Formulation of Influenza Therapeutic Agent

Abstract

Provided herein are compositions of antiviral therapeutic agents for treating influenza that are suitable for administration by inhalation.

Description

Background Art

[0001] Influenza spreads worldwide as a seasonal epidemic, resulting in hundreds of thousands of deaths every year and millions of deaths in pandemic years. For example, in the 20th century, three influenza pandemics occurred, killing tens of millions of people, each of which was caused by the emergence of a new virus strain in humans. Often, these new strains are due to the spread of existing influenza viruses from other animal species to humans.

[0002] Influenza is mainly transmitted from person to person through large virus-containing droplets produced when an infected person coughs or sneezes. These large droplets can then settle on the upper respiratory mucosa surface 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 spread influenza to others from 1 day before symptoms appear until about 5 days after symptoms begin. Children and people with weakened immune systems can be infectious for more than 10 days after the onset of symptoms.

[0003] Influenza viruses are RNA viruses of the family Orthomyxoviridae, including five genera: influenza A virus, influenza B virus, influenza C virus, Thogotovirus, and Isavirus.

[0004] The genus Orthomyxovirus A is responsible for seasonal influenza and pandemic influenza outbreaks. The genus Orthomyxovirus A has one species, influenza A virus, and wild waterfowl are the natural hosts of a very wide variety of influenza A. Occasionally, the virus can infect other species and then cause devastating outbreaks in poultry or human influenza pandemics. Influenza A virus is the most infectious human pathogen among the three influenza types and causes the most severe diseases. Influenza A viruses can be further divided into various serotypes based on the antibody response to these viruses. The serotypes identified in humans are ranked by the number of known human pandemic deaths, H1N1 (which caused the Spanish influenza in 1918), H2N2 (which caused the Asian Influenza in 1957), H3N2 (which caused the Hong Kong Flu in 1968), H5N1 (the threat of a pandemic during the 2007 - 2008 influenza season), H7N7 (a potential pandemic threat), H1N2 (an endemic in humans and pigs), H9N2, H7N2, H7N3, and H10N7.

[0005] The genus Orthomyxovirus B is responsible for 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 two to three times slower than influenza A, resulting in low genetic diversity and only one influenza B serotype. As a result of the lack of antigenic diversity, some immunity to influenza B is usually acquired in early childhood. However, influenza B mutates sufficiently to make sustained immunity impossible. Combined with its limited host range (which prevents interspecies antigenic shift), this low rate of antigenic change ensures that influenza B does not cause pandemics.

[0006] The genus Influenza C virus has one species, Influenza C virus, which infects humans and pigs and can cause severe illness and local epidemics. However, Influenza C is less common than the other types and is generally thought to cause mild disease, usually in children.

[0007] Influenza viruses have very similar structures across serotypes and genera. The influenza virus genome consists of eight single-stranded RNAs packaged into rod-shaped structures of various sizes, known as ribonucleoprotein complexes (RNPs). Each RNP contains a unique viral RNA, multiple copies of the scaffold nucleoprotein, and a heterotrimeric viral polymerase consisting of the PA, PB1, and PB2 subunits that 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 the 5’ cap. Then, the PA subunit, which is an endonuclease subunit, cleaves 10 to 13 nucleotides of the captured pre-mRNA downstream of the cap. Subsequently, the PB2 subunit rotates approximately 70° to direct the capped primer towards the active site of the PB1 polymerase. The PB1 subunit interacts directly with both the PB2 and PA subunits. These subunits contain domains that are highly conserved among various influenza strains and have attracted attention as potential targets for anti-influenza drugs. 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 anti-viral drugs oseltamivir (Tamiflu®) and zanamivir (Relenza®). These drugs are sialic acid analogs that inhibit the enzymatic activity of NA and thus delay the release of progeny viruses from infected cells.

[0008] Influenza results in decreased productivity and direct costs associated with the consequent medical treatment, as well as indirect costs of preventive measures. In the United States, influenza is responsible for total costs of over $10 billion annually, but it is estimated that direct and indirect costs could reach hundreds of billions of dollars due to a future pandemic. The cost of prevention is also high. Governments around the world have spent billions of dollars on preparations and plans for a potential H5N1 avian influenza pandemic, involving the purchase of drugs and vaccines, as well as costs associated with the development of strategies for disaster training and improved border control.

[0009] Current treatment options for influenza include vaccination and chemotherapy or chemoprevention with antiviral drugs. Vaccination against influenza using the influenza vaccine is often recommended for high-risk groups such as children and the elderly, or people with asthma, diabetes, or heart disease. However, it is still possible to contract influenza even after vaccination. The vaccine is reformulated each flu season for some specific influenza strains, but it cannot include all the strains that are actively infecting people around the world during that season. It takes manufacturers about six months to formulate and produce the millions of doses needed to address seasonal epidemics. Sometimes, new or overlooked strains become prominent during that period and infect people despite vaccination (such as due to H3N2 Fujian influenza during the 2003 - 2004 influenza season). Since the vaccine takes about two weeks to become effective, it is also possible to be infected immediately before vaccination and become ill with the very strain that the vaccine is supposed to prevent.

[0010] Furthermore, the effectiveness of these influenza vaccines is variable. Due to the high mutation rate of the virus, certain influenza vaccines usually provide protection for only a few years. The influenza virus changes rapidly over time, and different strains become dominant, so a vaccine formulated for a particular year may become ineffective the following year.

