Formulations of influenza therapeutic agents

Abstract

The present invention relates to formulations of influenza therapeutic agents. The present invention provides formulations comprising or consisting essentially of Compound 1, or a pharmaceutically acceptable salt thereof, and a filler; methods of using the formulations to treat or prevent influenza virus infection or replication in an individual in need thereof; and a method of preparing the formulation. The formulations, after administration by inhalation, exhibit a high level of drug exposure in the lung as compared to exposure in plasma.

Description

[0001] This application is a divisional application of International Application PCT / US2019 / 061065, which entered the Chinese national phase on May 12, 2021, with application number 201980074528.4 and invention title "Formulations for Treating Influenza".

[0002] Technical Field and Background Art

[0003] Influenza spreads worldwide in a seasonal epidemic pattern, resulting in hundreds of thousands of deaths annually and millions of deaths in pandemic years. For example, there were three influenza pandemics in the 20th century, killing tens of millions of people, each caused by the emergence of novel virus strains of human viruses. Generally, these novel virus strains are generated due to the transmission of existing influenza viruses from other animal species to humans.

[0004] Influenza is mainly transmitted from person to person by larger virus - carrying droplets produced when an infected person coughs or sneezes; these larger droplets can then settle on the mucosal surfaces of the upper respiratory tract of susceptible individuals near the infected person (e.g., within about 6 feet). Transmission can also occur through direct or indirect contact with respiratory secretions, such as touching a surface contaminated with influenza virus and then touching the eyes, nose, or mouth. Adults can transmit influenza to others from 1 day before symptoms appear to about 5 days after symptom onset. Young children and people with weakened immune systems may remain infectious for 10 days or more after symptom onset.

[0005] Influenza viruses are RNA viruses of the family Orthomyxoviridae, which includes five genera: influenza A virus, influenza B virus, influenza C virus, infectious salmon anemia virus (Isavirus), and Thogotovirus.

[0006] The genus influenza A virus is responsible for seasonal and pandemic influenza epidemics. It has one species, influenza A virus, and wild waterfowl are the natural hosts of various influenza A viruses. Occasionally, the virus spreads to other species and can then cause devastating outbreaks in poultry or lead to human influenza pandemics. Influenza A virus is the most virulent human pathogen among the three influenza types and causes the most severe diseases. Influenza A viruses can be further divided into different serotypes based on the antibody response to these viruses. The serotypes identified in humans ranked by the number of known human pandemic deaths are: H1N1 (which caused the Spanish flu in 1918), H2N2 (which caused the Asian flu in 1957), H3N2, H5N1 (pandemic threat in the 2007 - 2008 flu season), H7N7 (which is a potential pandemic threat), H1N2 (endemic in humans and pigs), H9N2, H7N2, H7N3, and H10N7.

[0007] The genus Orthomyxovirus B is the cause of seasonal influenza and has one species, influenza B virus. Influenza B almost exclusively infects humans and is less common than influenza A. The only other animal known to be susceptible to influenza B infection is the seal. This type of influenza mutates at a rate 2 to 3 times slower than influenza A and thus has lower genetic diversity, with only one influenza B serotype. Due to this lack of antigenic diversity, a degree of immunity to influenza B is usually acquired at a very young age. However, influenza B mutates sufficiently that lasting immunity is not possible. This reduced rate of antigenic change combined with its limited host range (which inhibits cross-species antigen transfer) ensures that pandemics of influenza B do not occur.

[0008] The genus Orthomyxovirus C has one species, influenza C virus, which infects humans and pigs and can cause severe disease and local epidemics. However, influenza C is less common than the other types and generally seems to cause mild disease in children.

[0009] Influenza viruses are extremely similar structurally between serotypes and genera. The influenza virus genome consists of eight single-stranded RNAs packaged into rod-shaped structures of different sizes, which are called ribonucleoprotein complexes (RNPs). Each RNP contains a unique viral RNA, multiple copies of the scaffolding nucleoprotein, and a heterotrimeric viral polymerase composed of the PA, PB1, and PB2 subunits, which catalyze the transcription and replication of the viral genome. Recent biochemical and structural studies of the influenza polymerase complex have provided insights into the mechanisms of cap-snatching and RNA synthesis by the influenza polymerase. Briefly, the PB2 cap-binding domain first sequesters the host pre-mRNA by binding to its 5' cap. PA, the endonuclease subunit, then cleaves the captured pre-mRNA 10 to 13 nucleotides downstream of the cap. The PB2 subunit then rotates approximately 70° to direct the capped primer into the PB1 polymerase active site. The PB1 subunit interacts directly with both the PB2 and PA subunits. These subunits contain highly conserved domains in different influenza virus strains and have attracted attention as potential anti-influenza drug targets. In addition to the polymerase complex, the influenza genome encodes its own neuraminidase (NA), hemagglutinin (HA), nucleoprotein (NP), matrix proteins M1 and M2, and non-structural proteins NS1 and NS2. NA is the target of the antiviral drugs oseltamivir (Tamiflu®) and zanamivir (Relenza®). These drugs are sialic acid analogs that inhibit the enzymatic activity of NA and thus slow the release of progeny virus from infected cells.

[0010] Influenza incurs direct costs due to productivity loss and associated medical treatment, as well as indirect costs due to preventive measures. In the United States, influenza causes a total cost of over $10 billion annually, and it is estimated that a future pandemic could result in direct and indirect costs in the hundreds of billions of dollars. The costs of prevention and control are also high. Billions of dollars have been spent worldwide to prepare for and plan for a potential H5N1 avian influenza pandemic, with costs associated with purchasing drugs and vaccines, as well as conducting disaster drills and improving border control strategies.

[0011] Current treatment options for influenza include vaccination and chemotherapy or chemoprevention using antiviral drugs. Vaccination against influenza using influenza vaccines is generally recommended for high-risk groups such as children and the elderly, or those with asthma, diabetes, or heart disease. However, it is possible to contract influenza even after vaccination. The vaccines are reformulated each season against several specific influenza virus strains, but may not include all the virus strains that are effectively infecting the population worldwide during that season. Manufacturers spend approximately six months formulating and producing the millions of doses needed to combat seasonal epidemics; occasionally, new or overlooked virus strains become prominent during that period and infect vaccinated individuals (such as the H3N2 Fujian influenza during the 2003-2004 influenza season). It is also possible to be infected just before vaccination and with the specific virus strain that the vaccine was originally supposed to prevent, as the vaccine takes approximately two weeks to become effective.

