RU2826285C2 - Compound and method for preventing transmission of influenza virus - Google Patents

Abstract

FIELD: medicine; pharmaceutics.

SUBSTANCE: invention relates to the field of medicine and pharmaceutics, specifically to the use of a compound of formula I or II or a pharmaceutically acceptable salt thereof for reducing the rate of transmission of influenza virus infection, wherein the compound or a pharmaceutically acceptable salt thereof is administered to a null patient infected with the influenza virus.

EFFECT: use of the invention enables to effectively reduce the rate of transmission of influenza virus infection from an infected patient to a healthy person, preventing the spread of viral infection.

49 cl, 18 dwg, 5 ex

Description

The present invention relates to a method for preventing the transmission of an influenza virus, wherein said method comprises administering to a patient having an influenza virus infection, hereinafter referred to as "patient zero", an effective amount of a compound, wherein said compound has one of formulas (I) and (II), or a pharmaceutically acceptable salt thereof. The compound to be used in the present invention reduces the infectivity of the influenza virus of patient zero and, therefore, reduces the risk of patient zero starting an influenza epidemic or an influenza pandemic compared to a control patient. Therefore, one aspect of the present invention relates to a method for preventing an influenza epidemic or an influenza pandemic, wherein said method comprises administering to patients having an influenza virus infection (patient zero) an effective amount of a compound, wherein said compound is administered to at least 10% of all influenza-infected individuals of a city or country, and wherein said compound has one of formulas (I) and (II), or a pharmaceutically acceptable salt thereof.

Influenza virus infection is an acute respiratory infectious disease caused by influenza viruses and is spread primarily by airborne transmission, particularly droplet infection. Influenza A and B viruses are highly infectious (Vanderlinden, Med Res Rev 2014; 34(2):301–339). Consequently, influenza epidemics often occur due to seasonal influenza viruses and constitute a major burden on the global health system. There are an estimated 3–5 million cases of severe disease worldwide and approximately 290,000–650,000 people die from influenza each year (WHO News Release, Geneva, Switzerland, 14 December 2017; WHO Fact Sheet, Geneva, Switzerland, 31 January 2018; Baxter D, Hum Vaccin Immunother. 2016; 12(10):2712–2717). Influenza also places significant demands on health services each season and impacts society through loss of workforce productivity (WHO Fact Sheet, Geneva, Switzerland, 31 January 2018).

Influenza pandemics are unexpected and, until now, inevitable events. Over the past century, they have caused global public health emergencies. The first and most severe of these is estimated to have caused more than 40-50 million deaths worldwide (Francis, Am J Hyg., 1945, 42:1-11). Experts predict that the next pandemic will be associated with a large number of casualties and high levels of morbidity requiring hospitalization, thus placing considerable strain on health care resources. Pandemics are global in nature, and few countries are likely to remain free of them. In developing countries, where health care resources are already strained and large segments of the population are often weakened by poor health and nutrition, the impact is likely to be enormous.

The conditions surrounding the 1997 Hong Kong “bird flu” outbreak highlight the need for advance planning to ensure an adequate response to a public health emergency that is distinctly unpredictable, complex, rapidly evolving, and associated with significant public concern. The onset of pandemics in the past has been too late to implement many of the key activities required to minimize the impact. Therefore, planning and implementation of preparedness activities must begin well in advance. Planning for a pandemic will also enhance the ability to respond to other large-scale public health emergencies, including bioterrorism threats that require widespread access to preventive and therapeutic interventions, and strong national plans that include a risk communication component to help assuage public fears. The impact of pandemic influenza is likely to be significantly greater—orders of magnitude—than typical bioterrorism scenarios. Unlike most other public health emergencies, pandemics occur in multiple waves and last one to two years. Consequently, response efforts will need to be sustained over a long period. In addition, preparing for an influenza pandemic will strengthen the response to an influenza epidemic, which occurs every year and is believed to kill between 500,000 and 1 million people worldwide each year. Investing in pandemic preparedness therefore has a direct and immediate benefit as a measure to reduce the impact of a specific and recurring event.

Annual vaccination programs are the cornerstone of efforts to prevent influenza virus infections, but the effectiveness of these programs is variable due to both suboptimal vaccination uptake and mismatches between the vaccine and circulating influenza strains (Centers for Disease Control and Prevention "Vaccine effectiveness - how well does the flu vaccine work?" 3 ^rd October 2017). M2 inhibitors such as amantadine and neuraminidase inhibitors such as oseltamvir are typical drugs used against influenza. However, a modeling study of M2 inhibitor use in a large outbreak setting did not find that either treatment significantly affected the course of the outbreak, indicating that this influenza drug treatment does not reduce transmission (Stilianakis, Journal of Infectious Diseases, 1 998, 177:863-873). There is currently no treatment with evidence of reducing influenza transmission. Therefore, an important unmet medical need is the identification of antiviral agents that reduce the transmission of influenza virus and thereby prevent or attenuate the outbreak of influenza epidemics during seasonal influenza or an influenza pandemic.

Thus, the technical problem underlying the present invention is to provide means and methods for maintaining health in society.

This technical problem is solved by proposing embodiments characterized in the invention formula.

Accordingly, the present invention relates to a method for preventing the transmission of influenza, said method comprising administering an effective amount of a compound to a patient having an influenza virus infection (patient zero), wherein said compound has one of the following formulas I and II:

or its pharmaceutically acceptable salt

Thus, the present invention relates to a compound for use in the treatment of a patient (patient zero) infection with an influenza virus, wherein the compound has one of formulas (I) and (II), or a pharmaceutically acceptable salt thereof, and wherein the compound prevents (e.g. reduces) the transmission of an influenza virus. Also encompassed by the present invention is a pharmaceutical composition comprising a compound that has one of formulas (I) and (II), or a pharmaceutically acceptable salt thereof, and optionally comprising a pharmaceutically acceptable carrier, wherein the pharmaceutical composition prevents (e.g. reduces) the transmission of an influenza virus. The pharmaceutical composition is particularly useful for the treatment of a patient infected with influenza (patient zero), and prevents (e.g. reduces) the transmission of an influenza virus to said patient.

As shown in the attached Examples, the compound to be used in the present invention has a beneficial effect in that it reduces transmission. Therefore, the compound reduces the likelihood that an epidemic or pandemic of influenza will occur, or reduces the spread of an existing epidemic or pandemic of influenza. The compound to be used in the present invention is also a valuable tool for reducing the effects of a bioterrorist attack that deals with an influenza virus, such as an epidemic or pandemic influenza virus. Thus, the compound is particularly useful in the treatment of a specific type of influenza virus infection, i.e., an infection that is caused by an influenza virus strain that has epidemic or pandemic potential. The unexpected technical effect of the compound to be used in the present invention to reduce the transmission of influenza virus clearly leads to a new clinical situation. Indeed, having in mind this beneficial technical effect, the treating physician will in particular treat with this compound those patients who may cause (e.g. economic or social) damage if his/her contagiousness is not reduced. Such patients are, for example, patients who have personal contact with individuals in the risk group for influenza virus infection, such as individuals who are at high risk of getting an influenza virus infection or who have an increased risk of getting severe complications associated with influenza (e.g. the elderly, small children, seriously ill people or people with weakened immunity). For example, treatment with the compound to be used in the present invention is very useful for influenza patients who stay at home during their influenza virus infection, but have contact with household members during their influenza virus infection, such as small children. In addition, due to its beneficial property of reducing the transmission of influenza, the compound of the present invention can be used in the treatment of individuals who are important for the functioning of society, which would allow these important individuals to continue working during their infection with the influenza virus without infecting colleagues or other contacts. For example, the compound to be used in the present invention can be advantageously used to treat medical personnel, in particular at the beginning of an epidemic or pandemic situation of influenza. This compound can also be used to treat a patient who will have many personal contacts during his/her infection with the influenza virus, for example, because he/she must fly on an airplane. Indeed, in the past, several airplanes had to be quarantined due to a sudden outbreak of the disease, in particular during long-distance flights. Thus, the means and methods provided herein advantageously reduce the social and economic consequences associated with influenza virus infections.

It is demonstrated that no conclusion can be drawn about the effect of a drug in reducing transmission from the efficacy of the drug against influenza (e.g., from the efficacy of the drug in reducing influenza symptoms, viral load, viral shedding, or viral RNA levels). Indeed, the attached Examples demonstrate that oseltamivir, which is a well-known and effective drug against influenza, does not prevent transmission of the influenza virus at all. The attached Examples also demonstrate that the viral RNA present in the nasal wash of subjects infected with influenza is identical in subjects treated with a compound of the invention (in particular, baloxavir marboxil), oseltamivir, or placebo (see, e.g., Figs. 6, 9 and 12). However, for an unknown reason, viral transmission was significantly reduced in subjects treated with baloxavir compared with subjects treated with oseltamivir or placebo.

The fact that an influenza drug reduces influenza symptoms does not necessarily mean that the drug is also suitable for reducing influenza virus transmission. Influenza symptoms may persist even after viral clearance, viral load, and viral shedding have occurred, and therefore efficacy in reducing transmission cannot be predicted in the absence of a well-designed non-clinical study that mimics human models. Many factors influence transmission (e.g., in a household). Therefore, the fact that a drug reduces viral shedding or viral load cannot be directly inferred that the drug also reduces transmission.

Furthermore, there is no absolute correlation between nasal/pharyngeal viral shedding and transmission. In humans, for example, H5N1 viruses reach very high titers in infected patients but are not transmitted through the air or between people. A ferret model has shown a narrow window of transmissibility that does not necessarily correlate with the temporal pattern of peak viral shedding (Roberts, PLoS One 7.8 (2012): e43303). Furthermore, we have shown that transmission does not correlate with clinical signs, so a reduction in symptoms does not necessarily mean a reduction in transmission. Indeed, there is consensus that a significant proportion of influenza virus transmission is asymptomatic or presymptomatic (Fraser, Proceedings of the National Academy of Sciences 101.16 (2004): 6146-6151). Thus, the fact that a known drug is effective in treating influenza does not necessarily indicate that the drug is also effective in reducing the transmission of that influenza virus. However, in the context of the present invention, it has been surprisingly found that baloxavir marboxil significantly reduces the transmission of the influenza virus.

As described above, the present invention is directed to the treatment of a patient with influenza, the infectivity of which should be prevented or reduced. Indeed, according to the present invention, the term "prevents transmission" includes that the compound "reduces transmission" compared to a control patient to whom the compound to be used in the present invention is not administered. In this document, a patient with influenza, the infectivity of which should be prevented or reduced, is called "patient zero" (or "patient zero"). This patient, which is further defined below, can be any person who has an infection with an influenza virus and whose infectivity should be prevented or reduced for any reason.

The compound to be used in the present invention reduces the infectivity of the influenza virus of patient zero. As used herein, the term "infectivity" means the ability of a pathogen, such as an influenza virus, to establish an infection in another host. As used herein, the term "infectivity" and "transmissibility" have the same meaning and define the frequency of spread of the pathogen among hosts, including hosts that are in a parent-child relationship and hosts that are not in a parent-child relationship. Thus, according to the present invention, the compound reduces the ability of the influenza virus of the treated patient zero to establish an infection in another host. Or, in other words, the compound to be used in the present invention reduces the frequency with which the influenza virus of patient zero infects other hosts.

The attached Examples unexpectedly demonstrate that the transmission of the influenza virus can be reduced immediately after the administration of the compound to be used in the present invention. In addition, the attached Examples also document that the infectivity of the treated patient zero is even further reduced within 24 hours from the time of administration of the compound. Therefore, in the context of the present invention, it is preferred that the patient zero has no personal contact with other persons (i.e., contacts) until 1 hour or until 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30 or 32 hours after the administration of the compound to be used in the present invention. The time intervals mentioned herein refer to minimum time intervals. For example, the time interval "2 hours after administration" also includes longer periods such as 2.5 hours after administration, etc. Indeed, the present invention encompasses that patient zero has no personal contact with contacts until one or two days (or one day and one or two nights) after administration of the compound to be used in the present invention. For example, patient zero may remain in isolation in a hospital for one day (or one day and one or two nights), such that personal contact with other persons is prevented during this period of time.

Accordingly, in one aspect of the present invention, the rate of influenza virus transmission from patient zero is reduced within one hour to 24 hours from the 1st administration of the compound. The rate of influenza virus transmission from patient zero can also be reduced within 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30 or 32 hours from the 1st administration of the compound. Accordingly, this period of time can be extended. Thus, according to the present invention, the rate of influenza virus transmission from patient zero is reduced within one hour to 32 hours (for example, within 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30 or 32 hours) from the 1st administration of the compound.

Thus, the compound to be used in the present invention reduces the rate of transmission of influenza virus from a treated patient zero to a contact person of the treated patient zero. The rate of transmission of influenza virus from the treated patient zero can be reduced to 70% or less, preferably to 50% or less, more preferably to 30% or less compared to the rate of transmission of influenza virus from a control patient.

In this document, the "transmission rate" (also referred to as the "spread rate") corresponds to the percentage of infected subjects among the contacts of a patient infected with influenza (i.e., patient zero or control patient, respectively) within a specified period of time. Thus, the "transmission rate" is the number of new cases (infection with the influenza virus strain that originates from patient zero or from the control patient, respectively) within a specified period of time, divided by the population at risk. The "population at risk" is preferably the number of persons who had personal contact with patient zero or with the control patient, respectively, during his/her infection with the influenza virus. Influenza virus can usually be transmitted from the day before the onset of influenza symptoms, during the duration of influenza symptoms and until recovery from the disease. Therefore, the "population at risk" may be the number of persons who had personal contact with patient zero or with the control patient, respectively, from the day before the onset of influenza symptoms and during the duration of influenza symptoms. Preferably, the "population at risk" does not include individuals who have received an influenza vaccination. As described herein above and below, the "transmission rate" decreases after administration of a compound to be used in the present invention. Therefore, the effect of the compound on the transmission rate is measured after administration of the compound. In particular, to assess the effect of the compound on the transmission rate, the "population at risk" can be defined as the number of individuals (e.g., individuals who are not vaccinated against influenza) who have had personal contact with the zero patient during the period of time from administration of the compound to the end of influenza symptoms. Accordingly, the "population at risk" of the control patient can be defined as the number of individuals (e.g., individuals who are not vaccinated against influenza) who have had personal contact with the control patient during the period of time from administration of an influenza drug other than the compound, or, if no influenza drug is administered, from an appropriate time point taking into account the pathogenesis of the influenza disease to the end of influenza symptoms and recovery from the disease.

As described above, according to the present invention, the compound reduces the transmission rate of the influenza virus of patient zero (i.e., the infectivity of patient zero) to 70% or less, preferably to 50% or less, more preferably to 30% or less, compared to the transmission rate of the influenza virus of a control patient who was not treated with the compound (i.e., compared to the infectivity of a control patient who was not treated with the compound). In seasonal influenza, including an influenza epidemic, the influenza virus is transmitted from one patient to approximately 1.35 subjects in 5 days. Thus, if patient zero is infected with a seasonal influenza strain (including an epidemic influenza strain, but not a pandemic influenza strain), then the compound reduces the number of subjects to whom the virus is transmitted to an average of about 0.945 subjects (i.e., 70% transmission) or less over 5 days, preferably to an average of about 0.675 subjects (i.e., 50% transmission) or less over 5 days, more preferably to an average of about 0.405 subjects (i.e., 30% transmission) or less over 5 days. In pandemic influenza, the influenza virus is transmitted from one patient to about 2.9 subjects over 6 days. Thus, if patient zero is infected with a pandemic influenza strain, then the present compound reduces the number of subjects to whom the virus is transmitted to an average of about 2.03 subjects (i.e., 70% transmission) or less over 6 days, preferably to an average of about 1.45 subjects (i.e., 50% transmission) or less over 6 days, more preferably to an average of about 0.87 subjects (i.e., 30% transmission) or less over 6 days.

Most individuals with influenza infection are advised to stay home until they have been fever-free for at least 24 hours, which puts other household members at risk of infection (Centers for Disease Control and Prevention. The flu: what to do if you get sick. 9th March 2018). Once one household member becomes infected with influenza, the risk of transmission to a household contact can be as high as 38%, with a delay between onset in patient zero and infection of a household contact of about 3 days (Tsang, Trends Microbiol 2016; 24(2): 123-133). As described above, the compound to be used in the present invention reduced the rate of influenza virus transmission from treated patient zero to 70% or less, preferably to 50% or less, more preferably to 30% or less. Thus, treatment of patient zero with said compound reduces the risk of transmission of influenza virus to a household contact to about 27% or less (i.e. 70%), preferably to about 19% or less (i.e. 50%), more preferably to about 11.4% or less (i.e. 30%). This is a significant advantage in preventing an influenza epidemic or pandemic.

The attached Examples demonstrate that the compound to be used in the present invention reduces the load of virus excreted in the upper respiratory tract. Thus, according to the present invention, this compound reduces the load of virus excreted in the upper respiratory tract of the zero patient compared to the load of virus excreted in the upper respiratory tract of the control patient, where this control patient has an influenza virus infection and has not been administered this compound.