[0011] Due to the absence of RNA proofreading enzymes, the RNA-dependent RNA polymerase of influenza vRNA introduces one nucleotide insertion error approximately every 10,000 nucleotides, which is the approximate length of influenza vRNA. Therefore, all newly produced influenza viruses exhibit mutant-antigenic drift. By separating the genome into eight separate segments of vRNA, it becomes possible to mix or reclassify vRNA when two or more virus strains infect a single cell. The resulting rapid change in viral genetics leads to antigenic shift, enabling the virus to infect new host species and rapidly overcome protective immunity.

[0012] Antiviral drugs can also be used to treat influenza, and NA inhibitors are particularly effective. However, the virus can develop resistance to approved NA antiviral drugs. The emergence of multi-drug resistant pandemic influenza A viruses has been well demonstrated. Drug-resistant pandemic influenza A poses a major public health threat. In addition to drug-resistant influenza A viruses, NA inhibitors are approved for the treatment of early (within 48 hours of the onset of influenza symptoms) influenza infections.

[0013] Therefore, there is a need for a formulation of an antiviral agent against influenza virus that can be administered via pulmonary delivery. SUMMARY OF THE INVENTION

[0014] This specification provides formulations of Compound 1 and fillers. In some cases, the formulation comprises (a) Compound 1 or a pharmaceutically acceptable salt thereof, and (b) a filler. In various cases, the formulation consists essentially of (a) Compound 1 or a pharmaceutically acceptable salt thereof, and (b) a filler. In various cases, the formulation is a powder formulation for inhalation comprising (a) Compound 1 or a pharmaceutically acceptable salt thereof and (b) a filler consisting essentially of lactose monohydrate, and the formulation has a volume median diameter (VMD) of 1 - 2 μm, D 10 of 0.5 μm - 0.7 μm, D 50 of 1 μm - 1.4 μm, and D 90 of 2.5 μm - 2.8 μm. In some cases, the VMD is 1.5 μm, D 10 is 0.6 μm 、 D 50 is 1.3 μm, and D 90 is 2.8 μm.

[0015] In various embodiments, the filler comprises lactose, and more specifically, lactose monohydrate. In some cases, the filler is micronized. The filler can have a volume median diameter (VMD) of 0.5 μm - 10 μm. In some cases, the filler has a VMD of 1.5 - 5 μm.

[0016] In various embodiments, Compound 1 or its salt is micronized. Compound 1 can crystallize (in a crystalline form) and in some cases exists as micronized crystals. In some cases, the crystalline form of Compound 1 is Form B and has an X-ray powder diffraction (XRPD) pattern showing 2θ values of 5.6 ± 0.2°, 6.8 ± 0.2°, 8.4 ± 0.2°, 10.1 ± 0.2°, 10.6 ± 0.2°, 11.3 ± 0.2°, 15.1 ± 0.2°, 15.8 ± 0.2°, 18.0 ± 0.2°, 18.5 ± 0.2°, 19.1 ± 0.2°, 20.4 ± 0.2°, and 20.9 ± 0.2°. In various cases, Compound 1 (e.g., as Form B) has a melting point of 280°C - 283°C. In various cases, Compound 1 can be in Form A or Form C.

[0017] Compound 1 or a salt thereof may have a volume median diameter (VMD) of 0.5 μm to 10 μm. In some cases, the VMD of Compound 1 is 1.5 to 5 μm. The formulations disclosed herein may have a weight ratio of Compound 1 or a salt thereof to the filler of 1:3 to 1:5. In some cases, the weight ratio is 1:4.

[0018] The formulations disclosed herein may be suitable as inhalation formulations. The formulations are contemplated as formulations for delivering Compound 1 or a salt thereof to a subject via inhalation. The formulations disclosed herein can provide a pulmonary drug concentration that is at least 50 times the plasma drug concentration 1 hour after inhalation upon administration by inhalation. In various cases, the pulmonary drug concentration is at least 100 times the plasma drug concentration 1 hour after inhalation. In various cases, the pulmonary drug concentration is at least 50 times the plasma drug concentration 24 hours after inhalation. In various cases, the pulmonary drug concentration is at least 100 times the plasma drug concentration 24 hours after inhalation. In various cases, the pulmonary drug concentration is at least 50 times the plasma drug concentration 48 hours after inhalation. In various cases, the pulmonary drug concentration is at least 100 times the plasma drug concentration 48 hours after inhalation.

[0019] Also provided herein is a method of treating or preventing influenza virus infection or replication in a subject in need thereof, the method comprising administering to the subject a formulation disclosed herein.

[0020] Also provided is a method of making a formulation disclosed herein by (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) combining the micronized Compound 1 or a salt thereof and, optionally, the micronized filler to form a formulation. In various cases, the micronization of Compound 1 or a salt thereof or the filler is performed via manual grinding or a jet mill.

[0021] In various cases, this method may further include crystallizing Compound 1 or a salt thereof before micronization. In certain cases, crystallization includes mixing Compound 1 or a salt thereof with ethanol at a temperature of at least 50 °C, cooling to room temperature to enable crystallization of Compound 1 or a salt thereof, collecting the crystals by filtration, and optionally drying the crystals before micronization. The temperature of the mixture can be 75 °C. In certain cases, the mixing occurs over a period of 4 to 10 hours.

