WO2022112399A1

WO2022112399A1 - Compositions for the inactivation of sars-cov-2

Info

Publication number: WO2022112399A1
Application number: PCT/EP2021/082964
Authority: WO
Inventors: Florian FÖGER; Martin Werle; Kurt Zatloukal; Julia RIEGER
Original assignee: Cyprumed GmbH
Current assignee: Cyprumed GmbH
Priority date: 2020-11-25
Filing date: 2021-11-25
Publication date: 2022-06-02
Anticipated expiration: 2023-05-25

Abstract

The present invention relates to an aqueous composition having a pH in the range of 8.5 to 11 for use in inactivating SARS-CoV-2 and, thus, for use in treating or preventing COVID-19.

Description

Compositions for the inactivation of SARS-CoV-2

The present invention relates to an aqueous composition having a pH in the range of 8.5 to 11 for use in inactivating SARS-CoV-2 and, thus, for use in treating or preventing COVID-19. The invention further relates to an aqueous composition having a pH in the range of 8.5 to 11 for use in inactivating an enveloped virus and, accordingly, for use in treating or preventing a disease caused by, or associated with, an enveloped virus.

The family of enveloped viruses includes many of the most dangerous pathogenic viruses for humans and livestock, such as, e.g., human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HBC), and influenza virus (see, e.g., Rey FA et al., Cell , 2018, 172(6): 1319-34; or Vaney MC et al., Cell Microbiol, 2011, 13(10): 1451-9).

A particularly prominent member of this family is the recently emerged novel coronavirus called "severe acute respiratory syndrome coronavirus 2” (SARS-CoV-2) which causes an acute respiratory disease that has been named "coronavirus disease 2019” (COVID-19) and has led to a devastating pandemic (Li H et al., Lancet, 2020, 395(10235): 1517-20), with more than 250 million infections and more than 5 million deaths worldwide until November 2021 alone. There is hence an urgent need for novel and effective medical approaches to combat enveloped viruses, including in particular SARS-CoV-2.

The scientific literature teaches that SARS-CoV-2 is extremely stable over a wide range of pH values, i.e. at least from pH 3 to pH 10 (Chin AWH et al., Lancet Microbe, 2020, 1 (1): e10). A study on the viability of SARS-CoV-2 on environmental surfaces (Chan KH et al., J Hosp Infect, 2020, 106(2): 226-31) likewise concluded that "the virus remains viable under a wide range of pH and environmental conditions”. It has further been reported that SARS-CoV can only be inactivated at pH >12 which, however, does not allow any therapeutic application (Darnell MER et al., J Virol Methods, 2004, 121 (1): 85-91).

In the context of the present invention, it was surprisingly found that SARS-CoV-2 (and other enveloped viruses) can be inactivated by moderate alkaline pH compositions, such as aqueous compositions having a pH of 8.5 to 11. This finding was entirely unexpected and provides an advantageously simple and highly effective novel approach for inactivating SARS-CoV-2 (and other enveloped viruses) and, thus, for treating or preventing COVID-19 (as well as diseases caused by, or associated with, other enveloped viruses). Moreover, it was found that SARS-CoV-2 can be very rapidly inactivated using the aqueous composition according to the invention (as also demonstrated in Example 2), which makes this composition particularly well-suited for therapeutic or prophylactic use, e.g., as a nasal spray, a pharyngeal spray, or for inhalation.

In addition, it was found that aqueous compositions having a pH between 8.5 and 11 are well suitable for therapeutic applications (including for nasal, pharyngeal or pulmonary administration), as also demonstrated in Examples 7 and 8. These findings are further supported by prior reports that ciliary beat frequencies (CBF) of bronchi and bronchioles sampled from human lung resections were not significantly modified when the pH was increased up to 10.5 (Clary- Meinesz C et al., EurRespirJ, 1998, 11(2): 330-3). The present invention thus provides an aqueous composition having a pH in the range of 8.5 to 11 for use in inactivating SARS-CoV-2 or for use in treating or preventing COVID-19 in a subject.

In particular, the invention relates to an aqueous composition having a pH in the range of 8.5 to 11 for use in inactivating SARS-CoV-2 (e.g., in a human subject). The invention also relates to an aqueous composition having a pH in the range of 8.5 to 11 for use in treating or preventing a SARS-CoV-2 infection (e.g., in a human subject). The invention further relates to an aqueous composition having a pH in the range of 8.5 to 11 for use in treating or preventing COVID-19 (e.g., in a human subject).

The present invention likewise relates to the use of an aqueous composition having a pH in the range of 8.5 to 11 for the preparation of a medicament (or pharmaceutical composition) for inactivating SARS-CoV-2 or for treating or preventing COVID-19.

Moreover, the present invention provides a method of inactivating SARS-CoV-2, or treating or preventing COVID-19, the method comprising administering an aqueous composition having a pH in the range of 8.5 to 11 to a subject in need thereof (preferably a human). It will be understood that a therapeutically effective amount of the aqueous composition should be administered.

The aqueous composition according to the invention can further be used as a disinfectant for inactivating SARS-CoV- 2 on inanimate (non-living) surfaces, inanimate objects, in the air, or in a room. Accordingly, the invention relates to the non-therapeutic use of an aqueous composition having a pH in the range of 8.5 to 11 for inactivating SARS-CoV- 2. The invention also relates to the non-therapeutic use of an aqueous composition having a pH in the range of 8.5 to 11 as a disinfectant for inactivating SARS-CoV-2. In particular, the aqueous composition can be applied to an inanimate object, an inanimate surface, in the air, or as a room disinfectant for inactivating SARS-CoV-2.

The present invention furthermore relates to the inactivation of enveloped viruses other than SARS-CoV-2 and the treatment or prevention of diseases caused by, or associated with, such other enveloped viruses. Accordingly, in addition to the above-discussed aspects, the invention also provides an aqueous composition having a pH in the range of 8.5 to 11 for use in inactivating an enveloped virus or for use in treating or preventing a disease caused by or associated with an enveloped virus in a subject. In particular, the present invention relates to an aqueous composition having a pH in the range of 8.5 to 11 for use in inactivating an enveloped virus (e.g., in a human subject). The invention also relates to an aqueous composition having a pH in the range of 8.5 to 11 for use in treating or preventing an enveloped virus infection (e.g., in a human subject). The invention further relates to an aqueous composition having a pH in the range of 8.5 to 11 for use in treating or preventing a disease caused by or associated with an enveloped virus (e.g., in a human subject). The invention likewise relates to the use of an aqueous composition having a pH in the range of 8.5 to 11 for the preparation of a medicament (or pharmaceutical composition) for inactivating an enveloped virus or for treating or preventing a disease caused by or associated with an enveloped virus. Moreover, the present invention provides a method of inactivating an enveloped virus, or treating or preventing a disease caused by or associated with an enveloped virus, the method comprising administering an aqueous composition having a pH in the range of 8.5 to 11 to a subject in need thereof (preferably a human). It will be understood that a therapeutically effective amount of the aqueous composition should be administered. The aqueous composition according to the invention can further be used as a disinfectant for inactivating an enveloped virus on inanimate (non-living) surfaces, inanimate objects, in the air, or in a room. Accordingly, the invention also relates to the non-therapeutic use of an aqueous composition having a pH in the range of 8.5 to 11 for inactivating an enveloped virus. The invention furthermore relates to the non-therapeutic use of an aqueous composition having a pH in the range of 8.5 to 11 as a disinfectant for inactivating an enveloped virus. In particular, the aqueous composition can be applied to an inanimate object, an inanimate surface, in the air, or as a room disinfectant for inactivating an enveloped virus.

The following description relates each of the above-described aspects of the present invention (including each aspect relating to SARS-CoV-2 or COVID-19, and each aspect relating to enveloped viruses or enveloped virus infections).

The aqueous composition to be used in accordance with the invention has a pH in the range of 8.5 to 11 . For example, the aqueous composition may have a pH of 8.5, 9.0, 9.5, 10.0, 10.5, or 11.0, or any range in between any of the aforementioned pH values. It is preferred that the aqueous composition has a pH in the range of 8.5 to 10.5. More preferably, the aqueous composition has a pH in the range of 8.5 to 10.0. Even more preferably, the aqueous composition has a pH in the range of 9.0 to 10.0 (e.g., 9.0 to 9.5, or 9.5 to 10.0).

The aqueous composition may comprise any pharmaceutically acceptable buffer system. Preferably, the aqueous composition comprises a bicarbonate buffer, an arginine buffer, a lysine buffer, a tris(hydroxymethyl)aminomethane (“Tris” or trometamol) buffer, a citrate buffer, a phosphate buffer, a urea buffer, an ammonium chloride buffer, or any combination thereof. More preferably, the aqueous composition comprises a bicarbonate buffer, an arginine buffer, a lysine buffer, a Tris buffer, or any combination thereof. It is particularly preferred that the aqueous composition comprises a bicarbonate buffer. The aqueous composition may also comprise a bicarbonate buffer in combination with, e.g., arginine and/or lysine. The pH of the aqueous composition can be adjusted to the desired value, e.g., by addition of NaOH or HCI.

It is preferred that the aqueous composition comprises sodium bicarbonate (NaHCOs), arginine (particularly arginine free base, preferably L-arginine), lysine (particularly lysine free base, preferably L-lysine), and/or tris(hydroxymethyl)aminomethane (Tris). It is particularly preferred that the aqueous composition comprises sodium bicarbonate. As described in Examples 8 and 11, it was found that aqueous compositions containing sodium bicarbonate are particularly well tolerated by nasal cells and are therefore highly suitable for nasal administration. It is furthermore preferred that the aqueous composition comprises tris(hydroxymethyl)aminomethane (Tris), which was likewise found to be particularly well tolerated by nasal cells, as described in Example 10.