The control patient may be any patient who has an influenza virus infection and who has not been treated with a compound to be used in the present invention. Thus, according to the present invention, the control patient has an influenza virus infection and has not been administered the compound. However, the control patient may be administered an anti-influenza drug, except for a compound of formula (I) or (II), or a pharmaceutically acceptable salt thereof. For example, the control patient may be administered oseltamivir. Thus, in one aspect of the present invention, the control patient was administered an anti-influenza drug, except for a compound of formula (I) or (II), or a pharmaceutically acceptable salt thereof. For example, the control patient may be administered oseltamivir. In another aspect of the present invention, the control patient was not administered an anti-influenza drug, for example, was not administered oseltamivir. Oseltamivir may be administered at a dose of 75 mg twice daily for 5 days. The human dose of oseltamivir is generally known in the art. For example, for adults and adolescents (13 years and older), 75 mg is usually given twice daily for 5 days. The dose for pediatric patients who are 2 weeks to less than 1 year of age is usually 3 mg/kg per day for 5 days. The control patient has an influenza virus strain that is identical to the influenza virus strain of the patient zero to whom the control patient is being compared. For example, if patient zero has a pandemic influenza virus strain, then the control patient to whom patient zero is being compared has the same pandemic influenza virus strain.

A person skilled in the art can easily recognize whether transmission of the influenza virus has occurred from a (treated) patient zero or from a control patient. For example, transmission has probably occurred when at least one contact person of a treated patient zero or a control patient becomes infected with the influenza virus after having personal contact with the treated patient zero or the control patient, respectively. A contact person of a treated patient zero is preferably a person who:

(i) did not have an influenza virus infection at the time the compound was first administered to patient zero; and

(ii) had personal contact with a treated patient zero during that patient zero's influenza virus infection.

With respect to (ii) immediately above, it is preferred that the contact person has had personal contact with the treated patient zero within 10 days of the administration of the compound to the patient zero. Thus, with respect to (ii) immediately above, it is preferred that the contact person has had personal contact with the treated patient zero within a period of time beginning with the administration of the compound and ending 5-10 days (e.g. 9 days or 5 days) after the administration of the compound.

The contact person of the control patient is preferably a person who:

(i) did not have an influenza virus infection at the time when the anti-influenza medicinal product was first administered to the control patient; or, if the control patient had not been administered an anti-influenza medicinal product, at an appropriate time point given the pathogenesis of the influenza disease; and

(ii) had personal contact with a control patient during the control patient's influenza virus infection.

With respect to point (ii) immediately above, it is preferred that the contact person has had personal contact with the control patient within 10 days of the administration of the anti-influenza medicinal product to the control patient, or, if no anti-influenza medicinal product has been administered, within an appropriate time period taking into account the pathogenesis of the influenza disease. Thus, with respect to point (ii) immediately above, it is preferred that the contact person has had personal contact with the control patient within a time period beginning with the administration of the anti-influenza medicinal product to the control patient; or, if no anti-influenza medicinal product has been administered, beginning with an appropriate time point taking into account the pathogenesis of the influenza disease and ending 5 to 10 days (e.g. 9 days or 5 days) after the administration of the anti-influenza medicinal product; or, if no anti-influenza medicinal product has been administered, ending at an appropriate time point taking into account the pathogenesis of the influenza disease.

Naturally, a contact person does not necessarily have to have personal contact with patient zero or the control patient, respectively, on every day within the 10 days mentioned above. Rather, a contact person may, for example, have personal contact with patient zero or the control patient, respectively, on only one day within these 10 days. For example, a contact person may have personal contact with patient zero or the control patient (e.g. due to shared household management) on approximately 7 out of 9 days.

In this document, when counting the day after the administration of the compound to the zero patient or the other influenza drug to the control patient, the day of administration is considered as "day 0". Thus, day 1 (i.e., one day after the administration of the compound or the other influenza drug, respectively) is the day after the day of administration, and so on. Accordingly, 10 days after the administration of the compound or the other influenza drug refers to the day that begins approximately 240 hours after the administration of the compound or the other influenza drug, respectively.

As mentioned above, according to the present invention, the contact person of the treated zero patient or control patient is a person who:

(i) with respect to patient zero: was not infected with influenza virus at the time when the compound was first administered to patient zero; or

relative to the control patient: was not infected with influenza virus at the time when the anti-influenza medicinal product was first administered to the control patient; or, if the control patient had not been administered the anti-influenza medicinal product, at the appropriate time given the pathogenesis of the influenza disease; and

(ii) in relation to patient zero: had personal contact with a treated patient zero during that patient zero's influenza virus infection; or

in relation to the control patient: had personal contact with the control patient during the control patient's influenza virus infection.

According to the present invention, the absence of an influenza virus infection preferably means PCR-negativity for the influenza virus (for example in a nasopharyngeal swab). Thus, it is preferable that at the time point as defined in (i) above, the contact person was PCR-negative for influenza.

Detection and quantification of influenza virus by PCR is well known in the art. For example, real-time reverse transcription-PCR amplification (RT-PCR) can be used as a method to determine the presence or absence or to quantify influenza RNA. Isolation and purification of influenza virus RNA is an established technique and can be performed, for example, using the MagNA Pure LC 1.0 or 2.0 Extraction Station (Roche Applied Science, product no. 05197686001). For this assay, nucleic acids are isolated from swab specimen aliquots using the MagNA Pure LC Extraction Station and the MagNA Pure LC Nucleic Acid Isolation Kit according to the manufacturer's instructions (Roche Applied Science). Reverse transcription and amplification reactions can be performed using the Taqman Fast Virus Master Mix. During clinical testing, a 4-point (low, medium and high) influenza A and B virus standard curve with known particle/mL counts can be used as a control and can be run with each run. A universal internal control, seal distemper virus (PDV), can be added to each isolate to monitor the entire process from isolation to detection in real time. Additionally, a non-amplification control (NAC) can be included with each PCR mixture that is run to monitor contamination with each isolation. Positive controls should give a positive signal that lies between defined reaction limits. If the positive control value lies outside the reaction limit, all samples run with the same PCR mixture should be re-assayed. The result of the influenza RT-PCR assay is what is known as the threshold cycle or Ct value and the Ct value is recorded for each assay. Ct values are converted to quantitative values of viral particles/mL with standard curves run concomitantly with the samples.

For influenza A positive subjects, PCR analysis for the influenza A subtype can also be performed. More specifically, for influenza A positive subjects, subtyping can be performed directly from the subject's swab specimen using a real-time RT-PCR assay. RNA can be isolated from clinical isolates as described above using the Roche MagNA Pure Total Nucleic Acid Kit and amplified using a one-step RT-PCR with influenza A subtype-specific primers. Other methods for detecting specific influenza virus subtypes, including suitable primer sequences, are well known in the art and are described, for example, in the "WHO information on the molecular detection of influenza viruses" of July 2017.

A person skilled in the art can easily determine the time point within an influenza virus infection that corresponds to a given influenza disease state (i.e., that corresponds to a given state within the pathogenesis of influenza disease). More specifically, it is common knowledge in the art that the pathogenesis of influenza disease can be monitored. For example, the pathogenesis of influenza disease can be monitored by monitoring clinical symptoms and/or virology indicators. Clinical symptoms can be monitored by the following:

(i) determination of the cough and nasal symptoms score (items 14 and 15 of the Canadian Acute Respiratory Illness and Influenza Scale (CARIFS);

(ii) asking the patient or his/her caregiver whether and when the patient was able to return to outpatient care/school/work or resume his/her normal daily activities in the same manner as before the development of influenza virus infection; and/or

(iii) if the patient has already returned to a temperature-free state (and preferably has remained so for at least 21.5 hours), determining the point in time when the patient first returns to a temperature-free state (i.e., tympanic temperature less than or equal to 37.2°C).

CARIFS is well known in the art and is shown in Fig. 18.

Virological measurements can be monitored, for example by measuring viral titer or viral shedding.

According to the present invention, personal contact between a contact person and a patient zero or a control patient, respectively, is a personal contact that ensures the transmission of the influenza virus. For example, this personal contact can be a contact that ensures airborne transmission and/or direct contact transmission, preferably only airborne transmission. Contact transmission refers to the direct transfer of the virus from an infected person to a susceptible individual, for example via contaminated hands (direct contact transmission), or to the indirect transfer of the virus via intermediate objects (fomites, indirect contact transmission). Transmission of the virus through the air (i.e., airborne transmission) can occur via droplets or aerosols. The generally accepted cutoff size between large droplets and small aerosols is 5 μm (Kutter, Current opinion in virology, 28 (2018): 142-151). Droplets generated by coughing, sneezing, or speaking do not remain suspended in the air. They typically remain airborne for less than 17 minutes and travel less than 1 m before depositing on the mucosa of close contacts (i.e., people who have face-to-face contact with an infected patient) or on surrounding surfaces. Aerosols have a low deposition velocity, so they remain suspended in the air longer and can travel long distances greater than 1 m (Kutter, Current opinion in virology, 28 (2018): 142-151). In studies analyzing influenza virus transmissibility, influenza virus RNA was detected in the air up to 3.7 m from patients, with most of the viral RNA contained in aerosols (less than 5 μm) (Kutter, Current opinion in virology, 28 (2018): 142-151).

Thus, according to the present invention, the personal contact that provides direct contact transmission is physical contact between an influenza-infected patient (i.e., patient zero or control patient, respectively) and a contact person, such as shaking hands, etc. Influenza A viruses have been shown to survive for up to 3 days when inoculated on banknotes (Thomas, Appl. Environ. Microbiol. 74.10 (2008): 3002-3007). The same inoculum in the presence of respiratory mucus showed a striking increase in survival time (up to 17 days, Thomas, Appl. Environ. Microbiol. 74.10 (2008): 3002-3007). Using nasopharyngeal secretions from naturally infected children, influenza virus survived for at least 48 hours in one third of cases (Thomas, Appl. Environ. Microbiol. 74.10 (2008): 3002-3007). Accordingly, in the context of the present invention, personal contact between an influenza-infected patient (i.e., patient zero or control patient) can provide indirect contact transmission. Said personal contact that provides indirect contact transmission can be skin-to-skin contact of a contact person with a fomite after an influenza-infected patient (i.e., patient zero or control patient) has had personal contact with the fomite. For example, the influenza-infected patient may have touched the fomite with his/her hands or may have breathed on the fomite. The time interval between personal contact between an influenza-infected patient and a fomite and personal contact between a contact and a fomite may be 3 days or less, preferably 48 hours or less.

Respiratory viruses such as influenza virus replicate in the respiratory tract, where they are then excreted and transmitted via respiratory secretions by airborne transmission. In the present invention, personal contact that enables airborne transmission is a distance between a patient infected with influenza (i.e., patient zero or control patient, respectively) and a contact person, which is up to 3.7 meters. Influenza is mainly transmitted by transmission with respiratory droplets. Therefore, it is preferable that during personal contact between a contact person and a patient zero or control patient, respectively, these persons (i.e., a contact person and a patient zero or control patient, respectively) have a distance of up to 1 meter and, more preferably, less than 1 meter.

Using the improved equipment, the transmission of the influenza virus can be detected immediately after the transmission. Typically, the transmission is detected one day after the transmission occurred. In one aspect of the present invention, the transmission from the treated patient zero has occurred when the influenza virus can be detected in at least one contact of the treated patient zero. In a preferred aspect of the present invention, the transmission from the treated patient zero has occurred if the influenza virus can be detected in at least one contact of the treated patient zero within 15 days from the time of administration of the compound. In one aspect of the present invention, the transmission from the treated patient zero has occurred if the influenza virus can be detected in at least one contact of the treated patient zero within 10 days from the time of administration of the compound to the patient zero. If the transmission has occurred, the influenza virus can be detected in the contact person even within a longer period of time, for example, within 15 days from the time of administration of the compound to the patient zero. In particular, an even longer course of the disease is expected, for example up to three weeks or longer if the virus mutates.

In one aspect of the present invention, transmission from a control patient has occurred if the influenza virus can be detected in at least one contact of the control patient. In a preferred aspect of the present invention, transmission from a control patient has occurred if the influenza virus can be detected in at least one contact of the control patient within 15 days of administration of another influenza drug to the control patient; or, if the control patient has not been administered an influenza drug, within an appropriate period of time taking into account the pathogenesis of the influenza disease. In one aspect of the present invention, transmission from a control patient has occurred if the influenza virus can be detected in at least one contact of the control patient within 10 days of administration of another influenza drug; or, if the control patient has not been administered an influenza drug, within an appropriate period of time taking into account the pathogenesis of the influenza disease. As mentioned above, if transmission has occurred, the influenza virus may be detected in the contact person even within a longer period of time, for example, within 15 days from the administration of another influenza drug to the control patient; or, if the control patient has not been administered an influenza drug, within an appropriate period of time taking into account the pathogenesis of the influenza disease. As also mentioned, in particular, an even longer course of the disease is expected, for example up to three weeks or longer if the virus mutates.

In the context of the present invention, the transmission rate can be measured by analyzing at least one contact person of the treated patient zero or the control patient, respectively, for an influenza virus infection. The transmission rate can be measured by analyzing at least one contact person of the treated patient zero or the control patient, respectively, for an influenza virus infection within 10 days of the administration of the compound to the treated patient zero, or within 10 days of the administration of another influenza drug to the control patient, or, if the control patient has not been administered an influenza drug, within 10 days of the relevant time point, taking into account the pathogenesis of the influenza disease.

Influenza is a severe disease and recovery may take from several days to weeks. Therefore, according to the present invention, the transmission rate can also be measured by analyzing at least one (e.g. at least 2) contacts of a treated patient zero or a control patient, respectively, for influenza virus infection within 20 days from the administration of the compound to the treated patient zero or within 20 days from the administration of another influenza drug to the control patient, or, if the control patient has not been administered an influenza drug, within 20 days from the relevant time point, taking into account the pathogenesis of the influenza disease. For example, the transmission rate can be measured by analyzing several contacts (e.g. at least 2) of the patient zero and the control patient, respectively, for influenza virus infection. For example, contacts may be tested for influenza virus infection at different time points, such as approximately 3 days, 5 days, 10 days, 15 days, and/or 20 days after administration of the compound to a null patient or after administration of another influenza drug to a control patient, or, if the control patient has not been administered an influenza drug, at the appropriate time point(s) based on the pathogenesis of the influenza disease.

In one aspect of the present invention, an influenza virus infection is present if the influenza virus can be detected (e.g., in a contact person of patient zero or a control patient, respectively). The influenza virus can be detected by PCR. Additionally or alternatively, the influenza virus can be detected by using an influenza assay kit. For example, rapid influenza diagnostic tests (RIDTs) can be used to detect the influenza virus. RIDTs are immunoassays that can identify the presence of influenza A or B nucleoprotein antigens in respiratory samples and they show a qualitative result (positive versus negative) (Ali T, Clin Infect Dis. 2004 Mar 1; 38(5):760-2). RIDT assays are ELISA (enzyme-linked immunosorbent assay) based assays that are less accurate than PCR, but have the advantage of being cheaper and faster, which is a significant advantage, in particular in an epidemic or pandemic situation.

Influenza virus can be additionally detected using the Roche cobas ^® Liat ^® point of care (POC) polymerase chain reaction (PCR) system (Chen, Eur J Microbiol Immunol (Bp). 2015; 5(4):236-245). The cobas ^® Liat ^® system provides rapid and accurate diagnosis of influenza A or B virus in nasopharyngeal swab specimens. This system includes the cobas ^® Liat ^® analyzer and the cobas ^® Influenza A/B Assay. Detection of influenza virus can also be accomplished using a PCR-based molecular assay (Prodesse ProFlu+ assay, Chen, Eur J Microbiol Immunol (Bp). 2015; 5(4):236-245) or the Alere i Influenza A & B rapid PCR system (Merckx, Ann Intern Med. 2017; 167(6):394-409).

The detection of the influenza virus can be further carried out by analyzing whether infectious viral particles are present, for example by measuring the viral titer and/or viral excretion. The viral titer (for example in the nasal mucosa) can be determined by means of a plaque assay (for example as carried out in the attached Examples and as described below).

Determination of infectious titer of virus is generally known in the art. For example, infectious titer of virus can be measured by quantitative culture assay, i.e., "plaque assay". Specifically, infectious titer of viruses can be determined using quantitative culture assay that expresses the amount of virus present as tissue culture median infectious dose (TCID ₅₀ ) per milliliter. In order to determine infectious titer of a given sample, clinical samples can be serially diluted and incubated on MDCK cells. The primary endpoint for this assay utilizes the ability of influenza viruses to bind to sialylated glycans on red blood cells, causing agglutination. Measurement of agglutination mediated by addition of red blood cells to a dilution series of influenza virus can then be used to quantify the titer of virus. For circulating virus isolates that have lost the ability to agglutinate red blood cells (e.g., current isolates of the relevant season), an ELISA-based influenza virus nucleoprotein (NP) assay can be used. The TCI ₅₀ assay can be implemented as a standardized protocol that allows for NP-ELISA to be performed on the same assay plate if the virus is a non-agglutinating strain. For clinical studies, the MR-ENEA assay for influenza virus subtypes (e.g., H3) (and non-typeable viruses) can be used after the red blood cell agglutination assay. Negative controls should be negative and positive controls should be positive to ensure the plate is valid. To calculate the virus titer (TCID ₅₀ ), the Karber method can be used (Karber, G., 1931, Beitrag zur kollektiven Behandlung Pharmakologischer Reihenversuche, Archiv fur Experimented Pathologie und Pharmakologie, 162, 480-487).

