RU2842052C1 - Azelastine as antiviral treatment - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: group of inventions can be used for preventing or treating a pathological condition associated with SARS-CoV-2 infection. Disclosed is use of azelastine or a pharmaceutically acceptable salt thereof in amount of 0.1–500 mcg/dose for prophylactic or therapeutic treatment of a subject in need of treatment of a pathological condition caused by or associated with SARS-CoV-2 infection. What is also presented is using azelastine or its pharmaceutically acceptable salt for treating a biological surface for preventing SARS-CoV-2 infection and/or SARS-CoV-2 propagation.

EFFECT: group of inventions provides a strong antiviral effect on SARS-CoV-2 even at a very low concentration of azelastine.

16 cl, 10 dwg, 7 tbl, 8 ex

Description

AREA OF TECHNOLOGY

The present invention relates to new uses of an antihistamine drug compound for treating viral infections, in particular to methods and compounds for treating Coronaviridae infections (e.g. SARS virus or MERS virus), adenovirus infections, human respiratory syncytial virus infections and influenza infections.

LEVEL OF TECHNOLOGY

Coronaviruses are single-stranded RNA viruses with a diameter of about 120 nanometers. They are prone to mutation and recombination and are therefore very diverse. There are about 40 different varieties, which mainly infect humans and non-human mammals, as well as birds. Their hosts are bats and wild birds, and they can be transmitted to other animals and therefore to humans.

There are four main genera (alpha, beta, gamma, and delta coronaviruses) based on their genomic structure. Alpha and beta coronaviruses infect only mammals, typically causing respiratory symptoms in humans and gastroenteritis in other animals. As of December 2019, six different coronaviruses were known to infect humans. Four of these (HCoV-NL63, HCoV-229E, HCoV-OC43, and HKU1) typically cause mild, cold-like symptoms in immunocompetent individuals, while the other two have caused pandemics in the past two decades. In 2002–2003, severe acute respiratory syndrome coronavirus (SARS-CoV) caused the SARS epidemic, which resulted in a 10% mortality rate. Similarly, Middle East respiratory syndrome coronavirus (MERS-CoV) caused a pandemic in 2012 with a 37% mortality rate.

In late 2019 and early 2020, a new coronavirus, SARS coronavirus 2 (SARS-CoV-2), closely related to SARS-CoV, was discovered to be the cause of a large and rapidly spreading outbreak of respiratory illness, including pneumonia. Because the new coronavirus was identified, the disease it caused was named coronavirus disease 2019 (CoVID-19).

SARS-related coronaviruses are coated with spike proteins that contain a variable receptor-binding domain (RBD). This RBD binds to the angiotensin-converting enzyme-2 (ACE-2) receptor found in the heart, lungs, kidneys, and gastrointestinal tract, facilitating the virus’s entry into target cells.

The original Wuhan strain of the virus is considered the “wild-type” virus, which soon gave rise to variants, i.e. mutants that have evolved through natural selection for higher infectivity. In early March 2020, a variant with the D614G mutation in the spike protein (variant B.1) was identified in Europe and soon replaced the original Wuhan strain worldwide. Subsequently, SARS-CoV-2 variants B.1.1.7 (also known as 201/501Y.V1, VOC 202012/01) and B.1.351 (also known as 20H/501Y.V2) were identified in the United Kingdom and South Africa, respectively, and have since spread to many countries. These variants contain different mutations in the gene encoding the spike protein. One of them is in the receptor binding domain (RBD, which binds human ACE2) at position 501, in which the amino acid asparagine (N) has been replaced by tyrosine (Y) (N501Y mutation). The B.1.1.7 variant also has several other mutations, including: a 69/70 deletion, likely resulting in a conformational change in the spike protein, and P681H, near the S1/S2 furin cleavage site. The combination of these mutations results in higher receptor binding, more efficient spread, and a higher ability to cause disease compared to the original variant that emerged in Wuhan, China, in 2019. The B.1.351 variant also has, in addition to N501Y, the E484K and K417N mutations, but not the 69/70 deletion. Several lines of evidence indicate that this variant arose due to immune suppression, i.e., as an escape from the human immune response induced by natural infection or passive immune therapy or active immunization with various vaccines. The E484K mutation appears to be responsible for the reduced vaccine efficacy. This mutation (along with 16 other mutations N501Y and K417T) was also found in the Brazilian variant, designated P.1. The P.1 variant caused widespread infection in Brazil and was later detected worldwide. This mutant is thought to cause more severe disease in younger people, lead to higher mortality, and escape the immune response induced by the previous variant and some vaccines.

The B.1.617 mutant was first discovered and distributed in India. It has 13 mutations out of 3 mutations associated with immune escape: E484Q, L452R and P681R. The B.1.618 lineage has emerged as one of the dominant mutants in West Bengal, carrying a two-amino acid deletion (H146del and Y145del) and E484K and D614G mutations in the spike protein. It is predicted to have higher infectivity and pose a threat to escape natural or vaccine-induced immunity.

It is highly likely that large-scale vaccination will induce the emergence of additional variants with different combinations of currently known and new mutations.

Accelerated global vaccination with simultaneous intense viral dissemination creates enormous evolutionary pressure for the emergence of escape mutants. All vaccines rely almost exclusively on triggering an immune response against the spike protein. In addition, according to current thinking, a neutralizing immune response against the receptor-binding domain of the spike protein is considered to be the main factor of protection. Based on these two facts, naturally occurring virus variants will be selected for those expressing mutated spike proteins that have a selective advantage (higher receptor binding and/or evasion of the neutralizing immune response). Subsequent variants may accumulate several successive mutations with higher overall virulence/transmissibility and/or higher probability of immune escape. In addition to the variants mentioned above, additional new virus variants are expected to emerge, potentially requiring second-generation vaccines for optimal protection against them.

Thus, antiviral compounds active against the currently dominant UK variant (B1.1.7) and the SA variant (B.1.351), as well as other emerging variants that are associated with significant reductions in vaccine-induced protection, are highly relevant for both prevention and treatment.

Adenoviruses are large, nonenveloped viruses with double-stranded genomic DNA. The family Adenoviridae contains six genera of viruses with broad host specificity. All seven human adenovirus families (A through G) belong to the genus Mastadenovirus. Human viruses are currently classified into 88 different (sero)types, which are responsible for different mucosal infections. Types in families B (HAdV-B) and C (HAdV-C) are responsible for upper respiratory tract infections, while types HAdV-F (mainly types 40 and 41) and HAdV-G (mainly type 52) cause gastroenteritis. Conjunctivitis is associated with HAdV-B and HAdV-D. These mucosal infections are usually self-limiting but may be more severe in an immunocompromised host. Adenovirus serotype 14 is an emerging pathogen that causes outbreaks of severe respiratory infections that can be fatal even in immunocompetent hosts.

Adenoviruses have a classical icosahedral capsid composed of 240 hexon proteins and 12 penton proteins. The penton bases are linked to protruding fibrils that bind to host cell receptors: CD46 (family B) and the coxsackievirus/adenovirus receptor (CAR, for all other families). The initial binding is followed by interaction of the viral penton base structures with αV integrins, which stimulates endocytosis of viral particles. Inside the cell, the capsid is destabilized, the endosome is degraded, and viral DNA enters the nucleus through the nuclear pore. Following association with histone proteins, the host transcription machinery is used to express viral genes without integration of the viral genome into the host genome. Viral proteins are expressed in early (mainly regulatory proteins) and late (structural proteins) phases separated by genome replication. Finally, viral genomes are packaged into a protein coat and released from the host cell following a virus-induced lysis procedure.

Adenoviruses are relatively resistant to disinfectants and detergents (not encapsulated) and survive for a long time on surfaces and in water. There are no approved antiviral drugs for the treatment of adenovirus infections, so treatment is largely symptomatic. There is currently no adenovirus vaccine available to the general public, but the US military has used a vaccine for types 4 and 7. Recently engineered adenoviruses have been successfully used in gene therapy and as viral vectors for heterologous vaccine antigen delivery (Ebola and Covid).

Human respiratory syncytial virus (hRSV) is an enveloped virus with a negative-sense, single-stranded RNA genome. The genome is linear and has 10 genes encoding 11 proteins. RSV is divided into two antigenic subtypes, A and B, with 16 and 22 clades (or strains), respectively.

RSV is highly contagious and can cause outbreaks by both contact and hospital transmission. Each year, RSV causes approximately 30 million cases of acute respiratory illness and more than 60,000 childhood deaths worldwide. Transmission occurs via contaminated aerosolized droplets landing on mucosal surfaces of the nose, mouth, or eyes. Infection of the ciliary cells of the upper respiratory tract is followed by dissemination to the lower respiratory tract. It is one of the most common childhood infections, with severity ranging from mild upper respiratory tract infection to bronchiolitis and viral pneumonia, the most severe of which may require mechanical ventilation. Immunocompromised individuals (including premature infants) are at higher risk of more severe outcomes. Therapeutic options are generally limited to supportive care, although ribavirin has been licensed for the treatment of RSV infections in children. Vaccines are not available (despite significant development efforts), but passive immunization with monoclonal antibodies (palivizumab) has become available as a prophylactic option.

Influenza virus infection, commonly referred to as "flu", is an infectious disease caused by influenza viruses. Each season (winter months in the Northern Hemisphere), 5-15% of the population becomes ill with influenza, with an estimated 3-5 million severe cases. More than half a million deaths occur each year in high-risk groups, including young children, the elderly, and people with chronic medical conditions. After an incubation period of 1-4 days, symptoms begin suddenly and include fever, chills, headache, muscle pain or aches, loss of appetite, fatigue, and confusion. Pneumonia may be caused by primary viral infection or secondary bacterial infection, such as pneumococcus or S. aureus. Transmission of influenza is mediated by aerosolized droplets. The primary site of infection is the upper respiratory tract, with subsequent progression to the lower respiratory tract and invasive infection.

There are four types (species) of influenza viruses: A, B, C, and D. Seasonal epidemics (i.e., flu season) are caused by human influenza A viruses (IAV) and B viruses (IBV), while C and D are rarely associated with symptomatic human infections. Influenza A viruses are divided into subtypes based on two proteins on the surface of the virus: hemagglutinin (H) and neuraminidase (N). Although there are potentially 198 different combinations of influenza A subtypes, the H1N1 and H3N2 types are currently circulating worldwide. Influenza B viruses are not divided into subtypes, but are instead further classified into two lineages: B/Yamagata and B/Victoria. For this reason, seasonal influenza vaccines typically contain a combination of two A (H1N1 and H3N2) and one or two B strains. Influenza viruses have a negative-sense, single-stranded RNA genome that is segmented. Both IAV and IBV contain eight segments that can combine with the genome of other influenza viruses when they coinfect the same cell. This process (called reassortment) produces progeny with significantly altered viral antigen compositions. Such reassorted virus variants are new to the human population and can cause pandemics (e.g., Spanish flu in 1918, H1N1). Reassortment can also occur between viruses specific to different hosts. For example, the 2009 "swine flu" pandemic was caused by a triple reassorted virus carrying a combination of pig-, bird-, and human-specific viral sequences (H1N1). Similarly, strains of avian influenza that are common in wild waterfowl sometimes infect humans (avian or bird flu, H5N1). When such avian viruses reassort with human influenza viruses, variants can arise that spread from person to person and thus have the potential to cause global pandemics.

Therapy for individuals at lower risk is mainly focused on symptomatic treatment (fever) and isolation. Patients with severe or progressive clinical illness associated with suspected or confirmed influenza virus infection are treated with antiviral drugs (eg, oseltamivir or other neuraminidase inhibitors) and supportive care (eg, anti-inflammatory drugs). High-risk groups (the elderly, pregnant women, immunocompromised individuals with underlying chronic diseases) should receive annual seasonal influenza vaccines as prophylaxis.

Azelastine, a phthalazine derivative, is an antihistamine available as an intranasal spray for the treatment of allergic and vasomotor rhinitis and as an ophthalmic solution for the treatment of allergic conjunctivitis. It is a racemic mixture, although there is no noted difference in pharmacological activity between the enantiomers, and was first approved by the U.S. Food and Drug Administration (FDA) in 1996.

Gysi et al. (“Network Medicine Framework for Identifying Drug Repurposing Opportunities for COVID-19,” arXiv:2004.07229 [q-bio.MN] 2020-04-15) disclose a network toolkit for COVID-19 to derive some drug candidates based on their likely efficacy in COVID-19 patients, including azelastine, but without any indication of whether the antihistamine would have a direct effect on the virus underlying the disease.