It will be understood that if a buffer is used in the aqueous composition, the buffer should be provided in a suitable buffer strength (buffer concentration), preferably in a buffer strength that is sufficient to maintain the corresponding pH when the aqueous composition is applied to a target environment, such as the nasal cavity of a subject/patient where the aqueous composition can come into contact with the subject's nasal fluid. An adequate buffer strength/buffer concentration can be readily determined by a person skilled in the art, e.g., using the approach described in Examples 13 and 14. Thus, by way of example, a bicarbonate (NaHCOs) buffer can be employed at a concentration of, e.g., >0.6% (w/v), preferably >1.0% (w/v), >1.5% (w/v), >2.0% (w/v), >3.0% (w/v), or >5.0% (w/v). Similarly, a Tris buffer can be employed at a concentration of, e.g., >3.5% (w/v), preferably >4.0% (w/v), >4.5% (w/v), >5.0% (w/v), >7.0% (w/v), or >10.0% (w/v).

While the use of a buffer is preferred (as described above), it is to be understood that this is not obligatory. Thus, the invention also encompasses aqueous compositions that do not contain any buffer system. For example, a diluted sodium hydroxide solution having a concentration of 0.01 mM NaOH (and a pH of 9) or of 0.1 mM NaOH (and a pH of 10) can also be used.

The aqueous composition may have an osmolality of, e.g., about 200 mOsm/kg to about 800 mOsm/kg, preferably an osmolality of about 250 mOsm/kg to about 500 mOsm/kg, more preferably an osmolality of about 280 mOsm/kg to about 315 mOsm/kg, even more preferably an osmolality of about 280 mOsm/kg to about 310 mOsm/kg, yet even more preferably an osmolality of about 285 mOsm/kg to about 305 mOsm/kg, and still more preferably an osmolality of about 290 mOsm/kg to about 300 mOsm/kg (e.g., about 296 mOsm/kg). The osmolality of the aqueous composition can be adjusted (e.g., to any of the aforementioned osmolality ranges or values) using, e.g., sodium chloride and/or any other suitable osmolality adjusting agent(s).

It will be understood that the aqueous composition according to the invention comprises water. Preferably, the aqueous composition at least about 60% (v/v) water, more preferably at least about 70% (v/v) water, even more preferably at least about 80% (v/v) water, even more preferably at least about 90% (v/v) water, yet even more preferably at least about 95% (v/v) water, and still more preferably at least about 98% (v/v) water, with respect to the total volume of the aqueous composition.

The aqueous composition may be, e.g., an aqueous solution or an oil-in-water emulsion. An example of a corresponding oil-in-water emulsion is an aqueous composition containing one or more essential oils (such as, e.g., eucalyptus oil, peppermint oil, tea tree oil, rosemary oil, thyme oil, lavender oil, rose oil, oregano oil, clary sage oil, garlic oil, Aloe vera oil, or any combination thereof). In general, it is preferred that the aqueous composition has an oil content of less than about 5% (v/v), more preferably of less than about 3% (v/v), even more preferably of less than about 2% (v/v), even more preferably of less than about 1% (v/v), even more preferably of less than about 0.5% (v/v), and yet even more preferably it does not contain any oil. Accordingly, it is preferred that the aqueous composition is an aqueous solution.

Moreover, the aqueous composition may further comprise: one or more sulfated polysaccharides, particularly a carrageenan (e.g., iota carrageenan and/or kappa carrageenan; preferably iota carrageenan); one or more anti inflammatory agents (e.g., dexamethasone and/or dexpanthenol); one or more vasoconstrictory agents (e.g., xylometazoline and/or oxymetazoline); one or more antiviral agents (e.g., remdesivir, favipiravir, APN01 (rhsACE2) and/or an antiviral antibody or an antiviral antibody fragment); one or more proteolytic enzymes (e.g., trypsin); one or more immune modulators; one or more minerals or trace elements (e.g., "Emser Salz”®; or a mixture containing the following ions: lithium (about 0.21 g/kg), sodium (about 308.7 g/kg), potassium (about 6.11 g/kg); magnesium (about 0.291 g/kg), calcium (about 0.016 g/kg), manganese (about 0.0001 g/kg), iron(ll) / iron(lll) (about 0.003 g/kg), fluoride (about 0.078 g/kg), chloride (about 188.4 g/kg), bromide (about 0.202 g/kg), iodide (about 0.005 g/kg), nitrate (about 0.355 g/kg), sulfate (about 9.24 g/kg), bicarbonate (about 474.4 g/kg), and carbonate (about 14.0 g/kg)); /oe vera\ honey; and/or hyaluronic acid or a pharmaceutically acceptable salt thereof (particularly sodium hyaluronate). It is preferred that the aqueous composition comprises a carrageenan, particularly iota carrageenan, which has been reported to inhibit SARS-CoV-2 (Bansal S et al., bioRxiv, 2020, doi: 10.1101/2020.08.19.225854) and has been demonstrated to be compatible with the aqueous composition according to the invention (see Examples 5 and 6). It is furthermore preferred that the aqueous composition comprises hyaluronic acid or a pharmaceutically acceptable salt thereof, particularly sodium hyaluronate.

The aqueous composition according to the invention can also be administered to a subject in combination with one or more further compositions (i.e., one or more separately formulated pharmaceutical compositions) comprising one or more sulfated polysaccharides (particularly a carrageenan, such as iota carrageenan and/or kappa carrageenan; preferably iota carrageenan), one or more anti-inflammatory agents (e.g., dexamethasone and/or dexpanthenol), one or more vasoconstrictory agents (e.g., xylometazoline and/or oxymetazoline), one or more antiviral agents (e.g., remdesivir, favipiravir, APN01 (rhsACE2) and/or an antiviral antibody or an antiviral antibody fragment), one or more proteolytic enzymes (e.g., trypsin), and/or one or more immune modulators. The respective compositions may be administered simultaneously/concomitantly or sequentially.

The aqueous composition according to the invention optionally further comprises one or more pharmaceutically acceptable excipients. Pharmaceutically acceptable excipients are well known in the art and include, e.g., carriers, diluents, fillers, disintegrants, lubricating agents, binders, colorants, pigments, stabilizers, preservatives, antioxidants, solubility enhancers etc., and can be suitably chosen depending on the intended pharmaceutical dosage form and the intended route of administration.

In particular, the aqueous composition may comprise one or more antimicrobial preservatives, such as, e.g., ethanol, benzyl alcohol, chlorobutanol, 2-ethoxyethanol, m-cresol, chlorocresol (e.g., 2-chloro-3-methyl-phenol or 4-chloro-3- methyl-phenol), benzalkonium chloride, benzethonium chloride, benzoic acid (or a pharmaceutically acceptable salt thereof), sorbic acid (or a pharmaceutically acceptable salt thereof), chlorhexidine, thimerosal, or any combination thereof.

The aqueous composition can be formulated as a pharmaceutical by techniques known to the person skilled in the art, such as, e.g., the techniques described in "Remington: The Science and Practice of Pharmacy”, Pharmaceutical Press, 22^nd edition. In particular, the aqueous composition can be formulated as a dosage form for nasal, pharyngeal, or pulmonary administration (e.g., through mouth and/or nose). Dosage forms for nasal administration include, e.g., a nasal spray (e.g., a nasal pump spray) or nasal drops. Dosage forms for pharyngeal administration include, e.g., a pharyngeal spray or pharyngeal drops. Dosage forms for pulmonary administration can be administered, e.g., via inhalation or insufflation, for example using an inhalation device, such as a metered dose inhaler (e.g., a metered dose inhaler releasing a fixed dose of medication in aerosol form into a patient's lungs), a dry powder inhaler, a soft mist inhaler, or a nebulizer (e.g., a nebulizer that delivers medication through a mouthpiece or face mask, or a nebulizer with a vibrating mesh that turns liquid medication into a fine particle mist). Administration of the aqueous composition into the lungs can also be achieved via a mechanical ventilator, application in a face mask, application in an open breathing mask (e.g., which actively sprays an aerosol at specific times of the respiratory cycle), or application as a solution or pre-concentrate.

It is preferred that the aqueous composition is administered to the subject via the nasal route, the pharyngeal route, or the pulmonary route. Accordingly, it is preferred that the aqueous composition is provided in the form of a nasal spray, nasal drops, a pharyngeal spray, pharyngeal drops, or a lung inhalation composition.

The aqueous composition according to the invention may be used to irreversibly inactivate SARS-CoV-2. Moreover, the aqueous composition is highly advantageous in that it allows to inactivate SARS-CoV-2 extremely rapidly (e.g., within 5 minutes or less, or even within 1 minute), which makes the aqueous composition according to the invention particularly well suited for therapeutic use, i.e. to treat COVID-19 or a SARS-CoV-2 infection, as well as for prophylactic use, i.e. to prevent COVID-19 or a SARS-CoV-2 infection. The aqueous composition is also suitable for use in an intensive care unit, including in the treatment or prevention of severe COVID-19 (or severe SARS-CoV-2 infections). Moreover, the aqueous composition can also advantageously be used for inactivating mutant forms of SARS-CoV-2 or other related viruses having at least 50% (preferably at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%) sequence identity to SARS-CoV-2 at the whole genome sequence level (for the genome sequence of SARS-CoV-2, see, e.g., Wang H et al., Eur J Clin Microbiol Infect Dis, 2020, 39(9): 1629-163 and references cited therein).

The subject or patient to be treated in accordance with the present invention may be an animal (e.g., a non-human animal). Preferably, the subject/patient is a mammal. More preferably, the subject/patient is a human (e.g., a male human or a female human) or a non-human mammal (such as, e.g., a guinea pig, a hamster, a rat, a mouse, a rabbit, a dog, a cat, a horse, a monkey, an ape, a marmoset, a baboon, a gorilla, a chimpanzee, an orangutan, a gibbon, a sheep, cattle, a pig, or a mink). Most preferably, the subject/patient to be treated in accordance with the invention is a human.

The term "treatment” of a disorder or disease as used herein (e.g., "treatment” of COVID-19) is well known in the art. "Treatment” of a disorder or disease implies that a disorder or disease is suspected or has been diagnosed in a patient/subject. A patient/subject suspected of suffering from a disorder or disease typically shows specific clinical and/or pathological symptoms which a skilled person can easily attribute to a specific pathological condition (i.e., diagnose a disorder or disease).