[0012] In addition, the effectiveness of these influenza vaccines is variable. Due to the high mutation rate of the virus, specific influenza vaccines generally provide protection for no more than a few years. The vaccine formulated for a particular year may be ineffective the following year because the influenza virus changes rapidly over time and different virus strains become dominant.

[0013] Due to the absence of RNA proofreading enzymes, the RNA-dependent RNA polymerase of influenza vRNA makes a single nucleotide insertion error approximately once every ten thousand nucleotides (which is the approximate length of influenza vRNA). Thus, almost every newly produced influenza virus is a mutant with antigenic drift. If more than one virus lineage infects a single cell, then the separation of the genome into eight independent segments of vRNA allows for vRNA mixing or reassortment. The resulting rapid changes in viral genetics produce antigenic shift and allow the virus to infect new host species and quickly overcome protective immunity.

[0014] Antiviral drugs can also be used to treat influenza, with NA inhibitors being particularly effective, but the virus can develop resistance to approved NA antiviral drugs. Similarly, the emergence of multi-drug resistant pandemic influenza A viruses has been well documented. Drug-resistant pandemic influenza A has become a major public health threat. In addition to drug-resistant influenza A viruses, NA inhibitors are also approved for the treatment of early influenza infections (within 48 hours of the onset of influenza symptoms).

[0015] Accordingly, there is a need for a formulation of an antiviral agent against influenza virus, which can be administered by pulmonary delivery. SUMMARY OF THE INVENTION

[0016] Provided herein are formulations of Compound 1 and a bulking agent. In some cases, the formulation comprises (a) Compound 1 or a pharmaceutically acceptable salt thereof; and (b) a bulking agent. In various cases, the formulation consists essentially of: (a) Compound 1 or a pharmaceutically acceptable salt thereof; and (b) a bulking agent. In various cases, the formulation is a powder formulation for inhalation administration, which comprises (a) Compound 1 or a pharmaceutically acceptable salt thereof; and (b) a bulking agent consisting essentially of lactose monohydrate, wherein the formulation has a particle size distribution characterized by a volume median diameter (VMD) of 1 to 2 µm, wherein D 10 is 0.5 µm to 0.7 µm, D 50 is 1 µm to 1.4 µm and D 90 is 2.5 µm to 2.8 µm. In some cases, the VMD is 1.5 µm, wherein D 10 is 0.6 µm, D 50 is 1.3 µm, and D 90 is 2.8 µm.

[0017] In various embodiments, the bulking agent comprises lactose, or particularly lactose monohydrate. In some cases, the bulking agent is micronized. The volume median diameter (VMD) of the bulking agent can be 0.5 µm to 10 µm. In some cases, the VMD of the bulking agent is 1.5 to 5 µm.

[0018] In various embodiments, Compound 1 or its salt is micronized. Compound 1 can be crystalline (in crystal form), and in some cases, exists in a micronized crystal form. In some cases, the crystal form of Compound 1 is Form B and has an X-ray powder diffraction (XRPD) pattern presenting 2θ values of 5.6, 6.8, 8.4, 10.1, 10.6, 11.3, 15.1, 15.8, 18.0, 18.5, 19.1, 20.4 and 20.9 ± 0.2°. In various cases, the melting point of Compound 1 (e.g., Form B) is 280°C to 283°C. In various cases, Compound 1 can be in Form A or Form C.

[0019] The volume median diameter (VMD) of Compound 1 or its salt can be 0.5 µm to 10 µm. In some cases, the VMD of Compound 1 is 1.5 to 5 µm. The formulations disclosed herein can have a weight ratio of Compound 1 or its salt to the bulking agent of 1:3 to 1:5. In some cases, the weight ratio is 1:4.

[0020] The formulations disclosed herein can be adapted as inhalation formulations. It is contemplated to be a formulation for delivering Compound 1 or a salt thereof to an individual by inhalation. When the formulations disclosed herein are administered by inhalation, they can provide a drug concentration in the lung that is at least 50 times the drug concentration in the plasma at 1 hour after inhalation. In various cases, the drug concentration in the lung is at least 100 times the drug concentration in the plasma at 1 hour after inhalation. In various cases, at 24 hours after inhalation, the drug concentration in the lung is at least 50 times the drug concentration in the plasma. In various cases, at 24 hours after inhalation, the drug concentration in the lung is at least 100 times the drug concentration in the plasma. In various cases, at 48 hours after inhalation, the drug concentration in the lung is at least 50 times the drug concentration in the plasma. In various cases, at 48 hours after inhalation, the drug concentration in the lung is at least 100 times the drug concentration in the plasma.

[0021] The present disclosure further provides a method of treating or preventing influenza virus infection or replication in an individual in need thereof, which comprises administering to the individual a formulation as disclosed herein.

[0022] Also provided is a method of preparing a formulation as disclosed herein by the following steps: (a) micronizing Compound 1 or a salt thereof to form particles of Compound 1; (b) optionally micronizing a filler to form particles of the filler; and (c) blending the micronized Compound 1 or a salt thereof with the optionally micronized filler to form a formulation. In various cases, the micronization of Compound 1 or a salt thereof or the filler is carried out by manual grinding or jet milling.

[0023] In various cases, the method can further comprise crystallizing Compound 1 or a salt thereof prior to micronization. In some cases, crystallization comprises mixing Compound 1 or a salt thereof with ethanol at a temperature of at least 50 °C, cooling to room temperature to allow Compound 1 or a salt thereof to crystallize, collecting the crystals by filtration, and optionally drying the crystals prior to micronization. The mixing temperature can be 75 °C. In some instances, the mixing is carried out for 4 to 10 hours.

[0024] The present disclosure further provides a crystalline form of Compound 1. In some cases, Compound 1 is in Form B, and the crystals can exhibit an X-ray powder diffraction (XRPD) pattern with 2θ values of 5.6, 6.8, 8.4, 10.1, 10.6, 11.3, 15.1, 15.8, 18.0, 18.5, 19.1, 20.4 and 20.9 ± 0.2°. In some cases, Form B has an XRPD substantially as Figure 1 shown. In various cases, the melting point of Form B is 280 °C to 283 °C. In some cases, Compound 1 is in Form C, and the crystals can exhibit an XRPD pattern (mid-spectrum) substantially as Figure 3 shown. Description of the Drawings

[0025] Figure 1 Show the XRPD (X-ray powder diffraction) pattern of crystalline Compound 1 in Form B.

[0026] Figure 2 Show the DSC (differential scanning calorimetry) thermogram of crystalline Compound 1 in Form B.

[0027] Figure 3 Show the XRPD pattern (mid-spectrum) of crystalline Compound 1 in Form C.