Hasanain Abdulhameed Odhar et al. (Bioinformation 2020, 16(3):236–244) report molecular docking and dynamic modeling of FDA-approved drugs using the 2019 novel coronavirus core protease. Drugs were ranked according to their minimum binding activity to the 2019-nCoV core protease crystal, with azelastine ranking less favorably than conivaptan.

Xia Xiao et al. (bioRxiv July 6, 2020, DOI:10.1101/2020.07.06.188953) describe the antiviral activity of a number of compounds against OC43, including azelastine, as an antihistamine.

Fu et al. (Cell Prolif. 2014, 47:326-335) disclose azelastine hydrochloride, a sodium taurocholate cotransporting polypeptide (NTCP)-targeted protein, in the treatment of hepatitis B virus (HBV). NTCP is a transmembrane protein expressed at high levels in human hepatocytes that mediates bile acid transport and plays a key role in HBV entry into hepatocytes.

M.W. Simon (Pediatric Asthma, Allergy & Immunology, 2003, Vol.16:275-282) describes azelastine as a potent and selective second-generation histamine _H1 receptor antagonist that negatively regulates the expression of the intracellular adhesion molecule-1 (ICAM-1) receptor, which plays an important role in the mucosal attachment of human rhinovirus, coxsackievirus type A, adenovirus type 5, human parainfluenza virus types 2 and 3, and respiratory syncytial virus, as well as in the mobilization of immune effector cells.

BRIEF DESCRIPTION OF THE INVENTION

The objective of the present invention is to provide new antiviral treatments, medical and pharmaceutical products that can be used to prevent viral infection and/or the spread of the virus, in particular in subjects who have been exposed to the virus or infected with it, or who are at risk of infection. The objective is achieved by the subject matter of the claims of the present invention and as further described herein.

According to the present invention, there is provided an azelastine compound in an antiviral effective amount for use as an antiviral substance in a pharmaceutical preparation for use in the prophylactic or therapeutic treatment of a subject in need of antiviral treatment.

In particular, the azelastine compound is azelastine or a pharmaceutically acceptable salt thereof, such as azelastine hydrochloride.

According to a particular aspect, the pharmaceutical preparation is a medical product or a medicinal product. In particular, the pharmaceutical preparation comprises an azelastine compound and a pharmaceutically acceptable carrier.

In particular, the subject requires antiviral treatment targeting a respiratory virus such as influenza virus, respiratory syncytial virus, adenovirus, coronavirus, or an upper respiratory tract virus such as rhinovirus.

In this document, a respiratory virus specifically refers to a virus that causes a respiratory disease. Although some of the target viruses described later in this document may not only cause a respiratory disease but also infect other parts of the body, such viruses are still referred to as a "respiratory virus" in this document. In particular, the antiviral treatment is targeted at one or more human viruses, in particular a human respiratory virus, such as one selected from the Coronaviridae, Adenoviridae, Paramyxoviridae or Orthomyxoviridae virus families.

In particular, they treat a pathological condition that is caused by or associated with an infection by one or more of:

a) Coronaviridae viruses, preferably selected from the group consisting of β-coronavirus such as SARS-CoV-2, MERS-CoV, SARS-CoV-1, HCoV-OC43, HCoV-HKU1, and α-coronavirus such as HCoV-NL63, HCoV-229E or PEDV, including naturally occurring variants or mutants of any of the above, or

b) Adenoviridae, preferably human adenoviruses such as HAdVB, HAdVC or HAdVD;

c) human respiratory syncytial viruses (RSV), such as RSV subtype A or B; or

d) influenza viruses, such as human influenza viruses, preferably influenza A virus (IVA), such as H1N1, H3N3 or H5N1, or influenza B virus (IVB), or influenza C virus (IVC), or influenza D virus (IVD).

In particular, a pathological condition is treated that is caused by or associated with infection with one or more Coronaviridae viruses, in particular one or more different Coronaviridae viruses.

According to a particular aspect, said Coronaviridae viruses are preferably selected from the group consisting of a β-coronavirus, such as SARS-CoV-2, MERS-CoV, SARS-CoV-1, HCoV-OC43 or HCoV-HKU1, or an α-coronavirus, such as human coronavirus NL63 (HCoV-NL63, New Haven coronavirus), HCoV-229E or porcine epidemic diarrhea virus (PEDV), including naturally occurring variants or mutants of any of the above.

In particular, said one or more different Coronaviridae viruses referred to herein are naturally occurring variants or mutants of SARS-CoV-2, in particular those that contain one or more mutations of the spike protein (SARS-CoV-2 S protein), such as any one or more of the following mutations: K417N, L452R, N501Y, D614G, P681H, P681R, E484K, E484Q, or deletion 69/70.

In particular, the spike protein comprises or consists of the amino acid sequence identified as SEQ ID NO: 4 (the sequence is shown in Fig. 5, NCBI accession number QII57161.1, SARS-CoV-2 S protein).

Preferably, said one or more different Coronaviridae viruses are natural variants or mutants of SARS-CoV-2 selected from the group consisting of the UK variant (B1.1.7), the South African variant (B.1.351), the Brazilian variant (P.1), the Indian variant (B.1.617) and the Bengal variant (B.1.618).

The formulations, methods and uses described herein specifically include an azelastine compound in an antiviral effective amount. Such an antiviral effective amount may target one specific virus or more than one of different viruses, virus mutants or virus variants, such as those further described herein.

The target virus of the antiviral effect is in particular understood to be a virus that is (directly or indirectly) related to the indication for treatment described herein, in particular for treatment for the purpose of prevention or therapy of a pathological condition or disease that is caused by or otherwise related to such target virus.

In particular, the target of the preparations, methods and uses described herein, and in particular the target of the antiviral effect described herein, may be any one or more of the different types of viruses, mutants of viruses or variants of viruses, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 different types of viruses, mutants or variants of viruses.

In particular, the target may be any two or more viruses, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 different virus species, virus mutants or virus variants, wherein at least 2, 3 or 4 of the viruses belong to different virus families, such as those selected from the families:

a) Coronaviridae (β-coronavirus such as SARS-CoV-2, MERS-CoV, SARS-CoV-1, HCoV-OC43, HCoV-HKU1; or α-coronavirus such as HCoV-NL63, HCoV-229E or PEDV, including natural variants or mutants of any of the above);

b) Adenoviridae (such as adenoviruses or human adenoviruses, e.g. HAdVB, HAdVC or HAdVD);

c) Paramyxoviridae (such as RSV or human RSV, e.g. hRSV subtype A or B); or

d) Orthomyxoviridae (such as influenza viruses or human influenza viruses, preferably influenza A virus (IVA) such as H1N1, H3N3 or H5N1, or influenza B virus (IVB), or influenza C virus (IVC), or influenza D virus (IVD)).

In particular, such viruses listed in paragraphs (a) to (d) above are referred to in this document as exemplary “respiratory viruses”.

According to a specific example, the target may be at least one virus selected from a) and at least one virus selected from b), c) or d). In particular, the target may be at least one β-coronavirus, such as the SARS virus, in particular SARS-CoV-2, and at least one influenza virus.

In particular, the target may be at least one β-coronavirus, such as the SARS virus, in particular SARS-CoV-2, and at least one adenovirus HAdVB, HAdVC or HAdVD.

In particular, the target may be at least one β-coronavirus, such as the SARS virus, in particular SARS-CoV-2, and at least one hRSV subtype A or B.

According to a specific example, the target may be at least one virus selected from d) and at least one virus selected from a), b) or c).

In particular, the target may be at least one of influenza viruses or human influenza viruses and at least one adenovirus HAdVB, HAdVC or HAdVD.

In particular, the target may be at least one of the influenza viruses or human influenza viruses and at least one hRSV subtype A or B.

According to a specific example, the target may be at least one virus selected from b) and at least one virus selected from a), c) or d).

In particular, the target may be at least one HAdVB, HAdVC or HAdVD adenovirus and at least one hRSV subtype A or B.

According to a specific example, the target may be at least one virus selected from a) and at least one virus selected from b) and optionally at least one virus selected from c) or d). In particular, the target may be at least one β-coronavirus, such as the SARS virus, in particular SARS-CoV-2, and at least one influenza virus, and optionally at least one HAdVB, HAdVC or HAdVD adenovirus and/or at least one hRSV subtype A or B.

The present invention further provides an azelastine compound (as further described herein) in an antiviral effective amount for use as an antiviral substance in a pharmaceutical preparation for the prophylactic or therapeutic treatment of a pathological condition caused by or associated with an infection by one or more different viruses that are Coronaviridae viruses, preferably selected from the group consisting of a β-coronavirus such as SARS-CoV-2, MERS-CoV, SARS-CoV-1, HCoV-OC43 or HCoV-HKU1, or an α-coronavirus such as HCoV-NL63, HCoV-229E or PEDV, including naturally occurring variants or mutants of any of the above, such as any one or more of the naturally occurring variants or mutants of SARS-CoV-2 mentioned herein, preferably selected from the group consisting of the UK variant (B1.1.7), the South African variant (B.1.351), the Brazilian variant (P.1), the Indian variant (B.1.617) and the Bengal variant (B.1.618).

In particular, the pathological condition associated with or caused by the target virus or respiratory virus described herein, in particular where the target virus is a virus from the Coronaviridae, Adenoviridae, Paramyxoviridae or Orthomyxoviridae family, is a common cold, nasal infection, sinusitis, throat and larynx infection, bronchiolitis, diarrhea, skin rash or pneumonia, acute respiratory distress syndrome (ARDS). In particular, the pathological condition may be a symptom associated with any one or more of the above. In particular, the symptom to be treated may be any of cough, sore throat, runny nose, sneezing, headache and fever.

According to a specific aspect, the antiviral effective amount effectively prevents the infection of susceptible cells by the virus, which ensures the treatment of the pathological condition. In particular, the susceptible cells are in the body of the subject or on a biological surface or in the subject.

According to a particular aspect, the antiviral effective amount is 0.1-500 μg/dose, preferably lower than any of 100, 90, 80, 70, 50, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1 or 1 μg/dose. In particular, the number of doses is up to 1-10 per day.

According to a particular aspect, the antiviral effective amount is from 15 mcg to 150 mcg per dose, preferably less than 100 mcg or less than 50 mcg.

In particular, the antiviral effective amount may be even lower, such as lower than any of 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2 or 0.1% of an amount in the range of 15 μg to 150 μg. In particular, such lower amounts have been shown to be effective against coronaviruses (or viruses from the Coronaviridae family) and/or viruses other than coronaviruses (or other than viruses from the Coronaviridae family), such as viruses from the Adenoviridae, Paramyxoviridae or Orthomyxoviridae families.

For example, the antiviral effect against any of the target viruses described in this paper has been shown to be highly effective with 0.1%, i.e. 2.39 mM, and 5-fold reduced (0.02%, i.e. 478 μM) dose of the commercial nasal spray formulation azelastine, reducing the viral load in humans.

For example, the anti-influenza virus effect was shown to be highly effective at 5-fold dilution (0.02%, i.e. 478 μM) and 10-fold dilution (0.01%, i.e. 239 μM) of the commercial drug azelastine (0.1%, 2.39 mM).

For example, the anti-RSV effect has been shown to be highly effective in the range of 0.4-6.4 μM in vitro.

According to a particular aspect, said pharmaceutical preparation is adapted for local administration, such as topical or local mucosal administration, preferably for use in the upper and lower respiratory tract, for nasal, pulmonary, intraoral, ocular or dermal administration, or for systemic administration, preferably by intravenous, intramuscular, subcutaneous, intradermal, transdermal or oral administration. Typically, for parenteral administration, intravenous or oral administration is preferred.

According to a particular aspect, said pharmaceutical preparation is administered to a subject in the form of a spray, such as a nasal spray, a powder, such as a soluble powder or an inhalation powder, or by means of a medical device, such as, for example, comprising a surface or fabric impregnated with an azelastine compound for inhalation, a gel, ointment, cream, foam or liquid solution, lotion, mouthwash, aerosolized powder, aerosolized liquid form, granules, capsules, drops, a tablet, syrup, lozenge, eye drops or a preparation for infusion or injection.

In particular, antiviral formulations and administration forms are provided, for example, for veterinary and human use. In particular, the formulations comprise a predetermined amount of an azelastine compound as an active ingredient; for example, in the form of a powder or granules; as a solution or suspension in an aqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion.

