cetylpyridinium chloride. The oromucosal spray medicine is intended for anti-inflammatory, analgesic and antiseptic treatment of irritations in the throat, mouth and gums, in gingivitis, pharyngitis and laryngitis and before and after tooth extractions. The oromucosal spray disinfects the mouth and throat and reduces signs of inflammation of throat such as pain, redness, swelling, heat and impaired function.

[0016] The fixed dose combination is further provided in form of lozenges containing 3 mg benzyda- mine hydrochloride and 1 mg cetylpyridinium chloride. The lozenge is anti-inflammatory, analgesic and antiseptic medicine for local oral use. The lozenge disinfects the mouth and throat and reduces signs of inflammation of throat such as pain, redness, swelling, heat and impaired function. The lozenge medicine is medicine is intended for anti-inflammatory, analgesic and antiseptic treatment of irritations in the throat, mouth and gums, in gingivitis, pharyngitis and laryngitis.

[0017] Throat lozenges with a fixed combination of benzydamine hydrochloride and cetylpyridinium chloride and other selected substances have been shown to provide effective symptomatic relief of irritations in the throat, mouth and gums. However, the effect of such combinations on viruses has not been investigated to date. The antiseptic cetylpyridinium chloride has already been described as a successful bactericide. In addition, there are some studies suggesting its efficacy against certain enveloped viruses.

[0018] After the emergence of more virulent coronaviruses, such as Severe Acute Respiratory Syndrome (SARS)-CoV-l in 2002, and Middle East Respiratory Syndrome (MERS)-CoV in 2012, it was recognized that coronaviruses can also cause severe pneumonias with longer incubation periods and often fatal outcome (E. de Wit et al., Nat Rev Microbiol. 2016, 14(8):523-34). As of late 2019, Coronavirus Disease 2019 (COVID- 19), caused by a novel corona virus, SARS-CoV-2, has resulted in a global pandemic with more than 144 million people infected and over three million deaths globally (Worldometer. Coronavirus Cases. 2021. https://www.worldometers.info/coronavirus/?zarsrc=130). Symptoms of COVID- 19 may vary from no symptoms being present to fever, cough, and severe illness, with difficulty breathing. Furthermore, the disease can cause serious medical conditions and can lead to death (S. Wan et al., J Med Virol., 2020, https://doi.org/ 10. 1002/jmv.25783; S.A. Hassan et al., Cureus, 2020, 12(3). https://doi.org/10. 7759/cureus. 7355).

[0019] Respiratory viruses are mainly transmitted via respiratory secretions during exhalation in the form of droplets, but indirect contact with contaminated fomites has also been shown to spread infection. A reduction in the viral load on the site of infection reduces the risk of transmission via both routes, while simultaneously reducing the symptoms of the patients and the potential spread of infection to the lower respiratory tract (M. Morokutti-Kurz et al., Int J Gen Med. 2017, 10:53-60. LRA138592). [0020] SARS-CoV-2 is transmited from human-to-human by either direct transmission such as cough, sneeze, and droplet inhalation or contact transmission like saliva, contact through mucous membranes of the mouth, nose, and eyes. SARS-CoV-2 uses ACE2 as the receptor and human proteases as entry activators; subsequently, it fuses the viral membrane with the cell membrane and achieves invasion. ACE-2 expression highly occurs in the oral mucosa epithelium, and the expression is more in the dorsum of the tongue (E. de Wit et al., Nat Rev Microbiol. 2016, 14(8):523-34; H. Xu et al., International Journal of Oral Science (2020) 12:8; B. Hu et al., Nature Reviews Microbiology, Vol. 19, 2021, 141-154; S. Senel et al., Drug Delivery and Translational Research (2021) 11: 1703-1734).

[0021] S. Perez-Errazuriz et al., Int. J. Odontostomat., 15(l):27-30, 2021 relates to cetylpyridinium chloride as a tool against COVID-19.

[0022] CN 112 043 701 A relates to application of three long -chain quaternary ammonium salt compounds or pharmaceutically acceptable salts thereof in preparing a medicament for inhibiting novel coronavirus 3 CL protease or SARS coronavirus 3 CL protease. The long-chain quaternary ammonium salt compound can be used for treating novel coronavirus or SARS coronavirus infection.

[0023] It has further been reported that toothpastes containing zinc or stannous and mouthwash formulas with cetylpyridinium chloride (CPC) neutralize the virus that causes COVID-19 by 99.9%. In the laboratory studies toothpaste neutralized 99.9% of the virus after two minutes of contact. Mouthwashes were similarly effective after 30 seconds (British Dental Journal, Vol. 229, No. 11, 2020, 753).

[0024] The route of SARS-CoV-2 infection is currently considered to be via respiratory droplets, and the virus particle is viable in aerosols for up to three hours (L. Zou et al., N Engl J Med 2020;382(12): 1177-1179; M. Varia et al., CMAJ 2003;169(4):285-292). Preventive measures such as hand hygiene, facial masks and social distancing have been the primary means of public infection control. To date, the relationship between the lung and throat viral load in terms of disease severity is unclear, nor is it known how the reduction in the viral load in the throat may affect the resulting lung disease or viral transmission. If it is expected that higher concentrations of the virus in the throat might increase the possibility of infecting others, then strategies to reduce the number of infective virus particles in mucous membranes could help reduce the risk of transmission. Therefore, when assuming that the throat is the major site of replication in early stages, the use of topical agents that could damage or destroy the lipid envelope of the virus has the potential to reduce the viral load in the oropharynx.

[0025] M.V. Mateos-Moreno et al., Journal of Hospital Infection 113 (2021) 30-43 relates to in vitro clinical evidence regarding oral antiseptics against coronavirus. [0026] A. Green et al., relates to an in vitro assessment of the virucidal activity of four mouthwashes containing cetylpyridinium chloride against human coronavirus.

[0027] F. Farrer et al., S Afir Pharm J 2011 78(4) 26-31 relates to sprays and lozenges for sore throats. Viral infections are said to be among the conditions that may cause sore throat symptoms. Andolex-C® is mentioned among other commercial throat lozenges.

[0028] Androlex-C® are commercialized lozenges against sore throat containing 3 mg benzydamine hydrochloride, 1.33 mg cetylpyridinium chloride, sugar (isomalt) and sweetener (sucrose).

[0029] IT MI 950 380 discloses lozenges comprising 1.0 mg cetylpyridinium chloride, 3.0 mg benzydamine hydrochloride, sorbitol, mannitol, magnesium stearate, mint flavor and aspartame.

[0030] The formulations of the prior art that are capable of neutralizing the virus that causes COVID- 19 are not satisfactory in every respect and there is a demand for improved formulations.

[0031] Reduction of coronavirus in the throat in early phases might prevent higher concentrations of the coronavirus in the throat and thereby prevent a more severe course of disease.

[0032] It is an object of the invention to provide specific formulations that are capable of very effectively neutralizing the virus that causes COVID-19 and other corona viruses and that have advantages compared to the formulations of the prior art. It is a further object of the invention to provide a specific combination that is capable of very effectively neutralizing the virus that causes COVID-19 and other corona viruses and that has advantages compared to the formulations or combinations of the prior art.

[0033] This object has been achieved by the subject matter of the patent claims.

[0034] It has been surprisingly found that in virucidal tests, conducted in controlled laboratory conditions for suspensions, the pharmaceutical dosage form according to the invention in a high concentration achieves a 10,000-fold reduction in infective virus concentration (99.99% reduction), i.e., a 4-log reduction.

[0035] At high concentration (lozenge dissolved in 4.0 ml, acidic pH value), the pharmaceutical dosage form according to the invention provided in form of a lozenge shows a significantly faster virucidal effect compared to cetylpyridinium chloride provided as a free active substance alone (neutral pH value). While the inventive formulation, particularly the inventive combination of cetylpyridinium salt and ben- zydamine or a physiologically acceptable salt thereof, e.g. the inventive lozenge, achieves a 4-log reduction in virus concentration just one minute after contact, cetylpyridinium chloride as a free active substance alone achieves a similar reduction only 15 minutes after contact.