[0022] Further provided herein is a crystalline form of Compound 1. In certain cases, Compound 1 is Form B, and the crystals can exhibit an X-ray powder diffraction (XRPD) pattern having 2θ values of 5.6 ± 0.2°, 6.8 ± 0.2°, 8.4 ± 0.2°, 10.1 ± 0.2°, 10.6 ± 0.2°, 11.3 ± 0.2°, 15.1 ± 0.2°, 15.8 ± 0.2°, 18.0 ± 0.2°, 18.5 ± 0.2°, 19.1 ± 0.2°, 20.4 ± 0.2°, and 20.9 ± 0.2°. In certain cases, Form B has an XRPD substantially shown in FIG. 1. In various cases, the melting point of Form B is 280 °C to 283 °C. In certain cases, Compound 1 is Form C, and the crystals can substantially exhibit the XRPD pattern shown in FIG. 3 (central spectrum).

Brief Description of the Drawings

[0023]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Mode for Carrying Out the Invention

[0024] This specification discloses a composition of an anti-influenza compound and the use of these compositions in the inhibition of influenza virus activity. In some embodiments, the present disclosure generally relates to inhibiting the replication of influenza virus in a biological sample or patient, reducing the amount of influenza virus in a biological sample or patient (reducing the virus titer), and the use of the compositions described herein for treating or preventing influenza in a patient. The compositions disclosed herein can be, for example, for pulmonary administration to a subject, patient, or host via inhalation.

[0025] The compositions disclosed herein are useful as a treatment for influenza virus infection. Thus, in some embodiments, there is provided the use of a therapeutically effective amount of a composition disclosed herein for the treatment or prevention of 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.

[0026] In various cases, there is provided a method of inhibiting the endonuclease activity of influenza polymerase in influenza A or B virus, including contacting the influenza virus with a composition disclosed herein. In certain cases, there is provided a method for treating or preventing influenza A or influenza B infection in a host, including administering a therapeutically effective amount of a composition 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, including administering a therapeutically effective amount of a composition disclosed herein to the host. In certain cases, there is provided a method for reducing influenza virus replication in a host, including administering a therapeutically effective amount of a composition disclosed herein to the host.

[0027] Compound 1 The compositions disclosed herein include, inter alia, 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, which is herein alternatively referred to as “Compound 1”. The active moiety of Compound 1 is believed to be a CAP-binding PB2 domain inhibitor.

[0028] Compound 1 may exist in free form or, where appropriate, as a salt. Their pharmaceutically acceptable salts are of particular interest since they are useful for administering the compounds which are components of the described combinations for medical purposes. Pharmaceutically unacceptable salts are useful in the manufacturing processes for isolation and purification purposes and, in some cases, for use in the separation of stereoisomers of the compounds described herein or their intermediates.

[0029] As used herein, the term “pharmaceutically acceptable salt” refers to salts of the compounds which are suitable for use in contact with the tissues of humans and lower animals within the scope of sound medical judgment, without undue side effects such as toxicity, irritation, allergic response, etc., and which are commensurate with a reasonable benefit / risk ratio.

[0030] Pharmaceutically acceptable salts are well known in the art. For example, S.M. Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, which is incorporated herein by reference. Pharmaceutically acceptable salts of the compounds described herein include those 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.

[0031] When the compounds described in this specification contain a basic group or a sufficiently basic bioisostere, acid addition salts can be prepared by 1) reacting the purified compound in the free base form with a suitable organic or inorganic acid and 2) isolating the salt thus formed. In practice, the acid addition salts may be a more convenient form for use, and the use of the salts is equivalent to the use of the free base form.

[0032] Examples of pharmaceutically acceptable non-toxic acid addition salts are salts of amino groups formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid, or formed by using other methods used in the art such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, glycolate, gluconate, glycolate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, salicylate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, and the like.

[0033] When the compounds described herein contain a carboxylic acid group or a sufficiently acidic bioisostere, a base addition salt can be prepared by 1) reacting the purified compound in its acid form with a suitable organic or inorganic base and 2) isolating the salt so formed. In practice, the use of the base addition salt 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 metals (e.g., sodium, lithium, and potassium), alkaline earth metals (e.g., magnesium, calcium), ammonium, and N + (C1-4 alkyl)4 salts. The present 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.

[0034] Basic addition salts include pharmaceutically acceptable metal salts and amine salts. Suitable metal salts include sodium, potassium, calcium, barium, zinc, magnesium, and aluminum. Sodium and potassium salts are usually preferred. Further pharmaceutically acceptable salts include, where appropriate, amine cations formed using non-toxic ammonium, quaternary ammonium, and counterions, such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkylsulfonates, and arylsulfonates. Suitable inorganic base addition salts are prepared from metal bases such as sodium hydride, sodium hydroxide, potassium hydroxide, calcium hydroxide, aluminum hydroxide, lithium hydroxide, magnesium hydroxide, zinc hydroxide, etc. Suitable amine base addition salts are prepared from amines frequently used in pharmaceutical chemistry due to their low toxicity and acceptability for medical use. Ammonia, ethylenediamine, N-methyl-glucamine, 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.

[0035] Other acids and bases, although not themselves pharmaceutically acceptable, may be used in the preparation of salts useful as intermediates in obtaining the compounds described herein and their pharmaceutically acceptable acid or base addition salts.

[0036] 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. Such solvent molecules are those commonly used in the pharmaceutical field known to be harmless to the recipient, for example, water, ethanol, dimethyl sulfoxide, acetone, and other common organic solvents. The term "hydrate" refers to a molecular complex containing a compound and water.

[0037] Compound 1, or a salt or solvate thereof, can be micronized for use in the compositions disclosed herein. Micronization refers to a solid having particles less than 15 μm. In various cases, Compound 1, or a salt or solvate thereof, can exist as particles of 0.5 μm to 10 μm, for example, 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.