[0028] Figure 4 Show the comparison of XRPD patterns of Forms C, C, B, B, and A (from top to bottom) of crystalline Compound 1 formed by the slurry method.

[0029] Figure 5 Show the comparison of XRPD patterns of Forms C, E, and A (from top to bottom) of crystalline Compound 1 formed by the anti-solvent method. Detailed Description

[0030] The present disclosure relates to compositions of anti-influenza compounds and the use of these compositions in inhibiting influenza virus activity. In some aspects, the present disclosure generally relates to the use of the compositions described herein for inhibiting the replication of influenza virus in a biological sample or a patient, for reducing the amount of influenza virus in a biological sample or a patient (reducing virus titer), and for treating or preventing influenza in a patient. The compositions disclosed herein can be used, for example, for administration to the lungs of an individual, patient, or host by inhalation.

[0031] The compositions disclosed herein are suitable as a therapy for anti-influenza virus infection. Thus, in some aspects, there is provided the use of a therapeutically effective amount of a composition as disclosed herein for treating or preventing influenza virus infection or replication in a human patient. For example, the influenza virus can be a pandemic or drug-resistant pandemic / seasonal influenza virus.

[0032] In various cases, there is provided a method of inhibiting the endonuclease activity of influenza polymerase in influenza A or B virus, which comprises contacting the virus with a composition as disclosed herein. In some cases, there is provided a method of treating or preventing influenza A or B infection in a host, which comprises administering a therapeutically effective amount of a composition as disclosed herein to the host. In various cases, there is provided a method for reducing the endonuclease activity of influenza polymerase in influenza A or B virus in a host, which comprises administering a therapeutically effective amount of a composition as disclosed herein to the host. In some cases, there is provided a method for reducing the replication of influenza virus in a host, which comprises administering a therapeutically effective amount of a composition as disclosed herein to the host.

[0033] Compound 1

[0034] The compositions disclosed herein particularly include 3-(2-(5-chloro-1H-pyrrolo[2,3-b]pyridin-3-yl)-5-fluoro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)bicyclo[2.2.2]octane-2-carboxylic acid, alternatively referred to herein as "Compound 1". It is believed that the active moiety of Compound 1 is a CAP-binding PB2 domain inhibitor.

[0035] Compound 1 may exist in free form or, where appropriate, in the form of a salt. Pharmaceutically acceptable salts are of particular interest because they are suitable for administering the compounds described as components of the combinations for medical purposes. Non-pharmaceutically acceptable salts are suitable for manufacturing processes, for separation and purification purposes, and in some cases, for separating stereoisomeric forms of the compounds or intermediates described herein.

[0036] As used herein, the term "pharmaceutically acceptable salt" refers to salts of compounds that are within the scope of sound medical judgment, suitable for contact with the tissues of humans and lower animals without undue side effects (such as toxicity, irritation, allergic reactions, etc.) and commensurate with a reasonable benefit / risk ratio.

[0037] Pharmaceutically acceptable salts are well known in the art. For example, pharmaceutically acceptable salts are described in detail by S. M. Berge et al. in Journal of Pharmaceutical Sciences, 1977, 66, 1-19, which is incorporated herein by reference. Pharmaceutically acceptable salts of the compounds described herein include salts derived from suitable inorganic and organic acids and bases. These salts can be prepared in situ during the final isolation and purification of the compounds.

[0038] In the case where the compounds described herein contain a basic group or a bioisostere that is sufficiently basic, acid addition salts can be prepared by 1) reacting the purified compound in free base form with a suitable organic or inorganic acid and 2) isolating the salt thus formed. In practice, acid addition salts may be the more convenient form to use, and the use of such salts is equivalent to the use of the free base form.

[0039] Examples of pharmaceutically acceptable non-toxic acid addition salts are salts formed by reacting the amino group with an inorganic acid such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid, or with an organic acid such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid, or salts formed by using other methods such as ion exchange used in the art. Other pharmaceutically acceptable salts include adipates, alginates, ascorbates, aspartates, benzenesulfonates, benzoates, bisulfates, borates, butyrates, camphorates, camphorsulfonates, citrates, cyclopentanepropionates, digluconates, dodecylsulfates, ethanesulfonates, formates, fumarates, glucoheptanoates, glycerophosphates, glycolates, glucuronates, glucopyranosides, heptanoates, hexanoates, hydrochlorides, hydrobromides, hydroiodides, 2-hydroxyethanesulfonates, lactates, lactobionates, laurates, lauryl sulfates, malates, maleates, malonates, methanesulfonates, 2-naphthalenesulfonates, nicotinates, nitrates, oleates, oxalates, palmitates, pamoates, pectates, persulfates, 3-phenylpropionates, phosphates, picrates, pivalates, propionates, salicylates, stearates, succinates, sulfates, tartrates, thiocyanates, p-toluenesulfonates, undecanoates, valerates, etc.

[0040] In cases where the compounds described herein contain a carboxylic acid group or a bioisostere with sufficient acidity, the base addition salts can be prepared by 1) reacting the purified compound in acid form with a suitable organic or inorganic base and 2) isolating the salt thus formed. In fact, the use of base addition salts may be more convenient, and the use of the salt form is essentially equivalent to the use of the free acid form. Salts derived from suitable bases include alkali metal (e.g., sodium, lithium and potassium) salts, alkaline earth metal (e.g., magnesium and calcium) salts, ammonium salts and N + (C 1-4 alkyl) 4 salts. This disclosure also contemplates the quaternization of any basic nitrogen-containing group of the compounds disclosed herein. Water-soluble or oil-soluble or dispersible products can be obtained by such quaternization.

[0041] The base addition salts include pharmaceutically acceptable metal salts and ammonium salts. Suitable metal salts include sodium, potassium, calcium, barium, zinc, magnesium and aluminum. Sodium salts and potassium salts are generally preferred. Where appropriate, other pharmaceutically acceptable salts include non-toxic ammonium, quaternary ammonium and amine cations formed using counter ions such as halide ions, hydroxide, carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate and aryl sulfonate. Suitable inorganic base addition salts are prepared from metal bases, which include sodium hydride, sodium hydroxide, potassium hydroxide, calcium hydroxide, aluminum hydroxide, lithium hydroxide, magnesium hydroxide, zinc hydroxide, etc. Suitable amine base addition salts are often used in amine preparations in pharmaceutical chemistry due to their low toxicity and acceptability for medical use. Ammonia, ethylenediamine, N-methyl-glucosamine, lysine, arginine, ornithine, choline, N,N'-dibenzylethylenediamine, chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine, diethylamine, piperazine, tris(hydroxymethyl)-aminomethane, tetramethylammonium hydroxide, triethylamine, dibenzylamine, ephenamine, dehydroabietylamine, N-ethylpiperidine, benzylamine, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, ethylamine, basic amino acids, dicyclohexylamine, etc.