The "treatment” of a disorder or disease may, for example, lead to a halt in the progression of the disorder or disease (e.g., no deterioration of symptoms) or a delay in the progression of the disorder or disease (in case the halt in progression is of a transient nature only). The "treatment” of a disorder or disease may also lead to a partial response (e.g., amelioration of symptoms) or complete response (e.g., disappearance of symptoms) of the subject/patient suffering from the disorder or disease. Accordingly, the "treatment” of a disorder or disease may also refer to an amelioration of the disorder or disease, which may, e.g., lead to a halt in the progression of the disorder or disease or a delay in the progression of the disorder or disease. Such a partial or complete response may be followed by a relapse. It is to be understood that a subject/patient may experience a broad range of responses to a treatment (such as the exemplary responses as described herein above). The treatment of a disorder or disease may, inter alia, comprise curative treatment (preferably leading to a complete response and eventually to healing of the disorder or disease) and palliative treatment (including symptomatic relief).

The term "prevention” of a disorder or disease as used herein (e.g., "prevention” of COVID-19) is also well known in the art. For example, a patient/subject suspected of being prone to suffer from a disorder or disease may particularly benefit from a prevention of the disorder or disease. The subject/patient may have a susceptibility or predisposition for a disorder or disease, including but not limited to hereditary predisposition. Such a predisposition can be determined by standard methods or assays, using, e.g., genetic markers or phenotypic indicators. It is to be understood that a disorder or disease to be prevented in accordance with the present invention has not been diagnosed or cannot be diagnosed in the patient/subject (for example, the patient/subject does not show any clinical or pathological symptoms). Thus, the term "prevention” comprises the use of a compound of the present invention before any clinical and/or pathological symptoms are diagnosed or determined or can be diagnosed or determined by the attending physician.

As explained above, the present invention particularly relates to the inactivation of SARS-CoV-2 and the treatment or prevention of COVID-19 in a subject, but it also relates more generally to the inactivation of enveloped viruses (including SARS-CoV-2 but also other enveloped viruses) as well as the treatment or prevention of diseases caused by, or associated with, such enveloped viruses.

The enveloped virus to be inactivated in accordance with the present invention is preferably a human or animal pathogenic enveloped virus, such as, e.g., a leukemia virus, a herpes virus (or a virus of the Herpesviridae family), a pox virus (or a virus of the Poxviridae family), a hepadnavirus (or a virus of the Hepadnaviridae family), a flavivirus (or a virus of the Flaviviridae family), a togavirus (or a virus of the Togaviridae family), a coronavirus (or a virus of the Coronaviridae family, particularly a virus of the Orthocoronavirinae subfamily), a hepatitis virus, a retrovirus (or a virus of the Retroviridae family), an orthomyxovirus (or a virus of the Orthomyxoviridae family), a paramyxovirus (or a virus of the Paramyxoviridae family), a rhadovirus (or a virus of the Rhabdoviridae family), a bunyavirus (or a virus of the Bunyavirales family), or a filovirus (or a virus of the Filoviridae family).

Exemplary leukemia viruses to be inactivated include, in particular, human T-cell lymphotropic virus type 1 (HTLV-1), human T-cell lymphotropic virus type 2 (HTLV-2), human T-cell lymphotropic virus type 3 (HTLV-3), or human T-cell lymphotropic virus type 4 (HTLV-4). Exemplary herpes viruses to be inactivated include, in particular, herpes simplex virus 1 (HSV-1), herpes simplex virus 2 (HSV-2), varicella-zoster virus (VZV), Epstein-Barr virus (EBV), human cytomegalovirus (HCMV), human herpesvirus 6A (HHV-6A), human herpesvirus 6B (HHV-6B), human herpesvirus 7 (HHV-7), or Kaposi's sarcoma-associated herpesvirus (KSHV). An exemplary hepadnavirus to be inactivated is hepatitis B virus (HBV). Exemplary flaviviruses to be inactivated include, in particular, hepatitis C virus (HBC), West Nile virus, dengue virus, tick-borne encephalitis virus, yellow fever virus, or Zika virus. An exemplary togavirus to be inactivated is Chikungunya virus (CHIKV). Exemplary coronaviruses to be inactivated include, in particular, severe acute respiratory syndrome coronavirus (SARS-CoV or SARS-CoV-1), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Middle East respiratory syndrome-related coronavirus (MERS-CoV), human coronavirus OC43 (HCoV-OC43), human coronavirus HKU1 (HCoV-HKUI), human coronavirus 229E (HCoV-229E), or human coronavirus NL63 (HCoV-NL63). Exemplary hepatitis viruses to be inactivated include, in particular, hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV), or hepatitis E virus (HEV). Exemplary retroviruses to be inactivated include, in particular, human immunodeficiency virus (HIV), HTLV-1, HTLV-2, HTLV-3, or HTLV-4. Exemplary orthomyxoviruses to be inactivated include, in particular, influenza viruses, e.g., influenza A virus (e.g., serotype H1N1, H1N2, H2N2, H3N1, H3N2, H3N8, H5N1, H5N2, H5N3, H5N8, H5N9, H7N1, H7N2, H7N3, H7N4, H7N7, H7N9, H9N2, or H10N7), influenza B virus, influenza C virus, or influenza D virus. Exemplary paramyxoviruses to be inactivated include, in particular, human parainfluenza virus type 1 (HPIV-1), human parainfluenza virus type 2 (HPIV-2), human parainfluenza virus type 3 (HPIV-3), human parainfluenza virus type 4 (HPIV-4), Hendra virus (HeV), or Nipah virus (NiV). An exemplary rhadovirus to be inactivated is Rabies lyssavirus (or Rabies virus). Exemplary bunyaviruses to be inactivated include, in particular, California encephalitis virus, La Crosse encephalitis virus, Jamestown Canyon virus, Snowshoe hare virus, Lassa mammarenavirus (LASV, or Lassa virus), or a hantavirus. Exemplary filoviruses to be inactivated include, in particular, an Ebola virus (e.g., Zaire ebolavirus (ZEBOV), Sudan ebolavirus (SUDV), Reston ebolavirus (RESTV), Tai Forest ebolavirus (TAFV), or Bundibugyo ebolavirus (BDBV)), Marburg virus (MARV), Ravn virus (RAW), Mengla dianlovirus (MLAV), or Lloviu virus (LLOV).

More preferably, the enveloped virus to be inactivated is an enveloped virus that causes a respiratory tract infection (e.g., a coronavirus or an influenza virus). Even more preferably, the enveloped virus to be inactivated is a coronavirus, yet even more preferably SARS-CoV-1, SARS-CoV-2, or MERS-CoV, still more preferably SARS-CoV- 2. While it is particularly preferred that the enveloped virus to be inactivated is SARS-CoV-2, the present invention also specifically relates to the inactivation of an enveloped virus which is not SARS-CoV-2 (i.e., which is different from SARS-CoV-2).

It will be understood that the invention also specifically encompasses the inactivation of more than one enveloped virus (including, in particular, any of the above-described exemplary viruses). Accordingly, the enveloped virus to be inactivated may also be a plurality of enveloped viruses (which may be selected, e.g., from any of the above-described exemplary viruses). The terms "virus(es)” and "viral particle(s)” are used herein interchangeably.

Unless specifically indicated otherwise, all properties and parameters referred to herein, including any pH values as well as any amounts/concentrations (indicated, e.g., in mg/ml, in % w/v or in % v/v), are preferably to be determined at standard ambient temperature and pressure conditions, particularly at a temperature of 25°C (298.15 K) and at an absolute pressure of 101.325 kPa (1 atm). Accordingly, it is preferred that any pH indicated herein is to be determined at a temperature of 25°C, more preferably at a temperature of 25°C and an absolute pressure of 1 atm. As used herein, unless explicitly indicated otherwise or contradicted by context, the terms "a”, "an” and "the” are used interchangeably with "one or more” and "at least one”. Thus, for example, a composition comprising "an” excipient can be interpreted as referring to a composition comprising "one or more” excipients.

It is to be understood that wherever numerical ranges are provided/disclosed herein, all values and subranges encompassed by the respective numerical range are meant to be encompassed within the scope of the invention. Accordingly, the present invention specifically and individually relates to each value that falls within a numerical range disclosed herein, as well as each subrange encompassed by a numerical range disclosed herein.

As used herein, the term "about” preferably refers to ±10% of the indicated numerical value, more preferably to ±5% of the indicated numerical value, and in particular to the exact numerical value indicated. If the term "about” is used in connection with the endpoints of a range, it preferably refers to the range from the lower endpoint -10% of its indicated numerical value to the upper endpoint +10% of its indicated numerical value, more preferably to the range from of the lower endpoint -5% to the upper endpoint +5%, and even more preferably to the range defined by the exact numerical values of the lower endpoint and the upper endpoint.

As used herein, the term "comprising” (or "comprise”, "comprises”, "contain”, "contains”, or "containing”), unless explicitly indicated otherwise or contradicted by context, has the meaning of "containing, inter alia”, i.e., "containing, among further optional elements, In addition, this term also includes the narrower meanings of "consisting essentially of and "consisting of”. For example, the term "A comprising B and C” has the meaning of "A containing, inter alia, B and C”, wherein A may contain further optional elements (e.g., "A containing B, C and D” would also be encompassed), but this term also includes the meaning of "A consisting essentially of B and C” and the meaning of "A consisting of B and C” (i.e., no other components than B and C are comprised in A).

As used herein, the terms "optional”, "optionally” and "may” denote that the indicated feature may be present but can also be absent. Whenever the term "optional”, "optionally” or "may” is used, the present invention specifically relates to both possibilities, i.e., that the corresponding feature is present or, alternatively, that the corresponding feature is absent. For example, if a component of a composition is indicated to be "optional”, the invention specifically relates to both possibilities, i.e., that the corresponding component is present (contained in the composition) or that the corresponding component is absent from the composition.