[0036] At medium concentration (lozenge dissolved in 20 ml, acidic pH value), the pharmaceutical dosage form according to the invention provided in form of a lozenge achieves a 4-log reduction in virus concentration between 5 and 15 minutes after contact. This is a concentration that can theoretically be achieved in the actual use of a throat lozenge when dissolved in saliva. If taking the whole saliva flow rate of approximately 4.0-5.0 ml/min into account during eating, chewing and other stimulating activities (e.g., in lozenge application) (G. lorgulescu et al., J Med Life 2009, 2(3) :303— 307) and considering the time needed to dissolve the lozenge (on average, 5 minutes) and the constant release of active substances from the lozenge, this medium concentration of test products (test products suspended in 20 ml) could hypothetically be achieved in the actual use of a throat lozenge in the mouth.

[0037] Further, it has been surprisingly found that the highly effective virucidal activity of the inventive combination, either as lozenge or free active substance, has a more rapid effect compared to cetylpyridinium chloride alone. Without wishing to be bound to any scientific theory, it seems that benzydamine hydrochloride, i.e., the additional component within the inventive combination, has an unexpected effect on the destabilization and loss of infectivity of viral particles.

[0038] Still further, it has been surprisingly found that acidic conditions influence the virucidal dynamics. It seems that the low pH value of the inventive combination of cetylpyridinium chloride and benzydamine hydrochloride lozenge suspension additionally contributes with a synergistic effect on the very rapid loss of virus infectivity.

[0039] It has been surprisingly found that the lozenge medicine that conventionally is intended for antiinflammatory, analgesic and antiseptic treatment of irritations in the throat, mouth and gums, in gingivitis, pharyngitis and laryngitis is also suitable to reduce viral activities

[0040] A first aspect of the invention relates to a pharmaceutical dosage form comprising

(i) a physiologically acceptable cetylpyridinium salt and

(ii) benzydamine or a physiologically acceptable salt thereof for use in the prevention and/or treatment of a coronavirus infection. [0041] Another aspect of the invention relates to the use of (i) a physiologically acceptable cetylpyridinium salt and (ii) benzydamine or a physiologically acceptable salt thereof for the manufacture of a pharmaceutical dosage form for the prevention and/or treatment of a coronavirus infection.

[0042] Another aspect of the invention relates to a method for the prevention and/or treatment of a coronavirus infection, the method comprising administering to a subject in need thereof a pharmaceutical dosage form comprising (i) an effective amount of a physiologically acceptable cetylpyridinium salt and (ii) an effective amount of benzydamine or a physiologically acceptable salt thereof.

[0043] The pharmaceutical dosage form according to the invention contains a physiologically acceptable cetylpyridinium salt, preferably cetylpyridinium chloride.

[0044] Cetylpyridinium (1-hexadecylpyridin-l-ium) is a cation that is typically provided in form of its salt with chloride, i.e. as cetylpyridinium chloride (C21H38CIN, CAS 123-03-5, ATC B05CA01):

[0045] In preferred embodiments, the weight content of cetylpyridinium salt, expressed on the basis of the equivalent weight of cetylpyridinium chloride, is at least 0.2 mg, preferably at least 0.4 mg, more preferably at least 0.6 mg, still more preferably at least 0.8 mg.

[0046] In preferred embodiments, the weight content of cetylpyridinium salt, expressed on the basis of the equivalent weight of cetylpyridinium chloride, is at most 1.8 mg, preferably at most 1.6 mg, more preferably at most 1.4 mg, still more preferably at most 1.2 mg.

[0047] The pharmaceutical dosage form according to the invention contains benzydamine or a physiologically acceptable salt thereof, preferably benzydamine hydrochloride.

[0048] Benzydamine (l-Benzyl-3-dimethylaminopropoxy-lH-indazole, C19H23N3O, CAS 642.72-8, ATC A01AD02, G02CC03, M01AX07, M02AA05, R02AX03) has the following structure:

[0049] It is available as the hydrochloride salt. It is a locally acting nonsteroidal anti-inflammatory drug (NSAID) with local anesthetic and analgesic properties for pain relief and anti-inflammatory treatment of inflammatory conditions of the mouth and throat.

[0050] In preferred embodiments, the weight content of benzydamine, expressed on the basis of the equivalent weight of benzydamine hydrochloride, is at least 1.0 mg, preferably at least 1.5 mg, more preferably at least 2.0 mg, still more preferably at least 2.5 mg.

[0051] In preferred embodiments, the weight content of benzydamine, expressed on the basis of the equivalent weight of benzydamine hydrochloride, is at most 5.0 mg, preferably at most 4.5 mg, more preferably at most 4.0 mg, still more preferably at most 3.5 mg.

[0052] In preferred embodiments, particularly when the pharmaceutical dosage form according to the invention is provided in form of lozenges, the weight content of benzydamine expressed on the basis of the equivalent weight of benzydamine hydrochloride is greater than the weight content of cetylpyridinium salt expressed on the basis of the equivalent weight of cetylpyridinium chloride.

[0053] In other preferred embodiments, particularly when the pharmaceutical dosage form according to the invention is provided in form of a sprayable liquid, the weight content of cetylpyridinium salt expressed on the basis of the equivalent weight of benzydamine hydrochloride is greater than the weight content of benzydamine expressed on the basis of the equivalent weight of benzydamine hydrochloride.

[0054] In preferred embodiments, the relative weight ratio of cetylpyridinium salt to benzydamine, expressed on the basis of the equivalent weight of benzydamine hydrochloride and on the basis of the equivalent weight of cetylpyridinium chloride, is within the range of from 11: 1 to 1:7, preferably 10: 1 to 1:6, more preferably 9: 1 to 1:5, still more preferably 8: 1 to 1:4, yet more preferably 7: 1 to 1:3, even more preferably 6 : 1 to 1:2, most preferably 5 : 1 to 1: 1, and in particular from 4 : 1 to 2 : 1.

[0055] While it is contemplated that besides the cetylpyridinium salt and the benzydamine or physiologically acceptable salt thereof the pharmaceutical dosage form according to the invention may contain additional active ingredients, the cetylpyridinium salt and the benzydamine or physiologically acceptable salt thereof are preferably the sole active ingredients that are contained in the pharmaceutical dosage form according to the invention.

[0056] The pharmaceutical dosage form according to the invention is for use in the prevention and/or treatment of a coronavirus infection. Preferably, the pharmaceutical dosage form according to the invention is likewise for use in the prevention and/or treatment of a coronavirus disease. [0057] While it is contemplated that the pharmaceutical dosage form according to the invention may be used for the prevention and/or treatment of a coronavirus disease, the invention preferably targets the prevention and/or treatment of a coronavirus infection per se, i.e. an aspect preceding the development of an infectious coronavirus disease. Of course, any prevention and/or treatment of a coronavirus infection in a subject who does (not yet) suffer from an infectious coronavirus disease also prevents such infectious coronavirus disease, because the infectious coronavirus disease requires a preceding coronavirus infection.

[0058] For the purpose of the invention, "coronavirus infection" preferably involves the invasion of an organism's body tissues by a coronavirus. Preferably, "coronavirus infection" additionally involves the multiplication of the coronavirus by the host's cells. Preferably, "coronavirus disease" is a disease or disorder resulting from the coronavirus infection and thus refers to a different, subsequent aspect of a coronavirus infection.

[0059] In preferred embodiments, the coronavirus is selected from the group consisting of the coronaviruses NL63, 229E, HKU1, OC43, SARS-CoV, and MERS-CoV.

[0060] In particularly preferred embodiments, the coronavirus is a severe acute respiratory syndrome- related coronavirus (SARS-CoV); preferably a severe acute respiratory syndrome coronavirus 2 (SARS- CoV-2).

