SPRAY DRIED INHALATABLE BIOTHERAPEUTICS FOR THE TREATMENT OF DISEASES - Patent application

Abstract

The technology described herein is directed to spray-dried biotherapeutic matrix compositions containing bacterial preparations that are formulated for administration by inhalation. Also described herein are unit dosage forms of such spray-dried biotherapeutic matrix compositions, devices containing such pharmaceutical compositions, methods of producing such pharmaceutical compositions, and methods of using such spray-dried biotherapeutic matrix compositions to treat diseases, such as bronchopulmonary diseases, among others.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 63 / 290,289, filed December 16, 2021, the contents of which are incorporated herein by reference in their entirety.

[0002] The technology described herein relates to spray dried inhalable biotherapeutics for the treatment of disease. [Background technology]

[0003] Inhalation drug delivery has an advantage over other routes of administration in that therapeutic agents delivered directly to the lungs have been shown to have improved clinical efficacy compared to other routes of administration.See, for example, Jackson (1995) British Journal of General Practice, 45(401):683:687, the contents of which are incorporated by reference in their entirety.Due to this improved clinical efficacy, many drugs are formulated to be inhaled by patients to achieve a reduced drug loading requirement for efficacy, resulting in lower systemic exposure and reduced side effects.

[0004] There are many routes of administration of drugs that can be used to treat indications such as chronic bronchopulmonary disorders, including parenteral administration (e.g., XOLAIR-omalizumab), intravenous delivery (e.g., CINQAIR-reslizumab), oral capsules (e.g., DALIRESP-roflumilast), metered dose inhalers (e.g., Ventolin-albuterol), liquid nebulizers (e.g., XOPENEX-levalbuterol), and dry powder inhalers (e.g., SPIRIVA-tiotropium). Some drugs that are administered systemically via ingestion (e.g., roflumilast for COPD) have a high risk of side effects due to systemic exposure and gastrointestinal interactions. See, e.g., Garnock-Jones, Drugs 75, 1645-1656 (2015), the contents of which are incorporated herein by reference in their entirety. Drugs prescribed for injectable use are typically approved for specific phenotypes of asthma or psoriasis that can be difficult to diagnose, but do not include other chronic respiratory diseases.

[0005] Nebulized therapeutic agents are typically given to patients in small containers of drug solution. The container is then inserted into a nebulizer device, which produces a mist of drug solution for the patient to inhale, usually over a sustained inhalation period. This route of administration is useful for dosing patients (children, infants, elderly) who are unable to inhale forcefully. However, drugs formulated in nebulized solutions have stability challenges that can make new drug development difficult.

[0006] To avoid some of the stability problems of formulated drugs for use in nebulizer systems, pressurized inhalers have been developed for use in inhaling therapeutic agents. These metered dose inhalers are commonly used to deliver bronchodilators and corticosteroids in patients suffering from asthma and COPD. Originally, these pressurized inhalers worked by pressurizing a container containing a drug solution and chlorofluorocarbons. Over time, it was discovered that inhaling CFCs affects the central nervous system, so more recent MDIs utilize hydrofluoroalkanes to pressurize the canister and deliver therapeutic agents. See, for example, Muralidharan et al. (2015) Expert Opinion on Drug Delivery, 12:6, 947-962, the contents of which are incorporated herein by reference in their entirety.

[0007] Dry powder inhalers have recently been used, in the past decade, to deliver drugs that would be difficult to formulate in a nebulizer or MDI (drugs such as tiotropium bromide and itraconazole). These therapeutics take the form of a dry, aerosolizable powder in a breakable container (such as a cellulose-based capsule or sachet). The patient breaks the drug container and inhales the dry powder through the broken housing via a mouthpiece attached to the dry powder inhaler itself. An exemplary dry powder inhaler device is the PLASTIAPE RS01 inhaler - it is publicly available and is an excellent candidate for delivery development studies. In this inhaler, the drug capsule is located within a chamber in the base of the inhaler. The user breaks the capsule by pressing two "triggers" on either side of the inhaler. The user then turns the inhaler horizontally and inhales through the mouthpiece, emptying the capsule of its powder contents while it rapidly rotates, increasing the amount of powder administered to the user. Many inhalation dry powder processes utilize custom inhalers with the powder pre-loaded into a chamber (eg, SPRIVIA HANDIHALER or BREO ELLIPTA) that corresponds to the dosing regimen.

[0008] This delivery route offers many advantages over other routes of administration, including more predictable particle fraction delivered to the lungs, improved drug stability, the ability to use hydrophobic active ingredients, and the absence of chemical propellants. Uses of dry powder inhalers include therapy for treating chronic obstructive pulmonary disease (COPD), asthma, and pulmonary infections. In addition to these pulmonary diseases, additional indications may benefit from dry powder delivery of therapeutic agents, such as idiopathic pulmonary fibrosis, cystic fibrosis, bronchiectasis, chronic cough, or infectious respiratory diseases. Furthermore, disease profiles other than chronic respiratory diseases, including central nervous system (CNS) diseases such as Parkinson's disease and migraine, osteoporosis, and pulmonary hypertension, may also benefit from pulmonary administration. See, e.g., Noymer et al. (2011) Ther Deliv 2(9):1125-1140, Abdou et al. (2019) Drug Deliv 26(1):689-699, Yu et al. (2021) J Control Release 338:486-504, Hill et al. (2015) Respir Care 60(6):794-802; discussion 802-795, Hosang et al. Nature 603:138-144(2022), the contents of each of which are incorporated by reference in their entirety.

[0009] Dysbiosis in the lungs can lead to chronic neutrophilic inflammation. After injury to the airway epithelium, collagen is exposed and cleaved by matrix metalloproteinase 9 (MMP-9). MMP-9 and prolyl endopeptidase (PE) cleave collagen fragments to liberate prolylglycylproline (PGP) peptide PGP in its acetylated form (Ac-PGP). Ac-PGP induces neutrophilic inflammation by binding to a receptor on the CXC chemokine receptor 2 (CXCR2). Both live bacteria and their extracts and metabolites can modulate the pathway leading to Ac-PGP production. Many chronic and infectious bronchopulmonary disorders are characterized by symptoms from the body's inflammatory response. Dysbiosis has been shown to be a contributing factor to chronic inflammatory lung diseases, including asthma, cystic fibrosis, bronchopulmonary dysplasia, and chronic obstructive pulmonary disease. For example, Gaggar et al. (2008) J Immunol 180(8):5662-5669, Malik et al. (2007) J Immunol 178(2):1013-1020, Lin et al. (2008) Am J Pathol 173(1):144-153, Weathington et al. (2006) Nature Medicine 12(3):317-323., Hilty et al. (2010) PLoS One 5(1):e8578, Marri et al. (2013) J Allergy Clin Immunol 131(2):346-352 e341-343, Zhao et al. (2012) Proc Natl Acad Sci USA 109(15):5809-5814, Coburn et al. (2015) Sci Rep 5:10241, Lal et al. (2016) Scientific Reports 6(1):31023, Bowerman et al. (2020) Nat Commun 11(1):5886, Huang et al. (2014) J Clin Microbiol 52(8):2813-2823, the contents of each of which are incorporated by reference in their entirety.

[0010] Therefore, there is a need to develop easily administrable therapeutic agents for chronic inflammatory lung diseases. Summary of the Invention

[0011] The present disclosure describes the unexpected discovery that biotherapeutic agents comprising microbiota and / or their extracts or metabolites can be formulated for administration by inhalation. Thus, the technology described herein is directed to spray-dried biotherapeutic matrix compositions comprising bacterial preparations, which are formulated for administration by inhalation. Also described herein are unit dosage forms of such spray-dried biotherapeutic matrix compositions, devices comprising such pharmaceutical compositions, methods of producing such pharmaceutical compositions, and methods of using such spray-dried biotherapeutic matrix compositions to treat diseases, such as bronchopulmonary diseases, among others.

[0012] In one aspect, described herein is a spray-dried biotherapeutic matrix composition comprising a bacterial preparation, wherein the matrix composition is formulated for administration by inhalation.

[0013] In some embodiments of any of the aspects, the bacterial preparation comprises viable or non-viable bacteria.

[0014] In some embodiments of any of the aspects, the viable bacteria are capable of actively metabolizing and / or multiplying in the lungs of the subject.

[0015] In some embodiments of any of the aspects, the non-viable bacteria are heat-killed.

[0016] In some embodiments of any of the aspects, the bacteria is gram negative.

[0017] In some embodiments of any of the aspects, the bacteria is gram positive.

[0018] In some embodiments of any of the aspects, the bacteria is spore forming.

[0019] In some embodiments of any of the aspects, the bacteria is in a spore form.

[0020] In some embodiments of any of the aspects, the bacteria is aerobic.

[0021] In some embodiments of any of the aspects, the bacteria is anaerobic.

[0022] In some embodiments of any of the aspects, the bacteria produces at least one immunomodulatory factor.

[0023] In some embodiments of any of the aspects, the bacterium belongs to a genus selected from the group consisting of Carnobacterium, Lactiplantibacillus, Lactobacillus, Lacticaseibacillus, Ligilactobacillus, Oenococcus, Leuconostoc, Pedicoccus, Enterococcus, Lactococcus, Staphylococcus, Streptococcus, Streptomyces, Bifidobacterium, Propionibacterium, and Moraxella.

[0024] In some embodiments of any of the aspects, the bacterium is Lacticaseibacillus rhamnosus, Lactobacillus acidophilus, or Lactiplantibacillus plantarum.

[0025] In some embodiments of any of the aspects, the bacteria is non-pathogenic.

[0026] In some embodiments of any of the aspects, the bacteria is at least 10 1 Colony forming units / gram (CFU / g), at least 10 2 CFU / g, at least 10 3 CFU / g, at least 10 4CFU / g, at least 10 5 CFU / g, at least 10 6 CFU / g, at least 10 7 CFU / g, at least 10 8 CFU / g, at least 10 9 CFU / g, at least 10 10 CFU / g, at least 10 11 CFU / g, or at least 10 12 It is present at a concentration of CFU / g.

[0027] In some embodiments of any of the aspects, the bacteria is at least 10 6 It is present in a concentration of colony forming units / g (CFU / g).

[0028] In some embodiments of any of the aspects, the bacteria is at least 10 8 It is present in a concentration of colony forming units / g (CFU / g).

[0029] In some embodiments of any of the aspects, the bacteria is resistant to at least one antibiotic.

[0030] In some embodiments of any of the aspects, the composition comprises at least 0.5% by weight of the bacterial preparation.

[0031] In some embodiments of any of the aspects, the bacterial preparation comprises a bacterial extract or a bacterial metabolite preparation.

[0032] In some embodiments of any of the aspects, the bacterial extract or bacterial metabolite preparation is selected from the group consisting of bacterial exosomes, bacterial cell wall, peptidoglycan, teichoic acid, lipoteichoic acid, bacterial S-layer, exopolysaccharides, polysaccharides, lactic acid polymers, lactic acid derivatives, lactic acid intermediates, hydrogen peroxide, bacteriocins, salivaricin, reuterin, and bacterial growth supernatant.

[0033] In some embodiments of any of the aspects, the composition further comprises at least one excipient.

[0034] In some embodiments of any of the aspects, the composition further comprises at least two excipients.

[0035] In some embodiments of any of the aspects, the excipient is selected from the group consisting of De Man, Rogosa and Sharpe (MRS) growth medium, gelatin, whey isolate, sweet whey, reconstituted skim milk powder, maltodextrin, gluco-oligosaccharides, lacto-oligosaccharides, fructooligosaccharides, inulin, sodium caseinate, goat milk, cow's milk, proline, carnitine, acetylcarnitine, propionylcarnitine, glutamate, glycine betaine, glycogen, trehalose, mannose, xylose, mannitol, sorbitol, maltose, dextrose, starch, lactose, sucrose, glucose, leucine, trileucine, sodium salts, potassium salts, lithium salts, and calcium salts.

[0036] In some embodiments of any of the aspects, the excipient is leucine and / or trehalose.

[0037] In some embodiments of any of the aspects, the composition comprises at least 0.5% by weight of an excipient.

[0038] In some embodiments of any of the aspects, the composition further comprises at least one stabilizer.

[0039] In some embodiments of any of the aspects, the stabilizer comprises a surfactant.

[0040] In some embodiments of any of the aspects, the stabilizer is a polysorbate, a poloxamer, or polyvinyl alcohol.

[0041] In some embodiments of any of the aspects, the stabilizer is polysorbate 20, polysorbate 40, polysorbate 60, or polysorbate 80.

[0042] In some embodiments of any of the aspects, the stabilizer is polysorbate 80.

[0043] In some embodiments of any of the aspects, the stabilizer is poloxamer 184, poloxamer 185, poloxamer 188, poloxamer 234, poloxamer 235, poloxamer 238, poloxamer 333, poloxamer 334, poloxamer 335, poloxamer 338, poloxamer 403, or poloxamer 407.

[0044] In some embodiments of any of the aspects, the composition comprises at least 0.25% by dry weight of a stabilizer.

[0045] In some embodiments of any of the aspects, the composition comprises at least one excipient and at least one stabilizer.

[0046] In some embodiments of any of the aspects, the composition further comprises at least one additional therapeutic agent.

[0047] In some embodiments of any of the aspects, the at least one additional therapeutic agent is selected from the group consisting of an anti-inflammatory agent, an antibacterial agent, an antiviral agent, an antifungal agent, a vasodilator, and a bronchodilator.

[0048] In some embodiments of any of the aspects, the at least one additional therapeutic agent is incorporated into the composition using microencapsulation, co-formulation, or covalent attachment to the composition with a degradable linker.

[0049] In some embodiments of any of the aspects, the matrix composition comprises a plurality of dry particles.

[0050] In some embodiments of any of the aspects, the dry particles have a Dv50 of at least 0.5 μm.

[0051] In some embodiments of any of the aspects, the dry particles have a median mass aerodynamic diameter (MMAD) of at least 1.5 μm and up to 7.5 μm.

[0052] In some embodiments of any of the aspects, the dry particles have a dispersibility of less than 2.0.

[0053] In some embodiments of any of the aspects, the dry particles have a dispersibility of at least 0.5 to 1.0.

[0054] In some embodiments of any of the aspects, the dry particles have a delivered dose to the target tissue of at least 25.0% and up to 125% by weight of the bacterial preparation.

[0055] In some embodiments of any of the aspects, the dry particles have a delivered dose to the target tissue that is at least 60% by weight of the bacterial preparation.

[0056] In some embodiments of any of the aspects, the target tissue is a target bronchopulmonary tissue.

[0057] In some embodiments of any of the aspects, the target bronchiolopulmonary tissue is the lung, trachea, bronchi, bronchioles, and / or alveoli.

[0058] In some embodiments of any of the aspects, the target tissue is a tissue site distal to the lung, delivered via the cardiovascular or lymphatic system.

[0059] In some embodiments of any of the aspects, the dry particles have a density of at least 0.1 g / cm 3 ~0.8g / cm 3 It has a bulk density of

[0060] In some embodiments of any of the aspects, the dry particles have a density of at least 0.5 g / cm 3 It has a bulk density of

[0061] In some embodiments of any of the aspects, the dry particles have a density of at least 0.1 g / cm 3 ~1.0g / cm 3 The tap density is

[0062] In some embodiments of any of the aspects, the dry particles have a density of at least 0.6 g / cm 3 The tap density is

[0063] In some embodiments of any of the aspects, the dry particles have a moisture content of at least 1.0% to 7.0% water by weight according to Karl Fischer.

[0064] In some embodiments of any of the aspects, the dry particles have a moisture content of at least 2.5% water by weight according to Karl Fischer.

[0065] In some embodiments of any of the aspects, the composition is formulated for delivery to the trachea, bronchi, bronchioles, and / or alveoli.

[0066] In some embodiments of any of the aspects, the composition is formulated for pulmonary delivery.

[0067] In some embodiments of any of the aspects, the composition is formulated as a capsule.

[0068] In some embodiments of any of the aspects, the capsule contains at least 10 mg of the spray-dried biotherapeutic matrix composition.

[0069] In some embodiments of any of the aspects, the composition is formulated for delivery by an inhaler.

[0070] In some embodiments of any of the aspects, the composition is formulated for delivery by a dry powder inhaler (DPI), a metered dose inhaler (MDI), or a soft mist inhaler (SMI).

[0071] In some embodiments of any of the aspects, the composition is combined with an inhaler.

[0072] In one aspect, described herein is an inhalation device for bronchopulmonary delivery, the inhalation device comprising: (a) an inhaler; and (b) a container containing a spray-dried biotherapeutic matrix composition comprising a bacterial preparation.

[0073] In some embodiments of any of the aspects, the inhaler is a dry powder inhaler (DPI), a metered dose inhaler (MDI), or a soft mist inhaler (SMI).

[0074] In some embodiments of any of the aspects, the inhaler comprises (a) a mouthpiece including an opening, and (b) a means for aerosolizing or dispersing the spray-dried biotherapeutic matrix composition within the container.

[0075] In one aspect, described herein is a method of preparing a spray-dried biotherapeutic matrix composition comprising a bacterial preparation, the method comprising: (a) preparing a liquid feedstock comprising the bacterial preparation; (b) introducing droplets of the liquid feedstock into a drying chamber via a spray nozzle; (c) exposing the liquid feedstock droplets to heated and pressurized gas in the drying chamber to produce dry particles; and (d) isolating dry particles of a predetermined range of diameters in a cyclone chamber, wherein the isolated dry particles comprise the bacterial preparation.

[0076] In one aspect, described herein is a method of preparing a spray-dried biotherapeutic matrix composition comprising a bacterial preparation, the method comprising: (a) obtaining a liquid feedstock comprising the bacterial preparation; (b) introducing droplets of the liquid feedstock into a drying chamber via a spray nozzle; (c) exposing the liquid feedstock droplets to heated and pressurized gas in the drying chamber to produce dry particles; and (d) isolating dry particles of a predetermined range of diameters in a cyclone chamber, wherein the isolated dry particles comprise a spray-dried biotherapeutic matrix composition.

[0077] In some embodiments of any of the aspects, the step of preparing the liquid ingredient includes dissolving a solid ingredient in an aqueous solution.

[0078] In some embodiments of any of the aspects, the solid feedstock comprises (a) at least 3.5% by weight of the bacterial preparation, (b) at least 5% by weight of an excipient, and / or (c) at least 0.25% by weight of a stabilizer.

[0079] In some embodiments of any of the aspects, the solid feedstock comprises (a) at least 3.5% by weight of the bacterial preparation, (b) at least 5% by weight of a first excipient, (c) at least 5% by weight of a second excipient, and / or (d) at least 0.25% by weight of a stabilizer.

[0080] In some embodiments of any of the aspects, the solid feedstock comprises at least 1% and up to 5% by weight of the bacterial preparation.

[0081] In some embodiments of any of the aspects, the solid ingredient comprises at least 45% and up to 95% by weight of an excipient.

[0082] In some embodiments of any of the aspects, the solid ingredient comprises at least 5%-60% by weight of a first excipient and at least 5%-60% by weight of a second excipient.

[0083] In some embodiments of any of the aspects, the solid feedstock comprises at least 0.25% by weight and up to 10% by weight of a stabilizer.

[0084] In some embodiments of any of the aspects, the liquid ingredient comprises at least 1 g / L of a solid ingredient dissolved in an aqueous solution.

[0085] In some embodiments of any of the aspects, the liquid ingredient comprises at least 25 g / L of a solid ingredient dissolved in an aqueous solution.

[0086] In some embodiments of any of the aspects, the liquid ingredient comprises at least 0.1% and up to 10% of a solid ingredient dissolved in an aqueous solution.

[0087] In some embodiments of any of the aspects, the liquid ingredient comprises at least 1% of a solid ingredient dissolved in an aqueous solution.

[0088] In some embodiments of any of the aspects, 1 L of the liquid feedstock comprises (a) at least 1.050 g of the bacterial preparation, (b) at least 1.5 g of an excipient, (b) at least 0.075 g of a stabilizer, and / or (c) at least 970 g of an aqueous solution.

[0089] In some embodiments of any of the aspects, 1 L of the liquid feedstock comprises (a) at least 1.050 g of the bacterial preparation, (b) at least 0.75 g of a first excipient, (c) at least 0.75 g of a second excipient, (d) at least 0.075 g of a stabilizer, and / or (e) at least 970 g of an aqueous solution.

[0090] In some embodiments of any of the aspects, 1 L of the liquid feedstock comprises (a) at least 1.050 g of the bacterial preparation, (b) at least 0.5 g of a first excipient, (c) at least 0.5 g of a second excipient, (d) at least 0.5 g of a third excipient, (e) at least 0.075 g of a stabilizer, and / or (f) at least 970 g of an aqueous solution.

[0091] In some embodiments of any of the aspects, 1 L of the liquid feedstock comprises at least 750 g and up to 999 g of aqueous solution.

[0092] In some embodiments of any of the aspects, the liquid feedstock comprises (a) at least 0.105% of the bacterial preparation, (b) at least 0.15% of an excipient, (c) at least 0.0075% of a stabilizer, and / or (d) at least 97% of an aqueous solution.

[0093] In some embodiments of any of the aspects, the liquid feedstock comprises (a) at least 0.105% by weight of the bacterial preparation, (b) at least 0.075% by weight of a first excipient, (c) at least 0.075% by weight of a second excipient, (d) at least 0.0075% by weight of a stabilizer, and / or (e) at least 97% by weight of an aqueous solution.

[0094] In some embodiments of any of the aspects, the liquid feedstock comprises (a) at least 0.105% by weight of the bacterial preparation, (b) at least 0.05% by weight of a first excipient, (c) at least 0.05% by weight of a second excipient, (d) at least 0.05% by weight of a third excipient, (e) at least 0.0075% by weight of a stabilizer, and / or (f) at least 97% by weight of the aqueous solution.

[0095] In some embodiments of any of the aspects, the liquid feedstock comprises at least 0.01% and up to 10% by weight of the bacterial preparation.

[0096] In some embodiments of any of the aspects, the liquid ingredient comprises at least 1.0% and up to 20% by weight of an excipient.

[0097] In some embodiments of any of the aspects, the liquid ingredient comprises at least 0.1% and up to 19.8% by weight of a first excipient and at least 0.1% and up to 19.8% by weight of a second excipient.

[0098] In some embodiments of any of the aspects, the liquid ingredient comprises at least 0.1% and up to 19.8% by weight of a first excipient, at least 0.1% and up to 19.8% by weight of a second excipient, and at least 0.1% and up to 19.8% by weight of a third excipient.

[0099] In some embodiments of any of the aspects, the liquid feedstock comprises at least 0.01% and up to 1.0% by weight of a stabilizer.

[0100] In some embodiments of any of the aspects, the liquid feedstock comprises at least 75% and up to 99.9% by weight of an aqueous solution.

[0101] In some embodiments of any of the aspects, the bacterial preparation comprises viable or non-viable bacteria.

[0102] In some embodiments of any of the aspects, the bacterium belongs to a genus selected from the group consisting of Carnobacterium, Lactiplantibacillus, Lactobacillus, Lacticaseibacillus, Ligilactobacillus, Oenococcus, Leuconostoc, Pedicoccus, Enterococcus, Lactococcus, Staphylococcus, Streptococcus, Streptomyces, Bifidobacterium, Propionibacterium, and Moraxella.

[0103] In some embodiments of any of the aspects, the bacterium is Lacticaseibacillus rhamnosus, Lactobacillus acidophilus, or Lactiplantibacillus plantarum.

[0104] In some embodiments of any of the aspects, the bacteria is non-pathogenic.

[0105] In some embodiments of any of the aspects, the bacteria is at least 10 1 Colony forming units / gram (CFU / g), at least 10 2 CFU / g, at least 10 3 CFU / g, at least 10 4 CFU / g, at least 10 5 CFU / g, at least 10 6 CFU / g, at least 10 7 CFU / g, at least 10 8 CFU / g, at least 10 9 CFU / g, at least 10 10CFU / g, at least 10 11 CFU / g, or at least 10 12 It is present at a concentration of CFU / g.

[0106] In some embodiments of any of the aspects, the bacteria is at least 10 6 It is present in a concentration of colony forming units / g (CFU / g).

[0107] In some embodiments of any of the aspects, the bacteria is at least 10 8 It is present in a concentration of colony forming units / g (CFU / g).

[0108] In some embodiments of any of the aspects, the excipient is selected from the group consisting of De Man, Rogosa and Sharpe (MRS) growth medium, gelatin, whey isolate, sweet whey, reconstituted skim milk powder, maltodextrin, gluco-oligosaccharides, lacto-oligosaccharides, fructooligosaccharides, inulin, sodium caseinate, goat milk, cow's milk, proline, carnitine, acetylcarnitine, propionylcarnitine, glutamate, glycine betaine, glycogen, trehalose, mannose, xylose, mannitol, sorbitol, maltose, dextrose, starch, lactose, sucrose, glucose, leucine, trileucine, sodium salts, potassium salts, lithium salts, and calcium salts.

[0109] In some embodiments of any of the aspects, the excipient is leucine and / or trehalose.

[0110] In some embodiments of any of the aspects, the stabilizer is a polysorbate, a poloxamer, or polyvinyl alcohol.

[0111] In some embodiments of any of the aspects, the stabilizer is polysorbate 80.

[0112] In some embodiments of any of the aspects, the aqueous solution is water.

[0113] In some embodiments of any of the aspects, the liquid ingredient further comprises at least one additional therapeutic agent.

[0114] In some embodiments of any of the aspects, the at least one additional therapeutic agent is selected from the group consisting of an anti-inflammatory agent, an antibacterial agent, an antiviral agent, an antifungal agent, a vasodilator, and a bronchodilator.

[0115] In some embodiments of any of the aspects, the spray nozzle to the drying chamber has a diameter of at least 1.2 um.

[0116] In some embodiments of any of the aspects, the droplets of the liquid feedstock produced by the spray nozzle into the drying chamber have a diameter of at least 1.5 um.

[0117] In some embodiments of any of the aspects, the droplets of the liquid feedstock have a flow rate through the drying chamber of at least 0.5 g / min.

[0118] In some embodiments of any of the aspects, the droplets of the liquid feedstock have a flow rate through the drying chamber of at least 15 g / min.

[0119] In some embodiments of any of the aspects, the droplets of the liquid feedstock have a flow rate through the drying chamber of up to 1000 g / min.

[0120] In some embodiments of any of the aspects, the heated pressurized gas is heated prior to being introduced into the drying chamber.

[0121] In some embodiments of any of the aspects, the heated pressurized gas is introduced into the drying chamber at a temperature of at least 100°C.

[0122] In some embodiments of any of the aspects, the heated pressurized gas is introduced into the drying chamber at a temperature of up to 195°C.

[0123] In some embodiments of any of the aspects, the heated pressurized gas is exhausted from the drying chamber at a temperature of at least 40°C.

[0124] In some embodiments of any of the aspects, the heated pressurized gas is exhausted from the drying chamber at a temperature of at least 48°C.

[0125] In some embodiments of any of the aspects, the heated pressurized gas is exhausted from the drying chamber at a temperature of up to 85°C.

[0126] In some embodiments of any of the aspects, the heated pressurized gas is pressurized prior to being introduced into the drying chamber.

[0127] In some embodiments of any of the aspects, the heated pressurized gas in the drying chamber has an atomizing gas pressure of at least 10 pounds per square inch gauge (psig).

[0128] In some embodiments of any of the aspects, the heated pressurized gas in the drying chamber has an atomizing gas pressure of at least 20 pounds per square inch gauge (psig).

[0129] In some embodiments of any of the aspects, the heated pressurized gas in the drying chamber has an atomizing gas pressure of up to 150 pounds per square inch gauge (psig).

[0130] In some embodiments of any of the aspects, the heated pressurized gas has a flow rate through the drying chamber of at least 5 kg / hr.

[0131] In some embodiments of any of the aspects, the heated pressurized gas has a flow rate through the drying chamber of at least 18 kg / hr.

[0132] In some embodiments of any of the aspects, the heated pressurized gas has a flow rate through the drying chamber of up to 150 kg / hr.

[0133] In some embodiments of any of the aspects, the heated pressurized gas is discharged through a cyclone chamber.

[0134] In some embodiments of any of the aspects, exposing the liquid feedstock droplets to heated and pressurized gas in the drying chamber takes up to 8 hours.

[0135] In some embodiments of any of the aspects, the dried particles isolated in the cyclone chamber have a median mass aerodynamic diameter (MMAD) of at least 1.5 μm and up to 7.5 μm.

[0136] In some embodiments of any of the aspects, the dried particles isolated in the cyclone chamber have a median mass aerodynamic diameter (MMAD) of at least 4.0 μm.

[0137] In some embodiments of any of the aspects, the step of isolating the dry particles of the predetermined range of diameters in the cyclone chamber is performed continuously.

[0138] In some embodiments of any of the aspects, the spray dried biotherapeutic matrix composition comprises at least 5% bacterial viability after the step of isolating the dried particles.

[0139] In one aspect, described herein is a method of delivering a spray dried biotherapeutic matrix composition comprising a bacterial preparation to a subject, the method comprising: (a) obtaining an inhalation device for bronchopulmonary delivery, the inhalation device comprising: (i) an inhaler; and (ii) a container containing a spray dried biotherapeutic matrix composition comprising a bacterial preparation; (b) activating the inhaler to cause aerosolization or dispersion of the spray dried biotherapeutic matrix composition; and (c) inhaling the aerosolized or dispersed spray dried biotherapeutic matrix composition.

[0140] In one aspect, described herein is a method of delivering a spray dried biotherapeutic matrix composition comprising a bacterial preparation to a subject, the method comprising: (a) obtaining an inhalation device for bronchopulmonary delivery, the inhalation device comprising: (i) an inhaler; and (ii) a container containing a spray dried biotherapeutic matrix composition as described herein; (b) activating the inhaler to cause aerosolization or dispersion of the spray dried biotherapeutic matrix composition; and (c) inhaling the aerosolized or dispersed spray dried biotherapeutic matrix composition.

[0141] In one aspect, described herein is a method of delivering a spray dried biotherapeutic matrix composition comprising a bacterial preparation to a subject, the method comprising: (a) obtaining an inhalation device as described herein; (b) activating the inhaler to cause aerosolization or dispersion of the spray dried biotherapeutic matrix composition; and (c) inhaling the aerosolized or dispersed spray dried biotherapeutic matrix composition.

[0142] In some embodiments of any of the aspects, the inhaler is a dry powder inhaler (DPI), a metered dose inhaler (MDI), or a soft mist inhaler (SMI).

[0143] In some embodiments of any of the aspects, the inhaler comprises (a) a mouthpiece including an opening, and (b) a means for aerosolizing or dispersing the spray-dried biotherapeutic matrix composition within the container.

[0144] In some embodiments of any of the aspects, the inhaler has an inhalation flow rate of at least 15 L / min.

[0145] In some embodiments of any of the aspects, at least 25% and up to 100.0% by weight of the spray-dried biotherapeutic matrix composition is delivered to the target bronchopulmonary tissue.

[0146] In some embodiments of any of the aspects, at least 60.0% by weight of the spray-dried biotherapeutic matrix composition is delivered to the target bronchopulmonary tissue.

[0147] In some embodiments of any of the aspects, the target bronchiolopulmonary tissue is the lung, trachea, bronchi, bronchioles, and / or alveoli.

[0148] In some embodiments of any of the aspects, the spray dried biotherapeutic matrix composition is delivered to a tissue site distal to the bronchopulmonary tissue via the cardiovascular or lymphatic system.

[0149] In one aspect, described herein is a method of treating a subject in need of treatment, the method comprising administering by inhalation an effective dose of a spray-dried biotherapeutic matrix composition comprising a bacterial preparation.

[0150] In one aspect, described herein is a method of treating a subject in need of treatment, the method comprising administering by inhalation an effective dose of a spray dried biotherapeutic matrix composition described herein.

[0151] In some embodiments of any of the aspects, the subject has been diagnosed with or is at risk for developing a chronic bronchopulmonary disease.

[0152] In some embodiments of any of the aspects, the chronic bronchopulmonary disease is selected from the group consisting of chronic obstructive pulmonary disease (COPD), lung cancer, asthma, bronchiectasis, emphysema, cystic fibrosis (CF), bronchopulmonary dysplasia (BPD), acute respiratory distress syndrome (ARDS), idiopathic pulmonary fibrosis (IPF), pulmonary arterial hypertension (PAH), silicosis, interstitial lung disease (ILD), and pleural effusion (PE).

[0153] In some embodiments of any of the aspects, the lung cancer is non-small cell lung cancer (SCLC) or small cell lung cancer (NSCLC).

[0154] In some embodiments of any of the aspects, the subject has been diagnosed with or is at risk for developing an infectious pulmonary disease.

[0155] In some embodiments of any of the aspects, the infectious bronchopulmonary disease is caused by or associated with an infectious agent selected from adenovirus, coronavirus, influenza virus, parainfluenza virus, parvovirus, respiratory syncytial virus, rhinovirus, enterovirus, measles virus, rubella virus, varicella virus, Corynebacterium diphtheriae, Haemophilus influenzae, Legionella pneumophila, Bordetella pertussis, Mycobacterium tuberculosis, Streptococcus species, Pseudomonas species, Escherichia coli, Aspergillus species, Cryptococcus species, and Pneumocystis species.

[0156] In some embodiments of any of the aspects, the spray dried biotherapeutic matrix composition is administered in conjunction with a standard of care treatment for a chronic or infectious bronchopulmonary disease.

[0157] In some embodiments of any of the aspects, the spray dried biotherapeutic matrix composition is administered using an inhaler.

[0158] In some embodiments of any of the aspects, the inhaler is a dry powder inhaler (DPI), a metered dose inhaler (MDI), or a soft mist inhaler (SMI).

[0159] In some embodiments of any of the aspects, an effective dose of the spray dried biotherapeutic matrix composition comprises at least 10 4 It is a CFU.

[0160] In some embodiments of any of the aspects, an effective dose of the spray dried biotherapeutic matrix composition comprises at least 10 4 CFU of viable bacteria.

[0161] In some embodiments of any of the aspects, the spray dried biotherapeutic matrix composition reduces neutrophilic inflammation in the target tissue.

[0162] In some embodiments of any of the aspects, the spray dried biotherapeutic matrix composition increases lactate concentration in the target tissue by at least 25%.

[0163] In some embodiments of any of the aspects, the method further comprises administering at least one additional therapeutic agent.

[0164] In some embodiments of any of the aspects, the at least one additional therapeutic agent is selected from the group consisting of an anti-inflammatory agent, an antibacterial agent, an antiviral agent, an antifungal agent, a vasodilator, and a bronchodilator.

[0165] In some embodiments of any of the aspects, the spray-dried biotherapeutic matrix composition comprises a bacterial preparation and at least one additional therapeutic agent.

[0166] In some embodiments of any of the aspects, the spray dried biotherapeutic matrix composition is co-administered with at least one additional therapeutic agent.

[0167] In some embodiments of any of the aspects, the co-administration comprises administering using a combination delivery device.

[0168] In one aspect, described herein is a unit dosage form comprising at least 1 mg of a spray-dried biotherapeutic matrix composition comprising a bacterial preparation.

[0169] In one embodiment, described herein is a unit dosage form comprising at least 1 mg of a spray dried biotherapeutic matrix composition described herein.

[0170] In one aspect, described herein is a unit dosage form comprising at least 1 mg of a spray dried biotherapeutic matrix composition prepared by the methods described herein.

[0171] In one embodiment, at least 10 per unit dose 4 Described herein is a unit dosage form comprising at least 1 mg of a spray-dried biotherapeutic matrix composition containing CFU of bacteria.

[0172] In some embodiments of any of the aspects, the dosage is at least 30 mg of the spray-dried biotherapeutic matrix composition.

[0173] In some embodiments of any of the aspects, the dosage is at least 10 4 Contains CFU of bacteria.