It is to be understood that the present invention specifically relates to each and every combination of features and embodiments described herein, including any combination of general and/or preferred features/embodiments.

In this specification, a number of documents including patent applications and scientific literature are cited. The disclosure of these documents, while not considered relevant for the patentability of this invention, is herewith incorporated by reference in its entirety. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference. The reference in this specification to any prior publication (or information derived therefrom) is not and should not be taken as an acknowledgment or admission or any form of suggestion that the corresponding prior publication (or the information derived therefrom) forms part of the common general knowledge in the technical field to which the present specification relates.

The invention is also described by the following illustrative figures. The appended figures show:

Figure 1: pH dependent inhibition of Sars-CoV-2 replication. Ct values of Sars-CoV-2 samples pre-incubated 15 minutes with various buffered media; all bars show the Ct values after 48 hours. See Example 1 .

Figure 2: Ct values of Sars-CoV-2 samples pre-incubated for 1 minute and 15 minutes with a pH 7.3 and a pH 10 solution, respectively; black bars: t₀, white bars: t₂4. See Example 2.

Figure 3: Duplicates of Ct values of Sars-CoV-2 samples incubated for 15 minutes with 25% arginine in citrate buffer (pH 3, 3.5, 4.0 and 4.5) and citrate buffer only (pH 3, 3.5, 4.0 and 4.5); black bars: to, white bars: Ϊ48· See Example 3.

Figure 4: Duplicates of Ct values of Sars-CoV-2 samples incubated for 15 minutes with 25% arginine in NH₄CI buffer (pH 9.5 - 11.0) and NH₄CI buffer only (pH 9.5 - 11.0); black bars: to, white bars: Ϊ48. See Example 3.

Figure 5: Duplicates of Ct values of Sars-CoV-2 samples incubated for 15 minutes with isoosmolar solutions of arginine base, lysine base, urea and NaHC03 at pH 9, 9.5 and 10 after 48 hours. See Example 4.

Figure 6: Duplicates of Ct values of Sars-CoV-2 samples incubated for 15 minutes with a hypoosmolar, an isoosmolar and a hyperosmolar solution of NH₄CI at pH 9, 9.5 and 10 after 48 hours. See Example 4.

Figure 7: Slope of viability against time. See Example 7.

Figure 8: Cell viability 0 hours compared to 24 hours; black bars represent t₀, light grey bars represent t₂4. See Example 7.

Figure 9: Effect of NaHC03 solutions of different pH values on RPMI 2650 cells after 5 minutes (black bars) and 15 minutes (grey bars) of incubation; each value represents the mean of n=6 ± S.D. See Example 8.

Figure 10: Effect of isosomolar solutions (arginine base, NaHC03 and lysine base) and a hyperosmolar solution (NH4CI) buffered to pH 10 on RPMI 2650 cells after 5 minutes (black bars) and 15 minutes (grey bars) of incubation; each value represents the mean of n=6 ± S.D. See Example 8.

Figure 11: pH and time dependent effect of solutions of NH₄CI (white bars) and TRIS (black bars) on RPMI 2650 cells after (A) 5 minutes and (B) 15 minutes of incubation; grey bars show controls; each value represents the mean of n=6 ± S.D. See Example 10. Figure 12: pH and time dependent effect of isosomolar solutions of Arginine base (white bars) and NaHC03 (black bars) on RPMI 2650 cells after (A) 5 minutes and (B) 15 minutes of incubation; grey bars show controls; each value represents the mean of n=6 ± S.D. See Example 11 .

Figure 13: pH of artificial nasal fluid after addition of different concentrations of NaHC0₃ solutions with pH 9.5. See Example 13.

Figure 14: pH of artificial nasal fluid after addition of different concentrations of TRIS solutions with pH 9.5. See Example 14.

Figure 15: Time dependent intranasal pH in Cynomolgus macaques after CYP-CM001 nasal spray application; each data points shows the mean of n=3 ± S.D. See Example 15.

Figure 16: Intranasal pH in Cynomolgus macaques before (to, white bar) and 15 minutes after CYP-CM002 nasal spray application (tis, grey bar); each data points shows the mean of n=3 ± S.D. See Example 15.

The invention will now be described by reference to the following examples which are merely illustrative and are not to be construed as a limitation of the scope of the present invention.

EXAMPLES

Example 1 : pH dependent inhibition of Sars-CoV-2 replication using alkaline buffer media

Materials:

Viral SARS-CoV-2 strain: Human 2019-nCoV Isolate

Product Description - Ref-SKU: 026V-03883 Infectious cellculture supernatant of human 2019-nCoV; Product Risk Group: RG3; ICTV Taxonomy: ssRNA(+) / Nidovirales / Coronaviridae / Coronavirinae / Betacoronavirus; Virus name: Human 2019-nCoV ex China; Strain: BavPat1/2020; Isolate: Germany ex China.

Based on this stock solution of 2.2 E+06 PFU/mL of Human 2019-nCoV Isolate, a viral working stock (VP2) was grown in Vero CCL81 cells using fetal calf serum (FCS)-free cell culture medium (OptiPro from Gibco). The working stock aliquots were used in all experiments.

The European Commission classifies SARS-CoV-2 as a risk group 3 pathogen. Experiments with active virus and high concentration virus stocks require working under biosafety level 3 conditions (BSL-3). At the Institute of Pathology at the Medical University of Graz (Austria), all working steps with the active virus were performed under those required BSL-3 conditions and additionally with an increased personal safety equipment, to avoid transmission of virus via aerosols. Experimental Procedures:

Vero-cells CCL81 (3 E+04 cells/well in serum free Gibco OptiPro) were seeded into 48 well plates 24 hours prior to infection.

The virus is stored at -80°C with cells. To purify the virus suspension is centrifuged for 1 min at 13,000 rpm. The cell pellet stays in the vial and the pure virus supernatant is used for the experiment.

The virus has to be pre-incubated in a ratio of 1 :10 virus to substance for 15 minutes at room temperature (RT). During that time 180 mI of new cell culture medium is applied to the seeded cells. With 20 mI of the virus substance mix, there will be again an 1 :10 mixture on the cells (= final 1 :100 dilution) for the infection. The plate is incubated for 60 min at 37°C with 5 % CO2.

1-hour post infection the cells were washed two times with phosphate-buffered saline (PBS) and covered with 440 mI_ fresh pre-warmed cell culture medium.

After 10 minutes of incubation at RT, 140 mI_ from the cell culture medium supernatant will be removed in order to determine the starting concentration of viral copy numbers (t = 0 h). After 48 hours of incubation again under previous conditions (37°C with 5 % C0₂), further 140 mI_ of cell culture medium supernatant is obtained to determine virus copy numbers.

The experiment included internal controls for the efficiency of infection. Cells infected with virus without any substance addition (positive control) and cells not infected with virus (negative control). Both were handled exactly the same as the substance samples in the view of dilution, time, conditions and earning supernatant for further treatment.

Viral RNA was isolated from cell culture medium supernatant by using QIAamp® Viral RNA Mini Kit, as recommended by the Centers for Disease Control and Prevention (CDC).

The RT-qPCR, to detect the viral load of the samples, was performed based on the CDC recommendation using QuantiTect Multiplex RT-PCR Kit with a Rotor Gene Q cycler:

2019-nCoV_N1-F 2019-nCoV_N1 Forward Primer: 5’-GAC CCC AAA ATC AGC GAA AT-3'

2019-nCoV_N1-R 2019-nCoV_N1 Reverse Primer: 5’-TCT GGT TAC TGC CAG TTG AAT CTG-3’ 2019-nCoV_N1-P 2019-nCoV_N1 Probe: 5’-FAM-ACC CCG CAT TAC GTT TGG TGG ACC-BHQ1-3'

("FAM” refers to the fluorescent dye 5(6)-carboxyfluorescein (i.e., a mixture of the two isomers 5-carboxyfluorescein and 6-carboxyfluorescein), and "BHQ-1” refers to Black Hole Quencher 1).

To quantify which Ct-value represents a certain number of viral RNA copies, the inventors analysed the internal used virus stock VP1 (3.15 E+05 copies /mI_ virus-stock) and the certified RNA-standard (Certificate of Analysis: ATCC Cat # VR-1986D Lot# 70035624) with a defined copy number (1 mί STD contains 4730 Copies), based on that the inventors calculated the viral input from the assay (2 mI_) as 6.3 E+05 copies, calculated with the number of seeded cells (3.0 E+04 cells per well) they infected every cell with 21 RNA copies. It should be mentioned that not every viral RNA copy must represent an infectious viral particle.

All Ct-values higher than 40 are considered negative. Virus replication was assessed by comparing Ct values after infection (t = 0 h) with Ct values after different time-periods of culturing. If there is a difference of minimum 4 cycles (t = 48 is higher), an inhibition of the virus replication in the cells can be seen.

Briefly, under BSL2 conditions, the pH-stock solutions were prepared and measured (pH was adjusted with NaOH and HCI). Detailed information on the pH is provided in the table below:

Table: Buffer system, target pH values and measured pH values of the solutions used for the experiment

The following procedure was used:

Vero cells were seeded 30,000 cells/well 24 h before under BSL2 conditions: pH-stock solutions were sterile filtrated under BSL3 conditions: 300 mI pH-solution were mixed with 30 mI virus suspension (Wuhan strain VP2) One pre-incubation time: 15 min (RT)

New cell culture medium was prepared in the wells (180 mI/well) and mixed with 20 mI virus-pH-solution (1:100 dilution)

Infection for 1 h at 37°C

After infection: 2 washing steps with PBS and addition of 440 mI cell culture medium

Waited for 10 minutes to take 140 mI as t=0 value for the qPCR

Plate stayed for 48 h at 37°C

Took 140 mI as t=48 value for the qPCR

To the samples AVL-Buffer were added to pass them through the sample shower with CIO2 (back to BSL2 conditions)

RNA-lsolation - qPCR

Results:

The results obtained are shown in Figure 1. Conclusion:

In conclusion, it was shown that in a pH buffered system, Sars-CoV-2 replication cannot be inhibited in an acidic or neutral pH range. In contrast, Sars-CoV-2 replication can be fully inhibited in a basic buffered system (NH₄CI) at a pH range of 8.5 to 9.5. According to recommended standards, the virus is not infectious anymore above a Ct value of 30.