[0042] Other acids and bases can be used to prepare salts that are suitable as intermediates in obtaining the compounds described herein and their pharmaceutically acceptable acid or base addition salts when they themselves are not pharmaceutically acceptable.

[0043] The components of the combination can exist in the form of solvates. The term "solvate" refers to a molecular complex of a compound (including its salts) with one or more solvent molecules. These solvent molecules are solvent molecules known to be harmless to recipients and commonly used in pharmaceutical technology, such as water, ethanol, dimethyl sulfoxide, acetone and other common organic solvents. The term "hydrate" refers to a molecular complex including a compound and water.

[0044] Compound 1 or its salt or its solvate can be micronized for use in the compositions disclosed herein. Micronization refers to a solid form having particles smaller than 15 µm. In various cases, Compound 1 or its salt or its solvate can exist in the form of particles of 0.5 µm to 10 µm, such as 1 µm to 10 µm, 2 µm to 10 µm, 3 µm to 10 µm, 4 µm to 10 µm, 5 µm to 10 µm, 6 µm to 10 µm, 1 µm to 7 µm, 2 µm to 7 µm, 3 µm to 7 µm, 2 µm to 6 µm, 2 µm to 5 µm, 3 µm to 7 µm or 3 µm to 6 µm.

[0045] Compound 1 or its salt or solvate can be micronized using any known technique. In some cases, micronization is carried out by jet milling or hand grinding.

[0046] Compound 1 can exist in crystalline form in the disclosed compositions.

[0047] Form B: In various cases, the crystalline form can be characterized by X-ray powder diffraction, such as the crystalline form obtained as described in the examples, which has peaks at 2θ of approximately 5.6, 6.8, 8.4, 10.1, 10.6, 11.3, 15.1, 15.8, 18.0, 18.5, 19.1, 20.4 and 20.9 ± 0.2° using Cu Kα radiation, and is referred to as "Form B". In some embodiments, crystalline Compound 1 can be substantially characterized by the X-ray powder diffraction pattern as depicted in Figure 1 wherein "substantially" means that the reported peaks can vary by approximately ±0.2°. It is well known in the field of XRPD that although the relative peak heights in the pattern depend on multiple factors such as sample preparation and instrument geometry, the peak positions are relatively insensitive to experimental details.

[0048] In some cases, crystalline Compound 1 can be characterized by, for example, a differential scanning calorimetry (DSC) thermogram substantially as shown in Figure 2 In some cases, the melting point of crystalline Compound 1 is from 280 °C to 283 °C or about 282 °C.

[0049] Form A: In various cases, the crystalline form can be characterized by X-ray powder diffraction, such as the crystalline form obtained as described in the examples, which has 2θ peaks substantially as shown in Figure 4 and is referred to as "Form A". "Substantially" means that the reported peaks can vary by approximately ±0.2°. It is well known in the field of XRPD that although the relative peak heights in the pattern depend on multiple factors such as sample preparation and instrument geometry, the peak positions are relatively insensitive to experimental details.

[0050] Form C: In various cases, the crystalline form can be characterized by X-ray powder diffraction, such as the crystalline form obtained as described in the examples, which has 2θ peaks (mid-spectrum) substantially as shown in Figure 3 and is referred to as "Form C". "Substantially" means that the reported peaks can vary by approximately ±0.2°. It is well known in the field of XRPD that although the relative peak heights in the pattern depend on multiple factors such as sample preparation and instrument geometry, the peak positions are relatively insensitive to experimental details.

[0051] Compound 1 can also exist as Form D or Form E, as discussed in the following Examples section.

[0052] Filler

[0053] The compositions disclosed herein include fillers. The fillers can include microcrystalline cellulose, dicalcium phosphate, lactose (including lactose monohydrate), trehalose, sucrose, mannose, mannitol, sorbitol, calcium carbonate, starch, and magnesium stearate or zinc stearate. In some cases, the filler is one or more of lactose, glucose, and sodium starch glycolate. In some cases, the filler includes lactose, such as lactose monohydrate. In some cases, the filler is crystalline lactose monohydrate, such as Inhalac®, for example Inhalac® 400.

[0054] The filler can be micronized for use in the compositions disclosed herein. Micronization refers to a solid form having particles smaller than 15 μm. In various cases, the filler can be in the form of particles from 0.5 µm to 10 µm, such as 1 µm to 10 µm, 2 µm to 10 µm, 3 µm to 10 µm, 4 µm to 10 µm, 5 µm to 10 µm, 6 µm to 10 µm, 1 µm to 7 µm, 2 µm to 7 µm, 3 µm to 7 µm, 2 µm to 6 µm, 2 µm to 5 µm, 3 µm to 7 µm, or 3 µm to 6 µm.

[0055] Any known technique can be used to micronize the filler. In some cases, micronization is carried out by jet milling or hand milling.

[0056] In various cases, the compositions disclosed herein include Compound 1 and the filler in a weight ratio of 1:3 to 1:5. In various cases, the weight ratio is about 1:4.

[0057] Pulmonary Administration and Devices

[0058] In some embodiments, the compositions described herein are suitable for administration to the lower respiratory tract (e.g., the lungs) directly through the airway by inhalation. The compositions administered by inhalation can be inhalable powders and can be administered using a powder inhaler device. These devices are well known.

[0059] Inhalable compositions can be formulated for unit dose or multi-dose delivery. For example, the compositions can be formulated for multi-dose delivery in a manner similar to that described in the following documents: GB 2242134, U.S. Patent Nos. 6,632,666, 5,860,419, 5,873,360, and 5,590,645 (all describing the "Diskus" device); or GB2178965, GB2129691, GB2169265, U.S. Patent Nos. 4,778,054, 4,811,731, and 5,035,237 (which describe the "Diskhaler" device); or EP 69715 (the "Turbuhaler" device) or GB 2064336 and U.S. Patent No. 4,353,656 (the "Rotahaler" device). The multi-doses can be stored in a reservoir or as multiple individually encapsulated doses in, for example, blisters or capsules. Examples of suitable devices include, but are not limited to: TURBUHALER (AstraZeneca), CLICKHALER (Innovata Biomed), EASYHALER (Orion), ACCUHALER, DISKUS, DISKHALER, ROTAHALER (GlaxoSmithKline), HANDIHALER, INHALATOR, AEROHALER (Boehringer Ingelheim), AEROLIZER (Schering Plough), and NOVOLIZER (ASTA Medica).