[0038] Compound 1, or a salt or solvate thereof, can be micronized using any known technique. In some cases, micronization is by jet milling or manual grinding.

[0039] Compound 1 can exist in crystalline form in the disclosed compositions.

[0040] Type B: In various cases, the crystal form is obtained as described in the examples and can be characterized by an X-ray powder diffraction pattern having 2θ peaks at approximately 5.6 ± 0.2°, 6.8 ± 0.2°, 8.4 ± 0.2°, 10.1 ± 0.2°, 10.6 ± 0.2°, 11.3 ± 0.2°, 15.1 ± 0.2°, 15.8 ± 0.2°, 18.0 ± 0.2°, 18.5 ± 0.2°, 19.1 ± 0.2°, 20.4 ± 0.2°, and 20.9 ± 0.2° when using Cu-Kα radiation, and is referred to as "Type B". In some embodiments, crystalline compound 1 can be characterized by an X-ray powder diffraction pattern substantially as shown in Figure 1, where "substantially" means that the reported peaks can vary by approximately ±0.2°. Although the relative peak heights of the spectrum depend on several factors such as sample preparation and the shape of the instrument, it is well known in the field of XRPD that the peak positions are relatively unaffected by the details of the experiment.

[0041] In some cases, crystalline compound 1 can be characterized by a differential scanning calorimetry (DSC) thermogram, for example, substantially as shown in Figure 2. In some cases, crystalline compound 1 has a melting temperature of 280°C to 283°C, or approximately 282°C.

[0042] Type A: In various cases, the crystal form is obtained as described in the examples and can be characterized by an X-ray powder diffraction pattern having 2θ peaks substantially as shown in Figure 4, and is referred to as "Type A". "Substantially" means that the reported peaks can vary by approximately ±0.2°. Although the relative peak heights of the spectrum depend on several factors such as sample preparation and the shape of the instrument, it is well known in the field of XRPD that the peak positions are relatively unaffected by the details of the experiment.

[0043] Form C: In various cases, the crystal form can be obtained as described in the Examples and characterized by an X-ray powder diffraction pattern having 2θ peaks substantially as shown in Figure 3 (central spectrum), and is referred to as "Form C". "Substantially" means that the reported peaks can vary by about ±0.2°. Although the relative peak heights of the spectrum depend on several factors such as sample preparation and the shape of the instrument, it is well known in the field of XRPD that the peak positions are relatively unaffected by the details of the experiment.

[0044] As described in the Examples section below, Compound 1 may also exist as Form D or Form E.

[0045] Filler The compositions disclosed herein contain a filler. Examples of fillers include microcrystalline cellulose, dicalcium phosphate, lactose (including lactose monohydrate), trehalose, sucrose, mannose, mannitol, sorbitol, calcium carbonate, starch, and magnesium or zinc stearate. In some cases, the filler is one or more of lactose, glucose, and sodium starch glycolate. In some cases, the filler contains lactose, for example lactose monohydrate. In some cases, the filler is crystalline lactose monohydrate such as Inhalac® (e.g., Inhalac® 400).

[0046] The filler can be micronized for use in the compositions disclosed herein. Micronization refers to a solid having particles less than 15 μm. In various cases, the filler can be present as particles of 0.5 μm to 10 μm, for example, 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.

[0047] The filler can be micronized using any known technique. In some cases, micronization is by jet milling or manual grinding.

[0048] In various cases, the compositions disclosed herein contain 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.

[0049] Lung Administration and Devices In some embodiments, the compositions described herein are adapted to be administered directly to the lower respiratory tract (e.g., the lungs) via the airway by inhalation. Compositions for administration by inhalation can be inhalable powders and can be administered using a powder inhaler device. Such devices are well known.

[0050] Inhalable compositions can be packaged for unit dose or multiple dose delivery. For example, the composition can be packaged for multiple dose delivery in a manner similar to the methods described in GB2242134, US Patent Nos. 6,632,666, 5,860,419, 5,873,360, and 5,590,645 (all of which refer to "Diskus" devices), or GB2178965, GB2129691, GB2169265, US Patent Nos. 4,778,054, 4,811,731, and 5,035,237 (which refer to "Diskhaler" devices), or EP69715 (the "Turbuhaler" device), or GB2064336, and US Patent No. 4,353,656 (the "Rotahaler" device). The multiple doses can be stored in a reservoir or, for example, multiple individually packaged doses can be stored in blisters or capsules. Examples of suitable devices include, but are not limited to, TURBUHALER (Astra Zeneca), CLICKHALER (Innovata Biomed), EASYHALER (Orion), ACCUHALER, DISKUS, DISKHALER, ROTAHALER (GlaxoSmithKline), HANDIHALER, INHALATOR, AEROHALER (Boehringer Ingelheim), AEROLIZER (Schering Plough), and NOVOLIZER (ASTA Medica).

[0051] For example, upon administration via inhalation, the compositions disclosed herein exhibit high levels of drug exposure in the lung compared to exposure in the plasma. These high drug exposure levels are useful for several reasons. First, lung administration provides rapid delivery of the therapeutic agent to the site of infection. Second, by maintaining the therapeutic agent in the lung while minimizing plasma exposure, minimal therapeutic agent moves away from the site of infection, thereby reducing systemic adverse events. Third, by concentrating the exposure in the lung, the therapeutic utility at the site of infection (e.g., the lung) can be maximized.