Example 2: Rapid inhibition of Sars-CoV-2 replication at pH 10

Materials:

Viral SARS-CoV-2 strain: Human 2019-nCoV Isolate

Product Description - Ref-SKU: 026V-03883 Infectious cellculture supernatant of human 2019-nCoV; Product Risk Group: RG3; ICTV Taxonomy: ssRNA(+) / Nidovirales / Coronaviridae / Coronavirinae / Betacoronavirus; Virus name: Human 2019-nCoV ex China; Strain: BavPat1/2020; Isolate: Germany ex China

The European Commission classifies SARS-CoV-2 as a risk group 3 pathogen. Experiments with active virus and high concentration virus stocks require working under biosafety level 3 conditions (BSL-3). At the Institute of Pathology at the Medical University of Graz (Austria), all working steps with the active virus were performed under those required BSL-3 conditions and additionally with an increased personal safety equipment, to avoid transmission of virus via aerosols.

Experimental Procedures:

Vero-cells CCL81 (3x104 cells/well in serum free Gibco OptiPro) were seeded into 48 well plates 24 hours prior to infection.

The virus has to be pre-incubated in a ratio of 1 :10 virus to substance for 1 and 15 minutes at RT. During that time 180 mI of cell culture medium is applied to the seeded cells. With 20 mI of the virus substance mix, there will be again an 1 :10 mixture on the cells (= final 1 :100 dilution) for the infection. The plate is incubated for 60 min at 37°C with 5 % C0₂. 1-hour post infection the cells were washed two times with PBS and covered with 440 mI_ fresh pre-warmed cell culture medium.

After 10 minutes of incubation at RT, 140 mI_ from the cell culture medium supernatant will be removed in order to determine the starting concentration of viral copy numbers (t = 0 h). After 24 hours of incubation again under previous conditions (37°C with 5 % CO2), further 140 mI_ of cell culture medium supernatant is obtained to determine virus copy numbers.

Viral RNA was isolated from cell culture medium supernatant by using QIAamp® Viral RNA Mini Kit, as recommended by CDC.

2019-nCoV_N1-F 2019-nCoV_N1 Forward Primer: 5’-GAC CCC AAA ATC AGC GAA AT-3'

2019-nCoV_N1-R 2019-nCoV_N1 Reverse Primer: 5’-TCT GGT TAC TGC CAG TTG AAT CTG-3’ 2019-nCoV_N1-P 2019-nCoV_N1 Probe: 5’-FAM-ACC CCG CAT TAC GTT TGG TGG ACC- B FI Q 1-3' FAM, BHQ-1

To quantify which Ct-value represents a certain number of viral RNA copies, the inventors analysed the internal used virus stock VP1 (3.15 E+05 copies /mI_ virus-stock) and the certified RNA-standard (Certificate of Analysis: ATCC Cat # VR-1986D Lot# 70035624) with a defined copy number (1 mί STD contains 4730 Copies), based on that they calculated the viral input from the assay (2 mί) as 6.3 E+05 copies, calculated with the number of seeded cells (3.0 E+04 cells per well) they infected every cell with 21 RNA copies. It should be mentioned that not every viral RNA copy must represent an infectious viral particle.

All Ct-values higher than 40 are considered negative. Virus replication was assessed by comparing Ct values after infection (t = 0 h) with Ct values after different time-periods of culturing (t = 24 hours). If there is a difference of minimum 4 cycles (t = 24 is higher) an inhibition of the virus replication in the cells can be seen.

Briefly, the following procedure was used:

Under BSL2 conditions: the pH-stock solutions were prepared and measured (pH was adjusted with NaOFI and HCI)

Target pH of the alkaline stock solution was pH 10.0; measured pH was 10.75

Vero cells were seeded 30,000 cells/well 24 h before under BSL3 conditions: pH-stock solutions were sterile filtrated

194.6 mI pFI-Medium were mixed with 5.4 mI virus suspension (Wuhan strain VP1) Two pre-incubation times: 1 min (RT) and 15 min (37°C)

New cell culture medium was prepared in the wells (225 mI/well) and mixed with 75 mI virus-pH-solution (1 :4 dilution) Infection for 1 h at 37°C

Waited for 10 minutes to take 140 mI as t=0 value for the qPCR

Plate stayed for 24 h at 37°C

Took 140 mI as t=24 value for the qPCR

RNA-lsolation - qPCR

Results:

Sars-CoV-2 replication was not inhibited when the virus suspension was pre-incubated for up to 15 minutes at pH 7.3. Contrary thereto, full inhibition of Sars CoV-2 replication was observed already after 1 minute as well as after 15 minutes of incubation with a pH 10 solution. The results are also shown in Figure 2.

Conclusion:

In conclusion, the data show that already after 1 minute of virus pre-incubation at pH 10, Sars-CoV-2 virus replication can be fully inhibited.

Example 3: pH dependent effect on Sars-CoV-2 replication using citrate, citrate/arginine, NH4CI and arginine/NH₄CI buffer systems

Materials:

Viral SARS-CoV-2 strain: Human 2019-nCoV Isolate

Product Description - Ref-SKU: 026V-03883 Infectious cell culture supernatant of human 2019-nCoV; Product Risk Group: RG3; ICTV Taxonomy: ssRNA(+) / Nidovirales / Coronaviridae / Coronavirinae / Betacoronavirus; Virus name: Human 2019-nCoV ex China; Strain: BavPat1/2020; Isolate: Germany ex China

Experimental Procedures:

The virus has to be pre-incubated in a ratio of 1 :10 virus to substance for 15 minutes at RT. During that time 180 mI of cell culture medium is applied to the seeded cells. With 20 mI of the virus substance mix, there will be again an 1 :10 mixture on the cells (= final 1 :100 dilution) for the infection. The plate is incubated for 60 min at 37°C with 5 % C0₂.

1-hour post infection the cells were washed two times with PBS and covered with 440 mί fresh pre-warmed cell culture medium.

After 10 minutes of incubation at RT, 140 mί from the cell culture medium supernatant will be removed in order to determine the starting concentration of viral copy numbers (t = 0 h). After 48 hours of incubation again under previous conditions (37°C with 5 % CO2), further 140 mί of cell culture medium supernatant is obtained to determine virus copy numbers.

2019-nCoV_N1-F 2019-nCoV_N1 Forward Primer: 5’-GAC CCC AAA ATC AGC GAA AT-3'

2019-nCoV_N1-R 2019-nCoV_N1 Reverse Primer: 5’-TCT GGT TAC TGC CAG TTG AAT CTG-3’ 2019-nCoV_N1-P 2019-nCoV_N1 Probe: 5’-FAM-ACC CCG CAT TAC GTT TGG TGG ACC-BHQ1-3' FAM, BHQ-1

To quantify which Ct-value represents a certain number of viral RNA copies, the inventors analysed the internal used virus stock VP1 (3.15 E+05 copies /mI_ virus-stock) and the certified RNA-standard (Certificate of Analysis: ATCC Cat # VR-1986D Lot# 70035624) with a defined copy number (1 mί STD contains 4730 Copies), based on that they calculated the viral input from the assay (2 mI_) as 6.3 E+05 copies, calculated with the number of seeded cells (3.0 E+04 cells per well) they infected every cell with 21 RNA copies. It should be mentioned that not every viral RNA copy must represent an infectious viral particle.

All Ct-values higher than 40 are considered negative. Virus replication was assessed by comparing Ct values after infection (t = 0 h) with Ct values after different time-periods of culturing (t=48 hrs). If there is a difference of minimum 4 cycles (t = 48 is higher), an inhibition of the virus replication in the cells can be seen.

Table: measured pH values of citrate (pH 3 - 4.5) and NH₄CI (9.5 - 11.0) solutions after preparation (stock solution) and after dilution with the virus suspension

The following procedure was used:

194.6 mI pH-Medium were mixed with 5.4 mI virus suspension (Wuhan strain VP1)

Two pre-incubation times: 1 min (RT) and 15 min (37°C)

Waited for 10 minutes to take 140 mI as t=0 value for the qPCR

Plate stayed for 48 h at 37°C

Took 140 mI as t=24 value for the qPCR

RNA-lsolation qPCR

Results:

Under acidic conditions in the pH range of 3.0 - 4.5 in a citrate buffer system, no inactivation of Sars-CoV-2 virus replication was seen, regardless of the presence or absence of 25% arginine (see also Figure 3).

Under basic conditions in the pH range of 9.5 to 11 .0 in a NH₄CI buffer system, full inactivation of Sars-CoV-2 virus replication was seen, regardless of the presence or absence of 25% arginine (see also Figure 4).

Conclusion:

In conclusion, it was shown that in a pH buffered system, Sars-CoV-2 replication cannot be inhibited in a pH range of 3.0 - 4.5. However, Sars-CoV-2 replication can be fully inhibited in a buffered system (NH4CI and NH4CI/25% arginine) at a pH range of 9.5 - 11.0.

Example 4: pH dependent inhibition of Sars-CoV-2 replication using isoosmolar buffer systems covering a pH range from 9.0 to 10.0

Materials:

Viral SARS-CoV-2 strain: Human 2019-nCoV Isolate

Experimental Procedures:

1-hour post infection the cells were washed two times with PBS and covered with 440 mI_ fresh pre-warmed cell culture medium.

After 10 minutes of incubation at RT, 140 mI_ from the cell culture medium supernatant will be removed in order to determine the starting concentration of viral copy numbers (t = 0 h). After 48 hours of incubation again under previous conditions (37°C with 5 % CO2), further 140 mI_ of cell culture medium supernatant is obtained to determine virus copy numbers.