[0174] In some embodiments of any of the aspects, the dosage is at least 10 4 Contains CFU of viable bacteria. [Brief description of the drawings]

[0175] [Figure 1] 1 is a schematic diagram of an exemplary spray dryer and associated methods, as further described herein. [Diagram 2] 1 is an image of an exemplary dry powder inhaler. [Diagram 3] 1 is a bar graph showing the assay of three bacterial strains, AB101, AB102, and AB103, for their growth and viability in vitro over a 24-hour period. The left-right order of the bars corresponds to the top-to-bottom order in the graph legend. [Figure 4A]FIG. 4 is a series of bar graphs and tables showing a study in which healthy mice were inoculated intratracheally with a blend of live bacterial strains AB101, 102, and 103 in a 1:1:1 ratio or a negative control. FIG. 4A is a bar graph showing the bacterial load in lung tissue of mice at 0, 4, 8, 12, 16, 24, and 72 hours after inoculation. FIG. 4B is a table showing the bacterial load from FIG. 4A. FIG. 4C is a bar graph showing the lactate output in bronchoalveolar lavage (BAL) fluid of mice at the indicated time points. [Figure 4B] FIG. 4 is a series of bar graphs and tables showing a study in which healthy mice were inoculated intratracheally with a blend of live bacterial strains AB101, 102, and 103 in a 1:1:1 ratio or a negative control. FIG. 4A is a bar graph showing the bacterial load in lung tissue of mice at 0, 4, 8, 12, 16, 24, and 72 hours after inoculation. FIG. 4B is a table showing the bacterial load from FIG. 4A. FIG. 4C is a bar graph showing the lactate output in bronchoalveolar lavage (BAL) fluid of mice at the indicated time points. [Figure 4C] FIG. 4 is a series of bar graphs and tables showing a study in which healthy mice were inoculated intratracheally with a blend of live bacterial strains AB101, 102, and 103 in a 1:1:1 ratio or a negative control. FIG. 4A is a bar graph showing the bacterial load in lung tissue of mice at 0, 4, 8, 12, 16, 24, and 72 hours after inoculation. FIG. 4B is a table showing the bacterial load from FIG. 4A. FIG. 4C is a bar graph showing the lactate output in bronchoalveolar lavage (BAL) fluid of mice at the indicated time points. [Figure 5A] FIG. 5 is a series of tables showing testing of spray-dried powders containing bacteria for bacterial viability. F4 solution contained 0.05% polysorbate 80 in water for injection, and F6 medium contained 5:55 MRS:F4. Liquid feed solution was the solution before drying. FIG. 5A is a table showing the spray-drying conditions for each batch. FIG. 5B is a table showing the viability of bacteria in each batch of spray-dried powder. As used herein, "API" stands for active pharmaceutical ingredient. [Figure 5B]FIG. 5 is a series of tables showing testing of spray-dried powders containing bacteria for bacterial viability. F4 solution contained 0.05% polysorbate 80 in water for injection, and F6 medium contained 5:55 MRS:F4. Liquid feed solution was the solution before drying. FIG. 5A is a table showing the spray-drying conditions for each batch. FIG. 5B is a table showing the viability of bacteria in each batch of spray-dried powder. As used herein, "API" stands for active pharmaceutical ingredient. [Figure 6] 1 is a table of particle characteristics of two exemplary formulations of spray-dried live biotherapeutic product AB1000 (AB1000 includes spray-dried formulations of strains AB101, AB102, and AB103 with excipients; see, e.g., Table 12). [Figure 7] 1 is a table showing the viability of two spray-dried formulations of AB1000 in each batch of spray-dried powder as measured by the International Organization for Standardization (ISO) 7889 enumeration standard. ISO 7889 specifies a method for the enumeration of characteristic microorganisms in yogurt by colony counting techniques at 37° C. [Figure 8] 1 is a table showing the resistance and susceptibility profiles of live bacterial strains AB101, AB102, and AB103 to 51 antibiotics. [Figure 9] 1 is a table showing group assignments for testing mice exposed to porcine pancreatic elastase (PPE) with or without lipopolysaccharide (LPS) administered a representative drug powder of biotherapeutic agent AB1000. This testing scheme is referred to herein as the "PPE model mouse." [Figure 10A] Figure 10 is a series of images and bar graphs showing changes in lung structure of mice in a PPE model. Figure 10A is a series of histological images showing changes in lung structure from the mice shown in Figure 9. Figure 10B is a series of bar graphs showing radial alveolar count (RAC) and mean alveolar diameter (MLI) of mouse lung tissue from Figure 10A. [Figure 10B]Figure 10 is a series of images and bar graphs showing changes in lung structure of mice in a PPE model. Figure 10A is a series of histological images showing changes in lung structure from the mice shown in Figure 9. Figure 10B is a series of bar graphs showing radial alveolar count (RAC) and mean alveolar diameter (MLI) of mouse lung tissue from Figure 10A. [Figure 11] FIG. 1 is a bar graph of pulmonary function tests using pulmonary resistance measured in cm H2O / mL / s in PPE model mice. [Figure 12] 1 is a bar graph showing MMP-9 mRNA transcription levels in lung tissues of PPE model mice. [Figure 13A] 13A is a series of bar graphs showing changes in protein levels of inflammatory markers in bronchoalveolar lavage (BAL) fluid of PPE model mice. FIG. 13A is a bar graph showing a significant decrease in MMP-9 protein in both PPE and PPE+LPS mice treated with AB1000. FIG. 13B is a bar graph showing a significant decrease in neutrophil elastase (NE) protein in the BAL of PPE+LPS mice treated with AB1000. FIG. 13C is a bar graph showing a significant decrease in C-reactive protein (CRP) in PPE+LPS mice treated with AB1000. FIG. 13D is a bar graph showing a significant decrease in interleukin 8 (IL-8) in PPE and PPE+LPS mice treated with AB1000. FIG. 13E is a bar graph showing a significant increase in immunoglobulin A (IgA) in PPE and PPE+LPS mice treated with AB1000. [Figure 13B]13A is a series of bar graphs showing changes in protein levels of inflammatory markers in bronchoalveolar lavage (BAL) fluid of PPE model mice. FIG. 13A is a bar graph showing a significant decrease in MMP-9 protein in both PPE and PPE+LPS mice treated with AB1000. FIG. 13B is a bar graph showing a significant decrease in neutrophil elastase (NE) protein in the BAL of PPE+LPS mice treated with AB1000. FIG. 13C is a bar graph showing a significant decrease in C-reactive protein (CRP) in PPE+LPS mice treated with AB1000. FIG. 13D is a bar graph showing a significant decrease in interleukin 8 (IL-8) in PPE and PPE+LPS mice treated with AB1000. FIG. 13E is a bar graph showing a significant increase in immunoglobulin A (IgA) in PPE and PPE+LPS mice treated with AB1000. [Figure 13C] 13A is a series of bar graphs showing changes in protein levels of inflammatory markers in bronchoalveolar lavage (BAL) fluid of PPE model mice. FIG. 13A is a bar graph showing a significant decrease in MMP-9 protein in both PPE and PPE+LPS mice treated with AB1000. FIG. 13B is a bar graph showing a significant decrease in neutrophil elastase (NE) protein in the BAL of PPE+LPS mice treated with AB1000. FIG. 13C is a bar graph showing a significant decrease in C-reactive protein (CRP) in PPE+LPS mice treated with AB1000. FIG. 13D is a bar graph showing a significant decrease in interleukin 8 (IL-8) in PPE and PPE+LPS mice treated with AB1000. FIG. 13E is a bar graph showing a significant increase in immunoglobulin A (IgA) in PPE and PPE+LPS mice treated with AB1000. [Figure 13D]13A is a series of bar graphs showing changes in protein levels of inflammatory markers in bronchoalveolar lavage (BAL) fluid of PPE model mice. FIG. 13A is a bar graph showing a significant decrease in MMP-9 protein in both PPE and PPE+LPS mice treated with AB1000. FIG. 13B is a bar graph showing a significant decrease in neutrophil elastase (NE) protein in the BAL of PPE+LPS mice treated with AB1000. FIG. 13C is a bar graph showing a significant decrease in C-reactive protein (CRP) in PPE+LPS mice treated with AB1000. FIG. 13D is a bar graph showing a significant decrease in interleukin 8 (IL-8) in PPE and PPE+LPS mice treated with AB1000. FIG. 13E is a bar graph showing a significant increase in immunoglobulin A (IgA) in PPE and PPE+LPS mice treated with AB1000. [Figure 13E] 13A is a series of bar graphs showing changes in protein levels of inflammatory markers in bronchoalveolar lavage (BAL) fluid of PPE model mice. FIG. 13A is a bar graph showing a significant decrease in MMP-9 protein in both PPE and PPE+LPS mice treated with AB1000. FIG. 13B is a bar graph showing a significant decrease in neutrophil elastase (NE) protein in the BAL of PPE+LPS mice treated with AB1000. FIG. 13C is a bar graph showing a significant decrease in C-reactive protein (CRP) in PPE+LPS mice treated with AB1000. FIG. 13D is a bar graph showing a significant decrease in interleukin 8 (IL-8) in PPE and PPE+LPS mice treated with AB1000. FIG. 13E is a bar graph showing a significant increase in immunoglobulin A (IgA) in PPE and PPE+LPS mice treated with AB1000. [Figure 14A]A series of bar graphs showing changes in protein levels of inflammatory markers in serum of PPE model mice. Figure 14A is a bar graph showing a significant reduction in MMP-9 protein in PPE mice treated with AB1000. Figure 14B is a bar graph showing a significant reduction in MMP-9 protein in PPE+LPS mice treated with AB1000. Figure 14C is a bar graph showing a significant reduction in NE protein in PPE mice treated with AB1000. Figure 14D is a bar graph showing a significant reduction in NE protein PPE+LPS mice treated with AB1000. Figure 14E is a bar graph showing a significant reduction in C-reactive protein (CRP) in PPE+LPS mice treated with AB1000. [Figure 14B] A series of bar graphs showing changes in protein levels of inflammatory markers in serum of PPE model mice. Figure 14A is a bar graph showing a significant reduction in MMP-9 protein in PPE mice treated with AB1000. Figure 14B is a bar graph showing a significant reduction in MMP-9 protein in PPE+LPS mice treated with AB1000. Figure 14C is a bar graph showing a significant reduction in NE protein in PPE mice treated with AB1000. Figure 14D is a bar graph showing a significant reduction in NE protein PPE+LPS mice treated with AB1000. Figure 14E is a bar graph showing a significant reduction in C-reactive protein (CRP) in PPE+LPS mice treated with AB1000. [Figure 14C]A series of bar graphs showing changes in protein levels of inflammatory markers in serum of PPE model mice. Figure 14A is a bar graph showing a significant reduction in MMP-9 protein in PPE mice treated with AB1000. Figure 14B is a bar graph showing a significant reduction in MMP-9 protein in PPE+LPS mice treated with AB1000. Figure 14C is a bar graph showing a significant reduction in NE protein in PPE mice treated with AB1000. Figure 14D is a bar graph showing a significant reduction in NE protein PPE+LPS mice treated with AB1000. Figure 14E is a bar graph showing a significant reduction in C-reactive protein (CRP) in PPE+LPS mice treated with AB1000. [Figure 14D] A series of bar graphs showing changes in protein levels of inflammatory markers in serum of PPE model mice. Figure 14A is a bar graph showing a significant reduction in MMP-9 protein in PPE mice treated with AB1000. Figure 14B is a bar graph showing a significant reduction in MMP-9 protein in PPE+LPS mice treated with AB1000. Figure 14C is a bar graph showing a significant reduction in NE protein in PPE mice treated with AB1000. Figure 14D is a bar graph showing a significant reduction in NE protein PPE+LPS mice treated with AB1000. Figure 14E is a bar graph showing a significant reduction in C-reactive protein (CRP) in PPE+LPS mice treated with AB1000. [Figure 14E]A series of bar graphs showing changes in protein levels of inflammatory markers in serum of PPE model mice. Figure 14A is a bar graph showing a significant reduction in MMP-9 protein in PPE mice treated with AB1000. Figure 14B is a bar graph showing a significant reduction in MMP-9 protein in PPE+LPS mice treated with AB1000. Figure 14C is a bar graph showing a significant reduction in NE protein in PPE mice treated with AB1000. Figure 14D is a bar graph showing a significant reduction in NE protein PPE+LPS mice treated with AB1000. Figure 14E is a bar graph showing a significant reduction in C-reactive protein (CRP) in PPE+LPS mice treated with AB1000. [Figure 15A] Figure 15 is a series of images and bar graphs showing changes in lung histology in mice exposed to PPE and LPS, and treated with AB1000 or the inhaled steroid fluticasone furoate. Figure 15A is a series of histological images showing changes in lung histology across exposure and treatment groups. Figure 15B is a bar graph of mean alveolar size (MLI) showing improvement in histology upon treatment with AB1000. [Figure 15B] Figure 15 is a series of images and bar graphs showing changes in lung histology in mice exposed to PPE and LPS, and treated with AB1000 or the inhaled steroid fluticasone furoate. Figure 15A is a series of histological images showing changes in lung histology across exposure and treatment groups. Figure 15B is a bar graph of mean alveolar size (MLI) showing improvement in histology upon treatment with AB1000. [Figure 16] 1 is a bar graph showing that ABlOO and Fluticasone Furoate performed comparably in reducing MMP-9 expression in lung tissue of mice exposed to PPE and LPS. [Figure 17A]17A-17I are a series of bar graphs showing significant increases in protein levels of the anti-inflammatory cytokines Exodus2 (FIG. 17A), macrophage inflammatory protein-3b (MIP-3b) (FIG. 17B), interleukin-11 (IL-11) (FIG. 17C), monocyte chemoattractant protein 5 (MCP-5) (FIG. 17D), thymus and activation-regulated chemokine (TARC) (FIG. 17E), MIP-3a (FIG. 17F), IL-16 (FIG. 17G), tissue inhibitor of metalloproteinase 1 (TIMP1) (FIG. 17H), and macrophage-derived chemokine (MDC) (FIG. 17I) in bronchoalveolar lavage fluid (BAL) of PPE+LPS-exposed mice treated with AB1000. [Figure 17B] 17A-17I are a series of bar graphs showing significant increases in protein levels of the anti-inflammatory cytokines Exodus2 (FIG. 17A), macrophage inflammatory protein-3b (MIP-3b) (FIG. 17B), interleukin-11 (IL-11) (FIG. 17C), monocyte chemoattractant protein 5 (MCP-5) (FIG. 17D), thymus and activation-regulated chemokine (TARC) (FIG. 17E), MIP-3a (FIG. 17F), IL-16 (FIG. 17G), tissue inhibitor of metalloproteinase 1 (TIMP1) (FIG. 17H), and macrophage-derived chemokine (MDC) (FIG. 17I) in bronchoalveolar lavage fluid (BAL) of PPE+LPS-exposed mice treated with AB1000. [Figure 17C] 17A-17I are a series of bar graphs showing significant increases in protein levels of the anti-inflammatory cytokines Exodus2 (FIG. 17A), macrophage inflammatory protein-3b (MIP-3b) (FIG. 17B), interleukin-11 (IL-11) (FIG. 17C), monocyte chemoattractant protein 5 (MCP-5) (FIG. 17D), thymus and activation-regulated chemokine (TARC) (FIG. 17E), MIP-3a (FIG. 17F), IL-16 (FIG. 17G), tissue inhibitor of metalloproteinase 1 (TIMP1) (FIG. 17H), and macrophage-derived chemokine (MDC) (FIG. 17I) in bronchoalveolar lavage fluid (BAL) of PPE+LPS-exposed mice treated with AB1000. [Figure 17D]17A-17I are a series of bar graphs showing significant increases in protein levels of the anti-inflammatory cytokines Exodus2 (FIG. 17A), macrophage inflammatory protein-3b (MIP-3b) (FIG. 17B), interleukin-11 (IL-11) (FIG. 17C), monocyte chemoattractant protein 5 (MCP-5) (FIG. 17D), thymus and activation-regulated chemokine (TARC) (FIG. 17E), MIP-3a (FIG. 17F), IL-16 (FIG. 17G), tissue inhibitor of metalloproteinase 1 (TIMP1) (FIG. 17H), and macrophage-derived chemokine (MDC) (FIG. 17I) in bronchoalveolar lavage fluid (BAL) of PPE+LPS-exposed mice treated with AB1000. [Figure 17E] 17A-17I are a series of bar graphs showing significant increases in protein levels of the anti-inflammatory cytokines Exodus2 (FIG. 17A), macrophage inflammatory protein-3b (MIP-3b) (FIG. 17B), interleukin-11 (IL-11) (FIG. 17C), monocyte chemoattractant protein 5 (MCP-5) (FIG. 17D), thymus and activation-regulated chemokine (TARC) (FIG. 17E), MIP-3a (FIG. 17F), IL-16 (FIG. 17G), tissue inhibitor of metalloproteinase 1 (TIMP1) (FIG. 17H), and macrophage-derived chemokine (MDC) (FIG. 17I) in bronchoalveolar lavage fluid (BAL) of PPE+LPS-exposed mice treated with AB1000. [Figure 17F] 17A-17I are a series of bar graphs showing significant increases in protein levels of the anti-inflammatory cytokines Exodus2 (FIG. 17A), macrophage inflammatory protein-3b (MIP-3b) (FIG. 17B), interleukin-11 (IL-11) (FIG. 17C), monocyte chemoattractant protein 5 (MCP-5) (FIG. 17D), thymus and activation-regulated chemokine (TARC) (FIG. 17E), MIP-3a (FIG. 17F), IL-16 (FIG. 17G), tissue inhibitor of metalloproteinase 1 (TIMP1) (FIG. 17H), and macrophage-derived chemokine (MDC) (FIG. 17I) in bronchoalveolar lavage fluid (BAL) of PPE+LPS-exposed mice treated with AB1000. [Figure 17G]17A-17I are a series of bar graphs showing significant increases in protein levels of the anti-inflammatory cytokines Exodus2 (FIG. 17A), macrophage inflammatory protein-3b (MIP-3b) (FIG. 17B), interleukin-11 (IL-11) (FIG. 17C), monocyte chemoattractant protein 5 (MCP-5) (FIG. 17D), thymus and activation-regulated chemokine (TARC) (FIG. 17E), MIP-3a (FIG. 17F), IL-16 (FIG. 17G), tissue inhibitor of metalloproteinase 1 (TIMP1) (FIG. 17H), and macrophage-derived chemokine (MDC) (FIG. 17I) in bronchoalveolar lavage fluid (BAL) of PPE+LPS-exposed mice treated with AB1000. [Figure 17H] 17A-17I are a series of bar graphs showing significant increases in protein levels of the anti-inflammatory cytokines Exodus2 (FIG. 17A), macrophage inflammatory protein-3b (MIP-3b) (FIG. 17B), interleukin-11 (IL-11) (FIG. 17C), monocyte chemoattractant protein 5 (MCP-5) (FIG. 17D), thymus and activation-regulated chemokine (TARC) (FIG. 17E), MIP-3a (FIG. 17F), IL-16 (FIG. 17G), tissue inhibitor of metalloproteinase 1 (TIMP1) (FIG. 17H), and macrophage-derived chemokine (MDC) (FIG. 17I) in bronchoalveolar lavage fluid (BAL) of PPE+LPS-exposed mice treated with AB1000. [Figure 17I] 17A-17I are a series of bar graphs showing significant increases in protein levels of the anti-inflammatory cytokines Exodus2 (FIG. 17A), macrophage inflammatory protein-3b (MIP-3b) (FIG. 17B), interleukin-11 (IL-11) (FIG. 17C), monocyte chemoattractant protein 5 (MCP-5) (FIG. 17D), thymus and activation-regulated chemokine (TARC) (FIG. 17E), MIP-3a (FIG. 17F), IL-16 (FIG. 17G), tissue inhibitor of metalloproteinase 1 (TIMP1) (FIG. 17H), and macrophage-derived chemokine (MDC) (FIG. 17I) in bronchoalveolar lavage fluid (BAL) of PPE+LPS-exposed mice treated with AB1000. [Figure 18]FIG. 1 is a series of dot plots showing a significant decrease in MMP-9 expression in lung tissue, a significant decrease in MMP-9 protein in serum, and a significant increase in IgA protein in BAL from mice exposed to cigarette smoke or control air and treated with AB1000 or control saline treatment. [Figure 19A] FIG. 19A is a series of bar graphs showing that bacterial administration (e.g., AB101, AB102, and / or AB103) reduced the expression of fibrotic and fibrotic markers. FIG. 19A is a series of bar graphs showing a significant reduction in the expression of markers of in vitro fibrosis (smooth muscle alpha-actin (αSMA)) and fibrosis development (collagen type I alpha 1 (COL1A1), COL1A2, fibronectin) in LL29 idiopathic pulmonary fibrosis (IPF) fibroblasts treated with lacto-blend. "Lacto-blend" refers to a blend of Lactobacillus bacterial strains AB101, AB102, and AB103, also referred to interchangeably herein as "AB-blend" (see, e.g., Table 12). FIG. 19B is a series of bar graphs showing that the blend of strains (AB101, AB102, AB103) performed better than the individual strains in reducing fibrotic markers. [Figure 19B] FIG. 19A is a series of bar graphs showing that bacterial administration (e.g., AB101, AB102, and / or AB103) reduced the expression of fibrotic and fibrotic markers. FIG. 19A is a series of bar graphs showing a significant reduction in the expression of markers of in vitro fibrosis (smooth muscle alpha-actin (αSMA)) and fibrosis development (collagen type I alpha 1 (COL1A1), COL1A2, fibronectin) in LL29 idiopathic pulmonary fibrosis (IPF) fibroblasts treated with lacto-blend. "Lacto-blend" refers to a blend of Lactobacillus bacterial strains AB101, AB102, and AB103, also referred to interchangeably herein as "AB-blend" (see, e.g., Table 12). FIG. 19B is a series of bar graphs showing that the blend of strains (AB101, AB102, AB103) performed better than the individual strains in reducing fibrotic markers. [Figure 20A] Figure 20 is a series of images and a bar graph showing that lactoblend delayed epithelial to mesenchymal transition in human bronchial epithelial cells exposed to bleomycin. Figure 20A is a series of images measuring the wound healing rate 17 hours after scratching of cells. Figure 20B is a bar graph showing the measured wound healing rate from Figure 20A. [Figure 20B] Figure 20 is a series of images and a bar graph showing that lactoblend delayed epithelial to mesenchymal transition in human bronchial epithelial cells exposed to bleomycin. Figure 20A is a series of images measuring the wound healing rate 17 hours after scratching of cells. Figure 20B is a bar graph showing the measured wound healing rate from Figure 20A. [Figure 21] 1 is a series of bar graphs showing that Lactoblend reduced profibrotic markers (transforming growth factor beta-1 (TGF beta-1)), protein found in inflammatory zones 1 (FIZZ1; also called resistin-like molecule alpha), and upregulated anti-fibrotic markers (IL-6, tumor necrosis factor alpha (TNF-alpha)) in THP1 monocytes (human leukemia monocytic cell line) exposed to bleomycin. [Figure 22] FIG. 1 is a series of bar graphs showing that Lactoblend reduced Influenza A (IAV) viral hemagglutinin (HA) protein and C-reactive protein (CRP) in BAL in IAV-infected mice. [Figure 23] 1 is a series of bar graphs showing that Lacto Blend ("Lacto") reduced markers of neutrophilic inflammation in a double-hit hyperoxia (HO)+E. coli mouse model of bronchopulmonary dysplasia (BPD). Neutrophilic inflammation markers measured included: MMP-9 mRNA in lung tissue, and the following proteins in BAL: MMP-9, myeloperoxidase (MPO), NE, CRP, and IL-6. [Figure 24] 1 is a table showing antibiotic resistance of a blend of live bacterial strains AB101, AB102, and AB103 (see also FIG. 8). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0176] The present disclosure describes the unexpected discovery that biotherapeutic agents comprising microbiota and / or their extracts or metabolites can be formulated for administration by inhalation. Thus, the technology described herein is directed to spray-dried biotherapeutic matrix compositions comprising bacterial preparations, which are formulated for administration by inhalation. Also described herein are unit dosage forms of such spray-dried biotherapeutic matrix compositions, devices comprising such pharmaceutical compositions, methods of producing such pharmaceutical compositions, and methods of using such spray-dried biotherapeutic matrix compositions to treat diseases, such as bronchopulmonary diseases, among others.

[0177] Providing a dose of commensal bacteria to the lungs of chronically ill patients can provide significant benefits to the patient, whether this means direct colonization of the patient's lungs with bacteria (e.g., live biotherapeutics) or administration of bacterial extracts and / or metabolites (e.g., biotherapeutics). Dysbiosis occurs when there is an imbalance between commensal (beneficial) and pathogenic (harmful) bacteria. The basis for addressing dysbiosis by administering commensal bacteria to obtain health benefits has been well established with the implementation of fecal transplants (FMT). Data also show that active bacterial extracts and metabolites can produce anti-inflammatory effects systemically. See, for example, Jang et al. (2020) Experimental & Molecular Medicine 52(7):1128-1139, Weingarden and Vaughn BP (2017) Gut Microbes 8(3):238-252, Arpaia et al. (2013) Nature 504(7480):451-455, Iraporda et al. (2015) Immunobiology 220(10):1161-1169, Vinolo et al. (2011) Nutrients 3(10):858-876 (the contents of each of which are incorporated by reference in their entirety).

[0178] Preparations and delivery forms for therapeutic agents including bacterial preparations Delivery of biotherapeutics directly to the lung requires inhalation administration techniques such as nebulized delivery, metered dose inhalers (MDIs), dry powder inhalers (DPIs), or soft mist inhalers (SMIs). In particular, administration of biotherapeutics is difficult due to stability concerns. The presence of moisture makes storage and use of powdered live biotherapeutics difficult. Typically, during production of live organisms (usually by fermentation), the "finishing" step of production involves freeze drying or lyophilization to remove moisture from the powdered organisms while in cold conditions to maximize shelf life. The use of nebulizers to deliver bacteria or bacterial products via inhalation can be considered, but stability becomes an issue when nebulizers use drug preparations in liquid (i.e., containing water) form, as well as the fact that typical nebulizers must use a small mesh to generate droplets for aerosolization. Many live biotherapeutics, especially bacteria, have a size that is too large to reliably pass through this mesh, creating additional challenges for the use of nebulizers to deliver bacteria or bacterial products.

[0179] In metered dose inhalers, many interactions come into play - i.e. the drug content to be administered is stored under high pressure with a propellant. Stability in this case is of concern not only for living organisms, but also for sensitive biotherapeutics that contain specific genes and proteins that require careful extraction and gentle processing. Propellant and high pressure generally do not help maintain the viability of bacteria, but MDIs can be used to deliver non-viable bacteria (e.g., heat-killed bacteria), bacterial extracts, and / or bacterial products.

[0180] To circumvent these challenges, delivery of inhalable dry powders through DPIs can be used. However, the therapeutic agent must be formulated as an inhalable dry powder to be successfully used in a DPI. This powder must have a specific moisture content (usually very low, 1-5%) for stability purposes, as well as specific aerodynamic properties to ensure proper and reliable delivery of the powder. As described in further detail herein, a process known as spray drying can be used to "engineer" this type of dry powder for use in a DPI.

[0181] spray drying Spray drying is a technique in which multiple ingredients are dissolved, sprayed into droplets, and rapidly dried in a drying chamber to produce custom dry, aerosolizable powders with tailored particle characteristics. Spray drying can be used to produce inhalable dry powders to treat COPD, cystic fibrosis (CF), asthma, and non-respiratory disorders such as diabetes and migraines.

[0182] Spray drying involves the production of a feedstock containing dissolved solid components intended to be present in the dried particles at some defined concentration (e.g., percent ratio of solvent weight to solvent weight, % w / w). The feedstock is then fed through a nozzle at a specified pressure to produce droplets. The droplets are dried by heated gas flowing through a drying chamber to rapidly produce dry particles. The particles are then collected at the bottom of a cyclone. A cyclone is a device designed to capture a reduced range of particle diameters, while the remaining "waste" particles are collected at the end of the process. The particles captured in these cyclones represent the pharmaceutical bulk powder intended or designed to be inhaled for the treatment or prevention of disease. See, for example, FIG. 1 for a schematic diagram of an exemplary spray drying process.

[0183] The production of an effective spray-dried powder can include more components than just the active pharmaceutical ingredient itself. The powder may include the active ingredient, one or more excipients, residual solvents, and / or emulsion stabilizers. These ingredients are dissolved or suspended in the material to be dried before the start of spray drying. The homogenous solution or suspension is then dried to produce the inhalable dry powder itself.

[0184] The included excipients are used to provide some advantage to the spray-dried powder; that is, they are included to provide certain thermodynamic and physical properties. Excipients are often responsible for the shape of the spray-dried particles themselves, due to their solubility properties. In the source solvent, the solubility of the excipients determines how quickly the sprayed droplets form solid particles and how quickly the solid molecules move toward the center of the droplets during drying. As the particles dry, the chemical and thermodynamic properties of the powders and their stability affect the solubility of the final powder, i.e., over time and beyond temperature and humidity exposure, depending on the polymorphism (or lack thereof) of the final powder. Appropriate selection of excipients results in dried powders that exhibit stable crystallinity, high particle density, consistent shape, and high dispersibility. Often, amino acids such as leucine are combined with salts or sugars to optimize this effect.

[0185] Surfactant stabilizers are often used when spray drying formulations with hydrophobic or insoluble particles. In some embodiments, the formulation includes stabilizers when spray drying the suspension, so that the suspension is uniformly emulsified and dispersed, resulting in a uniform suspension for consistent droplet spraying. These surfactants are often included in low percentages to minimize their impact on the final dry powder. Polysorbates of various purities are frequently used, but other organic acid combinations are possible, along with steric surfactants such as Pluronic F68.

[0186] These spray-dried powders can be encapsulated and used in several forms of dry powder delivery devices that allow the patient to inhale the particles deeply and deposit at a target depth in the airways. These spray-dried powders can be filled into crushable capsules or sachets that can be crushed when used inside the delivery device. Once the enclosure is broken, the patient can inhale deeply through the mouthpiece of the inhaler and the powder can be deposited in the patient's throat, esophagus, and lungs. The purpose of dry powders for inhalation is to generate a powder of a specific aerodynamic size and density so that a predictable and safe dose of drug is deposited in the appropriate portion of the lung airways. See, for example, FIG. 2 for non-limiting examples of inhalation dry powder delivery devices.

[0187] For the treatment of chronic bronchopulmonary disorders characterized by inflammation, a drug delivery mechanism is described herein that allows a patient to administer a biotherapeutic agent (e.g., a living or non-living extract, or polymerized metabolite) directly to the lungs via inhalation of a spray-dried powder that contains all or a portion of the components in a drug delivery matrix, as exemplified in Tables 1A-1C or 10A-10B below. [Table 1A] [Table 1B] [Table 1C] [Table 10A] [Table 10B]

[0188] Spray-dried biotherapeutic matrix compositions Described herein is an inhaled biotherapeutic product that delivers a matrix of components directly to the lungs for the treatment of chronic diseases. In one aspect, described herein is a spray-dried biotherapeutic matrix composition that includes a bacterial preparation, where the matrix composition is formulated for administration by inhalation. As used herein, the term "spray-dried biotherapeutic matrix composition" refers to a composition that includes a matrix of a biotherapeutic agent that can be produced using the spray-drying process described herein. The present disclosure encompasses embodiments of this inhaled biotherapeutic product (either in a dry crystalline solid form, a dry amorphous solid form, or a mixture) that contains a matrix of one or more of the following components: (a) a bacterial preparation, (b) at least one excipient, and / or (c) at least one stabilizer.

[0189] In some embodiments, the spray dried biotherapeutic matrix composition comprises (a) a bacterial preparation. In some embodiments, the spray dried biotherapeutic matrix composition comprises (b) at least one excipient. In some embodiments, the spray dried biotherapeutic matrix composition comprises (c) at least one stabilizer. In some embodiments, the spray dried biotherapeutic matrix composition comprises (a) a bacterial preparation, and (b) at least one excipient. In some embodiments, the spray dried biotherapeutic matrix composition comprises (a) a bacterial preparation, and (c) at least one stabilizer. In some embodiments, the spray dried biotherapeutic matrix composition comprises (b) at least one excipient, and (c) at least one stabilizer. In some embodiments, the spray dried biotherapeutic matrix composition comprises (a) a bacterial preparation, (b) at least one excipient, and (c) at least one stabilizer. In some embodiments, the spray dried biotherapeutic matrix composition comprises (a) a bacterial preparation, (b) at least one excipient, and (c) at least one stabilizer.

[0190] bacterial preparation The spray dried biotherapeutic matrix compositions described herein comprise at least one bacterial preparation. In some embodiments, the spray dried biotherapeutic matrix compositions comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more bacterial preparations. As used herein, the term "bacterial preparation" refers to a preparation that comprises live bacteria (i.e., a "live biotherapeutic") and / or non-living bacteria or components thereof (i.e., a "non-living biotherapeutic").

[0191] In some embodiments, the bacterial preparation comprises viable or non-viable bacteria. In some embodiments, the bacterial preparation comprises viable bacteria. In some embodiments, the viable bacteria are capable of actively metabolizing and / or growing in the lungs of the subject. In some embodiments, the bacterial preparation (e.g., in a spray-dried biotherapeutic matrix composition, e.g., after spray drying) comprises bacteria with a viability of at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% (see, e.g., FIG. 7). In some embodiments, the bacterial preparation comprises non-viable bacteria. In some embodiments, the non-viable bacteria are heat-killed. In some embodiments, the non-viable bacteria are killed with ultraviolet light, ethanol, or bleach. In some embodiments, the non-viable bacteria are killed with an antibiotic.

[0192] In some embodiments, the bacteria is aerobic. In some embodiments, the bacteria is anaerobic. In some embodiments, the bacteria is an aerobe. In some embodiments, the bacteria is an obligate aerobe. In some embodiments, the bacteria is an anaerobe. In some embodiments, the bacteria is an obligate anaerobe. In some embodiments, the bacteria is a facultative anaerobe. In some embodiments, the bacteria is an aerotolerant anaerobe. In some embodiments, the bacteria is a capnophylate. In some embodiments, the bacteria is microaerophilic.

[0193] In some embodiments, the bacteria produce at least one immunomodulator. As used herein, the term "immunomodulator" refers to a substance that affects the function of the immune system, e.g., increases or decreases immune function. It should be understood that the bacteria or its components can increase or induce an adaptive immune response that produces antibodies against the bacteria or its components, which technically affects the function of the immune system, but the immunomodulation encompassed by "immunomodulator" as used herein does not encompass an adaptive response that produces antibodies. Rather, the "immunomodulators" produced by the bacteria described herein generally modulate innate immune function, including, but not limited to, inflammatory responses (or suppression thereof), cytokine or chemokine production (or suppression thereof), and the like.

[0194] In some embodiments, the bacterial immunomodulator increases immune function. Non-limiting examples of bacterial immunomodulators that increase immune function include bacterial toxins (e.g., cholera toxin, pertussis toxin, etc.), flagellin, or lipopolysaccharide (LPS). Such bacterial immunomodulators that increase immune function may be used for indications involving immune deficiency, infection, or cancer, or any other need for an increased immune response. In some embodiments, the bacterial immunomodulator decreases immune function. Non-limiting examples of bacterial immunomodulators that decrease immune function include lipopeptides (e.g., amphomycin, polymyxin, teicoplanin, or bacitracin) that can suppress the expression of inflammatory cytokines, or daptomycin. Such bacterial immunomodulators that decrease immune function may be used for indications involving autoimmunity, cytokine storm, or any other need for a decreased immune response.