For example, to determine the infectious titer of a virus, the virus can first be grown from a nasal swab sample and then the infectious titer of the virus can be measured as follows:

Virus growth from a nasal wash specimen can be accomplished as follows. MDCK cells can be maintained in cell culture medium until used. One day prior to assay, MDCK cells can be plated in 96-well flat-bottomed plates at 3.5 x 10 ⁴ cells/well in cell culture medium. Trypsin-EDTA may be required to detach the cells from the culture flask. 24 h of incubation at 37°C, 5% CO ₂ results in cell monolayers that are 80-100% confluent. 20 µl of thawed nasal wash specimen in triplicate can be added to 180 µl of infection medium to make a 10-fold serial dilution. MDCK monolayers can then be washed twice with PBS and 100 µl of the diluted samples added to the cells for a 2 h incubation at 37°C, 5% CO ₂ . The last row of this plate can be left without sample to act as a negative control. The virus inoculum can then be removed and replaced with 200 µl of fresh infection medium. The plates can then be incubated at 37°C, 5% _CO2 for 96 hours.

Measurement of viral titers in nasal swabs can be performed as follows: After 96 hours of incubation, viral titers of nasal swab samples cultured in MDCK plates can be read by hemagglutination assay. Twenty-five µl of MDCK supernatant from each well can be transferred to the corresponding wells of a new 96-well U-bottom plate and mixed with 25 µl of 1% (v/v) turkey red blood cells by gentle tapping. The plates can be incubated at room temperature for 30 min and the agglutination pattern can be recorded. Viral titers can be calculated using the method described by Reed and Muench (Reed, LJ and H. Muench, American Journal of Epidemiology, 1938. 27(3): p. 493-497) and can be expressed as log ₁₀ TCID ₅₀ /ml.

Also, the presence of at least one flu symptom indicates that an influenza virus infection is present. Therefore, according to the present invention, an influenza virus infection is present if:

(i) the influenza virus can be detected; and/or

(ii) at least one symptom of influenza virus is present.

In one aspect of the present invention, an influenza virus infection is present if both characteristics apply, i.e., an influenza virus can be detected and at least one symptom of an influenza virus infection is present. Thus, the transmission rate (i.e., infectivity) of a treated null patient or a control patient can be measured by analyzing whether an influenza virus can be detected and, optionally, by further analyzing whether symptoms of an influenza virus are present in a person(s) who has had personal contact with the treated null patient or the control patient, respectively. For example, it can be analyzed whether at least one symptom of an influenza virus infection is present. The at least one symptom of an influenza virus infection can be a sudden onset of fever, chills, headache, muscle and/or joint pain, cough, weakness, sore throat and/or nasal congestion. It is also possible to analyze whether the body temperature reaches 38°C-40°C within 24 hours from the onset of influenza symptoms (Wright, Fields Virology. 5th ed. (2). Wolters Kluwer Health/Lippincott Williams &Wilkins; 2007. P. 1691-1740; Monto, Arch Intern Med. 2000; 160:3243-3247). Based on these symptoms, influenza virus infection tends to be a severe disease and can be distinguished from the common cold.

In this document, the onset of influenza symptoms may be defined as one of two things:

(i) the time of the first increase in body temperature (an increase of at least 1°C from normal body temperature); or

(ii) the point in time when the patient experiences at least one general or respiratory symptom.

Preferably, the time of onset of influenza symptoms is confirmed by checking that within 24 hours of time points (i) and (ii) above, the body temperature reaches between 38°C and 40°C.

Additionally or alternatively, a diagnosis of influenza may be confirmed by all of the following:

(i) Fever (38°C or greater) (axillary) on examination prior to dosing or more than 4 hours after dosing of antipyretic agents, if taken.

(ii) At least one of the following general systemic symptoms associated with influenza, with moderate or greater severity:

(ii)-1 headache;

(ii)-2 fever or chills;

(ii)-3 Muscle or joint pain;

(ii)-4 Weakness.

(iii) At least one of the following influenza-associated respiratory symptoms of moderate or greater severity:

(iii)-1 Cough;

(iii)-2 Sore throat;

(iii)-3 Nasal congestion;

(iii)-4 Influenza A or B infection confirmed by ROS PCR analysis.

Transmission of influenza virus from a patient zero or a control patient to a contact can be tested in a number of ways. For example, transmission can be assumed to have occurred if influenza virus is detected in a contact (e.g. by PCR) and the patient zero or control patient was the first (or even the only) influenza-infected person with whom the contact had personal contact immediately before the identification of influenza virus in the contact, e.g. within the last four weeks (preferably within the last two weeks or, more preferably, within the last 7 days) immediately before the identification of influenza virus in the contact. In one aspect of the present invention, transmission from a treated patient zero has occurred if an influenza virus strain can be detected in at least one contact of the treated patient zero, and when said influenza virus strain is identical to the influenza virus strain of the treated patient zero, and transmission from a control patient has occurred if an influenza virus strain can be detected in at least one contact of the control patient, and when said influenza virus strain is identical to the influenza virus strain of the control patient. Thus, in one aspect of the present invention, transmission from a treated patient zero has occurred if at least one contact of the treated patient zero has an influenza virus infection with an influenza virus strain that is identical to the influenza virus strain of the treated patient zero, and transmission from a control patient has occurred when at least one contact of the control patient has an influenza virus infection with an influenza virus strain that is identical to the influenza virus strain of the control patient.

For example, if a contact (e.g., a household contact) develops influenza A or B confirmed by PCR, a nasopharyngeal swab of the contact can be used to sequence the viral genome to analyze whether the influenza virus originated (i.e., was transmitted) from patient zero or a control patient, respectively. Methods for sequencing the viral genome are well known in the art and are also described below. A person skilled in the art can easily determine whether the influenza virus originated from patient zero or a control patient, respectively. For example, a person skilled in the art will know that transmission has occurred if the viral genome of the influenza virus of the contact is identical to the viral genome of the influenza virus of patient zero or a control patient, respectively. However, the influenza virus can also mutate during or after transmission from patient zero or a control patient to a contact patient. Therefore, transmission also occurred if the viral genome of the influenza virus of the contact person is identical to the viral genome of the influenza virus of the null patient or the control patient, respectively, except for one or more nucleotide differences (i.e. point mutations).

In order to determine whether the transmission of the influenza virus from the zero patient to the contact person or from the control patient to the contact person has occurred, it is possible to obtain a phylogenetic tree and measure the genetic distance. More specifically, in order to determine whether the transmission has occurred, it is possible to use the methods as described in Example 4.

For example, phylogenetic analysis (WHT with bootstrap tree topology and clustering support) and/or genetic distance measurements between paired populations (e.g. L1-Norm) can be performed to identify transmission pairs. The outcome of the analysis is influenced by two factors: first, the amount of sequence data available. To maximize this, whole genome next generation sequencing (WGNGS) can be performed to increase the resolution and discriminatory power (compared to Sanger sequencing and/or single gene sequencing). Second, the diversity within the sampled community/metapopulation can be determined with high confidence, such as assessing that viruses within housemates are more similar to each other compared to the outgroup. More sequences from a community increase the diversity within a given group and thus facilitate the identification of actual transmission pairs.

For example, 15 or more IPs, preferably at least 30-40 IPs, can be used to develop a community distribution of pairwise distances that reflects community diversity, such that true transmission events (e.g., household transmission events) can be inferred from the sequence data, such as described in McCrone, John T., et al. "Stochastic processes constrain the within and between host evolution of influenza virus." Elife 7 (2018): e35962. Methods as described in McCrone (Elife 7 (2018): e35962) can be used, such as the method described there for sequencing. In the context of the present invention, the analysis is preferably focused on the analysis of influenza A virus samples.

Both epidemiological linkage and genetic relatedness of viruses (e.g., within households) can be used to define transmission pairs and to exclude confounding due to background diversity in the community. A pair of individuals (e.g., within a household) can be considered an epidemiologically linked transmission pair if they have had personal contact and both test positive for the same influenza virus subtype within 7 days of each other. For example, all individuals who have had personal contact with each other (e.g., within a household) with symptom onset within a 7-day window can be considered epidemiologically linked. The donor in each putative pair can be defined as the individual with the earlier symptom onset. The transmission event can be ignored if there were multiple possible donors with the same symptom onset day. The donor and recipients may not be allowed to have symptom onset on the same day unless both individuals were case zero (e.g., for the household being analyzed).

The sequence data can then be used to determine which of the epidemiologically linked pairs represent true transmission events, as opposed to coincident community-acquired infections. The genetic distance between influenza populations from each putative transmission pair can be measured using the L1-norm, and these distances can be compared with the distances of randomly assigned community pairs within each season. Individuals can be considered a true transmission pair only if they have a genetic distance less than the 5th percentile of the community distribution of randomly assigned pairs.

As mentioned above, the influenza virus is mainly transmitted by airborne transmission, in particular by droplet infection. Therefore, in one aspect of the present invention, the transmission of the influenza virus is direct contact transmission and/or airborne transmission, preferably airborne transmission. More preferably, the transmission is droplet infection.

As provided herein, the compound to be used in the present invention has the advantageous characteristic that it significantly reduces the transmission of influenza virus. Accordingly, said compound is particularly useful for treating specific patients infected with influenza whose infectivity is to be reduced. For example, it is particularly important to reduce the infectivity of patients who may cause significant (e.g., social or economic) harm due to the transmission of their influenza virus. In one aspect of the present invention, the treated patient zero has personal contact with at least one person from a risk group for influenza or with at least one person who is important for the functioning of society, after administration of the compound to the patient zero and during his/her infection with the influenza virus. The at least one person from a risk group for influenza may be at least one person who has an increased risk of getting an infection with the influenza virus or who has an increased risk of getting a complication associated with influenza. Said influenza-related complication may be at least one complication selected from the group consisting of hospitalization, sinusitis, otitis media, bronchitis and pneumonia. Said at least one person having an increased risk of developing an influenza-related complication may have an increased risk of dying due to an influenza virus infection. In one aspect of the present invention, said at least one person having an increased risk of developing an influenza-related complication is an individual:

(i) having a chronic cardiovascular disease, a chronic pulmonary disease, a chronic metabolic disease, a chronic renal disease, a cardiopulmonary disorder, and/or who is immunocompromised; and/or

(ii) who is at least 65 years of age or less than 5 years of age.

Additionally or alternatively, at least one person at increased risk of developing an influenza-related complication may be a seriously ill (e.g., hospitalized) patient.

As mentioned above, in one aspect of the present invention, the treated patient zero has personal contact with at least one person from the risk group for influenza after the administration of the compound to the patient zero and during his/her infection with the influenza virus, wherein said person from the risk group for influenza may be at least one person having an increased risk of getting an infection with the influenza virus. In the context of the present invention, said at least one person having an increased risk of getting an infection with the influenza virus may be an individual who is contraindicated for vaccination against influenza. Said at least one person having an increased risk of getting an infection with the influenza virus may also be a person at high risk of getting an infection with the influenza virus, a young child (for example, a child who is under 5 years old), an elderly person (for example, a person 65 years old or older) or an immunocompromised person.

As mentioned above, the contact person of the zero patient may be at least one person who is important for the functioning of society. Such a person who is important for the functioning of society may be a person who is important for the functioning of a city or a state. According to the present invention, said at least one person who is important for the functioning of society may work as an important service provider, including a person who belongs to the firefighters, police personnel, health care personnel, emergency rescue personnel, the army or the government.

Staying at home during an influenza virus infection can be harmful if the infected patient is an essential service provider. This is especially applicable during an emergency. Therefore, the patient zero itself can be a person who is important for the functioning of society. Treating such a patient zero with a compound to be used in the present invention will reduce (or even prevent) the virus from being transmitted from the patient zero to other people, so that the patient zero does not necessarily have to stay at home during his/her infection with the influenza virus. For example, this patient zero can work as an essential service provider, for example, can be a person belonging to the firefighting personnel, the police personnel, the health care personnel or the emergency rescue personnel.

Patient zero may be a person in an influenza epidemic or influenza pandemic situation. Patient zero may also be a person in a bioterrorism threat or bioterrorism attack situation, where the influenza virus plays a role in the bioterrorism threat or bioterrorism attack, respectively. Indeed, the impact of an influenza pandemic is likely to be significantly greater, by orders of magnitude, than typical bioterrorism scenarios. Therefore, preventing or reducing influenza transmission would be an important benefit during an epidemic, pandemic, or bioterrorism situation. As mentioned above, experts predict that the next influenza pandemic will be associated with a large death toll and a high rate of illness requiring hospitalization, thus placing a significant strain on healthcare resources. Thus, patient zero may be a member of the healthcare workforce, and treatment of said patient zero with the compound may be administered during an influenza epidemic or an influenza pandemic. As also mentioned above, in developing countries, where health system resources are already strained and the general population is often weakened by poor health and nutrition, the impact of an influenza pandemic is likely to be greatest. Therefore, patient zero may belong to the population of a developing country.

Naturally, the reduction of the transmission rate is particularly useful (or even necessary) if the patient zero infected with influenza has or will have personal contact with a large number of people. Preferably, this patient zero has personal contact with a large number of people after administration of the compound to be used in the present invention. In one aspect of the present invention, the treated patient zero has personal contact with many people, for example, more than 10 people, after administration of the compound (to the patient zero) and during his/her infection with the influenza virus. For example, patient zero may have personal contact with many people in his/her household or at his/her work. In one aspect of the present invention, a treated patient zero visits a healthcare facility (such as a nursing home, a nursery, or a hospital), an educational facility (such as a school or university), a public facility, a vehicle, a car, an airplane, and/or a store, and has personal contact with many people, such as more than 10 people, after administering the compound to patient zero and/or during his/her infection with the influenza virus.

Preferably, the rate of transmission of the influenza virus from the treated patient zero to household contacts is reduced. Accordingly, it is preferred that the treated patient zero has household contacts. For example, the treated patient zero may live in the same household with children (preferably with children who are not older than 5 years). In addition or alternatively, the treated patient zero may have a job with many contacts with people. It is preferred that at least one person of the household contact(s) and/or work contact(s) of the treated patient zero is not vaccinated against influenza.

As mentioned above, the treated patient zero may have personal contact with many people, for example, more than 10 people (preferably more than 30, more preferably more than 50, even more preferably more than 100 people) after administration of the compound and during his/her infection with the influenza virus. The attached Examples show that the transmission rate is reduced in the treated subject immediately after administration of the compound to be used in the present invention. According to the present invention, the compound can be administered at least one hour before said personal contact. In one aspect of the present invention, the compound is administered at least 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30 or 32 hours before said personal contact. In this regard, longer time intervals are preferred over shorter ones. In the context of the present invention, the present compound can be administered within 48 hours from the onset of influenza symptoms, preferably within 24 hours from the onset of influenza symptoms.

The present invention is not limited to any particular dosage of the compound of the present invention. An effective amount of the compound can be selected and/or adjusted by the attending physician according to his/her experience and applicable guidance. However, the compound is preferably administered once as a single treatment. The compound can be administered to the patient in a suitable dose. The compound can be administered in a dosage range varying depending on the patient's body weight, age, sex, health condition, diet, time of administration, route of administration, excretion rate and severity of the disease. The dosage schedule will be determined by the attending physician and clinical factors. As is well known in the medical field, dosages for any patient depend on many factors including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health condition and other concomitantly administered drugs. However, an effective amount of the compound to be used in the present invention is preferably about 40 mg for patients lighter than 80 kg and about 80 mg for patients weighing 80 kg or more. The dosing of baloxavir marboxil in pediatric patients may be as follows. In a patient who is less than 1 year old: (a) if the patient is less than 4 weeks old, then the effective amount may be approximately 1 mg/kg body weight; (b) if the patient is 4 weeks or older but less than 3 months old, then the effective amount may be approximately 1 mg/kg body weight; (c) if the patient is 3 months or older but less than 12 months old, then the effective amount may be approximately 2 mg/kg body weight. In a patient who is 1 year or older but less than 12 years old, dosing may be based on body weight. Two phase III clinical trials have been conducted in Japan in pediatric patients aged 6 months to 12 years (studies 1618T0822 and 1705T0833). In the first study, 1618T0822, one tablet of baloxavir marboxil was administered to pediatric subjects with influenza aged 6 months to less than 12 years. The baloxavir marboxil dosing was as follows: 40 kg or more: 40 mg dose, 20 kg - 40 mg: 20 mg dose, 10 kg - 20 mg: 10 mg dose, 5 kg - less than 10 kg: 5 mg. This dosing may also be used for pediatric nulliparous patients.