[0061] In preferred embodiments, the coronavirus is a mutant of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) selected from WHO labels Alpha, Beta, Gamma and Delta.

[0062] In preferred embodiments, the coronavirus is a mutant of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) selected from the group consisting of PANGO Cluster 5, Lineage B.l.1.7, Lineage B.1.1.207, Lineage B. 1.1.317, Lineage B. 1.1.318, Lineage B.1.351, Lineage B.1.429, Lineage B.1.525, Lineage B.1.526, Lineage B.1.617.2, Lineage B.1.618, Lineage P. l, and Lineage P.3.

[0063] In preferred embodiments, the prevention and/or treatment of a coronavirus infection according to the invention involves administering the pharmaceutical dosage form to a subject who has

(a) neither signs nor symptoms of a coronavirus disease, nor detectable viral load; or

(b) neither signs nor symptoms of a coronavirus disease, but a detectable viral load; or

(c) signs and/or symptoms of a coronavirus disease as well as detectable viral load. [0064] According to above embodiment (a), administering the pharmaceutical dosage form according to the invention preferably serves the purpose of neutralizing released coronavirus in oral cavity, the throat and/or the pharynx , and preferably also preventing development of a coronavirus disease. When the subject being treated in accordance with the invention is infected by a coronavirus, the pharmaceutical dosage form according to the invention preferably neutralizes free coronavirus, at least locally in the oral cavity, the throat and/or the pharynx, thereby preventing the coronavirus infection. Preferably, as a consequence of the treatment according to the invention, less coronavirus invades the organism's body tissues.

[0065] According to above embodiment (b), administering the pharmaceutical dosage form according to the invention preferably serves the purpose of reducing the viral load in oral cavity, the throat and/or the pharynx, and preventing the spread of coronavirus infection, and preferably also reducing the development of a coronavirus disease. The subject being treated in accordance with the invention is already infected by the coronavirus and the coronavirus is multiplicated by the host's cells such that a viral load is detectable. The pharmaceutical dosage form according to the invention preferably reduces the coronavirus load, at least locally in the oral cavity, the throat and/or the pharynx, thereby reducing the quantity of the coronavirus, which could invade the host's cells. Therefore, the pharmaceutical dosage form according to the invention preferably prevents the development of a coronavirus disease in the subject that is being treated and preferably also reduces the risk that said subject infects other subjects with the coronavirus.

[0066] According to above embodiment (c), administering the pharmaceutical dosage form according to the invention preferably also serves the purpose of reducing the viral load in oral cavity, the throat and/or the pharynx, and preventing the spread of coronavirus infection,, and preferably also reducing the symptoms of a coronavirus disease. The subject being treated in accordance with the invention is already infected by the coronavirus and the coronavirus is multiplicated by the host's cells such that a viral load is detectable. Further, the coronavirus disease has already developed such that the subject has signs and/or symptoms of a coronavirus disease. The pharmaceutical dosage form according to the invention preferably reduces the viral load, at least locally in the oral cavity, the throat and/or the pharynx, thereby reducing the quantity of coronavirus which could invade the host's cells. Therefore, the pharmaceutical dosage form according to the invention preferably could reduce the symptoms of the coronavirus disease in the subject that is being treated and preferably also reduces the risk that said subject infects other subjects with the coronavirus.

[0067] The pharmaceutical dosage form according to the invention preferably reduces the coronavirus load thereby reducing or terminating the quantity of the coronavirus which could invade the host's cells. Therefore, the pharmaceutical dosage form according to the invention neutralizes or reduces the viral load in the oral cavity, the throat and/or the pharynx in the subject that is being treated and preferably also reduces the risk that said subject infects other subjects with the coronavirus.

[0068] Suitable means for detecting coronavirus are known to the skilled person and include but are not limited to antibody tests, such as SARS-CoV-2 rapid antibody tests based upon chromatographic immune assays. Other tests are PCR test. Preferably, coronavirus and SARS CoV-2 virus in a subject are determined by means of PCR tests.

[0069] In preferred embodiments, the prevention and/or treatment according to the invention involves reducing viral load.

[0070] In preferred embodiments, the prevention and/or treatment according to the invention involves reducing the infectability of the subject receiving the pharmaceutical dosage form according to the invention.

[0071] In preferred embodiments, the prevention and/or treatment according to the invention involves reducing the infectiousness (infectivity) of the subject receiving the pharmaceutical dosage form according to the invention, i.e. reducing the risk that said subject infects other subjects with the coronavirus.

[0072] In preferred embodiments, the prevention and/or treatment involves improving signs and/or symptoms of a coronavirus disease.

[0073] Characteristic signs and symptoms of corona virus disease (COVID) and COVID- 19 are known to the skilled person and include but are not limited to severe acute respiratory syndrome, cough, fever, cold, disturbance of olfactory sense, disturbance of gustatory sense, pneumonia, sore throat, breathlessness (dyspnea), headache, limb pain, anorexia, weight loss, nausea, vomiting, diarrhea, stomach ache, conjunctivitis, skin rash, sentinel node swelling, apathy, and somnolence.

[0074] In a preferred embodiment, the subject suffers from chronic corona virus disease syndrome (CCS), which is also known as "long COVID", "post-acute sequelae of SARS-CoV-2 infection", "postacute sequelae of COVID-19 (PASC)", or "long-haul COVID" .

[0075] In preferred embodiments, the subject suffers from (i) acute COVID-19 (signs and symptoms of COVID- 19 for up to 4 weeks); (ii) ongoing symptomatic COVID- 19 (signs and symptoms of COVID- 19 from 4 to 12 weeks); (iii) post-COVID-19 syndrome (signs and symptoms that develop during or after an infection consistent with COVID- 19, continue for more than 12 weeks and are not explained by an alternative diagnosis); or (iv) long COVID (signs and symptoms that continue or develop after acute COVID-19; includes both ongoing symptomatic COVID-19 and post-COVID-19 syndrome, i.e. (ii) and (iii) defined above). The above stages (i) to (iv) of COVID-19 are defined in accordance with the COVID-19 rapid guideline: managing the long-term effects of COVID-19, NICE guideline [NG188], published on 18 December 2020.

[0076] In preferred embodiments, the combination of cetylpyridinium chloride and benzydamine hydrochloride exhibits virucidal activity in in vitro environment on SARS CoV-2 virus strain, i.e. exhibits significant reduction of the infective virus concentration. Moreover, virucidal test in controlled laboratory conditions for combination of cetylpyridinium chloride and benzydamine hydrochloride exhibits faster (minutes wise) in vitro virucidal activity compared to the cetylpyridinium chloride suspension alone.

[0077] The combination of cetylpyridinium chloride and benzydamine hydrochloride is indicated for anti-inflammatory, analgesic and antiseptic treatment of irritations in the throat, mouth and gums, in gingivitis, pharyngitis and laryngitis and before and after tooth extractions.

[0078] Preferably, the pharmaceutical dosage form according to the invention is administered locally and/or topically; preferably to the pharynx, the throat and/or the oral cavity; more preferably to a region of the oral cavity selected from teeth, buccal mucosa, sublingual mucosa, gingival mucosa, soft palate, hard palate, tongue, and any combination thereof.

[0079] The pharmaceutical dosage form according to the invention may contain conventional excipients that are known to the skilled person such as carriers, fillers, diluents, binders, matrix polymers, glidants, lubricants, disintegrants, surfactants, pH modifiers, osmolarity modifiers, moisture scavengers, humidifiers, antioxidants, preservatives, chelating agents, flavoring agents, sweetening agents, taste masking agents, colorants, and the like.

[0080] Excipients are typically contained in conventional amounts.

[0081] In preferred embodiments, the pharmaceutical dosage form according to the invention comprises a flavoring agent.