According to a particular aspect, the azelastine compound is administered in an antiviral effective amount to provide a peak concentration (or maximum concentration) in blood or plasma that is about 0.01-2 mcg/mL or up to any of 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 mcg/mL.

According to a particular aspect, the azelastine compound is used in the formulation at a concentration of 1 μM - 10 mM, preferably up to any of 1 mM, 100 μM, 90 μM, 80 μM, 70 μM, 60 μM or 50 μM. According to a particular embodiment, the concentration is 3-50 μM.

In particular, the liquid solution or dispersion is used for nasal administration, for example by means of nasal drops or a nasal spray, wherein preferably the antiviral effective amount is 1-1000 μg per nostril, preferably 1-500 μg per nostril or up to any of 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, more preferably up to 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 5, 4, 3, 2 or 1 μg per nostril.

In particular, the dose can be administered by nasal administration and is about 2-2000 mcg per dose, preferably 2-1000 mcg per dose or up to any of 400, 380, 360, 340, 320, 300, 280, 260, 240, 220, more preferably up to 200, 180, 160, 140, 120, 100, 80, 60, 40, 20, 10, 5, 4, 3 or 2 mcg per dose.

The composition is preferably administered in the form of a nasal spray, nasal drops, an aerosol such as an aerosolized liquid or powder, for example as a throat spray or for intrapulmonary administration, or as eye drops.

Exemplary formulations may comprise an azelastine compound as an active ingredient in an amount of, for example, 0.001 to 2% (w/w), such as about 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01%, 0.02%, 0.05%, 0.1%, 0.15%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9% or 2.0% (w/w).

In particular, a volume of 100-1000 μl per dose can be applied in a spray formulation, for example, up to 500 μl of spray volume. According to a specific example, a nasal spray can deliver a volume of about 100-150 μl per spray. Typically, two sprays are administered per nostril once or twice a day.

When using a spray, a metered dose spray is preferably used to administer a specific volume of spray or dose per spray.

Formulations suitable for intrapulmonary administration may have a particle size in the range of 0.1 to 500 microns and may be administered by inhalation through the nasal passage or by inhalation through the mouth to reach the alveolar sacs. Suitable formulations include aqueous or oil solutions of the azelastine compound. Formulations suitable for administration as an aerosol or dry powder may be prepared according to conventional methods and may be delivered with other therapeutic agents, such as compounds used in the treatment or prevention of pulmonary inflammation or lung diseases.

In particular, the liquid solution or dispersion is used for parenteral administration, for example by infusion or injection, wherein preferably the antiviral effective amount provides a dose of about 1-500 mg.

In particular, a single loading dose of about 1-500 mg may be administered parenterally, followed by maintenance doses of about 10-200 mg, or about 100 mg, or about 200 mg, for example, by daily administration for 1-10 days or until a specified clinical response is achieved.

In particular, the tablet, gel or lozenge is used for oral administration, wherein preferably the antiviral effective amount is from 1 mcg to 12 mg per dose, preferably up to any of 5, 4, 3, 2 or 1 mg; or up to 100 mcg per dose.

In particular, a tablet containing the compound azelastine can be used, which can be administered one to three times a day.

Formulations suitable for topical administration in the mouth include lozenges containing the active ingredient in a flavoured base such as sucrose, acacia or tragacanth, lozenges containing the azelastine compound in an inert base such as gelatin and glycerol, sucrose or acacia, or mouth rinses containing the azelastine compound in a suitable liquid carrier.

When formulated as a gel or ointment for topical application, the active ingredients may be applied with a paraffin or water-miscible ointment base. Alternatively, the active ingredients may be formulated as a cream with an oil-in-water cream base.

Formulations suitable for topical administration to the eye may include eye drops, gel or cream, wherein the azelastine compound is dissolved or suspended in a suitable vehicle, particularly an aqueous solvent or an oil/water emulsion.

According to specific examples, such formulations for topical administration in the mouth or a topically applied form such as a gel or ointment may contain an azelastine compound in a concentration of, for example, from 0.001 to 20% (w/w), such as about 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01%, 0.02%, 0.05%, 0.1%, 0.15%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 9 or 20% (w/w).

According to a particular aspect, treatment with an azelastine compound may be combined with additional treatment by administering an azelastine compound (which may be the same or a different compound) in another form of administration. For example, treatment with an intranasal or throat spray containing azelastine hydrochloride may be combined with a tablet containing an azelastine compound (which may be azelastine hydrochloride or may be different from azelastine hydrochloride).

According to a particular aspect, the azelastine compound is administered as a single antiviral agent, or wherein the treatment is combined with additional treatment, such as additional antiviral, anti-inflammatory and/or antibiotic treatment, for example comprising the administration of one or more antiviral agents or substances, and/or one or more anti-inflammatory and/or antibiotic agents or substances, via one or more different formulations and/or one or more different routes of administration.

In particular, the azelastine compound can be combined with one or more additional active therapeutic agents in a single dosage form for simultaneous, co-administration, or sequential administration to a subject. Combination therapy, for example, can be administered as a simultaneous, parallel, or sequential regimen. When administered sequentially, the combination can be administered as two or more administrations.

According to a specific aspect, a subject is treated that has been infected or is at risk of becoming infected with said virus, preferably a human or a non-human mammal such as a dog, cat, horse, camelid, cattle or pig.

In a particular case, the subject is or has been exposed to the virus, or is otherwise at risk of becoming infected with the virus.

In a particular case, the subject has a weakened immune system and is at higher risk of developing a viral disease or increased severity of a viral disease.

In a particular case, the subject has been diagnosed or established to be infected with the virus.

In specific embodiments, a subject is treated that is a sick subject or patient suffering from a disease caused by a Coronaviridae virus, such as a disease caused by a SARS virus, upon exposure to a pathogen such as COVID19 or COVID19-associated pneumonia.

According to additional specific embodiments, a subject is treated that is a sick subject or patient suffering from a disease caused by an influenza virus, such as influenza.

According to the present invention, there is further provided an azelastine compound described herein for use as an antiviral substance in a medical product for treating a biological surface to prevent viral infection and/or the spread of a virus.

According to a specific aspect, said medical product is designed for topical application, preferably for application in the upper and lower respiratory tract, nasal, pulmonary, oral, ocular or dermal application.

Topical application generally refers to the skin surface, wound, and/or mucosal cells or tissues (e.g. alveolar, buccal, lingual, masticatory or nasal mucosa, etc.).

According to a particular aspect, said medicinal product is used in a formulation that is suitably used for topical administration, in particular for administration through the mucous membrane, such as a spray, solution, dispersion, dry powder or aerosolized liquid or powder.

According to a particular aspect, the azelastine compound is applied to a biological surface in an antiviral effective amount, wherein the amount is preferably from 1 ng to 1000 ng per ^cm2 , preferably 10-800 ng/ ^cm2 or up to any of 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50 ng/ ^cm2 .

Any of the medical products that are suitably used for topical treatment as described herein may be used to treat a biological surface.

According to a particular aspect, the biological surface comprises or consists of a mucosal surface that is infected or at risk of becoming infected with said virus.

According to the present invention, there is further provided the use of the azelastine compound described herein as a viral disinfectant, in particular suitable for treating a biological surface or a non-biological surface, such as sanitary devices, face masks, etc. In particular, the disinfectant can be used to treat living or non-living surfaces.

In particular, a viral disinfectant is an antiviral drug such as a medical product.

According to a particular aspect of the present invention, there is provided an azelastine compound as described herein for use in the prevention or treatment of Coronaviridae virus infection, in particular SARS virus infection, in a human or non-human mammal.

According to a particular aspect of the present invention, there is provided an azelastine compound as described herein for use in the prevention or treatment of Coronaviridae virus infection, in particular SARS virus infection, in a human or non-human mammal.

According to a particular aspect of the present invention, there is provided an azelastine compound as described herein for use in the prevention or treatment of Adenoviridae virus infection, in particular HAdVB, HAdVC or HAdVD virus infection in humans.

According to a particular aspect of the present invention, there is provided an azelastine compound as described herein for use in the prevention or treatment of Paramyxoviridae virus infection, in particular human RSV virus infection, in humans.

According to a particular aspect of the present invention, there is provided an azelastine compound as described herein for use in the prevention or treatment of an Orthomyxoviridae viral infection, in particular an influenza virus infection, in a human or non-human mammal.

According to a particular aspect, a kit is provided that comprises one or more individual dosage units of an azelastine compound as further described herein and instructions for use thereof in the treatment of a Coronaviridae virus infection or a disease caused by a Coronaviridae virus in a human or non-human mammal.

According to another specific aspect, a kit is provided that comprises one or more individual dosage units of an azelastine compound as further described herein and instructions for use thereof in the treatment of an Adenoviridae virus infection, in particular an HAdVB, HAdVC or HAdVD virus infection, in a human.

According to another specific aspect, a kit is provided that comprises one or more individual dosage units of an azelastine compound as further described herein and instructions for use thereof in the treatment of a Paramyxoviridae viral infection, in particular a human RSV virus infection, in a human.

According to another specific aspect, a kit is provided that comprises one or more individual dosage units of an azelastine compound as further described herein and instructions for use thereof in the treatment of an Orthomyxoviridae viral infection, in particular an influenza virus infection, in a human or non-human mammal.

According to a particular aspect of the present invention, there is provided an antiviral pharmaceutical preparation comprising an azelastine compound as further described herein and a pharmaceutically acceptable carrier.

In particular, the pharmaceutical preparation is proposed for medical use, in particular for use in the prophylactic or therapeutic treatment of a pathological condition caused by the Coronaviridae virus, such as COVID19.

In particular, the pharmaceutical preparation is proposed for medical use, in particular for use in the prophylactic or therapeutic treatment of a pathological condition caused by the Coronaviridae virus and/or the Adenoviridae virus and/or the Paramyxoviridae virus and/or the Orthomyxoviridae virus.

According to a particular aspect of the present invention, there are further provided methods of treating a subject infected or at risk of infection with one or more different viruses, such as one or more of Coronaviridae viruses and/or Adenoviridae viruses and/or Paramyxoviridae viruses and/or Orthomyxoviridae viruses, comprising administering an antiviral effective amount of an azelastine compound and corresponding medical products or pharmaceuticals, as further described herein.

According to a further specific aspect of the present invention, there is provided an antiviral preparation of the azelastine compound described herein (such as a medical product, a pharmaceutical preparation or a disinfectant), and methods for producing such an antiviral preparation, comprising formulating an antiviral effective amount of the azelastine compound with a pharmaceutically acceptable carrier to produce an antiviral preparation, in particular a medical product or a pharmaceutical preparation.

Topical application of any of the antiviral preparations described herein (such as a medical product, pharmaceutical preparation or disinfectant) to a biological surface is preferably such that at a specified contact time, such as from 10 minutes to 24 hours and/or up to 24, 18, 12, 6, 5, 4, 3, 2 or 1 hour, the contact results in at least a 0.5-fold (half) reduction or a 1-log, 2-log, 3-log, 4-log, 5-log reduction in the amount of virus on said surface.

GRAPHIC MATERIALS

Figure 1 shows an illustration of the Shannon entropy-based approach for identifying drug homologs with matching pathway profiles (A) and identifying corresponding SARS-CoV-2 pathways (B). Pathway profiles were calculated for hydroxychloroquine and SARS inhibitors with well-defined mechanisms and mode of action: SSAA09E2, a small molecule ACE-2 inhibitor, and SSAA09E3, a general host-virus membrane fusion inhibitor.

Figure 2 shows the prevention of the cytopathic effect of SARS-CoV-2 induced in Vero E6 cells based on phase contrast microscopy images. A: Uninfected (control) culture; B: SARS-CoV-2 infected cells; C-F: SARS-CoV-2 infected cells in the presence of increasing concentrations of azelastine-HCl: 3.125, 6.25, 12.5, and 25 μM. Cells were infected with SARS-Co-V2 virus at an MOI of 0.1 for 30 min, then the culture medium was removed and replaced with fresh culture medium without virus (but containing azelastine), and microscopic images were taken 48 h after infection.

Figure 3 shows the reduction in the cytopathic effect of SARS-CoV-2 on reconstituted human nasal tissue (MucilAir). Human nasal tissue was infected with SARS-CoV-2 and then treated with a 5-fold dilution of 0.1% azelastine HCl nasal spray solution for 20 minutes every 24 hours for 3 days. The figure shows a low-resolution microscopic image of human nasal tissue at 48 and 72 hours post-infection. Mucin production (a sign of intact tissue function) is demonstrated as the presence of dark spots on the tissue.