[0052] In some cases, administration of the compositions disclosed herein by inhalation provides exposure of Compound 1 in the lung that is 50-fold greater than exposure in plasma after 1 hour. In various cases, the exposure after 1 hour is 60-fold, or 70-fold, or 80-fold, or 90-fold, or 100-fold, or 125-fold, or 150-fold greater in the lung than in plasma.

[0053] In some cases, administration of the compositions disclosed herein by inhalation results in exposure of Compound 1 in the lung that is 50-fold greater than exposure in plasma after 24 hours. In various cases, the exposure after 24 hours is 60-fold, or 70-fold, or 80-fold, or 90-fold, or 100-fold, or 125-fold, or 150-fold greater in the lung than in plasma.

[0054] In some cases, administration of the compositions disclosed herein by inhalation results in exposure of Compound 1 in the lung that is 50-fold greater than exposure in plasma at 48 hours. In various cases, the exposure at 48 hours is 60-fold, or 70-fold, or 80-fold, or 90-fold, or 100-fold, or 125-fold, or 150-fold greater in the lung than in plasma.

[0055] In various cases, even 4 days after administration by inhalation, the exposure of Compound 1 in the lung is at least 100-fold greater than the exposure in plasma.

[0056] Method of Use The compositions described herein can be used to reduce the viral titer in a biological sample (e.g., an infected cell culture) or in a human (e.g., the viral titer in a patient's lung).

[0057] As used herein, the terms “influenza virus-mediated condition,” “influenza infection,” or “influenza” are used interchangeably to mean a disease caused by infection with an influenza virus.

[0058] Influenza is an infection that affects birds and mammals and is caused by influenza viruses. Influenza viruses are RNA viruses of the family Orthomyxoviridae, which include five genera: influenza A virus, influenza B virus, influenza C virus, isavirus, and thogotovirus. The genus influenza A virus has one species, influenza A virus, which can be classified into different serotypes: H1N1, H2N2, H3N2, H5N1, H7N7, H1N2, H9N2, H7N2, H7N3, H7N9, and H10N7, based on the antibody response to these viruses. The genus influenza B virus has one species, influenza B virus. Influenza B almost exclusively infects humans and is less common than influenza A. The genus influenza C virus has one species, influenza C virus, which infects humans and pigs and can cause severe illness and local epidemics. However, influenza C is less common than the other types and usually appears to cause mild disease in children.

[0059] In some embodiments, the influenza or influenza virus is associated with an influenza A or B virus. In some embodiments, the influenza or influenza virus is associated with an influenza A virus. In some particular embodiments, the influenza A virus is H1N1, H2N2, H3N2, H7N9, or H5N1. In some embodiments, the disclosed combinations are effective to inhibit the growth or replication of pandemic or drug-resistant pandemic / seasonal influenza viruses.

[0060] In humans, the common symptoms of influenza are chills, fever, pharyngitis, myalgia, severe headache, cough, weakness, and general malaise. In more severe cases, influenza can cause pneumonia, which can be fatal especially in children and the elderly. Influenza is often confused with the common cold, but influenza is a much more serious disease and is caused by different types of viruses. Influenza can cause nausea and vomiting especially in children, but these symptoms are characteristic of unrelated gastroenteritis sometimes called "stomach flu" or "24-hour flu".

[0061] The symptoms of influenza can occur very suddenly, 1 to 2 days after infection. Usually, the first symptoms are chills or a feeling of coldness, but fever is also common early in the infection, with body temperature in the range of 38 to 39 °C (about 100 to 103 °F). Many people feel very ill, being bedridden for several days with pain and aches all over the body, which worsens the back and legs. Symptoms of influenza can include body aches, especially in the joints and throat, extreme cold and fever, fatigue, headache, inflamed and red eyes, skin (especially the face), mouth, throat and nose, and abdominal pain (in children with type B influenza). The symptoms of influenza are non-specific and overlap with many pathogens ("influenza-like illness"). Usually, laboratory data are needed to confirm the diagnosis.

[0062] The terms "disease", "disorder", and "condition" may be used interchangeably herein and refer to influenza virus-mediated medical or pathological conditions.

[0063] The terms "subject", "host", and "patient" are used interchangeably. The terms "subject", "host", and "patient" can refer to an animal (e.g., a bird such as a chicken, quail, or pigeon, or a mammal), specifically 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), more specifically a human. In some embodiments, the subject 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 subject is a "human".

[0064] As used herein, the term "biological sample" includes, but is not limited to, cell cultures or extracts thereof; biopsy materials obtained from mammals or extracts thereof; blood, saliva, urine, feces, semen, tears, or other body fluids or extracts thereof.

[0065] As used herein, the term "inhibition of influenza virus replication" includes both a decrease in the amount of virus replication (e.g., at least a 10% decrease) to a complete stop of virus replication (i.e., a 100% decrease in the amount of virus replication). In some embodiments, the replication of the influenza virus is inhibited by at least 50%, at least 65%, at least 75%, at least 85%, at least 90%, or at least 95%.