[0082] Preferably, the flavoring agent is selected from the group consisting of lime, orange, lemon, black current, blood orange, cranberry, cloudberry, goji berry, raspberry, strawberry, wild strawberry, sea buckthorn, cherry, melon, kiwi, papaya, pineapple, passion fruit, coconut, and other flavors such as honey, herbs, tea, anise, water grass, lemon grass, cooling agent ginger, coffee, mangosten, peppermint, spearmint, wintergreen, cinnamon, cacao/cocoa, vanilla, liquorice, salt, pepper, chili, menthol, aniseeds, mint, menthol, levomethol, menthyl acetate, menthyl lactate, camphor, eucalyptus, eucalyptus oil, eucalyptol, anethole, eugenol, cassia, oxanone, x-irisone, propenyl guaiethol, thymol, linalool, benzaldehyde, cinnamaldehyde, N-ethyl-p-menthan-3-carboxamine, N,2,3-trimethyl-2-isopropylbutanamide, 3- l-menthoxypropane-l,2-diol, cinnamaldehyde glycerol acetal (CGA), methone glycerol acetal (MGA) and mixtures thereof.

[0083] Preferably, the flavoring agent is eucalyptus oil and/or levomenthol.

[0084] In preferred embodiments, the pharmaceutical dosage form according to the invention comprises a salivary stimulating agent. The salivary stimulating agent may also serve additional purposes such as acidifying agent, chelating agent, preservative, and the like.

[0085] In preferred embodiments, the pharmaceutical dosage form according to the invention comprises an acid. The acid may be organic or inorganic. When the benzydamine is present in form of a physiologically acceptable salt thereof, it is typically present in form of an acid addition salt. Under these circumstances, the acid, which is preferably contained in the pharmaceutical dosage form according to the invention, preferably differs from the acid that forms the acid addition salt with the benzydamine.

[0086] In preferred embodiments, the acid is a carboxylic acid, preferably an aliphatic carboxylic acid, more preferably selected from the group consisting of aliphatic carboxylic acids, aliphatic hydroxy carboxylic acids, aliphatic dicarboxylic acids, aliphatic hydroxy dicarboxylic acids, aliphatic tricarboxylic acids, and aliphatic hydroxy tricarboxylic acids.

[0087] Preferably, the carboxylic acid is selected from citric acid, lactic acid, glycolic acid acetic acid, succinic acid, fumaric acid, and any combination thereof.

[0088] In particularly preferred embodiments, the carboxylic acid is citric acid, more preferably anhydrous citric acid (E330).

[0089] In preferred embodiments, the pharmaceutical dosage form according to the invention comprises a sweetening agent.

[0090] Preferably, the sweetening agent is selected from the group consisting of synthetic or natural sugars; artificial sweeteners including saccharin, sodium saccharin, aspartame, acesulfame, thaumatin, glycyrrhizin, sucralose, cyclamate, dihydrochalcone, alitame, miraculin and monellin; sugar alcohols including sorbitol, mannitol, glycerol, lactitol, maltitol, and xylitol; sucrose, dextrose, fructose and lactose; isomalt, Stevia, and mixtures thereof.

[0091] Preferably, the sweetening agent is sucralose (E955) and/or isomalt (E953).

[0092] In preferred embodiments, the pharmaceutical dosage form according to the invention is muco- adhesive.

[0093] In preferred embodiments, the pharmaceutical dosage form according to the invention is a liquid. Preferred liquids are selected from solutions, syrups, suspensions, emulsions, mouth washes, gargles, and sprayable liquids.

[0094] In a particularly preferred embodiment, the pharmaceutical dosage form according to the invention is provided in form of an oromucosal spray.

[0095] In preferred embodiments, the pharmaceutical dosage form according to the invention is solid or semisolid.

[0096] Preferred solids or semisolids are selected from oral therapeutic systems, chewing gums, pastes, gels, fdms, wafers, powders, lyophilizates, granules, pellets, tablets, orodispersible tablets, buccal tablets, sublingual tablets, capsules, troches, pastilles, and lozenges.

[0097] In a particularly preferred embodiments, the pharmaceutical dosage form according to the invention is provided in form of troches, pastilles, or lozenges.

[0098] In a particularly preferred embodiments, the pharmaceutical dosage form according to the invention is provided in form of lozenges, preferably selected from compressed lozenge tablets, hard candy lozenges, chewy or caramel based medicated lozenges, soft lozenges, and center filled hard lozenges.

[0099] In a particularly preferred embodiments, the pharmaceutical dosage form according to the invention is a lozenge comprising

3.0 mg benzydamine hydrochloride;

1.0 mg cetylpyridinium chloride; citric acid; optionally, sucralose; optionally, isomalt; optionally eucalyptus oil; optionally, levomenthol; and optionally, brilliant blue.

[0100] In preferred embodiments, the pharmaceutical dosage form according to the invention after being dissolved or dispersed in a volume of 4 ml purified water at 23 °C provides a pH value of at most 9.0, preferably at most 8.5, more preferably at most 8.0, still more preferably at most 7.5, yet more preferably at most 7.0, even more preferably at most 6.5, most preferably at most 6.0, and in particular at most 5.5.

[0101] In preferred embodiments, the pharmaceutical dosage form according to the invention after being dissolved or dispersed in a volume of 4 ml purified water at 23 °C provides a pH value of at most 5.0, preferably at most 4.5, more preferably at most 4.0, still more preferably at most 3.5, yet more preferably at most 3.0.

[0102] In preferred embodiments, the pharmaceutical dosage form according to the invention after being dissolved or dispersed in a volume of 4 ml purified water at 23 °C provides a pH value within the range of 4.0±3.5, preferably 3.5±3.0, more preferably 3.0±2.5, still more preferably 3.0±2.0, yet more preferably 3.0±1.5, even more preferably 3.0±1.0, and most preferably 3.0±0.5.

[0103] In preferred embodiments, the pharmaceutical dosage form according to the invention is administered once daily, twice daily, three times daily or four times daily.

[0104] Preferably, the pharmaceutical dosage form according to the invention is administered three to four times daily, preferably every 3 to 6 hours.

[0105] The following examples further illustrate the invention but are not to be construed as limiting its scope.

Examples

[0106] The virucidal activity on SARS-CoV-2 was examined in vitro for (a) cetylpyridinium chloride alone; (b) a combination of cetylpyridinium chloride and benzydamine hydrochloride as free substances; and (c) a combination of cetylpyridinium chloride and benzydamine hydrochloride contained in a lozenge.

[0107] Under in-laboratory simulated conditions of lozenge administration, SARS-CoV-2 was incubated for 1 min, 5 min and 15 min of contact time with three different concentrations of each of the active substances, (a) cetylpyridinium chloride alone, (b) the free combination of cetylpyridinium chloride and benzydamine hydrochloride, and (c) a lozenge suspension of the combination of cetylpyridinium chloride and benzydamine hydrochloride. Infective viral particles were detected in cell cultures and the viral titer was calculated accordingly.

Cell preparation

[0108] Seven-day-old Vero E6 cells (passage 13-16) were trypsinized and resuspended in the Dulbec- co's Minimal Essential Medium (DMEM) (Thermo Fisher Scientific, Waltham, MA), supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich, St. Louis, MO), to obtain a final concentration of IxlO⁵ cells/ml. A total of 100 pl of prepared cell suspension was then transferred in each well of the 96- well cell culture plate and incubated overnight at 37°C and 5% CO2 to reach the 80% cell confluence required for quantitative suspension tests.

Virus isolation and quantification

[0109] SARS-CoV-2 was isolated from a throat swab of a patient diagnosed with CO VID-19 in Vero E6 cells cultivated with DMEM, supplemented with 10% FBS. The virus was passaged 5 times on Vero E6, aliquoted and stored at -80°C until further use.

[0110] The virus concentration was determined with the endpoint dilution assay and expressed as fifty- percent Tissue Culture Infectious Dose/ml (TCID50/ml), calculated with the Spearman and Karber method. The stock concentration used in all experiments was 1.17xl0⁹ TCID50/ml.