[0195] In some embodiments, the bacteria is gram negative. In some embodiments, the bacteria is gram positive. In some embodiments, the bacteria is acid fast. In some embodiments, the bacteria is sporulating. In some embodiments, the bacteria is in spore form. In some embodiments, the spray dried biotherapeutic matrix composition comprises at least one (e.g., at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or more) bacterial preparations listed in Table 11. In some embodiments, the spray dried biotherapeutic matrix composition comprises three bacterial preparations. [Table 11]

[0196] In some embodiments, the bacterium belongs to a genus selected from the group consisting of Carnobacterium, Lactiplantibacillus, Lactobacillus, Lacticaseibacillus, Ligilactobacillus, Oenococcus, Leuconostoc, Pedicoccus, Enterococcus, Lactococcus, Staphylococcus, Streptococcus, Streptomyces, Bifidobacterium, Propionibacterium, and Moraxella. In some embodiments, the bacterium belongs to the genus Carnobacterium. In some embodiments, the bacterium belongs to the genus Lactiplantibacillus. In some embodiments, the bacterium belongs to the genus Lactobacillus. In some embodiments, the bacterium belongs to the genus Lacticaseibacillus. In some embodiments, the bacterium belongs to the genus Ligilactobacillus. In some embodiments, the bacterium belongs to the genus Oenococcus. In some embodiments, the bacterium belongs to the genus Leuconostoc. In some embodiments, the bacterium belongs to the genus Pedicoccus. In some embodiments, the bacterium belongs to the genus Enterococcus. In some embodiments, the bacterium belongs to the genus Lactococcus. In some embodiments, the bacterium belongs to the genus Staphylococcus. In some embodiments, the bacterium belongs to the genus Streptococcus. In some embodiments, the bacterium belongs to the genus Streptomyces. In some embodiments, the bacterium belongs to the genus Bifidobacterium. In some embodiments, the bacterium belongs to the genus Propionibacterium. In some embodiments, the bacterium belongs to the genus Moraxella.

[0197] In some embodiments, the bacterium is Lacticaseibacillus rhamnosus, Lactobacillus acidophilus, or Lactiplantibacillus plantarum. In some embodiments, the bacterium is Lacticaseibacillus rhamnosus (formerly known as Lactobacillus rhamnosus). In some embodiments, the bacterium is Lactobacillus acidophilus. In some embodiments, the bacterium is Lactiplantibacillus plantarum (formerly known as Lactobacillus plantarum). In some embodiments, the bacterium is Lacticaseibacillus rhamnosus and Lactobacillus acidophilus. In some embodiments, the bacterium is Lacticaseibacillus rhamnosus and Lactiplantibacillus plantarum. In some embodiments, the bacterium is Lactobacillus acidophilus and Lactiplantibacillus plantarum. In some embodiments, the bacteria is Lacticaseibacillus rhamnosus, Lactobacillus acidophilus, and Lactiplantibacillus plantarum. In some embodiments, the bacteria is Lacticaseibacillus rhamnosus strain LGG. In some embodiments, the bacteria is Lactiplantibacillus plantarum ATCC BAA-793™. In some embodiments, the bacteria is Lactobacillus acidophilus ATCC 4356™. In some embodiments, the bacteria is Lacticaseibacillus rhamnosus ATCC 53103™. In some embodiments, the bacteria is lactic acid bacteria (LAB), i.e., belongs to the order Lactobacillales, and produces lactic acid as the major metabolic end product of carbohydrate fermentation.

[0198] In some embodiments of any of the aspects, 16S rRNA gene sequencing is performed on or obtained from bacteria. 16S rRNA gene sequencing may also be referred to as "16S ribosomal RNA sequencing," "16S rDNA sequencing," or "16s rRNA sequencing." Sequencing of the 16S rRNA gene can be used for genetic research because it is highly conserved among different species of bacteria, but absent in eukaryotic species. In addition to the highly conserved regions, the 16S rRNA gene also contains nine hypervariable regions (V1-V9) that vary between species. 16S rRNA gene sequencing typically involves using multiple universal primers that bind to conserved regions of the 16S rRNA gene, PCR amplifying regions of the bacterial 16S rRNA gene (including hypervariable regions), and sequencing the amplified 16S rRNA gene using, for example, next-generation sequencing techniques described herein (see, e.g., U.S. Pat. Nos. 5,654,418, 6,344,316, and 8,889,358, as well as U.S. Patent Application Nos. 2013 / 0157265 and 2018 / 0195111, which are incorporated by reference in their entireties).

[0199] In some embodiments, the bacterium is selected from a species that comprises a 16S rRNA sequence, gene sequence, or genome sequence selected from a species listed in Table 12. In some embodiments, the bacterium is selected from a species that comprises a 16S rRNA sequence, gene sequence, or genome sequence that is at least 90% identical to a 16S rRNA sequence, gene sequence, or genome sequence from a species listed in Table 12. In some embodiments, the bacterium is selected from a species that comprises a 16S rRNA sequence, gene sequence, or genome sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identical to a 16S rRNA sequence, gene sequence, or genome sequence of a species listed in Table 12. For further details on the exemplary bacterial strains listed in Table 12, see, e.g., U.S. Pat. No. 11,141,443 B2, the contents of which are incorporated herein by reference in their entirety. [Table 12]

[0200] In some embodiments, the spray dried biotherapeutic matrix composition described herein comprises a combination of at least two bacterial species or strains described herein. In some embodiments, the spray dried biotherapeutic matrix composition described herein comprises a combination of at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 or more bacterial species or strains described herein. It is contemplated herein that such combinations may have synergistic or additive effects. In some embodiments, the spray dried biotherapeutic matrix composition comprises 20 or fewer bacterial species or strains. In some embodiments, the spray dried biotherapeutic matrix composition comprises 19 or fewer, 18 or fewer, 17 or fewer, 16 or fewer, 15 or fewer, 14 or fewer, 13 or fewer, 12 or fewer, 11 or fewer, 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, or only one bacterial species or strain.

[0201] In some embodiments, the bacteria is non-pathogenic. In some embodiments of any of the aspects, the bacteria is engineered to be non-pathogenic. In some embodiments of any of the aspects, the bacteria is genetically modified to produce, for example, a biomolecule in the target bronchopulmonary tissue. In some embodiments of any of the aspects, the bacteria contains / does not contain a virulence gene. In some embodiments of any of the aspects, the bacteria is engineered to inactivate at least one virulence gene.

[0202] In some embodiments, the spray dried biotherapeutic matrix composition is pathogen-free. As used herein, the term "pathogen" or "pathogenic" refers to any infectious microorganism that causes or can cause disease in an organism. In one embodiment, pathogens include bacteria, fungi, archaea (e.g., methanogens, halophiles, thermophiles, and psychrophiles), protozoans (e.g., Plasmodium, Entamoeba histolytica, Trypanosoma brucei, Giardia lamblia), viruses, prions (e.g., PrPres and PrPSc), microphytes (e.g., Shewanella algae, Shewanella putrefaciens, and Shewanella xiamenensis), and / or microanimals / parasites (e.g., plankton, planaria, helminths, schistosomes, and trypanosomes). In some embodiments, the pathogenic virus includes, but is not limited to, an RNA virus (e.g., a flavivirus, a picornavirus, a rhabdovirus, a filovirus, a retrovirus (including a lentivirus), or a DNA virus (e.g., an adenovirus, a poxvirus, a herpesvirus, a cytomegalovirus, a hepadnavirus, or other viruses).

[0203] Non-limiting examples of pathogenic bacteria include spirochetes (e.g., Borrelia), actinomycetes (e.g., Actinomyces), mycoplasmas, rickettsiae, gram-negative aerobic bacilli, gram-negative aerobic cocci, gram-negative facultative anaerobic bacilli (e.g., Erwinia and Yersinia), gram-negative cocci, gram-negative cocci, gram-positive cocci (e.g., Staphylococcus and Streptococcus), endospore-forming bacilli, and endospore-forming cocci. Further non-limiting examples of bacterial pathogens include certain species of Bacillus, Brucella, Burkholderia, Francisella, Yersinia, Streptococcus, Haemophilus, Nisseria, Listeria, Clostridium, Klebsiella, Legionella, Escherichia (e.g., E. coli), Mycobacterium, Staphylococcus, Campylobacter, Vibrio, and Salmonella, as well as drug-resistant and multi-drug resistant and hypervirulent strains of these pathogenic bacteria. Non-limiting examples of known food-borne bacterial pathogens include certain species of Salmonella, Clostridium, Campylobacter species, Staphylococcus, Salmonella, Escherichia (e.g., E. coli), and Listeria.In some embodiments, non-limiting examples of bacterial pathogens include Bacillus anthracis, Brucella abortus, Brucella melitensis, Brucella suis, Burkholderia mallei, Burkholderia pseudomallei, Francisella tularensis, Yersinia pestis, Streptococcus groups A and B, MRSA, Streptococcus pneumonia, Haemophilus influenza, Nisseria meningitides, Listeria monocytegenes, Clostridium difficile, Klebsiella, hypervirulent pathogenic strains of E. coli, Mycobacterium tuberculosis, Staphylococcus aureus, Campylobacter species, Salmonella species, and Clostridium perfringens, as well as drug and multi-drug resistant and hypervirulent strains of these pathogenic bacteria. In some embodiments, non-limiting examples of known food-borne bacterial pathogens include Salmonella, non-typhoidal Clostridium perfringens, Campylobacter spp., Staphylococcus aureus, Salmonella, non-typhoidal, Campylobacter spp., E. coli (STEC) 0157, and Listeria monocytogenes.

[0204] In some embodiments of any of the aspects, the spray dried biotherapeutic matrix composition is substantially free of human pathogens (e.g., as described above or known in the art). In some embodiments of any of the aspects, the spray dried biotherapeutic matrix composition is substantially free of non-human mammalian pathogens that can cause infection and / or disease in humans.

[0205] In some embodiments, the bacteria are present at a concentration of at least 101 colony forming units per gram (CFU / g), at least 102 CFU / g, at least 103 CFU / g, at least 104 CFU / g, at least 105 CFU / g, at least 106 CFU / g, at least 107 CFU / g, at least 108 CFU / g, at least 109 CFU / g, at least 1010 CFU / g, at least 1011 CFU / g, or at least 1012 CFU / g. In embodiments where the bacteria are non-viable, the concentration refers to the colony forming units of the bacteria prior to killing the bacteria. In embodiments where the bacterial preparation comprises non-living components of the bacteria, the concentration refers to the colony forming units of the bacteria prior to isolating the non-living components from the bacteria. In some embodiments, the bacteria are present at a concentration of at least 350 x 106 colony forming units per gram (CFU / g). In some embodiments, the bacteria is present at a concentration of at least 350 x 109 colony forming units per gram (CFU / g). In some embodiments, the bacteria is present at a concentration of at least 106 colony forming units per gram (CFU / g). In some embodiments, the bacteria is present at a concentration of at least 108 colony forming units per gram (CFU / g). In some embodiments, the bacteria is present at a concentration of at least 109 colony forming units per gram (CFU / g).

[0206] In some embodiments, such as those including viable bacteria, the bacteria are each resistant to at least one antibiotic. In some embodiments, the bacteria are collectively resistant to at least two antibiotics. In some embodiments, the ensemble model is (each, alone or collectively) resistant to at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, at least sixteen, at least seventeen, at least eighteen, at least nineteen, at least twenty, or more antibiotics. In some embodiments of any of the aspects, the antibiotic is selected from amikacin, aztreonam, cefepime, cefoxitin, ciprofloxacin, levofloxacin, metronidazole, trimethoprim / sulfa, vancomycin, extended spectrum beta-lactamase (ESBL) plazomycin, fosfomycin, ceftazidime, or ofloxacin.

[0207] In some embodiments, such as those including viable bacteria, the bacteria are each susceptible to at least one antibiotic. In some embodiments, the bacteria are collectively susceptible to at least two antibiotics. In some embodiments, the ensemble model is (each alone or collectively) susceptible to at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or more antibiotics. In some embodiments of any of the aspects, the antibiotic is selected from amikacin, aztreonam, cefepime, cefoxitin, ciprofloxacin, levofloxacin, metronidazole, trimethoprim / sulfa, trimethoprim, vancomycin, extended-spectrum beta-lactamase (ESBL) plazomycin, fosfomycin, ceftazidime, or ofloxacin (see, e.g., Figures 8, 24).

[0208] In some embodiments, the spray dried biotherapeutic matrix composition comprises at least 0.5% by dry weight of the bacterial preparation, hi some embodiments, the spray dried biotherapeutic matrix composition comprises at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1.0%, at least 2.0%, at least 3.0%, at least 4.0%, at least 5.0%, at least 6.0%, at least 7.0%, at least 8.0%, at least 9.0%, at least 10.0% or more by dry weight of the bacterial preparation.

[0209] In some embodiments, the spray-dried biotherapeutic matrix composition comprises at least 5 mg of bacterial preparation per unit dose. In some embodiments, the spray dried biotherapeutic matrix composition comprises at least 0.1 mg, at least 0.2 mg, at least 0.3 mg, at least 0.4 mg, at least 0.5 mg, at least 0.6 mg, at least 0.7 mg, at least 0.8 mg, at least 0.9 mg, at least 1.0 mg, at least 2.0 mg, at least 3.0 mg, at least 4.0 mg, at least 5.0 mg, at least 10 mg, at least 15 mg, at least 20 mg, at least 25 mg, at least 30 mg, at least 35 mg, at least 40 mg, at least 45 mg, at least 50 mg, at least 55 mg, at least 60 mg, at least 65 mg, at least 70 mg, at least 75 mg, at least 80 mg, at least 85 mg, at least 90 mg, at least 95 mg, at least 100 mg, at least 200 mg, at least 300 mg, at least 400 mg, at least 500 mg, or more of the bacterial preparation per unit dose.

[0210] In some embodiments, the bacterial preparation comprises a bacterial extract or a bacterial metabolite preparation, in some embodiments, the bacterial extract or bacterial metabolite preparation is selected from the group consisting of bacterial exosomes, bacterial cell walls, peptidoglycans, teichoic acid, lipoteichoic acid, bacterial S-layer, exopolysaccharides, polysaccharides, lactic acid polymers, lactic acid derivatives, lactic acid intermediates, hydrogen peroxide, bacteriocins, salivaricins, reuterin, and bacterial growth supernatants.

[0211] In some embodiments, the bacterial preparation comprises bacterial exosomes, which are membrane-bound extracellular vesicles (EVs) that contain cytoplasmic, periplasmic, or transmembrane components from bacterial cells.

[0212] In some embodiments, the bacterial preparation comprises a bacterial cell wall. In some embodiments, the bacterial preparation comprises components of a bacterial cell wall. Bacterial cell walls are made of peptidoglycan (also called murein), which is made of polysaccharide chains cross-linked by unusual peptides containing D-amino acids. In some embodiments, the bacterial preparation comprises peptidoglycan. Peptidoglycan is a polymer of sugars and amino acids that form a mesh-like peptidoglycan layer on the outside of the plasma membrane of most bacteria that form the cell wall. The sugar component consists of alternating residues of β-(1,4) linked N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM). Attached to the N-acetylmuramic acid is a peptide chain of 3-5 amino acids.

[0213] In some embodiments, the bacterial preparation comprises teichoic acid. Teichoic acids are bacterial copolymers of carbohydrates and glycerol phosphate or ribitol phosphate linked via phosphodiester bonds. Teichoic acids are found in the cell walls of most Gram-positive bacteria, such as species of Staphylococcus, Streptococcus, Bacillus, Clostridium, Corynebacterium, and Listeria, and can extend to the surface of the peptidoglycan layer. They can be covalently bound to N-acetylmuramic acid or terminal D-alanine in tetrapeptide bridges between N-acetylmuramic acid units of the peptidoglycan layer, or anchored to the cytoplasmic membrane with a lipid anchor. Teichoic acids anchored to lipid membranes are referred to as lipoteichoic acids (LTA), while teichoic acids covalently bound to peptidoglycans are referred to as cell wall teichoic acids (WTA). In some embodiments, the bacterial preparation comprises lipoteichoic acid.

[0214] In some embodiments, the bacterial preparation comprises a bacterial S-layer. S-layers are two-dimensional (2D) protein arrays frequently found on the surface of bacteria and archaea. S-layers contain one or more (glyco)proteins known as S-layer proteins (SLPs), which form a regularly spaced array on the surface of the cell. S-layers are usually composed of a single protein.

[0215] In some embodiments, the bacterial preparation comprises exopolysaccharides. Exopolysaccharides (EPS) are extracellular macromolecules excreted in microorganisms as tightly bound capsules or loosely bound slime layers. ESP extracellular polymeric substances are high molecular weight natural polymers secreted by microorganisms into the environment. EPS establish the functional and structural integrity of biofilms. Exopolysaccharides generally consist of monosaccharides and several non-carbohydrate substituents (e.g., acetate, pyruvate, succinate, and phosphate).

[0216] In some embodiments, the bacterial preparation comprises polysaccharides, i.e., polysaccharides isolated from bacteria. Bacterial polysaccharides can include capsular polysaccharides (CPS), exopolysaccharides (EPS), lipopolysaccharides (LPS), teichoic acid (TA), and peptidoglycan.

[0217] In some embodiments, the bacterial preparation comprises a lactic acid producing compound. In some embodiments, the lactic acid producing compound is selected from (i) lactic acid, (ii) a non-polymeric compound capable of producing lactic acid, or (iii) a polymeric compound capable of producing lactic acid. In some embodiments, the non-polymeric lactic acid producing compound is an inorganic salt of lactic acid, an ester of lactic acid, or lactide. In some embodiments, the polymeric lactic acid producing compound is polylactic acid (PLA). In some embodiments, the polylactic acid is poly(L-lactide) (PLLA), poly(D,L-lactide) (PDLLA), or poly(D-lactide) (PDLA). In some embodiments, the polylactic acid is poly(D,L-lactide) (PDLLA). In some embodiments, the bacterial preparation comprises a lactic acid polymer. In some embodiments, the bacterial preparation comprises a lactic acid derivative. In some embodiments, the bacterial preparation comprises a lactic acid intermediate.

[0218] In some embodiments, the bacterial preparation comprises hydrogen peroxide (H2O2). Hydrogen peroxide can function as a bacteriostatic agent.

[0219] In some embodiments, the bacterial preparation comprises a bacteriocin. A bacteriocin is a proteinaceous or peptide toxin produced by bacteria that inhibits the growth of similar or closely related bacterial strain(s). Non-limiting examples of bacteriocins include colicin, colicin-like bacteriocins, microcin, teylosin, class I bacteriocins, class II bacteriocins, class III bacteriocins, or class IV bacteriocins.

[0220] In some embodiments, the bacterial preparation comprises salivaricin. Salivaricin is a genetically encoded peptide containing an intramolecular ring structure (i.e., a lantibiotic) that was first shown to be produced by Streptococcus salivarius. Non-limiting examples of salivaricins include salivaricin A, salivaricin B, salivaricin C, salivaricin D, or salivaricin E.

[0221] In some embodiments, the bacterial preparation comprises reuterin. Reuterin (3-hydroxypropionaldehyde) is an organic compound with the formula HOCH2CH2CHO. The name reuterin comes from Lactobacillus reuteri, which produces the compound biosynthetically from glycerol as a broad-spectrum antibiotic (bacteriocin).

[0222] In some embodiments, the bacterial preparation comprises a bacterial growth supernatant, which may also be referred to as a bacterial culture supernatant. A bacterial supernatant may be prepared by growing bacteria in a liquid medium and then removing the liquid medium, any debris, or floating cells at a particular time point (which may be removed by centrifuging the medium and removing the resulting liquid). A bacterial supernatant may contain substances such as polypeptides or peptides secreted from the bacteria.

[0223] Bacterial preparations may be prepared by methods known in the art. For example, live bacteria may be prepared by growing a bacterial isolate in a liquid growth medium suitable for the growth of that particular bacterial species. Non-viable bacteria may be prepared by killing the bacteria, such as by heat killing or treatment with chemicals such as UR light or alcohol. Bacterial components may be prepared by methods such as overexpression, transformation, solvent extraction, lysis, bead milling, centrifugation, dissociation, percolation, reflux extraction, Soxhlet extraction, pressurized liquid extraction, supercritical fluid extraction, ultrasound-assisted extraction, microwave-assisted extraction, pulsed electric field extraction, enzyme-assisted extraction, chromatography, affinity columns, immunochemistry, etc., or any combination thereof.

[0224] In some embodiments, the composition comprises prebiotics (amino acids (e.g., arginine, glutaric acid, and ornithine), biotin, fructooligosaccharides, galactooligosaccharides, hemicelluloses (e.g., arabinoxylan, xylan, xyloglucan, and glucomannan), inulin, chitin, lactose, mannan oligosaccharides, oligofructose-enriched inulin, gums (e.g., guar gum, gum arabic, and carrageenan), oligofructose, oligodextrose, and the like). These further include, but are not limited to, sugar, tagatose, resistant maltodextrins (e.g., resistant starch), transgalactooligosaccharides, pectins (e.g., xylogalacturonan, citrus pectin, apple pectin, and rhamnogalacturonan I), dietary fiber (e.g., soy fiber, sugar beet fiber, pea fiber, corn bran, and oat fiber), xylooligosaccharides, polyamines (such as, but not limited to, spermidine and putrescine).

[0225] In some embodiments, the bacterial preparation comprises at least one bacterial species (or an extract or metabolite thereof) and at least one of the following: bulking agents / binders, cryoprotectants, ascorbic acid, and / or oligosaccharides (see, e.g., Table 1C). Non-limiting examples of bulking agents or binders include GLUCIDEX (e.g., corn maltodextrin II). Non-limiting examples of cryoprotectants include trehalose or sodium glutamate. Non-limiting examples of oligosaccharides include beta cyclodextrin or sucrose.

[0226] In some embodiments, the bacterial preparation (see, e.g., Table 1C) is freeze-dried prior to use to prepare the solid and / or liquid feedstocks described herein. In such embodiments, the bacteria are freeze-dried and then spray-dried during preparation of the spray-dried biotherapeutic matrix compositions described herein. In some embodiments, the bacterial preparation is spray-dried prior to use to prepare the solid and / or liquid feedstocks described herein. In such embodiments, the bacteria are spray-dried a second time during preparation of the spray-dried biotherapeutic matrix compositions described herein.

[0227] In some embodiments, the bacterial preparation comprises fresh, non-spray-dried bacteria that are added directly to the solid and / or liquid feedstocks described herein. In such embodiments, the bacteria are spray-dried once during the preparation of the spray-dried biotherapeutic matrix compositions described herein. In some embodiments, the bacteria are fermented prior to use to prepare the solid and / or liquid feedstocks described herein.

[0228] An exemplary procedure for preparing a bacterial preparation includes (a) inoculating a bacterial inoculum (e.g., from a working cell bank) into a liquid growth medium, (b) fermenting the bacteria in the liquid growth medium, (c) recovering the bacteria from the liquid growth medium (e.g., by centrifugation), and (d) lyophilizing the recovered bacteria. This lyophilization process for preparing a bacterial preparation can include at least one of the excipient(s) and / or at least one of the stabilizer(s) described herein, and additional amounts of at least one excipient(s) and / or at least one stabilizer(s) can be added to prepare a spray-dried biotherapeutic matrix composition (see, e.g., Tables 1A-1C).

[0229] Pharmaceutically acceptable excipients, stabilizers, and additives In some embodiments, the technology described herein relates to a spray-dried biotherapeutic matrix composition comprising a bacterial preparation described herein and, optionally, pharma- ceutically acceptable excipients, stabilizers, and / or additives. In some embodiments, the active ingredient of the spray-dried biotherapeutic matrix composition comprises a bacterial preparation described herein. In some embodiments, the active ingredient of the spray-dried biotherapeutic matrix composition consists essentially of a bacterial preparation described herein. In some embodiments, the active ingredient of the spray-dried biotherapeutic matrix composition consists of a bacterial preparation described herein. In some embodiments, the pharma-ceutically acceptable excipients, stabilizers, and / or additives include saline, aqueous buffer, solvents, and / or dispersion media. The use of such excipients, stabilizers, and / or additives is well known to those skilled in the art.Some non-limiting examples of materials that can function as pharma- ceutically acceptable excipients, stabilizers, and / or additives include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethylcellulose, methylcellulose, ethylcellulose, microcrystalline cellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricants, such as magnesium stearate, sodium lauryl sulfate, and talc; (8) excipients, such as cocoa butter and suppository wax; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) glycols, such as protease inhibitors, ... (11) polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol (PEG), (12) esters, such as ethyl oleate and ethyl laurate, (13) agar, (14) buffers, such as magnesium hydroxide and aluminum hydroxide, (15) alginic acid, (16) pyrogen-free water, (17) isotonic saline, (18) Ringer's solution, (19) ethyl alcohol, (20) pH buffer solutions, (21) polyesters, polycarbonates, and / or polyanhydrides, (22) bulking agents, such as polypeptides and amino acids, (23) serum components, such as serum albumin, HDL, and LDL, (24) C2-C12 alcohols, such as ethanol, and (25) other non-toxic, non-toxic, compatible substances used in pharmaceutical formulations. Wetting agents, colorants, release agents, coating agents, sweeteners, flavoring agents, fragrances, preservatives, and antioxidants may also be present in the formulation. In some embodiments, the pharma- ceutically acceptable excipients, stabilizers, and / or additives inhibit degradation of the active agent (e.g., the bacterial preparations described herein). In some embodiments, the spray-dried biotherapeutic matrix composition comprises at least one pharma- ceutically acceptable excipient and / or at least one pharma- ceutically acceptable stabilizer.

[0230] Excipients In some embodiments, the spray dried biotherapeutic matrix composition comprises at least one excipient. In some embodiments, the spray dried biotherapeutic matrix composition comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more excipients. As used herein, the term "excipient" refers to an inert substance that serves as a vehicle, diluent, or medium for an active agent (e.g., a bacterial preparation described herein). An excipient can facilitate the processability of a spray dried biotherapeutic matrix composition (e.g., formulated for respiratory administration) and maintain the physical structure of the composition to add long-term stability. An excipient can provide stability (e.g., of a biotherapeutic agent), protect viability, and improve the aerosol properties of a spray dried biotherapeutic matrix composition. In some embodiments, the composition comprises at least two excipients. In some embodiments, the composition comprises two excipients. In some embodiments, the composition comprises three excipients. In some embodiments, the composition comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or more excipients.

[0231] Typically, excipients are used when spray drying pharmaceuticals for two purposes: to add dispersibility to the final powder and to glass-stabilize high viscosity crystalline or amorphous particles. See, for example, Vehring (2008) Pharm Res 25(5):999-1022, the contents of which are incorporated herein by reference in their entirety. Amino acids (e.g., leucine and trileucine) can be used to add dispersibility to the final powder. Dispersible powders tend not to clump easily, are more flowable in bulk, and tend to facilitate encapsulation.

[0232] Because spray drying produces particles in energetically unfavorable conditions, excipients used for glass stabilization help provide long-term stability benefits and reliable solid-state chemical characteristics, such as stable crystallinity or co-crystallinity that results in consistent bioavailability in vivo. The effectiveness of these excipients is typically assessed by measuring the glass transition temperature of the dried particles, which is best maintained well above storage temperatures (e.g., frozen at -18°C, refrigerated at 4°C, or room temperature at 20-22°C) to ensure physical stability. In some embodiments, the minimum Tg is 35°C. In some embodiments, the Tg is at least 30°C, at least 35°C, at least 40°C, at least 45°C, or at least 50°C.

[0233] In some embodiments, the excipient is selected from the group consisting of De Man, Rogosa and Sharpe (MRS) growth medium, gelatin, whey isolate, sweet whey, reconstituted skim milk powder, maltodextrin, gluco-oligosaccharides, lacto-oligosaccharides, fructooligosaccharides, inulin, sodium caseinate, goat milk, cow milk, proline, carnitine, acetylcarnitine, propionylcarnitine, glutamate, glycine betaine, glycogen, trehalose, mannose, xylose, mannitol, sorbitol, maltose, dextrose, starch, lactose, sucrose, glucose, leucine, trileucine, sodium salts, potassium salts, lithium salts, and calcium salts.

[0234] In some embodiments, the excipient is selected from Table 2. In some embodiments, the excipient is selected from any combination of excipients listed in Table 2, for example, a combination of at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, or at least 39 excipients from Table 2. [Table 2]

[0235] In some embodiments, the excipient is leucine, trehalose, and / or sodium citrate. In some embodiments, the excipient is leucine and / or trehalose. In some embodiments, the excipient is leucine and / or sodium citrate. In some embodiments, the excipient is trehalose and / or sodium citrate.

[0236] In some embodiments, the excipient is leucine. In some embodiments, the excipient is L-leucine (see, e.g., Formula I below). In some embodiments, the excipient is D-leucine. In some embodiments, the excipient is a racemic mixture of L-leucine and D-leucine. When used herein, "racemic mixture" refers to a solution in which both enantiomers of a compound are present in a 50:50 ratio. Leucine (symbol Leu or L) is an essential amino acid for humans. In some embodiments, the excipient is trileucine (also referred to as Leu-Leu-Leu), a tripeptide composed of three leucine residues (see, e.g., Formula II below). In some embodiments, trileucine is used instead of leucine as an excipient. In some embodiments, the excipient is trehalose (see, e.g., Formula III below). Trehalose is a sugar composed of two glucose molecules. Trehalose is also known as mycose or tremalose. Trehalose has a high water-holding capacity. Some bacteria, fungi, plants, and invertebrates synthesize trehalose as an energy source and to survive freezing and water shortages. In some embodiments, the excipients are leucine and trehalose. In some embodiments, the first excipient is leucine and the second excipient is trehalose. In some embodiments, the second excipient is leucine and the first excipient is trehalose. In some embodiments, the excipient is sodium citrate (Na3C6H5O7, see e.g., Formula IV below). In some embodiments, the first excipient and the second excipient are leucine and trehalose, and the third excipient is sodium citrate. In some embodiments, the three excipients are leucine, trehalose, and sodium citrate, and the stabilizer is polysorbate 80. [ka] [ka] [ka] [ka]

[0237] In some embodiments, the spray dried biotherapeutic matrix composition comprises at least 0.5% by weight of an excipient, hi some embodiments, the spray dried biotherapeutic matrix composition comprises at least 0.01%, at least 0.02%, at least 0.03%, at least 0.04%, at least 0.05%, at least 0.06%, at least 0.07%, at least 0.08%, at least 0.09%, at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1.0%, at least 2.0%, at least 3.0%, at least 4.0%, at least 5.0%, at least 10%, at least 15%, at least 20%, at least 25%, or at least 30% or more by weight of an excipient.

[0238] In some embodiments, the spray dried biotherapeutic matrix composition comprises at least 0.5 wt% of the first excipient and at least 0.5 wt% of the second excipient, in some embodiments, the spray dried biotherapeutic matrix composition comprises at least 0.01 wt%, at least 0.02 wt%, at least 0.03 wt%, at least 0.04 wt%, at least 0.05 wt%, at least 0.06 wt%, at least 0.07 wt%, at least 0.08 wt%, at least 0.09 wt%, at least 0.1 wt%, at least 0.2 wt%, at least 0.3 wt%, at least 0.4 wt%, at least 0.5 wt%, at least 0.6 wt%, at least 0.7 wt%, at least 0.8 wt%, at least 0.9 wt%, at least 1.0 wt%, at least 2.0 wt%, at least 3.0 wt%, at least 4.0 wt%, or at least 5.0 wt%. %, or more of a first excipient, and at least 0.01%, at least 0.02%, at least 0.03%, at least 0.04%, at least 0.05%, at least 0.06%, at least 0.07%, at least 0.08%, at least 0.09%, at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1.0%, at least 2.0%, at least 3.0%, at least 4.0%, or at least 5.0% or more of a second excipient.

[0239] In some embodiments, the spray dried biotherapeutic matrix composition comprises at least 0.01 wt%, at least 0.02 wt%, at least 0.03 wt%, at least 0.04 wt%, at least 0.05 wt%, at least 0.06 wt%, at least 0.07 wt%, at least 0.08 wt%, at least 0.09 wt%, at least 0.1 wt%, at least 0.2 wt%, at least 0.3 wt%, at least 0.4 wt%, at least 0.5 wt%, at least 0.6 wt%, at least 0.7 wt%, at least 0.8 wt%, at least 0.9 wt%, at least 1.0 wt%, at least 2.0 wt%, at least 3.0 wt%, at least 4.0 wt%, or at least 5.0 wt% or more of a first excipient and at least 0.5 wt% of a second excipient. In some embodiments, the spray dried biotherapeutic matrix composition comprises at least 0.5 wt.% of a first excipient and at least 0.01 wt.%, at least 0.02 wt.%, at least 0.03 wt.%, at least 0.04 wt.%, at least 0.05 wt.%, at least 0.06 wt.%, at least 0.07 wt.%, at least 0.08 wt.%, at least 0.09 wt.%, at least 0.1 wt.%, at least 0.2 wt.%, at least 0.3 wt.%, at least 0.4 wt.%, at least 0.5 wt.%, at least 0.6 wt.%, at least 0.7 wt.%, at least 0.8 wt.%, at least 0.9 wt.%, at least 1.0 wt.%, at least 2.0 wt.%, at least 3.0 wt.%, at least 4.0 wt.%, or at least 5.0 wt.% or more of a second excipient.

[0240] In some embodiments, the spray dried biotherapeutic matrix composition comprises at least 5 mg of excipient per unit dose. In some embodiments, the spray dried biotherapeutic matrix composition comprises at least 0.1 mg, at least 0.2 mg, at least 0.3 mg, at least 0.4 mg, at least 0.5 mg, at least 0.6 mg, at least 0.7 mg, at least 0.8 mg, at least 0.9 mg, at least 1.0 mg, at least 2.0 mg, at least 3.0 mg, at least 4.0 mg, at least 5.0 mg, at least 10 mg, at least 15 mg, at least 20 mg, at least 25 mg, at least 30 mg, at least 35 mg, at least 40 mg, at least 45 mg, at least 50 mg, at least 55 mg, at least 60 mg, at least 65 mg, at least 70 mg, at least 75 mg, at least 80 mg, at least 85 mg, at least 90 mg, at least 95 mg, at least 100 mg, at least 200 mg, at least 300 mg, at least 400 mg, at least 500 mg, or more of excipient per unit dose.

[0241] In some embodiments, the spray dried biotherapeutic matrix composition comprises at least 5 mg of a first excipient and at least 5 mg of a second excipient per unit dose. In some embodiments, the spray dried biotherapeutic matrix composition comprises at least 0.1 mg, at least 0.2 mg, at least 0.3 mg, at least 0.4 mg, at least 0.5 mg, at least 0.6 mg, at least 0.7 mg, at least 0.8 mg, at least 0.9 mg, at least 1.0 mg, at least 2.0 mg, at least 3.0 mg, at least 4.0 mg, at least 5.0 mg, at least 10 mg, at least 15 mg, at least 20 mg, at least 25 mg, at least 30 mg, at least 35 mg, at least 40 mg, at least 45 mg, at least 50 mg, at least 55 mg, at least 60 mg, at least 65 mg, at least 70 mg, at least 75 mg, at least 80 mg, at least 85 mg, at least 90 mg, at least 95 mg, at least 100 mg, at least 200 mg, at least 300 mg, at least 400 mg, at least 5 In some embodiments, the composition comprises at least 0.00 mg or more of a first excipient and at least 0.1 mg, at least 0.2 mg, at least 0.3 mg, at least 0.4 mg, at least 0.5 mg, at least 0.6 mg, at least 0.7 mg, at least 0.8 mg, at least 0.9 mg, at least 1.0 mg, at least 2.0 mg, at least 3.0 mg, at least 4.0 mg, at least 5.0 mg, at least 10 mg, at least 15 mg, at least 20 mg, at least 25 mg, at least 30 mg, at least 35 mg, at least 40 mg, at least 45 mg, at least 50 mg, at least 55 mg, at least 60 mg, at least 65 mg, at least 70 mg, at least 75 mg, at least 80 mg, at least 85 mg, at least 90 mg, at least 95 mg, at least 100 mg, at least 200 mg, at least 300 mg, at least 400 mg, at least 500 mg, or more of a second excipient.