[0066] The replication of influenza virus 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 in a human (e.g., the viral titer in the lungs of a patient) can be measured. More specifically, for cell-based assays, in each case, cells are cultured in vitro, the virus is added to the culture in the presence or absence of a test agent, and virus-dependent evaluation items are evaluated after a suitable length of time. In a typical assay, Madin-Darby canine kidney cells (MDCK) and the influenza strain A / Puerto Rico / 8 / 34 adapted to standard tissue culture can be used. The first type of cell assay that can be used in the present disclosure is a process called the cytopathic effect (CPE), which depends on the death of infected target cells, and viral infection causes depletion of the cell source and eventual cell lysis. In the first type of cell assay, a low percentage (usually 1 / 10 to 1 / 1000) of the cells in the wells of a microtiter plate are infected, and the virus can replicate several times over 48 to 72 hours, and then the amount of cell death is measured using the decrease in cellular ATP content compared to uninfected controls. The second type of cell assay that can be used in the present disclosure depends on the growth of virus-specific RNA molecules in infected cells, and the RNA level is measured directly using the branched DNA hybridization method (bDNA). In the second type of cell assay, a small number of cells are first infected in the wells of a microtiter plate, the virus replicates in the infected cells, and is further spread to additional rounds of cells, and then the cells are lysed and the viral RNA content is measured. This assay stops early, usually after 18 to 36 hours, but all target cells are still alive. The viral RNA is quantified by hybridization to specific oligonucleotide probes immobilized on the wells of the assay plate, followed by amplification of the signal by hybridization to additional probes conjugated to a reporter enzyme.

[0067] As used herein, "viral titer" or "titer" is a measure of viral concentration. A titer test can obtain approximate quantitative information from an analytical procedure that is essentially evaluated only as positive or negative using serial dilutions. The titer corresponds to the highest dilution factor that still gives a positive reading. For example, positive readings at the first eight serial two-fold dilutions are converted to a titer of 1:256. A specific example is the viral titer. To determine the titer, several dilutions are prepared, for example, 10 -1 10 -2 10 -3 10 -8 and so on. The lowest concentration of virus that still infects cells is the viral titer.

[0068] As used herein, the terms "treat", "treatment", and "treating" refer to both therapeutic and prophylactic treatments. For example, therapeutic treatment includes a decrease or improvement in the progression, severity, and / or duration of an influenza virus-mediated condition, or an improvement in one or more symptoms (specifically, one or more recognizable symptoms) of an influenza virus-mediated condition, resulting from the administration of one or more therapies (e.g., one or more therapeutic agents such as the compounds or compositions described herein). In certain embodiments, therapeutic treatment includes an improvement in at least one measurable physical parameter of an influenza virus-mediated condition. In other embodiments, therapeutic treatment includes inhibiting the progression of an influenza virus-mediated condition, physically, e.g., by stabilizing recognizable symptoms, physiologically, e.g., by stabilizing physical parameters, or both. In other embodiments, therapeutic treatment includes a decrease or stabilization of influenza virus-mediated infection. Antiviral drugs can be used in a community setting to treat people who already have influenza in order to reduce the severity of symptoms and the number of days people are sick.

[0069] As used herein, the terms "prophylaxis," "prophylactic," "prophylactic use," and "prophylactic treatment" refer to any medical or public health procedure that is intended to prevent, rather than treat or cure, a disease. As used herein, the terms "prevent," "prevention," and "preventing" refer to the reduction of the risk of acquiring or developing a given condition, or the alleviation of the above condition or the inhibition of its recurrence, in a subject who may be near or who has been near a person having a disease but not yet ill. The term "chemoprevention" refers to the use of a pharmaceutical, such as a small molecule drug, rather than a vaccine, for the prevention of a disorder or disease.

[0070] Prophylactic use includes use in situations where an outbreak has been detected to prevent the transmission or spread of infection in places where many people at high risk of severe influenza complications live in close proximity to each other (e.g., hospital wards, day care centers, prisons, nursing homes, etc.). Prophylactic use also includes use in populations that need protection from influenza but do not obtain protection after vaccination (e.g., due to a weakened immune system, etc.), populations for which a vaccine is not available, or populations that cannot receive a vaccine due to side effects. Use two weeks after vaccination, or at any time after vaccination but before the vaccine is effective, is also included. Prophylactic use may also include treating a person who has influenza but is not ill or is not considered to be at high risk of complications to reduce the chance of transmitting influenza to persons at high risk of coming into close contact with them (e.g., healthcare workers, nursing home workers, etc.).

[0071] As used herein, and consistent with the usage of the United States Centers for Disease Control and Prevention (US CDC), an "outbreak" of influenza is defined as a sudden increase in acute febrile respiratory illness (AFRI) that occurs within 48 to 72 hours among groups of people who are in close proximity to one another (e.g., the same area of a long-term care facility, the same household, etc.) and that exceeds the normal background rate or when any subject of the population is analyzed as testing positive for influenza.

[0072] In some embodiments, the composition is useful as a prophylactic or preventive measure for patients, particularly humans, who are predisposed to complications resulting from infection by influenza virus. The composition may be useful in a method of prevention for preventing the spread of infection in the remainder of a region or population in the context of an identified index case or outbreak.

[0073] As used herein, "effective amount" refers to an amount sufficient to induce 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 remit the severity, duration, progression, or onset of influenza virus infection, prevent the progression of influenza virus infection, prevent the recurrence, occurrence, onset, or progression of symptoms associated with influenza virus infection, or improve or enhance the prophylactic or therapeutic effect(s) of another therapy used against influenza infection. The exact amount of the compound administered to a subject depends on the mode of administration, the type and severity of the infection, and the characteristics of the subject such as general health status, age, sex, weight, and drug tolerance. One of ordinary skill in the art will be able to determine the appropriate dosage depending 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 a subject in a dosage range of about 0.01 to 100 mg / kg body weight / day for therapeutic or prophylactic treatment.

[0074] 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 undue or harmful side effects in a patient.