[0060] After administration, for example by inhalation, the compositions disclosed herein exhibit high levels of drug exposure in the lung compared to the exposure in plasma. These high levels of drug exposure are beneficial for several reasons. First, pulmonary administration enables rapid delivery of the therapeutic agent to the site of infection. Second, maintaining the therapeutic agent in the lung while minimizing plasma exposure reduces systemic adverse events because very little of the therapeutic agent will move away from the site of infection. Third, concentrating the exposure at the lung allows for maximization of the therapeutic benefit at the site of infection.

[0061] In some cases, the compositions disclosed herein are administered by inhalation such that the exposure of Compound 1 provided in the lung after 1 hour is 50 times the exposure in plasma. In various cases, after 1 hour, the exposure in the lung is 60 times, or 70 times, or 80 times, or 90 times, or 100 times, or 125 times, or 150 times the exposure in plasma.

[0062] In some cases, the compositions disclosed herein are administered by inhalation such that the exposure of Compound 1 provided in the lung after 24 hours is 50 times the exposure in plasma. In various cases, after 24 hours, the exposure in the lung is 60 times, or 70 times, or 80 times, or 90 times, or 100 times, or 125 times, or 150 times the exposure in plasma.

[0063] In some cases, the compositions disclosed herein are administered by inhalation such that the exposure of Compound 1 provided in the lung after 48 hours is 50 times the exposure in plasma. In various cases, after 48 hours, the exposure in the lung is 60 times, or 70 times, or 80 times, or 90 times, or 100 times, or 125 times, or 150 times the exposure in plasma.

[0064] In various cases, even 4 days after administration by inhalation, the exposure of Compound 1 in the lung is still at least 100 times the exposure in plasma.

[0065] Method of Use

[0066] The compositions described herein can be used to reduce the viral titer in a biological sample (such as an infected cell culture) or the viral titer in a human body (such as the viral titer in the lungs of a patient).

[0067] As used herein, the terms "influenza virus-mediated condition", "influenza infection", or "influenza" can be used interchangeably to mean a disease caused by influenza virus infection.

[0068] Influenza is an infectious disease that affects birds and mammals and is caused by influenza viruses. Influenza viruses are RNA viruses of the family Orthomyxoviridae, which includes five genera: influenza A virus, influenza B virus, influenza C virus, infectious salmon anemia virus, and Thogoto virus. The genus Influenza A virus has one species, namely influenza A virus, which can be further divided into different serotypes based on the reaction of antibodies to these viruses: H1N1, H2N2, H3N2, H5N1, H7N7, H1N2, H9N2, H7N2, H7N3, H7N9, and H10N7. The genus Influenza B virus has one species, namely influenza B virus. Influenza B almost exclusively infects humans and is less common than influenza A. The genus Influenza C virus has one species, namely influenza C virus, which infects humans and pigs and can cause severe disease and local epidemics. However, influenza C virus is less common compared to other types and generally seems to cause mild disease in children.

[0069] In some embodiments, influenza or an influenza virus is associated with an influenza A or B virus. In some embodiments, influenza or an influenza virus is associated with an influenza A virus. In some specific embodiments, the influenza A virus is H1N1, H2N2, H3N2, H7N9, or H5N1. In some embodiments, the disclosed combination can effectively inhibit the growth or replication of pandemic or drug-resistant pandemic / seasonal influenza viruses.

[0070] For humans, common symptoms of influenza are chills, fever, pharyngitis, muscle pain, severe headache, cough, weakness, and general malaise. In more severe cases, influenza causes pneumonia, which can be fatal, especially for young children and the elderly. Although influenza is often confused with the common cold, influenza is a much more severe disease and is caused by different types of viruses. Influenza can cause nausea and vomiting, especially in children, but these symptoms are more characteristic of unrelated gastroenteritis, which is sometimes called "stomach flu" or "24-hour flu".

[0071] The symptoms of influenza can start rather suddenly one to two days after infection. Typically, the first symptom is chills or shivering, but fever is also common early in infection, with body temperatures ranging from 38°C to 39°C (about 100°F to 103°F). Many people are so ill that they are bedridden for several days, with general body aches and discomfort, which are more severe in their back and legs. The symptoms of influenza can include: body aches (especially joints and throat), extreme cold and fever, fatigue, headache, eye irritation with tearing, red eyes, redness of the skin (especially the face), redness of the mouth, redness of the throat and nose, abdominal pain (in children with influenza B). The symptoms of influenza are not specific and overlap with those of many pathogens ("influenza-like illnesses"). Typically, laboratory data are needed to confirm the diagnosis.

[0072] The terms "disease", "disorder", and "condition" may be used interchangeably herein to refer to influenza virus-mediated medical or pathological conditions.

[0073] As used herein, the terms "individual", "host", and "patient" may be used interchangeably. The terms "individual", "host", and "patient" refer to an animal (e.g., a bird such as a chicken, quail, or turkey, or a mammal), and more particularly to a mammal, such as a non-primate (e.g., a cow, pig, horse, sheep, rabbit, guinea pig, rat, cat, dog, or mouse) or a primate (e.g., a monkey, chimpanzee, or human), and even more particularly to a human. In some embodiments, the individual is a non-human animal, such as a domestic animal (e.g., a horse, cow, pig, or sheep) or a pet (e.g., a dog, cat, guinea pig, or rabbit). In a preferred embodiment, the individual is a human.

[0074] As used herein, the term "biological sample" includes, but is not limited to: cell cultures or extracts thereof; biopsy material or extracts thereof obtained from a mammal; blood, saliva, urine, feces, semen, tears or other body fluids or extracts thereof.

[0075] As used herein, the term "inhibiting influenza virus replication" includes both reducing the amount of virus replication (e.g., reducing by at least 10%) and completely suppressing virus replication (i.e., reducing the amount of virus replication by 100%). In some embodiments, influenza virus replication is inhibited by at least 50%, at least 65%, at least 75%, at least 85%, at least 90% or at least 95%.