2019-nCoV_N1-F 2019-nCoV_N1 Forward Primer: 5’-GAC CCC AAA ATC AGC GAA AT-3'

To quantify which Ct-value represents a certain number of viral RNA copies, the inventors analysed the internal used virus stock VP1 (3.15 E+05 copies /mI_ virus-stock) and the certified RNA-standard (Certificate of Analysis: ATCC Cat # VR-1986D Lot# 70035624) with a defined copy number (1 mί STD contains 4730 Copies), based on that they calculated the viral input from the assay (2 mί) as 6.3 E+05 copies, calculated with the number of seeded cells (3.0 E+04 cells per well) they infected every cell with 21 RNA copies. It should be mentioned, not every viral RNA copy must represent an infectious viral particle.

All Ct-values higher than 40 are considered negative. Virus replication was assessed by comparing Ct values after infection (t = 0 h) with Ct values after different time-periods of culturing (t = 48 hours). If there is a difference of minimum 4 cycles (t = 48 is higher) an inhibition of the virus replication in the cells can be seen.

Briefly, under BSL2 conditions, the pH-stock solutions were prepared and measured; samples containing arginine, lysine, urea, bicarbonate and NH₄CI were prepared in an isoosmotic concentration; in addition, hypo- and hyperosmolar NH₄CI solutions were prepared (see table below):

Table: Concentrations, pH values and osmolarities of solutions used in the experiment

The following procedure was used:

194.6 mI pH-Medium were mixed with 5.4 mI virus suspension (Wuhan strain VP1) - Two pre-incubation times: 1 min (RT) and 15 min (37°C)

New cell culture medium was prepared in the wells (225 mI/well) and mixed with 75 mI virus-pH-solution (1 :4 dilution) Infection for 1 h at 37°C After infection: 2 washing steps with PBS and addition of 440 mI cell culture medium

Waited for 10 minutes to take 140 mI as t=0 value for the qPCR

Plate stayed for 48 h at 37°C

Took 140 mI as t=24 value for the qPCR

To the samples AVL-Buffer were added to pass them through the sample shower with CI0₂ (back to BSL2 conditions)

RNA-lsolation - qPCR

Results:

Isoosmolar solutions of arginine base, lysine base, urea and NaHC0₃ at pH 9, 9.5 and 10 inhibited Sars-CoV-2 replication (see Figure 5). In the case of lysine, a lower efficacy was observed at pH 9 compared to pH 9.5 and 10. A similar, but not as pronounced trend was also observed for other buffer systems.

Isoosmolar solutions, but also hypo- and hyperosmolar solutions of NH₄CI at pH 9, 9.5 and 10 fully inhibited Sars- CoV-2 replication (see Figure 6).

Conclusion:

Isoosmolar solutions of arginine base, lysine base, urea, NaHCCh and NH4CI at pH 9, 9.5 and 10 inhibited Sars-CoV- 2 replication. The effect was most robust and consistent from pH 9.5 upwards. Hypo- and hyperosmolar solutions of NH4CI showed an activity comparable to the isoosmolar solution.

Example 5: Physical stability of a combination of a commercial carrageenan nasal spray and alkaline amino acids

In a clear, colorless aqueous solution containing 1.2 mg/ml iota carrageenan, 0.4mg/ml kappa carrageenan as well as NaCI and with an initial pH of 7.2 (exemplary commercial product: A!govis^® Effek , different amounts of arginine or lysine were dissolved and the pH was measured. Samples were kept at room temperature for 2 hours and analysed regarding their physical stability. Results are shown in the Table below.

Table: Physical compatibility and pH values of solutions containing arginine or lysine as well as carrageenan

Samples 3 and 5 of the table above were stored under light protection and room temperature for a total of 14 days. After pre-determined time-points, the samples were evaluated concerning their physical stability. Results are shown in the table below:

Table: Physical stability of solutions containing carrageenan and arginine when stored at room temperature

Conclusion: Combinations of carrageenan and arginine (HCI or base) and combinations of carrageenan and lysine (HCI and base) in aqueous solution resulted in clear, colorless solutions. Combinations of carrageenan and arginine in aqueous solution were physically stable for at least 14 days when stored at room temperature and under light protection. Example 6: Physical stability of a combination of a commercial carrageenan nasal spray and isoosmolar alkaline buffer systems

In a clear, colorless aqueous solution containing 1.2 mg/ml iota carrageenan, 0.4 mg/ml kappa carrageenan as well as NaCI and with an initial pH of 7.2 (exemplary commercial product: A!govis^® Effek , different amounts of arginine base, NaHC0₃ or NH₄CI were dissolved. Four solutions of each of the three buffer systems with different alkaline pH values were prepared. Samples were stored under light protection and room temperature for a total of 14 days. After pre-determined time-points, the samples were evaluated concerning their physical stability. Results are shown in the table below.

Table: Physical compatibility and pH values of solutions containing arginine, NaHCCh or NH₄CI, as well as carrageenan

Conclusion:

Alkaline combinations of carrageenan and either arginine base or NH4CI in aqueous solution resulted in clear, colorless solutions. Combinations of carrageenan and said alkaline buffer systems covering a pH range from 7.77 to 11.70 were physically stable for at least 14 days when stored at room temperature and under light protection. The physical stability of a combination of carrageenan and NaHC03 was pH dependent; samples between pH 7.8 and pH 9.4 appeared to be hazy, whereas samples stored at pH 11.7 appeared to be clear up to the end of the storage observation period of 14 days.

Example 7: Cell viability of Vero cell line CCL81 after incubation with pH solutions 3-10

Verocells CCL81

The Vero cell line was initiated from the kidney of a normal adult African green monkey. By default, cells were maintained in 75 cm² tissue culture flasks at 37 °C in a humidified atmosphere containing 5 % C0₂ with medium changes 2-3 times per week. The culture medium is a fetal calf serum (FCS)-free cell culture medium, Gibco OptiPro with 1 % L-glutamine and 2 % Penstrep (100 U/mL penicillin G sodium salt, 100 pg/mL streptomycin sulfate).

When confluence reached approximately 80 % (see Figure 7), cells were washed with PBS-solution and detached from the surface by Trypsin/EDTA (37 °C for 3 min). The cells were resuspended and counted using a Fuchs Rosenthal chamber. After resuspending, the cells were seeded at a density of 10,000 cells per well in a 96 well plate.

Resazurin assay (Cell viability)

Stock concentration: 1 mM resazurin (Sigma Aldrich, R7017-1G) in PBS (filter sterilized and stored at RT protected from light). Working concentration: 10 mM in culture medium (100x diluted). Working solution was pre-warmed. Culture medium was exchanged with working solution and incubated for 2 hours. Plate fluorescence (Exc. 530 nm / Em. 590 nm) was measured according to internal protocols. Medium supplemented with 10 mM resazurin incubated without cells was used as a blank. Blank values were subtracted from each measurement.

The assay was performed to determine the effects of the pH-solutions on viability of CCL81 cells. The experiment was conducted with n = 6 each. CCL81 cells were seeded onto a 96-well tissue culture test plate at a density of 10,000 cells per well for 24 h. Medium was removed from the cells and replaced by 90 pL excipient solution per well. Cells were incubated with solutions for 2 hours at 37 °C in humidified, 5 % C0₂ atmosphere. After the incubation period 90 mI Reagent were pipetted into each well of the 96-well assay plate containing 90 pL of the excipient solution. This step happens automatically by the Synergy protocol. The samples were analysed immediately as well as after hours. Results:

The results obtained are shown in Figures 7 and 8. Conclusion:

The cell viability values show a positive slope in the entire tested pH range (pH 3-10). This indicates that the CCL81 cells continue growing for at least 24 hours under the various tested conditions, indicating full viability.

Example 8: Cell viability of human nasal cell line RPMI 2650 after incubation with alkaline pH buffered solutions

A range of alkaline buffer systems based on arginine base, lysine base, NaHC0₃ and NH4CI as shown in the table below were prepared. Further, 0.9 % NaCI and KRB (Krebs-Ring buffer solution) were used as negative control; a 1% Triton X solution was used as positive control.

Table: Buffer systems used for cell viability studies with RPMI 2650 cells

RPMI 2650 Cells

The human RPMI 2650 cell line originated from a squamous cell carcinoma of the nasal septum. The cells were obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany, Cat. No. ACC 287). By default, cells were maintained in 25 cm² tissue culture flasks (Sarstedt, NCimbrecht, Germany) at 37 °C in a humidified atmosphere containing 5 % CO2 with medium changes three times per week. The culture medium consisted of Minimum Essential Medium (MEM), 10 % fetal calf serum (FCS), 1 % non-essential amino acids (NEAA, Biochrom, Germany), 1 % L-glutamine, 100 U/mL penicillin G sodium salt, 100 pg/mL streptomycin sulfate and 0.25 pg/mL amphotericin B.

When confluence reached approximately 80 % (see Figure 9), cells were washed with EDTA-solution and detached from the surface by trypsinization (37 °C for 5 min). The cells were resuspended and counted using a Z2 Coulter Counter (Beckman Coulter, Krefeld, Germany). After resuspending, the cells were seeded at a density of 40,000 cells per cm² in a new tissue culture flask.

CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS)

CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega, Mannheim, Germany) is based on a colorimetric method to determine the number of viable cells in a cytotoxicity assay. The MTS tetrazolium compound is bioreduced by viable cells into a colored formazan product. The quantity of formazan is directly proportional to the number of living cells and can be measured by absorbance at 490 nm.

The assay was performed to determine the effects of the alkaline buffer solutions shown in the table above on viability of RPMI 2650 cells. The experiment was conducted with n = 6 each. RPMI 2650 cells were seeded onto a 96-well tissue culture test plate at a density of 60,000 cells per well for 24 h. Medium was removed from the cells and replaced by 100 pL excipient solution per well. Cells were incubated with the buffer solutions for 5 min and 15 min. Furthermore, cells were incubated accordingly with 0.9% NaCI, KRB or 1.0 % Triton-X solution (V/V) as a negative and positive control, respectively. The CellTiter 96® AGueous One Solution Cell Proliferation Assay was carried out following the manufacturer's protocol. After the incubation period 20 pL CellTiter 96® AGueous One Solution Reagent were pipetted into each well of the 96-well assay plate containing 100 pL of the excipient solution. Plates were incubated for 3 h at 37 °C in humidified, 5 % CO2 atmosphere. After the incubation period the samples were analyzed immediately. Absorbance was recorded at 490 nm and measured by using a microplate reader Infinite® M Plex (Tecan, Switzerland).