[0075] Generally, the dosing regimen can be selected according to a variety of factors, including the disorder being treated and its severity; the activity of the particular compound used; the particular composition used; the age, weight, general health, sex, and diet of the patient; the dosing time, route of administration, and excretion rate of the particular compound used; the renal and hepatic function of the subject; as well as the particular compound or its salt used, the duration of treatment; drugs used in combination with or simultaneously with the particular compound used, and similar factors well known in the medical arts. One of ordinary skill in the art can readily determine and prescribe the effective amount of the compounds described herein necessary to treat, prevent, inhibit (completely or partially), or arrest the progression of the disease.

[0076] The dosage of Compound 1 can range from about 0.01 to about 100 mg / kg body weight per day, from about 0.01 to about 50 mg / kg body weight per day, from about 0.1 to about 50 mg / kg body weight per day, or from about 1 to about 25 mg / kg body weight per day. The total amount per day can be administered as a single dose or 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 (such as every 6 hours).

[0077] For therapeutic treatment, Compound 1 can be administered to a patient, for example, within 48 hours (or within 40 hours, or less than 2 days, or less than 1.5 days, or within 24 hours) from the onset of symptoms (e.g., nasal congestion, sore throat, cough, pain, fatigue, headache, chills / sweating). The therapeutic treatment can continue for any appropriate period, such as 5 days, 7 days, 10 days, 14 days, etc. For prophylactic treatment during an outbreak in a region, Compound 1 can be administered, for example, within 2 days of the onset of symptoms in an index case and can continue for any appropriate period, such as 7 days, 10 days, 14 days, 20 days, 28 days, 35 days, 42 days, etc.

Example

[0078] Polymorph Screening of Compound 1 (Slurry Method): Approximately 10 mg of Compound 1 was added to 200 μL of various solvents - methyl t-butyl ether (MTBE), methanol (MeOH), ethanol (EtOH), isopropyl alcohol (IPA), isopropyl and ethyl acetate in a 5 / 5 volume ratio (IPA / EtOAc), ethyl acetate (EtOAc), isopropyl alcohol and water in an 8 / 2 volume ratio, acetonitrile (ACN), acetone, and tetrahydrofuran (THF). Each suspension was stirred at 700 rpm for 24 hours at 40 °C. The residue of the compound was separated by centrifugation (14,000 rpm for 10 minutes) and further dried overnight in a vacuum oven at 30 °C. If a clear solution remained, the solution was dried under vacuum to produce a dry solid. The dry solid was analyzed by XRPD and the form was assigned. The results are shown in the following table.

Table 1

[0079] The XRPD patterns of the crystals were obtained using a Bruker D8 Advance instrument with the following parameters. The results of the Type B XRPD analysis are shown in Figure 1. The results of the Type C are shown in Figure 3. The results of Types A, B, and C are shown in Figure 4.

Table 2

[0080] The DSC of the Type B crystals was obtained using a TA Q2000 instrument with the following parameters. The results of the DSC are shown in Figure 2.

Table 3

[0081] Polymorph Screening of Compound 1 (Poor Solvent Method): Approximately 25 mg of Compound 1 was weighed into a glass vial, followed by the addition of 0.5 mL of dimethylacetamide (DMA) to achieve a concentration of 50 mg / mL as a clear solution. Then, a poor solvent was added dropwise to this solution while stirring at 700 rpm at room temperature. The resulting crystals were collected by centrifugation. The crystals were analyzed by XRPD, and the results are shown in Figure 5. In the figure, the top is the E form, the middle is the C form, and the bottom is the A form.

Table 4

[0082] Polymorph Formation at Various Temperatures - Slurry: Approximately 25 mg of Compound 1 was weighed into a glass vial, followed by the addition of 500 μL of various solvents. The solution was stirred at 700 rpm at 55 °C or 25 °C for 3 days. The residue of the compound was separated by a centrifuge (10,000 rpm for 10 minutes) and further dried in a vacuum oven at 30 °C for 2 days. The dried solid was analyzed by XRPD. An overview of the forms obtained under various conditions is shown below.

Table 5

[0083] Polymorph Formation at Various Temperatures: Approximately 50 mg of Compound 1 was weighed into a glass vial, followed by the addition of 500 μL of various solvents. Then, the solution was stirred at 700 rpm at 55 °C for 3 days. The solid was separated by a centrifuge (10,000 rpm for 10 minutes) and further dried in a vacuum oven at 30 °C for 2 days. The solid was analyzed by XRPD.

[0084] Approximately 25 mg of Compound 1 was weighed into a glass vial and stored at 25 °C / 60% RH or 40 °C / 75% RH for 1 week. Then, the sample was analyzed by XRPD.

[0085] The results of XRPD are summarized below.

Table 6

[0086] Polymorph formation at various temperatures (poor solvent): Weighed approximately 50 mg of Compound 1 into a glass vial, then added 1 mL of DMA, and then sonicated to obtain a clear solution. Then, the solution was stirred at 700 rpm and 55 °C, and then a poor solvent was added either with fast precipitation or slow precipitation. Fast precipitation: Added a certain amount of poor solvent at a fast rate and filtered the solid within 1 hour. Slow precipitation: Added a certain amount of poor solvent at a low rate, slurried for 3 days, and then filtered the solid. The obtained solid was separated by a centrifuge (10,000 rpm for 10 minutes) and further dried in a vacuum oven at 30 °C for 2 days. The solid was analyzed by XRPD. The XRPD results are shown below.

Table 7

Table 8

Table 9

[0087] Micronization of Compound 1: Crystalline Compound 1 (Form B) was gradually added to a jet mill with an injector gas pressure of 4.5 bar and a milling gas of 4 bar. The micronized product showed the same characteristic peaks as the compound before micronization. Furthermore, according to the DSC results, a continuous exothermic peak at 198.27 °C and a single endothermic peak at 280.40 °C before decomposition were confirmed to be the same as those observed in the sample before micronization. The particle size distribution (PSD) of the dry dispersion results 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.