The present invention is also not limited to any particular route of administration of the compound. All possible routes of administration that the attending physician considers useful or necessary are within the scope of the present invention. For example, the compound can be administered orally, rectally, nasally, topically, intradermally, as an aerosol, vaginally or parenterally, such as intramuscularly, intravenously, subcutaneously, intraarterially or intracardiacly. It is preferred that the compound is administered orally. Dosage forms for oral administration include coated and uncoated tablets, soft gelatin capsules, hard gelatin capsules, troches, lozenges, solutions, emulsions, suspensions, syrups, elixirs, powders and granules for dissolution, dispersible powders and granules, medicinal chewing gums, chewable tablets and effervescent tablets. Dosage forms for parenteral administration include solutions, emulsions, suspensions, dispersions, powders and granules for dissolution. Dosage forms for rectal and vaginal administration include suppositories and ovules. Dosage forms for nasal administration can be administered by inhalation and insufflation, for example, with a metered dose inhaler. Dosage forms for topical administration include creams, gels, ointments, balms, patches and transdermal delivery systems. However, it is preferable that the compound to be used in the present invention is administered in the form of a tablet, more preferably a tablet for oral administration. For example, the compound to be used in the present invention can be administered in the form of a 20 mg tablet and/or a 40 mg tablet. The compound to be used in the present invention can also be administered in the form of granules. Granules are particularly useful when the compound is administered to people who are unable to swallow tablets, such as children or people with a nasogastric tube. Thus, a person weighing less than 80 kg may receive two 20 mg tablets or one 40 mg tablet as a single oral dose. A patient weighing 80 kg or more may receive four 20 mg tablets or two 40 mg tablets as a single oral dose.

The patient zero may be a human subject having an influenza virus infection. It is preferred that the patient zero is at least 2 years old. More preferably, the patient zero is at least 12 years old. For example, the patient zero may be a human patient who is preferably 12 years old or older and 64 years old or younger. The treated patient zero is preferably healthy except for the influenza virus infection. As mentioned above, it is preferred that the treated patient zero has at least one contact person (e.g., a household contact) who is not vaccinated against influenza. Even more preferably, the treated patient zero has at least two contacts (e.g., household contacts) who are not vaccinated against influenza. Said contacts may be 2 years old or older.

A control patient is a patient infected with influenza that is as similar to the zero patient as possible, except that the control patient is not treated with a compound to be used in the present invention. Thus, the control patient has an identical or at least similar (i.e. comparable) age and health condition as the zero patient, and this control patient and this zero patient are infected with the same strain of influenza virus.

Influenza viruses cause seasonal epidemics and, in very rare cases, global pandemics. The term "world pandemic" (from the Greek "pan" meaning "all" and "demos" meaning "people") describes an epidemic that affects the entire population. The compound to be used in the present invention has the advantage that it reduces the transmission of the influenza virus and, therefore, can reduce the risk that the treated patient zero will cause (or contribute to) the development of an influenza epidemic or an influenza pandemic. Thus, in one aspect of the present invention, the treatment (of the patient zero) has the effect that the treated patient zero has a reduced risk of starting an influenza epidemic or an influenza pandemic compared to a control patient. Thus, according to the present invention, the treated patient zero has a reduced risk of starting an influenza epidemic or an influenza pandemic compared to a control patient.

In this document, an "influenza pandemic" is a global epidemic caused by an influenza virus for which there is little or no pre-existing immunity in the human population. A pandemic is defined as an epidemic occurring worldwide or over a very large area, such as an entire country. A pandemic often crosses international boundaries and typically affects large numbers of people. A true influenza pandemic occurs when nearly simultaneous transmission occurs, such as worldwide. Simultaneous worldwide transmission of influenza is sufficient to define an influenza pandemic and is consistent with the classic definition of "an epidemic occurring worldwide." For example, a pandemic may be caused by a newly evolved influenza virus that results from changes in an existing influenza virus. Such changes are discussed in more detail below. A pandemic may be relatively mild or may cause severe illness or death. Severe illness may occur in certain risk groups, which may correspond to groups at risk of severe illness from seasonal influenza. However, healthy people are also likely to experience more severe illness than that caused by seasonal influenza.

An “influenza epidemic” is a sudden increase in the number of cases of influenza virus infection above what is normally expected. An “influenza epidemic” is the widespread occurrence of influenza in a community at a specific time. An epidemic is an event in which a disease is actively spreading. In contrast, the term “pandemic” refers to geographic spread and is used to describe a disease that affects an entire country or the entire world.

There are three types of influenza viruses: A, B and C. Types A and B cause widespread outbreaks of influenza disease almost every year. Influenza C is associated with sporadic, often asymptomatic infection with little or no mortality and, therefore, is not a threat to public health. Pandemic influenza occurs only with influenza A viruses. However, influenza B viruses can cause an influenza epidemic. Indeed, influenza B is part of the seasonal influenza epidemic. Thus, according to the present invention, the influenza virus can be an influenza A virus or an influenza B virus, preferably an influenza A virus. It is preferable that the influenza virus is an epidemic strain of an influenza virus or a pandemic strain of an influenza virus.

Influenza pandemics and epidemics in humans result from changes in surface glycoproteins, which is known as "antigenic shift" and "antigenic drift".

In particular, one way in which influenza viruses change is called “antigenic drift.” “Antigenic drift” is the small changes in the genes of influenza viruses that continually occur over time as the virus replicates. These small genetic changes typically produce viruses that are fairly closely related to each other, as can be illustrated by their proximity to each other on a phylogenetic tree. Viruses that are closely related to each other typically have the same antigenic properties, and the immune system exposed to a similar virus will usually recognize it and respond (this is sometimes called cross-protection). But these small genetic changes can accumulate over time and result in viruses that are antigenically different (i.e., further apart on the phylogenetic tree). When this happens, the body’s immune system may not recognize the viruses, which can lead to a flu epidemic.

Another type of change in the influenza virus is called an "antigenic shift." Pandemic influenza is the result of an antigenic shift, and it occurs only with influenza A viruses. An antigenic shift is an abrupt, large change in an influenza A virus that results in a new hemagglutinin (HA) and/or new HA and neuraminidase (NA) proteins in the influenza virus. Thus, an antigenic shift results in a new influenza A subtype or a virus with a hemagglutinin (HA) or a combination of hemagglutinin (HA) and neuraminidase (NA) that has emerged from an animal population that is too different from the same subtype in humans that most people have no immunity to the new virus. Such a "shift" occurred in the spring of 2009 when an H1N1 virus with a new combination of genes emerged, infected people, and spread rapidly, causing a pandemic. When a shift occurs, most people have little or no protection against the new virus. Thus, antigenic shift involves an abrupt change in HA and possibly NA antigens that are completely different from those that have circulated in humans for many years previously. Antigenic shift results in an entirely new virus that is serologically distinct from earlier viruses and could not have arisen from them by mutation. A pandemic is likely when large sections of the population worldwide have no immunity to the new virus (i.e., have no or little antibody to the HA of the new virus) and it is easily transmitted from person to person, causing serious disease. A pandemic is considered inevitable when a new virus spreads rapidly beyond the community in which it was first identified. While influenza viruses change all the time through antigenic drift, antigenic shift occurs only occasionally. Type A viruses undergo both types of change; type B viruses change only through the more gradual process of antigenic drift.

As mentioned above, an influenza pandemic is usually the result of an antigenic "shift" and an influenza epidemic is usually the result of an antigenic "drift". Therefore, in the context of the present invention, an influenza virus is antigenically different from a parent strain of an influenza virus as a result of antigenic drift and/or antigenic shift, preferably antigenic shift, or both.

One skilled in the art can readily determine whether a given influenza virus is the result of antigenic drift or antigenic shift. As described above, antigenic shift is an abrupt, large change in influenza A viruses that results in new combinations of hemagglutinin and neuraminidase proteins in influenza A viruses that infect humans. Thus, in order to determine whether an influenza A virus that infects humans is the result of antigenic shift, the hemagglutinin (HA) and neuraminidase (NA) protein combinations of a given influenza A virus can be compared to the hemagglutinin (HA) and neuraminidase (NA) protein combinations of known influenza A viruses that infect humans. If the hemagglutinin (HA) and neuraminidase (NA) protein combination of a given influenza A virus is not present in known influenza A viruses that infect humans, then the influenza A virus in question is the result of antigenic shift.

Antigenic drift is a point mutation within the viral genome. One skilled in the art can easily determine whether a given influenza virus is the result of antigenic drift by sequencing the genome of the given influenza virus and comparing the resulting sequence to the viral genome sequences of known influenza viruses. Accordingly, in order to determine whether an influenza virus that infects humans is the result of antigenic drift, the viral genome sequence of the given influenza virus can be compared to the viral genome sequence of known influenza viruses that infect humans. If the viral genome sequence of the influenza virus in question contains nucleotide substitutions (i.e., point mutations) compared to known influenza viruses that infect humans, then the influenza virus in question is the result of antigenic drift.

Antigenic drift is a normal evolutionary process whereby virus genomes mutate (slowly compared to antigenic shift) due to error-prone polymerases. These mutations can alter the functional characteristics of the virus, such as the resistance of enzymes to specific drugs. Therefore, the functional characteristics of the virus can be determined to determine whether a virus is the result of antigenic drift.

In addition, antigenically shifting viruses with pandemic potential may also arise through viral recycling. More specifically, by testing blood samples from people of varying ages, it can be shown whether a particular influenza subtype had previously circulated and, if so, when it approximately ceased circulating. This analysis, called seroarchaeology, supports the theory of viral recycling. Seroarchaeology has established beyond a reasonable doubt that the H2 and H3 subtypes recycled in humans throughout the 19th and 20th centuries. Furthermore, the H1 subtype, which circulated in humans from 1918 to 1957, re-emerged, or "recycled," in 1978. Analysis of RNA from viruses isolated during the 1977-78 epidemic showed that the virus was more closely related to viruses isolated in 1950 than to strains isolated since that time. Because of antigenic drift, it was assumed that the H1N1 virus must have re-emerged from a frozen state in nature or elsewhere. However, there is evidence that influenza viruses can remain unchanged for long periods in pigs, which can serve as a reservoir for human infection. Thus, in the context of the present invention, the influenza strain may be a recycled virus that has caused an epidemic or pandemic in the past.

For example, virus strains that can recycle are A/Hong Kong/97 (H5N1), A/New Jersey/8/76 (H1N1), A/USSR/90/77 (H1N1), A/Hong Kong/68 (H3N2), A/Hong Kong/68 (H3N2), A/Hong Kong/68 (H3N2), A/Hong Kong/68 (H3N2), A/USSR/77 (H1 N1), A/USSR/77 (H1 N1), or A/USSR/77 (H1 N1).

As mentioned above, according to the present invention, the influenza virus may be an influenza A virus or an influenza B virus (e.g. B/Minnesota/23/2015). The attached Examples surprisingly show that the compound to be used in the present invention can reduce the transmission of influenza virus strains A/Perth/265/2009 (H1 N1pdm09) and A/England/195/2009. Thus, according to the present invention, the influenza virus strain is preferably an influenza A virus, more preferably an influenza A virus of the H1N1 subtype (e.g. A/Perth/265/2009 (H1N1pdm09) or A/England/195/2009). However, the influenza A virus may also be of the H2N2, H3N2, H5N1 or H7N9 subtype.

In a preferred aspect of the present invention, the influenza virus strain does not carry the I38X mutation, which includes the I38T mutation. Thus, it is preferred that the influenza virus strain does not carry the I38T mutation. The I38T substitution is a mutation within the viral acidic polymerase (PA) protein of certain mutated influenza A strains. The sequence of the PA protein of an influenza A virus having the I38T mutation is shown in SEQ ID NO: 4. Thus, in a preferred aspect of the present invention, the influenza virus strain does not comprise a PA protein having the sequence of SEQ ID NO: 4. It is also preferred that the influenza virus strain does not comprise a PA protein having a sequence that has at least 80%, preferably at least 90%, more preferably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the sequence of SEQ ID NO: 4 and comprising a T at a position corresponding to position 38 of SEG ID NO: 4. A fraction of the PA protein of an influenza A virus containing the 13 mutation VT, shown in SEG ID NO: 5. Thus, in a preferred aspect of the present invention, the influenza virus strain does not comprise a PA protein comprising a sequence as shown in SEQ ID NO: 5.

As mentioned above, the compound to be used in the present invention can be used to prevent an influenza epidemic or an influenza pandemic. Thus, the present invention relates to a method for preventing an influenza epidemic or an influenza pandemic, wherein the method comprises administering to patients having an influenza virus infection (null patients) an effective amount of the compound, wherein the compound is administered to at least 13% of all influenza-infected people in the population of a city or country,

and where the given compound has one of the following formulas I and II:

or a pharmaceutically acceptable salt thereof.

Thus, the present invention relates to a compound for use in the treatment of patients having an influenza virus infection (null patients), wherein the said compound is to be administered to at least 10% of all influenza-infected people in the population of a city or country, wherein the said compound prevents an influenza epidemic or influenza pandemic, and wherein the said compound has one of the following formulas I and II:

or a pharmaceutically acceptable salt thereof.

In this document, "prevent" an influenza epidemic or influenza pandemic also means to reduce the outbreak of an influenza epidemic or influenza pandemic, such as to reduce the impact of an influenza epidemic or influenza pandemic.

The attached Examples provide a simulation that demonstrates that treating 15% or 30% of the population with baloxavir marboxil would prevent an influenza epidemic or pandemic, respectively. The attached Examples also demonstrate that the compound to be used in the present invention has a beneficial effect in that it reduces transmission and therefore reduces the likelihood that an epidemic or pandemic will occur. Thus, the means and methods provided herein advantageously reduce the economic impact associated with influenza infections.

As described above, the conditions surrounding the Hong Kong outbreak of the 1997 pandemic "bird flu" emphasize the need for advance planning to ensure an adequate response to a public health emergency that is certainly unpredictable, complex, rapidly evolving and accompanied by significant public anxiety. Once a pandemic has begun, it has been too late in the past to implement many of the key activities required to minimize the impact. Therefore, planning and implementation of preparatory activities must begin well in advance. Accordingly, in order to prevent an influenza pandemic, it is necessary to treat infected patients with a compound to be used in the present invention at an early stage. Thus, according to the present invention, it is contemplated that even an influenza epidemic is prevented by treating a few (e.g., at least 10%) of influenza-infected people during the seasonal influenza. In one aspect of the present invention, the above-described method (i.e., the method for preventing an influenza epidemic or an influenza pandemic) serves to prevent an influenza epidemic, and at least 10%, preferably at least 15%, more preferably at least 20% of all people infected with influenza, the population of a city or country (preferably the population of a city) are treated with the compound. Accordingly, in one aspect of the present invention, the above-described compound (i.e., the compound that prevents an influenza epidemic or an influenza pandemic) prevents an influenza epidemic, and at least 10%, preferably at least 15%, more preferably at least 20% of all people infected with influenza, the population of a city or country (preferably the population of a city) are to be treated with the compound. For example, a compound to be used in the present invention can be used to prevent an influenza epidemic (e.g., during seasonal influenza) if at least 20% of the population has been effectively vaccinated and if the number of susceptible individuals is no more than 30% of the population.

Naturally, the effect of the compound to be used in the present invention for preventing an epidemic (for example, during seasonal flu) or a pandemic increases with the percentage of people infected with flu who are treated with the compound. Therefore, in order to prevent an epidemic of flu (for example, during seasonal flu), at least 30%, at least 40% or at least 50% of the infected people of the city or country (preferably the city population) can be treated with the compound.

In one aspect of the invention, the above-described method (i.e., the method for preventing an influenza epidemic or an influenza pandemic) serves to prevent an influenza pandemic, and at least 25%, preferably at least 30% and more preferably at least 35% of all people infected with influenza, of the population of the country are treated with the compound. Accordingly, in one aspect of the invention, the above-described compound (i.e., the compound that prevents an influenza epidemic or an influenza pandemic) serves to prevent an influenza pandemic, and at least 25%, preferably at least 30% and more preferably at least 35% of all people infected with influenza, of the population of the country are to be treated with the compound. For example, the compound to be used in the present invention can be used to prevent an influenza pandemic if there is no effective vaccination available and if the number of susceptible people is no more than 30% of the population. As mentioned above, the effect of the compound to be used in the present invention for preventing a pandemic increases with the percentage of people infected with influenza who are treated with the compound. Therefore, in order to prevent an influenza pandemic, at least 45%, at least 55% or at least 65% of the infected people of a city or country can be treated with the compound.

The means and methods provided herein are particularly useful if the influenza virus strain does not have resistance to the compound to be used in the present invention. However, the influenza virus strain may have resistance to other antiviral drugs (such as peremavir, laninamivir, oseltamivir, zanamivir, rimantadine, umifenovir, or amantadine). Assays for determining whether a virus has resistance to one or more drugs are well known in the art and include, for example, a phenotypic resistance assay and the NA-Star assay, both of which are described below.

Phenotypic resistance analysis can be performed as described below: Phenotypic resistance analysis (spot/spot reduction assay) can be performed using the sensitive Virospot detection technology, which combines classical virus culture in multiwell microtiter plates and virus-specific immunostaining with automated visualization, detection of infected cells using a UV (ultraviolet) CTL Immunospot analyzer equipped with the Biospot assay software. The Virospot technology platform determines the susceptibility of virus isolates to antiviral drugs by measuring the _IC50 (half-maximal inhibitory concentration)/ _IC90 (concentration inhibiting by 90%). Briefly, this method is based on the inoculation of infectious virus onto MDCK cell monolayers in 96-well plates in the presence of a range of drug concentrations. After incubation, cells are fixed and immunostained with virus-specific antibodies, followed by the addition of TrueBlue substrate and image capture using a UV analyzer.