Results:

Nasal cells, when incubated with an isoosmolar NaHC0₃ buffer (for details refer to the table above), showed no decrease in cell viability at alkaline pH values of pH 10.1 and pH 10.7 after 5 and 15 minutes of incubation. However, a sharp drop in cell viability was observed starting at pH 11.6 and was consequently also seen at pH 12.6. Negative controls did not lead to any cell damage after 5 and 15 minutes of incubation, whereas cell were completely damaged already after 5 minutes of incubation with the positive control Triton X.

When RPMI 2650 nasal cells were incubated with isoosmolar arginine base, NaHC03 and lysine base as well as with a hyperosmolar NH4CI solution (for details refer to the table above) buffered to pH 10, different levels of cell viability after 5 and 15 minutes of incubation were observed depending on the buffering agent. A NaHC0₃ buffer at pH 10 did not lead to any cell damage during the observed incubation duration of 15 minutes, whereas other buffer systems caused time dependent cell toxicity. In terms of the acute safety on human nasal cell lines, the buffering agents can be ranked as NaHC0₃ > lysine base = arginine base. Further, it was shown that cell viability was decreased in presence of a hyperosmolar NH4CI solution at pH 10.

These results are also illustrated in Figures 9 and 10.

Conclusion:

Nasal cells, when incubated with an isoosmolar NaHC0₃ buffer, did not show any decrease in cell viability at alkaline pH values of pH 10 and pH 10.7 during a 15-minute incubation period. However, a sharp drop in cell viability was observed at pH 11.6 and was consequently also seen with a hyperosmolar NaHCCh solution at pH 12. In terms of the acute safety on human nasal cell lines, the buffering agents can be ranked as NaHCC>₃> lysine base = arginine base. Cell viability at pH 10 was strongly decreased in presence of a hyperosmolar solution of NH4CI.

Example 9: Physical stability of the combination of different commercial nasal/oral sprays and various alkaline buffering agents

The impact of the addition of three alkaline buffering agents (arginine base, trometamol (TRIS) and NaHCOs) to various commercially available sprays regarding their physical stability was assessed. After addition of the buffering agent to the sprays, samples were kept at room temperature for 2 hours and analysed regarding their physical stability. The following sprays were tested (see table below):

Table: Composition and measured pH of the commercial sprays used in the study

The results thus obtained are shown in the Table below. Table: Physical compatibility and pH values of commercial spray solutions after addition of arginine base, TRIS or NaHCChand after 2 hours of storage at RT

Example 10: Cell viability of human nasal cell line RPMI 2650 after incubation with alkaline pH buffered solutions of NH₄CI and TRIS at different conditions

Buffer solutions of NH₄CI and TRIS, covering a pH range of pH 9.0 - 10.5 as shown in the table below were prepared. Further, KRB (Krebs-Ring buffer solution) was used as negative control; a 1% Triton X solution was used as positive control. Table: Buffer systems used for cell viability studies with RPMI 2650 cells

RPMI 2650 Cells The human RPMI 2650 cell line originated from a squamous cell carcinoma of the nasal septum. The cells were obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany, Cat. No. ACC 287). By default, cells were maintained in 25 cm² tissue culture flasks (Sarstedt, NCimbrecht, Germany) at 37 °C in a humidified atmosphere containing 5 % CO2 with medium changes three times per week. The culture medium consisted of Minimum Essential Medium (MEM), 10 % fetal calf serum (FCS), 1% non-essential amino acids (NEAA, Biochrom, Germany), 1 % L-glutamine, 100 U/mL penicillin G sodium salt, 100 pg/mL streptomycin sulfate and 0.25 pg/mL amphotericin B.

When confluence reached approximately 80 %, cells were washed with EDTA-solution and detached from the surface by trypsinization (37 °C for 5 min). The cells were resuspended and counted using a Z2 Coulter Counter (Beckman Coulter, Krefeld, Germany). After resuspending, the cells were seeded at a density of 40,000 cells per cm² in a new tissue culture flask.

CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS)

The assay was performed to determine the effects of the alkaline buffer solutions shown in the table above on viability of RPMI 2650 cells. The experiment was conducted with n=6 each. RPMI 2650 cells were seeded onto a 96-well tissue culture test plate at a density of 60,000 cells per well for 24 h. Medium was removed from the cells and replaced by 100 pL excipient solution per well. Cells were incubated with the buffer solutions for 5 min and 15 min. Furthermore, cells were incubated accordingly with 0.9% NaCI, KRB, culture medium or 1 .0 % Triton-X solution (V/V) as a negative and positive control, respectively. The CellTiter 96® AGueous One Solution Cell Proliferation Assay was carried out following the manufacturer^'s protocol. After incubation, the excipient solutions were removed and 100 mI_ Krebs Ringer Buffer pH 7.4 (KRB) was added to each well. Then 20 pL CellTiter 96® AGueous One Solution Reagent were pipetted into each well of the 96-well assay plate containing 100 pL of KRB. Plates were incubated for 3 h at 37 °C in humidified, 5 % CO2 atmosphere. After the incubation period the samples were analyzed immediately. Absorbance was recorded at 490 nm and measured by using a microplate reader Infinite® M Plex (Tecan, Switzerland).

Results:

Nasal cells, when incubated with a NH4CI buffer (for details refer to the table above), showed a clear time and pH dependent decrease in cell viability. Lowest viabilities were observed after the highest measured incubation period (15 min.) and the highest measured pH (pH 10.5). Notably, even at mild conditions (5 min. of incubation at pH 9.0), a mild decrease in cell viability was measured. Contrary to these findings, no decrease in cell viability at alkaline pH values between 9.0 and 10.5 and after both 5 and 15 minutes of incubation was observed with a TRIS solution. These results are also illustrated in Figure 11.

Conclusion: Nasal cells, when incubated with TRIS buffer, did not show any decrease in cell viability at alkaline pH values between pH 9.0 to 10.5 during a 5 and 15-minute incubation period. In contrast, solutions of NH₄CI showed a time and pH dependent decrease in cell viability. Even at mild conditions (5 min. of incubation at pH 9.0), a mild decrease in cell viability was measured.

Example 11: Cell viability of human nasal cell line RPMI 2650 after incubation with alkaline pH buffered solutions of Arginine base and NaHC03 at different conditions

Buffer solutions of Arginine base and NaHC0₃, covering a pH range of pH 9.0 - 10.5 as shown in the table below were prepared. Further, KRB (Krebs-Ring buffer solution) was used as negative control; a 1% Triton X solution was used as positive control.

Table: Buffer systems used for cell viability studies with RPMI 2650 cells

RPMI 2650 Cells

The human RPMI 2650 cell line originated from a squamous cell carcinoma of the nasal septum. The cells were obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany, Cat. No. ACC 287). By default, cells were maintained in 25 cm² tissue culture flasks (Sarstedt, NCimbrecht, Germany) at 37 °C in a humidified atmosphere containing 5 % C0₂ with medium changes three times per week. The culture medium consisted of Minimum Essential Medium (MEM), 10 % fetal calf serum (FCS), 1% non-essential amino acids (NEAA, Biochrom, Germany), 1 % L-glutamine, 100 U/mL penicillin G sodium salt, 100 pg/mL streptomycin sulfate and 0.25 pg/mL amphotericin B.

CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS)

Results:

Nasal cells, when incubated with an Arginine base buffer (for details refer to the table above), showed a clear time and pH dependent decrease in cell viability. Lowest viabilities were observed after the highest measured incubation period (15 min.) and the highest measured pH (pH 10.5). Notably, even at mild conditions (5 min. of incubation at pH 9.0), a mild decrease in cell viability was measured. Contrary to these findings, no statistically significant decrease in cell viability at alkaline pH values between 9.0 and 10.5 and after both 5 and 15 minutes of incubation was observed with a NaHC03 solution.

These results are also illustrated in Figure 12.

Conclusion:

Nasal cells, when incubated with NaHC03 buffer, did not show any decrease in cell viability at alkaline pH values between pH 9.0 to 10.5 during a 5 and 15-minute incubation period. In contrast, solution of Arginine base showed a time and pH dependent decrease in cell viability. Even at mild conditions (5 min. of incubation at pH 9.0), a mild decrease in cell viability was measured.

Example 12: Antimicrobial effectiveness testing - pH dependent effect on pathogenic microorganisms

The objective of the antimicrobial test was to investigate whether an isoosmolar, alkaline NaHC03 solution (CYP- NaHC0₃-9.5) with a target pH of 9.5 shows improved antimicrobial activity in comparison to a commercial nasal spray (Emser® Nasenspray, containing mainly NaHCOs as well as other salts, pH target of 9.0). Antimicrobial testing has been conducted in guidance of Ph.Eur. 5.1.3-1. The set-up was modified to serve for comparison of the two nasal spray solutions. One read-out time point after an incubation period of 14 days was selected.

Inoculum:

After measuring the initial pH of the two spray formulations, the following microorganisms (cool storage 2-8°C) have been added to the products in the mentioned amount.

Table: Microorganisms and quantities thereof added to the spray formulations

Media:

Caso Bouillon +0,3% Lecithin +3% Tween 80 Peptone

TSA - Casein-Soy-Agar Sabouraud-Agar All media used in the test were examined for growth promotion according to Ph.Eur. prior to execution of the test.