[0088] Micronization of lactose monohydrate: Several lactose monohydrate materials were examined and characterized as summarized in the following table.

Table 10

[0089] Preparation of formulation: The crystalline compound 1 (Form B) was manually pulverized and then mixed with lactose monohydrate (Inhalac400) in a ratio of 1:4. The mixture was mixed by manual pulverization for 10 minutes. Subsequently, the mixture was subjected to jet milling under the following conditions: injector gas pressure 4.5 bar, pulverizing gas 4 bar. The PSD of the dry dispersion data showed 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.

[0090] In vivo mouse PK study: To show the delivery of compound 1 to the lungs via the inhalation route, a mouse (BALB / C) pharmacokinetic study was conducted. Before collecting samples, the mice were treated with a single dose of approximately 1 mg of dry powder using an inhaler. Plasma and lung samples were collected at different time points, and the drug concentrations in the lungs and plasma of the mice were measured. As shown in the table below, a rapid accumulation of a high concentration of the drug in the lung tissue via the inhalation route was observed. In contrast, the drug concentration in the plasma was significantly lower than that detected in the lungs. These data indicate that the delivery of compound 1 can be effectively achieved via inhalation administration using the disclosed formulation, and that compound 1 can, for example, enable contact with the inflamed airways infected with influenza. Interestingly, the drug levels remained at least 100-fold in excess of the therapeutic dose (anti-influenza efficacy, EC 50 0.1 - 3 nM) on day 4. These results further confirmed the potential clinical use of the compound 1 dry powder for the treatment of influenza infection.

Table 11

Claims

Claim 1 (a) A crystal of 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 (Compound 1) or a pharmaceutically acceptable salt thereof having an X-ray powder diffraction (XRPD) pattern showing 2θ values of 5.6 ± 0.2°, 6.8 ± 0.2°, 8.4 ± 0.2°, 10.1 ± 0.2°, 10.6 ± 0.2°, 11.3 ± 0.2°, 15.1 ± 0.2°, 15.8 ± 0.2°, 18.0 ± 0.2°, 18.5 ± 0.2°, 19.1 ± 0.2°, 20.4 ± 0.2°, and 20.9 ± 0.2° (referred to as Form B) using Cu-Kα radiation, (b) An excipient, and a pharmaceutical preparation comprising the same, wherein the crystal of Compound 1 referred to as Form B is present in a micronized form, and the preparation is adapted to be an inhalable preparation. Claim 2 Comprising an excipient consisting essentially of lactose monohydrate, The preparation has a volume median diameter (VMD) of 1 to 2 μm, a D of 0.5 μm to 0.7 μm １０ , a D of 1 μm to 1.4 μm ５０ , and a D of 2.5 μm to 2.8 μm ９０ The preparation according to claim 1, having a particle size distribution characterized thereby. Claim 3 The preparation according to claim 1, wherein the excipient comprises lactose. Claim 4 The preparation according to claim 1, wherein the excipient is micronized. Claim 5 The preparation according to claim 1, wherein the crystal of Compound 1 referred to as Form B has a melting point of 280°C to 283°C. Claim 6 The preparation according to claim 1, wherein the crystal of Compound 1 referred to as Form B or a pharmaceutically acceptable salt thereof has a volume average particle size of 0.5 to 10 µm. Claim 7 The preparation according to claim 1, wherein the excipient has a volume average particle size of 0.5 to 10 µm. Claim 8 The preparation according to claim 1, wherein the crystal of Compound 1 referred to as Form B or a pharmaceutically acceptable salt thereof and the excipient are provided in a weight ratio of 1:3 to 1:

5. Claim 9 Use of the preparation according to claim 1 for the manufacture of a therapeutic or prophylactic agent for the treatment or prevention of influenza virus infection or replication in a subject. Claim 10 A method for preparing the preparation according to claim 1, comprising: (a) micronizing a crystal of Compound 1 referred to as Form B or a salt thereof to form particles of the crystal of Compound 1 referred to as Form B; (b) optionally, micronizing the excipient to form particles of the excipient; and (c) mixing the micronized crystal of Compound 1 referred to as Form B or a salt thereof and the optionally micronized excipient to form the preparation. Claim 11 The method according to claim 10, further comprising crystallizing compound 1 or a salt thereof before the micronization step (a).

12. The method according to claim 11, wherein the crystallizing comprises mixing compound 1 or a salt thereof and ethanol at a temperature of at least 50 °C, cooling to room temperature to enable crystallization of compound 1 or a salt thereof, collecting the crystals by filtration, and optionally drying the crystals before the micronization step (a).

13. The method according to claim 12, wherein compound 1 or a salt thereof and ethanol are mixed at a temperature of 75 °C.

14. Crystals of 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 (compound 1), called Form B, having an X-ray powder diffraction (XRPD) pattern showing 2θ values of 5.6 ± 0.2°, 6.8 ± 0.2°, 8.4 ± 0.2°, 10.1 ± 0.2°, 10.6 ± 0.2°, 11.3 ± 0.2°, 15.1 ± 0.2°, 15.8 ± 0.2°, 18.0 ± 0.2°, 18.5 ± 0.2°, 19.1 ± 0.2°, 20.4 ± 0.2°, and 20.9 ± 0.2° using Cu-Kα radiation.

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