Figure 4 (In vitro inhibition of SARS-CoV-2 B.1.351 and B.1.1.7 infection by azelastine) shows the inhibition effect of azelastine on infection of Vero-TMPRSS2/ACE-2 cells with SARS-CoV-2 B.1.351 (A) or B.1.1.7 (B). Cells were infected with SARS-CoV-2 at an MOI of 0.01 for 30 min, azelastine was added to the infected cells either immediately before infection (prophylactic condition) or 30 min after infection (therapeutic condition). At 48 h post-infection, virus copy number was determined by qPCR. Inhibition of infection is expressed as virus copy number relative to wells containing virus only (without azelastine treatment). Graphs show the mean and standard deviation calculated from 9 (A) or 6 (B) individual values. The azelastine concentration inhibiting 50% of infection (EC50) was calculated using nonlinear regression (1og(agonist) versus normalized response - variable slope) using GraphPad Prism 8.4.3.

Figure 5: SEQ ID NO: 4 (NCBI accession number QII57161.1, SARS-CoV-2 S protein).

Figure 6: Antiviral effect of azelastine at 2 different concentrations determined in human Calu-3 cells infected with SARS-CoV-2 at an MOI of 0.01, as well as the cytotoxicity of the same concentrations of the compound measured in identical cells without viral infection. The graph shows the average values of the results of three replicates of one typical experiment.

Figure 7: Viability of uninfected Hep-2 cells treated with the indicated final concentration range of azelastine-HCl and appropriately diluted DMSO vehicle for 48 hours. The mean and SEM were calculated for 3 independent experiments. The threshold for statistically significant difference was P<0.05. ****P<0.0001

Figure 8: Antiviral effect of azelastine-HCl at the indicated final concentration range on RSV infection of HEp-2 cells. Azelastine-HCl was applied before (A), simultaneously (B), or after (C) viral infection, and replication was assessed by imaging RSV-infected cells as described in the text. DMSO, the solvent of azelastine-HCl stock solution, was diluted accordingly for use as a control.

Experiments were repeated twice with two replicates. Bars represent the mean with standard error of the mean as error bars.

Figure 9 shows the effect of two different concentrations of azelastine HCl as well as oseltamivir on virus copy numbers recovered from apical washes of recovered nasal tissues 24 hours after infection with H1N1 influenza virus. Statistically significant differences are marked with asterisks. The graph shows the average results of three replicates of one typical experiment. *P<0.05; **P<0.01.

Figure 10: Effect of two different concentrations of azelastine as well as oseltamivir on the levels of IL-8 (panel A) and RANTES (panel B) secreted by reconstituted nasal tissues infected with H1N1 influenza virus. Statistically significant differences are marked with asterisks. Graph shows the mean values of three replicates of one typical experiment. **P<0.01; **P<0.001; ***P<0.0001.

DETAILED DESCRIPTION OF THE INVENTION

The terms "contain", "consist", "have" and "include" may be used as synonyms in the context of this document and are to be understood as an open definition allowing for additional members or parts or elements. "Consisting" is considered the closest definition without additional elements of the feature of a compound definition. Thus, the term "containing" is broader and includes the definition of "consisting".

The term “about” as used herein refers to the same value or a value that differs by +/- 10% or +/- 5% from a specified value.

The term "azelastine compound" as used herein refers to azelastine or a salt thereof.

The molecular formula of azelastine is C ₂₂ H ₂₄ ClN ₃ O with the following chemical structure (I):

IUPAC name: 4-[(4-chlorophenyl)methyl]-2-(1-methylazepan-4-yl)phthalazin-1-one.

CAS number: 58581-89-8.

The azelastine salt is preferably a pharmaceutically acceptable salt, such as, for example, the hydrochloride.

Azelastine hydrochloride is the hydrochloride salt form of azelastine and is used as an antihistamine medicinal compound formulated as a metered-dose spray solution for intranasal administration. Commercial products containing azelastine hydrochloride are presented as a nasal spray containing 0.1% or 0.15% azelastine hydrochloride USP in an aqueous solution at pH 6.8±0.3.

Azelastine hydrochloride is a white or almost white crystalline powder. It is sparingly soluble in water and soluble in ethanol and dichloromethane.

Molecular formula: C ₂₂ H ₂₅ Cl ₂ N ₃ O.

IUPAC name: 4-[(4-chlorophenyl)methyl]-2-(1-methylazepan-4-yl)phthalazin-1-one; hydrochloride.

CAS numbers: 58581-89-8; 37932-96-0; 79307-93-0.

The choice of azelastine salt is determined primarily by how acidic or basic the chemical is (pH), the safety of the ionized form, the intended use of the drug, the route of administration of the drug (such as by mouth, injection, or skin), and the type of dosage form (such as tablet, capsule, or liquid).

The term "pharmaceutically acceptable", also referred to as "pharmacologically acceptable", means compatible with the treatment of animals, in particular humans. The term "pharmacologically acceptable salt" includes both pharmacologically acceptable acid addition salts and pharmacologically acceptable base addition salts.

The term "pharmacologically acceptable acid addition salt" as used herein means any non-toxic organic or inorganic salt of any basic compound of the present invention or any of its intermediates. Basic compounds of the present invention that can form an acid addition salt include, for example, compounds that contain a basic nitrogen atom. Illustrative inorganic acids that form suitable salts include hydrochloric, hydrobromic, sulfuric and phosphoric acids, as well as metal salts such as sodium monohydrogen phosphate and potassium hydrogen sulfate. Illustrative organic acids that form suitable salts include mono-, di-, and tricarboxylic acids such as glycolic, lactic, pyruvic, malonic, succinic, glutaric, fumaric, malic, tartaric, citric, ascorbic, maleic, benzoic, phenylacetic, cinnamic, and salicylic acids, as well as sulfonic acids such as p-toluenesulfonic and methanesulfonic acids. Mono-, di-, or tri-acid salts may be formed, and such salts may exist in hydrated, solvated, or substantially anhydrous form. In general, the acid addition salts of the compounds of the present invention are more soluble in water and various hydrophilic organic solvents and generally exhibit higher melting points than their free base forms. One skilled in the art will recognize the selection of a suitable salt. Other pharmacologically unacceptable acid addition salts, such as oxalates, can be used, for example, for the isolation of the compounds of the present invention, for laboratory use, or for subsequent conversion into a pharmacologically acceptable acid addition salt.

The term "pharmacologically acceptable base salt" as used herein means any non-toxic organic or inorganic base addition salt of any acidic compound of the present invention or any of its intermediates that is suitable or compatible with the treatment of animals, in particular humans. The acidic compounds of the present invention that can form a base addition salt include, for example, compounds that contain a carboxylic acid, a sulfonic acid, a sulfinic acid, a sulfonamide, an N-unsubstituted tetrazole, an ester of phosphoric acid or an ester of sulfuric acid. Illustrative inorganic bases that form suitable salts include lithium, sodium, potassium, calcium, magnesium or barium hydroxide. Illustrative organic bases that form suitable salts include aliphatic, alicyclic or aromatic organic amines such as methylamine, trimethylamine and picoline or ammonia. The person skilled in the art will know how to select a suitable salt. Other pharmacologically unacceptable base addition salts can be used, for example, for the isolation of the compounds of the present invention, for laboratory use or for subsequent conversion into a pharmacologically acceptable base addition salt. The formation of the desired salt of the compound is achieved using standard techniques. For example, the neutral compound is treated with a base in a suitable solvent, and the resulting salt is isolated by filtration, extraction or any other suitable method.

The term "antiviral" as used herein refers to any substance, drug or preparation that affects the biology of a virus and reduces or inhibits viral attachment, entry, replication, detachment, latency or a combination thereof, resulting in a reduction in viral load or infectivity. The terms "reduction", "inhibition", "reduction" or "prevention" or any variation of these terms when used in the claims and/or description include any measurable reduction or complete inhibition to achieve a desired result, such as a reduction in the risk of viral infection (prior to exposure) or a reduction in viral survival, load or growth following exposure.

Exemplary antiviral drugs described herein are medical products, pharmaceuticals, or disinfectants for in vivo, ex vivo, or in vitro use.

The term "biological surface" as used herein shall refer to a surface containing viable cells, such as mammalian (human or non-human) cells, including, for example, a biological tissue surface such as an epithelial or dermal tissue surface (e.g. skin), mucosal tissue, or membrane tissue.

The term "effective amount" in relation to an antiviral effect in the context of this document shall refer to an amount (in particular a predetermined amount) that has a proven antiviral effect. An amount is generally an amount or potency that, when applied to a surface or administered to a subject, results in the achievement of beneficial or desired results, including antiviral or clinical results, and as such, an effective amount or its synonym depends on the context in which it is used.

An effective amount of a pharmaceutical preparation or drug is intended to mean that amount of the compound that is sufficient to treat, prevent or inhibit a disease, pathological condition or disorder. Such an effective dose specifically refers to that amount of the compound that is sufficient to heal, prevent or alleviate the conditions associated with the diseases or disorders described herein.

In the context of a disease, effective amounts (particularly prophylactically or therapeutically effective amounts) of an azelastine compound described herein are specifically used to treat, modulate, alleviate, reverse or affect a disease or condition that will benefit from an antiviral effect. The amount of the compound that will correspond to such an effective amount will vary depending on various factors such as the specific drug or compound, formulation, route of administration, type of disease or disorder, characteristics of the subject or host being treated, assessment of medical situations and other relevant factors, but can nevertheless be routinely determined by a person skilled in the art.

A regimen for the treatment or prophylaxis of a subject using an effective amount of an azelastine compound described herein may consist of a single application or administration, or, alternatively, may include a number of applications and administrations, respectively. For example, the azelastine compound may be administered at least once a month, or at least once a week, or at least once a day. However, in some acute phase situations, such as suspected or confirmed exposure to a virus, or after a viral infection has been determined, the azelastine compound may be administered more frequently, such as 1-10 times a day.

In particular, a combination therapy is proposed that includes treatment with the drug described herein and standard therapy for a disease caused by the Coronaviridae virus and/or standard therapy for a disease caused by any of the other target viruses.

Doses may be used in combination with other active agents such as antiviral agents, anti-inflammatory drugs or antibiotics, for example, when there is a risk of viral spread in the subject, to prevent a pathogen-related reaction.

The treatment may be combined with antiviral, anti-inflammatory or antibiotic treatment, whereby the pharmaceutical preparation is preferably administered before, during (e.g. by co-administration or in parallel) or after said antiviral, anti-inflammatory or antibiotic treatment.

In particular, the azelastine compound described herein can be combined with an additional antiviral agent, which can be an azelastine compound, such as the same or a different compound. Particular embodiments relate to additional antiviral agents selected from an ACE-2 inhibitor, an inhibitor of the ion channel formed by the viral protein M2, a neuraminidase inhibitor, an inhibitor of RNA replication and translation, and a polymerase inhibitor. The antiviral agent can be amantadine or rimantadine. In particular, the antiviral agent can be oseltamivir, zanamivir, peramivir, ribavirin, lopinavir or ritonavir. Specific additional antiviral examples are those that are suitably used to treat a biological surface, such as carrageenan, or those that are currently being studied for the treatment of SARS-Cov2 infections, such as hydroxychloroquine or remdesivir.

In particular, the azelastine compound is combined with an anti-inflammatory agent such as standard steroidal anti-inflammatory drugs, glucocorticoids, and non-steroidal anti-inflammatory drugs (NSAIDs). Suitable NSAIDs include, but are not limited to, ibuprofen, naproxen, fenoprofen, ketoprofen, flurbiprofen, oxaprozin, indomethacin, sulindac, etodolac, ketorolac, diclofenac, nabumetone, piroxicam, meloxicam, tenoxicam, droxicam, lornoxicam, isoxicam, mefenamic acid, meclofenamic acid, flufenamic acid, tolfenamic acid, and celecoxib. Suitable steroidal anti-inflammatory agents include, but are not limited to, corticosteroids such as synthetic glucocorticoids. Specific examples are fluticasone, COX-2 inhibitors, ibuprofen, hydroxychloroquine, heparin, low molecular weight heparin, hirudin, or immunosuppressants such as azathioprine, cyclosporine A, or cyclophosphamide.