[0242] In some embodiments, the spray dried biotherapeutic matrix composition comprises at least 0.1 mg, at least 0.2 mg, at least 0.3 mg, at least 0.4 mg, at least 0.5 mg, at least 0.6 mg, at least 0.7 mg, at least 0.8 mg, at least 0.9 mg, at least 1.0 mg, at least 2.0 mg, at least 3.0 mg, at least 4.0 mg, at least 5.0 mg, at least 10 mg, at least 15 mg, at least 20 mg, at least 25 mg, at least 30 mg, at least 35 mg, at least 40 mg, at least 45 mg, at least 50 mg, at least 55 mg, at least 60 mg, at least 65 mg, at least 70 mg, at least 75 mg, at least 80 mg, at least 85 mg, at least 90 mg, at least 95 mg, at least 100 mg, at least 200 mg, at least 300 mg, at least 400 mg, at least 500 mg, or more of a first excipient and at least 5 mg of a second excipient per unit dose.

[0243] In some embodiments, the spray dried biotherapeutic matrix composition comprises at least 5 mg of a first excipient and at least 0.1 mg, at least 0.2 mg, at least 0.3 mg, at least 0.4 mg, at least 0.5 mg, at least 0.6 mg, at least 0.7 mg, at least 0.8 mg, at least 0.9 mg, at least 1.0 mg, at least 2.0 mg, at least 3.0 mg, at least 4.0 mg, at least 5.0 mg, at least 10 mg, at least 15 mg, at least 20 mg, at least 25 mg, at least 30 mg, at least 35 mg, at least 40 mg, at least 45 mg, at least 50 mg, at least 55 mg, at least 60 mg, at least 65 mg, at least 70 mg, at least 75 mg, at least 80 mg, at least 85 mg, at least 90 mg, at least 95 mg, at least 100 mg, at least 200 mg, at least 300 mg, at least 400 mg, at least 500 mg, or more of a second excipient per unit dose.

[0244] In some embodiments, the spray dried biotherapeutic matrix composition comprises at least 5 mg of a first excipient, at least 5 mg of a second excipient, and at least 1 mg of a third excipient per unit dose. In some embodiments, the spray dried biotherapeutic matrix composition comprises at least 5 mg of a first excipient, at least 5 mg of a second excipient, and at least 0.1 mg, at least 0.2 mg, at least 0.3 mg, at least 0.4 mg, at least 0.5 mg, at least 0.6 mg, at least 0.7 mg, at least 0.8 mg, at least 0.9 mg, at least 1.0 mg, at least 2.0 mg, at least 3.0 mg, at least 4.0 mg, at least 5.0 mg, at least 10 mg, at least 25 mg, at least 30 mg, at least 40 mg, at least 50 mg, at least 60 mg, at least 70 mg, at least 80 mg, at least 90 mg, at least 100 mg, at least 15 ... at least 15 mg, at least 20 mg, at least 25 mg, at least 30 mg, at least 35 mg, at least 40 mg, at least 45 mg, at least 50 mg, at least 55 mg, at least 60 mg, at least 65 mg, at least 70 mg, at least 75 mg, at least 80 mg, at least 85 mg, at least 90 mg, at least 95 mg, at least 100 mg, at least 200 mg, at least 300 mg, at least 400 mg, at least 500 mg, or more of a third excipient.

[0245] Stabilizers In some embodiments, the spray dried biotherapeutic matrix composition comprises at least one stabilizer. In some embodiments, the spray dried biotherapeutic matrix composition comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more stabilizers. As used herein, the term "stabilizer" refers to a substance that prevents or reduces degradation of the spray dried biotherapeutic matrix composition. Surfactant stabilizers can provide initial wettability during feedstock preparation, emulsification of feedstock during spray drying, and protection of the solid state of the dried powder by surface moisture protection of the therapeutic agent.

[0246] The use of stabilizers aids in the process of wetting hydrophobic materials to create a homogenous suspension for spray drying. For example, stabilizers can be included if a wetting agent is needed to bring any poorly soluble compounds into suspension. These stabilizers act as emulsifiers to increase consistency across the bulk spray-dried powder. In addition, some stabilizers (e.g., poloxamers) can also adjust the size of the wet particles down to the nanoscale and can be used to pre-disperse macromolecules in the feedstock solution. See, for example, Da Silva et al. (2019) Front Bioeng Biotechnol 7:137, the contents of which are incorporated herein by reference in their entirety.

[0247] In some embodiments, the stabilizer comprises a surfactant. A surfactant is a substance that tends to reduce the surface tension of the liquid in which it is dissolved. In some embodiments, the stabilizer is selected from the group consisting of mannitol, carboxymethylcellulose (CMC), polyvinyl alcohol (PVA), polysorbate, and poloxamer. In some embodiments, the stabilizer is polysorbate, poloxamer, or polyvinyl alcohol.

[0248] In some embodiments, the stabilizer comprises polysorbate. Polysorbate is a synthetic non-ionic surfactant and emulsifier. Polysorbate is also called MONTANOX, ALKEST TW, TWEEN, or PS. In some embodiments, the stabilizer is polysorbate 20, polysorbate 40, polysorbate 60, or polysorbate 80. The number following the "polysorbate" portion relates to the type of fatty acid associated with the polyoxyethylene sorbitan portion of the molecule. Monolaurate is designated by 20 (e.g., polysorbate 20), monopalmitate is designated by 40 (e.g., polysorbate 40), monostearate is designated by 60 (e.g., polysorbate 60), and monooleate is designated by 80 (e.g., polysorbate 80).

[0249] In some embodiments, the stabilizer is polysorbate 80 (see, for example, Formula V below). Polysorbate 80 is also referred to as polyoxyethylene (20) sorbitan monooleate (the number 20 after the "polyoxyethylene" portion refers to the total number of oxyethylene-(CH2CHO)- groups found in the molecule), (x)-sorbitan mono-9-octadecenoate poly(oxy-1,2-ethanediyl), MONTANOX80, ALKEST TW80, TWEEN80, or PS80. Polysorbate 80 is derived from polyethoxylated sorbitan and oleic acid. The hydrophilic group in this compound is a polyether, also known as a polyoxyethylene group, which is a polymer of ethylene oxide. In the nomenclature of polysorbates, the numerical designation after polysorbate refers to the lipophilic group, in this case oleic acid. The critical micelle concentration of polysorbate 80 in pure water is reported as 0.012 mM. [ka]

[0250] In some embodiments, the stabilizer is a poloxamer. Poloxamers are non-ionic triblock copolymers consisting of a central hydrophobic chain of polyoxypropylene (poly(propylene oxide)) flanked by two hydrophilic chains of polyoxyethylene (poly(ethylene oxide)). Poloxamers are also called PLURONIC, KOLLIPHOR, or SYNPERONIC. Because the length of the polymer block can be customized, there are many different poloxamers with slightly different properties. With respect to the general term poloxamer, these copolymers are generally named with the letter P (for poloxamer) followed by three numbers, the first two numbers are multiplied by 100 to obtain the approximate molecular weight of the polyoxypropylene core, and the last number is multiplied by 10 to obtain the polyoxyethylene content (e.g., P407 = poloxamer with a polyoxypropylene molecular weight of 4000 g / mo and a polyoxyethylene content of 70%). As for the PLURONIC and SYNPERONIC designations, the coding of these copolymers begins with a letter to define its physical form at room temperature (L=liquid, P=paste, F=flake (solid)), followed by two or three numbers. The first number (or two of the three numbers) of the numerical designation multiplied by 300 indicates the approximate molecular weight of the hydrophobe, and the last number multiplied by 10 indicates the polyoxyethylene content (for example, L61 indicates a polyoxypropylene molecular weight of 1800 g / mol and a polyoxyethylene content of 10%). In the given example, Poloxamer 181 (P181) = PLURONIC L61 and SYNPERONIC PE / L61.

[0251] One characteristic of poloxamer solutions is their temperature-dependent self-assembly and thermogelation behavior. Concentrated aqueous solutions of poloxamers are liquid at low temperatures and form gels at higher temperatures in a reversible process. The transitions that occur in these systems depend on the polymer composition. Due to their amphiphilic structure, poloxamers have surfactant properties. Among other things, poloxamers can be used to increase the water solubility of hydrophobic, oily substances, or to otherwise increase the miscibility of two substances with different hydrophobicities. See, for example, Table 3 for the physicochemical properties of exemplary poloxamers. [Table 3]

[0252] In some embodiments, the stabilizer is poloxamer 184 (i.e., Pluronic L64), poloxamer 185 (i.e., Pluronic P65), poloxamer 188 (i.e., Pluronic F68), poloxamer 234 (i.e., Pluronic P84), poloxamer 235 (i.e., Pluronic P85), poloxamer 238 (i.e., Pluronic F88), poloxamer 333 (i.e., Pluronic P103), poloxamer 334 (i.e., Pluronic P104), poloxamer 335 (i.e., Pluronic P105), poloxamer 338 (i.e., Pluronic F108), poloxamer 403 (i.e., Pluronic P123), or poloxamer 407 (i.e., Pluronic F127). In some embodiments, the stabilizer is poloxamer 184, poloxamer 185, poloxamer 234, poloxamer 235, poloxamer 238, poloxamer 333, poloxamer 334, poloxamer 335, poloxamer 338, poloxamer 403, or poloxamer 407. In some embodiments, the stabilizer is Pluronic L64, Pluronic P65, Pluronic P84, Pluronic P85, Pluronic F88, Pluronic P103, Pluronic P104, Pluronic P105, Pluronic F108, Pluronic P123, or Pluronic F127.

[0253] In some embodiments, the stabilizer is poloxamer 188, also referred to as Pluronic F68 or polyoxyethylene-polyoxypropylene block copolymer (linear formula: (C3H6O.C2H4O)x, see, e.g., Formula VI below). In some embodiments, the spray dried biotherapeutic matrix composition comprises polysorbate 80. In some embodiments, the spray dried biotherapeutic matrix composition comprises poloxamer 188 (i.e., Pluronic F68). In some embodiments, the spray dried biotherapeutic matrix composition comprises polysorbate 80 and poloxamer 188 (i.e., Pluronic F68). [ka]

[0254] In some embodiments, the stabilizer is polyvinyl alcohol (PVA, see, for example, Formula VII below). PVA is a water-soluble synthetic polymer with the idealized formula [CH2CH(OH)]n. PVA can be used as a thickener and emulsion stabilizer. PVA exhibits biocompatibility, low tendency for protein adhesion, and low toxicity. PVA is prepared by hydrolysis of polyvinyl acetate or other vinyl ester-derived polymers with formate or chloroacetate groups instead of acetate. Conversion of polyvinyl esters can be carried out by base-catalyzed transesterification with ethanol. [ka]

[0255] In some embodiments, the spray dried biotherapeutic matrix composition comprises at least 0.05% stabilizer by weight. In some embodiments of any of the aspects, the composition comprises at least 0.25% stabilizer by weight. In some embodiments of any of the aspects, the composition comprises at least 0.5% stabilizer by weight (e.g., dry weight). In some embodiments, the spray dried biotherapeutic matrix composition comprises at least 0.01%, at least 0.02%, at least 0.03%, at least 0.04%, at least 0.05%, at least 0.06%, at least 0.07%, at least 0.08%, at least 0.09%, at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1.0% or more stabilizer by weight.

[0256] In some embodiments, the spray dried biotherapeutic matrix composition comprises at least 1 mg of stabilizer per unit dose, hi some embodiments, the spray dried biotherapeutic matrix composition comprises at least 0.1 mg, at least 0.2 mg, at least 0.3 mg, at least 0.4 mg, at least 0.5 mg, at least 0.6 mg, at least 0.7 mg, at least 0.8 mg, at least 0.9 mg, at least 1.0 mg, at least 2.0 mg, at least 3.0 mg, at least 4.0 mg, at least 5.0 mg, or at least 10 mg of stabilizer per unit dose.

[0257] In some embodiments, the spray dried biotherapeutic matrix composition comprises at least one excipient and at least one stabilizer. In some embodiments, the spray dried biotherapeutic matrix composition comprises one excipient and one stabilizer. In some embodiments, the spray dried biotherapeutic matrix composition comprises at least two excipients and at least one stabilizer. In some embodiments, the spray dried biotherapeutic matrix composition comprises two excipients and one stabilizer. In some embodiments, the spray dried biotherapeutic matrix composition comprises leucine and trehalose as excipients and poloxamer 188 as stabilizer. In some embodiments, the spray dried biotherapeutic matrix composition comprises leucine and trehalose as excipients and polysorbate 80 as stabilizer.

[0258] Additives In some embodiments, the spray dried biotherapeutic matrix composition further comprises at least one additive. In some embodiments, the spray dried biotherapeutic matrix composition comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more additives. As described herein, some additives can be added to the polymeric particles, and these additives can be incorporated by mixing at the molecular level, dry blending, coating on the particles, or co-administration. In some embodiments, the spray dried biotherapeutic matrix composition further comprises at least one of the following: (a) a pore former, (b) an adhesive, and / or (c) a pH adjusting agent.

[0259] In some embodiments, the spray dried biotherapeutic matrix composition further comprises a pore-forming agent. The pore-forming agent can reduce the density of the particle (i.e., by creating voids or "pores" within the particle), allowing for more rapid water uptake, decomposition, and acid production. In some embodiments, the pore-forming agent is selected from the group consisting of NaCl, sucrose, polyethylene glycol (PEG), and polyvinylpyrrolidone (PVP).

[0260] In some embodiments, the spray dried biotherapeutic matrix composition further comprises an adhesive. The adhesive can enhance the bioadhesion of the polymer to biological tissue. In some embodiments, the adhesive is selected from the group consisting of a sugar, an adhesive polymer, and an amine-containing compound. Non-limiting examples of such adhesive sugars include trehalose, mannitol, lactose, or glucose.

[0261] In some embodiments, the spray dried biotherapeutic matrix composition further comprises a pH adjusting agent. The pH adjusting agent can increase or decrease the degradation of the bacterial preparation. In some embodiments, the pH adjusting agent is a buffer such as sodium citrate. In some embodiments, the pH adjusting agent is an acid. In some embodiments, the pH adjusting agent is a base (e.g., NaOH), and such bases can be neutralizing agents when preparing the feedstock for spray drying for inhalation powders.

[0262] In some embodiments, the spray dried biotherapeutic matrix composition includes at least one additional therapeutic agent, for example for chronic or infectious bronchopulmonary disorders. A range of drug classes include anti-inflammatory agents, antibacterial agents, antiviral agents, antifungal agents, vasodilators, and bronchodilators. These drugs can be incorporated using techniques such as microencapsulation, co-administration, or covalent attachment with degradable linkers.

[0263] In some embodiments, the spray dried biotherapeutic matrix composition comprises an anti-inflammatory agent selected from the group consisting of nonsteroidal anti-inflammatory drugs (NSAIDs), corticosteroids, glucocorticoids, methotrexate, sulfasalazine, leflunomide, anti-tumor necrosis factor (TNF) drugs, cyclophosphamide, inflammation-resolving lipid mediators, mycophenolates, opiates, and barbiturates.

[0264] In some embodiments, the spray dried biotherapeutic matrix composition comprises an antibacterial, antiviral, and / or antifungal agent, hi some embodiments, the antibacterial agent is selected from the group consisting of aminoglycosides, ansamycins, beta-lactams, bis-biguanides, carbacephems, carbapenems, cationic polypeptides, cephalosporins, fluoroquinolones, glycopeptides, iron-sequestering glycoproteins, linosamides, lipopeptides, macrolides, monobactams, nitrofurans, oxazolidinones, penicillins, polypeptides, quaternary ammonium compounds, quinolones, silver compounds, sulfonamides, and tetracyclines.

[0265] Some exemplary specific antibacterial agents include broad spectrum penicillins, amoxicillins (e.g., ampicillin, bacampicillin, carbenicillin indanyl, mezlocillin, piperacillin, ticarcillin), penicillins and beta-lactamase inhibitors (e.g., amoxicillin-clavulanate, ampicillin-sulbactam, benzylpenicillin, cloxacillin, dicloxacillin, methicillin, oxacillin, penicillin G, penicillin V, piperacillin-tazobactam, ticarcillin-clavulanate, nafcillin), cephalosporins (e.g., first generation cephalosporins, cephalosporins (e.g., cefadroxil, cefazolin, cephalexin, cephalothin, cephapirin, cephradine), second-generation cephalosporins (e.g., cefaclor, cefamandole, cefonicid, cefotetan, cefoxitin, cefprozil, cefmetazole, cefuroxime, loracarbef), third-generation cephalosporins (e.g., cefdinir, ceftibuten, cefoperazone, cefixime, cefotaxime, cefpodoxime proxetil, ceftazidime, ceftizoxime, ceftriaxone), fourth-generation cephalosporins (e.g., cefe pim), macrolides and lincosamides (e.g., azithromycin, clarithromycin, clindamycin, dirithromycin, erythromycin, lincomycin, troleandomycin), quinolones and fluoroquinolones (e.g., cinoxacin, ciprofloxacin, enoxacin, gatifloxacin, grepafloxacin, levofloxacin, lomefloxacin, moxifloxacin, nalidixic acid, norfloxacin, ofloxacin, sparfloxacin, trovafloxacin, oxolinic acid, gemifloxacin, perfloxacin), Carbapenems (e.g., imipenem-cilastatin, meropenem), monobactams (e.g., aztreonam), aminoglycosides (e.g., amikacin, gentamicin, kanamycin, neomycin, netilmicin, streptomycin, tobramycin, paromomycin), glycopeptides (e.g., teicoplanin, vancomycin), tetracyclines (e.g., demeclocycline, doxycycline, methacycline, minocycline, oxytetracycline, tetracycline, chlortetracycline), sulfonamides (e.g.,Mafenide, silver sulfadiazine, sulfacetamide, sulfadiazine, sulfamethoxazole, sulfasalazine, sulfisoxazole, trimethoprim-sulfamethoxazole, sulfamethizole), rifampin (e.g., rifabutin, rifampin, rifapentine), oxazolidinone (e.g., linezolid, streptogramin, quinupristin dalfopristin), bacitracin, chloramphenicol, fosfomycin, isoniazid, methenamine, metronidazole, mupirocin, nitrofurantoin, nitrofurazone, novobiocin, polymyxin, spectinomycin, trimethoprim, colistin, cycloserine, capreomycin, ethionamide, pyrazinamide, para-aminosalicylic acid, erythromycin ethylsuccinate, etc.

[0266] Non-limiting examples of antiviral agents include abacavir, acyclovir, adefovir, amantadine, ampligen, amprenavir, antiretroviral agents, arbidol, atazanavir, atripla, cidofovir, combivir, darunavir, delavirdine, didanosine, docosanol, dolutegravir, ecolieber, edoxudine, efavirenz, emtricitabine, enfuvirtide, entecavir, famciclovir, fomivirsen, fosamprenavir, foscarnet, phosphonet, fusion inhibitors, ibacitabine, idoxuridine, imiquimod, immunovir, indinavir, inosine, integrase inhibitors, interferon, interferon type I, interferon type II, interferon type III, lamivudine, lopinavir, loviride, maraviroc, metisazolidine, naphthalene, naphthalene, naphthalene, naphthalene serotonin, moroxydine, nelfinavir, nevirapine, nexavir, nitazoxanide, norvir, nucleoside analogs, oseltamivir (Tamiflu), peginterferon alfa-2a, penciclovir, peramivir, pleconaril, podophyllotoxin, viral protease inhibitors, pyramidine, raltegravir, reverse transcriptase inhibitors, ribavirin, rimantadine, ritonavir, saquinavir, sovosbuvir, stavudine, synergistic enhancers (antiretroviral agents), telaprevir, tenofovir, tenofovir disoproxil, tipranavir, trifluridine, trizivir, tromantadine, truvada, valacyclovir (Valtrex), valganciclovir, vicriviroc, vidarabine, pyramidine, zalcitabine, zanamivir (Relenza), or zidovudine.

[0267] Non-limiting examples of antifungal agents (also called antifungal agents) include polyene antifungals, amphotericin B, candicidin, filipin, hamycin, natamycin, nystatin, rimocidin, imidazole antifungals, triazole antifungals, thiazole antifungals, bifonazole, butoconazole, clotrimazole, econazole, fenticonazole, isoconazole, ketoconazole, luliconazole, miconazole, omoconazole, oxiconazole, sertaconazole, sulconazole, tioconazole, triazole, albaconazole, efinaconazole, epoxiconazole, fluconazole, isavuconazole, itraconazole, posaconazole, propiconazole, Ravuconazole, terconazole, voriconazole, abafungin, allylamine, amorolfine, butenafine, naftifine, terbinafine, echinocandin, anidulafungin, caspofungin, micafungin, aurone, benzoic acid, ciclopirox, flucytosine, 5-fluorocytosine, griseofulvin, haloprogin, tolnaftate, undecylenic acid, triacetin, crystal violet, castellani paint, orotomide, miltefosine, potassium iodide, coal tar, copper(II) sulfate, selenium disulfide, sodium thiosulfate, piroctone olamine, iodoquinol, clioquinol, acrisorcin, zinc pyrithione, and sulfur.

[0268] In some embodiments, the spray dried biotherapeutic matrix composition comprises at least one bacteriophage. Non-limiting examples of bacteriophages include Caudovirales (e.g., Myoviridae, Siphoviridae, and Podiviridae), Ligamenvirales (e.g., Lipothrixviridae, Rudiviridae, Ampullaviridae, Bicaudaviridae, Clavaviridae, Corticoviridae, Cystoviridae, and Fuselloviridae), and other families (e.g., Globuloviridae, Inoviridae, Leviviridae, Microviridae, Plasmaviridae, and Techtiviridae).

[0269] In some embodiments, the spray dried biotherapeutic matrix composition comprises a vasodilator, hi some embodiments, the vasodilator is selected from the group consisting of angiotensin converting enzyme (ACE) inhibitors, angiotensin receptor blockers (ARBs), calcium channel blockers (CCBs), and nitric oxide generating compounds.

[0270] In some embodiments, the vasodilator is an ACE inhibitor selected from the group consisting of benazepril (LOTENSIN), captopril (CAPOTEN), enalapril (VASOTEC, EPANED), fosinopril (MONOPRIL), lisinopril (PRINIVIL, ZESTRIL), moexipril (UNIVASC), perindopril (ACEON), quinapril (ACCUPRIL), ramipril (ALTACE), and trandolapril (MAVIK).

[0271] In some embodiments, the vasodilator is an ARB selected from the group consisting of azilsartan (EDARBI), candesartan (ATACAND), eprosartan (TEVETEN), irbesartan (AVAPRO), telmisartan (MICARDIS), valsartan (DIOVAN), losartan (COZAAR), and olmesartan (BENICAR).

[0272] In some embodiments, the vasodilator is a CCB selected from the group consisting of amlodipine (NORVASC), clevidipine (CLEVIPREX), diltiazem (CARDIZEM), felodipine (CARDENE, CARDENE SR), isradipine, nicardipine, nimodipine, nisoldipine (SULAR), and verapamil (CALAN).

[0273] In some embodiments, the vasodilator is a nitric oxide generating compound selected from the group consisting of nitroglycerin (GONITRO, NITROBID, NITROMIST, NITROLINGUAL, NITROSTAT, NITROBID), isosorbide mononitrate (ISMO, MONOKET), isosorbide dinitrate (IMDUR, ISORDIL), hydrazine (APRESOLINE), minoxidil, fenoldopam (CARLOPAM), and nitroprusside (NIPRIDE, NITROPRESS).

[0274] In some embodiments, the spray dried biotherapeutic matrix composition further comprises a bronchodilator as an additional therapeutic agent. In some embodiments, the bronchodilator is selected from the group consisting of short acting beta-adrenergic bronchodilators (e.g., albuterol, levalbuterol, epinephrine), long acting beta-adrenergic bronchodilators (e.g., salmeterol, formoterol), anticholinergic bronchodilators (e.g., ipratropium, tiotropium), and xanthine derivatives (e.g., theophylline, aminophylline). In some embodiments of any of the aspects, the bronchodilator is albuterol.

[0275] In some embodiments, at least one additional therapeutic agent is microencapsulated. In some embodiments, the additional therapeutic agent is microencapsulated in smaller capsules within the overall spray dried biotherapeutic matrix composition (i.e., physically separated from the bacterial preparation). In some embodiments, the spray dried biotherapeutic matrix composition contains separate particles or microspheres for the therapeutic agent and the bacterial preparation that are mixed together, e.g., during administration. In some embodiments, the additional therapeutic agent is microencapsulated together with the bacterial preparation.

[0276] In some embodiments, at least one additional therapeutic agent is covalently attached to the bacterial preparation component using a linker that is biodegradable, degradable, cleavable, or otherwise reversible. A cleavable linker means that the linker can be cleaved to release the two moieties that the linker holds together. In some embodiments, the cleavable linker is polyethylene glycol. The cleavable linker can be sensitive to a cleaving agent, such as, but not limited to, an enzyme, pH, redox potential, or the presence of a degradable molecule. Examples of such cleaving agents include oxidoreductases or reductases that are selective for a particular substrate or have no substrate specificity, such as mercaptans present in cells that can degrade the oxidatively cleavable linking group by reduction, esterases, amidases, endosomes or agents that can create an acidic environment (e.g., those that result in a pH of 5 or less), general acids, peptidases (which can be substrate specific), proteases, and enzymes that can hydrolyze or degrade the acid-cleavable linking group by acting as phosphatases.

[0277] In some embodiments, the spray dried biotherapeutic matrix composition is co-administered with, for example, at least one additional therapeutic agent for chronic bronchopulmonary disorder. In some embodiments, the spray dried biotherapeutic matrix composition is co-administered with, for example, at least one additional therapeutic agent for chronic bronchopulmonary disorder. In some embodiments, the spray dried biotherapeutic matrix composition is administered, for example, before, at least one additional therapeutic agent for chronic bronchopulmonary disorder. In some embodiments, the spray dried biotherapeutic matrix composition is administered, for example, after, at least one additional therapeutic agent for chronic bronchopulmonary disorder. In some embodiments, the spray dried biotherapeutic matrix composition is administered in alternation with, for example, at least one additional therapeutic agent for chronic bronchopulmonary disorder.

[0278] formulation In some embodiments, the spray dried biotherapeutic matrix composition is formulated for administration by inhalation. As used herein, the term "formulated for administration by inhalation" means that the composition or formulation is designed or adapted to deliver the active ingredient or agent to the bronchopulmonary tissues by inhaling small particles or droplets containing the active ingredient. Thus, a pharmaceutical composition formulated for administration by inhalation generally includes such particles (e.g., as a powder) or can easily generate such droplets (e.g., via a nebulizer). In some embodiments, the spray dried biotherapeutic matrix composition is formulated for delivery to the trachea, bronchi, bronchioles, and / or alveoli. In some embodiments, the spray dried biotherapeutic matrix composition is formulated for delivery to the trachea. In some embodiments, the spray dried biotherapeutic matrix composition is formulated for delivery to the bronchi. In some embodiments, the spray dried biotherapeutic matrix composition is formulated for delivery to the bronchioles. In some embodiments, the spray dried biotherapeutic matrix composition is formulated for delivery to the alveoli. In some embodiments, the composition is formulated for delivery to the lungs.

[0279] In some embodiments, the compositions are formulated as a capsule or tablet for administration by inhalation (e.g., using an inhaler). In some embodiments, the capsule contains at least 10 mg of the spray dried biotherapeutic matrix composition. In some embodiments, the capsule contains at least 30 mg of the spray dried biotherapeutic matrix composition. In some embodiments, the capsule contains at least 10 mg, at least 15 mg, at least 20 mg, at least 25 mg, at least 30 mg, at least 40 mg, at least 50 mg, at least 60 mg, at least 70 mg, at least 80 mg, at least 90 mg, or at least 100 mg or more of the spray dried biotherapeutic matrix composition.

[0280] In some embodiments, the composition is formulated for delivery by an inhaler. In some embodiments, the composition is formulated for delivery by a metered dose inhaler (MDI). A metered dose inhaler (MDI) is a device that delivers a specific amount of drug to the lungs, usually in the form of a short burst of aerosolized drug that is self-administered by the patient via inhalation. In some embodiments, the composition is formulated for delivery by a dry powder inhaler (DPI). A dry powder inhaler (DPI) is a device that delivers drug to the lungs in the form of a dry powder. In some embodiments, the composition is formulated for delivery by a soft mist inhaler (SMI). A soft mist inhaler (SMI) is a device that delivers drug to the lungs in the form of a mist.

[0281] In some embodiments, the spray dried biotherapeutic matrix composition comprises at least 106 CFU of the bacterial preparation per unit dose deliverable to the target tissue. In some embodiments, the spray dried biotherapeutic matrix composition comprises at least 200 x 106 CFU of the bacterial preparation per unit dose deliverable to the target tissue. In some embodiments, the spray dried biotherapeutic matrix composition comprises at least 101 CFU, at least 102 CFU, at least 103 CFU, at least 104 CFU, at least 105 CFU, at least 106 CFU, at least 107 CFU, at least 108 CFU, at least 109 CFU, at least 1010 CFU, at least 1011 CFU, or at least 1012 CFU of the bacterial preparation per unit dose deliverable to the target tissue.

[0282] In some embodiments, the spray dried biotherapeutic matrix composition comprises at least 5 mg of bacterial preparation per unit dose deliverable to a target tissue, in some embodiments, the spray dried biotherapeutic matrix composition comprises at least 0.1 mg, at least 0.2 mg, at least 0.3 mg, at least 0.4 mg, at least 0.5 mg, at least 0.6 mg, at least 0.7 mg, at least 0.8 mg, at least 0.9 mg, at least 1.0 mg, at least 2.0 mg, at least 3.0 mg, at least 4.0 mg, at least 5.0 mg, at least 10 mg, at least 15 mg, at least 20 mg, at least 25 mg, at least 30 mg, at least 35 mg, at least 40 mg, at least 45 mg, at least 50 mg, at least 55 mg, at least 60 mg, at least 65 mg, at least 70 mg, at least 75 mg, at least 80 mg, at least 85 mg, at least 90 mg, at least 95 mg, or at least 100 mg of bacterial preparation per unit dose deliverable to a target tissue.

[0283] In some embodiments, the spray dried biotherapeutic matrix composition comprises at least 15 mg of bacterial preparation per unit dose, hi some embodiments, the spray dried biotherapeutic matrix composition comprises at least 0.1 mg, at least 0.2 mg, at least 0.3 mg, at least 0.4 mg, at least 0.5 mg, at least 0.6 mg, at least 0.7 mg, at least 0.8 mg, at least 0.9 mg, at least 1.0 mg, at least 2.0 mg, at least 3.0 mg, at least 4.0 mg, at least 5.0 mg, at least 10 mg, at least 15 mg, at least 20 mg, at least 25 mg, at least 30 mg, at least 35 mg, at least 40 mg, at least 45 mg, at least 50 mg, at least 55 mg, at least 60 mg, at least 65 mg, at least 70 mg, at least 75 mg, at least 80 mg, at least 85 mg, at least 90 mg, at least 95 mg, or at least 100 mg of bacterial preparation per unit dose.

[0284] In some embodiments, the spray dried biotherapeutic matrix composition is formulated as a microsphere. In some embodiments, the microsphere has a median mass aerodynamic diameter (MMAD) of at least 1 μm up to 1 mm. In some embodiments, the microsphere has a median mass aerodynamic diameter (MMAD) of at least 1 μm, at least 2 μm, at least 3 μm, at least 4 μm, at least 5 μm, at least 6 μm, at least 7 μm, at least 8 μm, at least 9 μm, at least 10 μm, at least 20 μm, at least 30 μm, at least 40 μm, at least 50 μm, at least 60 μm, at least 70 μm, at least 80 μm, at least 90 μm, at least 100 μm, at least 200 μm, at least 300 μm, at least 400 μm, at least 500 μm, at least 600 μm, at least 700 μm, at least 800 μm, at least 900 μm, or at least 1 mm. In some embodiments, the microspheres have a median mass aerodynamic diameter (MMAD) of at most 1 μm, at most 2 μm, at most 3 μm, at most 4 μm, at most 5 μm, at most 6 μm, at most 7 μm, at most 8 μm, at most 9 μm, at most 10 μm, at most 20 μm, at most 30 μm, at most 40 μm, at most 50 μm, at most 60 μm, at most 70 μm, at most 80 μm, at most 90 μm, at most 100 μm, at most 200 μm, at most 300 μm, at most 400 μm, at most 500 μm, at most 600 μm, at most 700 μm, at most 800 μm, at most 900 μm, or at most 1 mm.

[0285] dry particles In some embodiments, the spray dried biotherapeutic matrix composition comprises a plurality of dry particles comprising the same type and / or multiple types of dry particles (e.g., exemplary particle characteristics defined in Tables 4A-4B or Tables 5A-5B). In some embodiments, the spray dried biotherapeutic matrix composition is a dry powder comprising particles having the physical characteristics set forth in Tables 4A-4B or Tables 5A-5B. [Table 4A] [Table 4B] [Table 5A] [Table 5B]

[0286] In some embodiments, the dry particles have a Dv50 of at least 0.5 μm. In some embodiments, the dry particles have a Dv50 of at least 2.0 μm. Dv50 is the size below which 50% of the particles are smaller. Dv50 is substantially the median particle diameter, as measured by the technique used. In some embodiments, Dv50 is measured via laser diffraction techniques. Dv50 can be applied to aerosolized particles or liquid suspended particles. Dv50 is essentially a pure volumetric measurement and does not take into account the aerosol properties of the particles. Dv50 can be a useful measure to estimate the dispersibility of a powder. Dispersibility refers to how easily a powder aerosolizes at different pressures. A fully dispersible powder can be consistently aerosolized regardless of the pressure applied. In some embodiments, the dry particles have a Dv50 of at least 0.5 um, at least 0.6 um, at least 0.7 um, at least 0.8 um, at least 0.9 um, 1 μm, at least 1.5 μm, at least 2 μm, at least 2.5 μm, at least 3 μm, at least 3.5 μm, at least 4 μm, at least 4.5 μm, at least 5 μm, at least 5.5 μm, at least 6 μm, at least 6.5 μm, at least 7 μm, at least 7.5 μm, at least 8 μm, at least 8.5 μm, at least 9 μm, at least 9.5 μm, or at least 10 μm.

[0287] In some embodiments, the dry particles have a median mass aerodynamic diameter (MMAD) of at least 1.5 μm to a maximum of 7.5 μm. In some embodiments, the dry particles have a median mass aerodynamic diameter (MMAD) of at least 3.5 μm to a maximum of 7.5 μm. In some embodiments, the dry particles have a median mass aerodynamic diameter (MMAD) of at least 2.0 μm to a maximum of 7.5 μm. In some embodiments, the dry particles have a median mass aerodynamic diameter (MMAD) of at least 2.5 μm to a maximum of 7.5 μm. In some embodiments, the dry particles have a median mass aerodynamic diameter (MMAD) of at least 1.0 μm to a maximum of 10 μm. As used herein, the term "MMAD" refers to the aerodynamic diameter value at which 50% of a given volume in a given aerosol is associated with particles smaller than the MMAD and 50% of the volume is associated with particles larger than the MMAD. In some embodiments, the dry particles have a median mass aerodynamic diameter (MMAD) of at least 4.0 μm. In some embodiments, the dry particles have a mass median aerodynamic diameter (MMAD) of at least 4.2 μm. In some embodiments, the dry particles have a mass median aerodynamic diameter (MMAD) of at least 1 μm, at least 1.5 μm, at least 2 μm, at least 2.5 μm, at least 3 μm, at least 3.5 μm, at least 4 μm, at least 4.5 μm, at least 5 μm, at least 5.5 μm, at least 6 μm, at least 6.5 μm, at least 7 μm, at least 7.5 μm, at least 8 μm, at least 8.5 μm at least 9 μm, at least 9.5 μm, or at least 10 μm. In some embodiments, the dry particles have a median mass aerodynamic diameter (MMAD) of at most 1 μm, at most 1.5 μm, at most 2 μm, at most 2.5 μm, at most 3 μm, at most 3.5 μm, at most 4 μm, at most 4.5 μm, at most 5 μm, at most 5.5 μm, at most 6 μm, at most 6.5 μm, at most 7 μm, at most 7.5 μm, at most 8 μm, at most 8.5 μm, at most 9 μm, at most 9.5 μm, or at most 10 μm.