The NA-Star assay is particularly useful for detecting phenotypic resistance to neuraminidase inhibitors (such as oseltamivir) and can be performed as follows: The assay uses a chemiluminescent substrate to highly sensitively detect neuraminidase enzyme activity. Neuraminidase activity produces a luminescent compound that is quantified using a plate reader. Viral neuraminidase activity is determined in the presence of serial dilutions of the neuraminidase inhibitor. Sensitivity to the neuraminidase inhibitor is expressed as IC ₅₀ /IC ₉₀ values.

The compound to be used according to the present invention can be combined with other anti-influenza drugs. In the EU (European Union), four antiviral drugs are currently approved for the prevention and treatment of influenza: the M2 ion channel inhibitor amantadine and the NAIs (neuraminidase inhibitors) oseltamivir phosphate, zanamivir and peramivir. The second M2 inhibitor, rimantadine, has marketing authorizations in the Czech Republic, France and Poland, but is not marketed in these countries. Therefore, the compound to be used in the present invention can be administered as a combination therapy with amantadine, oseltamivir phosphate, zanamivir, peramivir and/or rimantadine. Neuraminidase inhibitors (NAIs) are the mainstay of treatment of influenza infections. Therefore, if the compound to be used in the present invention is administered as a combination therapy, then it is preferably combined with oseltamivir phosphate or zanamivir. Both oseltamivir phosphate and zanamivir are administered twice daily for 5 days.

As described above, the present invention provides a means and method for reducing the transmission of an influenza virus, i.e. for reducing the infectivity of a patient infected with influenza (i.e., patient zero). Accordingly, the present invention also relates to the following aspects. All explanations, definitions and preferred aspects explained above also apply, mutatis mutandis, to the aspects of the invention described below.

The present invention also relates to a method of treating an influenza virus infection, wherein said method comprises administering an effective amount of a compound to a patient having an influenza virus infection (patient zero), wherein said compound has one of formulas (I) and (II), or a pharmaceutically acceptable salt thereof, and wherein said compound reduces transmission. Also covered by the present invention is a method of treating influenza, comprising: reading the dosing instructions on the package insert or on the package for a pharmaceutical preparation containing a compound having one of formulas (I) and (II) or being a pharmaceutically acceptable salt thereof; and administering an effective amount of the compound to a patient infected with influenza (patient zero), whose infectivity is to be reduced. The present invention also relates to the use of a compound having formulas (I) and (II), or a pharmaceutically acceptable salt thereof, for the preparation of a medicament for the treatment of a patient infected with influenza (patient zero), whose infectivity is to be reduced. The present invention also provides a package containing a pharmaceutical preparation containing a compound that has one of formulas (I) and (II) or a pharmaceutical salt thereof, and additionally containing dosage instructions for administering an effective amount of the compound to an influenza-infected patient (patient zero), whose infectivity is to be reduced.

As explained above, the patient zero is preferably healthy except for an influenza virus infection. It is preferable that the patient zero is not treated with any drug other than the compound to be used in the present invention. For example, it is preferable that the patient zero is not treated with an experimental therapy, a systemic antiviral drug (e.g., peramivir, laninamivir, oseltamivir, zanamivir, rimantadine, umifenovir or amantadine), immunosuppressants, corticosteroids, antifungal drugs, or a drug that is administered to the eyes, nose or ears, or by inhalation.

The term "influenza virus infection" or a variation thereof is a commonly used term in the art and refers to disease caused by influenza virus. More specifically, influenza virus infection is an acute respiratory infectious disease caused by a virus of the orthomyxoviridae family. Principally, two forms are known to infect humans and cause disease in humans: influenza A virus and influenza B virus. Influenza viruses have a segmented, negative-sense, single-stranded, lipid-encapsulated ribonucleic acid (RNA) genome; they range in size from 80 to 100 nm. Subtypes are defined according to the glycoproteins hemagglutinin (HA) and neuraminidase (NA) present in the viral lipid envelope. Influenza viruses enter the respiratory epithelial cell via the attachment of viral HA to sialic acid-containing receptors on the cell membrane, followed by internalization of the virus into an acidic endosome. In the acidic environment of the endosome, HA undergoes a conformational change that releases the fusion peptide and results in the fusion of the viral envelope with the endosomal membrane. At the same time, matrix protein 2 (M2) acts as an ion channel, allowing hydrogen ions to enter the virion from the endosome. This allows viral gene segments to leave the virion and enter the cytoplasm, a process known as "uncoating". The viral gene segments are transported to the nucleus where the viral polymerase complex, composed of the proteins polymerase basic protein 1 (PB1), polymerase basic protein 2 (PB2), and polymerase acidic protein (PA), directs the synthesis of plus-sense messenger RNA (mRNA) and, via plus-sense full-length complementary RNA, the synthesis of negative-sense full-length copies that will serve as progeny genomic RNA. The polymerase proteins also play a role in disrupting host cell protein synthesis. Assembly of progeny virions occurs at the plasma membrane, and the viral NA protein plays a role in the release of virus from the cell surface by cleaving surface sialic acid.

The term “compound” as used in the present invention is a compound that has one of the following formulas I and II:

or a pharmaceutically acceptable salt thereof (i.e., a compound having formula (I) or (II)). The compound to be used in the present invention is also referred to herein as a "compound", "a compound for use", "a compound to be used (herein/in the present invention)" or "a compound of the present invention".

The compound to be used in the present invention acts as a selective cap-dependent endonuclease inhibitor (CEN) that inhibits the "NEP grip" function of the PA subunit of influenza polymerase, which is used to cleave 5' cap structures from host cell mRNA that are used as primers for transcription of viral mRNA. By inhibiting this essential function, the compound as used herein suppresses the replication of influenza viruses.

The compound to be used in the present invention has a broad spectrum of activity against seasonal: (e.g. A/H1N1, A/H3N2 and B) and highly pathogenic avian influenza viruses (e.g. A/H5N1, A/H7N9) with more potent antiviral activity (lower half-maximal inhibitory concentration [IC ₅₀ ]) compared to other conventional antiviral drugs such as oseltamivir, zanamivir or peramivir. The ability of the compound to be effective as a single dose administration simplifies treatment and improves patient compliance compared to neuraminidase inhibitors (NAIs). Preferably, the compound has formula (I) or (II), most preferably (I). The compound of formula (I) can also be shown as follows:

This compound (i.e. the compound of formula (I)) has the molecular formula C ₂₇ H ₂₃ F ₂ N ₃ O ₇ S. This compound is a prodrug, which is known as baloxavir marboxil. Baloxavir marboxil is known in the art and is described, for example, in Noshi, Antiviral research 160 (2018): 109-117.

Baloxavir marboxil (i.e., the compound of formula (I)) is an anti-influenza virus drug with a novel mechanism of action. It was discovered and is being developed by Shionogi & Co., Ltd. and F. Hoffman-La Roche, Ltd. Baloxavir marboxil (S-033188) is a prodrug, and it is converted into the active form (S-033447) through metabolism (hydrolysis). This active form is shown herein as formula (II). This active form (S-033447) selectively inhibits the activity of cap-dependent endonuclease (CEN), which is essential for the replication of influenza viruses (Omoto, Sci Rep. 2018; 8(1):9633). Broad-spectrum activity against seasonal influenza viruses and alleviation of influenza symptoms have been demonstrated in non-clinical efficacy studies and clinical trials in patients with influenza, including a phase 2 proof-of-concept and dose-discovery study, a phase 3 double-blind study in otherwise healthy patients (Portsmouth S, Kawaguchi K, Arai M, Tsuchiya K, Uehara T. Cap-dependent endonuclease inhibitor S-033188 for the treatment of influenza: results from a phase 3, randomized, double-blind, placebo- and active-controlled study in otherwise healthy adolescents and adults with seasonal influenza. Abstract LB-2. Oral presentation at ID Week 2017, October 4-8 2017, San Diego, CA, USA.) and an open-label Phase 3 study in otherwise healthy pediatric patients.

The compound as shown in formula (II) is the active form of baloxavir marboxil (i.e., a prodrug of formula (I)). The compound of formula (I) can also be shown as follows:

The compound of formula (II) is also known as baloxavir acid. Baloxavir acid is known in the art and is described, for example, in Noshi, Antiviral research 160 (2013): 109-117.

Pharmaceutically acceptable salts of the compounds used in the present invention include, for example, salts with an alkali metal (e.g. lithium, sodium, potassium or the like), an alkaline earth metal (e.g. calcium, barium or the like), magnesium, a transition metal (e.g. zinc, iron or the like), ammonia, organic bases (e.g. trimethylamine, triethylamine, dicyclohexylamine, ethanolamine, diethanolamine, triethanolamine, meglumine, ethylenediamine, pyridine, picoline, quinoline or the like) or amino acids, or salts with inorganic acids (e.g. hydrochloric acid, sulfuric acid, nitric acid, carbonic acid, hydrobromic acid, phosphoric acid, hydroiodic acid or the like) or organic acids (e.g. formic acid, acetic acid, propionic acid, trifluoroacetic acid, citric acid, lactic acid, tartaric acid, oxalic acid, maleic acid, fumaric acid, mandelic acid, glutaric acid, malic acid, benzoic acid, phthalic acid, ascorbic acid, benzene sulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, ethanesulfonic acid or the like). Particularly, salts with sodium, potassium, calcium, magnesium, iron and the like are included. These salts can be formed by conventional methods.

The preparation of the compound of the present invention is well known in the art. For example, the compound of the present invention can be prepared by the methods described in patent application PCT/JP2016/063139, which is published as WO 2016/175224 A1.

As mentioned above, according to the present invention, it is preferable that the influenza virus strain does not comprise a PA protein having a sequence that has at least 80%, preferably at least 90%, more preferably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with the sequence of SEQ ID NO: 4 and comprises T at a position corresponding to position 38 of SEQ ID NO: 4. In particular, FASTA sequences of two PA viral protein sequences can be obtained and aligned in order to evaluate the degree of identity between these two PA viral proteins. To determine the percentage of identity of two sequences, these sequences are aligned for the purpose of optimal comparison (for example, gaps can be introduced into one or both of the first and second amino acid sequences for optimal alignment, and non-homologous sequences can be ignored for the purpose of comparison). The percentage identity between two sequences is a function of the number of identical positions that divide the sequences, taking into account the number of gaps and the length of each gap that must be introduced to optimally align the two sequences. The percentage identity between two polypeptide/amino acid sequences is determined in a variety of ways known to those skilled in the art, for example, using a publicly available computer program such as Smith Waterman Alignment (Smith, T. F. and M. S. Waterman (1981) J Mol Biol 147:195-7); "BestFit" (Smith and Waterman, Advances in Applied Mathematics, 482-489 (1981)), as incorporated into GeneMatcher Plus™, Schwarz and Dayhof (1979), Atlas of Protein Sequence and Structure, Dayhof, M. O., Ed, pp 353-358; BLAST (Basic Local Alignment Search Tool; (Altschul, S. F., W. Gish, et al. (1990) J Mol Biol 215: 403-10), BLAST-2, BLAST-P, BLAST-N, BLAST-X, WU-BLAST-2, ALIGN, ALIGN-2, CLUSTAL, or the Megalign (DNASTAR) program can also be used. In addition, those skilled in the art can determine suitable parameters for measuring alignment, including any algorithms necessary to achieve maximum alignment over the length of the sequences being compared. Preferably, the PA viral protein sequences are compared over their entire length. For the purposes of the present invention, sequence comparison and determination of percent identity between two sequences can be performed using the Blossum 62 scoring matrix (with a gap penalty of 12, a gap extension penalty of 4, and a gap penalty with frameshift of 5).

As mentioned above, one aspect of the present invention relates to a pharmaceutical composition comprising a compound having one of the formulas (I) and (II), or a pharmaceutically acceptable salt thereof, and optionally comprising a pharmaceutically acceptable carrier, wherein the pharmaceutical composition prevents (e.g. reduces) the transmission of an influenza virus. These pharmaceutical compositions can be prepared with a pharmaceutically acceptable carrier by known methods. For example, these compositions can be prepared by appropriately combining the ingredients with a pharmaceutically acceptable carrier or medium, in particular, with sterile water or physiological solution, vegetable oils, emulsifiers, suspending agents, surfactants, stabilizers, flavoring agents, excipients, carriers, preservatives, binding agents, and as, for example, by mixing them in a unit dose and form required by conventional pharmaceutical embodiments. Specific examples of carriers include light anhydrous silicic acid, lactose, crystalline cellulose, mannitol, starch, calcium carmellose, sodium carmellose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinyl acetal diethylaminoacetate, polyvinylpyrrolidone, gelatin, medium chain triglyceride, polyoxyethylene-hardened castor oil, sucrose, carboxymethylcellulose, corn starch, inorganic salt and the like. The content of the active ingredient in such a preparation is adjusted so that a suitable dose can be obtained within the required range.

This pharmaceutical composition may optionally contain one or more pharmaceutically acceptable excipients such as carriers, diluents, fillers, disintegrants, lubricants, binders, dyes, pigments, stabilizers, preservatives, antioxidants or solubility enhancers. Also, these pharmaceutical compositions can contain one or more solubility enhancers, such as, for example, poly(ethylene glycol), including poly(ethylene glycol) having a molecular weight in the range of from about 200 to about 5000 Da, ethylene glycol, propylene glycol, nonionic surfactants, tyloxapol, polysorbate 80, macrogol-15-hydroxystearate, phospholipids, lecithin, dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, cyclodextrins, hydroxyethyl-β-cyclodextrin, hydroxypropyl-β-cyclodextrin, hydroxyethyl-γ-cyclodextrin, hydroxypropyl-γ-cyclodextrin, hydroxypropyl-β-cyclodextrin, glucosyl-α-cyclodextrin, glucosyl-β-cyclodextrin, diglucosyl-β-cyclodextrin, maltosyl-α-cyclodextrin, maltosyl-β-cyclodextrin, maltosyl-γ-cyclodextrin, maltotriosyl-β-cyclodextrin, maltotriosyl-γ-cyclodextrin, dimaltosyl-β-cyclodextrin, methyl-β-cyclodextrin, carboxyalkyl thioethers, hydroxypropyl methylcellulose, hydroxypropyl cellulose, polyvinylpyrrolidone, vinyl acetate copolymers, vinylpyrrolidone, sodium lauryl sulfate, dioctyl sodium sulfosuccinate, or any combination thereof.

These pharmaceutical compositions are not limited to the means and methods described herein. The skilled person can use his/her knowledge available in the art to create a suitable composition. In particular, these pharmaceutical compositions can be prepared by methods known to the skilled person, such as the methods published in Remington's Pharmaceutical Sciences, ^20th Edition.

The contents of any documents cited in this document, whether above or below, are incorporated by reference in their entirety.

The present invention is further described by reference to the following non-limiting Drawings and Examples.

These Graphic Materials demonstrate:

Fig. 1. Time course and study design of Example 1. Time course of the experiment of Example 1. Baloxavir was administered once as a s.c. (subcutaneous) suspension at a dosage of 1 mg/kg to 4 different sites on the back of the ferret. Oseltamivir phosphate (5 mg/kg) was administered orally twice daily for 5 days. See text for details. DF - donor ferret; SF - sentinel ferret; DC - direct contact; RD - respiratory droplet.

Fig. 2. Plaque assay results of Example 1. (A) Control ferrets. All control ferrets were positive for influenza by plaque assay (donor: 4/4; indicator DC: 4/4; indicator RD: 3/4, here all (4/4) were positive by RT-PCR). (B) Oseltamivir-treated ferrets. All oseltamivir-treated ferrets were positive for influenza by plaque assay (donor: 4/4; indicator DC: 4/4; indicator RD: 3/4, here all (4/4) were positive by RT-PCR). (C) Baloxavir-treated ferrets. Only one baloxavir-treated sentinel ferret was infected with respiratory droplet infection by plaque assay (donor: 3/4, here all (4/4) were positive by RT-PCR; sentinel DC: 4/4; sentinel RD: 1/4). DF - donor ferret; SF - sentinel ferret; DC - direct contact; RD - respiratory droplet.

Fig. 3. RT-qPCR results of Example 1. (A) Control ferrets. All control ferrets were positive for influenza by RT-qPCR (donor: 4/4; indicator DC: 4/4; indicator RD: 4/4). (B) Oseltamivir-treated ferrets. All oseltamivir-treated ferrets were positive for influenza by RT-qPCR (donor: 4/4; indicator DC: 4/4; indicator RD: 4/4). (C) Baloxavir-treated ferrets. Only one baloxavir-treated sentinel ferret was infected with respiratory droplet infection by RT-qPCR (donor: 4/4; indicator DC: 4/4; indicator RD: 1/4). (D) Summary of RT-qPCR results. Only one baloxavir-treated sentinel ferret was infected with respiratory droplet infection as detected by qRT-PCR. DF - donor ferret; SF - sentinel ferret; DC - direct contact; RD - respiratory droplet.

Fig. 4. Time course of Experiment 1, Example 2. Baloxavir was administered in a single treatment consisting of 1 mg/kg s.c. injections at 4 separate sites on the back of the ferret (4 mg/kg total). Placebo (1 ml/kg vehicle only suspension) was administered in the same manner. Oseltamivir phosphate (5 mg/kg) was administered orally twice daily until the endpoint (excluding the endpoint). Donor ferret treatment: 24 h after donor inoculation. Recipient ferret co-housing: 24 h after donor inoculation. See text for details. BCA, baloxavir; OST, oseltamivir.