Preparation of reagents

[0111] The following reagents were tested:

(a) cetylpyridinium chloride (CPC) alone;

(b) a combination of cetylpyridinium chloride (CPC) and benzydamine hydrochloride (BH) as free substances;

(c) a combination of cetylpyridinium chloride (CPC) and benzydamine hydrochloride (BH) contained in a lozenge; and

(d) placebo lozenge.

[0112] The interfering substance was prepared by first dissolving 3 g of bovine albumin in 97 ml of water. Next, 97 ml of the prepared and filtrated bovine albumin solution was mixed with 3 ml of sheep erythrocytes (BioSap SEA, BioGnost) to achieve the final composition of interfering substance. Hard water was freshly prepared under aseptic conditions on the day of the experiment according to SIST EN 14476:2013+A2:2019 “Chemical disinfectants and antiseptics - Quantitative suspension test for the evaluation of virucidal activity in the medical area” (standard) and was used within 12h (SIST EN 14476:2013+A2:2019 Chemical disinfectants and antiseptics - Quantitative suspension test for the evaluation of virucidal activity in the medical area - Test method and requirements (Phase 2/Step 1); valid from 01.09.2019).

[0113] For each active substance, three different concentrations were prepared by dissolving the tested substance in either 4 ml (high concentration), 20 ml (medium concentration) or 30 ml (low concentration) of hard water. All active substance compositions and experimental conditions are presented in the table here below (in each case test temperature: 37°C; interfering substance 2: 3.0 g/1 bovine albumin solution and 3.0 ml/1 erythrocytes; contact time: 1, 5 and 15 min):

BH - benzydamine hydrochloride; CPC - cetylpyridinium chloride

Quantitative suspension test for the evaluation of virucidal activity

[0114] To mimic the physicochemical properties of the human oral cavity, several factors, such as temperature, saliva composition (interfering substance) and the amount of saliva produced during lozenges dissolution the dynamics of lozenges dissolution (different virus-substance contact time), were taken into consideration. Experimental procedures were carried out, in accordance with the standard requirements.

[0115] An active substance was first dissolved in hard water and then added to a suspension of 1: 10 ratio of SARS-CoV-2 and an interfering substance. The mixture was maintained at 37°C for different incubation periods (1 min, 5 min and 15 min). At the end of each contact time, an aliquot of incubated suspension was diluted in an ice-cold cell maintenance medium DMEM with 2% FBS to immediately suppress the virucidal action of the active substance used. The prepared dilutions, from 1: 10 to 1: 10⁸ of the initial virus-active substance suspension, were transferred onto Vero E6 cell monolayer in 96-well microtiter plates and incubated for 7 days at 37°C and 5% CO2. After incubation, the plates were screened for CPE and the titers of infectivity were calculated according to Spearman and Karber (Karber, G., 1931. Beitrag zur kollektiven Behandlung pharmakologischer Reihenversuche. Arch. Exp. Path. Pharmak. Vol.162, pp. 480-487. Springer doi: 10.1007/BF01863914).

[0116] For every SARS-CoV-2 aliquot used, a virus titration test was performed immediately after thawing, and 15 min later to evaluate the effect of thawing on virus concentration and to determine the exact working virus concentrations for each experiment.

Control experiments

[0117] In addition to experiments evaluating the virucidal effect of active substances on SARS-CoV- 2, control experiments were also carried out in accordance with the standard.

[0118] Each experiment included untreated cells that served as a negative control, providing information on cell viability throughout whole incubation period.

[0119] Virus viability test was performed for each quantitative suspension test to examine the stability of the virus in the maintenance medium and to evaluate the virus infectivity under experimental conditions during all contact times. A mixture of 0.1 ml of viral suspension, 0.1 ml of interfering substance and 0.8 ml of hard water was incubated at 37°C for 15 minutes. A volume of 0. 1 ml was sampled at the beginning and after 15 minutes of incubation. Ten-fold serial dilutions were prepared in DMEM supplemented with 1% FBS, and 100 pl of prepared serial dilutions were added per well in eight replicas.

[0120] To avoid false positive results due to the morphological changes caused by the cytotoxic effect of the active substance on Vero E6 cells under experimental conditions, we determined the highest concentration of the tested substance that causes no visible morphological changes in the cells. First, 10- fold dilutions of the tested substance were prepared in the maintenance medium and 100 pl of each dilution was transferred to the cell monolayer in eight replicates. Cells were incubated for 48h and screened for any morphological changes.

[0121] The inhibiting effect of ice-cold media on the virucidal activity of the tested product was also determined. A suspension of 0. 1 ml of interfering substance, 0. 1 ml of maintenance medium and 0.8 ml of test product was prepared. Then, 0.1 ml of prepared mixture was transferred to 0.8 ml of ice-cold maintenance medium and placed at 4°C. Next, 0.1 ml of viral suspension was added to the ice-cold suspension and incubated on ice for 15 minutes. After incubation, serial dilutions were prepared and transferred onto cell culture in eight replicas.

[0122] To determine the impact of the highest non-cytotoxic concentration of the active substance on the cell susceptibility to the virus, we prepared 10-fold dilutions of the tested substances in maintenance medium, removed the culture medium and added 100 pl of the prepared active substance dilution onto cell monolayers in eight replicates. After Ih of incubation at 37 ° C and 5% CO2, supernatants were removed, and cells were washed with maintenance medium and inoculated with prepared 10-fold serial dilutions of SARS-CoV-2.

[0123] All the control experiments were carried out at the same time and under the same conditions as the quantitative suspension tests.

Electron microscopy

[0124] The effect of cetylpyridinium chloride as a free active substance, and of combination of cetylpyridinium chloride and benzydamine hydrochloride as a lozenge, on virus morphology was additionally evaluated with electron microscopy. Virus particles were visualized under the transmission electron microscope after exposure to cetylpyridinium chloride and a combination of cetylpyridinium chloride and benzydamine hydrochloride lozenge in high-concentration suspensions. An 800-pl test-product suspension (or maintenance medium for negative control) was prepared, and 100 pl of the interfering substance and 100 pl of the virus suspension were added. After a contact time of 15 minutes at 37 °C, samples were prepared for electron microscopy. Electron-microscopy grids (400-mesh copper grids, coated with Formvar and carbon-supported) were prepared with ultracentrifugation in the Airfuge system. The prepared grids with concentrated viral particles were negatively contrasted with 2% phosphotungstic acid and examined under the transmission electron microscope at from 30,000- to 100,000-fold magnification.

Statistical analysis

[0125] To evaluate the effect of active substances on SARS-CoV-2 concentration and infectivity, the calculated virus concentrations after all three incubation periods were compared with different active substances using an independent t-test in the SPSS software tool.

Results of control procedures [0126] Cell and virus control were performed continuously for each test. All controls passed the criteria.

[0127] Virus test suspension was incubated for 15 minutes, which is the total incubation time in the test procedure. It was performed in parallel with each virucidal test. In the observed time period, there was no significant reduction in the infective virus concentration. When testing the low concentration for a combination of benzydamine hydrochloride and cetylpyridinium chloride as a free active substance and placebo lozenge, a critical decrease in the concentration of infectious viruses (value 1 of the logarithmic level) was achieved. Results are shown in the table here below:

CPC - cetylpyridinium chloride; BH - benzydamine hydrochloride; CI - confidence interval; TCID50

- 50% Tissue Culture Infectious Dose

[0128] The test for product activity suppression in ice-cold media was performed for a high concentration of the test products. Results are shown in table below. The inhibitory effect of the ice-cold media on the action of the test product, used in a high concentration against viral infectivity, was confirmed. The virus titer before and after exposure did not differ significantly, nor did it approach the difference of 1 log concentration value given as the cut-off value by the standard guidelines.