In particular, the azelastine compound is combined with an antibiotic such as a beta-lactam antibiotic, an aminoglycoside antibiotic, ansamycin, carbacephem, carbapenem, cephalosporin, glycopeptide, lincosamide, lipopeptide, macrolide, monobactam, nitrofuran, oxazolidinone, polypeptide, sulfonamide, clofazimine, dapsone, capreomycin, cycloserine, ethambutol, ethionamide, isoniazid, pyrazinamide, rifampicin, rifabutin, rifapentine, streptomycin, arsphenamine, chloramphenicol, fosfomycin, mupirocin, platensimycin, quinupristin/dalfopristin, thiamphenicol, tigecycline, tinidazole, trimethoprim, teixobactin, malacidins, halicin, clindamycin, vancomycin, metronidazole, fusidic acid, thiopeptides, fidaxomicin, quinolones, tetracyclines, omadacycline, rifamycin, kibdelomycin, oxazolidinone, ketolides, thiazolides, amixicil, teicoplanin, ramoplanin, oritavancin, lantibiotics, capuramycin, surotomycin, thuricin, endolysin, avidocin CD, kadazolide, ramizole, defensins, ridinilazole, medium-chain fatty acids, phages, berberine, lactoferrin.

In particular, treatment with the azelastine compound described herein may be combined with treatment comprising the administration of at least one other therapeutic agent selected from the group consisting of a corticosteroid, an anti-inflammatory signal transduction modulator, a 2-adrenergic receptor agonist bronchodilator, an anticholinergic, a mucolytic, a hypertonic saline solution, and other drugs for the treatment of Coronaviridae viral infections and/or infections caused by any of the other target viruses described herein; or mixtures thereof. Specific pharmaceutical compositions may in particular include one or more anti-inflammatory agents and/or analgesics, PPAR-γ agonists, and immune response modulators.

The duration of the treatment period depends on many factors, such as the severity of the disease, whether the disease is acute or chronic, the age of the patient and the concentration of the azelastine compound. It should also be understood that the effective dosage used for treatment or prophylaxis may be increased or decreased during a particular treatment or prophylaxis regimen. Dosage changes may result and may become apparent using standard diagnostic tests known in the art.

According to a particular aspect, the medical product or pharmaceutical composition described herein comprises an effective amount of an azelastine compound as defined herein. The preparation described herein may be provided for use in single or multiple doses.

Single-dose or multi-dose containers, such as sealed ampoules and vials or multi-use sprays, may be used and may be stored, including the liquid or dry phase, such as in a sublimated (lyophilized) state, requiring only the addition of a sterile liquid carrier, such as water for injection, immediately before use. Preferred unit dose formulations are those containing a daily dose or a fraction of a single daily dose or multiple doses of the azelastine compound.

The term "single dose" as used herein is understood as follows. A single dose or single use quantity is a quantity intended for administration that is intended for use in a single subject, such as a patient, whether human or animal, for a single occasion/procedure/administration. Packages containing a single dose are generally labeled as such by the manufacturer. A single dose quantity is specifically understood to be a daily dose for an individual, such as a child or an adult, that provides an effective amount.

The medical product or pharmaceutical composition described herein is specifically proposed as a medical product or pharmaceutical composition for use in humans or animals. Medical products are understood to mean substances that are used to treat diseases, to alleviate complaints, or to prevent such diseases or complaints in the first place. This definition applies regardless of whether the medical product is administered to humans or animals. Substances can act both in the body and on the surface of the body.

The medical product or pharmaceutical composition described herein preferably comprises one or more pharmaceutically acceptable excipients and is in a pharmaceutical form that allows the active pharmaceutical compound to be administered with high bioavailability. Suitable excipients can be, for example, based on cyclodextrins. Suitable formulations can include, for example, synthetic polymeric nanoparticles formed from a polymer selected from the group consisting of acrylates, methacrylates, cyanoacrylates, acrylamides, polylactates, polyglycolates, polyanhydrates, polyorthoesters, gelatin, albumin, polystyrenes, polyvinyls, polyacrolein, polyglutaraldehyde and their derivatives, copolymers and mixtures.

Specific medicinal products or pharmaceutical compositions described herein comprise an azelastine compound and a pharmaceutically acceptable carrier or excipient.

"Pharmaceutically acceptable carrier" refers to an ingredient in a composition for medical or therapeutic use, other than the active ingredient, that is non-toxic to the subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative, and in particular, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like.

The azelastine compound in the context of this document may be formulated with conventional carriers and excipients, which will be selected in accordance with normal practice.

Pharmaceutically acceptable carriers generally include any and all suitable solvents, dispersion media, coatings, antiviral, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like that are physiologically compatible with the antiviral small molecule compound or corresponding composition or combination preparation described herein.

According to a particular aspect, the azelastine compound can be combined with one or more carriers suitable for the desired route of administration. The azelastine compound can be mixed with, for example, any of lactose, sucrose, starch, cellulose esters of alkanoic acids, stearic acid, talc, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric and sulfuric acids, gum arabic, gelatin, sodium alginate, polyvinylpyrrolidine, polyvinyl alcohol, and optionally further tableted or encapsulated for conventional administration. Alternatively, the azelastine compound may be dispersed or dissolved in saline, water, polyethylene glycol, propylene glycol, colloidal solutions of carboxymethylcellulose, ethanol, corn oil, peanut oil, cottonseed oil, sesame oil, gum tragacanth and/or various buffers. Other carriers, adjuvants and routes of administration are well known in the pharmaceutical art. The carrier may include a controlled release material or a time delay material such as glyceryl monostearate or glyceryl distearate alone or with a wax, or other materials well known in the art.

The compounds described herein may be provided in controlled release pharmaceutical formulations (“controlled release formulations”) in which the release of the azelastine compound is controlled and adjusted to provide less frequent dosing or to improve the pharmacokinetic or toxicity profile of the active ingredient.

The pharmaceutical compositions may also be coated by conventional means, typically pH-dependent or time-dependent coatings, so that the agent in question is released in the gastrointestinal tract in close proximity to the desired topical application or at different times to prolong the desired action. Such dosage forms typically include, but are not limited to, one or more of cellulose acetate phthalate, polyvinyl acetate phthalate, hydroxypropyl methylcellulose phthalate, ethyl cellulose, waxes, and shellac.

Additional pharmaceutically acceptable carriers are known in the art and are described, for example, in Remington: The Science and Practice of Pharmacy, 22nd revised edition (Allen Jr, LV, ed., Pharmaceutical Press, 2012). Liquid formulations may be solutions, emulsions, or suspensions and may include excipients such as suspending agents, solubilizers, surfactants, preservatives, and chelating agents.

The preferred drug is in a ready-to-use, shelf-stable form with a shelf life of at least one to two years.

The term "formulation" as used herein refers to a preparation ready for use in a specific manner. In particular, the compositions described herein comprise an azelastine compound and a pharmaceutically acceptable diluent, carrier, or excipient.

According to a particular aspect, compositions are provided that contain pharmaceutically acceptable carriers for nasal, pulmonary, oral, topical, mucosal or parenteral administration. Administration may also be intradermal or transdermal. In addition, the present invention includes such compounds that have been lyophilized and which can be reconstituted to form pharmaceutically acceptable formulations for administration.

Specific medical products or pharmaceutical compositions described herein are formulated for intranasal administration or other topical routes, such as to biological surfaces, including, for example, mucous membranes or skin. Pharmaceutical carriers suitable for facilitating such routes of administration are well known in the art.

In particular, a nasal spray can be used containing 0.001% or 0.15% (w/w) of an azelastine compound in an aqueous solution at a pH of 6.8±0.3, optionally further containing any one or more of citric acid monohydrate, sodium hydrogen phosphate dodecahydrate, disodium edetate, hypromellose, purified water, sodium chloride and a preservative such as benzalkonium chloride.

Administration of the azelastine compound by any route other than parenteral administration may require coating the active agent with a material to prevent its inactivation or co-administering the active agent with it. For example, a suitable carrier such as liposomes or a diluent may be used. Pharmaceutically acceptable diluents include saline and aqueous buffer solutions.

The azelastine compound can be administered orally, for example, by means of an inert diluent or an assimilable or edible carrier. For example, the preparation can be enclosed in a hard or soft gelatin capsule or compressed into tablets. For oral therapeutic administration, the azelastine compound can be included with excipients and can be used in the form of swallowable tablets, buccal tablets, lozenges, capsules, elixirs, suspensions, syrups, strips and the like. The percentage of the compound in the compositions and preparations can, of course, vary. The amount of the azelastine compound in such therapeutically useful compositions is such that a suitable dosage will be obtained.

The tablets will contain excipients, glidants, fillers, binders, disintegrants, lubricants, flavors, and the like. Granules may be prepared using isomaltose. In addition, preferably provided is a preparation made to act in a mucosal area, such as in mucosal areas (such as the nose, mouth, eyes, oesophagus, throat, lungs), for example locally without systemic action. Aqueous formulations are prepared in sterile form, and if intended for delivery by a route other than oral, they will typically be isotonic.

The term "mucosal" in relation to the administration or use or other mucosal application of a preparation for the treatment of a subject or a formulation thereof refers to administration via the mucosal route, including systemic or topical administration, wherein the active ingredient is absorbed by contact with mucosal surfaces. This includes nasal, pulmonary, oral or peroral administration and formulations such as a liquid, syrup, troche, eye drop, tablet, spray, powder, soluble powder, granules, capsules, cream, gel, drops, suspension or emulsion.

Oral formulations may include liquid solutions, emulsions, suspensions and the like. Pharmaceutically acceptable carriers suitable for preparing such compositions are well known in the art. Typical components of carriers for syrups, elixirs, emulsions and suspensions include ethanol, glycerol, propylene glycol, polyethylene glycol, liquid sucrose, sorbitol and water. For suspension, typical suspending agents include methyl cellulose, sodium carboxymethyl cellulose, tragacanth and sodium alginate; typical wetting agents include lecithin and polysorbate 80; and typical preservatives include methyl paraben and sodium benzoate. Oral liquid compositions may also contain one or more components such as the sweetening, flavoring and coloring agents described above.

Other compositions that can be used to provide systemic delivery of the azelastine compound or related drugs include sublingual, buccal and nasal dosage forms. Such compositions typically contain one or more soluble fillers such as sucrose, sorbitol and mannitol; and binding agents such as acacia, microcrystalline cellulose, carboxymethylcellulose and hydroxypropylmethylcellulose, or glidants, lubricants, sweeteners, colorants, antioxidants and flavoring agents.

The azelastine compound or formulations thereof may also be administered to a subject topically, such as by directly applying to or distributing a composition containing the compound onto epidermal or epithelial tissue of the subject, or transdermally via a "patch." Such compositions include, for example, lotions, creams, solutions, gels, and solids. Said topical compositions may contain an effective amount, typically at least about 0.001% by weight, or even from about 0.1% by weight to 5% by weight, or from 1% by weight to about 5% by weight, of the azelastine compound. Suitable carriers for topical administration typically remain in place on the skin as a continuous film and are resistant to removal by perspiration or immersion in water. Typically, the carrier is organic in nature and is capable of dispersing or dissolving the therapeutic agent therein. The carrier may include pharmaceutically acceptable softening agents, emulsifiers, thickening agents, solvents and the like.

Pharmaceutical compositions suitable for injection use include sterile aqueous solutions (particularly if the compounds or pharmaceutically acceptable salts are water-soluble) or dispersions and sterile powders for the preparation of sterile injection solutions or dispersions for immediate use. In particular, the composition is, in particular, sterile and fluid so that it can be easily administered via a syringe; it is stable under the conditions of manufacture and storage and is preserved against the contaminating action of microorganisms such as bacteria and fungi.

Suitable pharmaceutically acceptable carriers include, but are not limited to, any non-immunogenic pharmaceutical adjuvants suitable for oral, parenteral, nasal, mucosal, transdermal, intravascular (IV), intraarterial (IA), intramuscular (IM), and subcutaneous (SC) routes of administration, such as phosphate buffered saline (PBS).

The term "subject" as used herein refers to a warm-blooded mammal, in particular a human or a non-human animal, including, for example, dogs, cats, rabbits, horses, cattle and pigs. In particular, the treatment and medical use described herein relates to a subject in need of prevention or therapy of a pathological condition associated with a Coronaviridae virus infection and/or infection with any of the other target viruses described herein. In particular, the treatment may be accomplished by interfering with the pathogenesis of a pathological condition in which a Coronaviridae virus and/or an Adenoviridae virus and/or a Paramyxoviridae virus and/or an Orthomyxoviridae virus is the causative agent of the condition. A subject may be a patient at risk of such a pathological condition or suffering from a disease. In particular, the subject may have a weakened immune system or an existing respiratory or cardiac disease that is more likely than others to increase the risk of disease or the severity of disease.