[0288] In some embodiments, the dry particles described herein have a fine particle fraction (FPF) of <5.0 μm of 94%. In some embodiments, the dry particles described herein have a fine particle fraction of <5.0 μm of at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more. In some embodiments, the dry particles described herein have a fine particle fraction of <3.3 μm of 73%. In some embodiments, the dry particles described herein have a fine particle fraction of <3.3 μm of at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more.

[0289] Fine particle fraction (FPF) is a value obtained from cascade impaction testing of dry powders. In cascade impaction testing, the powder is run through different stages of gradually decreasing size. The amount of powder deposited at each stage represents a percentage of the size cutoff for the overall powder. Two such methods are Next Generation Impaction (NGI), which flows the powder horizontally across the stages, and Anderson Cascade Impaction (ACI), which flows the powder vertically across the stages. Typically, two values ​​are provided: the percentage of delivered dose smaller than 5.0 μm, and the percentage of delivered dose smaller than 3.3 um. Typically, <5.0 μm is the size cutoff considered for deposition in the central and peripheral airways, and typically <3.3 μm for deposition in the peripheral airways. See, e.g., Bianco et al., Respiratory Research volume 22, Article number: 71 (2021) (see, e.g., Figure 4 in Bianco); Darquenne, J Aerosol Med Pulm Drug Deliv. 2012, 25(3):140-147, Description of the Next Generation Impactor (available worldwide at sanyo-si.com / wp-content / uploads / NGI.pdf), the contents of each of which are incorporated herein by reference in their entirety.

[0290] In some embodiments, the dry particles have a dispersibility of less than 2.0. In some embodiments, the dry particles have a dispersibility of at least 0.5 to 1.0. As used herein, the term "dispersibility" refers to the ability of particles to be spatially well-dispersed in a liquid, in terms of size and concentration, after a controlled dispersion process. Dispersibility is unitless. Dispersibility represents the ratio of particle sizes measured for the same powder at different air pressures used to deliver the particles to the system. Specifically, dispersibility is a measurement of the Dv50 of the powder delivered to the system at "low" pressure divided by the Dv50 of the powder delivered to the system at "high" pressure. In some embodiments, the dispersibility of the dry particles is measured by dividing the Dv50 measured at a pressure of 2 kPa by the Dv50 measured at a pressure of 4 kPa (such a measurement may be referred to herein as "2 / 4KPA"). Dispersibility may be measured for a delivered (e.g., emitted) dose. Dv50 can be measured by impaction, laser diffraction (e.g., RODOS or MASTERSIZER), or both. Low pressure systems may have a higher Dv50 when measured using laser diffraction. Bulk and particle density affect dispersibility, as well as the "stickiness" and shape of the dry particles, and vice versa. Dispersibility is a good dimensionless measure of how "well" a particle will hang in the air, even though the pressure from the air applied to the particle is not constant, since it includes so many other attributes. In some embodiments, an "optimal" powder has a dispersibility of 1 or close to 1, meaning that its Dv50 does not change across air pressures in the aerodynamic system. As a ratio, the minimum value of dispersibility is zero, and dispersibility can be less than 1 or greater than 1.

[0291] In some embodiments, the dry particles have a dispersibility of at least 0.9. In some embodiments, the dry particles have a dispersibility of at least 0.50, at least 0.55, at least 0.60, at least 0.65, at least 0.70, at least 0.75, at least 0.80, at least 0.85, at least 0.90, at least 0.91, at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, at least 1.0, at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, or 2.0. In some embodiments, the dry particles have a viscosity of 0.50-0.55, 0.55-0.60, 0.60-0.65, 0.65-0.70, 0.70-0.75, 0.75-0.80, 0.80-0.85, 0.85-0.90, 0.90-0.91, 0.91-0.92, 0.92-0.93, 0.93-0.94, 0.94-0.95, 0.95-0.96, 0.96-0.97, 0. The dispersibility is 97 to 0.98, 0.98 to 0.99, 0.99 to 1.0, 1.0 to 1.1, 1.1 to 1.2, 1.2 to 1.3, 1.3 to 1.4, 1.4 to 1.5, 1.5 to 1.6, 1.6 to 1.7, 1.7 to 1.8, 1.8 to 1.9, 1.9 to 2.0, 0.5 to 0.75, 0.75 to 1.0, 0.75 to 1.25, 1.0 to 1.25, 1.25 to 1.5, or 0.5 to 1.5.

[0292] In some embodiments, the dry particles have a delivered dose of at least 25.0% to up to 125% by weight of the composition to the target tissue. In some embodiments, the dry particles have a delivered dose of at least 25.0% to up to 80.0% by weight of the composition to the target tissue. As used herein, the term "delivered dose" refers to the percentage of the composition or its active ingredients (e.g., bacterial preparation) that is expelled from a delivery device (e.g., an inhaler) and / or delivered to a target tissue. In some embodiments, the delivered dose may be greater than 100% when the expected dose (e.g., 1.0 mg) in the delivery device is less than the actual dose (e.g., 1.25 mg, e.g., due to overfilling) in the delivery device, and when the expected dose (e.g., >1.0 mg, e.g., >100%) is greater, but the actual dose (e.g., 1.25 mg, e.g., <125%) is delivered to the target tissue. Factors that influence the delivered dose ultimately relate to dispersibility, powder density, hygroscopicity, and electrostatic charge (e.g., some of these factors affect dispersibility more than others). As a non-limiting example, high density particles may aerosolize and disperse more than low density particles that may disperse unpredictably or be too large. As another non-limiting example, excipients that are too hygroscopic and therefore make the powder less "flowable" or sticky may cause less powder to exit the capsule when the inhaler (e.g., DPI) is actuated. It is expected that not all of the powder from a capsule containing a spray-dried biotherapeutic matrix composition described herein will be able to reach the target tissue (e.g., alveoli). Certain portions of the composition may not be delivered because they remain in the capsule, remain in the device, or hit the tongue or esophagus. In some embodiments, the delivered dose refers to the amount of spray-dried biotherapeutic matrix composition described herein that exits the device and capsule.

[0293] The delivered dose can be measured as the mass % of powder that escapes the delivery system and reaches the target area(s). The emitted dose is a good measure of what escapes the delivery device in a real system. The content uniformity of the emitted dose can be measured to ensure that what is emitted from the administration device is consistent. In some embodiments, the dry particles have a delivered dose of at least 60% by mass of the bacterial preparation to the target tissue. In some embodiments, the dry particles have a delivered dose of at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80% by mass of the bacterial preparation that is expelled from the delivery device (e.g., an inhaler) and / or delivered to the target tissue.

[0294] In some embodiments, the particles have a bulk density of at least 0.1 g / cm3 to 0.8 g / cm3. In some embodiments, the particles have a bulk density of at least 0.2 g / cm3 to 0.8 g / cm3. As used herein, "bulk density" is defined as the mass of many particles of a material divided by the total volume they occupy, which includes particle volume, interparticle void volume, and internal pore volume. In some embodiments, the particles have a bulk density of at least 0.5 g / cm3. In some embodiments, the particles have a bulk density of at least 0.1 g / cm3, at least 0.2 g / cm3, at least 0.3 g / cm3, at least 0.4 g / cm3, at least 0.5 g / cm3, at least 0.6 g / cm3, at least 0.7 g / cm3, or at least 0.8 g / cm3.

[0295] In some embodiments, the particles have a tap density of at least 0.1 g / cm3 to 1.0 g / cm3. In some embodiments, the particles have a tap density of at least 0.2 g / cm3 to 1.0 g / cm3. In some embodiments, the particles have a tap density of at least 0.3 g / cm3 to 1.0 g / cm3. In some embodiments, the particles have a tap density of at least 0.1 g / cm3. In some embodiments, the particles have a tap density of at least 0.3 g / cm3. As used herein, "tap density" refers to the bulk density of a powder after a particular compaction process (e.g., involving vibration of a container containing a powder of dry particles). In some embodiments, the particles have a tap density of at least 0.6 g / cm3. In some embodiments, the particles have a tap density of at least 0.1 g / cm3, at least 0.2 g / cm3, at least 0.3 g / cm3, at least 0.4 g / cm3, at least 0.5 g / cm3, at least 0.6 g / cm3, at least 0.7 g / cm3, at least 0.8 g / cm3, at least 0.9 g / cm3, or at least 1.0 g / cm3.

[0296] In some embodiments, the particles have a moisture content of at least 1.0% to 7.0% water by weight. As used herein, "moisture content" refers to the amount of water present in a product, measured as a percentage (%). Moisture can affect the physical properties of a substance, including weight, density, viscosity, conductivity, and the like. Moisture content can be determined by weight loss on drying (loss on drying, LOD). In some embodiments, moisture content is measured using Karl Fischer titration. Karl Fischer uses coulometric or volumetric titration to determine trace amounts of water in a sample. The elementary reaction responsible for the quantification of water in Karl Fischer titration is the oxidation of sulfur dioxide by iodine: H2O+SO2+I2→SO3+2HI. This elementary reaction consumes exactly one molar equivalent of water compared to iodine. Iodine is added to the solution until it is present in excess (indicating the endpoint of the titration), which can be detected potentiometrically. The reaction is carried out in an alcoholic solution containing a base which consumes the sulfur trioxide and hydroiodic acid produced. The endpoint is most commonly detected by a two-potentiometric titration method.

[0297] In some embodiments, the particles have a moisture content of at least 1.0% water by weight (e.g., as measured by Karl Fischer). In some embodiments, the particles have a moisture content of at least 2.5% water by weight (e.g., as measured by Karl Fischer). In some embodiments, the particles have a moisture content of up to 2.5% water by weight. In some embodiments, the particles have a moisture content of at least 2.8% water by weight. In some embodiments, the particles have a moisture content of up to 2.8% water by weight. In some embodiments, the particles have a moisture content of at least 1%, at least 1.5%, at least 2%, at least 2.5%, at least 3%, at least 3.5%, at least 4%, at least 4.5%, at least 5%, at least 5.5%, at least 6%, at least 6.5%, at least 7% water by weight. In some embodiments, the particles have a moisture content of up to 1%, up to 1.5%, up to 2%, up to 2.5%, up to 3%, up to 3.5%, up to 4%, up to 4.5%, up to 5%, up to 5.5%, up to 6%, up to 6.5%, up to 7% water by weight.

[0298] Device In one aspect, the spray dried biotherapeutic matrix composition described herein is combined with a delivery device such as an inhaler. In one aspect, an inhalation device for bronchopulmonary delivery is described herein, comprising (a) an inhaler and (b) a container containing a spray dried biotherapeutic matrix composition described herein. In some embodiments, the inhaler comprises (a) a mouthpiece comprising an opening and (b) a means for aerosolizing or dispersing the pharmaceutical composition within the container. In some embodiments, the means for aerosolizing or dispersing includes, but is not limited to, a propellant, pressurized air, a spring, or another chemical or mechanical generation of pressure. In some embodiments, the inhaler is a dry powder inhaler (DPI). In some embodiments, the inhaler is a metered dose inhaler (MDI). In some embodiments, the inhaler is a soft mist inhaler (SMI).

[0299] In some embodiments, the spray dried biotherapeutic matrix compositions described herein are administered as aerosolized compositions. Aerosolization is the process or act of converting any physical substance (e.g., a solid) into the form of particles small enough and light enough to be carried in the air. In some embodiments, the spray dried biotherapeutic matrix compositions described herein can also be administered directly to the airways in the form of a dry powder. For use as a dry powder, the spray dried biotherapeutic matrix compositions described herein can be administered by use of an inhaler. The inhaler is a portable handheld device that can be available as a metered dose inhaler (MDI), a dry powder inhaler (DPI), or a soft mist inhaler (SMI).

[0300] A metered dose inhaler or "MDI" is a pressure-resistant canister or container filled with a product such as a pharmaceutical composition dissolved in a liquefied propellant or micronized particles suspended in a liquefied propellant. Propellants that can be used include chlorofluorocarbons, hydrocarbons, or hydrofluoroalkanes. Particularly preferred propellants are P134a (tetrafluoroethane) and P227 (heptafluoropropane), each of which can be used alone or in combination. They are optionally used in combination with one or more other propellants, and / or one or more surfactants, and / or one or more other excipients, such as ethanol, lubricants, antioxidants, and / or stabilizers. Metered dose inhalers allow the correct dosage of the composition to be delivered to the patient. Although propellants and high pressure generally do not help maintain the viability of bacteria, MDIs can be used to deliver non-viable bacteria (e.g., heat-killed bacteria), bacterial extracts, and / or bacterial products. In embodiments involving viable bacteria, a DPI can be used instead of an MDI.

[0301] Dry powder inhalers (ie, TURBUHALER (ASTRA AB)) are systems operable with a pressurized air source to generate dry powder particles of a pharmaceutical composition compressed into a very small volume.

[0302] Dry powder aerosols for inhalation therapy are generally produced with average diameters mainly in the range of <5 μm. As the particle diameter increases beyond 3 μm, it is less and less phagocytosed by macrophages. However, increasing the particle size has also been found to minimize the probability of particles (with standard mass density) entering the airways and acini due to excessive deposition in the oropharynx or nasal region.

[0303] Suitable powder compositions include, by way of example, a powder preparation of the spray-dried biotherapeutic matrix composition described herein thoroughly mixed with lactose or other inert powder acceptable for intrabronchial administration. The powder composition may be administered via an aerosol dispenser or may be enclosed in a frangible capsule that can be punctured by the patient and inserted into a device that expels the powder in a steady stream suitable for inhalation. The composition may include propellants, surfactants, and co-solvents and may be filled into conventional aerosol containers that are closed by a suitable metering valve.

[0304] Soft mist inhalers (SMIs, e.g., Respimat® Soft Mist™ inhalers) are multi-dose, propellant-free, handheld liquid inhalers. The aerosol clouds generated by SMIs contain a higher percentage of microspheres than most pressurized metered dose inhalers (pMDIs) and dry powder inhalers (DPIs), and the aerosol spray leaves the inhaler more slowly and for a longer period of time than pMDIs. This leads to higher drug deposition in the lungs and lower deposition in the oropharynx, allowing for a lower nominal dose of drug delivered without reducing efficacy. In some embodiments, the inhaler formulation does not contain a propellant. The drug is stored as a solution in an SMI drug cartridge (e.g., an aluminum cylinder containing a double-walled plastic collapsible bag that shrinks when the solution is used). SMI solutions can be formulated with ethanol or water, and preservatives (e.g., benzalkonium chloride or ethylenediaminetetraacetic acid (EDTA)). The amount of preservative in each dose (eg, puff) can be about 0.44 μg of benzalkonium chloride, or about 2.2 μg of EDTA.

[0305] In some embodiments of any of the aspects, the spray dried biotherapeutic matrix composition described herein is administered using a nasal spray or nebulizer. In some embodiments of any of the aspects, the spray dried biotherapeutic matrix composition is formulated as a nasal spray. In one aspect, the spray dried biotherapeutic matrix composition described herein is combined with a nebulizer. In some embodiments of any of the aspects, the spray dried biotherapeutic matrix composition is formulated for delivery by an inhaler.

[0306] Nasal sprays typically contain a saline solution containing the spray dried biotherapeutic matrix composition described herein, which is sprayed as a mist into the nasal passages using a mechanical spray nozzle. A nebulizer is an electric or battery-powered machine that converts the liquid spray dried biotherapeutic matrix composition described herein into a fine mist that is inhaled into the lungs. The inhalers or nebulizers described herein may further include a mouthpiece or face mask. Nasal sprays typically administer the spray dried biotherapeutic matrix composition described herein to the upper respiratory tract, while inhalers or nebulizers typically administer the spray dried biotherapeutic matrix composition described herein to the lower respiratory tract.

[0307] The spray dried biotherapeutic matrix compositions described herein can be administered directly to the airways of a subject in the form of an aerosol or by nebulization. For use as an aerosol, the spray dried biotherapeutic matrix compositions described herein in solution or suspension can be packaged in a pressurized aerosol container with a suitable propellant (e.g., a hydrocarbon propellant such as propane, butane, or isobutane) using conventional adjuvants. The spray dried biotherapeutic matrix compositions described herein can also be administered in non-pressurized forms, such as with a nebulizer or atomizer.

[0308] The term "nebulization" is well known to include reducing a liquid to a fine spray. Preferably, such nebulization produces small droplets of uniform size from larger droplets of liquid in a controlled manner. Nebulization can therefore be achieved by any suitable means, including using many nebulizers known and commercially available today. For example, the AEROMIST pneumatic nebulizer available from Inhalation Plastic, Inc., Niles, Illinois. When active ingredients are adapted to be administered either together or individually via a nebulizer(s), they can be in the form of nebulized aqueous suspension or solution, with or without suitable pH or tonicity adjustment, either as a unit dose or multi-dose device. Nebulized solutions containing active pharmaceutical ingredients are well suited for infant or adolescent lungs, as well as patients with weaker lung capacity. Nebulized solutions face stability challenges and have size limitations that may prevent aerosolization during administration.

[0309] As is well known, any suitable gas can be used to apply pressure during spraying. Preferred gases are those that are thus far chemically inert to the spray-dried biotherapeutic matrix compositions described herein. Exemplary gases can be used to great advantage, including, but not limited to, nitrogen, argon, or helium.

[0310] Aerosols for delivery to the respiratory tract are known in the art.See, for example, Adjei, A. and Garren, J. Pharm. Res., 1:565-569(1990); Zanen, P. and Lamm, J.-WJ Int. J. Pharm., 114:111-115(1995); Gonda, I. "Aerosols for delivery of therapeutic and diagnostic agents to the respiratory tract," in Critical Reviews in Therapeutic Drug Carrier Systems, 6:273-313(1990); Anderson et al., Am. Rev. Respir. Dis., 140:1317-1324(1989)).It also has the potential for systemic delivery of peptides and proteins (Patton and Platz, Advanced Drug Delivery Reviews, 8:179-196 (1992)); Timsina et.al., Int. J. Pharm., 101:1-13 (1995); and Tansey, IP, Spray Technol. Market, 4:26-29 (1994); French, DL, Edwards, DA and Niven, RW, Aerosol Sci., 27:769-783 (1996); Visser, J., Powder Technology 58:1-10 (1989)); Rudt, S. and RH Muller, J. Controlled Release, 22:263-272 (1992); Tabata, Y, and Y. Ikada, Biomed. Mater. Res., 22:837-858 (1988), Wall, DA, Drug Delivery, 2:10 1-20 1995), Patton, J. and Platz, R., Adv. Drug Del.Rev.,8:179-196(1992), Bryon,P.,Adv.Drug.Del.Rev.,5:107-132(1990),Patton,JS,et al.,Controlled Release,28:15 79-85(1994),Damms,B.and Bains,W.,Nature Biotechnology (1996), Niven, RW, et al., Pharm. Res., 12(9), 1343-1349 (1995), and Kobayashi, S., et al. al., Pharm. Res., 13(1):80-83 (1996), the contents of each of which are incorporated herein by reference in their entirety.

[0311] Spray drying method Described herein are methods of preparing a spray dried biotherapeutic matrix composition comprising, for example, a bacterial preparation as described herein. In one aspect, described herein is a method of preparing a spray dried biotherapeutic matrix composition comprising a bacterial preparation, the method comprising: (a) preparing a liquid feedstock comprising the bacterial preparation; (b) introducing droplets of the liquid feedstock into a drying chamber via a spray nozzle; (c) exposing the liquid feedstock droplets to heated pressurized gas in the drying chamber to produce dry particles; and (d) isolating dry particles of a predetermined range of diameters in a cyclone chamber, wherein the isolated dry particles comprise the bacterial preparation.

[0312] In one aspect, described herein is a method of preparing a spray dried biotherapeutic matrix composition comprising a bacterial preparation, the method comprising: (a) obtaining a liquid feedstock comprising the bacterial preparation; (b) introducing droplets of the liquid feedstock into a drying chamber via a spray nozzle; (c) exposing the liquid feedstock droplets to heated and pressurized gas in the drying chamber to produce dry particles; and (d) isolating dry particles of a predetermined range of diameters in a cyclone chamber, wherein the isolated dry particles comprise the bacterial preparation. In some embodiments, the method of preparing a spray dried pharmaceutical composition comprising a bacterial preparation is performed using at least one of the conditions set forth in Table 6 or Tables 7A-7B. [Table 6] [Table 7A] [Table 7B]

[0313] The process conditions shown in Tables 6 and 7A-7B above provide exemplary ranges across the operating sizes of spray units, for example, the higher values ​​of gas pressure and flow rate represent conditions closer to a full-scale (i.e., large-scale) production unit, while the lower values ​​represent conditions closer to a bench-top or lab-scale (i.e., small-scale) process unit.

[0314] Drying gas flow rates can be expressed in either kg / hr or L / min. The kg / hr value can be calculated from the L / min value using the following formula: Mass (kg / hr) = [[Density of air (g / L) / Volume (L / min)] * (1 kg / 1000 g) * (60 min / 1 hr)], where the density of air is approximately 1.225 g / L (e.g., may vary slightly with temperature, altitude, etc.). For example, 600 L / min corresponds to 44.1 kg / hr. The L / min value can be calculated from the kg / hr value using the following formula: Volume (L / min) = [[Mass (kg / hr) / Density of air (g / L)] * (1000 g / 1 kg) * (1 hr / 60 min)]. For example, 18 kg / hr corresponds to 245 L / min.

[0315] Feed flow rates can be expressed in g / min or mL / min, and such flow rates can be converted depending on the density of the liquid feed (e.g., g / mL). Atomization pressures can be expressed in psig, kPa, or other known units of pressure (1 psig = 6.89475728 kPa, 345 kPa = approximately 50 psig).

[0316] To spray dry the sensitive biotherapeutics described herein, target processing conditions are optimized for three characteristics: (1) component viability / activity / assay, (2) powder flow and stability, and (3) aerodynamic size and characteristics (see, e.g., Tables 4 and 5 for exemplary characteristics of spray-dried particles).

[0317] To protect the viability of the components, the powder is spray dried at the lowest possible outlet temperature, which is the effective temperature that the dried powder will experience. During drying, a phenomenon known as evaporative cooling keeps the solid components relatively cool compared to the heated surroundings. Once particle formation is complete, the particles are heated to a temperature between room temperature and the temperature of the inlet drying gas. This temperature (measured at the outlet of the spray drying chamber) needs to be minimized. Working in the opposite direction is the moisture content of the powder, which can also be minimized to improve long-term stability and physical properties. However, higher outlet temperatures of the same formulation are typically attributed to dryer particles. These two process conditions can be optimized to maximize particle viability and minimize moisture content. Furthermore, since reducing the atomization pressure favors component viability, and higher pressures stress living organisms and sensitive therapeutic agents, the atomization pressure used to create the droplets needs to be kept as low as reasonably possible to protect the components.

[0318] Aerodynamic properties are governed by the included excipients, particle density, and particle size. Excipients can be selected to allow dispersibility (e.g., by adding excipients such as amino acids such as leucine) and stability (e.g., by adding high glass transition temperatures, strongly soluble sugars, polyols, or ionic salts, e.g., by adding stabilizers as described herein). Including agents to enhance dispersibility also enhances bulk powder properties such as flowability, making the capsule filling process more efficient.

[0319] During spray drying, particle formation is driven by two factors: (1) the evaporation rate of the solvent, and (2) the solubility of the solute. A dimensionless number called the Peclet number (Pe) provides an indication of which of these two phenomena dominates the droplet drying process in the spray drying chamber. Equation VIII:

number

[0320] At high Peclet numbers, particles tend to be very low density, large, hollow, and brittle. This is because evaporation of the droplets dominates solute transport to the center of the droplet (in most systems), leaving behind a "shell" of solute. Morphology tends to be spherical. At low Peclet numbers, particles are small and dense. Solute mass transfer dominates the system as these solids migrate to the center of the slowly evaporating droplet. Morphology tends to be crumpled.

[0321] In many cases, high process temperatures result in faster evaporation rates, while low process temperatures tend to do the opposite, resulting in dense, crumpled particles, which is the target morphology for our application.

[0322] A lower amount of dissolved solids in the feedstock allows for faster diffusion of solutes, which also contributes to smaller, denser, crumpled particles. A higher amount of solids limits the rate at which solute diffusion can occur, but contributes to higher material yields and improved processing.

[0323] Raw material preparation In some embodiments, the method of preparing a spray-dried biotherapeutic matrix composition includes, for example, preparing a liquid feedstock including a bacterial preparation. In some embodiments, the step of preparing the liquid feedstock includes dissolving a solid feedstock in a solution. In some embodiments, the step of preparing the liquid feedstock includes dissolving a solid feedstock in an aqueous solution. An example of a solid feedstock for use in preparing an inhaled bacterial preparation formulation at optimal processing conditions on a lab scale can be found in Table 8 below. [Table 8A] [Table 8B]

[0324] In some embodiments, the solid feedstock comprises at least 3.5% by weight of the bacterial preparation, at least 91.5% by weight of the excipient, and at least 5% by weight of the stabilizer. In some embodiments, the solid feedstock comprises at least 3.5% by weight of the bacterial preparation. In some embodiments, the solid feedstock comprises at least 91.5% by weight of the excipient. In some embodiments, the solid feedstock comprises at least 5% by weight of the stabilizer.

[0325] In some embodiments, the solid feedstock comprises at least 3.5% by weight of the bacterial preparation, at least 45.75% by weight of the first excipient, at least 45.75% by weight of the second excipient, and at least 5% by weight of the stabilizer, hi some embodiments, the solid feedstock comprises at least 45.75% by weight of the first excipient, and at least 45.75% by weight of the second excipient.

[0326] In some embodiments, the solid feedstock comprises at least 3.5% by weight of the bacterial preparation, at least 5% by weight of the excipient, and at least 0.25% by weight of the stabilizer. In some embodiments, the solid feedstock comprises at least 25% by weight of the bacterial preparation. In some embodiments, the solid feedstock comprises at least 50% by weight of the excipient. In some embodiments, the solid feedstock comprises at least 1% by weight of the stabilizer.

[0327] In some embodiments, the solid feedstock comprises at least 3.5% by weight of the bacterial preparation, at least 5% by weight of the first excipient, at least 5% by weight of the second excipient, and at least 0.25% by weight of the stabilizer. In some embodiments, the solid feedstock comprises at least 30% by weight of the first excipient, and at least 30% by weight of the second excipient. In some embodiments, the solid feedstock comprises at least 30% by weight of the first excipient, at least 30% by weight of the second excipient, and at least 2.5% by weight of the third excipient. In some embodiments, the three excipients are leucine, trehalose, and / or sodium citrate.

[0328] In some embodiments, the solid feedstock comprises at least 1% and up to 50% by weight of the bacterial preparation. In some embodiments, the solid feedstock comprises at least 1% and up to 5% by weight of the bacterial preparation. In some embodiments, the solid feedstock comprises at least 1%, at least 1.5%, at least 2%, at least 2.5%, at least 3%, at least 3.5%, at least 4%, at least 4.5%, or at least 5% by weight of the bacterial preparation.

[0329] In some embodiments, the solid ingredient comprises at least 45% up to 95% by weight of excipients, hi some embodiments, the solid ingredient comprises at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% by weight of excipients.

[0330] In some embodiments, the solid ingredient comprises at least 5% to 60% by weight of the first excipient and at least 5% to 60% by weight of the second excipient. In some embodiments, the solid ingredient comprises at least 30% to 60% by weight of the first excipient and at least 30% to 60% by weight of the second excipient. In some embodiments, the solid ingredient comprises at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, or at least 60% by weight of the first excipient and at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, or at least 60% by weight of the second excipient. In some embodiments, the solid ingredient comprises at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, or at least 60% by weight of the first excipient and at least 30% by weight of the second excipient. In some embodiments, the solid ingredient comprises at least 30% by weight of a first excipient and at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, or at least 60% by weight of a second excipient.

[0331] In some embodiments, the solid feedstock comprises at least 0.25% to at most 10% stabilizer by weight. In some embodiments, the solid feedstock comprises at least 0.5% to at most 10% stabilizer by weight. In some embodiments, the solid feedstock comprises at least 0.25%, at least 0.5%, at least 1%, at least 1.5%, at least 2%, at least 2.5%, at least 3%, at least 3.5%, at least 4%, at least 4.5%, at least 5%, at least 5.5%, at least 6%, at least 6.5%, at least 7%, at least 8%, at least 9%, or at least 10% stabilizer by weight.

[0332] An exemplary liquid feed formulation is provided below in Tables 9A-9B. In some embodiments, this exemplary liquid feed formulation is 3% w / w solids, or 1000 g feed suspension at 30 g / L (i.e., approximately 1 L, assuming approximately 1 g per mL; thus, "1 L liquid feed" may be used interchangeably with "1000 g liquid feed"). Such an exemplary liquid feed can be scaled up to a full-scale production unit, for example, using the same or substantially the same ratios of components. [Table 9A] [Table 9B]

[0333] In some embodiments, the liquid feedstock comprises at least 1.0 g / L of solid feedstock dissolved in an aqueous solution. In some embodiments, the liquid feedstock comprises at least 1.0 g / L and up to 100 g / L of solid feedstock dissolved in an aqueous solution. In some embodiments, the liquid feedstock comprises at least 25 g / L of solid feedstock dissolved in an aqueous solution. In some embodiments, the liquid feedstock comprises at least 30 g / L of solid feedstock dissolved in an aqueous solution. In some embodiments, the liquid feedstock comprises at least 1 g / L, at least 2 g / L, at least 3 g / L, at least 4 g / L, at least 5 g / L, at least 6 g / L, at least 7 g / L, at least 8 g / L, at least 9 g / L, at least 10 g / L, at least 20 g / L, at least 30 g / L, at least 40 g / L, at least 50 g / L, at least 60 g / L, at least 70 g / L, at least 80 g / L, at least 90 g / L, or at least 100 g / L of solid feedstock dissolved in an aqueous solution.

[0334] In some embodiments, the liquid feedstock comprises at least 0.1% up to 10% of a solid feedstock dissolved in an aqueous solution. In some embodiments, the liquid feedstock comprises at least 1% of a solid feedstock dissolved in an aqueous solution. In some embodiments, the liquid feedstock comprises at least 3% of a solid feedstock dissolved in an aqueous solution. In some embodiments, the liquid feedstock comprises at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, or at least 10% or more of a solid feedstock dissolved in an aqueous solution.

[0335] In some embodiments, 1 L of the liquid feed comprises (a) at least 1.050 g of the bacterial preparation, (b) at least 1.5 g of an excipient, (c) at least 0.075 g of a stabilizer, and / or (d) at least 970 g of an aqueous solution. In some embodiments, 1 L of the liquid feed comprises (a) at least 1.050 g of the bacterial preparation, (b) at least 0.75 g of a first excipient, (c) at least 0.75 g of a second excipient, (d) at least 0.075 g of a stabilizer, and / or (e) at least 970 g of an aqueous solution.

[0336] In some embodiments, 1 L of the liquid feedstock comprises (a) at least 1.050 g of the bacterial preparation, (b) at least 0.5 g of a first excipient, (c) at least 0.5 g of a second excipient, (d) at least 0.5 g of a third excipient, (e) at least 0.075 g of a stabilizer, and / or (f) at least 970 g of an aqueous solution. In some embodiments, 1 L of the liquid feedstock comprises (a) at least 1.050 g of the bacterial preparation, (b) at least 0.75 g of a first excipient, (c) at least 0.75 g of a second excipient, (d) at least 0.75 g of a third excipient, (e) at least 0.075 g of a stabilizer, and / or (f) at least 970 g of an aqueous solution.

[0337] In some embodiments, 1 L of the liquid feed comprises (a) at least 1.050 g of the bacterial preparation, (b) at least 27.45 g of an excipient, (c) at least 1.5 g of a stabilizer, and / or (d) at least 970 g of an aqueous solution. In some embodiments, 1 L of the liquid feed comprises (a) at least 1.050 g of the bacterial preparation, (b) at least 13.725 g of a first excipient, (c) at least 13.725 g of a second excipient, (d) at least 1.5 g of a stabilizer, and / or (e) at least 970 g of an aqueous solution.

[0338] In some embodiments, 1 L of the liquid feed comprises at least 0.1 g and up to 15 g of the bacterial preparation (e.g., considering 50% API load). In some embodiments, 1 L of the liquid feed comprises at least 0.1 g and up to 10 g of the bacterial preparation. In some embodiments, 1 L of the liquid feed comprises at least 1 g of the bacterial preparation. In some embodiments, 1 L of the liquid feed comprises at least 1.05 g of the bacterial preparation. In some embodiments, 1 L of the liquid feed comprises at least 2 g of the bacterial preparation. In some embodiments, 1 L of the liquid feed comprises at least 0.1 g, at least 0.2 g, at least 0.3 g, at least 0.4 g, at least 0.5 g, at least 0.6 g, at least 0.7 g, at least 0.8 g, at least 0.9 g, at least 1 g, at least 2 g, at least 3 g, at least 4 g, at least 5 g, at least 6 g, at least 7 g, at least 8 g, at least 9 g, or at least 10 g of the bacterial preparation.

[0339] In some embodiments, 1 L of liquid feed comprises at least 10 g up to 50 g of excipients. In some embodiments, 1 L of liquid feed comprises at least 0.75 g of excipients. In some embodiments, 1 L of liquid feed comprises at least 25 g of excipients. In some embodiments, 1 L of liquid feed comprises at least 20 g of excipients. In some embodiments, 1 L of liquid feed comprises at least 27.45 g of excipients. In some embodiments, 1 L of liquid feed comprises at least 10 g, at least 15 g, at least 20 g, at least 25 g, at least 30 g, at least 35 g, at least 40 g, at least 45 g, or at least 50 g of excipients.

[0340] In some embodiments, 1 L of liquid ingredient comprises at least 0.75 g to up to 25 g of a first excipient, and at least 0.75 g to up to 25 g of a second excipient. In some embodiments, 1 L of liquid ingredient comprises at least 0.75 g to up to 25 g of a first excipient, at least 0.75 g to up to 25 g of a second excipient, and at least 0.75 g to up to 25 g of a third excipient. In some embodiments, 1 L of liquid ingredient comprises at least 5 g to up to 25 g of a first excipient, and at least 5 g to up to 25 g of a second excipient. In some embodiments, 1 L of liquid ingredient comprises at least 5 g to up to 25 g of a first excipient, at least 5 g to up to 25 g of a second excipient, and at least 5 g to up to 25 g of a third excipient. In some embodiments, 1 L of the liquid ingredient comprises at least 9.840 g of the first excipient and at least 1.440 g of the second excipient. In some embodiments, 1 L of the liquid ingredient comprises at least 13.725 g of the first excipient and at least 13.725 g of the second excipient. In some embodiments, 1 L of the liquid ingredient comprises at least 5 g, at least 10 g, at least 15 g, at least 20 g, or at least 25 g of the first excipient and at least 5 g, at least 10 g, at least 15 g, at least 20 g, or at least 25 g of the second excipient. In some embodiments, 1 L of the liquid ingredient comprises at least 5 g, at least 10 g, at least 15 g, at least 20 g, or at least 25 g of the first excipient and at least 5 g of the second excipient. In some embodiments, 1 L of the liquid ingredient comprises at least 5 g of a first excipient and at least 5 g, at least 10 g, at least 15 g, at least 20 g, or at least 25 g of a second excipient.