[0129] The suppression efficiency of test product's activity is shown in the table here below:

CPC - cetylpyridinium chloride; BH - benzydamine hydrochloride; CI - confidence interval; TCID50

- 50% Tissue Culture Infectious Dose

[0130] Interference control was performed in two separate procedures: first for cetylpyridinium chloride as a free active substance and a combination of cetylpyridinium chloride and benzydamine hydrochloride lozenge, and then for combination of cetylpyridinium chloride and benzydamine hydrochloride as a free active substance and a placebo lozenge. Virus concentration in the exposed cells was slightly lower compared to virus concentration in non-exposed cells. However, the difference was still acceptable and within the 1 log difference. The results indicated that the test products had no major impact on cell susceptibility to virus infection.

[0131] With regard to cytotoxic effects, dilutions 10 ¹ to 10’¹¹ of the initial concentration were tested, and the cytotoxic effect (deformation of cells and destruction of cell monolayer) was observed in dilutions 10 ¹ and 10’². The virus propagation effect was checked only in the cells inoculated with test product dilutions ranging from 10’³ to 10’¹¹. This observation was considered in the interpretation of results (cytotoxic level). The level of cytotoxicity with respect to virus dilution was at 4.5 loglO(c) (TCID50/ml) for undiluted combination of cetylpyridinium chloride and benzydamine hydrochloride as a free active substance, the combination of cetylpyridinium chloride and benzydamine hydrochloride lozenge, and the placebo lozenge. For all other test products in medium and at a low concentration, and cetylpyridinium chloride as a free active substance in a high concentration, the level of cytotoxicity was 3.5 loglO(c) (TCID50/ml).

Results of test product effectiveness on virus inactivation

[0132] The virus was exposed to each of the three tested substance concentrations for contact times of 1, 5 and 15 min. Each test was performed in duplicate on different days. The graphical results of SARS- CoV-2 exposure to the test product suspension at a high concentration and for long contact times are presented in Figure 1, showing the virucidal effects of a specific test product.

[0133] Figure 1 shows the results with respect to SARS-CoV-2 exposure to the test product suspension in high concentration and contact times; CPC — cetylpyridinium chloride; BH — benzydamine hydrochloride; TCID50 — 50% Tissue Culture Infectious Dose. [0134] Numerical test results of virucidal activity against SARS-CoV-2 are shown in the table here below:

¹ TCIDso/ml, CL - cytotoxic level, CPC - cetylpyridinium chloride; BH - benzydamine hydrochloride; CI - 95% confidence interval

[0135] With regards to effects of test products in high concentration, the following was observed:

[0136] Free active substance: Cetylpyridinium chloride exhibited a mean loglO reduction in viral titer of 0.00±1.06 at 1 minute, 1.88±0.96 at 5- minutes and 4.94±0.75 loglO at 15 minutes of exposure, combination of cetylpyridinium chloride and benzydamine hydrochloride exhibited results with a mean loglO reduction of 1.57±0.86, >4.49±0.67 and >4.49±0.67 at 1, 5 and 15 minutes of exposure, respectively.

[0137] Lozenge: Combination of cetylpyridinium chloride and benzydamine hydrochloride exhibited a mean loglO reduction in viral titer of 4.32±0.82 as early as at 1 minute of exposure; at 5 and 15 minutes of exposure, the reductions were >4.44±0.75. [0138] The placebo lozenge exhibited considerably smaller reductions. At 1 minute of exposure, an increase in viral titer was observed, with values 0.25±l .01, followed by the reduction of 2.00±0.92 and 2.88±0.83 at 5 and 15 minutes, respectively.

[0139] The negative control did not exhibit any negative effect on virus titer.

[0140] Test product concentrations with pH values and log reduction of infective virus concentration for each of the contact times are shown in the table here below:

CPC - cetylpyridinium chloride; BH - benzydamine hydrochloride; CI - confidence interval; * statistical significant reduction of virus titer (p<0.05)

[0141] With regard to effects of test products in medium concentration, the following was observed:

[0142] Free active substance: With cetylpyridinium chloride as a free active substance, a mean loglO reduction of 0.06±0.99, 0.75±0.98 and 2.44±0.79 was observed at 1, 5 and 15 minutes of exposure, respectively. With the combination of cetylpyridinium chloride and benzydamine hydrochloride, values were 1.96±0.95, 3.30±0.96 and 5.63±0.77 at 1, 5 and 15 minutes of exposure, respectively.

[0143] Lozenge: Combination of cetylpyridinium chloride and benzydamine hydrochloride exhibited a mean loglO reduction of 1.69±0.96, 3.56±0.86 and 5.00±0.68 at 1, 5 and 15 minutes of exposure, respectively. The placebo lozenge exhibited reductions of 0.81±0.92, 1.06±0.98 and 0.65±1.05 at 1, 5 and 15 minutes of exposure, respectively. The negative control did not exhibit any negative effect on virus titer.

[0144] With regard to effects of test products in low concentration, the following was observed:

[0145] Free active substance: Cetylpyridinium chloride exhibited loglO reductions of 0.32±1.08, 0.70±1.04 and 1.69±1.03 at 1, 5 and 15 minutes of exposure, respectively. With the combination of cetylpyridinium chloride and benzydamine hydrochloride combination, reductions were 0.89±1.03, 1.58±0.98 and 2.44±0.94.

[0146] Lozenge: With of the combination of cetylpyridinium chloride and benzydamine hydrochloride, a mean loglO reduction of 0.88±l.l l, 1.19±1.03 and 3.01±1.05 was observed at 1, 5 and 15 minutes of exposure, respectively. The placebo lozenge exhibited an increase in viral titer of 0. 13±1.06, at 1 minute of exposure, and reduction of 0.46±0.98 and 1.46±0.97 at 5 and 15 minutes, respectively. The negative control did not exhibit any negative effect on the virus titer.

Electron microscopy analysis

[0147] The negative control samples observed under the electron microscope contained intact virus particles with no visible morphological changes or damage; clearly expressed peplomers of the S protein (corona) were present, and an intact viral envelope was recorded (Figure 2A,B). On the contrary, in samples exposed to high concentration of test products, cetylpyridinium chloride (as a free active substance) (Figure 2C,D) or a combination of cetylpyridinium chloride and benzydamine hydrochloride lozenge (Figure 2E,F) a destruction of the viral envelope is visible, with unclear peplomers formation or even without peplomers and envelope. Virus exposure to the test products severely damaged the outer layer of the viral envelope, which is the most likely the reason for loss of viral infectivity. In some damaged viruses, the internal nucleocapsid is exposed and visible. The viral envelope destruction is also noted, with a contrast accumulation in the virus internal site (black virus center).

[0148] Figure 2 shows the electron micrographs of viral particles after ultracentrifugation and negative contrast. A, B — viral particles in maintenance media, not exposed to test product; 50,000-fold (A) and 80,000-fold (B) magnification; intact SARS-CoV-2 particles with peplomers (corona). C, D — 15 minute exposure to high cetylpyridinium chloride concentration (0.25 mg/ml); 80,000-fold (C) and 100,000- fold (D) magnification; damaged viral particles, rarely visible peplomers, disrupted envelope and exposed internal nucleocapsid. E, F — 15 minute exposure to high concentration of a combination of cetylpyridinium chloride and benzydamine hydrochloride lozenge (1 lozenge in 4 ml); 60,000-fold (E) and 100,000-fold (F) magnification; outer-layer damage and accumulation of negative contrast agent inside the nucleocapsid (black virus center), rare peplomers, destruction of outer layer and nucleocapsid (F).

Summary of results and conclusions

[0149] As demonstrated, all active substances in high-concentration suspensions, as well as a medium concentration of the combination of cetylpyridinium chloride and benzydamine hydrochloride combination, exhibited a 4-log reduction in viral titer. Additionally, the highest concentration of combination of cetylpyridinium chloride and benzydamine hydrochloride as a lozenge containing an acid had a faster virucidal effect compared to cetylpyridinium chloride as a free active substance alone, since a contact time as short as 1 minute reduced the initial virus concentration by more than 4 log.