The term "at risk" for certain pathological conditions refers to a subject who is likely to develop such a pathological condition, for example, due to a certain predisposition, exposure to a virus or virus-infected subjects, or who already suffers from such a pathological condition at various stages, in particular associated with other causative pathological conditions or other conditions or complications arising as a consequence of a viral infection. The determination of risk is especially important in a subject who has not yet been diagnosed with a disease. Thus, this determination of risk includes early diagnosis to ensure preventive therapy. In particular, the azelastine compound is used in subjects with a high risk, for example, a high probability of developing a disease.

The term "patient" includes human and other mammalian subjects who receive either prophylactic or therapeutic treatment. The term "patient" in the context of this document always includes healthy subjects. The term "treatment" thus includes both prophylactic and therapeutic treatment.

In particular, the term "prophylaxis" refers to preventive measures and is intended to include prevention of the onset of pathogenesis or preventive measures to reduce the risk of pathogenesis.

The term "therapy" as used herein with respect to the treatment of subjects refers to medical treatment of a subject with the purpose of curing, reducing, stabilizing, decreasing the incidence of, or preventing a disease, condition, or disorder, individually or collectively understood as a "condition". The term includes active treatment aimed specifically at ameliorating a condition, prophylaxis aimed specifically at preventing a condition, and also includes causal treatment aimed at eliminating the cause of a related condition. In addition, the term includes palliative treatment aimed at alleviating symptoms rather than curing a condition, and adjunctive treatment of a condition aimed at minimizing or partially or completely inhibiting the progression of a related condition, and supportive treatment used to complement other specific therapy aimed at ameliorating a related condition.

The above description will be more clearly understood with reference to the following examples. However, such examples are merely typical of the ways of implementing one or more embodiments of the present invention and should not be considered as limiting the scope of the present invention.

EXAMPLES

Example 1: Identification of an antiviral drug

The development of mathematical representations of small molecules (drugs) is an area of research of great value for modern pharmaceutical research and therefore numerous molecular descriptors have been developed that exploit 2-dimensional and/or 3-dimensional features of chemical structures. These descriptors have been very valuable in assessing quantitative structure-activity relationships. In particular, atom-centered feature pairs have proven to be of great value for drug discovery programs as they provide cost-effective approaches for high-throughput structure-activity relationship analysis, compound selection, virtual chemical screening and pharmacological profiling. One of the widely used chemical descriptors is called SHED - Shannon entropy descriptor (1). In this approach, topological distributions of atom-centered feature pairs are quantified based on information theory (Shannon entropy). A particular advantage is that chemically distinct but topologically related chemical scaffolds can be identified using the SHED approach.

A pathway-based intervention strategy was used to identify novel antiviral drugs. Clinically approved drugs that match a predefined mechanistic profile (mode of action) were identified as candidate antiviral drugs using a Shannon entropy-based description of small molecule (drug) chemical signatures. The rationale for this approach is that ligands with similar Shannon entropy vectors will bind to similar protein targets.

In this case, we used the DRUGBANK database (2), a repository of approved drugs and their experimentally validated protein targets. Drug similarity was assessed by calculating Euclidean distances (0.25 was used as a cutoff value).

As a starting point for the analysis, available mechanistic information on SARS-CoV-2 infection was extracted from a recent bioinformatics analysis (3) together with the analysis of some query antiviral compounds. Pathway profiles of query antiviral compounds with known activity against SARS-CoV2 (hydroxychloroquine (RS)-2-[{4-[(7-chloro-4-quinolinyl)amino]pentyl}(ethyl)amino]ethanol; SSAA09E2 {N-[[4-(4-methylpiperazin-1-yl)phenyl]methyl]-1,2-oxazole-5-carboxamide}; and SSAA09E3 [N-(9,10-dioxo-9,10-dihydroanthracen-2-yl)benzamide]) were predicted using the Shannon entropy-based approach and interestingly showed significant mutual overlap.

Second, pathway profiles were predicted for SARS inhibitors with well-defined mechanisms and mode of action: SSAA09E2, a small molecule ACE-2 inhibitor, and SSAA09E3, a general host-virus membrane fusion inhibitor (4). Both inhibitors were found to share a significant number of pathways with hydroxychloroquine. In summary, the analysis revealed that similar pathways are involved in SARS-CoV-2 infection, which are targeted by hydroxychloroquine and affected by inhibitors with well-defined mechanisms. Thus, it was concluded that these pathways are of high importance for antiviral activity and may serve as a basis for repurposing a new drug for antiviral activity by searching for clinically approved drugs that were not previously known to have antiviral activity with corresponding pathway profiles.

Clinically approved drugs were screened for overlap in their individual pathway profiles. The predicted pathway profiles obtained for different query compounds (hydroxychloroquine, SSAA09E2, and SSAA09E3) were used to screen the SELLECKCHEM database of approved (and commercially available) drugs. The rationale for candidate selection was based on the co-occurrence of approved drugs in different (predicted) datasets. Hydroxychloroquine and SSAA09E2 (an ACE-2 inhibitor) showed significant overlap with each other and with drugs derived from the SARS-CoV-2 pathway profile. After exclusion of drugs based on chemical composition, two approved drugs were selected as antiviral candidates for further testing in an in vitro SARS-CoV-2 infection model: azelastine and maraviroc.

References

(1) Gregori-Puigjane, E. and Mestres, J. SHED: Shannon Entropy Descriptors from Topological Feature Distributions. J. Chem. Inf. Model. 46, 1615-1622 (2006)

(2) Wishart, D.S., Knox, C., Guo, A.C.:, Cheng, D., Shrivastava, S., Tzur, D., Gautam, B. and Hassanali, M. DrugBank: a knowledgebase for drugs, drug actions and drug targets. Nucleic Acids Res. D901-D906, (2008)

(3) Zhou, Y., Hou, Y., Shen, J., Huang, Y., Martin, W. and Cheng, F. Network-based drug repurposing for novel coronavirus 2019-nCoV/SARS-CoV-2. Cell Discovery 6,14-32, (2020)

(4) Adedeji, AO., Severson, W., Jonsson, C., Singh, K., Weiss SR. and Sarafanos, S.G. Novel Inhibitors of Severe Acute Respiratory Syndrome Coronavirus Entry That Act by Three Distinct Mechanisms. J Virol. 87, 8017-8028, (2013).

Example 2: Prevention of SARS-CoV-2 infection of Vero E6 cells

To detect the effect of azelastine HCl on SARS-CoV-2 infection, Vero E6 cells (monkey kidney) expressing ACE2 were infected with SARS-CoV-2 in the absence or presence of azelastine HCl, and the cytopathogenic effect was assessed by microscopic examination of the cells.

Experimental methodology:

Vero E6 cells (ATCC CRL-1586) were seeded in 96-well plates. After 2 days, the cell cultures reached confluency and formed a homogeneous monolayer. The cells were fed with fresh culture medium (DMEM + FBS?). Azelastine HCl (Seleckchem, Cat. No. S2552, 10 mM stock solution dissolved in DMSO) and maraviroc (Seleckchem, Cat. No. S2003, 10 mM stock solution dissolved in DMSO), an antiviral agent against HIV, were added to the cell culture medium at final concentrations of 50, 25, 12.5, 6.25, and 3.125 μM (dilutions were prepared in the culture medium). For viral infection, SARS-CoV-2 virus (hCoV-19/Hungary/SRC_isolate_2/2020, accession number: EPI_ISL_483637) was added to the supernatant at an MOI of 0.1 (multiplicity of infection: 1 virus particle per 10 cells) immediately after replacing the culture medium (essentially simultaneously). The stock virus suspension was obtained by propagation in Vero E6 cells and the infectious titer was determined. After 30 min of incubation with the virus, the culture medium was removed and replaced with fresh culture medium containing azelastine HCl or maraviroc at the concentrations indicated above (co-administration: prevention model). Experiments were also performed with azelastine under conditions where the drug was administered only after 30 min of incubation with the virus but not during this period (post-infection: post-exposure/therapeutic condition). At 48 h post infection, cells were assessed by microscopic observation, and then supernatants were collected and stored at -80°C for quantitative PCR analysis. Viral RNA was extracted from culture supernatant samples using the Monarch Total RNA Isolation Mini Preparative Kit (New England BioLabs, Cat.# T2010S) according to the manufacturer's instructions. Briefly, 300 μl of lysis buffer was mixed with 100 μl of culture supernatant, gDNA contamination was removed using a DNA retention column, and the flow-through fraction containing RNA was applied to an RNA binding column. After washing the column, RNA was eluted with _H2O and samples were stored at -80°C until analysis. Following the reverse transcription reaction, DNA was amplified with primers F, R, and P2 from the RdRp gene

The primers and probes used were specific for the SARS-CoV-2 RdRp gene:

Reverse primer: CARATGTTAAASACACTATTAGCATA (SEQ ID NO: 1),

Forward primer: GTGARATGGTCATGTGTGGCGG (SEQ ID NO: 2),

Probe: FAM-CAGGTGGAACCTCATCAGGAGATGC-BBQ (SEQ ID NO: 3).

A droplet PCR kit (BioRad ddPCR™, Bio-Rad Laboratories GmbH, Germany) was used. The RT-PCR reaction results were quantified and calculated as viral particles/µl.

Results:

The confluent homogeneous layer of cells (uninfected, Fig. 2A) is disrupted and “holes” appear, indicating cell death due to the virus (Fig. 2B). In the presence of azelastine-HCl at all concentrations tested, SARS-CoV-2-infected cells were significantly protected from cell death, indicating a direct antiviral effect (Figs. 2C-F). Maraviroc was not effective at all at lower concentrations, and even higher concentrations (12.5 to 50 μM) had only a slight protective effect with high cytopathogenicity scores (Table 1). Azelastine was unexpectedly an effective antiviral agent that was at least as effective as hydroxychloroquine.

These data suggest that the compound azelastine was able to stop SARS-CoV-2 infection immediately after application to cells. Since the virus must enter cells to replicate and spread throughout the body, azelastine is expected to prevent COVID-19 right where the virus infects the human body - the surface of the respiratory tract mucosa.

Ratings:

0: no cytopathic effect (CPE), cells were the same as in the uninfected control

1: very small areas showed low CPE

2: CPE was observed in small areas of cell culture

3: stronger CPE, but not as strong as in infected control

4: CPE is as strong as in infected controls.

Quantitative PCR analysis showed that azelastine was highly effective in reducing viral loads in both co-administration (modeling prevention) by >99% and in the post-infection setting (modeling post-exposure or treatment) by >97% (Table 2), suggesting that azelastine may be used in both prophylactic and therapeutic settings. As expected, co-administration is more effective, but the low viral load at 25 μM azelastine demonstrates that ongoing infection may be stopped rather than just prevented.

Numbers represent median values of up to 5 replicate samples/concentration with 2-3 technical replicates (qPCR repeated from the same biological sample).

Example 3: Efficacy against SARS-CoV-2 viral infection of reconstituted human nasal tissue

To confirm the efficacy of azelastine HCl against SARS-CoV2 in human cells, reconstituted human 3D nasal tissue (MucilAir, in vitro reconstituted 3D human airway epithelium) was infected with SARS-CoV-2 and treated with a commercial nasal spray containing azelastine. The cytopathogenic effect was assessed by microscopic examination of the tissue, and the number of viral particles was determined by droplet PCR.

Experimental methodology:

Human MucilAir nasal tissue from healthy donors (Epithelix Sàri (Geneva, Switzerland), catalog no. EP02MP) was infected with SARS-CoV-2 (SARS-CoV-2 virus, hCoV-19/Hungary/SRC_isolate_2/2020, accession number: EPI_ISL_483637) at a multiplicity of infection (MOI) of 0.01 on the apical side. After 20 min of incubation at 37°C in 5% _CO2 , the virus-containing medium was completely removed. Then, a 5-fold diluted (in MucilAir culture medium) solution of Allergodil nasal spray (0.1% azelastine-HCl, Mylan) was added to the apical side (in 200 μl volume) for 20 min. After treatment, the diluted nasal spray was completely removed from the cell surface to ensure an air-liquid interface and incubated for 24 h. The 20-min treatment with diluted Allergodil was repeated at 24 and 48 h post infection (hpi). At 24, 48, and 72 hpi, the apical sides of the cells were washed for 15 min with MucilAir culture medium and the solution was collected for quantification of infectious virus particles. The cells were also observed under an inverted microscope at 48 and 72 hpi.