[0076] Influenza virus replication can be measured by any suitable method known in the art. For example, the influenza virus titer in a biological sample (e.g., an infected cell culture) or the influenza virus titer in a human (e.g., the lung virus titer in a patient) can be measured. More specifically, for cell-based assays, under various conditions where cells are cultured in vitro, the virus is added to the culture in the presence or absence of a test agent, and the virus-dependent endpoint is evaluated after an appropriate length of time. For a typical assay, Madin-Darby canine kidney cells (MDCK) and an influenza virus strain A / Puerto Rico / 8 / 34 adapted to standard tissue culture can be used. The first type of cell assay that can be used depends on the death of the infected target cells, which is a process called the cytopathic effect (CPE), in which virus infection causes depletion of cellular resources and eventual lysis of the cells. In the first type of cell assay, a smaller proportion of the cells in the wells of a microtiter plate are infected (usually 1 / 10 to 1 / 1000), allowing the virus to undergo several rounds of replication over 48 to 72 hours, and then the amount of cell death is measured using the decrease in cellular ATP content compared to an uninfected control. The second type of cell assay that can be employed depends on the proliferation of virus-specific RNA molecules in the infected cells, where the branched-chain DNA hybridization method (bDNA) is used to directly measure the RNA content. In the second type of cell assay, a lower number of cells are initially infected in the wells of a microtiter plate, allowing the virus to replicate in the infected cells and spread to additional rounds of cells, and then the cells are lysed and the virus RNA content is measured. This assay is typically stopped prematurely after 18 to 36 hours, when all target cells are still viable. The virus RNA is quantified by hybridization to specific oligonucleotide probes immobilized in the wells of the assay plate, followed by amplification of the signal by hybridization to additional probes linked to a reporter enzyme.

[0077] As used herein, "viral titer" or "titer" is a measure of the concentration of a virus. Titer testing can use serial dilutions to obtain approximate quantitative information from an analytical procedure that is essentially only positive or negative. The titer corresponds to the highest dilution factor that still gives a positive reading; for example, a positive reading conversion in the first 8 serial two-fold dilutions is a titer of 1:256. A specific example is viral titer. To determine the titer, a number of dilutions will be prepared, such as 10 -1 、10 -2 、10 -3 、……、10 -8 . The lowest virus concentration that still infects cells is the viral titer.

[0078] As used herein, the terms "treat / treatment / treating" refer to both therapeutic treatment and prophylactic treatment. For example, therapeutic treatment includes alleviating or improving the progression, severity, and / or duration of an influenza virus-mediated condition, or improving one or more symptoms of an influenza virus-mediated condition (specifically, one or more distinguishable symptoms), caused by the administration of one or more therapies (such as one or more therapeutic agents, such as the compounds or compositions described herein). In a specific embodiment, therapeutic treatment includes improving at least one measurable physical parameter of an influenza virus-mediated condition. In other embodiments, therapeutic treatment includes physically inhibiting the progression of an influenza virus-mediated condition, such as by stabilizing distinguishable symptoms, and physiologically, such as by stabilizing physical parameters, or both. In other embodiments, therapeutic treatment includes alleviating or stabilizing an influenza virus-mediated infection. Antiviral drugs can be used in a community setting to treat people who already have influenza, to reduce the severity of symptoms and reduce the number of days they are ill.

[0079] As used herein, the terms "prevent", "prophylactic", "prophylactic use", and "prophylactic treatment" refer to any medical or public health procedure aimed at preventing rather than treating or curing a disease. As used herein, the term "prevent / prevention / preventing" refers to reducing the risk of developing or acquiring a given condition, or reducing or inhibiting the recurrence of a condition in an individual who is not ill but has become or may be close to becoming an ill person. The term "chemoprevention" refers to the prevention of a condition or disease using a drug, such as a small molecule drug (as opposed to a vaccine).

[0080] As used herein, prophylactic use includes use in situations where an outbreak has been detected to prevent the infection from spreading or transmitting in places where a large number of people at high risk of severe influenza complications live in close contact with each other. It also includes use in populations that need to be protected from influenza infection but have not received protection after vaccination (e.g., due to a weakened immune system), in populations where vaccines are not available, or in populations that cannot receive vaccines due to side effects. It also includes use during the two-week period after vaccination or at any time after vaccination but before the vaccine becomes effective. Prophylactic use may also include treating individuals who do not have influenza or are not considered to be at high risk of complications to reduce the chance of infecting influenza and transmitting it to high-risk individuals (e.g., healthcare workers, nursing home workers, etc.) who are in close contact with him / her.

[0081] As used herein and consistent with the practice of the United States Centers for Disease Control and Prevention (US CDC), an influenza "outbreak" is defined as a sharp increase in acute febrile respiratory illness (AFRI) relative to the normal background rate or any individual in the analyzed group testing positive for influenza within a 48- to 72-hour period in a population in close proximity to each other (e.g., in the same area of an assisted living facility, in the same household, etc.).

[0082] In some embodiments, the composition is suitable for prophylactic or therapeutic measures for patients (especially humans) who are prone to complications caused by influenza virus infection. The composition can be applied in a therapeutic method in the case of a confirmed index case or outbreak to prevent the infection from spreading in the community or the rest of the population.

[0083] As used herein, an "effective amount" means an amount sufficient to elicit a desired biological response. In the present disclosure, the desired biological response is to inhibit the replication of influenza virus, reduce the amount of influenza virus, or alleviate or improve the severity, duration, progression, or onset of influenza virus infection, prevent the development of influenza virus infection, prevent the recurrence, development, onset, or progression of symptoms associated with influenza virus infection, or enhance or improve the prophylactic or therapeutic effect of another therapy used against influenza infection. The precise amount of the compound administered to an individual will depend on the mode of administration, the type and severity of the infection, and the characteristics of the individual, such as general health status, age, sex, weight, and tolerance. Those skilled in the art will be able to determine an appropriate dosage based on these and other factors. For example, 3-(2-(5-chloro-1H-pyrrolo[2,3-b]pyridin-3-yl)-5-fluoro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)bicyclo[2.2.2]octane-2-carboxylic acid or a pharmaceutically acceptable salt or solvate thereof can be administered to an individual in a dosage range of from about 0.01 to 100 mg / kg body weight / day.

[0084] As used herein, a "safe and effective amount" of a compound or composition described herein is an effective amount of the compound or composition that does not cause excessive or harmful side effects in a patient.

[0085] In general, a dosing regimen can be selected based on a variety of factors, including: the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, weight, general health status, sex, and diet of the patient; the time of administration, route of administration, and excretion rate of the specific compound employed; the renal and hepatic function of the individual; and the duration of treatment with the specific compound or its salt, the drugs used in combination with or concomitantly with the specific compound employed; and similar factors well known in the medical arts. Those skilled in the art can readily determine and prescribe a safe and effective amount of the compounds described herein required for the treatment, prevention, inhibition (fully or in part), or arrest of the development of a disease.