Fig. 5. _TCID50 of donor ferrets in Experiment 1 of Example 2. 0/4 of baloxavir-treated donors shed detectable infectious virus at the endpoint of 3 days post infection (DPI). The titer of the baloxavir group was significantly lower than the placebo group at 2 DPI (*p less than or equal to 0.05) and 3 DPI (***p less than or equal to 0.001). 4/4 of oseltamivir-treated and 4/4 of placebo-treated donors shed infectious virus at the endpoint of 3 DPI. There was no significant difference in titer groups observed between oseltamivir-treated and placebo-treated donors. BCA, baloxavir; OST, oseltamivir.

Fig. 6. qRT-PCR results from donor ferrets of Experiment 1, Example 2. Viral RNA was present in the nasal wash of all animals at all sampling times. Viral copy numbers in the baloxavir group (*) and the oseltamivir group (*) were significantly reduced compared to placebo at 3 DPI (p less than or equal to 0.05). The copy number threshold for positive detection is based on the lowest RNA copy number of the standard resulting in a Ct value less than 35 per assay. The pharmacokinetics (PK) of the baloxavir group at 48 hours post-treatment was 10.2 plus or minus 2.8 ng/mL baloxavir in plasma plus or minus SD. BCA, baloxavir; OST, oseltamivir.

Fig. 7. Results from ferret recipients of Experiment 1, Example 2. (A) TCID ₅₀ results. Only 1/4 of the baloxavir group of ferret recipients excreted detectable infectious virus after contact exposure to the donor, compared with 4/4 of the oseltamivir group and 4/4 of the placebo group. (B) RT-qPCR results. Influenza virus RNA was detected in only 2/4 of the baloxavir group recipients, compared with 4/4 of the oseltamivir group and 4/4 of the placebo group. BCA, baloxavir; OST, oseltamivir.

Fig. 8. Time course of Experiment 2 Example 2. Baloxavir was administered in a single treatment consisting of 1 mg/kg s.c. injections into 4 separate sites on the back of the ferret (total 4 mg/kg). Placebo (1 ml/kg vehicle only suspension) was administered in the same manner. Oseltamivir phosphate (5 mg/kg) was administered orally twice daily until the end point (excluding the last day). Donor treatment: 24 h after donor inoculation.

Recipient co-housing: 48 h after donor inoculation. See text for details. BCA - baloxavir; OST - oseltamivir.

Fig. 9. Results from ferret donors of Experiment 2, Example 2. (A) _TCID50 results. 0/4 of baloxavir-treated donors excreted detectable infectious virus at the final time point of 4 DPI. The titer of the baloxavir group was significantly lower than that of the placebo group at 2 DPI (*p less than or equal to 0.05), 3 DPI (**p less than or equal to 0.001), and 4 DPI (***p less than or equal to 0.001). The titer of the baloxavir group was significantly lower than that of the oseltamivir group at 3-4 DPI (p less than or equal to 0.01). However, immediately before treatment (i.e., at 1 DPI), titers were significantly higher in the baloxavir group than in the placebo group (p less than or equal to 0.001). Four-quarters of oseltamivir-treated and four-quarters of placebo-treated donors were shedding infectious virus at the final time point of 4 DPI. The titer of the oseltamivir group was significantly lower than the titer of the placebo group at 1 DPI (p < 0.05), but there was no difference at 2–4 DPI. (B) RT-qPCR results. Viral RNA was present in the nasal washes of all animals at all sampling times. The mean viral copy number was reduced in the baloxavir group at 3 DPI, but no differences were observed at any other day. The pharmacokinetics (PK) of the baloxavir group at 72 h post-treatment was 12.5 ± 2.4 ng/mL baloxavir in plasma ± SD. BCA, baloxavir; OST, oseltamivir.

Fig. 10. Results of ferret recipients of Experiment 2 Example 2.

(A) _TCID50 results: Only 1/4 of baloxavir-treated recipient ferrets shed detectable infectious virus following donor exposure compared with 4/4 oseltamivir-treated and 4/4 placebo-treated ferrets.

(B) RT-qPCR. Influenza virus RNA was detected in 1/4 of the baloxavir recipients compared with 4/4 of the oseltamivir and 4/4 of the placebo groups. (C) Serology. 2/4 of the baloxavir recipients demonstrated antibody responses to the donor virus compared with 4/4 of the oseltamivir and 4/4 of the placebo ferrets. The antibody response detected in the TCO ₅₀ -negative ferret was reduced (>32-fold change) compared with the TCO ₅₀ -positive ferrets. "-" no antibody response was observed.

Fig. 11. Time course of Experiment 3 of Example 2. Baloxavir was administered in a single treatment consisting of 1 mg/kg s.c. injections into 4 separate sites on the back of the ferret (4 mg/kg total). Placebo (1 ml/kg vehicle suspension only) was administered in the same manner. Oseltamivir phosphate (5 mg/kg) was administered orally twice daily until the final time point (excluding the last day). Donor treatment: 48 h after donor inoculation. Recipient co-housing: 48 h after donor inoculation. See text for details. BCA, baloxavir; OST, oseltamivir.

Fig. 12. Results of donor ferrets from Experiment 3 Example 2. (A)

_TCID50 results: None (i.e., 0/4) of the baloxavir-treated donors shed infectious virus at the 4 DPI endpoint. The baloxavir group titer was significantly lower than the placebo group titer at 3 and 4 DPI (*p less than or equal to 0.001) and less than the oseltamivir group at 4 DPI (***p less than or equal to 0.001). All (i.e., 4/4) of the oseltamivir-treated and all (i.e., 4/4) of the placebo-treated donors shed infectious virus at the 4 DPI endpoint. The oseltamivir group titer was significantly lower than the placebo group titer at 3 DPI (p less than or equal to 0.05). (B) RT-qPCR results. Viral RNA present in nasal washes of all animals was measured at all sampling times. There were no significant differences between the mean copy numbers of the groups. The pharmacokinetics (PK) of the baloxavir group at 72 h post-treatment was 24.5 plus or minus 4.03 ng/mL baloxavir in plasma plus or minus SD. BCA, baloxavir; OST, oseltamivir.

Fig. 13. Results from ferret recipients of Experiment 3 of Example 2. (A) _TCID50 results. 2/4 of baloxavir-treated donors shed infectious virus at the 4 DPI endpoint compared to 4/4 of oseltamivir and 4/4 of placebo donors. (B) RT-qPCR. Viral RNA was detected in 3/4 of baloxavir-treated donors compared to 4/4 of oseltamivir and 4/4 of placebo donors. (C) Serology. Sera from all recipients in all groups demonstrated antibody responses to donor virus. TCID ₅₀ -negative baloxavir group recipients (2/4) demonstrated reduced antibody titers (>16-fold change) compared to TCID ₅₀ -positive ferrets. "-" no antibody response was observed. *Ferret 11333 (placebo) achieved the human expectation at 4 DPE and was therefore excluded from the serology analysis.

Fig. 14. Summary of the effect of baloxavir on donor ferrets in Experiments 1-3 of Example 2. Baloxavir treatment rapidly reduces the shedding of infectious virus in the upper respiratory tract. No baloxavir-treated ferret shed detectable infectious virus at the donor endpoint in each experiment.

Fig. 15. Summary of the effect of baloxavir on contact transmission in Experiments 1-3 of Example 2. The placebo group demonstrates that the contact transmission rate of A/Perth/265/2009 to naive ferrets is 100% in the model used. Oseltamivir treatment does not reduce contact transmission to naive ferrets. 100% of oseltamivir-treated recipients demonstrate evidence of infection by TCID ₅₀ , qRT-PCR and serology under all experimental conditions. Baloxavir treatment reduces the contact transmission rate compared to oseltamivir or placebo. Baloxavir treatment at 1 DPI reduces the shedding of infectious virus in recipients by up to 25%. No difference was observed when recipients were delayed in co-housing for 24 h. Treatment with baloxavir at 2 DPI reduced the incidence of infectious virus shedding in recipients by up to 50%. However, virus copies and serum antibody responses were detected in baloxavir-treated recipients who did not show evidence of infectious virus shedding in the upper respiratory tract (URT), indicating viral exposure but not productive infection.

Fig. 16. Simulation of influenza epidemic prevention during seasonal influenza by treatment with baloxavir. See text for details.

Fig. 17. Simulation of influenza pandemic prevention by baloxavir treatment. See text for details.

Fig. 18. Canadian Acute Respiratory Illness and Influenza Scale (CARIFS).

The examples illustrate the present invention.

Example 1: Baloxavir marboxil prevents respiratory droplet transmission

Study design

The design of this study is shown in Fig. 1(A) and (B). In this study, bacloxavir was administered to ferrets once as a 1 mg/kg s.c. suspension at 4 different sites on the back. Oseltamivir phosphate (5 mg/kg) was administered orally twice daily for 5 days.

This experiment is planned with 3 groups: baloxavir, oseltamivir and no drug. For each group there were 4 groups of animals. In each group there were donor animals and sentinel animals that are exposed to infected donors either through direct contact or airborne droplets.

This experiment was carried out as follows:

Day 0:

All animals were implanted with a chip for temperature monitoring and identification. Four ferrets (donors) were infected with influenza A virus. Wild type (RG) A/England/195/2009 at a dose of 10 ⁴ PFU was given intranasally under light anesthesia (ketamine/xylazine) in a total volume of 200 microliters.

Day 1:

At 24 h post-infection, nasal washes were obtained from 4 infected ferrets. Nasal washes were analyzed immediately by plaque loading onto fresh MDCK cells to detect infectious virus and RT-PCR to determine viral RNA load. Each donor ferret was then treated with one of baloxavir, oseltamivir phosphate, or vehicle alone. Baloxavir was administered subcutaneously once to 4 dorsal sites using 1 mg/1 mL/kg per injection (total 4 mg/4 mL/kg). Following baloxavir dosing, blood samples (minimum 200 µL) were collected from donor animals at 48 h and 144 h, respectively, for pharmacokinetic (PK) analysis.

A 5 mg/kg dose of oseltamivir phosphate was administered orally twice daily for 5 days, which is considered equivalent to the standard human adult treatment dose of 75 mg (J Antimicrob Chemother 2014; 69: 2458–2469). Following drug administration on day 1, one sentinel animal was introduced into the same cage as each infected donor (DC animal) and one sentinel animal was introduced into an adjacent cage (RD animal). Sentinel animals were not treated with any drug.

Day 2:

All ferrets were given nasal swabs on day 2 and every day thereafter until the end of the experiment, beginning with RD animals, then DC animals, and ending with donors. Nasal swabs were immediately subjected to plaque assay. Viral RNA was also monitored by RT-PCR. Clinical signs, including temperature, weight loss, and respiratory signs, were monitored daily. In addition, exhaled virus was monitored in 4 donor animals by placing them in a chamber for 10 minutes and sampling air using MDCK indicator cells.

Day 4:

In the morning, 4 DC and 4 RD animals were removed and housed separately. Thus, sentinel animals were exposed on days 1-4 after donor infection (72 h).

This experiment was terminated when all animals had two consecutive days of virus-negative nasal swabs.

Materials and methods

Animal studies

Female ferrets (20–24 weeks old) weighing 750–1000 g were used. After acclimatization, sera were obtained and assayed by HAI for antibodies against A/England/195/09, the pandemic H1N1. All ferrets were negative for influenza virus antibodies at the start of the experiments. Body weights were measured daily. Strict procedures were followed to prevent inappropriate cross-contamination between animals. Sentinel animals were handled prior to inoculated animals, and handling surfaces and gloves were disinfected between animals. For inoculation, ferrets were lightly anesthetized with ketamine (22 mg kg ^-1 ) and xylazine (0.9 mg kg ^-1 ) and inoculated intranasally with virus diluted in PBS (0.1 ml per nostril). All conscious animals were given daily nasal lavage by dropping 2 ml of PBS into the nostrils and expectorated secretions were collected in modified 250 ml centrifuge tubes. The expectorated product from the nasal lavage was used for virus titration by plaque assay. The limit of detection of virus in plaque assays was 10 PFU ml ^-1 .

Ferret tracking and activity measurement

Ferrets were weighed daily after infection. Body temperature was measured daily using a subcutaneous IPTT-300 transponder (Plexx B.V, The Netherlands). Ferrets' activity was counted manually at the same time each day. This count was based on a previous publication by Oh DY, Barr IG, Hurt AC (2015) PLoS ONE 10(3): e0118780. Ferrets were observed in their cages for at least 4 hours after nasal swab collection to minimize disturbance to their activity.

Cells

MDCK cells were maintained in Dulbecco's modified Eagle's medium (DMEM; Gibco, Invitrogen) supplemented with 10% FBS and 1% penicillin/streptomycin (Sigma-Aldrich).

Plaque analysis

Plaque assay was performed using MDCK cells. Cell layers inoculated with 100 μl of serially diluted samples were overlaid with 0.6% agarose (Oxoid) in DMEM supplemented with 2 μg trypsin (Worthington) ml ^-1 and incubated at 37°C for 3 days.

Sample preparation for real-time PCR

140 µl of ferret nasal wash was used for RNA extraction using the Qiagen Viral RNA Mini Kit (Cat#/ID: 52904) according to the manufacturer's instructions.

Real-time PCR

Real-time PCR was performed using the 7500 Real-Time PCR System (ABI) in 20 µl reactions with AgPath-ID™ One-Step RT-PCR reagents, 10 µl RT-PCR buffer (Thermo Fisher, Cat. #4387391), 4 µl RNA, 0.8 µl forward (5'GACCRATCCTGTCACCTCTGA 3', SEQ ID NO:1) and reverse primers (5' AGGGCATTYTGGACAAAKCGTCTA3', SEQ ID NO:2), and 0.4 µl probe (5' FAM-TCGAGTCCTCGCTCACTGGGCACG-BHQ1 3', SEQ ID NO:3). Reverse transcription was performed at 45°C for 10 min. The PCR amplification conditions consisted of 95°C for 15 s and 60°C for 1 min after an initial denaturation step of 95°C for 10 min. A total of 40 cycles were performed. The Ct value for the target M gene was determined for each sample. The M gene is an influenza virus gene encoding 2 matrix proteins, M1 and M2. The M gene is detectable by PCR and can be used to quantify influenza virus. Absolute copy numbers were calculated from standard curves to identify influenza A positive ferrets.

Declaration of Ethics

All work was approved by the local Genetic Manipulation (GM) Safety Committee of Imperial College London, St. Mary's Campus (centre number GM77) and the UK Health and Safety Executive. All animal studies described in this work were carried out under UK Home Office approval P48DAD9B4.

All of the above methods are well known in the art and are described, for example, in the document "Contact transmission of influenza virus between ferrets imposes a looser bottleneck than respiratory droplet transmission allowing propagation of antiviral resistance." Frise R, Bradley K, van Doremalen N, Galiano M, Elderfield RA, Stilwell P, Ashcroft JW, Fernandez-Alonso M, Miah S, LackenbyA, Roberts KL, Donnelly CA, Barclay WS. Sci Rep. 2016 Jul 19;6:29793. doi: 10.1038/srep29793.PMID:27430528.

Results and discussion

The results of Example 1 are shown in Figs. 2 and 3. These results indicate that airborne transmission occurred less frequently in subjects treated with baloxavir compared to subjects treated with oseltamivir or placebo (i.e., 25% transmission relative to 100% transmission).

In addition, a delay in time to virus shedding was observed for sentinel animals exposed to donors treated with either oseltamivir or baloxavir compared to controls. The median time to virus shedding was 4.25 days compared to 5.5 days for sentinel animals exposed to direct contact and treated with either oseltamivir or baloxavir. For sentinel animals exposed to airborne transmission, the median time to virus shedding was 4.5 days from untreated donor animals compared to 9.3 days for oseltamivir-treated animals and 8 days from the one baloxavir-treated sentinel animal that acquired infection via the RD route.

Overall, this shows that treatment of donor ferrets infected with 2009 N1 RNA virus with baloxavir delayed transmission to housed sentinel ferrets and reduced the likelihood of airborne transmission.

Example 2: Baloxavir marboxil prevents direct contact transmission

Materials and methods

Administration of Antiviral Drugs and Medicines The active form baloxavir acid (S-033447, hereinafter referred to as BCA) and the prodrug oseltamivir phosphate (hereinafter referred to as OST) were kindly provided by Shionogi & Co., Ltd., Japan. The delivery vehicle for BCA was methylcellulose (Sigma-Aldrich, Australia) in 0.5% (w/v) aqueous solution prepared using sterile water (hereinafter referred to as MC solution). BCA in suspension with MC solution (1 mg/mL) was prepared using an agate mortar and pestle and delivered to reversibly anesthetized animals in a single treatment consisting of 4 subcutaneous injections at 4 sites on the dorsal region (total dose 4 mg/kg per animal). Animals in the placebo group were dosed identically using MC solution alone (1 mL/kg). Doses of OST (10 mg/mL) were prepared in sugar solution (1 g/mL) and delivered twice daily (8 hours apart) to naive ferrets via the oral route (total dose 10 mg/kg/day).

Cells and virus

Madin-Darby canine kidney (MDCK) cells were maintained in Dulbecco's modified Eagle's medium (DMEM, Gibco) supplemented with 10% (v/v) fetal bovine serum, 2 mM GlutaMAX, 0.05% sodium bicarbonate, 100 μM MEM (minimal essential medium) with nonessential amino acids, 20 mM HEPES, and 50,000 U penicillin-streptomycin (complete mixture, referred to as "growth medium").