[0150] The lower pH value due to the presence of an acid in the lozenge also seems to have an effect. In the free active substance suspensions of cetylpyridinium chloride and combination of cetylpyridinium chloride and benzydamine hydrochloride, the pH value in all tested concentrations was, on average, 7.65 and 7.78, which is near neutral. However, for the combination of cetylpyridinium chloride and benzydamine hydrochloride lozenge, the pH value was, on average, 3.90, which is acidic.

[0151] The virucidal effect of cetylpyridinium chloride and the combination of cetylpyridinium chloride and benzydamine hydrochloride lozenge was confirmed in the morphology analysis of virus particles. After virus incubation in suspensions of cetylpyridinium chloride as a free active substance and of combination of cetylpyridinium chloride and benzydamine hydrochloride as a lozenge, a similar effect was noted, i.e., destruction of the virus envelope. With the combination of cetylpyridinium chloride and benzydamine hydrochloride lozenge, a destabilization of the nucleocapsid was observed.

[0152] The results demonstrate the effective strong virucidal effect of the lozenge, with the possibility of viral load reduction in the oral cavity and, consequently, reduced risk of viral transmission.

Claims

26 Patent claims:

1. A pharmaceutical dosage form comprising

(i) a physiologically acceptable cetylpyridinium salt and

(ii) benzydamine or a physiologically acceptable salt thereof for use in the prevention and/or treatment of a coronavirus infection.

2. The pharmaceutical dosage form for use according to claim 1, wherein the coronavirus is a severe acute respiratory syndrome-related coronavirus.

3. The pharmaceutical dosage form for use according to claim 1, wherein the coronavirus is a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

4. The pharmaceutical dosage form for use according to any of the preceding claims, wherein the prevention and/or treatment involves reducing viral load.

5. The pharmaceutical dosage form for use according to any of the preceding claims, wherein the prevention and/or treatment involves improving signs and/or symptoms of a coronavirus disease.

6. The pharmaceutical dosage form for use according to any of the preceding claims, wherein the pharmaceutical dosage form is administered locally.

7. The pharmaceutical dosage form for use according to any of the preceding claims, wherein the pharmaceutical dosage form is administered locally to the pharynx, the throat and/or the oral cavity.

8. The pharmaceutical dosage form for use according to any of the preceding claims, wherein the pharmaceutical dosage form is administered locally to a region of the oral cavity selected from teeth, buccal mucosa, sublingual mucosa, gingival mucosa, soft palate, hard palate, tongue, and any combination thereof.

9. The pharmaceutical dosage form for use according to any of the preceding claims, wherein

(i) the physiologically acceptable cetylpyridinium salt is cetylpyridinium chloride; and/or