[0341] In some embodiments, 1 L of the liquid feedstock comprises at least 0.075 g and up to 10 g of stabilizer. In some embodiments, 1 L of the liquid feedstock comprises at least 0.1 g and up to 10 g of stabilizer. In some embodiments, 1 L of the liquid feedstock comprises at least 0.075 g of stabilizer. In some embodiments, 1 L of the liquid feedstock comprises at least 1.5 g of stabilizer. In some embodiments, 1 L of the liquid feedstock comprises at least 0.075 g, at least 0.1 g, at least 0.2 g, at least 0.3 g, at least 0.4 g, at least 0.5 g, at least 0.6 g, at least 0.7 g, at least 0.8 g, at least 0.9 g, at least 1 g, at least 2 g, at least 3 g, at least 4 g, at least 5 g, at least 6 g, at least 7 g, at least 8 g, at least 9 g, or at least 10 g of stabilizer.

[0342] In some embodiments, 1 L of the liquid feedstock comprises at least 750 g and up to 999 g of aqueous solution. In some embodiments, 1 L of the liquid feedstock comprises at least 900 g and up to 999 g of aqueous solution. In some embodiments, 1 L of the liquid feedstock comprises at least 750 g of aqueous solution. In some embodiments, 1 L of the liquid feedstock comprises at least 970 g of aqueous solution. In some embodiments, 1 L of the liquid feedstock comprises at least 900 g, at least 905 g, at least 910 g, at least 915 g, at least 920 g, at least 925 g, at least 930 g, at least 935 g, at least 940 g, at least 945 g, at least 950 g, at least 955 g, at least 960 g, at least 965 g, at least 970 g, at least 975 g, at least 980 g, at least 985 g, at least 990 g, at least 995 g, or at least 999 g of aqueous solution.

[0343] In some embodiments, the liquid feedstock comprises (a) at least 0.105% of the bacterial preparation, (b) at least 0.15% of an excipient, (c) at least 0.0075% of a stabilizer, and / or (d) at least 97% of an aqueous solution. In some embodiments, the liquid feedstock comprises (a) at least 0.105% by weight of the bacterial preparation, (b) at least 0.075% by weight of a first excipient, (c) at least 0.075% by weight of a second excipient, (d) at least 0.0075% by weight of a stabilizer, and / or (e) at least 97% by weight of an aqueous solution.

[0344] In some embodiments, the liquid feedstock comprises (a) at least 0.105% by weight of the bacterial preparation, (b) at least 0.05% by weight of the first excipient, (c) at least 0.05% by weight of the second excipient, (d) at least 0.05% by weight of the third excipient, (e) at least 0.0075% by weight of the stabilizer, and / or (f) at least 97% by weight of the aqueous solution. In some embodiments, the liquid feedstock comprises (a) at least 0.105% by weight of the bacterial preparation, (b) at least 0.075% by weight of the first excipient, (c) at least 0.075% by weight of the second excipient, (d) at least 0.075% by weight of the third excipient, (e) at least 0.0075% by weight of the stabilizer, and / or (f) at least 97% by weight of the aqueous solution.

[0345] In some embodiments, the liquid feedstock comprises (a) at least 0.105% of the bacterial preparation, (b) at least 2.745% of an excipient, (c) at least 0.15% of a stabilizer, and / or (d) at least 97% of an aqueous solution. In some embodiments, the liquid feedstock comprises (a) at least 0.105% by weight of the bacterial preparation, (b) at least 1.3725% by weight of a first excipient, (c) at least 1.3725% by weight of a second excipient, (d) at least 0.15% by weight of a stabilizer, and / or (e) at least 97% by weight of an aqueous solution.

[0346] In some embodiments, the liquid feed comprises at least 0.01% and up to 10% by weight of the bacterial preparation. In some embodiments, the liquid feed comprises at least 0.01% and up to 1.0% by weight of the bacterial preparation. In some embodiments, the liquid feed comprises at least 0.105% by weight of the bacterial preparation. In some embodiments, the liquid feedstock comprises at least 0.01%, at least 0.02%, at least 0.03%, at least 0.04%, at least 0.05%, at least 0.06%, at least 0.07%, at least 0.08%, at least 0.09%, at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, or at least 10% by weight of the bacterial preparation.

[0347] In some embodiments, the liquid ingredient comprises at least 1% and up to 5% by weight of excipients. In some embodiments, the liquid ingredient comprises at least 0.15% by weight of excipients. In some embodiments, the liquid ingredient comprises at least 1%, at least 1.5%, at least 2%, at least 2.5%, at least 3%, at least 3.5%, at least 4%, at least 4.5%, or at least 5% by weight of excipients.

[0348] In some embodiments, the liquid ingredient comprises at least 0.075% and up to 2.5% by weight of a first excipient and at least 0.075% and up to 2.5% by weight of a second excipient. In some embodiments, the liquid ingredient comprises at least 0.075% by weight of a first excipient and at least 0.075% by weight of a second excipient.

[0349] In some embodiments, the liquid ingredient comprises at least 1% and up to 20% by weight of excipients. In some embodiments, the liquid ingredient comprises at least 1% and up to 5% by weight of excipients. In some embodiments, the liquid ingredient comprises at least 2.745% by weight of excipients. In some embodiments, the liquid ingredient comprises at least 1%, at least 1.5%, at least 2%, at least 2.5%, at least 3%, at least 3.5%, at least 4%, at least 4.5%, at least 5% by weight of excipients, at least 10%, at least 15%, or at least 20% by weight.

[0350] In some embodiments, the liquid ingredient comprises at least 0.1% and up to 19.8% by weight of a first excipient and at least 0.1% and up to 19.8% by weight of a second excipient. In some embodiments, the liquid ingredient comprises at least 0.5% and up to 2.5% by weight of a first excipient and at least 0.5% and up to 2.5% by weight of a second excipient. In some embodiments, the liquid ingredient comprises at least 1.3725% by weight of a first excipient and at least 1.3725% by weight of a second excipient.

[0351] In some embodiments, the liquid ingredient comprises at least 0.075 wt%, at least 0.5 wt%, at least 1 wt%, at least 1.5 wt%, at least 2 wt%, or at least 2.5 wt%, or more of a first excipient and at least 0.075 wt%, at least 0.5 wt%, at least 1 wt%, at least 1.5 wt%, at least 2 wt%, or at least 2.5 wt%, or more of a second excipient. In some embodiments, the liquid ingredient comprises at least 0.5 wt%, at least 1 wt%, at least 1.5 wt%, at least 2 wt%, or at least 2.5 wt%, or more of a first excipient and at least 0.5 wt% of a second excipient. In some embodiments, the liquid ingredient comprises at least 0.5 wt% of a first excipient and at least 0.5 wt%, at least 1 wt%, at least 1.5 wt%, at least 2 wt%, or at least 2.5 wt%, or more of a second excipient.

[0352] In some embodiments, the liquid feed comprises at least 0.0075% and up to 1.0% stabilizer by weight. In some embodiments, the liquid feed comprises at least 0.01% and up to 1.0% stabilizer by weight. In some embodiments, the liquid feed comprises at least 0.15% stabilizer by weight. In some embodiments, the liquid feed comprises at least 0.0075% stabilizer by weight. In some embodiments, the liquid feed comprises at least 0.05% stabilizer by weight. In some embodiments, the liquid feed comprises at least 0.045% stabilizer by weight. In some embodiments, the liquid feedstock comprises at least 0.0075% by weight, at least 0.01%, at least 0.02%, at least 0.03%, at least 0.04%, at least 0.05%, at least 0.06%, at least 0.07%, at least 0.08%, at least 0.09%, at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1%, at least 2%, at least 3% by weight, at least 4% by weight, at least 5% by weight, at least 6% by weight, at least 7% by weight, at least 8% by weight, at least 9% by weight, or at least 10% by weight or more of a stabilizer.

[0353] In some embodiments, the liquid feedstock comprises at least 75% and up to 99.9% by weight of an aqueous solution. In some embodiments, the liquid feedstock comprises at least 90% and up to 99.9% by weight of an aqueous solution. In some embodiments, the liquid feedstock comprises at least 97% by weight of an aqueous solution. In some embodiments, the liquid feedstock comprises at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, 99.5% or more, 99.9% or more by weight of an aqueous solution.

[0354] In some embodiments, the bacterial preparation comprises viable or non-viable bacteria. In some embodiments, the bacteria belongs to a genus selected from the group consisting of Carnobacterium, Lactiplantibacillus, Lactobacillus, Lacticaseibacillus, Ligilactobacillus, Oenococcus, Leuconostoc, Pedicoccus, Enterococcus, Lactococcus, Staphylococcus, Streptococcus, Streptomyces, Bifidobacterium, Propionibacterium, and Moraxella. In some embodiments, the bacteria is Lacticaseibacillus rhamnosus, Lactobacillus acidophilus, or Lactiplantibacillus plantarum. In some embodiments, the bacteria is Lacticaseibacillus rhamnosus. In some embodiments, the bacteria is Lactobacillus acidophilus. In some embodiments, the bacteria is Lactiplantibacillus plantarum. In some embodiments, the bacteria is Lacticaseibacillus rhamnosus and Lactobacillus acidophilus. In some embodiments, the bacteria is Lacticaseibacillus rhamnosus and Lactiplantibacillus plantarum. In some embodiments, the bacteria is Lactobacillus acidophilus and Lactiplantibacillus plantarum. In some embodiments, the bacteria is Lacticaseibacillus rhamnosus, Lactobacillus acidophilus, and Lactiplantibacillus plantarum. In some embodiments, the bacteria is Lacticaseibacillus rhamnosus strain LGG.In some embodiments, the bacterium is Lactiplantibacillus plantarum ATCC BAA-793™. In some embodiments, the bacterium is Lactobacillus acidophilus ATCC 4356™. In some embodiments, the bacterium is Lacticaseibacillus rhamnosus ATCC 53103™. In some embodiments, the bacterium is a lactic acid bacterium (LAB), i.e., belongs to the order Lactobacillales, and produces lactic acid as the primary metabolic end product of carbohydrate fermentation.

[0355] In some embodiments, the bacteria are non-pathogenic. In some embodiments, the bacteria are present at a concentration of at least 101 colony forming units per gram (CFU / g), at least 102 CFU / g, at least 103 CFU / g, at least 104 CFU / g, at least 105 CFU / g, at least 106 CFU / g, at least 107 CFU / g, at least 108 CFU / g, at least 109 CFU / g, at least 1010 CFU / g, at least 1011 CFU / g, or at least 1012 CFU / g. In some embodiments, the bacteria are present at a concentration of at least 106 colony forming units per gram (CFU / g). In some embodiments, the bacteria are present at a concentration of at least 350 x 106 colony forming units per gram (CFU / g).

[0356] In some embodiments, the excipient is selected from the group consisting of De Man, Rogosa and Sharpe (MRS) growth medium, gelatin, whey isolate, sweet whey, reconstituted skim milk powder, maltodextrin, gluco-oligosaccharides, lacto-oligosaccharides, fructooligosaccharides, inulin, sodium caseinate, goat milk, cow milk, proline, carnitine, acetylcarnitine, propionylcarnitine, glutamate, glycine betaine, glycogen, trehalose, mannose, xylose, mannitol, sorbitol, maltose, dextrose, starch, lactose, sucrose, glucose, leucine, trileucine, sodium salts, potassium salts, lithium salts, and calcium salts. In some embodiments, the excipient is leucine and / or trehalose.

[0357] In some embodiments, the stabilizer is a polysorbate, a poloxamer, or polyvinyl alcohol. In some embodiments, the stabilizer is polysorbate 80.

[0358] In some embodiments, the aqueous solution is water. In some embodiments, the aqueous solution comprises water.

[0359] In some embodiments, the liquid feedstock further comprises at least one additional therapeutic agent. Non-limiting examples of the at least one additional therapeutic agent include an anti-inflammatory agent, an antibacterial agent, an antiviral agent, an antifungal agent, a vasodilator, or a bronchodilator as described herein. In some embodiments, the bacterial preparation and the at least one additional therapeutic agent are spray dried together. In some embodiments, the bacterial preparation and the at least one additional therapeutic agent are spray dried separately.

[0360] spray The present specification describes a method for spray-drying a composition comprising a bacterial preparation. Such spray-drying can be carried out using spray-drying equipment or techniques known in the art. Methods of spray-drying are known in the art and are not limited thereto. For example, see U.S. Patent Nos. 7,258,873, 7,378,110, 8,273,374, 8,293,275, 9,238,005, 9,044,497, U.S. Patent Publication Nos. 2013 / 0022728, 2014 / 0086965, and 2018 / 0027855 (the contents of each of which are incorporated herein by reference in their entirety).

[0361] In one aspect, a method of preparing a spray dried pharmaceutical composition includes: (a) introducing droplets of a liquid feedstock described herein into a drying chamber via a spray nozzle; (b) exposing the liquid feedstock droplets to heated and pressurized gas in the drying chamber to produce dry particles; and (c) isolating dry particles of a predetermined range of diameters in a cyclone chamber, wherein the isolated dry particles comprise a bacterial preparation.

[0362] In some embodiments, the method of preparing a spray-dried pharmaceutical composition includes introducing droplets of a liquid ingredient described herein into a drying chamber through a spray nozzle. In some embodiments, the spray nozzle into the drying chamber has a diameter of at least 1.2 mm. Some commercially available full-scale nozzles may have a nozzle diameter much larger than 1.2 mm, but can be sprayed at much higher spray pressures (e.g., greater than 150 psig) to reproduce the ideal droplet size and dry particle characteristics described herein. In some embodiments, the spray nozzle into the drying chamber has a diameter of at least 0.5 mm, at least 1 mm, at least 1.5 mm, at least 2 mm, at least 2.5 mm, at least 3 mm, at least 3.5 mm, at least 4 mm, at least 4.5 mm, or at least 5 mm.

[0363] In some embodiments, the droplets of the liquid feedstock produced by the spray nozzle into the drying chamber have a diameter of at least 1.5 um. In some embodiments, the droplets of the liquid feedstock produced by the spray nozzle into the drying chamber have a diameter of at least 3.5 um. In some embodiments, the droplets of the liquid feedstock produced by the spray nozzle into the drying chamber have a diameter of at least 0.5 um, at least 1 um, at least 1.5 um, at least 2 um, at least 2.5 um, at least 3 um, at least 3.5 um, at least 4 um, at least 4.5 um, or at least 5 um.

[0364] Drying Chamber In some embodiments, a method of preparing a spray-dried pharmaceutical composition comprises exposing liquid feed droplets to heated and pressurized gas in a drying chamber to produce dry particles.

[0365] In some embodiments, the droplets of the liquid feedstock have a flow rate through the drying chamber of at least 0.5 g / min. In some embodiments, the droplets of the liquid feedstock have a flow rate through the drying chamber of at least 5 g / min. In some embodiments, the droplets of the liquid feedstock have a flow rate through the drying chamber of at least 15 g / min. In some embodiments, the droplets of the liquid feedstock have a flow rate through the drying chamber of up to 1000 g / min. In some embodiments, the droplets of the liquid feedstock have a flow rate through the drying chamber of at least 0.5 g / min, at least 1 g / min, at least 2 g / min, at least 3 g / min, at least 4 g / min, at least 5 g / min, at least 10 g / min, at least 15 g / min, at least 20 g / min, at least 25 g / min, at least 30 g / min, at least 35 g / min, at least 40 g / min, at least 45 g / min, at least 50 g / min, at least 60 g / min, at least 70 g / min, at least 80 g / min, at least 90 g / min, at least 100 g / min, at least 200 g / min, at least 300 g / min, at least 400 g / min, at least 500 g / min, at least 600 g / min, at least 700 g / min, at least 800 g / min, at least 900 g / min, or at least 1000 g / min.

[0366] In some embodiments, the heated pressurized gas is filtered before being introduced into the drying chamber. In some embodiments, the filter is a high efficiency particulate air (HEPA) filter. In some embodiments, the filter removes contaminants of at least 0.3 um. In some embodiments, the filter removes contaminants of at least 0.01 um, at least 0.02 um, at least 0.03 um, at least 0.04 um, at least 0.05 um, at least 0.06 um, at least 0.07 um, at least 0.08 um, at least 0.09 um, at least 0.1 um, at least 0.2 um, at least 0.3 um, at least 0.4 um, at least 0.5 um, at least 0.6 um, at least 0.7 um, at least 0.8 um, at least 0.9 um, or at least 1 um.

[0367] In some embodiments, the heated pressurized gas is heated prior to being introduced into the drying chamber. In some embodiments, the heated pressurized gas is introduced into the drying chamber at a temperature of at least 100° C. In some embodiments, the heated pressurized gas is introduced into the drying chamber at a temperature of at least 135° C. In some embodiments, the heated pressurized gas is introduced into the drying chamber at a temperature of up to 195° C. In some embodiments, the heated pressurized gas is introduced into the drying chamber at a temperature of at least 100° C., at least 105° C., at least 110° C., at least 115° C., at least 120° C., at least 125° C., at least 130° C., at least 135° C., at least 140° C., at least 145° C., at least 150° C., at least 155° C., at least 160° C., at least 165° C., at least 170° C., at least 175° C., at least 180° C., at least 185° C., at least 190° C., or at least 195° C.

[0368] In some embodiments, the heated pressurized gas is discharged from the drying chamber at a temperature of at least 40° C. In some embodiments, the heated pressurized gas is discharged from the drying chamber at a temperature of at least 48° C. In some embodiments, the heated pressurized gas is discharged from the drying chamber at a temperature of at least 50° C. In some embodiments, the heated pressurized gas is discharged from the drying chamber at a temperature of up to 50° C. In some embodiments, the heated pressurized gas is discharged from the drying chamber at a temperature of at least 60° C. In some embodiments, the heated pressurized gas is discharged from the drying chamber at a temperature of up to 60° C. In some embodiments, the heated pressurized gas is discharged from the drying chamber at a temperature of up to 85° C. In some embodiments, the heated pressurized gas is discharged from the drying chamber at a temperature of at least 40° C., at least 45° C., at least 50° C., at least 55° C., at least 60° C., at least 65° C., at least 70° C., at least 75° C., at least 80° C., or at least 85° C.

[0369] In some embodiments, the heated pressurized gas is pressurized before being introduced into the drying chamber. In some embodiments, the heated pressurized gas in the drying chamber has an atomizing gas pressure of at least 10 pounds per square inch gauge (psig). In some embodiments, the heated pressurized gas in the drying chamber has an atomizing gas pressure of at least 20 pounds per square inch gauge (psig). In some embodiments, the heated pressurized gas in the drying chamber has an atomizing gas pressure of at least 150 pounds per square inch gauge (psig). In some embodiments, the heated pressurized gas in the drying chamber has an atomizing gas pressure of at least 10 psig, at least 20 psig, at least 30 psig, at least 40 psig, at least 50 psig, at least 60 psig, at least 70 psig, at least 80 psig, at least 90 psig, at least 100 psig, at least 110 psig, at least 120 psig, at least 130 psig, at least 140 psig, at least 150 psig.

[0370] In some embodiments, the heated pressurized gas has a flow rate through the drying chamber of at least 5 kg / hr. In some embodiments, the heated pressurized gas has a flow rate through the drying chamber of at least 18 kg / hr. In some embodiments, the heated pressurized gas has a flow rate through the drying chamber of at least 44 kg / hr. In some embodiments, the heated pressurized gas has a flow rate through the drying chamber of at least 150 kg / hr. In some embodiments, the heated pressurized gas has a flow rate through the drying chamber of at least 5 kg / hr, at least 6 kg / hr, at least 7 kg / hr, at least 8 kg / hr, at least 9 kg / hr, at least 10 kg / hr, at least 11 kg / hr, at least 12 kg / hr, at least 13 kg / hr, at least 14 kg / hr, at least 15 kg / hr, at least 16 kg / hr, at least 17 kg / hr, at least 18 kg / hr, at least 19 kg / hr, at least 20 kg / hr, at least 30 kg / hr, at least 40 kg / hr, at least 50 kg / hr, at least 60 kg / hr, at least 70 kg / hr, at least 80 kg / hr, at least 90 kg / hr, at least 100 kg / hr, at least 110 kg / hr, at least 120 kg / hr, at least 130 kg / hr, at least 140 kg / hr, at least 150 kg / hr.

[0371] Spray drying can be done in batches, depending on the stability of the feedstock and the availability to run the dryer for a certain amount of time. In some embodiments, a spray drying batch lasts for 8 hours a day, but may be longer depending on the manufacturer. After this, the equipment is cleaned and prepared for another run using "new" equipment. This batch time depends on the size of the drying chamber, which determines the scale that can be spray dried (assuming the flow rate and temperature of the drying gas can be lengthened to match). In some embodiments, the step of exposing the liquid feedstock droplets to the heated pressurized gas in the drying chamber takes up to 8 hours, e.g., 8 hours per batch cycle.

[0372] In some embodiments, exposing the liquid feed droplets to the heated pressurized gas in the drying chamber takes at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 11 hours, at least 12 hours, at least 18 hours, or at least 24 hours. In some embodiments, exposing the liquid feed droplets to the heated pressurized gas in the drying chamber takes up to 1 hour, up to 2 hours, up to 3 hours, up to 4 hours, up to 5 hours, up to 6 hours, up to 7 hours, up to 8 hours, up to 9 hours, up to 10 hours, up to 11 hours, up to 12 hours, up to 18 hours, or up to 24 hours.

[0373] To establish the powder production rate in grams / minute, this can be converted from the flow rate of solids into the drying chamber by two values: 1) the mass concentration in the feedstock, and 2) the mass flow rate of the feedstock into the drying chamber.

[0374] Cyclone Chamber In some embodiments, a method of preparing a spray-dried pharmaceutical composition includes isolating dry particles of a predetermined range of diameters in a cyclone chamber, hi some embodiments, heated pressurized gas is discharged through the cyclone chamber.

[0375] In some embodiments, the step of isolating the dry particles of a predetermined range of diameters in the cyclone chamber is performed continuously. The powder that settles into the cyclone chamber and into the collection vessel at the bottom of the cyclone chamber settles continuously. The isolation does not necessarily take a fixed time.

[0376] In some embodiments, the dry particles isolated in the cyclone chamber have a median mass airdynamic diameter (MMAD) of at least 1.5 μm to a maximum of 7.5 μm. In some embodiments, the dry particles isolated in the cyclone chamber have a median mass airdynamic diameter (MMAD) of at least 2.5 μm to a maximum of 7.5 μm. In some embodiments, the dry particles isolated in the cyclone chamber have a median mass airdynamic diameter (MMAD) of at least 1.0 μm to a maximum of 10.0 μm. In some embodiments, the dry particles isolated in the cyclone chamber have a median mass airdynamic diameter (MMAD) of at least 2.3 μm. In some embodiments, the dry particles isolated in the cyclone chamber have a median mass airdynamic diameter (MMAD) of about 2.4 μm to about 2.5 μm (see, e.g., FIG. 6). In some embodiments, the dry particles isolated in the cyclone chamber have a median mass airdynamic diameter (MMAD) of at least 4.0 μm. In some embodiments, the dry particles isolated in the cyclone chamber have a mass median aerodynamic diameter (MMAD) of at least 4.2 μm. In some embodiments, the dry particles isolated in the cyclone chamber have a mass median aerodynamic diameter (MMAD) of at least 5.0 μm. In some embodiments, the dry particles isolated in the cyclone chamber have a mass median aerodynamic diameter (MMAD) of at least 1 μm, at least 1.5 μm, at least 2 μm, at least 2.5 μm, at least 3 μm, at least 3.5 μm, at least 4.0 μm, at least 4.5 μm, at least 5.0 μm, at least 5.5 μm, at least 6.0 μm, at least 6.5 μm, at least 7.0 μm, at least 7.5 μm, at least 8.5 μm at least 9 μm, at least 9.5 μm, or at least 10 μm.In some embodiments, the dried particles isolated in the cyclone chamber have a median mass aerodynamic diameter (MMAD) of at most 1 μm, at most 1.5 μm, at most 2 μm, at most 2.5 μm, at most 3 μm, at most 3.5 μm, and finally at most 4.0 μm, at most 4.5 μm, at most 5.0 μm, at most 5.5 μm, at most 6.0 μm, at most 6.5 μm, at most 7.0 μm, at most 7.5 μm, at most 8.5 μm, at most 9 μm, at most 9.5 μm, or at most 10 μm.

[0377] In some embodiments involving a viable bacterial preparation, the spray dried biotherapeutic matrix composition comprises at least 5% bacterial viability after preparation using the spray drying methods described herein (see, e.g., FIG. 5B). In some embodiments, the viability is determined after the step of isolating the dried particles. In some embodiments, the viability in the spray dried biotherapeutic matrix composition (or dried particles thereof) is compared to the number of viable bacteria in the liquid feedstock prior to spray drying. In an embodiment, the spray dried biotherapeutic matrix composition comprises at least 7.4% bacterial viability after preparation using the spray drying methods described herein. In an embodiment, the spray dried biotherapeutic matrix composition comprises at least 8.9% bacterial viability after preparation using the spray drying methods described herein. In an embodiment, the spray dried biotherapeutic matrix composition comprises at least 5% bacterial viability after preparation using the spray drying methods described herein. In an embodiment, the spray dried biotherapeutic matrix composition comprises at least 8.9% bacterial viability after preparation using the spray drying methods described herein.

[0378] In some embodiments, the spray dried biotherapeutic matrix composition, after preparation using the spray drying methods described herein, comprises at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or more bacterial viability. In some embodiments, the spray dried biotherapeutic matrix composition, after preparation using the spray drying methods described herein, comprises between 5%-10%, 10%-15%, 15%-20%, 20%-25%, 25%-30%, 30%-35%, 35%-40%, 40%-45%, 45%-50%, 50%-55%, 55%-60%, 60%-65%, 65%-70%, 70%-75%, 75%-80%, 80%-85%, 85%-90%, 90%-95%, or 95%-100% bacterial viability.

[0379] In embodiments, such as those involving administration using a DPI, the spray-dried particles may be encapsulated (i.e., encapsulated) after they are isolated from the cyclone chamber of the spray dryer.

[0380] Administration In some embodiments, the methods described herein relate to the treatment of subjects with bronchopulmonary disease or subjects diagnosed with bronchopulmonary disease. In some embodiments, the subject has or has been diagnosed with chronic bronchopulmonary disease. In some embodiments, the subject has or has been diagnosed with infectious bronchopulmonary disease. Subjects with chronic bronchopulmonary disease can be identified by physicians using current methods of diagnosing chronic bronchopulmonary disease. Symptoms and / or complications of chronic bronchopulmonary disease that characterize and aid in diagnosis of these conditions are well known in the art and include, but are not limited to, shortness of breath, especially during physical activity, wheezing, chest tightness, chronic coughing that may produce mucus (phlegm), frequent respiratory infections, lack of energy, unintentional weight loss, and / or swelling of the ankles, feet, or legs. For example, tests that may be useful in diagnosing chronic bronchopulmonary disease include, but are not limited to, lung (pulmonary) function tests, chest x-rays, lung CT scans, arterial blood gas analysis, and / or laboratory tests (e.g., genetic testing for gene signatures associated with certain chronic bronchopulmonary diseases). A family history of chronic bronchopulmonary disease or exposure to risk factors for chronic bronchopulmonary disease (e.g., long-term exposure to irritating gases or particulate matter, e.g., exposure to tobacco smoke) may also be useful in determining whether a subject is likely to have chronic bronchopulmonary disease or in making a diagnosis of chronic bronchopulmonary disease.

[0381] Subjects with infectious bronchopulmonary disease can be identified by physicians using current methods for diagnosing infectious bronchopulmonary disease. The symptoms and / or complications of infectious bronchopulmonary disease that characterize these conditions and aid in diagnosis are well known in the art and include, but are not limited to, chest pain when breathing or coughing, confusion or changes in mental awareness (e.g., in adults over 65 years old), cough that may produce phlegm, fatigue, fever, sweating and chills, lower than normal body temperature (e.g., in adults over 65 years old or people with weakened immune systems), nausea, vomiting or diarrhea, and / or shortness of breath. For example, tests that can aid in the diagnosis of infectious bronchopulmonary disease include, but are not limited to, blood tests, blood cultures, oximetry, arterial blood gases, bronchoscopy, transtracheal mucus culture, lung biopsy, thoracentesis, and / or computed tomography (CT) scan. A family history of infectious bronchopulmonary disease, or exposure to risk factors for infectious bronchopulmonary disease (e.g., long-term exposure to irritating gases or particulate matter, exposure to tobacco smoke, autoimmunity, allergy, asthma, pregnancy) may also be helpful in determining whether a subject is likely to have infectious bronchopulmonary disease or in making a diagnosis of infectious bronchopulmonary disease.

[0382] In some embodiments, the subject has been diagnosed with or is at risk of developing a bronchopulmonary disease. In some embodiments, the subject has been diagnosed with or is at risk of developing a chronic bronchopulmonary disease. In some embodiments, the chronic bronchopulmonary disease is selected from the group consisting of chronic obstructive pulmonary disease (COPD), lung cancer, asthma, bronchiectasis, emphysema, cystic fibrosis (CF), bronchopulmonary dysplasia (BPD), acute respiratory distress syndrome (ARDS), idiopathic pulmonary fibrosis (IPF), pleural effusion (PE), pulmonary hypertension (e.g., pulmonary arterial hypertension, PAH), interstitial lung disease (ILD), and silicosis. In some embodiments, the lung cancer is small cell lung cancer (SCLC) or non-small cell lung cancer (NSCLC).

[0383] In some embodiments, the infectious bronchopulmonary disease is caused by or associated with an infectious agent selected from the following: adenovirus, coronavirus (e.g., common cold virus, severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1), SARS-CoV-2, Middle East Respiratory Syndrome (MERS) CoV), influenza virus (e.g., influenza), parainfluenza virus, parvovirus B19 (e.g., parvovirus B19, fifth disease), respiratory syncytial virus (RSV), rhinovirus (e.g., common cold), enterovirus (e.g., EV-D68), measles virus, rubella virus, varicella virus (e.g., chickenpox), Corynebacterium diphtheriae (e.g., diphtheria), Haemophilus influenzae (e.g., type B), Legionella pneumophila (e.g., Legionnaires' disease), Bordetella pertussis (e.g., whooping cough), Mycobacterium tuberculosis (e.g., tuberculosis), Streptococcus species (e.g., Streptococcus pneumoniae, Streptococcus pyogenes, e.g., pneumonia), Pseudomonas species (e.g., Pseudomonas aeruginosa, pulmonary infections, chronic recurrent respiratory infections), Escherichia coli (e.g., community-acquired pneumonia), Aspergillus species (e.g., Aspergillus fumigatus, Aspergillus flavus, aspergillosis), Cryptococcus species (e.g., Cryptococcus neoformans, Cryptococcus gattii, cryptococcal pneumonia), and Pneumocystis species (e.g., Pneumocystis jirovecii, pneumocystis). See, e.g., Dasaraju and Liu, "Chapter 93: Infections of the Respiratory System," Medical Microbiology. 4th edition (1996), for non-limiting examples of infectious bronchopulmonary diseases.

[0384] In some embodiments, the chronic bronchopulmonary disease is chronic obstructive pulmonary disease (COPD). In some embodiments, the chronic bronchopulmonary disease is lung cancer. In some embodiments, the chronic bronchopulmonary disease is asthma. In some embodiments, the chronic bronchopulmonary disease is bronchiectasis. In some embodiments, the chronic bronchopulmonary disease is emphysema. In some embodiments, the chronic bronchopulmonary disease is cystic fibrosis (CF). In some embodiments, the chronic bronchopulmonary disease is bronchopulmonary dysplasia (BPD). In some embodiments, the chronic bronchopulmonary disease is acute respiratory distress syndrome (ARDS). In some embodiments, the chronic bronchopulmonary disease is idiopathic pulmonary fibrosis (IPF). In some embodiments, the chronic bronchopulmonary disease is interstitial lung disease (ILD). In some embodiments, the chronic bronchopulmonary disease is pleural effusion (PE). In some embodiments, the chronic bronchopulmonary disease is pulmonary hypertension (PAH). In some embodiments, the chronic bronchopulmonary disease is silicosis. In some embodiments, the chronic bronchopulmonary disease is small cell lung cancer (SCLC). In some embodiments, the chronic bronchopulmonary disease is non-small cell lung cancer (NSCLC). In some embodiments, the chronic bronchopulmonary disease is non-cystic fibrosis (CF) bronchiectasis.

[0385] In one aspect, described herein is a method of delivering a spray dried pharmaceutical composition comprising a bacterial preparation to a subject, the method comprising: (a) obtaining an inhalation device for bronchopulmonary delivery, the inhalation device comprising: (i) an inhaler; and (ii) a container containing the spray dried pharmaceutical composition comprising the bacterial preparation; (b) activating the inhaler to cause aerosolization or dispersion of the spray dried pharmaceutical composition; and (c) inhaling the aerosolized or dispersed spray dried pharmaceutical composition.

[0386] In one aspect, described herein is a method of delivering a spray dried pharmaceutical composition comprising a bacterial preparation to a subject, the method comprising: (a) obtaining an inhalation device for bronchopulmonary delivery, the inhalation device comprising: (i) an inhaler; and (ii) a container containing the spray dried pharmaceutical composition described herein; (b) activating the inhaler to cause aerosolization or dispersion of the spray dried pharmaceutical composition; and (c) inhaling the aerosolized or dispersed spray dried pharmaceutical composition.

[0387] In one aspect, described herein is a method of delivering a spray dried pharmaceutical composition comprising a bacterial preparation to a subject, the method comprising: (a) obtaining an inhalation device as described herein; (b) activating the inhaler to cause aerosolization or dispersion of the spray dried pharmaceutical composition; and (c) inhaling the aerosolized or dispersed spray dried pharmaceutical composition.

[0388] In some embodiments, the inhaler is a dry powder inhaler (DPI). In some embodiments, the inhaler is a metered dose inhaler (MDI). In some embodiments of any of the aspects, the inhaler is a soft mist inhaler (SMI). In some embodiments, the inhaler comprises (a) a mouthpiece comprising an opening, and (b) a means for aerosolizing or dispersing the spray-dried pharmaceutical composition in the container. In some embodiments, the inhaler has an inhalation flow rate of at least 15 L / min. In some embodiments, the inhaler has an inhalation flow rate of at least 15 L / min and up to 60 L / min. In some embodiments, the inhaler has an inhalation flow rate of at least 15 L / min and up to 120 L / min. In some embodiments, the inhaler has an inhalation flow rate of at least 15 L / min, at least 20 L / min, at least 25 L / min, at least 30 L / min, at least 35 L / min, at least 40 L / min, at least 45 L / min, at least 50 L / min, at least 55 L / min, at least 60 L / min, at least 70 L / min, at least 80 L / min, at least 90 L / min, at least 100 L / min, at least 110 L / min, or up to 120 L / min.

[0389] In some embodiments, at least 25% to up to 125.0% by weight of the spray dried pharmaceutical composition is delivered to the target bronchopulmonary tissue. In some embodiments, at least 25% to up to 80.0% by weight of the spray dried pharmaceutical composition is delivered to the target bronchopulmonary tissue. In some embodiments, at least 25% to up to 100.0% by weight of the spray dried pharmaceutical composition is delivered to the target bronchopulmonary tissue. In some embodiments, at least 60.0% by weight of the spray dried pharmaceutical composition is delivered to the target bronchopulmonary tissue. In some embodiments, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, or at least 125% by weight of the spray dried biotherapeutic matrix composition is delivered to the target bronchopulmonary tissue.