The influenza virus A/Perth/265/2009 H1N1pdm09 was used for inoculation of animals. This virus was isolated from a clinical specimen (Lab#910894) at the World Health Organization Collaborating Centre for Influenza Reference and Research (WHOCCRRI, Melbourne, Australia). Viruses were plaque purified and propagated in MDCK cells.

Animals

Outbred male ferrets (Mustela purtorius furo), 12 weeks or older and weighing 600–1800 g, were obtained from independent animal breeders and confirmed to be seronegative against recently circulating influenza antigens (A/Michigan/45/2015, A/Singapore/INFIMH-16-0019/2016, B/Phuket/3073/2013, B/Brisbane/60/2008) by hemagglutination inhibition (HI) assay. Identification/temperature tracking chips (LifeChip, Bio-Thermo) were implanted subcutaneously on the dorsal region of each ferret. Animals were monitored at least daily for changes in temperature and weight and were provided with ad libitum access to pelleted food (Eukanuba) and water with additional wet food (Hill's Pet Nutrition, Australia) as required for ethical reasons (up to 90% of initial weight). Routine sampling was performed under sedation using intramuscular injection of xylazine (5 mg/kg) (Troy Laboratories), whereas virus inoculation and subcutaneous drug delivery were performed under reversible anesthesia using intramuscular injection of ketamine (10 mg/kg) (Troy Laboratories) in combination with midazolam (0.5 mg/kg) (Troy Laboratories) and midetomidine (0.02 mg/kg) (Troy Laboratories), antagonized by atipamezole (0.01 mg/kg) (Troy Laboratories). Ferrets were injected i.m. (intramuscularly) at the final time point with an irreversible anesthetic consisting of ketamine (up to 25 mg/kg) and xylazine (up to 5 mg/kg) before being killed with an overdose of sodium pentobarbitone (Troy Laboratories) (up to 1000 mg/kg) via intracardiac injection.

Ferret studies were conducted in a bioresource facility (physical containment level 2) at the Peter Doherty Institute, Melbourne, Australia, in accordance with the Australian Government, National Health and Medical Research Council Code of Practice for the Care and Use of Animals for Scientific Purposes (8th edition). The study procedures were approved (AEC#1714278) by the Biochemistry & Molecular Biology, Dental Science, Medicine, Microbiology & Immunology, and Surgery Animal Ethics Committee of The University of Melbourne.

Virology

Nasal swab samples were stored with 1% (w/v) bovine serum albumin (BSA) at -80°C until viral titer was determined by _TCID50 assay. Briefly, samples were serially diluted 10-fold in serum-free growth medium containing TPCK-treated trypsin (4 μg/ml; Sigma-Aldrich) and added in triplicate to flat-bottomed 96-well plates containing a confluent monolayer of MDCK cells in each well. Following infection, cells were maintained in serum-free growth media (containing 4 μg/ml TPCK-treated trypsin) for 96 h at 37°C, 5% _CO2 , before assessing virus growth by hemagglutination assay using 1% turkey red blood cells (RBC). TCID ₅₀ titers were calculated according to the method of Reed and Muench (1938) (Reed, L.J., and Muench, H. (1938), American Journal of Epidemiology 27, 493-497).

Quantitative real-time RT-PCR

Viral RNA was isolated from 200 μl of nasal swab samples using the NucleoMag VET Isolation Kit (Macherey Nagel) on the KingFisher Flex Platform (ThermoFisher Scientific) according to the manufacturer's instructions. The number of influenza M gene copies in 4 μl of RNA was determined by quantitative real-time RT-PCR performed using the SensiFAST Probe Lo-ROX One-Step qRT-PCR System (Bioline) on an ABI 7500 Real-Time PCR System (Applied Biosystems) under the following cycling conditions: 45°C for 10 min, 1 cycle; 95°C for 2 min, 1 cycle; 95°C for 5 s, then 60°C for 30 s, 40 cycles. Sample RNA was quantified using influenza A virus RNA standards of known copy number kindly provided by Seqirus, Australia. Influenza A virus real-time assay universal primer/probe sets were kindly provided by CDC Influenza Branch (Atlanta, USA) (sequences available upon request). Results were analyzed using 7500 Fast System SDS v1.5.1.

Serology

Sera were collected from recipient blood samples at the final time point by centrifugation and non-specific inhibitors of agglutination were removed by treatment with receptor degrading enzyme (RDE) (Denka Seiken) according to the manufacturer's instructions. Treated sera were adsorbed using turkey RBCs prior to the HI assay to remove non-specific agglutination factors. Briefly, sera (initial dilution 1:20) were serially diluted two-fold in PBS in 96-well V-bottom plates (the last row was left as a negative control - PBS). A/Perth/265/2009 virus adjusted to 4 hemagglutinating units in 25 μl was mixed in all wells with samples for 1 h incubation at room temperature, followed by 45 min incubation in 1% turkey RBCs. Positive inhibition was defined as the appearance of a running "tear" pattern comparable to the negative control, PBS. The HI titer was calculated as the reciprocal of the highest serum dilution that inhibited agglutination.

Antiviral Pharmacokinetic Analysis

The final time-point blood samples from the VCA- and placebo-treated donors were collected in heparinized tubes and plasma was isolated by centrifugation. Plasma samples were stored at -80°C until transport to the assay facilities at Shionogu & Co., Ltd.

Statistical analyses

Data analysis was performed using Graphpad Prism (GraphPad software, v5.01). Nasal wash viral titers from all ferrets at each day post-inoculation/exposure were compared by paired two-way analysis of variance (ANOVA) followed by Tukey's multiple comparisons test. Quantitative real-time RT-PCR was performed on control influenza RNA of known copy number to generate a standard curve (linear regression of influenza cycle threshold (Ct) values against copy number). Viral copy numbers in nasal wash RNA samples in the same assay were calculated using the standard curve formula. Influenza A virus copy numbers for each group of ferrets were compared by paired two-way ANOVA followed by Tukey's multiple comparisons test. P < 0.05 was considered statistically significant.

Study design

The dynamics of Experiments 1-3 are shown in Figs. 4, 8 and 11.

Experimental groups and treatment schemes

This study consisted of three separate experiments using different antiviral treatment regimens and naive animal co-housing schedules. In each experiment, twenty-four ferrets were randomly assigned to three groups (n = 8): BCA, OST, and placebo. Within each group, n = 4 ferrets were assigned as treated donor ferrets and n = 4 as naive recipients. At 0 DPI for each experiment, all donor ferrets (each housed individually) were inoculated intranasally with influenza A/Perth/265/2009 at ¹⁰³ 50% tissue culture challenge doses ( _TCID50 ) in 40 µl phosphate-buffered saline (PBS). As described above, donors in the VCA and placebo groups received a single antiviral treatment, whereas donors in the OST group were treated daily until the final time point (excluding the day of sacrifice). At the designated time point, naive recipient ferrets were co-housed with treated donor ferrets (1:1 per cage) for a 48-h exposure period.

Experiment 1: Antiviral treatment of all donors was initiated at 1 DPI, and naive recipient ferrets were housed together immediately after treatment. Body weight and temperature were monitored daily for all animals.

Daily nasal washing samples were collected in 1 ml PBS from 1 DPI to 3 DPI for donor animals and from 1 day post exposure (DPE) to 10 DPE for recipient animals. Donors were sacrificed at 3 DPI (2 DPE) and blood was collected from BCA- and vehicle-treated ferrets via cardiac puncture. Recipients were sacrificed at 10 DPE.

Experiment 2: Antiviral treatment was initiated at 1 DPI and co-housing with the recipient was delayed until 24 h post-treatment (2 DPI). All animals were monitored daily for body weight and temperature. Daily nasal washing samples were collected with 1 mL PBS from 1 DPI to 4 DPI for donor animals and from 1 DPI to 10 DPI for recipient animals. Donors were sacrificed at 4 DPI (2 DPI) and blood was collected from BCA- and placebo-treated ferrets via cardiac puncture. Recipients were maintained until 16 DPE, at which time blood was collected via cardiac puncture.

Experiment 3: Antiviral treatment was initiated at 2 DPI and recipients were housed together immediately thereafter. All animals were monitored daily for body weight and temperature. Daily nasal wash samples were collected with 1 mL PBS from 1 DPI to 4 DPI for donor animals and from 1 DPI to 10 DPI for recipient animals. Donors were sacrificed at 4 DPI (2 DPI) and blood was collected from BCA- and placebo-treated ferrets via cardiac puncture. Recipients were maintained until 16 DPE, at which time blood was collected via cardiac puncture.

Results and discussion

In three independent experiments, single BCA treatment of H1N1pdm09-infected ferrets significantly reduced pathogenic virus titers in the URT compared with both placebo and oseltamivir (Fig. 14). Mean viral titers in the baloxavir group were significantly reduced compared with the placebo group at 24 h post-treatment across all experimental conditions (p less than or equal to 0.05). While high infectious titers were maintained at the donor endpoint for both oseltamivir and placebo donors, baloxavir-treated donors were not (i.e., 0/4) TCID ₅₀ positive at the donor endpoint across all experimental regimens. The effect of baloxavir was superior to that of oseltamivir in treated donors (Fig. 15). All (i.e. 4/4) oseltamivir-treated donors were TCID ₅₀ positive at the final time point under all conditions - there was no reduction compared with all (i.e. 4/4) placebo-treated donors.

In the ferret model used, placebo recipients demonstrated 100% contact transmission of A/Perth/265/2009 to naive ferrets (Figs. 14-15). Placebo recipients demonstrated robust viral kinetics as measured by both TCID ₅₀ and qRT-PCR and high titer serum antibody responses (greater than 1280) at the recipient end point (Figs. 7, 10, 13). We observed that oseltamivir treatment did not reduce contact transmission to naive ferrets. 100% of oseltamivir-treated recipients demonstrated infection parameters that were very similar to placebo, regardless of the antiviral treatment schedule (Figs. 7, 10, 13). In contrast, baloxavir treatment within 24 h of infection resulted in a 75% reduction in transmission. In both Exps 1 and 2, only 1/4 of the baloxavir group recipients demonstrated a viral TCID ₅₀ kinetic curve indicative of placebo-like infection (Figs 7, 10). When baloxavir treatment was administered 48 h after infection, a 50% reduction in transmission was still observed (Fig 13).

The amount of viral RNA in the URT of baloxavir-treated ferrets does not correlate with changes in pathogenic virus titer measured by cell culture.

While viral RNA was detectable in 2/4 of baloxavir group recipients in Exp. 1, the copy number approached the threshold for positive detection and the viral kinetics were not similar to the placebo group viral curve (Fig. 7).

Despite the presence of virus shedding in URT detected by qRT-PCR, these viruses appear to be unable to produce detectable infection in cell culture.

Antibody responses were detected in _TCID50 -negative baloxavir recipients in Exp. 2 (Fig. 10, 15563) and 3 (Fig. 13, 96642, 14779), but they were reduced by 16-32 times compared with titers in placebo recipients.

The mean plasma concentration of baloxavir peaked at 3 hours post-dose (26.1 ng/mL) and then declined gradually until the last sampling time (168 hours post-dose, 9.08 ng/mL). This result indicates that a single subcutaneous administration of 4 mg/kg baloxavir suspension (4 sites, 1 mg/kg per site) could maintain baloxavir plasma concentrations in ferrets for one week or more.

Furthermore, the data clearly show that baloxavir treatment reduces contact transmission.

Example 3: Modeling influenza transmission for baloxavir

Based on the unexpected finding that baloxavir marboxil can prevent influenza virus transmission, it was possible to perform modeling that shows the effect of baloxavir treatment on influenza outbreaks. This modeling also considered the effect of baloxavir on the time to viral shedding (Tshed) observed in the Phase 3 clinical trial (NCT02954354).

This modeling was performed by providing the expected percentage of infected patients during an influenza epidemic and influenza pandemic by developing a specific model that takes into account

- flu/social characteristics

- various processing effects

About the model

Examples 1 and 2, as described above, show that baloxavir treatment not only reduces viral shedding, but also unexpectedly reduces transmission. The surprising and unexpected finding that baloxavir marboxil can be used to reduce transmission is the background to the modeling below, which simulates the effect of baloxavir marboxil treatment during an (emerging) influenza epidemic or influenza pandemic.

The epidemiological SEIR model was used for this simulation. First described in the early 20th century (Kermack and McKendrick), this model characterizes the number of infections in a population by integrating different types of individuals: susceptible, exposed, infected, and recovered. This model is in fact widely used for influenza pandemic planning, policy decisions regarding stockpiling and movement of antiviral agents, and other interventions, and is described, for example, in Murillo, J TheorBiol. 2013. 332:267–290.

Assumptions

1) Virus shedding correlates with infectivity with persistent infectivity during the shedding period.

2) Phase 3 results provide correct estimates of expected viral shedding times for baloxavir and oseltamivir treatments.

3) All patients will have:

- the same latent period

- the same duration of natural disease

- the same rate of infectivity during the disease

- there is no specific intervention other than treatment

- no effects of age, geography or health status on sensitivity.

Results and discussion

Significant reduction in the percentage of infected patients with baloxavir compared with placebo or oseltamivir in both seasonal (see Fig. 15) and pandemic (see Fig. 16) scenarios.

The results indicate that the % of treated patients required to halve the number of infected patients is approximately 15% treated or 30% treated in an epidemic or pandemic, respectively. The exact threshold for classifying an influenza outbreak as "epidemic" or "pandemic" varies each year. However, in view of the results obtained, it is highly likely that treating at least 15% or at least 30% of infected people with baloxavir marboxil would prevent an influenza epidemic or influenza pandemic, respectively.

Example 4: Sequence-based transmission identification

To identify transmission pairs, phylogenetic analysis (WHT with bootstrap support for tree topology or clustering) and/or measurement of genetic distance between paired populations (L1-Norm) can be performed. The outcome of this analysis is influenced by two factors: firstly, the amount of sequence data available. To maximise this, whole genome next generation sequencing (WGNGS) can be performed to increase the level of detail and discrimination (compared to Sanger sequencing and/or single gene sequencing). Secondly, diversity within the community/metapopulation is analysed so that it can be estimated with high confidence that viruses within pairs in a household are more similar to each other compared to the outgroup. More sequences from the community increase the diversity within the group and thus facilitate the identification of actual transmission pairs. Since sequencing cannot be performed for individuals not enrolled in this study, we will perform this analysis on a subset of patients, focusing on sites that have enrolled an adequate number of index patients (IPs) with unique sequences. We will also attempt to obtain sequences from public databases for the same season (this route is expected to be low yielding, but will be pursued).

In a previous study examining influenza virus evolution in an HIVE cohort, 47 transmission pairs were analyzed and could be distinguished based on sequence. Thirty-four of these pairs were from the same season (H3N2, 2014/2015). Based on these results, it is expected that 15 or more index patients (IPs), preferably at least 30-40 IPs, would be sufficient to develop a community distribution of pairwise distances that reflects community diversity, such that actual household transmission events can be inferred from sequence data as described in McCrone, John T., et al. "Stochastic processes constrain the within and between host evolution of influenza virus." Elife 7 (2018): e35962. The methods described in McCrone (Elife 7 (2018): e35962) can be used, such as the method described in that paper for sequencing. In the context of the present invention, the assay is preferably focused on the analysis of influenza A virus samples.

In order to better understand the method used to identify transmission pairs and the basic aspects of this method, the content of McCrone's paper (Elife 7 (2018): e35962) is briefly discussed below.

McCrone (Elife 7 (2018): e35962) used both epidemiological linkage and genetic relatedness of viruses within households to define transmission pairs and exclude interference from background diversity in the community.

More specifically, McCrone (Elife 7 (2018): e35962) described that studies of influenza A virus (IAV) populations in animal and human systems indicate that most within-host single nucleotide variants (iSNVs) are rare and that host populations are subject to strong purifying selection. McCrone (Elife 7 (2018): e35962) used within-host next-generation sequencing of influenza virus populations to determine the evolutionary dynamics of IAV within and across human hosts. A comparative analysis pipeline was used to identify iSNVs and characterize the genetic diversity of H3N2 and H1N1 populations sampled over five post-pandemic seasons from individuals enrolled in a prospective household influenza study. In McCrone (Elife 7 (2018): e35962), the authors find that within-host populations are dynamic and constrained by genetic drift and purifying selection. In the McCrone (Elife 7 (2018): e35962) study, positive selection rarely amplified a beneficial variant de novo to a frequency greater than 2%. In contrast to what has been previously reported for human influenza transmission, but consistent with what has been observed in many other viruses with distinct modes of transmission, McCrone (Elife 7 (2018): e35962) identified a very narrow effective transmission bottleneck that limits the transmission of low-frequency variants.

In McCrone (Elife 7 (2018): e35962), individuals within a household were considered an epidemiologically linked transmission pair if they were both positive for the same influenza virus subtype within 7 days of each other. Several households had 3 or 4 symptomatic cases within this 1-week window, suggesting longer chains of transmission.