(ii) the benzydamine is present in form of a physiologically acceptable salt thereof; preferably as benzydamine hydrochloride. The pharmaceutical dosage form for use according to any of the preceding claims, wherein the relative weight ratio of cetylpyridinium salt to benzydamine, expressed on the basis of the equivalent weight of benzydamine hydrochloride and on the basis of the equivalent weight of cetylpyridinium chloride, is within the range of from 11 : 1 to 1:7. The pharmaceutical dosage form for use according to any of the preceding claims, wherein the relative weight ratio of cetylpyridinium salt to benzydamine, expressed on the basis of the equivalent weight of benzydamine hydrochloride and on the basis of the equivalent weight of cetylpyridinium chloride, is within the range of from 10: 1 to 1:6. The pharmaceutical dosage form for use according to any of the preceding claims, wherein the relative weight ratio of cetylpyridinium salt to benzydamine, expressed on the basis of the equivalent weight of benzydamine hydrochloride and on the basis of the equivalent weight of cetylpyridinium chloride, is within the range of from 9: 1 to 1:5. The pharmaceutical dosage form for use according to any of the preceding claims, wherein the relative weight ratio of cetylpyridinium salt to benzydamine, expressed on the basis of the equivalent weight of benzydamine hydrochloride and on the basis of the equivalent weight of cetylpyridinium chloride, is within the range of from 8: 1 to 1:4. The pharmaceutical dosage form for use according to any of the preceding claims, wherein the relative weight ratio of cetylpyridinium salt to benzydamine, expressed on the basis of the equivalent weight of benzydamine hydrochloride and on the basis of the equivalent weight of cetylpyridinium chloride, is within the range of from 7: 1 to 1:3. The pharmaceutical dosage form for use according to any of the preceding claims, wherein the relative weight ratio of cetylpyridinium salt to benzydamine, expressed on the basis of the equivalent weight of benzydamine hydrochloride and on the basis of the equivalent weight of cetylpyridinium chloride, is within the range of from 6: 1 to 1:2. The pharmaceutical dosage form for use according to any of the preceding claims, wherein the relative weight ratio of cetylpyridinium salt to benzydamine, expressed on the basis of the equivalent weight of benzydamine hydrochloride and on the basis of the equivalent weight of cetylpyridinium chloride, is within the range of from 5 : 1 to 1: 1. The pharmaceutical dosage form for use according to any of the preceding claims, wherein the relative weight ratio of cetylpyridinium salt to benzydamine, expressed on the basis of the equivalent weight of benzydamine hydrochloride and on the basis of the equivalent weight of cetylpyridinium chloride, is within the range of from 4: 1 to 2: 1. The pharmaceutical dosage form for use according to any of the preceding claims, wherein the weight content of benzydamine, expressed on the basis of the equivalent weight of benzydamine hydrochloride, is at least 1.0 mg. The pharmaceutical dosage form for use according to any of the preceding claims, wherein the weight content of benzydamine, expressed on the basis of the equivalent weight of benzydamine hydrochloride, is at least 1.5 mg. The pharmaceutical dosage form for use according to any of the preceding claims, wherein the weight content of benzydamine, expressed on the basis of the equivalent weight of benzydamine hydrochloride, is at least 2.0 mg. The pharmaceutical dosage form for use according to any of the preceding claims, wherein the weight content of benzydamine, expressed on the basis of the equivalent weight of benzydamine hydrochloride, is at least 2.5 mg. The pharmaceutical dosage form for use according to any of the preceding claims, wherein the weight content of benzydamine, expressed on the basis of the equivalent weight of benzydamine hydrochloride, is at most 5.0 mg. The pharmaceutical dosage form for use according to any of the preceding claims, wherein the weight content of benzydamine, expressed on the basis of the equivalent weight of benzydamine hydrochloride, is at most 4.5 mg. The pharmaceutical dosage form for use according to any of the preceding claims, wherein the weight content of benzydamine, expressed on the basis of the equivalent weight of benzydamine hydrochloride, is at most 4.0 mg. The pharmaceutical dosage form for use according to any of the preceding claims, wherein the weight content of benzydamine, expressed on the basis of the equivalent weight of benzydamine hydrochloride, is at most 3.5 mg. 29 The pharmaceutical dosage form for use according to any of the preceding claims, wherein the weight content of cetylpyridinium salt, expressed on the basis of the equivalent weight of cetylpyridinium chloride, is at least 0.2 mg. The pharmaceutical dosage form for use according to any of the preceding claims, wherein the weight content of cetylpyridinium salt, expressed on the basis of the equivalent weight of cetylpyridinium chloride, is at least 0.4 mg. The pharmaceutical dosage form for use according to any of the preceding claims, wherein the weight content of cetylpyridinium salt, expressed on the basis of the equivalent weight of cetylpyridinium chloride, is at least 0.6 mg. The pharmaceutical dosage form for use according to any of the preceding claims, wherein the weight content of cetylpyridinium salt, expressed on the basis of the equivalent weight of cetylpyridinium chloride, is at least 0.8 mg. The pharmaceutical dosage form for use according to any of the preceding claims, wherein the weight content of cetylpyridinium salt, expressed on the basis of the equivalent weight of cetylpyridinium chloride, is at most 1.8 mg. The pharmaceutical dosage form for use according to any of the preceding claims, wherein the weight content of cetylpyridinium salt, expressed on the basis of the equivalent weight of cetylpyridinium chloride, is at most 1.6 mg. The pharmaceutical dosage form for use according to any of the preceding claims, wherein the weight content of cetylpyridinium salt, expressed on the basis of the equivalent weight of cetylpyridinium chloride, is at most 1.4 mg. The pharmaceutical dosage form for use according to any of the preceding claims, wherein the weight content of cetylpyridinium salt, expressed on the basis of the equivalent weight of cetylpyridinium chloride, is at most 1.2 mg. The pharmaceutical dosage form for use according to any of the preceding claims, which comprises a flavoring agent. The pharmaceutical dosage form for use according to claim 34, wherein the flavoring agent is selected from the group consisting of lime, orange, lemon, black current, blood orange, cranberry, 30 cloudberry, goji berry, raspberry, strawberry, wild strawberry, seabuckthorn, cherry, melon, kiwi, papaya, pineapple, passion fruit, coconut, and other flavors such as honey, herbs, tea, anise, water grass, lemon grass, cooling agent ginger, coffee, mangosten, peppermint, spearmint, wintergreen, cinnamon, cacao/cocoa, vanilla, liquorice, salt, pepper, chili, menthol, aniseeds, mint, menthol, levomethol, menthyl acetate, menthyl lactate, camphor, eucalyptus, eucalyptus oil, eucalyptol, anethole, eugenol, cassia, oxanone, x-irisone, propenyl guaiethol, thymol, linalool, benzaldehyde, cinnamaldehyde, N-ethyl-p-menthan-3-carboxamine, N,2,3-trimethyl-2-isopropylbutanamide, 3- l-menthoxypropane-l,2-diol, cinnamaldehyde glycerol acetal (CGA), methone glycerol acetal (MGA) and mixtures thereof; preferably eucalyptus oil and/or levomenthol. The pharmaceutical dosage form for use according to any of the preceding claims, which comprises a sweetening agent. The pharmaceutical dosage form for use according to claim 36, wherein the sweetening agent is selected from the group consisting of synthetic or natural sugars; artificial sweeteners including saccharin, sodium saccharin, aspartame, acesulfame, thaumatin, glycyrrhizin, sucralose, cyclamate, dihydrochalcone, alitame, miraculin and monellin; sugar alcohols including sorbitol, mannitol, glycerol, lactitol, maltitol, and xylitol; sucrose, dextrose, fructose and lactose; isomalt, Stevia, and mixtures thereof; preferably sucralose (E955) and/or isomalt (E953). The pharmaceutical dosage form for use according to any of the preceding claims, which comprises an acid. The pharmaceutical dosage form for use according to claim 38, wherein the acid is a carboxylic acid. The pharmaceutical dosage form for use according to claim 38 or 39, wherein the acid is an aliphatic carboxylic acid. The pharmaceutical dosage form for use according to any of claims 38 to 40, wherein the acid is selected from the group consisting of aliphatic carboxylic acids, aliphatic hydroxy carboxylic acids, aliphatic dicarboxylic acids, aliphatic hydroxy dicarboxylic acids, aliphatic tricarboxylic acids, and aliphatic hydroxy tricarboxylic acids. The pharmaceutical dosage form for use according to any of claims 38 to 41, wherein the acid is selected from the group consisting of citric acid, lactic acid, glycolic acid acetic acid, succinic 31 acid, fumaric acid, and any combination thereof; preferably citric acid; more preferably anhydrous citric acid (E330). The pharmaceutical dosage form for use according to any of the preceding claims, which after being dissolved or dispersed in a volume of 4 ml purified water at 23 °C provides a pH value of at most 9.0. The pharmaceutical dosage form for use according to any of the preceding claims, which after being dissolved or dispersed in a volume of 4 ml purified water at 23 °C provides a pH value of at most 8.5. The pharmaceutical dosage form for use according to any of the preceding claims, which after being dissolved or dispersed in a volume of 4 ml purified water at 23°C provides a pH value of at most 8.0. The pharmaceutical dosage form for use according to any of the preceding claims, which after being dissolved or dispersed in a volume of 4 ml purified water at 23 °C provides a pH value of at most 7.5. The pharmaceutical dosage form for use according to any of the preceding claims, which after being dissolved or dispersed in a volume of 4 ml purified water at 23 °C provides a pH value of at most 7.0. The pharmaceutical dosage form for use according to any of the preceding claims, which after being dissolved or dispersed in a volume of 4 ml purified water at 23°C provides a pH value of at most 6.5. The pharmaceutical dosage form for use according to any of the preceding claims, which after being dissolved or dispersed in a volume of 4 ml purified water at 23 °C provides a pH value of at most 6.0. The pharmaceutical dosage form for use according to any of the preceding claims, which after being dissolved or dispersed in a volume of 4 ml purified water at 23 °C provides a pH value of at most 5.5. 32 The pharmaceutical dosage form for use according to any of the preceding claims, which after being dissolved or dispersed in a volume of 4 ml purified water at 23°C provides a pH value within the range of 4.0±3.5. The pharmaceutical dosage form for use according to any of the preceding claims, which after being dissolved or dispersed in a volume of 4 ml purified water at 23°C provides a pH value within the range of 3.5±3.0. The pharmaceutical dosage form for use according to any of the preceding claims, which after being dissolved or dispersed in a volume of 4 ml purified water at 23°C provides a pH value within the range of 3.0±2.5. The pharmaceutical dosage form for use according to any of the preceding claims, which after being dissolved or dispersed in a volume of 4 ml purified water at 23°C provides a pH value within the range of 3.0±2.0. The pharmaceutical dosage form for use according to any of the preceding claims, which after being dissolved or dispersed in a volume of 4 ml purified water at 23°C provides a pH value within the range of 3.0± 1.5. The pharmaceutical dosage form for use according to any of the preceding claims, which after being dissolved or dispersed in a volume of 4 ml purified water at 23°C provides a pH value within the range of 3.0± 1.0. The pharmaceutical dosage form for use according to any of the preceding claims, which after being dissolved or dispersed in a volume of 4 ml purified water at 23°C provides a pH value within the range of 3.0±0.5. The pharmaceutical dosage form for use according to any of the preceding claims, which exhibits virucidal activity on SARS CoV-2 virus strain; preferably reduces the infective virus concentration. The pharmaceutical dosage form for use according to any of the preceding claims, which is additionally for use in anti-inflammatory, analgesic or antiseptic treatment of irritations in the throat, mouth and gums, in gingivitis, pharyngitis and laryngitis and before and after tooth extractions. 33 The pharmaceutical dosage form for use according to any of the preceding claims, which is a liquid. The pharmaceutical dosage form for use according to claim 60, wherein the liquid is selected from solutions, syrups, suspensions, emulsions, mouth washes, gargles, and sprayable liquids. The pharmaceutical dosage form for use according to any of claims 1 to 59, which is solid or semisolid. The pharmaceutical dosage form for use according to claim 62, wherein the solid or semisolid is selected from oral therapeutic systems, chewing gums, pastes, gels, fdms, wafers, powders, lyophilizates, granules, pellets, tablets, orodispersible tablets, buccal tablets, sublingual tablets, capsules, troches, pastilles, and lozenges. The pharmaceutical dosage form for use according to claim 62 or 63, wherein the solid or semisolid is selected from troches, pastilles, and lozenges. The pharmaceutical dosage form for use according to any of claims 62 to 64, wherein the solid or semisolid is selected from compressed lozenge tablets, hard candy lozenges, chewy or caramel based medicated lozenges, soft lozenges, and center filled hard lozenges. The pharmaceutical dosage form for use according to any of the preceding claims, which is a lozenge comprising

3.0 mg benzydamine hydrochloride;

1.0 mg cetylpyridinium chloride; citric acid; optionally, sucralose; optionally, isomalt; optionally eucalyptus oil; optionally, levomenthol; and optionally, brilliant blue.