[0390] In some embodiments, at least 25% to up to 125% by weight of the spray dried pharmaceutical composition is discharged from the inhalation device (e.g., inhaler). In some embodiments, at least 25% to up to 80.0% by weight of the spray dried pharmaceutical composition is discharged from the inhalation device (e.g., inhaler). In some embodiments, at least 60.0% by weight of the spray dried pharmaceutical composition is discharged from the inhalation device (e.g., inhaler). In some embodiments, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, or at least 125% by weight of the spray dried pharmaceutical composition is discharged from the inhalation device (e.g., inhaler).

[0391] In some embodiments, the target bronchopulmonary tissue is a lung, trachea, bronchi, bronchioles, or alveoli. In some embodiments, the target bronchopulmonary tissue is a lung. In some embodiments, the target bronchopulmonary tissue is a trachea. In some embodiments, the target bronchopulmonary tissue is a bronchi. In some embodiments, the target bronchopulmonary tissue is a bronchioles. In some embodiments, the target bronchopulmonary tissue is an alveolus.

[0392] In some embodiments, the dry particles have a median mass aerodynamic diameter (MMAD) of at least 0.5 μm to a maximum of 10 μm, which may affect the bronchopulmonary regions that the particles can reach. Particles of about 0.5 um to about 2.0 um are deposited in small airways such as alveoli and bronchioles, which are therapeutically useful. Particles of about 2.0 um to about 10.0 um are deposited in the bronchial regions of the airways, which are therapeutically useful. Particles below 0.5 um are essentially exhaled, which are not therapeutically useful. Particles above about 10.0 um are retained in the oropharyngeal region and larynx due to impaction, which are not therapeutically useful. See, for example, Thakur et al., Chapter 22: Mucoadhesive drug delivery systems in respiratory diseases, in Targeting Chronic Inflammatory Lung Diseases Using Advanced Drug Delivery Systems, 2020, pp. 475-491, the contents of which are incorporated by reference in their entirety.

[0393] In some embodiments, the spray-dried pharmaceutical composition is delivered from the bronchopulmonary tissue to a distal tissue site (i.e., a non-pulmonary tissue) via the cardiovascular or lymphatic system. Having a bacterial preparation (e.g., a live or non-live bacterial biotherapeutic) in the cardiovascular or lymphatic system is not necessarily harmful to the subject, especially at low doses. For example, studies show that blood exhibits microbial signatures. See, for example, Castillo et al., Front. Cell. Infect. Microbiol., 9:148, 2019, the contents of which are incorporated by reference in their entirety. In some embodiments, the distal tissue site (i.e., a non-pulmonary tissue) is in the gastrointestinal system, the cardiovascular system, the lymphatic system, the musculoskeletal system, the nervous system, the urinary system, the reproductive system, the endocrine system, or the integumentary system. In some embodiments, the distal tissue site (i.e., a non-pulmonary tissue) is a connective tissue, an epithelial tissue, a muscle tissue, or a nervous tissue.

[0394] The compositions and methods described herein may be administered to a subject who has or has been diagnosed with a bronchopulmonary disease (e.g., chronic bronchopulmonary disease). In some embodiments, the methods described herein include administering to a subject an effective amount of a composition described herein, such as a spray-dried pharmaceutical composition described herein (e.g., comprising a bacterial preparation), to alleviate a symptom of chronic bronchopulmonary disease. As used herein, "alleviating a symptom of chronic bronchopulmonary disease" refers to improving any condition or symptom associated with chronic bronchopulmonary disease. Compared to an equivalent untreated control, such reduction is at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, 99%, or more, as measured by any standard technique.

[0395] Compared to other routes of administration, inhalation offers many unique advantages (see, e.g., Borghardt et al. 2018, the contents of which are incorporated herein by reference in their entirety). Inhalation delivers bacterial preparations directly to lung tissue, resulting in high lung concentrations and low systemic concentrations with minimal systemic side effects. Inhalation formulations containing bacterial preparations can be produced via several processing routes, including spray drying to produce dry powder inhalation formulations, compounding to produce nebulizable formulations, or compounding and pressurization in metered dose inhalers. Each of these routes of administration offers benefits. Dry powder formulations are composed of engineered particles with specific size distributions, allowing for more precisely tailored lung deposition, aerodynamic properties, long-term stability, and relatively easy administration in patients with normal inhalation capabilities. Nebulized solutions containing active pharmaceutical ingredients are well suited to infant or adolescent lungs, as well as patients with weaker lung volumes. Nebulized solutions face stability challenges and size limitations that may prevent aerosolization during administration.

[0396] Metered dose inhalers are common in the bronchodilator and inhaled corticosteroid market, but typically require pressurized containers and chemical propellants to function effectively. Some of these propellants have unknown side effects, and the sustained pressure on drug-containing solutions can pose stability challenges, especially when formulated with more sensitive biotherapeutics. Although propellants and high pressure generally do not aid in maintaining bacterial viability, MDIs can be used to deliver non-viable bacteria (e.g., heat-killed bacteria), bacterial extracts, and / or bacterial products.

[0397] As used herein, the term "effective amount" refers to the amount of the spray-dried pharmaceutical composition (e.g., containing a bacterial preparation) described herein necessary to alleviate at least one or more symptoms of a disease or disorder, and relates to a sufficient amount of the pharmacological composition to provide the desired effect. Thus, the term "therapeutically effective amount" refers to an amount of the spray-dried pharmaceutical composition (e.g., containing a bacterial preparation) described herein that is sufficient to provide an effect against a particular bronchopulmonary disease when administered to a typical subject. In various contexts, an effective amount as used herein will also include an amount sufficient to delay the onset of a disease symptom, alter the disease course of a disease symptom (e.g., but not limited to, delay the progression of a disease symptom), or ameliorate a disease symptom. Thus, in general, it is not practical to specify an exact "effective amount". However, in any given case, an appropriate "effective amount" can be determined by one of ordinary skill in the art using only routine experimentation.

[0398] In some embodiments, an effective amount of the spray-dried inhalable biotherapeutic agent described herein can be determined using biodistribution and / or translocation studies to determine where the administered bacteria travel and / or deposit in the body, kinetic studies on how quickly the bacteria are cleared from the target tissue and whether the bacteria engraft or colonize the target tissue, and / or studies to evaluate changes in metabolic output (e.g., of a subject) as a result of bacteria-derived by-products. Effective amounts, toxicity, and therapeutic effects can also be determined by standard pharmaceutical procedures in cell cultures or experimental animals, including, for example, determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). Dosages can vary depending on the dosage form used and the route of administration utilized. The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as the ratio LD50 / ED50. Compositions and methods that exhibit a high therapeutic index are preferred. The therapeutically effective dose can be initially estimated from cell culture assays. Doses may also be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the metabolite(s) produced by the administered bacterium that achieves half-maximal inhibition of symptoms) as determined in cell culture. Plasma levels (e.g., levels of the metabolite(s) produced by the administered bacterium) can be measured by high performance liquid chromatography. The effect of any particular dosage can be monitored by a suitable bioassay, such as pH or apoptosis assays, among others. Dosages are determined by a physician and can be adjusted, if necessary, to the observed effects of the treatment.

[0399] In some embodiments of any of the aspects, a spray dried biotherapeutic matrix composition described herein (e.g., comprising a bacterial preparation) is administered as a monotherapy, e.g., no other treatment for a chronic bronchopulmonary disease is administered to the subject.

[0400] In some embodiments of any of the aspects, the methods described herein can further include administering to the subject a second agent and / or treatment, e.g., as part of a combination therapy. Non-limiting examples of second agents and / or treatments can include cancer therapies selected from the group consisting of radiation therapy, surgery, gemcitabine, cisplatin, paclitaxel, carboplatin, bortezomib, AMG479, vorinostat, rituximab, temozolomide, rapamycin, ABT-737, PI-103, alkylating agents such as thiotepa and CYTOXAN® cyclophosphamide, alkylsulfonates such as busulfan, improsulfan, and piposulfan, bevacizumab, cyclophosphamide ... Aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylmelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine; acetogenins (especially bullatacin and bullatacinone); camptothecins (including the synthetic analog topotecan); bryostatin, kallistatin, CC-1065 (including the synthetic analogs adozelesin, carzelesin, and bizelesin); cryptophycins (especially cryptophycin 1 and cryptophycin 8), dolastatins, duocarmycins (including synthetic analogs, KW-2189 and CB1-TM1), erytherobin, pancratistatin, sarcodictin, spongiostatin, nitrogen mustards, e.g., chlorambucil, chlornaphazine, clophosfamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembitine, phenesterine, prednimustine, trofosfamide, and u nitrosoureas, such as rasil mustard, carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; antibiotics, such as enediyne antibiotics (e.g., the calicheamicins, particularly calicheamicin gamma 1I and calicheamicin omega 11) (see, e.g., Agnew, Chem. Intl. Ed. Engl., 33:183-186 (1994)); dynemicins (including dynemicin A); bisphosphonates, such as clodronate; esperamicin;and neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomycin, actinomycin, ausramycin, azaserine, bleomycin, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® doxorubicin (morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, and deoxydoxorubicin). xorubicin), mitomycins such as epirubicin, esorubicin, idarubicin, marcelomycin, and mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfilomycin, puromycin, keramycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin, antimetabolites such as methotrexate and 5-fluorouracil (5-FU), folic acid analogs such as denopterin, methotrexate, pteropterin, and trimetrexate. purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens such as calsterone, dromostanolone propionate, epithiostanol, mepitiostane, and testolactone; antiadrenal agents such as aminoglutethimide, mitotane, and trilostane; folic acid supplements such as floric acid; aceglatone, and aldophosphamide glycosides. , aminolevulinic acid, eniluracil, amsacrine, bestravcil, bisantrene, edatraxate, defofamine, demecolcine, diaziquone, erformitin, elliptinium acetate, epothilone, etoglucide, gallium nitrate, hydroxyurea, lentinan, lonidynin, maytansinoids such as maytansine and ansamitocin, mitoguazone, mitoxantrone, mopidamol, nitraelin, pentostatin, phenamet, pirarubicin, rosoxantrone, podophyllic acid, 2-ethylhydrazide, procarbazine,PSK® polysaccharide complex (JHS Natural Products, Eugene, Oreg.), razoxane, rhizoxin, schizofuran, spirogermanium, tenuazonic acid, triazicon, 2,2',2''-trichlorotriethylamine, trichothecenes (especially T-2 toxin, veracrine A, roridin A, anguidin), urethanes, vindesine, dacarbazine, mannomustine, mitobronitol, mitolactol, pipobroman, gacytosine, arabinoside ("Ara-C"), cyclophosphamide, thiotepa, taxoids such as TAXOL® paclitaxel (Bristol-Myers Squibb Oncology, Princeton, NJ), ABRAXANE® cremophor-free, albumin engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERE® doxetaxel (Rhone-Poulenc Rorer, Antony, France), chlorambucil, GEMZAR® gemcitabine, 6-thioguanine, mercaptopurine, methotrexate, platinum analogs such as cisplatin, oxaliplatin, carboplatin, vinblastine, platinum, etoposide (VP-16), ifosfamide, mitoxantrone, vincristine, NAVELBINE® vinorelbine, novantrone, teniposide, edatrexate, daunomycin, aminopterin, xeloda, ibandronate, irinotecan (camptosar, CPT-11) (5-FU and leucovorin), irinotecan treatment regimens with riboflavin, topoisomerase inhibitor RFS2000, difluoromethylornithine (DMFO), retinoids such as retinoic acid, capecitabine, combretastatins, leucovorin (LV), oxaliplatin, including oxaliplatin treatment regimens (FOLFOX), lapatinib (Tykerb®), inhibitors of PKC-alpha, Raf, H-Ras, EGFR (e.g., erlotinib (Tarceva®)) and VEGF-A that reduce cell proliferation, and pharmacologic acceptable salts, acids, or derivatives of any of the above.

[0401] Those skilled in the art can easily identify the chemotherapeutic agent to be used (see, for example, Physicians' Cancer Chemotherapy Drug Manual 2014, Edward Chu, Vincent T., DeVita Jr., Jones&Bartlett Learning, Principles of Cancer Therapy, Chapter 85 in Harrison's Principles of Internal Medicine, 18th edition, Therapeutic Targeting of Cancer Cells: Era of Molecularly Targeted Agents and Cancer Pharmacology, Chs.28-29 in Abeloff's Clinical Oncology, 2013 Elsevier, and Fischer DS(ed): The Cancer Chemotherapy Handbook, 4th ed. St. Louis, Mosby-Year Book, 2003). In addition, the treatment method may further include the use of radiation or radiation therapy. In addition, the treatment method may further include the use of surgical treatment.

[0402] The methods described herein can further include administering a second agent and / or treatment to the subject, e.g., as part of a combination therapy. As a non-limiting example, when a subject is treated for pain or inflammation according to the methods described herein, the subject can also be administered a second agent and / or treatment known to be beneficial to subjects suffering from pain or inflammation. Examples of such drugs and / or treatments include, but are not limited to, nonsteroidal anti-inflammatory drugs (NSAIDs such as aspirin, ibuprofen, or naproxen), corticosteroids including glucocorticoids (e.g., cortisol, prednisolone, prednisolone, methylprednisolone, dexamethasone, betamethasone, triamcinolone, and beclomethasone), methotrexate, sulfasalazine, leflunomide, anti-TNF drugs, cyclophosphamide, inflammation-resolving drugs, mycophenolic acid, or opiates (e.g., enkephalins and dynorphins), steroids, analgesics, barbiturates, oxycodone, morphine, lidocaine, and the like.

[0403] In some embodiments, the spray dried biotherapeutic matrix composition is co-administered with at least one additional therapeutic treatment or intervention for a chronic bronchopulmonary disorder.

[0404] In some embodiments, the chronic bronchopulmonary disorder is chronic obstructive pulmonary disease (COPD) and the at least one additional therapeutic treatment or intervention is smoking cessation, bronchodilators, inhaled steroids, oral steroids, roflumilast (DALIRESP, a phosphodiesterase-4 inhibitor), theophylline (ELIXOPHYLLIN, THEO-24, THEOCHRON), antibiotics, oxygen therapy, pulmonary rehabilitation (e.g., education, breathing exercises, nutritional advice, and / or counseling), home non-invasive ventilation (e.g., bilevel positive airway pressure (BiPAP)), and / or surgery (e.g., lung volume reduction surgery, pulmonary bulla resection, or lung transplant).

[0405] In some embodiments, the chronic bronchopulmonary disorder is lung cancer and the at least one additional therapeutic treatment or intervention is surgery (e.g., wedge resection, segmentectomy, lobectomy, or pneumonectomy), radiation therapy, chemotherapy, stereotactic body radiation therapy, gene-targeted chemotherapy, or immunotherapy.

[0406] In some embodiments, the chronic bronchopulmonary disorder is asthma and the at least one additional therapeutic treatment or intervention is selected from the group consisting of bronchodilators, inhaled corticosteroids (e.g., fluticasone propionate (FLOVENT HFA, FLOVENT DISKUS, XHANCE), budesonide (PULMICORT FLEXHALER, PULMICORT RESPULES, RHINOCORT), ciclesonide (ALVESCO), beclomethasone (QVAR REDIHALER), mometasone (ASMANEX HFA, ASMANEX TWISTHALER), and fluticasone furoate (ARNUITY ELLIPTA), leukotriene modifiers (e.g., montelukast (SINGULAIR), zafirlukast (ACCOLATE), and zileuton (ZYFLO), long-acting beta agonists and corticosteroids (e.g., fluticasone-salmeterol (ADVAIR HFA, AIRDUO DIGIHALER), budesonide-formoterol (SYMBICORT), formoterol-mometasone (DULERA) and fluticasone furoate-vilanterol (BREO ELLIPTA), theophylline (ELIXOPHYLLIN, THEO-24, THEOCHRON), short-acting beta agonists (e.g., albuterol (PROAIR HFA, VENTOLIN HFA) and levalbuterol (XOPENEX, XOPENEX HFA), anticholinergics (e.g., ipratropium (ATROVENT HFA) and tiotropium (SPIRIVA, SPIRIVA RESPIMAT), oral and intravenous corticosteroids (e.g., prednisone (PREDNISONE These include treatments such as INTENSOL, RAYOS) and methylprednisolone (MEDROL, DEPO-MEDROL, SOLU-MEDROL)), allergy injections (e.g., injections of specific allergens to gradually reduce the immune system response), allergy biologics (e.g., omalizumab (XOLAIR), mepolizumab (NUCALA), dupilumab (DUPIXENT), reslizumab (CINQAIR) or benralizumab (FASENRA)), or bronchial thermoplasty.

[0407] In some embodiments, the chronic bronchopulmonary disorder is bronchiectasis and the at least one additional therapeutic treatment or intervention is an antibiotic, a macrolide, a mucus thinning agent (e.g., guaifenesin (MUCINEX)), an airway clearance device (e.g., a positive expiratory pressure (PEP) device, a percussive, wearable device such as a percussive vest), or chest physical therapy (e.g., chest clapping).

[0408] In some embodiments, the chronic bronchopulmonary disorder is emphysema and the at least one additional therapeutic treatment or intervention is a bronchodilator, an inhaled steroid, an antibiotic, pulmonary rehabilitation (e.g., breathing exercises), nutritional therapy, supplemental oxygen, smoking cessation, or surgery (e.g., lung volume reduction surgery, lung transplant).

[0409] In some embodiments, the chronic bronchopulmonary disorder is cystic fibrosis (CF) and the at least one additional therapeutic treatment or intervention is a cystic fibrosis transmembrane conductance regulator (CFTR) modulator (e.g., a combination containing elexacaftor, ivacaftor, and tezacaftor (TRIKAFTA), a combination containing tezacaftor and ivacaftor (SYMDEKO), a combination containing lumacaftor and ivacaftor (ORKAMBI), ivacaftor (KALYDECO), an airway clearance device (e.g., a positive expiratory pressure (PEP) device, a percussive wearable device such as a percussive vest), chest physical therapy (e.g., chest clapping), pulmonary rehabilitation (e.g., breathing exercises), nasal and sinus surgery, oxygen therapy, noninvasive ventilation, a feeding tube, intestinal surgery, a lung transplant, or a liver transplant.

[0410] In some embodiments, the chronic bronchopulmonary disorder is bronchopulmonary dysplasia (BPD) and the at least one additional therapeutic treatment or intervention is a diuretic (e.g., reducing the amount of fluid in and around the alveoli) to prevent or reduce respiratory tract infection, a bronchodilator, a corticosteroid, a cardiac medication, or a respiratory syncytial virus (RSV) immunization.

[0411] In some embodiments, the chronic bronchopulmonary disorder is acute respiratory distress syndrome (ARDS) and the at least one additional therapeutic treatment or intervention is supplemental oxygen, mechanical ventilation, intravenous fluids, smoking cessation, antibiotics, painkillers, anti-inflammatory agents, blood thinners, gastric reflux medications (e.g., esomeprazole (NEXIUM)), or sedatives.

[0412] In some embodiments, the chronic bronchopulmonary disorder is idiopathic pulmonary fibrosis (IPF) and the at least one additional therapeutic treatment or intervention is an antifibrotic agent (e.g., nintedanib (OFEV) or pirfenidone (ESBRIET)), a corticosteroid (e.g., prednisone), an immunosuppressant (e.g., azathioprine (IMURAN), cyclophosphamide (CYTOXAN), or mycophenolate mofetil (CELLCEPT)), an oral or spray antioxidant (e.g., N-acetylcysteine, NAC (MUCOMYST)), oxygen therapy, or pulmonary rehabilitation (e.g., breathing exercises).

[0413] In some embodiments, the chronic bronchopulmonary disorder is an interstitial lung disease (ILD) and the at least one additional therapeutic treatment or intervention is a corticosteroid (e.g., prednisone), an immunosuppressant, a drug to slow the progression of idiopathic pulmonary fibrosis such as pirfenidone (ESBRIET) or nintedanib (OFEV), an H-2 receptor antagonist or a proton pump inhibitor such as lansoprazole (PREVACID 24HR), omeprazole (Prilosec OTC), or pantoprazole (PROTONIX) for symptoms of gastroesophageal reflux disease (GERD) that affects the majority of people with idiopathic pulmonary fibrosis, oxygen therapy, pulmonary rehabilitation, and / or surgery such as a lung transplant.

[0414] In some embodiments, the chronic bronchopulmonary disorder is pleural effusion (PE) and the at least one additional therapeutic treatment or intervention is a diuretic, chemotherapy, radiation therapy, therapeutic thoracentesis, thoracic drainage, sclerosing agents (e.g., talc, doxycycline, and tetracycline), and / or surgery, such as video-assisted thoracoscopic surgery (VATS) or thoracotomy.

[0415] In some embodiments, the chronic bronchopulmonary disorder is pulmonary hypertension (PAH) and the at least one additional therapeutic treatment or intervention is a vasodilator, a guanylate cyclase (GSC) stimulator such as riociguat (ADEMPAS), an endothelin receptor antagonist such as bosentan (TRACLEER), macitentan (OPSUMIT), or ambrisentan (LETAIRIS), a phosphodiesterase 5 (PDE5) inhibitor such as sildenafil (REVATIO, VIAGRA) or tadalafil (ADCIRCA, CIALIS, ALYQ), a calcium channel blocker such as amlodipine (NORVASC), diltiazem (CARDIZEM, TIAZAC), or nifedipine (PROCARDIA), an anticoagulant such as warfarin (JANTOVEN), digoxin (LANOXIN), a diuretic, oxygen therapy, and / or surgery such as atrial septal septum resection, lung transplant, or heart-lung transplant.

[0416] In some embodiments, the chronic bronchopulmonary disorder is silicosis and the at least one additional therapeutic treatment or intervention is a bronchodilator, smoking cessation, supplemental oxygen, pulmonary rehabilitation, and / or surgery such as a lung transplant.

[0417] In some embodiments, the at least one additional therapeutic treatment or intervention for an infectious bronchopulmonary disorder is an antiviral agent, an antibiotic, or an antifungal agent, such as the non-limiting examples listed above.

[0418] In some embodiments, the methods described herein may further include administering an antibiotic to the subject, e.g., as part of a combination therapy. In some embodiments, the antibiotic is administered before, during, or after administration of the spray dried biotherapeutic matrix composition. In some embodiments, the antibiotic is administered to kill or reduce the growth of bacteria naturally growing in the target tissue, e.g., to allow the bacteria of the spray dried biotherapeutic matrix composition to grow and / or colonize the target tissue. In some embodiments, the antibiotic is administered to kill or reduce the growth of bacteria from the spray dried biotherapeutic matrix composition, e.g., after a sufficient time or growth of such bacteria. In some embodiments, the bacteria from the spray dried biotherapeutic matrix composition are resistant to the administered antibiotic (e.g., when the antibiotic is administered to kill or reduce the growth of native bacteria in the target tissue). In some embodiments, the bacteria from the spray dried biotherapeutic matrix composition are not resistant (i.e., susceptible) to the administered antibiotic (e.g., when the antibiotic is administered to kill or reduce the growth of bacteria from the spray dried biotherapeutic matrix composition).

[0419] In some embodiments of any of the aspects, the antibiotic is selected from amikacin, aztreonam, cefepime, cefoxitin, ciprofloxacin, levofloxacin, metronidazole, trimethoprim / sulfa, trimethoprim, vancomycin, the extended-spectrum β-lactamase (ESBL) plazomycin, fosfomycin, ceftazidime, or ofloxacin.

[0420] In some embodiments of any of the aspects, the antibacterial agent may be selected from aminoglycosides, ansamycins, beta-lactams, bis-biguanides, carbacephems, carbapenems, cationic polypeptides, cephalosporins, fluoroquinolones, glycopeptides, iron-sequestering glycoproteins, linosamides, lipopeptides, macrolides, monobactams, nitrofurans, oxazolidinones, penicillins, polypeptides, quaternary ammonium compounds, quinolones, silver compounds, sulfonamides, and tetracyclines.

[0421] Some exemplary antibiotics include broad spectrum penicillins, amoxicillins (e.g., ampicillin, bacampicillin, carbenicillin indanyl, mezlocillin, piperacillin, ticarcillin), penicillins and beta-lactamase inhibitors (e.g., amoxicillin-clavulanate, ampicillin-sulbactam, benzylpenicillin, cloxacillin, dicloxacillin, methicillin, oxacillin, penicillin G, penicillin V, piperacillin-tazobactam, ticarcillin-clavulanate, nafcillin), cephalosporins (e.g., first generation cephalosporins, cephalosporins (e.g., cefadroxil, cefazolin, cephalexin, cephalothin, cephapirin, cephradine), second-generation cephalosporins (e.g., cefaclor, cefamandole, cefonicid, cefotetan, cefoxitin, cefprozil, cefmetazole, cefuroxime, loracarbef), third-generation cephalosporins (e.g., cefdinir, ceftibuten, cefoperazone, cefixime, cefotaxime, cefpodoxime proxetil, ceftazidime, ceftizoxime, ceftriaxone), fourth-generation cephalosporins (e.g., cefepidimycin, cefadroxil, cefazolin, cephalexin, cephalothin, cephapirin, cephradine), ), macrolides and lincosamides (e.g., azithromycin, clarithromycin, clindamycin, dirithromycin, erythromycin, lincomycin, troleandomycin), quinolones and fluoroquinolones (e.g., cinoxacin, ciprofloxacin, enoxacin, gatifloxacin, grepafloxacin, levofloxacin, lomefloxacin, moxifloxacin, nalidixic acid, norfloxacin, ofloxacin, sparfloxacin, trovafloxacin, oxolinic acid, gemifloxacin, perfloxacin), Rubapenems (e.g., imipenem-cilastatin, meropenem), monobactams (e.g., aztreonam), aminoglycosides (e.g., amikacin, gentamicin, kanamycin, neomycin, netilmicin, streptomycin, tobramycin, paromomycin), glycopeptides (e.g., teicoplanin, vancomycin), tetracyclines (e.g., demeclocycline, doxycycline, methacycline, minocycline, oxytetracycline, tetracycline, chlortetracycline), sulfonamides (e.g.,Mafenide, silver sulfadiazine, sulfacetamide, sulfadiazine, sulfamethoxazole, sulfasalazine, sulfisoxazole, trimethoprim-sulfamethoxazole, sulfamethizole), rifampin (e.g., rifabutin, rifampin, rifapentine), oxazolidinone (e.g., linezolid, streptogramin, quinupristin dalfopristin), bacitracin, chloramphenicol, fosfomycin, isoniazid, methenamine, metronidazole, mupirocin, nitrofurantoin, nitrofurazone, novobiocin, polymyxin, spectinomycin, trimethoprim, colistin, cycloserine, capreomycin, ethionamide, pyrazinamide, para-aminosalicylic acid, erythromycin ethylsuccinate, etc.

[0422] In certain embodiments, a composition comprising an effective dose of the bacterial preparation described herein may be administered once to a patient. In certain embodiments, a composition comprising an effective dose of the bacterial preparation may be administered repeatedly to a patient. For systemic administration, a subject may be administered a therapeutic amount of a composition comprising a bacterial preparation, for example, 0.1 mg / kg, 0.5 mg / kg, 1.0 mg / kg, 2.0 mg / kg, 2.5 mg / kg, 5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, 40 mg / kg, 50 mg / kg, or more.

[0423] In some embodiments, after an initial treatment regimen, treatment may be administered less frequently. For example, after 3 months of biweekly treatment, treatment may be repeated once a month for 6 months or for a year or more. Treatment according to the methods described herein may reduce the level of a marker or symptom of chronic bronchopulmonary disease by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%, or more.

[0424] Dosages of the compositions described herein may be determined by a physician and adjusted as necessary to the observed therapeutic effects. With regard to duration and frequency of treatment, a skilled clinician will typically monitor the subject to determine when the treatment is providing therapeutic benefit and to determine whether to increase or decrease the dosage, increase or decrease the frequency of administration, discontinue treatment, resume treatment, or make other changes to the treatment regimen. Dosage schedules may vary from once a week to once a day, depending on several clinical factors, such as the subject's susceptibility to bacterial preparations. The desired dose or amount may be administered once or divided into subdoses (e.g., 2-4 subdoses) and administered over a period of time, e.g., at appropriate intervals throughout the day or on other suitable schedules. In some embodiments, administration may be chronic, e.g., one or more doses and / or treatments daily for a period of weeks or months. Examples of dosing and / or treatment schedules are administration once a day, twice a day, three times a day, or four times a day or more for a period of 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months or more. The composition comprising the bacterial preparation may be administered over a period of time (e.g., over 5 minutes, 10 minutes, 15 minutes, 20 minutes, or 25 minutes).

[0425] The dosage range for administration of the spray dried biotherapeutic matrix composition (e.g., including a bacterial preparation) according to the methods described herein depends, for example, on the form of the bacterial preparation, its potency, and the degree to which it is desired to reduce the symptoms, markers, or indicators of the conditions described herein. The dosage should not be so large as to cause adverse side effects, such as infection or sepsis. In general, dosage will vary with the age, condition, and sex of the patient, and can be determined by one of skill in the art. Dosage can also be adjusted by the individual physician in the event of any complications.

[0426] For example, the efficacy of a spray-dried biotherapeutic matrix composition (e.g., including a bacterial preparation) in treating a condition described herein can be determined by a skilled clinician. However, if one or more of the signs or symptoms of a condition described herein are altered in a beneficial manner and other clinically recognized symptoms are improved or even ameliorated, or if, for example, a desired response is induced by at least 10% following treatment according to the methods described herein, the treatment is considered to be an "effective treatment" as that term is used herein. Efficacy can be assessed, for example, by measuring the occurrence of markers, indicators, symptoms, and / or conditions being treated according to the methods described herein or any other suitable measurable parameters. Efficacy can also be measured by failure of an individual to deteriorate as assessed by hospitalization or need for medical intervention (i.e., progression of the disease is halted). Methods for measuring these indicators are known to those skilled in the art and / or described herein. Treatment includes any treatment of a disease in an individual or animal (some non-limiting examples include humans or animals), including (1) inhibiting the disease, e.g., preventing the worsening of symptoms, or (2) reducing the severity of the disease, e.g., causing regression of symptoms. An effective amount for the treatment of a disease means an amount that, when administered to a subject in need thereof, is sufficient to provide effective treatment for the disease, as that term is defined herein. The efficacy of an agent can be determined by assessing physical indicators of the condition or desired response. It is within the ability of one of ordinary skill in the art to monitor the efficacy of administration and / or treatment by measuring any one of such parameters, or any combination of parameters. Efficacy can be assessed in animal models of treatment of conditions described herein, such as bronchopulmonary dysplasia (COPD), lung cancer, asthma, bronchiectasis, emphysema, cystic fibrosis (CF), bronchopulmonary dysplasia (BPD), acute respiratory distress syndrome (ARDS), or idiopathic pulmonary fibrosis (IPF). When experimental animal models are used, efficacy of treatment is demonstrated when a statistically significant change in a marker is observed.

[0427] In vitro and animal model assays allow for the evaluation of a given dose of a spray dried biotherapeutic matrix composition (e.g., including a bacterial preparation). As non-limiting examples, the effect of a dose of a spray dried biotherapeutic matrix composition (e.g., including a bacterial preparation) can be evaluated in a lung epithelial cell dysbiosis model, a mouse model of dysbiosis, or A549 non-small cell lung cancer (NSCLC) adenocarcinoma cells.

[0428] Treatment method The compositions described herein can be administered to a subject in need thereof, for example, for the treatment of bronchopulmonary disease, including, but not limited to, chronic bronchopulmonary disease. In some embodiments, the treatment method can include first diagnosing a subject or patient that can benefit from treatment with a composition described herein. In some embodiments, the method further includes administering a composition described herein to the patient.

[0429] In one aspect, a method of treating a subject in need of treatment is described herein, the method comprising administering by inhalation an effective dose of a spray dried biotherapeutic matrix composition comprising a bacterial preparation.In one aspect, a method of treating a subject in need of treatment is described herein, the method comprises administering by inhalation an effective dose of a spray dried biotherapeutic matrix composition described herein.

[0430] In some embodiments, the spray dried biotherapeutic matrix composition (e.g., a bacterial preparation in a spray dried biotherapeutic matrix composition) reduces neutrophilic inflammation in a target tissue. In some embodiments, the spray dried biotherapeutic matrix composition reduces neutrophilic inflammation in a target tissue by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or more.

[0431] In some embodiments, administration of the spray dried biotherapeutic matrix composition results in acid production in a target tissue of the subject. In some embodiments, administration of the spray dried biotherapeutic matrix composition results in lactic acid (LA) production in the target tissue. In some embodiments, administration of the spray dried biotherapeutic matrix composition results in lactic acid production in a bronchopulmonary target tissue (e.g., lung). In some embodiments, increased lactic acid production may be associated with reduced neutrophilic inflammation in the target tissue. In some embodiments, lactic acid is produced by viable bacteria in the administered spray dried biotherapeutic matrix composition. In some embodiments, lactic acid is produced by cells of the subject, e.g., induced by at least one bacterial preparation (e.g., viable or non-viable bacteria or components thereof) in the administered spray dried biotherapeutic matrix composition.

[0432] In some embodiments, at least 4 nmol / mL of lactic acid is produced in the target tissue following administration (e.g., at least 2 hours, at least 8 hours, at least 12 hours, or at least 16 hours after administration) of a spray dried biotherapeutic matrix composition (see, e.g., FIG. 4C). In some embodiments, at least 10 nmol / mL of lactic acid is produced in the target tissue following administration of a spray dried biotherapeutic matrix composition. In some embodiments, at least 9 nmol / mL, at least 9.5 nmol / mL, at least 10 nmol / mL, at least 10.5 nmol / mL, at least 11 nmol / mL, at least 11.5 nmol / mL, at least 12 nmol / mL, at least 12.5 nmol / mL, at least 13 nmol / mL, at least 13.5 nmol / mL, at least 14 nmol / mL, at least 14.5 nmol / mL, at least 15 nmol / mL, or more of lactic acid is produced in the target tissue following administration of a spray dried biotherapeutic matrix composition. In some embodiments, the spray dried biotherapeutic matrix composition (e.g., a bacterial preparation in a spray dried biotherapeutic matrix composition) increases lactate concentration in a target tissue by at least 100%. In some embodiments, the spray dried biotherapeutic matrix composition increases lactate concentration in a target tissue by at least about 25%. In some embodiments, the spray dried biotherapeutic matrix composition increases lactate concentration in a target tissue by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, at least about 100%, at least about 150%, at least about 200%, or more.

[0433] In some embodiments, the target tissue is a target bronchopulmonary tissue. In some embodiments, the target bronchopulmonary tissue is a lung, trachea, bronchi, bronchioles, and / or alveoli. In some embodiments, the target tissue is a tissue site distal to the lung that is delivered via the cardiovascular or lymphatic system.

[0434] In some embodiments, the subject has been diagnosed with or is at risk of developing a chronic or infectious bronchopulmonary disease. In some embodiments, the subject has been diagnosed with or is at risk of developing a chronic bronchopulmonary disease. In some embodiments, the chronic bronchopulmonary disease is selected from the group consisting of chronic obstructive pulmonary disease (COPD), lung cancer, asthma, bronchiectasis, emphysema, cystic fibrosis (CF), bronchopulmonary dysplasia (BPD), acute respiratory distress syndrome (ARDS), and idiopathic pulmonary fibrosis (IPF), interstitial lung disease (ILD), pleural effusion (PE), pulmonary hypertension (PAH), and silicosis. In some embodiments, the lung cancer is small cell lung cancer (SCLC) or non-small cell lung cancer (NSCLC).