[0086] The dosage of Compound 1 can range from about 0.01 to about 100 mg / kg body weight / day, from about 0.01 to about 50 mg / kg body weight / day, from about 0.1 to about 50 mg / kg body weight / day, or from about 1 to about 25 mg / kg body weight / day. It is to be understood that the total daily amount can be administered in a single dose, or can be administered in multiple doses, such as twice a day (e.g., every 12 hours), three times a day (e.g., every 8 hours), or four times a day (e.g., every 6 hours).

[0087] For therapeutic treatment, Compound 1 can be administered to a patient within, for example, 48 hours (or within 40 hours, or less than 2 days, or less than 1.5 days or within 24 hours) of the onset of symptoms such as nasal congestion, sore throat, cough, pain, fatigue, headache, and chills / sweats. The therapeutic treatment can continue for any suitable duration, such as 5 days, 7 days, 10 days, 14 days, etc. For prophylactic treatment during a community outbreak, Compound 1 can be administered to a patient within, for example, 2 days of the onset of symptoms of the first case, and can continue for any suitable duration, such as 7 days, 10 days, 14 days, 20 days, 28 days, 35 days, 42 days, etc.

[0088] Example

[0089] Polymorph Screening of Compound 1 (Slurry Method): Approximately 10 mg of Compound 1 was added to 200 µL of each of the following solvents: methyl tert-butyl ether (MTBE), methanol (MeOH), ethanol (EtOH), isopropanol (IPA), isopropanol and ethyl acetate in a volume ratio of 5 / 5 (IPA / EtOAc), ethyl acetate (EtOAc), isopropanol and water in a volume ratio of 8 / 2, acetonitrile (ACN), acetone, and tetrahydrofuran (THF). Each suspension was stirred at 700 rpm at 40 °C for 24 hours. The residue of the compound was separated by centrifugation (10 minutes at 14,000 rpm) and further dried overnight in a vacuum oven at 30 °C. If a clear solution was retained, the solution was dried under vacuum to yield a dry solid. The dry solid was analyzed by XRPD and the form was specified. The results are shown in the table below.

[0090]

[0091] The XRPD pattern of the crystal was obtained using a Bruker D8 Advance instrument with the following parameters. The XRPD analysis results for type B are as Figure 1 shown. The results for type C are as Figure 3 shown. The results for types A, B, and C are as Figure 4 shown.

[0092]

[0093] The DSC of type B crystal was obtained using a TA Q2000 instrument with the following parameters, and the DSC results are as Figure 2 shown.

[0094]

[0095] Polymorph Screening of Compound 1 (Antisolvent Method):Weigh approximately 25 mg of Compound 1 into a glass bottle, then add 0.5 mL of dimethylacetamide (DMA) to obtain a clear solution with a concentration of 50 mg / mL. Then, add the anti-solvent dropwise to this solution while stirring at 700 rpm at room temperature. Then, collect the resulting crystals by centrifugation. Analyze the crystals by XRPD, and the results are as Figure 5 shown, where the top is Form E, the middle is Form C, and the bottom is Form A.

[0096]

[0097] Formation of Polymorph - Slurry at Different Temperatures: Weigh approximately 25 mg of Compound 1 into a glass bottle, then add 500 µL of different solvents. Stir the solution at 700 rpm at 55 °C or 25 °C for 3 days. Separate the residue of the compound by centrifugation (10 minutes at 10,000 rpm), and further dry it in a vacuum oven at 30 °C for 2 days. Analyze the dried solid by XRPD. An overview of the forms obtained under different conditions is shown below.

[0098]

[0099] Formation of Polymorphs at Different Temperatures: Weigh approximately 50 mg of Compound 1 into a glass bottle, then add 500 µL of different solvents. Then stir the solution at 700 rpm at 55 °C for 3 days. Separate the solid by centrifugation (10 minutes at 10,000 rpm), and further dry it in a vacuum oven at 30 °C for 2 days. Analyze the solid by XRPD.

[0100] Weigh approximately 25 mg of Compound 1 into a glass bottle, then store it at 25 °C / 60% RH or 40 °C / 75% RH for 1 week. Then analyze the sample by XRPD.

[0101] The XRPD results are summarized as follows.

[0102]

[0103] Formation of Polymorphs at Different Temperatures (Antisolvent): Weigh approximately 50 mg of Compound 1 into a glass bottle, then add 1 mL of DMA, and then perform sonication to obtain a clear solution. Then stir the solution at 700 rpm at 55 °C, and then add the anti-solvent under rapid precipitation or slow precipitation. Rapid precipitation: Rapidly add a certain amount of anti-solvent, and filter the solid within 1 hour. Slow precipitation: Slowly add a certain amount of anti-solvent, and filter the solid after slurrying for 3 days. Separate the resulting solid by centrifugation (10 minutes at 10,000 rpm), and further dry it in a vacuum oven at 30 °C for 2 days. Analyze the solid by XRPD. The XRPD results are shown below.

[0104]

[0105] Overview of the results of the polymorph study

[0106]

[0107]

[0108] Micronization of Compound 1: Crystalline compound 1 (Form B) was gradually added to jet milling, where the injector gas pressure was 4.5 bar and the milling gas pressure was 4 bar. The micronized product exhibited the same characteristic peaks as the compound before micronization. In addition, the DSC results confirmed that the continuous exothermic peak at 198.27 °C and the single endothermic peak at 280.40 °C before decomposition were the same as the peaks observed in the sample before micronization. The particle size distribution (PSD) results of the dry dispersion showed that the particle size of the micronized compound was VMD = 2.08 µm, D 10 = 0.65 µm, D 50 = 1.44 µm and D 90 = 4.21 µm.

[0109] Micronization of Lactose Monohydrate: Several lactose monohydrate substances were tested and characterized as outlined in the following table.

[0110]

[0111] Formulation Preparation: Crystalline compound 1 (Form B) was manually ground before mixing with lactose monohydrate (Inhalac 400) in a 1:4 ratio. The mixture was blended by manual grinding for 10 minutes. Subsequently, the mixture was jet milled under the following conditions: injector gas pressure 4.5 bar, milling gas pressure 4 bar. The PSD data of the dry dispersion confirmed that the particle size of the formulation was VMD = 1.52 µm, D 10 = 0.63 µm, D 50 = 1.26 µm and D 90 = 2.77 µm.