Intrahost single nucleotide variants (iSNVs) were identified in McCrone (Elife 7 (2018): e35962) 2014) using their empirically validated chain reaction assay. Consistent with previous studies of natural infections and other infections, the authors of McCrone (Elife 7 (2018): e35962) found that within-host diversity of seasonal influenza A virus (IAV) populations is low. Two hundred forty-three of 249 samples had fewer than 10 minor iSNVs (median 2, IQR 1-3). The number of minor iSNVs identified was not affected by day of infection, viral load, subtype, or vaccination status.

In McCrone (Elife 7 (2018): e35962), single nucleotide variants were evenly distributed across the genome. Minor variants were rarely shared among many individuals. Ninety-eight percent of minor iSNVs were detected only once, 2.3% were detected in two individuals, and no minor iSNVs were detected in three or more individuals. The low level of shared diversity between individuals suggests that populations within a host exploit distinct regions of sequence space with little evidence for parallel evolution. The ratio of nonsynonymous to synonymous variants was 0.75, and given the abundance of nonsynonymous sites across the genome and within the HA gene, these data suggest significant purifying selection within hosts.

McCrone (Elife 7 (2018): e35962) found that the majority of iSNVs (68%) detected in the second sample were either novel or previously present below the 2% detection limit. Overall, McCrone's (Elife 7 (2018): e35962) data suggest that the upper respiratory tract population is highly dynamic while maintaining a stable consensus, and that positive selection for novel variants within hosts is inefficient and rarely amplifies a newly generated variant to a frequency greater than 2%.

McCrone's host-wide data (Elife 7 (2018): e35962) suggest that emerging iSNVs with positive fitness effects are likely to be present at low frequencies (<2%) during acute infection. Maintenance of these mutations in host populations is therefore highly dependent on transmission bottlenecks.

In McCrone (Elife 7 (2018): e35962), all individuals in a household with symptom onset within a 7-day window were considered epidemiologically linked. The donor in each putative pair was defined as the individual with the earlier symptom onset. A transmission event was ignored if there were multiple possible donors with the same symptom onset day. Donors and recipients were not allowed to have symptom onset on the same day unless both individuals were index cases for the household. Based on these criteria, the McCrone (Elife 7 (2018): e35962) cohort had 124 putative transmission events in the household over five seasons. Of these, 52 pairs had samples of sufficient quality to reliably identify iSNVs from both individuals.

The sequence data were then used in McCrone (Elife 7 (2018): e35962) to determine which of the 52 epidemiologically linked pairs represented true household transmission events, as opposed to coincident community-acquired infections. The genetic distance between influenza populations from each household pair was measured using the L1-norm, and these distances were compared with the distances of randomly assigned community pairs within each season.

In McCrone (Elife 7 (2018): e35962), only individuals were considered a true transmission pair if they had a genetic distance less than the 5th percentile of the community distribution of randomly assigned pairs. Forty-seven transmission events in the household satisfied this event. Among these 47 transmission pairs with confirmed sequence, three had no iSNV in the donor and one additional donor appeared to have a mixed infection. These four transmission events were removed from the bottleneck analysis because donors without iSNV are not informative and mixed infections violate the model's assumptions of site independence. The transmission bottleneck in the remaining 43 high-quality pairs (37 H3N2, 6 H1N1) was assessed.

A transmission bottleneck limits the amount of genetic diversity that is shared between both members of a pair. McCrone (Elife 7 (2018): e35962) found that a small number of minor iSNVs were polymorphic in both donor and recipient populations. Minor iSNVs were either absent in the donor or fixed in the recipient. The lack of shared polymorphic sites suggests a strong effective bottleneck in which only one allele is transmitted from donor to recipient.

McCrone (Elife 7 (2018): e35962) conclude that acute influenza infections are characterized by low diversity, limited positive selection, and narrow transmission bottlenecks. Because they used viruses collected over five influenza seasons from individuals enrolled in a prospective household cohort, these dynamics are likely broadly representative of many seasonal influenza infections in temperate regions. McCrone's (Elife 7 (2018): e35962) data suggest that even if selection operates below the level of detection, such rare variants are unlikely to be transmitted. Given the size of the estimated bottleneck, the probability of transmission is approximately 1.7% for a variant at 1% frequency and 3.3% for a variant at 2%.

Example 5: Pharmacokinetic analysis in a ferret

Materials, methods and results for animals

Outbred female ferrets 28 months old and weighing 650–802 g were purchased from Japan SLC, Inc. Identification was performed by labeling with sequential numbers on the front of each cage. Animals were monitored at least once daily for clinical observations and had unlimited access to pelleted diet (LabDiet High Density Ferret Diet, Lab Supply, USA) and water. Ferrets were sacrificed at the final time point by exsanguination under isoflurane inhalation anesthesia (Pfizer). Ferret studies were performed at Shionogi Pharmaceutical Research Center, Shionogi & Co., Ltd. (Osaka, Japan) according to an animal study protocol that was approved by the Institutional Animal Care and Use Committee of Shionogi.

Administration of the drug and blood sampling

The active form of baloxavir acid (S-033447, hereinafter referred to as BCA) was provided by the Analyte Control Personnel, Shionogi & Co., Ltd., Japan. The delivery vehicle for BCA was methylcellulose (Fuji-Film Wako, Japan) in 0.5% w/v aqueous solution prepared with sterile water (hereinafter referred to as MC solution). BCA in suspension with MC solution (1 mg/mL) was prepared using an agate mortar and pestle and delivered to reversibly anesthetized animals in a single treatment consisting of 4 subcutaneous injections at 4 sites on the dorsal region (total dose of 4 mg/kg per animal). After dosing, a needle prick was performed to bleed from the hind paw vein for blood collection, and then blood samples (approximately 60-100 μl) were collected from the bleeding portion using heparinized capillary tubes (Drummond Scientific Company, USA) at the scheduled time, and plasma was collected by centrifugation. The plasma samples were stored in a freezer at approximately -80°C until used for analysis by a liquid chromatography tandem mass spectrometry (LC-MS/MS) system.

Determination of VHA in plasma

Plasma BCA concentration was determined by LC-MS/MS consisting of an LC-20A system (Shimadzu Corporation, Japan) and API 5000 (AB SCIEX, USA). Plasma samples were obtained by protein precipitation. Chromatographic separation was performed on a metal-free L-column 2 ODS (3 μm, id (internal diameter) 2.0 mm × 50 mm, Chemicals Evaluation and Research Institute, Japan) at 40°C. Binary mobile phases - 0.1% formic acid in water and 0.1% formic acid in acetonitrile - were delivered at a total flow rate of 0.6 ml/min in gradient mode. The mass spectrometer was operated in positive polarity electrospray ionization (ESI) mode using multiple reaction monitoring (MRM). The precursor/product transitions (m/z) 484/247 and 490/247 were monitored for VCA and the internal standard, VCA-racemate-d ₄ ¹⁸ O, respectively. Calculations were based on the peak area ratios of VCA to the internal standard. The analytical method was validated over the calibration range of 0.5 to 500 ng/mL for selectivity, yield, accuracy, precision, and stability under a variety of conditions.

Pharmacokinetic analysis

Plasma concentrations of BCA for each individual ferret were calculated using the Analyst program (AB SCIEX, USA). The mean and standard deviation were also calculated using individual data using Microsoft Excel (Microsoft Co., USA).

Result and discussion

The mean plasma concentration peaked at 3 hours post-dose (26.1 ng/mL) and then declined gradually to the last sampling point (168 hours post-dose, 9.08 ng/mL). This result indicated that a single subcutaneous administration of 4 mg/kg BCA suspension (4 sites, 1 mg/kg per site) could maintain plasma BCA concentrations in ferrets for a week or more.

The present invention relates to the following nucleotide and amino acid sequences:

SEQ ID NO: 1: Forward primer for real-time PCR

5'G ACC R ATCCTGTCACCTCTG A 3'

SEQ ID NO: 2: Reverse primer for real-time PCR

5' AGGGCATTYTGGACAAAKCGTCTA3'

SEQ ID NO: 3: Real-time PCR probe ("FAM" means fluorescein amidite, i.e., a fluorescent dye for labeling oligonucleotides; "BHQ1" means black hole trap 1, i.e., a trap; BHQ1 and FAM together are not fluorescent, only after cleavage of the probe will FAM be fluorescent)

5' FAM-TCG AGTCCTGCTCACTGGGCACG - B H Q1 3'

SEQ ID NO: 4: Influenza A virus (A/WSN/1933(H1N1)): GenBank: X17336.1, containing the I38T mutation. The I38T mutation is underlined and shown in bold.

SEQ ID NO: 5: Portion of influenza A virus sequence (A/WSN/1933(H1N1)): GenBank: X17336.1, containing the I38T mutation. The I38T mutation is underlined and shown in bold.

FAA T CTH

Claims (58)

1. The use of a compound or a pharmaceutically acceptable salt thereof for reducing the rate of transmission of an influenza virus infection, wherein the compound or a pharmaceutically acceptable salt thereof is administered to a patient zero infected with an influenza virus, wherein said compound has one of the following formulas I and II: 2. The use according to claim 1, wherein said compound reduces the infectivity of the influenza virus of patient zero. 3. Use according to claim 1 or 2, wherein the rate of transmission of the influenza virus from patient zero is reduced within one hour to 32 hours from the moment of the 1st administration of the said compound. 4. Use according to any one of claims 1-3, wherein the rate of transmission of the influenza virus from patient zero is reduced within 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30 or 32 hours from the moment of the 1st administration of the said compound. 5. The use according to any one of claims 1 to 4, wherein the rate of transmission of influenza virus from the treated null patient is reduced to 70% or less, preferably to 50% or less, more preferably to 30% or less compared to the rate of transmission of influenza virus from the control patient. 6. Use according to claim 5, wherein the control patient is infected with the influenza virus and has not been administered the said compound. 7. Use according to claim 5 or 6, wherein the control patient was administered an anti-flu drug, with the exception of a compound of formula (I) or (II), or a pharmaceutically acceptable salt thereof. 8. Use according to any of paragraphs 5-7, wherein the control patient was not administered the anti-flu drug. 9. The use according to any one of claims 1 to 8, wherein transmission occurs when at least one contact person of a treated patient zero or control patient becomes infected with an influenza virus following personal contact with the treated patient zero or control patient, respectively. 10. The use according to paragraph 9, where the contact person of the treated patient zero is a person who: (i) was not infected with influenza virus at the time when the compound was administered to patient zero for the first time; and (ii) had personal contact with a treated patient zero during the patient zero's influenza virus infection. 11. Use according to claim 10, where the contact person had personal contact with the treated patient zero within 10 days from the moment of administration of the said compound to the patient zero. 12. Use according to claim 10 or 11, wherein the contact person had personal contact with the treated patient zero within the time period starting with the administration of the said compound and ending 5-10 days after the administration of the said compound. 13. The use according to any of paragraphs 9-12, wherein the contact person of the control patient is a person who: (i) did not have an influenza virus infection at the time when the anti-influenza medicinal product was first administered to the control patient or, if the control patient had not been administered an anti-influenza medicinal product, at an appropriate time point given the pathogenesis of the influenza disease; and (ii) had personal contact with a control patient during the control patient's influenza virus infection. 14. Use according to item 13, where the contact person had personal contact with the control patient within 10 days from the moment of administration of the anti-influenza medicinal product to the control patient; or, if the anti-influenza medicinal product was not administered, within the appropriate period of time taking into account the pathogenesis of the influenza disease. 15. Use according to claim 13 or 14, where the contact person had personal contact with the control patient within the period of time starting from the moment of administration of the anti-influenza medicinal product to the control patient; or, if the anti-influenza medicinal product was not administered, starting from the appropriate time point taking into account the pathogenesis of the influenza disease and ending 5-10 days after administration of the anti-influenza medicinal product; or, if the anti-influenza medicinal product is not administered, ending at the appropriate time point taking into account the pathogenesis of the influenza disease. 16. Use according to claim 10 or 13, where at the time as defined in (i) the contact person was PCR negative for influenza. 17. The use according to any one of claims 9 to 16, wherein the personal contact is a contact that provides airborne transmission and/or direct contact transmission, preferably only airborne transmission. 18. The use according to any one of claims 9 to 17, wherein transmission from a treated patient zero has occurred if the influenza virus can be detected in at least one contact person of the treated patient zero. 19. The use according to claim 18, wherein transmission from a treated patient zero has occurred if the influenza virus can be detected in at least one contact person of the treated patient zero within 15 days of administration of the said compound. 20. The use according to any one of paragraphs 9-19, wherein transmission from a control patient has occurred if the influenza virus can be detected in at least one contact person of the control patient. 21. Use according to claim 20, wherein transmission from a control patient has occurred if the influenza virus can be detected in at least one contact person of the control patient within 15 days of the administration of another influenza medicinal product to the control patient; or, if the control patient has not been administered an influenza medicinal product, within an appropriate period of time taking into account the pathogenesis of the influenza disease. 22. The use according to any one of claims 3 to 21, wherein the rate of transmission is measured by testing at least one contact person of the treated patient zero or the control patient, respectively, for the presence of an influenza virus infection within 10 days of administration of said compound to the treated patient zero, or within 10 days of administration of another influenza medicinal product to the control patient, or, if the control patient has not been administered an influenza medicinal product, within 10 days of the relevant time point taking into account the pathogenesis of the influenza disease. 23. The use according to any one of claims 1-22, wherein an influenza virus infection is present if the influenza virus can be detected. 24. The use according to item 23, wherein the influenza virus is detected by means of polymerase chain reaction (PCR). 25. The use according to claim 23, wherein the influenza virus is detected by using an influenza analysis kit. 26. The use according to any one of claims 1 to 25, wherein transmission from a treated patient zero has occurred when at least one contact of the treated patient zero has been infected with an influenza virus strain of an influenza virus that is identical to the influenza virus strain of the treated patient zero, and wherein transmission from a control patient has occurred when at least one contact of the control patient has been infected with an influenza virus strain of an influenza virus that is identical to the influenza virus strain of the control patient. 27. Use according to any one of claims 1-26, wherein the transmission of the influenza virus is direct contact transmission and/or airborne transmission. 28. The use according to any one of claims 1 to 27, wherein after administration of said compound and during his/her infection with the influenza virus, the treated patient zero has personal contact with at least one person from a risk group for influenza or with at least one person who is important for the functioning of society. 29. The use according to claim 28, wherein at least one person from the influenza risk group is at least one person who has an increased risk of getting an influenza virus infection or who has an increased risk of getting a complication associated with influenza. 30. The use according to claim 29, wherein the complication associated with influenza is at least one complication selected from the group consisting of hospitalization, sinusitis, otitis media, bronchitis and pneumonia. 31. The use according to claim 29 or 30, wherein at least one person having an increased risk of developing a complication associated with influenza has an increased risk of death due to infection with the influenza virus. 32. The use according to any one of paragraphs 29-31, wherein at least one person having an increased risk of developing a complication associated with influenza is an individual: (i) has chronic cardiovascular disease, chronic pulmonary disease, chronic metabolic disease, chronic renal disease, cardiopulmonary disorder and/or who is immunocompromised; and/or (ii) who is at least 65 years of age or less than 5 years of age. 33. The use according to any one of paragraphs 29-32, wherein at least one person having an increased risk of getting an influenza virus infection is an individual for whom vaccination against influenza is contraindicated. 34. The use according to any one of paragraphs 28-33, wherein at least one person who is important for the functioning of society works as an important service provider, including a person who belongs to the fire personnel, the police personnel, the health care personnel, the emergency rescue personnel, the military or the government. 35. The use according to any one of claims 1-34, wherein after administration of said compound and during his/her infection with the influenza virus, the treated patient zero has personal contact with many people. 36. The use according to any one of claims 1 to 35, wherein after administration of said compound and during his/her infection with the influenza virus, the treated patient zero (i) visits a medical institution, educational institution, public institution, vehicle, car, airplane and/or store, and (ii) has personal contact with many people. 37. Use according to claim 35 or 36, wherein said compound is administered at least one hour before said personal contact. 38. Use according to any one of claims 35-37, wherein said compound is administered at least 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30 or 32 hours before said personal contact. 39. Use according to any one of claims 1-38, wherein said compound is administered within 48 hours of the onset of flu symptoms. 40. The use according to any one of claims 1-39, wherein said compound is administered once as a single treatment. 41. Use according to any one of claims 1-40, wherein the effective amount is about 40 mg for patients less than 80 kg and about 80 mg for patients 80 kg or more. 42. Use according to any one of claims 1-41, wherein said compound is administered orally. 43. Use according to any one of claims 1-42, wherein the zero patient is at least 12 years old. 44. The use according to any one of claims 1 to 43, wherein the treated zero patient has a reduced risk of triggering an influenza epidemic or influenza pandemic compared to the control patient. 45. The use according to any one of claims 1-44, wherein the influenza virus is an influenza A virus or an influenza B virus. 46. The use according to any one of claims 1-45, wherein the influenza virus is an epidemic strain of influenza virus or a pandemic strain of influenza virus. 47. The use according to any one of claims 1-46, wherein the influenza virus is antigenically different compared to the parent strain of influenza virus as a result of antigenic drift and/or antigenic shift. 48. The use according to any one of claims 1-47, wherein the influenza strain is a recycled virus that has caused an epidemic or pandemic in the past. 49. The use according to any one of claims 1-48, wherein the influenza virus strain does not carry the I38X mutation, including the I38T mutation.