Results:

Measured pH values of the formulations, prior to inoculation was:

CYP- NaHC0₃-9.5 pH = 9.78

Emser® Nasenspray pH = 9.11

Table: Actual read-outs on plate after an inoculation period of 14 days and after preparation of the dilution series

9 cfu: colony forming unit

²> a negative logarithmic reduction means an increase in cfu 3> too numerous to count

Conclusion:

In the case of CYP-NaHC0₃-9.5, no growth of any of the 3 tested microorganisms was observed. Contrary to this finding, in the reference formulation (Emser® Nasenspray) with a pH only -0.6 lower than that of CYP-NaHC0₃-9.5, growth of Pseudomonas aeruginosa and Candida albicans was observed. The antimicrobial effect against Staphylococcus aureus was more pronounced with CYP-NaHC0₃-9.5. In conclusion, CYP-NaHC0₃-9.5 showed higher antimicrobial activity against these 3 microorganisms compared to the reference spray (Emser® Nasenspray), indicating that increasing the pH from 9.11 to 9.78 leads to an increase of the antimicrobial activity.

Example 13: Effect of the nasal spray buffer strength of NaHC03 on the pH of artificial nasal fluid

The aim of the experiment was to assess how the buffer strength of the nasal spray affects the pH of the nasal fluid. In order to study this effect in vitro , artificial nasal spray according to a previously published report was prepared (Masiuk, T., Kadakia, P., & Wang, Z. (2016). Development of a physiologically relevant dripping analytical method using simulated nasal mucus for nasal spray formulation analysis. Journal of pharmaceutical analysis, 6(5), 283-291). In healthy subjects, the pH of the nasal mucosa is 5.5 - 6.5 (England, R. J. A., Homer, J. J., Knight, L. C., & Ell, S. R. (1999). Nasal pH measurement: a reliable and repeatable parameter. Clinical Otolaryngology & Allied Sciences, 24(1), 67-68). In the experiment, 500 mI of various nasal sprays were added to 500 mI of artificial nasal fluid (ANF) with a pH of 6.2. The pH values of the ANF/nasal spray mixtures were measured after the addition of the nasal sprays with a pH glass electrode.

Media:

Artificial nasal fluid (ANF): 75 mg NaCI, 13 mg KCI and 3 mg CaCh dihydrate were dissolved in 10 ml of aqua destillata. Subsequently, 800 mg of porcine mucus (mucin from porcine stomach) were added under stirring. The mixture was then incubated for one hour on a thermoshaker (500 rpm, 37°C) to yield a homogenous fluid. Finally, the pH was adjusted with 1 M NaOH to pH=6.2.

Nasal spray formulations:

The table below shows the spray formulations that were used in the study.

Results:

The measured pH of ANF after 1 :1 dilution with the spray solutions shown in the table above are summarized in the table below:

In addition, the correlation between the pH of ANF and the buffer strength of various added NaHCCh solutions with an initial pH of 9.5 are shown in Figure 13.

Conclusion:

It was demonstrated in vitro that an initial physiological nasal pH of 6.2 can be increased via the administration of alkaline nasal spray formulations. However, the pH increase depends on a) the pH value of the administered solution and b) the buffer strength (buffer concentration) of the alkaline buffer system. In order to achieve an antiviral activity against enveloped viruses such as Sars-CoV-2, a target pH above 8.5, preferably above 9.0 of the nasal fluid needs to be present. In the current experimental set-up, a NaHCCh solution with an initial pH of 9.5 and a buffer concentration of at least 0.65% (w/v), preferably of 1.3% (w/v) was needed to achieve an increase of the artificial nasal fluid value above 8.5 or 9.0, respectively.

Example 14: Effect of the nasal spray buffer strength of TRIS on the pH of artificial nasal fluid

Media:

Artificial nasal fluid (ANF): 75 mg NaCI, 13 mg KCI and 3 mg CaCh dihydrate were dissolved in 10 ml of aqua destillata. Subsequently, 800 mg of porcine mucus (mucin from porcine stomach) were added under stirring. The mixture was then incubated for one hour on a thermoshaker (500 rpm, 37°C) to yield a homogenous fluid. Finally, the pH was adjusted with 1 M NaOH to pH=5.7.

Nasal spray formulations:

The table below shows the spray formulations that were used in the study.

Results:

In addition, the correlation between the pH of ANF and the buffer strength of various added TRIS solutions with an initial pH of 9.5 are shown in Figure 14.

Conclusion:

It was demonstrated in vitro that an initial physiological nasal pH of 5.7 can be increased via the administration of alkaline nasal spray formulations. However, the pH increase depends on a) the pH value of the administered solution and b) the buffer strength (buffer concentration) of the alkaline buffer system. In order to achieve an antiviral activity against enveloped viruses such as Sars-CoV-2, a target pH above 8.5, preferably above 9.0 of the nasal fluid needs to be present. In the current experimental set-up, a TRIS solution with an initial pH of 9.48 and a buffer concentration of at least 3.7% (w/v) was needed to achieve an increase of the artificial nasal fluid value above 8.5. Example 15: In vivo nasal spray application in Cynomolgus macaques

Two different nasal spray formulations were prepared as shown in the table below.

In vivo experimental set-up

Each formulation was tested in a group of 3 animals (Cynomolgus macaques, body weight 3.9 - 5.4 kg). The animals were anesthetized and moved to monkey chairs in a sitting posture. Prior to nasal spray application dosing, a predose pH measurement (0 min. sample) was taken from each animal from the left nostril only. For measuring the nasal pH, a cotton swab was first moistened with water. Then it was inserted around 1-2 cm into the nostril where it was rubbed against the nasal mucosa while turning for around 3-5 seconds. Finally, the pH of the swab was tested with pH paper. Before administering the nasal spray, each spray was tested for possible obstruction or incomplete filling of the spray system by puffing 5 times (Aptar device) and by spraying around 0.3 ml of the formulation through the MAD Nasal™ device. Subsequently, 0.1 ml of the respective spray formulation was applied to each nostril of each animal, by slightly (around 0.5 cm) inserting the spray device into the nose. After applying the spray into the nostril, swab samples were taken at pre-determined time-points as described above. After the experiment, animals were returned to their cages.

Results:

As shown in Figure 15, the spray application of CYP-CM001 resulted in an intranasal pH increase from initially pH 6.9 to about pH 9.3. A pH above 8.5 was maintained for about 13 minutes.

As shown in Figure 16, the spray application of CYP-CM002 resulted in an intranasal pH increase from initially pH 7.3 to pH 9.2 after 15 minutes.

Conclusion:

The data of the in vivo experiment indicate that after application of 100 mI of a 1.3% (w/v) NaHCCh nasal spray solution (CYP-CM001), an intranasal pH above 8.5 can be maintained for around 10 - 15 minutes. In addition, it was shown that with an optimized device and formulation (1.3% NaHCC>3 and 0.05% sodium hyaluronate; CYP-CM002), the intranasal pH increase can be further sustained.

Claims

1. An aqueous composition having a pH in the range of 8.5 to 11 for use in inactivating SARS-CoV-2 or for use in treating or preventing COVID-19 in a subject.

2. The aqueous composition for use according to claim 1, wherein the aqueous composition has a pH in the range of 8.5 to 10.5.

3. The aqueous composition for use according to claim 1 or 2, wherein the aqueous composition has a pH in the range of 9.0 to 10.0.

4. The aqueous composition for use according to any one of claims 1 to 3, wherein the aqueous composition comprises a bicarbonate buffer, an arginine buffer, a lysine buffer, a tris(hydroxymethyl)aminomethane buffer, a citrate buffer, a phosphate buffer, a urea buffer, an ammonium chloride buffer, or any combination thereof.

5. The aqueous composition for use according to any one of claims 1 to 4, wherein the aqueous composition comprises a bicarbonate buffer, an arginine buffer, a lysine buffer, a tris(hydroxymethyl)aminomethane buffer, or any combination thereof, preferably wherein the aqueous composition comprises a bicarbonate buffer.

6. The aqueous composition for use according to any one of claims 1 to 5, wherein the aqueous composition comprises sodium bicarbonate.

7. The aqueous composition for use according to any one of claims 1 to 6, wherein the aqueous composition comprises carrageenan, preferably iota carrageenan and/or kappa carrageenan.

8. The aqueous composition for use according to any one of claims 1 to 7, wherein the aqueous composition is to be administered to the subject via the nasal route, the pharyngeal route, or the pulmonary route.

9. The aqueous composition for use according to any one of claims 1 to 8, wherein the aqueous composition is provided in the form of a nasal spray, nasal drops, a pharyngeal spray, pharyngeal drops, or a lung inhalation composition.

10. The aqueous composition for use according to any one of claims 1 to 9, wherein SARS-CoV-2 is irreversibly inactivated.

11. The aqueous composition for use according to any one of claims 1 to 10, wherein SARS-CoV-2 is inactivated within 5 minutes or less from the administration of the aqueous composition.

12. The aqueous composition for use according to any one of claims 1 to 11, wherein the subject to be treated is a human.

13. Non-therapeutic use of an aqueous composition having a pH in the range of 8.5 to 11 for inactivating SARS- CoV-2.

14. The use of claim 13, wherein the aqueous composition is applied to an inanimate object, an inanimate surface, or as a room disinfectant for inactivating SARS-CoV-2.

15. The use of claim 13 or 14, wherein the aqueous composition has a pH in the range of 8.5 to 10.5.

16. The use of any one of claims 13 to 15, wherein the aqueous composition has a pH in the range of 9.0 to

100

17. The use of any one of claims 13 to 16, wherein the aqueous composition comprises a bicarbonate buffer, an arginine buffer, a lysine buffer, a tris(hydroxymethyl)aminomethane buffer, a citrate buffer, a phosphate buffer, a urea buffer, an ammonium chloride buffer, or any combination thereof.

18. The use of any one of claims 13 to 17, wherein the aqueous composition comprises a bicarbonate buffer, an arginine buffer, a lysine buffer, a tris(hydroxymethyl)aminomethane buffer, or any combination thereof, preferably wherein the aqueous composition comprises a bicarbonate buffer.

19. The use of any one of claims 13 to 18, wherein the aqueous composition comprises sodium bicarbonate.

20. The use of any one of claims 13 to 19, wherein the aqueous composition comprises carrageenan, preferably iota carrageenan and/or kappa carrageenan.