[0112] In vivo mouse PK study: To demonstrate the delivery of Compound 1 to the lungs via the inhalation route, a pharmacokinetic study was conducted in mice (BALB / C). Prior to sample collection, the mice were treated with a single dose of approximately 1 mg of the dry powder using an insufflator. Plasma and lung samples were collected at different time points, and the drug concentrations in the mouse lungs and plasma were determined. As shown in the table below, rapid accumulation of high concentrations of the drug in the lung tissue was observed via the inhalation route. In contrast, the drug concentration in the plasma was significantly lower than the drug concentration detected in the lungs. These data demonstrate that the delivery of Compound 1 can be effectively carried out by inhalation using the disclosed formulation, thereby allowing Compound 1 to come into contact with the respiratory tract, such as in the case of influenza infection. Interestingly, the drug content was still at least 100-fold higher than the therapeutic dose (anti-influenza efficacy, EC 50 0.1 - 3 nM) on the fourth day. These results further confirm the potential clinical use of Compound 1 dry powder for the treatment of influenza infection.

[0113]

Claims

1. A formulation, which comprises: (a) Compound 1 or a pharmaceutically acceptable salt thereof; and (b) A filler.

2. A formulation, which consists essentially of the following components: (a) Compound 1 or a pharmaceutically acceptable salt thereof; and (b) A filler.

3. A powder formulation for inhalation administration, which comprises: (a) Compound 1 or a pharmaceutically acceptable salt thereof; and (b)A filler substantially composed of lactose monohydrate, wherein the formulation has a particle size distribution characterized by a volume median diameter (VMD) of 1 to 2 µm, where D 10 is 0.5 µm to 0.7 µm, D 50 is 1 µm to 1.4 µm and D 90 is 2.5 µm to 2.8 µm.

4. The formulation according to claim 1 or 2, wherein the filler comprises lactose.

5. The formulation according to claim 4, wherein the filler comprises lactose monohydrate.

6. The formulation according to any one of claims 1 to 5, wherein the filler is micronized.

7. The formulation according to any one of claims 1 to 6, wherein Compound 1 or its salt is micronized.

8. The formulation according to any one of claims 1 to 7, wherein Compound 1 or its salt is crystalline.

9. The formulation according to claim 8, wherein the crystalline Compound 1 or its salt has an X-ray powder diffraction (XRPD) pattern presenting 2θ values of 5.6, 6.8, 8.4, 10.1, 10.6, 11.3, 15.1, 15.8, 18.0, 18.5, 19.1, 20.4 and 20.9 ± 0.2°.

10. The formulation according to claim 8 or 9, wherein the melting point of the crystalline Compound 1 or its salt is 280°C to 283°C.

11. The formulation according to any one of claims 8 to 11, wherein Compound 1 or its salt exists in a micronized crystalline form.

12. The formulation according to any one of claims 1 to 11, wherein the volume average particle size diameter of Compound 1 or its salt is 0.5 to 10 µm.

13. The formulation according to claim 12, wherein the volume average particle size diameter of Compound 1 or its salt is 1.5 µm to 5 µm.

14. The formulation according to any one of claims 1 to 13, wherein the volume average particle size diameter of the filler is 0.5 to 10 µm.

15. The formulation according to claim 14, wherein the volume average particle size diameter of the filler is 1.5 µm to 5 µm.

16. The formulation according to any one of claims 1 to 15, wherein the weight ratio of Compound 1 or its salt to the filler is 1:3 to 1:

5.

17. The formulation according to any one of claims 1 to 16, which is suitable as an inhalable formulation.

18. The formulation according to claim 17, wherein when administered by inhalation, the drug concentration of Compound 1 in the lung 1 hour after inhalation is at least 50 times the drug concentration of Compound 1 in the plasma.

19. The formulation according to claim 18, wherein the drug concentration of Compound 1 in the lung 1 hour after inhalation is at least 100 times the drug concentration of Compound 1 in the plasma.

20. The formulation according to any one of claims 17 to 19, wherein when administered by inhalation, the drug concentration of Compound 1 in the lung 24 hours after inhalation is at least 50 times the drug concentration of Compound 1 in the plasma.

21. The formulation according to claim 20, wherein the drug concentration of Compound 1 in the lung 24 hours after inhalation is at least 100 times the drug concentration of Compound 1 in the plasma.

22. The formulation according to any one of claims 17 to 21, wherein when administered by inhalation, the drug concentration of Compound 1 in the lung 48 hours after inhalation is at least 50 times the drug concentration of Compound 1 in the plasma.

23. The formulation according to claim 22, wherein the drug concentration of Compound 1 in the lung 48 hours after inhalation is at least 100 times the drug concentration of Compound 1 in the plasma.

24. A method for treating or preventing influenza virus infection or replication in an individual in need thereof, which comprises administering to the individual an amount of the formulation according to any one of claims 1 to 23 effective to treat said influenza infection.

25. A method for preparing the formulation according to any one of claims 1 to 23, which comprises (a) micronizing Compound 1 or a salt thereof to form particles of Compound 1; (b) optionally micronizing a filler to form particles of said filler; and (c) blending the micronized Compound 1 or a salt thereof with the optionally micronized filler to form said formulation.

26. The method according to claim 25, wherein the micronization of Compound 1 or the filler is carried out by jet milling or manual milling.

27. The method according to claim 26, wherein the micronization of Compound 1 or the filler is carried out by jet milling.

28. The method according to any one of claims 25 to 27, which further comprises crystallizing Compound 1 or a salt thereof before the micronization step.

29. The method according to claim 28, wherein the crystallization comprises mixing Compound 1 or a salt thereof with ethanol at a temperature of at least 50 °C, cooling to room temperature to allow Compound 1 or a salt thereof to crystallize, and collecting the crystals by filtration, and optionally drying the crystals before micronization.

30. The method according to claim 29, wherein Compound 1 or a salt thereof is mixed with ethanol at a temperature of 75 °C.

31. The method according to claim 30, wherein Compound 1 or a salt thereof is mixed with ethanol at a temperature of 75 °C for 4 to 10 hours.

32. A crystalline Compound 1, which is designated as Form B.

33. The crystalline Compound 1 according to claim 32, which has an X-ray powder diffraction (XRPD) pattern exhibiting 2θ values of 5.6, 6.8, 8.4, 10.1, 10.6, 11.3, 15.1, 15.8, 18.0, 18.5, 19.1, 20.4 and 20.9 ± 0.2°.

34. The crystalline Compound 1 according to claim 32 or 33, which has a melting point of 280 °C to 283 °C.

35. A crystalline Compound 1, which is designated as Form A.

36. A crystalline Compound 1, which is designated as Form C.

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