Total RNA was extracted from apical washes (100 μl) using the Monarch Total RNA Isolation Mini Preparative Kit (Promega, Cat# T2010S) according to the manufacturer's instructions. As described in Example 2, droplet digital PCR technology (Bio-Rad Laboratories Inc. QX200 Droplet Digital PCR System) was used to quantify virus copy numbers.

Results:

Microscopic analysis of tissues at 48 and 72 hours revealed a decrease in mucin production in infected cells, observed as a complete absence of black spots in the microscopic image, compared to control cells (no virus or drug treatment, large amounts of black material observed above the cells) (Figure 3). Importantly, mucin production, demonstrated by the presence of black material in the microscopic image, was clearly demonstrated in the presence of 5-fold diluted allergodil and was comparable to that observed in the negative control (no virus or drug treatment). No differences in tissue morphology were detected in either control or azelastine-treated cells (no virus infection), and ciliary movement was detected in all tissues.

Digital droplet PCR analysis confirmed efficient SARS-CoV-2 infection and rapid viral replication, reaching several thousand copies per microliter by 72 hours post-infection in the apical compartment of tissue inserts. A 5-fold diluted nasal spray (0.02% azelastine HCl) administered daily for 20 minutes significantly reduced viral particles at 48 and 72 hours post-infection (>99.9% inhibition) (Table 3).

Example 4: Demonstration of in vitro efficacy of azelastine against SARS-CoV-2 variants of concern B.1.1.7 and B.1.351

To detect the antiviral effect of azelastine HCl on SARS-CoV-2 virus variants, Vero (monkey kidney) cell line expressing ACE2 and TMPRSS2 were infected with B.1.1.7 and B.1.351 variants of concern in the absence or presence of azelastine HCl, and the effect on virus spread was assessed by qPCR.

Experimental methodology:

Vero-TMPRSS2/ACE-2 SARS-CoV-2 infection assay Vero cells stably overexpressing the human serine protease TMPRSS2 and the ACE-2 receptor (Riepler et al., Comparison of Four SARS-CoV-2 Neutralization Assays. Vaccines (Basel), 2020;9(1):13) were seeded in 96-well plates at 10 ⁴ /well the day before infection. Azelastine hydrochloride (Sigma-Aldrich, PHR1636-1G, lot no. LRAC4832) dissolved in DMSO to a concentration of 10 mM was diluted in Dulbecco's modified Eagle's medium (Merck, Darmstadt, Germany) containing 2% FBS to final concentrations ranging from 50 μM to 0.4 μM (2-fold serial dilutions). Before cell infection, cell culture supernatant was aspirated and replaced with 50 μl of azelastine-HCl dilutions in the prophylactic condition (co-administration) and 50 μl of medium in the post-infection condition. Triplicate measurements were performed for each azelastine concentration. Cells were then infected with either type B.1.1.7 or type B.1.351 SARS-CoV-2 isolates at an MOI of 0.01 for 30 min at 37°C. For both experimental conditions, the supernatant was then aspirated and replaced with 50 μl fresh medium and 50 μl of the same concentrations of azelastine used previously, resulting in azelastine concentrations ranging from 25 μM to 0.2 μM. At 48 h post infection, the cytopathic effect was assessed and the supernatant was mixed at a 1:1 ratio with DLR buffer (0.5% IGEPAL, 25 mM NaCl in 10 mM Tris-HCl buffer, 15 μl RiboLock RNase inhibitor (ThermoScientific, 40 U/μl, EO0381 per ml DLR buffer) to isolate viral RNA. SARS-CoV-2 genome copies were quantified by qPCR using E gene-specific primers (5' ACA GGT ACG TTA ATA GTT AAT AGC GT 3' (SEQ ID NO: 5) and 5' ATA TTG CAG CAG TAC GCA CAC A3' (SEQ ID NO: 6)), FAM-labeled probe (FAM-ACA CTA GCC ATC CTT ACT GCG CTT CG-BHQ1 (SEQ ID NO: 7)) and the iTaq One-Step Universal Probe Kit (BioRad, Cat. #1725141). A home-made in vitro transcribed RNA (SARS-CoV-2 E gene) standard was used to quantify qPCR results. Wells containing virus only without azelastine treatment were set to 100%, and the percent inhibition relative to wells containing virus only was calculated for each sample.

Results

Quantitative PCR analysis showed that azelastine was highly effective in reducing viral particles under both prophylactic (co-administration) and post-infection (simulated post-exposure or treatment conditions) administration conditions (Tables 4 and 5 and Figure 4). Similar to the results for the wild-type virus, co-administration is slightly more effective, but the low viral particle numbers (>90% inhibition) at 12.5 μM azelastine demonstrates that ongoing infection can be not only stopped but also prevented. The effective concentration of azelastine required to inhibit 50% of infection (EC50) ranged from 4 to 6.5 μM depending on the conditions and virus mutation, which is in the range of the EC50 observed for the wild-type virus (~6 μM). This demonstrates that the efficacy of azelastine against SARS-CoV-2 is not affected by emerging high-risk mutations.

Conclusion:

These data indicate that the compound azelastine was able to stop infection with SARS-CoV-2 mutants B.1.351 and B.1.1.7 immediately after application to cells. Since the virus must enter cells to replicate and spread throughout the body, azelastine is expected to prevent COVID-19 right at the site where the virus infects the human body - the surface of the respiratory tract mucosa.

Example 5: Prevention of SARS-CoV-2 infection of Calu-3 cells

To confirm the antiviral effect of azelastine-HCl on cells expressing the surface protease TMPRSS2, the human lung adenocarcinoma cell line Calu-3 was infected with SARS-CoV-2 in the absence or presence of azelastine-HCl and the number of viral particles was measured after 48 hours.

Experimental methodology:

Calu-3 (ATCC® HTB-55™) were seeded in 96-well plates. After 2 days, when the cells reached confluency, the cultures were fed with fresh cell culture medium (EMEM+10% FBS+1% P/S, 1% L-glutamine, 1% nonessential amino acids). Azelastine-HCl (Seleckchem, Cat.# S2552, 10 mM stock solution dissolved in DMSO) and hydroxychloroquine sulfate (TCl, Cat.# H1306, 10 mM stock solution dissolved in _H2O and sterile filtered) were added to the cell culture medium at final concentrations of 50 and 25 μM (dilutions were prepared in the culture medium). Hydroxychloroquine sulfate induces a strong anti-SARS-CoV-2 effect in Vero E6 cells (which do not express TMPRSS2), but loses significant potency when tested in cells expressing the TMPRSS2 surface protease, including Calu-3 cells (see Hoffmann et al. Nature 2020, 585:588–590).

The virus stock suspension was generated by propagation in Vero E6 cells and infectious titer was determined. For viral infection, SARS-CoV-2 virus was added to the supernatant at an MOI of 0.01 (multiplicity of infection: 1 virus particle per 100 cells) immediately after replacing the culture medium (essentially simultaneously). After 30 min of incubation with the virus, the culture medium was removed and replaced with fresh culture medium containing azelastine HCl or hydroxychloroquine sulfate at the concentrations indicated above. At 48 h post infection, the supernatants were collected and stored at -80°C for quantitative PCR analysis. Viral RNA was extracted from culture supernatant samples using the Monarch Total RNA Isolation Mini Kit (New England BioLabs, Cat. #T2010S) according to the manufacturer's instructions. Briefly, 300 µl of lysis buffer was mixed with 100 µl of culture supernatant, gDNA contamination was removed using a dedicated (DNA retention) column, and the flow-through fraction containing RNA was applied to the RNA binding column. After washing the column, RNA was eluted with _H2O and samples were stored at -80°C until analysis. Following the reverse transcription reaction, DNA was amplified with primers F, R, and P2 from the RdRp gene using a one-step advanced RT-ddPCR probe kit (BioRad ddPCR™, cat. no. 1864021 and 1864022, https://www.bio-rad.com/de-at/product/1-step-rt-ddpcr-advanced-kit-for-probes?ID=NTGCRI15).

The RT-PCR reaction results were quantified and calculated as virus copy number/µl and compared with the copy number measured in mock-treated (buffer) infected cells (positive control).

Concurrently, cell viability was measured on uninfected Calu-3 cells treated with azelastine HCl or hydroxychloroquine sulfate for 48 hours using the CellTiter-Glo® 2.0 Cell Viability Assay (Promega®, Cat. #G9241) according to the manufacturer’s instructions. In general, CellTiter-Glo® 2.0 reagent was added in a volume equal to the volume of medium to the cells and incubated for 10 minutes at room temperature. The luminescent signal was measured and compared to the signal measured with untreated cells (negative control).

Results:

Quantitative PCR analysis showed that azelastine HCl was highly effective in reducing viral particles on human lung adenocarcinoma cells by >99% at 50 μM and >90% at 25 μM (Figure 6), confirming that azelastine exerts its anti-SARS-CoV-2 activity on human airway epithelial cells expressing the surface protease TMPRSS2 and most relevant for SARS-CoV-2 infection.

Example 6: Prevention and treatment of HEp-2 cell infection by respiratory syncytial virus (RSV)

To confirm the broad antiviral effect of azelastine-HCl, HEp-2 cells (ATCC CCL-23) were infected with RSV in the absence or presence of azelastine-HCl. Azelastine-HCl was tested under 3 conditions: prophylactic condition (azelastine-HCl treatment followed by infection), co-administration (cells infected and treated with azelastine-HCl simultaneously) and therapeutic condition (infected cells treated with azelastine-HCl 1 h after infection). Spot numbers and viral genome copy numbers were determined and compared with buffer-treated cells.

Experimental methodology:

Azelastine-HCl (Sigma, cat.# PHR1636-1G) was tested for activity against RSV over the concentration range of 0.4 to 25 μM using a 10 mM stock solution prepared in DMSO.

1× ¹⁰⁴ HEp-2 cells (ATCC CCL-23) were seeded in 96-well plates and cultured in 50 μl/well of DMEM supplemented with 10% FBS and 2 mM L-glutamine at 37°C, 5% _CO2 , and 100% humidity for 3 h. After 3 h, the cells achieved adherence and were infected with RSV Long strain (ATCC VR-26, kindly provided by T. Grunwald, Fraunhofer Institute for Cell Therapy and Immunology, Leipzig, Germany) at an MOI of 0.01. Azelastine-HCl was added to the cells either 1 h before infection, simultaneously with infection, or 1 h after infection. The azelastine-HCl stock solution was serially diluted with culture medium (DMEM/10% FCS/2 mM L-glutamine) to achieve the intended final concentration. DMSO was diluted in the same way and added to the cells as a solvent control. Cells infected and treated in a total volume of 200 μl were incubated for 48 h at 37°C under 5% _CO2 and 100% humidity. RSV-infected plaques were detected by immunocytochemical staining with goat polyclonal antibody against RSV (anti-RSV Gt X (IgG Frac), Merck) and HRP-conjugated rabbit polyclonal antibody against goat IgG (Novusbio). 3-amino-9-ethylcarbazole (AEC, Sigma) was used as a chromogen in immunohistochemistry to visualize RSV-infected cells. Spots were counted manually. Viral genome copy number was determined by RT-PCR using RSV-1 (5'-AGA TCA ACT TCT GTC ATC CAG CAA-3', SEQ ID NO: 18) and RSV-2 (5'-GCA CAT CAT AAT TAG GAG TAT CAA T-3', SEQ ID NO: 19) primers as previously described by Wilmschen et al. (Vaccines, 2019, 7:59). The Ct value obtained after azelastine-HCl treatment was compared with cells treated with vehicle only. The following equation was used to calculate the viral genome copy number in treated samples relative to vehicle: An increase of 1 Ct equals 50% fewer copies.

The cytotoxicity of azelastine HCl towards HEp-2 cells was determined by flow cytometric analysis using the vital staining dye propidium iodide.

Experiments were performed independently 3-4 times.

Results:

Azelastine HCl was cytotoxic to HEp-2 cells at 12.8 and 25.6 μM (Fig. 7). Therefore, the effect of azelastine HCl was evaluated at 6.4 μM and lower concentrations. RSV infection of cells, as determined by spot counting, was reduced to ~50% at 3.2 and 6.4 μM concentrations compared to vehicle (DMSO)-treated controls (Fig. 8) under all 3 conditions, indicating a potent prophylactic and therapeutic effect of azelastine HCl on RSV infection of HEp-2 cells. Viral genome copy number determination confirmed this effect at 6.4 μM azelastine HCl (Table 6).

Example 7. Prevention of MucilAir™ Influenza A H1N1 Virus Infection

The effect of repeated doses of azelastine HCl nasal spray (Pollival, UrsaPharm Arzneimittel) on H1N1 influenza infection was tested in fully differentiated human nasal epithelial cells cultured at the air-liquid interface (MucilAir™ pool, Epithelix Sarl - primary cells from a pool of 14 different normal nasal cell donors).

Experimental methodology:

The antiviral effect of azelastine HCl against H1N1 influenza was tested as described by Boda et al (Antiviral Research 156 (2018) 72-79). In general, after apical washing of the MucilAir™ pool with MucilAir™ culture medium (200 µl for 10 min), 10 µl of azelastine HCl at a concentration of 0.02 or 0.01% diluted from Pollival® nasal spray (Ursapharm Arzneimittel GmbH) with its own diluent was applied to the apical side of the MucilAir™ for 10 min. Then, 100 μl of H1N1 influenza virus (ATCC VR-95) from a stock suspension of 10 ⁶ genome copies per ml were applied to the apical side and incubated for 3 h. The inoculum was washed off by washing the apical side of the cells three times with 200 μl of MucilAir culture medium (20 min of incubation). After the 3rd wash, the same concentration of azelastine-HCl was added to the apical side of the cells in a volume of 10 μl and incubated for 21 h. After 24 h, 48 h, and 72 h, the MucilAir™ cell washes were repeated, after which azelastine was replaced on the apical side as before. The number of viral copies was determined in the apical washes at each time point. The basal culture medium was also removed and replaced daily with 500 μl of fresh culture medium. LDH release was measured in the main wash (to assess cell death at 96 h) and cytokine levels (IL-8 and RANTES) were determined at 48 and 96 h time points. All incubation steps were performed at 34°C, 5% _CO2 and 100% humidity. Viral copy number was determined in the apical wash: RNA was extracted using the QIAamp® Viral RNA Kit (Qiagen) and viral RNA was quantified by RT-PCR (QuantyTect RT-PCR Probe, Qiagen) using the qTOWER3 detection system. Ct data were plotted on a standard curve and expressed as genome copy number/mL.

Uninfected cells were included as a negative control. Cells infected with H1N1 influenza virus but treated with buffer only were used as a positive control. The antiviral effect of azelastine-HCl was compared with that of oseltamivir carboxylate (oseltamivir from Carbosynth, Compton, UK, at 10 μM) added to infected cells in the basolateral compartment. Compounds were tested in triplicate.

Results:

Treatment of MucilAir™ cells with azelastine-HCl at both concentrations resulted in statistically significant reductions in viral genome copy number at 24 hours post infection compared to buffer-treated cells. The reduction in viral copy number was ~1.9 log (98.74% reduction) with 0.02% azelastine-HCl and -0.9 log (87.4% reduction) with 0.01% azelastine-HCl (Figure 9).

Furthermore, azelastine HCl exhibited an anti-inflammatory effect during H1N1 MucilAir™ infection; IL-8 (Figure 10/A) and RANTES (Figure 10/B) levels were significantly reduced compared to buffer control at days 2 and 4 post-infection.

Azelastine HCl at 0.02% concentration showed slight cytotoxicity (6.5%) in uninfected cells but no cytotoxicity in infected cells or at lower (0.01%) concentrations.

Example 8: Prevention and treatment of A549 cell infection with adenovirus

To confirm the broad antiviral effect of azelastine-HCl, human lung carcinoma cells (A549) were infected with adenovirus in the absence or presence of azelastine-HCl. Azelastine-HCl was tested both under co-administration and under therapeutic conditions.

Experimental methodology:

Azelastine-HCl (Sigma, cat.# PHR1636-1G) was tested for activity against adenovirus over the concentration range of 0.78 to 50 μM using a 10 mM stock solution prepared in DMSO.

3× ¹⁰⁴ A549 cells (ATCC CCL-185™) were seeded in 96-well plates in 100 µl complete growth medium (DMEM, high glucose supplemented with GlutaMAX™ and 10% FBS, quality assurance, HI 500 ml, Cat#: 10500064, Lot: 08Q6291K) and incubated at 37°C with 5% _CO2 and 100% humidity. After 24 h, 25 µl fresh medium with an MOI of 0.01 TCID50 of hAdv5 adenovirus (ATCC VR-5) was added to the cells to initiate infection. Under the co-administration condition, 25 µl azelastine HCl diluted in complete growth medium was added to the cells simultaneously with the infection. In the therapeutic condition, azelastine-HCl was added to the cells 6 h after infection. The cells were incubated for 48 h after infection at 37°C, 5% _CO2 , and 100% humidity. The plate was then frozen at -80°C, and the virus was released from the cells by 3 freeze-thaw cycles. Virus titer was determined using the Adeno-X™ Rapid Titer Kit (3 replicates per well, Clontech, Takara Bio, Cat.# PT3651-2) according to the manufacturer's instructions.

The cytotoxicity of azelastine against uninfected A549 cells was determined using the CellTiter Glo assay (Promega, USA) using the same protocol as described above, but without infection of the cells with adenovirus.

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List of sequences

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Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys Val

1 5 10 15

Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr Asn Ser Phe

20 25 30

Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser Ser Val Leu

35 40 45

His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn Val Thr Trp

50 55 60

Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys Arg Phe Asp

65 70 75 80

Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser Thr Glu

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Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp Ser

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Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val Ile

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Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Gly Val Tyr

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Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe Arg Val Tyr

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Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe Leu

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Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu Phe

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Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His Thr

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Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser Ala Leu Glu

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Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln Thr

225 230 235 240

Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser

245 250 255

Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro

260 265 270

Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala

275 280 285

Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys

290 295 300

Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val

305 310 315 320

Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys

325 330 335

Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala

340 345 350

Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu

355 360 365

Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro

370 375 380

Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe

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Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly

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Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys

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Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe

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Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys

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Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly

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Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val

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Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys

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Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe Leu

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Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala Val

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Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser Phe

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Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr Ser Asn Gln Val

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Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val Pro Val Ala Ile

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His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly Ser

625 630 635 640

Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly Ala Glu His Val

645 650 655

Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys Ala

660 665 670

Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg Ala Arg Ser Val Ala

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Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly Ala Glu Asn Ser

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Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr Asn Phe Thr Ile

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Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr Lys Thr Ser Val

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Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ser Asn Leu

740 745 750

Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Thr

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Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu Val Phe Ala Gln

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Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp Phe Gly Gly Phe

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Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro Ser Lys Arg Ser

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Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly

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Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile Ala Ala Arg Asp

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Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu

850 855 860

Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala Leu Leu Ala Gly

865 870 875 880

Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile

885 890 895

Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr

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Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser Thr Ala Ser Ala

930 935 940

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Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val

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Leu Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys

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Arg Val Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro

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Gln Ser Ala Pro His Gly Val Val Phe Leu His Val Thr Tyr Val

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Pro Ala Gln Glu Lys Asn Phe Thr Thr Ala Pro Ala Ile Cys His

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Asp Gly Lys Ala His Phe Pro Arg Glu Gly Val Phe Val Ser Asn

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Gly Thr His Trp Phe Val Thr Gln Arg Asn Phe Tyr Glu Pro Gln

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Ile Ile Thr Thr Asp Asn Thr Phe Val Ser Gly Asn Cys Asp Val

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<212> DNA

<213> Artificial sequence

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<223> Probe

<220>

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<222> (9)..(9)

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<222> (18)..(18)

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<400> 10

ccaggtggwa crtcatcmgg tgatgc 26

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<211> 26

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<220>

<221> R

<222> (3)..(3)

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<222> (12)..(12)

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<223> Primer

<400> 12

acaggtacgt taatagttaa tagcgt 26

<210> 13

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<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 13

acactagcca tccttactgc gcttcg 26

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<212> DNA

<213> Artificial sequence

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<223> Primer

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<213> Artificial sequence

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<223> Primer

<400> 15

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<213> Artificial sequence

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<400> 16

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gaggaacgag aagaggcttg 20

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<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 18

agatcaactt ctgtcatcca gcaa 24

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<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 19

gcacatcata attaggagta tcaa 24

<---

Claims (16)

1. Use of azelastine or a pharmaceutically acceptable salt thereof in an amount of 0.1-500 mcg/dose for the prophylactic or therapeutic treatment of a subject in need of treatment of a pathological condition caused by or associated with SARS-CoV-2 infection. 2. The use according to claim 1, characterized in that said SARS-CoV-2 is a natural variant or mutant of SARS-CoV-2, such as one that comprises one or more mutations of the S protein of SARS-CoV-2, preferably K417N, L452R, N501Y, D614G, P681H, P681R, E484K, E484Q or the 69/70 deletion in SEQ ID NO: 4, wherein preferably said variant or mutant of SARS-CoV-2 is selected from the group consisting of mutants B.1.1.7 (British variant), B.1.351 (South African), P.1 (Brazilian), B.1.617 (Indian), B.1.618 (Bengal). 3. The use according to claim 1 or 2, characterized in that azelastine or a pharmaceutically acceptable salt thereof is contained in a pharmaceutical preparation, such as a medical product or a medicinal product, containing an effective amount of azelastine or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier. 4. Use according to any one of paragraphs 1-3, characterized in that the said pathological condition is a common cold, a nasal infection, sinusitis, an infection of the throat and larynx, bronchiolitis, diarrhea, a skin rash or pneumonia, acute respiratory distress syndrome (ARDS). 5. Use according to any one of paragraphs 1-4, characterized in that azelastine or its pharmaceutically acceptable salt is designed with the possibility of local administration, preferably for mucosal administration. 6. Use according to any one of claims 1 to 5, characterized in that azelastine or a pharmaceutically acceptable salt thereof is used for use in the upper and lower respiratory tract, for nasal, pulmonary, intraoral, ocular or dermal administration, or for systemic administration, preferably by intravenous, intramuscular, subcutaneous, intradermal, transdermal or oral administration. 7. Use according to any one of paragraphs. 1-6, characterized in that azelastine or a pharmaceutically acceptable salt thereof is used in the form of a spray, powder, gel, ointment, cream, foam or liquid solution, lotion, mouthwash, aerosolized powder, aerosolized liquid form, granules, capsules, drops, tablets, syrup, lozenges, eye drops or a preparation for infusion or injection. 8. Use according to any one of paragraphs 1-7, characterized in that azelastine or its pharmaceutically acceptable salt is used for nasal administration in an amount of 1-1000 mcg per nostril. 9. Use according to any one of claims 1-8, characterized in that azelastine or a pharmaceutically acceptable salt thereof is used as the only antiviral substance or is intended for administration in combination with one or more active substances, preferably selected from the group consisting of antiviral, anti-inflammatory and antibiotic substances. 10. The use according to any one of claims 1 to 9, characterized in that said subject is infected or at risk of infection with said virus, wherein preferably the subject is a human, a dog, a cat, a horse, a camelid, cattle or a pig. 11. Use of azelastine or a pharmaceutically acceptable salt thereof for treating a biological surface to prevent SARS-CoV-2 infection and/or the spread of SARS-CoV-2. 12. The use according to paragraph 11, characterized in that said biological surface is a mucous membrane surface that is infected or at risk of infection with SARS-CoV-2. 13. The use according to claim 11 or 12, characterized in that said SARS-CoV-2 is a natural variant or mutant of SARS-CoV-2, such as one that comprises one or more mutations of the S protein of SARS-CoV-2, preferably K417N, L452R, N501Y, D614G, P681H, P681R, E484K, E484Q or the 69/70 deletion in SEQ ID NO: 4, wherein preferably said variant or mutant of SARS-CoV-2 is selected from the group consisting of mutants B.1.1.7 (British variant), B.1.351 (South African), P.1 (Brazilian), B.1.617 (Indian), B.1.618 (Bengal). 14. Use according to any one of paragraphs. 11-13, characterized in that azelastine or a pharmaceutically acceptable salt thereof is in a medical product that is designed for local application, preferably for application in the upper and lower respiratory tract, nasal, pulmonary, intraoral, ocular or dermal application. 15. Use according to paragraph 14, characterized in that the said medical product is presented in the form of a solution, dispersion, dry powder or aerosolized liquid or powder. 16. The use according to any one of paragraphs. 11-15, characterized in that azelastine or a pharmaceutically acceptable salt thereof is intended for administration in an effective amount, wherein preferably said amount is 1 ng - 1000 ng/ ^cm2 .