[0435] In some embodiments, the subject has been diagnosed with or is at risk for developing infectious bronchopulmonary disease. In some embodiments, the infectious bronchopulmonary disease is caused by or associated with an infectious agent selected from the following: adenovirus, coronavirus (e.g., common cold virus, severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1), SARS-CoV-2, Middle East Respiratory Syndrome (MERS) CoV), influenza virus (e.g., influenza), parainfluenza virus, parvovirus B19 (e.g., parvovirus B19, fifth disease), respiratory syncytial virus (RSV), rhinovirus (e.g., common cold), enterovirus (e.g., EV-D68), measles virus, rubella virus, varicella virus (e.g., chickenpox), Corynebacterium diphtheriae (e.g., diphtheria), Haemophilus influenzae (e.g., type B), Legionella pneumophila (e.g., Legionnaires' disease), Bordetella pertussis (e.g., whooping cough), Mycobacterium tuberculosis (e.g., tuberculosis), Streptococcus species (e.g., Streptococcus pneumoniae, Streptococcus pyogenes, e.g., pneumonia), Pseudomonas species (e.g., Pseudomonas aeruginosa, pulmonary infections, chronic recurrent respiratory infections), Escherichia coli (e.g., community-acquired pneumonia), Aspergillus species (e.g., Aspergillus fumigatus, Aspergillus flavus, aspergillosis), Cryptococcus species (e.g., Cryptococcus neoformans, Cryptococcus gattii, cryptococcal pneumonia), and Pneumocystis species (e.g., Pneumocystis jirovecii, pneumocystis).

[0436] In some embodiments, an effective dose of the spray dried biotherapeutic matrix composition is at least 200 x 106 CFU of bacteria per unit dose. In some embodiments, an effective dose of the spray dried biotherapeutic matrix composition is at least 101 CFU, at least 102 CFU, at least 103 CFU, at least 104 CFU, at least 105 CFU, at least 106 CFU, at least 107 CFU, at least 108 CFU, at least 109 CFU, at least 1010 CFU, at least 1011 CFU, or at least 1012 CFU of bacteria per unit dose. In some embodiments, an effective dose of the spray dried biotherapeutic matrix composition is at least 200 x 106 CFU of viable bacteria per unit dose. In some embodiments, an effective dose of the spray dried biotherapeutic matrix composition has at least 101 CFU, at least 102 CFU, at least 103 CFU, at least 104 CFU, at least 105 CFU, at least 106 CFU, at least 107 CFU, at least 108 CFU, at least 109 CFU, at least 1010 CFU, at least 1011 CFU, or at least 1012 CFU of viable bacteria per unit dose.

[0437] In some embodiments, the spray-dried biotherapeutic matrix composition formulated for administration by inhalation is co-administered with at least one additional therapeutic agent for chronic or infectious bronchopulmonary disorders. In some embodiments, the at least one additional therapeutic agent is an anti-inflammatory agent, an antibacterial agent, an antiviral agent, an antifungal agent, a vasodilator, or a bronchodilator, as further described herein. In some embodiments, the spray-dried biotherapeutic matrix composition includes a bacterial preparation and at least one additional therapeutic agent in the same composition or unit dosage. As a non-limiting example, the bacterial preparation and the at least one additional therapeutic agent can be spray-dried together and formulated, for example, in a single capsule. In some embodiments, the bacterial preparation and the at least one additional therapeutic agent are each spray-dried separately and formulated, for example, for administration in the same or different capsules. In some embodiments, the bacterial preparation and the at least one additional therapeutic agent are co-administered using a combination delivery device, such as an inhalation device, that administers multiple different formulations at once.

[0438] In some embodiments, the spray dried biotherapeutic matrix composition is administered in conjunction with standard of care for chronic or infectious bronchopulmonary disease, as known to those of skill in the art. As used herein, the term "standard of care" refers to the level at which an average, conscientious provider in a given community would practice, for example, in treating a given indication.

[0439] As a non-limiting example, COPD standard of care varies based on the GOLD staging of severity, but may include a variety of therapies combining anti-inflammatory effects (e.g., inhaled steroids) with symptomatic relief (e.g., long-acting beta agonists (LABAs), long-acting muscarinic antagonists (LAMAs)). Standard of care for pulmonary fibrosis is typically at least one of two anti-fibrotic agents: nintedanib and pirfenidone. Standard of care for bronchopulmonary dysplasia includes a variety of treatments based on the patient's risk profile, including supplemental oxygen, surfactants, and bronchodilators. See, e.g., Safka et al., Chronic Obstr Pulm Dis. 2017;4(1):45-55 (see, e.g., Table 1 in Safka), the 2022 Gold Reports available on the World Wide Web at goldcopd.org / 2022-gold-reports (see, e.g., pages 49-57, Table 3.3 on page 49, Table 3.4 on page 51, and Table 3.5 on page 53 of the Gold Reports), and Maher et al. “Respiratory Research volume 20, Article number:205 (2019). Non-limiting examples of additional therapeutic treatments or interventions for certain chronic or infectious bronchopulmonary diseases are further described herein.

[0440] In some embodiments, the spray-dried biotherapeutic matrix composition is administered to treat, alleviate, or prevent central nervous system (CNS) diseases, non-limiting examples of which include Parkinson's disease, multiple sclerosis (MS), and migraine headaches. Of note, among the indicators of the lung-brain axis is the discovery that dysregulation of the lung microbiota significantly affected the susceptibility of rats to develop autoimmune diseases of the CNS. See, for example, Hosang et al., Nature 603:138-144 (2022), which reports that local treatment with neomycin, which shifts the lung microbiota to LPS-rich phyla, induces a type I interferon-stimulated state in brain-resident microglial cells, impairs autoimmune stimulation by type II interferon, and reduces proinflammatory responses and immune cell recruitment. In that study, inhibition of LPS-producing phyla in the lungs exacerbated disease, while the addition of LPS-rich phyla or LPS itself reproduced the effects of neomycin. Thus, inhalation delivery of the microbial products or microorganisms described herein may have an effect on the treatment, mitigation, or prevention of CNS diseases or disorders. See, e.g., Noymer et al. (2011) Ther Deliv 2(9):1125-1140, Abdou et al. (2019) Drug Deliv 26(1):689-699, Hosang et al. Nature 603:138-144(2022), the contents of each of which are incorporated herein by reference in their entirety.

[0441] Unit dosage form In one aspect, described herein are unit dosage forms comprising the spray dried biotherapeutic matrix compositions described herein. In some embodiments, the single unit dosage is one capsule comprising the spray dried biotherapeutic matrix compositions described herein. In some embodiments, the single unit dosage is multiple (e.g., 1, 2, 3, 4, 5, or more) capsules, each comprising the spray dried biotherapeutic matrix compositions described herein. In some embodiments, the unit dosage is administered (e.g., inhaled) during a single inhalation (e.g., less than 10, 20, or 30 seconds). In some embodiments, the unit dosage is administered (e.g., inhaled) over a period of time (e.g., more than 30, 60, or 120 seconds) and / or administered (e.g., inhaled) using multiple dose inhalers.

[0442] In some embodiments, a unit dosage containing the spray-dried biotherapeutic matrix composition described herein results in about 100×106 CFU of bacteria delivered to the patient's lungs from a 30 mg capsule. In some embodiments, a unit dosage containing the spray-dried biotherapeutic matrix composition described herein results in about 200×106 CFU of bacteria delivered to the patient's lungs from a 30 mg capsule. This is an example of a reasonable dose consisting of 4 doses of 1×108 CFU per day (400×106 CFU total) when considering the dose of bacterial spores to the gut for the treatment of C. difficile. See, for example, McGovern et al. Clin Infect Dis. 2021;72(12):2132-2140, the contents of which are incorporated herein by reference in their entirety. Once the final dry powder is produced, it can be loaded into a capsule for delivery by a dry powder inhaler. The dry powder blend itself can undergo several process steps that will result in a uniform blend and a robust solid dosage form.

[0443] In one aspect, described herein is a unit dosage form comprising a spray-dried biotherapeutic matrix composition comprising at least 1 mg to at most 50 mg of a bacterial preparation. In some embodiments, the unit dosage form comprises at least 7.5 mg of the bacterial preparation per unit dose. In some embodiments, the unit dosage form comprises at least 15 mg of the bacterial preparation per unit dose. In some embodiments, the unit dosage form comprises at least 1 mg, at least 2 mg, at least 3 mg, at least 4 mg, at least 5 mg, at least 10 mg, at least 15 mg, at least 20 mg, at least 25 mg, at least 30 mg, at least 35 mg, at least 40 mg, at least 45 mg, or at least 50 mg of the bacterial preparation per unit dose. In some embodiments, the unit dosage form comprises at most 1 mg, at most 2 mg, at most 3 mg, at most 4 mg, at most 5 mg, at most 10 mg, at most 15 mg, at most 20 mg, at most 25 mg, at most 30 mg, at most 35 mg, at most 40 mg, at most 45 mg, or at most 50 mg of the bacterial preparation per unit dose. In some embodiments, a unit dose may contain a small amount of bacterial preparation or spray dried biotherapeutic matrix composition when a high concentration of bacteria is present, hi some embodiments, a unit dose may contain a large amount of bacterial preparation or spray dried biotherapeutic matrix composition when a low concentration of bacteria is present in the bacterial preparation.

[0444] In one embodiment, described herein is a unit dosage form comprising at least 1 mg to at most 50 mg of a spray dried biotherapeutic matrix composition described herein. In one embodiment, described herein is a unit dosage form comprising at least 1 mg, at least 2 mg, at least 3 mg, at least 4 mg, at least 5 mg, at least 10 mg, at least 15 mg, at least 20 mg, at least 25 mg, at least 30 mg, at least 35 mg, at least 40 mg, at least 45 mg, or at least 50 mg of a spray dried biotherapeutic matrix composition described herein. In one embodiment, described herein is a unit dosage form comprising up to 1 mg, up to 2 mg, up to 3 mg, up to 4 mg, up to 5 mg, up to 10 mg, up to 15 mg, up to 20 mg, up to 25 mg, up to 30 mg, up to 35 mg, up to 40 mg, up to 45 mg, or up to 50 mg of a spray dried biotherapeutic matrix composition described herein.

[0445] In one aspect, described herein is a unit dosage form comprising at least 1 mg to at most 50 mg of a spray dried biotherapeutic matrix composition prepared by the methods described herein. In one aspect, described herein is a unit dosage form comprising at least 1 mg, at least 2 mg, at least 3 mg, at least 4 mg, at least 5 mg, at least 10 mg, at least 15 mg, at least 20 mg, at least 25 mg, at least 30 mg, at least 35 mg, at least 40 mg, at least 45 mg, or at least 50 mg of a spray dried biotherapeutic matrix composition prepared by the methods described herein. In one aspect, described herein is a unit dosage form comprising up to 1 mg, up to 2 mg, up to 3 mg, up to 4 mg, up to 5 mg, up to 10 mg, up to 15 mg, up to 20 mg, up to 25 mg, up to 30 mg, up to 35 mg, up to 40 mg, up to 45 mg, or up to 50 mg of a spray dried biotherapeutic matrix composition prepared by the methods described herein.

[0446] In one embodiment, described herein is a unit dosage form comprising at least 1 mg to up to 50 mg of a spray dried biotherapeutic matrix composition comprising at least 104 CFU of bacteria per unit dose. In one embodiment, described herein is a unit dosage form comprising at least 1 mg to up to 50 mg of a spray dried biotherapeutic matrix composition comprising at least 100×104 CFU of bacteria per unit dose. In one embodiment, described herein is a unit dosage form comprising at least 1 mg to up to 50 mg of a spray dried biotherapeutic matrix composition comprising at least 100×106 CFU of bacteria per unit dose. In one embodiment, described herein is a unit dosage form comprising at least 1 mg to up to 50 mg of a spray dried biotherapeutic matrix composition comprising at least 200×106 CFU of bacteria per unit dose. In one embodiment, described herein is a unit dosage form that comprises at least 1 mg, at least 2 mg, at least 3 mg, at least 4 mg, at least 5 mg, at least 10 mg, at least 15 mg, at least 20 mg, at least 25 mg, at least 30 mg, at least 35 mg, at least 40 mg, at least 45 mg, or at least 50 mg of a spray dried biotherapeutic matrix composition containing at least 200×10 CFU of bacteria per unit dose. In one embodiment, described herein is a unit dosage form that comprises up to 1 mg, up to 2 mg, up to 3 mg, up to 4 mg, up to 5 mg, up to 10 mg, up to 15 mg, up to 20 mg, up to 25 mg, up to 30 mg, up to 35 mg, up to 40 mg, up to 45 mg, or up to 50 mg of a spray dried biotherapeutic matrix composition containing up to 200×10 CFU of bacteria per unit dose.

[0447] In one embodiment, described herein is a unit dosage form that comprises at least 1 mg, at least 2 mg, at least 3 mg, at least 4 mg, at least 5 mg, at least 10 mg, at least 15 mg, at least 20 mg, at least 25 mg, at least 30 mg, at least 35 mg, at least 40 mg, at least 45 mg, or at least 50 mg of a spray dried biotherapeutic matrix composition containing at least 100×10 CFU of bacteria per unit dose. In one embodiment, described herein is a unit dosage form that comprises up to 1 mg, up to 2 mg, up to 3 mg, up to 4 mg, up to 5 mg, up to 10 mg, up to 15 mg, up to 20 mg, up to 25 mg, up to 30 mg, up to 35 mg, up to 40 mg, up to 45 mg, or up to 50 mg of a spray dried biotherapeutic matrix composition containing up to 100×10 CFU of bacteria per unit dose.

[0448] In some embodiments, the dosage is at least 30 mg of the spray dried biotherapeutic matrix composition, hi some embodiments, the dosage is at least 1 mg, at least 2 mg, at least 3 mg, at least 4 mg, at least 5 mg, at least 10 mg, at least 15 mg, at least 20 mg, at least 25 mg, at least 30 mg, at least 35 mg, at least 40 mg, at least 45 mg, at least 50 mg, at least 55 mg, at least 60 mg, at least 65 mg, at least 70 mg, at least 75 mg, at least 80 mg, at least 85 mg, at least 90 mg, at least 95 mg, or at least 100 mg of the spray dried biotherapeutic matrix composition. In some embodiments, the dosage is up to 1 mg, up to 2 mg, up to 3 mg, up to 4 mg, up to 5 mg, up to 10 mg, up to 15 mg, up to 20 mg, up to 25 mg, up to 30 mg, up to 35 mg, up to 40 mg, up to 45 mg, up to 50 mg, up to 55 mg, up to 60 mg, up to 65 mg, up to 70 mg, up to 75 mg, up to 80 mg, up to 85 mg, up to 90 mg, up to 95 mg, or up to 100 mg of spray dried biotherapeutic matrix composition.

[0449] In some embodiments, the dosage comprises at least 108 CFU of bacteria per unit dose. In some embodiments, the dosage comprises at least 200×106 CFU of bacteria per unit dose. In some embodiments, the dosage comprises at least 200×104 CFU of bacteria per unit dose. In some embodiments, the dosage comprises at least 101 CFU, at least 102 CFU, at least 103 CFU, at least 104 CFU, at least 105 CFU, at least 106 CFU, at least 107 CFU, at least 108 CFU, at least 109 CFU, at least 1010 CFU, at least 1011 CFU, or at least 1012 CFU of bacteria per unit dose. In some embodiments, the dosage comprises at least 104 CFU of viable bacteria per unit dose. In some embodiments, the dosage comprises at least 200×106 CFU of viable bacteria per unit dose. In some embodiments, the dosage comprises at least 101 CFU, at least 102 CFU, at least 103 CFU, at least 104 CFU, at least 105 CFU, at least 106 CFU, at least 107 CFU, at least 108 CFU, at least 109 CFU, at least 1010 CFU, at least 1011 CFU, or at least 1012 CFU of viable bacteria per unit dose. In some embodiments, the dosage comprises up to 101 CFU, up to 102 CFU, up to 103 CFU, up to 104 CFU, up to 105 CFU, up to 106 CFU, up to 107 CFU, up to 108 CFU, up to 109 CFU, up to 1010 CFU, up to 1011 CFU, or up to 1012 CFU of viable bacteria per unit dose. In some embodiments, the dosage comprises up to 1011 CFU of viable bacteria per unit dose. In some embodiments, the dosage comprises up to 200×106 CFU of viable bacteria per unit dose. In some embodiments, the dosage comprises up to 101 CFU, up to 102 CFU, up to 103 CFU, up to 104 CFU, up to 105 CFU, up to 106 CFU, up to 107 CFU, up to 108 CFU, up to 109 CFU, up to 1010 CFU, up to 1011 CFU, or up to 1012 CFU of viable bacteria per unit dose.

[0450] In some embodiments, the unit dosage comprises at least 0.5% by dry weight of the bacterial preparation. In some embodiments, the unit dosage comprises at least 25% by dry weight of the bacterial preparation. In some embodiments, the unit dosage comprises at least 0.5%, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or more by dry weight of the bacterial preparation. In some embodiments, the unit dosage comprises up to 0.5%, up to 1%, up to 2%, up to 3%, up to 4%, up to 5%, up to 6%, up to 7%, up to 8%, up to 9%, up to 10%, up to 15%, up to 20%, up to 25%, up to 30%, up to 35%, up to 40%, up to 45%, up to 50%, up to 55%, up to 60%, up to 65%, up to 70%, up to 75%, up to 80%, up to 85%, up to 90%, or up to 95% by dry weight of the bacterial preparation.

[0451] In some embodiments, the unit dosage comprises at least 0.5% excipient(s) by dry weight. In some embodiments, the unit dosage comprises at least 70% excipient(s) by dry weight. In some embodiments, the unit dosage comprises at least 73% excipient(s) by dry weight. In some embodiments, the unit dosage comprises at least 73.4% excipient(s) by dry weight. In some embodiments, the unit dosage comprises at least 0.5%, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or more excipient(s) by dry weight. In some embodiments, the unit dosage comprises up to 0.5%, up to 1%, up to 2%, up to 3%, up to 4%, up to 5%, up to 6%, up to 7%, up to 8%, up to 9%, up to 10%, up to 15%, up to 20%, up to 25%, up to 30%, up to 35%, up to 40%, up to 45%, up to 50%, up to 55%, up to 60%, up to 65%, up to 70%, up to 75%, up to 80%, up to 85%, up to 90%, or up to 95% excipient(s) by dry weight.

[0452] In some embodiments, the unit dose comprises at least 0.5% by dry weight of the first excipient, second excipient, and / or third excipient. In some embodiments, the unit dose comprises at least 5% by dry weight of the first excipient, second excipient, and / or third excipient. In some embodiments, the unit dose comprises at least 30% by dry weight of the first excipient, second excipient, and / or third excipient. In some embodiments, the unit dose comprises at least 34% by dry weight of the first excipient, second excipient, and / or third excipient. In some embodiments, the unit dose comprises at least 34.2% by dry weight of the first excipient, second excipient, and / or third excipient. In some embodiments, the unit dosage comprises at least 0.5%, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or more of the first excipient, second excipient, and / or third excipient by dry weight. In some embodiments, the unit dosage comprises up to 0.5%, up to 1%, up to 2%, up to 3%, up to 4%, up to 5%, up to 6%, up to 7%, up to 8%, up to 9%, up to 10%, up to 15%, up to 20%, up to 25%, up to 30%, up to 35%, up to 40%, up to 45%, up to 50%, up to 55%, up to 60%, up to 65%, up to 70%, up to 75%, up to 80%, up to 85%, up to 90%, or up to 95% by dry weight of the first excipient, the second excipient, and / or the third excipient.

[0453] In some embodiments, the unit dosage comprises at least 0.25% stabilizer(s) by dry weight. In some embodiments, the unit dosage comprises at least 0.5% stabilizer(s) by dry weight. In some embodiments, the unit dosage comprises at least 1.6% stabilizer(s) by dry weight. In some embodiments, the unit dosage comprises at least 0.5%, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or more stabilizer(s) by dry weight. In some embodiments, the unit dosage comprises up to 0.5%, up to 1%, up to 2%, up to 3%, up to 4%, up to 5%, up to 6%, up to 7%, up to 8%, up to 9%, up to 10%, up to 15%, up to 20%, up to 25%, up to 30%, up to 35%, up to 40%, up to 45%, up to 50%, up to 55%, up to 60%, up to 65%, up to 70%, up to 75%, up to 80%, up to 85%, up to 90%, or up to 95% stabilizer(s) by dry weight.

[0454] In some embodiments, the pharmaceutical composition is a spray-dried pharmaceutical composition. In some embodiments, the pharmaceutical composition comprises a spray-dried pharmaceutical composition. In some embodiments, the pharmaceutical composition essentially comprises a spray-dried composition.

[0455] definition For convenience, the meanings of some terms and phrases used in the specification, examples, and appended claims are provided below. Unless otherwise stated or implied from the context, the following terms and phrases include the meanings provided below. The definitions are provided to help explain certain embodiments and are not intended to limit the claimed invention, as the scope of the invention is limited only by the claims. Unless otherwise expressly stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In the event of an apparent discrepancy between the use of a term in the art and its definition provided herein, the definition provided in the specification shall prevail.

[0456] For convenience, certain terms employed in the specification, examples, and appended claims are collected here.

[0457] The terms "reduce", "reduced", "reduction", or "inhibit" are all used herein to mean a statistically significant amount of reduction. In some embodiments, "reduce", "reduce", or "reduce" or "inhibit" typically means a reduction of at least 10% compared to a reference level (e.g., the absence of a given treatment or agent), and can include, for example, a reduction of at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or more. As used herein, "reduce" or "inhibit" does not encompass complete inhibition or reduction compared to a reference level. "Complete inhibition" is 100% inhibition compared to a reference level. The decrease is preferably to a level that is accepted as being within the normal range, for example, for individuals without a given disorder.

[0458] The terms "increased," "increase," "enhance," or "activate" are all used herein to mean a statically significant amount of increase. In some embodiments, the terms "increased...

Claims

1. A spray-dried biotherapeutic matrix composition comprising a bacterial preparation, said matrix composition formulated for administration by inhalation.

2. The composition of claim 1 , wherein the bacterial preparation comprises viable or non-viable bacteria.

3. 3. The composition of claim 2, wherein the viable bacteria are capable of actively metabolizing and / or multiplying in the lungs of a subject.

4. The composition of claim 2 , wherein the non-viable bacteria are heat-killed.

5. The composition of claim 2 , wherein the bacterium is gram-negative.

6. The composition of claim 2 , wherein the bacterium is gram-positive.

7. The composition of claim 2 , wherein the bacterium is spore-forming.

8. The composition of claim 2 , wherein the bacteria is in spore form.

9. The composition of claim 2 , wherein the bacteria is aerobic.

10. The composition of claim 2 , wherein the bacteria is anaerobic.

11. The composition of claim 2 , wherein the bacterium produces at least one immunomodulatory factor.

12. 3. The composition of claim 2, wherein the bacterium belongs to a genus selected from the group consisting of Carnobacterium, Lactiplantibacillus, Lactobacillus, Lacticaseibacillus, Ligilactobacillus, Oenococcus, Leuconostoc, Pedicoccus, Enterococcus, Lactococcus, Staphylococcus, Streptococcus, Streptomyces, Bifidobacterium, Propionibacterium, and Moraxella.

13. 3. The composition of claim 2, wherein the bacterium is Lacticaseibacillus rhamnosus, Lactobacillus acidophilus, or Lactiplantibacillus plantarum.

14. The composition of claim 2 , wherein the bacteria is non-pathogenic.

15. The bacteria are at least 10 １ Colony forming units / gram (CFU / g) at least 10 ２ CFU / g, at least 10 ３ CFU / g, at least 10 ４ CFU / g, at least 10 ５ CFU / g, at least 10 ６ CFU / g, at least 10 ７ CFU / g, at least 10 ８ CFU / g, at least 10 ９ CFU / g, at least 10 １０ CFU / g, at least 10 １１ CFU / g, or at least 10 １２ 3. The composition of claim 2, wherein the composition is present at a concentration of CFU / g.

16. The bacteria are at least 10 ６ 3. The composition of claim 2, wherein the composition is present in a concentration of colony forming units per gram (CFU / g).

17. The bacteria are at least 10 ８ 3. The composition of claim 2, wherein the composition is present in a concentration of colony forming units per gram (CFU / g).

18. The composition of claim 2 , wherein the bacteria is resistant to at least one antibiotic.

19. The composition of claim 1 , wherein the composition comprises at least 0.5% by dry weight of the bacterial preparation.

20. The composition of claim 1 , wherein the bacterial preparation comprises a bacterial extract or a bacterial metabolite preparation.

21. 21. The composition of claim 20, wherein the bacterial extract or bacterial metabolite preparation is selected from the group consisting of bacterial exosomes, bacterial cell walls, peptidoglycans, teichoic acid, lipoteichoic acid, bacterial S-layers, exopolysaccharides, polysaccharides, lactic acid polymers, lactic acid derivatives, lactic acid intermediates, hydrogen peroxide, bacteriocins, salivaricin, reuterin, and bacterial growth supernatants.

22. The composition of claim 1 further comprising at least one excipient.

23. The composition of claim 1 further comprising at least two excipients.

24. 23. The composition of claim 22, wherein the excipient is selected from the group consisting of De Man, Rogosa and Sharpe (MRS) growth medium, gelatin, whey isolate, sweet whey, reconstituted skim milk powder, maltodextrin, glucooligosaccharides, lacto-oligosaccharides, fructooligosaccharides, inulin, sodium caseinate, goat milk, cow milk, proline, carnitine, acetylcarnitine, propionylcarnitine, glutamate, glycine betaine, glycogen, trehalose, mannose, xylose, mannitol, sorbitol, maltose, dextrose, starch, lactose, sucrose, glucose, leucine, trileucine, sodium salts, potassium salts, lithium salts, and calcium salts.

25. 23. The composition of claim 22, wherein the excipient is leucine and / or trehalose.

26. 10. The composition of claim 1, wherein the composition comprises at least 0.5% excipient by dry weight.

27. The composition of claim 1 further comprising at least one stabilizer.

28. 28. The composition of claim 27, wherein the stabilizer comprises a surfactant.

29. 28. The composition of claim 27, wherein the stabilizer is a polysorbate, a poloxamer, or a polyvinyl alcohol.

30. 28. The composition of claim 27, wherein the stabilizer is polysorbate 20, polysorbate 40, polysorbate 60, or polysorbate 80.

31. 28. The composition of claim 27, wherein the stabilizer is polysorbate 80.

32. 28. The composition of claim 27, wherein the stabilizer is poloxamer 184, poloxamer 185, poloxamer 188, poloxamer 234, poloxamer 235, poloxamer 238, poloxamer 333, poloxamer 334, poloxamer 335, poloxamer 338, poloxamer 403, or poloxamer 407.

33. 10. The composition of claim 1, wherein the composition comprises at least 0.25% by dry weight of a stabilizer.

34. The composition of claim 1 , wherein the composition comprises at least one excipient and at least one stabilizer.

35. The composition of claim 1 , wherein the composition further comprises at least one additional therapeutic agent.

36. 36. The composition of claim 35, wherein the at least one additional therapeutic agent is selected from the group consisting of anti-inflammatory agents, antibacterial agents, antiviral agents, antifungal agents, vasodilators, and bronchodilators.

37. 37. The composition of claim 36, wherein the at least one additional therapeutic agent is incorporated into the composition using microencapsulation, co-formulation, or covalent attachment to the composition by a degradable linker.

38. The composition of claim 1 , wherein the matrix composition comprises a plurality of dry particles.

39. 39. The composition of claim 38, wherein the dry particles have a Dv50 of at least 0.5 μm.

40. 39. The composition of claim 38, wherein the dry particles have a mass median aerodynamic diameter (MMAD) of at least 1.5 μm and at most 7.5 μm.

41. 39. The composition of claim 38, wherein the dry particles have a dispersibility of less than 2.

0.

42. 39. The composition of claim 38, wherein the dry particles have a dispersibility of at least 0.5 to 1.

0.

43. 39. The composition of claim 38, wherein the dry particles have a delivered dose to a target tissue of at least 25.0% and up to 125% by weight of the bacterial preparation.

44. 39. The composition of claim 38, wherein the dry particles have a delivered dose to a target tissue that is at least 60% by weight of the bacterial preparation.

45. 45. The composition of claim 44, wherein the target tissue is a target bronchopulmonary tissue.

46. 46. ​​The composition of claim 45, wherein the target bronchopulmonary tissue is the lung, trachea, bronchi, bronchioles, and / or alveoli.

47. 46. ​​The composition of claim 45, wherein the target tissue is a tissue site distal to the lung delivered via the cardiovascular or lymphatic system.

48. The dry particles have a density of at least 0.1 g / cm ３ 39. The composition of claim 38 having a bulk density of about 0.8 g / cm.

49. The dry particles have a density of at least 0.5 g / cm ３ 39. The composition of claim 38 having a bulk density of

50. The dry particles have a density of at least 0.1 g / cm ３ 39. The composition of claim 38 having a tap density of up to 1.0 g / cm.

51. The dry particles have a density of at least 0.6 g / cm ３ 39. The composition of claim 38 having a tap density of

52. 39. The composition of claim 38, wherein the dry particles have a moisture content of at least 1.0% to 7.0% water by weight according to Karl Fischer.

53. 39. The composition of claim 38, wherein the dry particles have a moisture content of at least 2.5% water by weight according to Karl Fischer.

54. 10. The composition of claim 1, wherein the composition is formulated for delivery to the trachea, bronchi, bronchioles, and / or alveoli.

55. The composition of claim 1 , wherein the composition is formulated for pulmonary delivery.

56. The composition of claim 1 , wherein the composition is formulated as a capsule.

57. 57. The composition of claim 56, wherein the capsule contains at least 10 mg of the spray-dried biotherapeutic matrix composition.

58. The composition of claim 1 , wherein the composition is formulated for delivery by an inhaler.

59. 10. The composition of claim 1, wherein the composition is formulated for delivery by a dry powder inhaler (DPI), a metered dose inhaler (MDI), or a soft mist inhaler (SMI).

60. The composition of claim 1 in combination with an inhaler.

61. 1. An inhalation device for bronchopulmonary delivery, comprising: a) an inhaler; b) a container containing a spray-dried biotherapeutic matrix composition comprising a bacterial preparation.

62. 62. The device of claim 61, wherein the inhaler is a dry powder inhaler (DPI), a metered dose inhaler (MDI), or a soft mist inhaler (SMI).

63. The inhaler comprises: a) a mouthpiece including an opening; and b) a means for aerosolizing or dispersing the spray-dried biotherapeutic matrix composition in the container.

64. 1. A method for preparing a spray-dried biotherapeutic matrix composition comprising a bacterial preparation, the method comprising: a) preparing a liquid feedstock containing the bacterial preparation; b) introducing droplets of said liquid feedstock into a drying chamber through a spray nozzle; c) exposing the liquid feedstock droplets to heated and pressurized gas in the drying chamber to produce dried particles; d) isolating dry particles of a predetermined range of diameters in a cyclone chamber, wherein the isolated dry particles comprise the spray-dried biotherapeutic matrix composition.

65. 1. A method for preparing a spray-dried biotherapeutic matrix composition comprising a bacterial preparation, the method comprising: a) obtaining a liquid feedstock containing the bacterial preparation; b) introducing droplets of said liquid feedstock into a drying chamber through a spray nozzle; c) exposing the liquid feedstock droplets to heated and pressurized gas in the drying chamber to produce dried particles; d) isolating dry particles of a predetermined range of diameters in a cyclone chamber, wherein the isolated dry particles comprise the spray-dried biotherapeutic matrix composition.

66. 65. The method of claim 64, wherein the step of preparing the liquid ingredient comprises dissolving a solid ingredient in an aqueous solution.

67. The solid raw material is a) at least 3.5% by weight of a bacterial preparation; b) at least 5% by weight of excipients, and / or 65. The method of claim 64, comprising: c) at least 0.25 wt. % of a stabilizer.

68. The solid raw material is a) at least 3.5% by weight of a bacterial preparation; b) at least 5% by weight of a first excipient; c) at least 5% by weight of a second excipient, and / or d) at least 0.25 wt. % of a stabilizer.

69. 65. The method of claim 64, wherein the solid feedstock comprises at least 1% and up to 5% by weight of the bacterial preparation.

70. 65. The method of claim 64, wherein the solid feedstock comprises at least 45% and up to 95% by weight of the excipient.

71. 65. The method of claim 64, wherein the solid ingredient comprises at least 5% to 60% by weight of the first excipient and at least 5% to 60% by weight of the second excipient.

72. 65. The method of claim 64, wherein the solid feedstock comprises at least 0.25% and up to 10% by weight of a stabilizer.

73. 65. The method of claim 64, wherein the liquid feedstock comprises at least 1 g / L of solid feedstock dissolved in an aqueous solution.

74. 65. The method of claim 64, wherein the liquid feedstock comprises at least 25 g / L of solid feedstock dissolved in an aqueous solution.

75. 65. The method of claim 64, wherein the liquid ingredient comprises at least 0.1% and up to 10% of a solid ingredient dissolved in an aqueous solution.

76. 65. The method of claim 64, wherein the liquid ingredient comprises at least 1% solid ingredient dissolved in an aqueous solution.

77. 1 L of the liquid raw material, a) at least 1.050 g of a bacterial preparation; b) at least 1.5 g of excipients; c) at least 0.075 g of a stabilizer, and / or d) at least 970 g of an aqueous solution.

78. 1 L of the liquid raw material, a) at least 1.050 g of a bacterial preparation; b) at least 0.75 g of a first excipient; c) at least 0.75 g of a second excipient; d) at least 0.075 g of a stabilizer, and / or 65. The method of claim 64, comprising: e) at least 970 g of an aqueous solution.

79. 1 L of the liquid raw material, a) at least 1.050 g of a bacterial preparation; b) at least 0.5 g of a first excipient; c) at least 0.5 g of a second excipient; d) at least 0.5 g of a third excipient; e) at least 0.075 g of a stabilizer, and / or 65. The method of claim 64, comprising: f) at least 970 g of an aqueous solution.

80. 65. The method of claim 64, wherein 1 L of the liquid feedstock comprises at least 750 g and up to 999 g of aqueous solution.

81. The liquid raw material is a) at least 0.105% of a bacterial preparation; b) at least 0.15% excipients; c) at least 0.0075% of a stabilizer, and / or d) at least 97% aqueous solution.

82. The liquid raw material is a) at least 0.105% by weight of a bacterial preparation; b) at least 0.075% by weight of a first excipient; c) at least 0.075% by weight of a second excipient; d) at least 0.0075% by weight of a stabilizer, and / or e) at least 97% by weight of an aqueous solution.

83. The liquid raw material is a) at least 0.105% by weight of a bacterial preparation; b) at least 0.05% by weight of a first excipient; c) at least 0.05% by weight of a second excipient; d) at least 0.05% by weight of a third excipient; e) at least 0.0075% by weight of a stabilizer, and / or 65. The method of claim 64, comprising: f) at least 97% by weight of an aqueous solution.

84. 65. The method of claim 64, wherein the liquid feedstock comprises at least 0.01% and up to 10% by weight of the bacterial preparation.

85. 65. The method of claim 64, wherein the liquid ingredient comprises at least 1.0% and up to 20% by weight of an excipient.

86. 65. The method of claim 64, wherein the liquid ingredient comprises at least 0.1% and up to 19.8% by weight of a first excipient and at least 0.1% and up to 19.8% by weight of a second excipient.

87. 65. The method of claim 64, wherein the liquid ingredient comprises at least 0.1% and up to 19.8% by weight of a first excipient, at least 0.1% and up to 19.8% by weight of a second excipient, and at least 0.1% and up to 19.8% by weight of a third excipient.

88. 65. The method of claim 64, wherein the liquid feedstock comprises at least 0.01% and up to 1.0% by weight of a stabilizer.

89. 65. The method of claim 64, wherein the liquid feedstock comprises at least 75% and up to 99.9% by weight of an aqueous solution.

90. 65. The method of claim 64, wherein the bacterial preparation comprises viable or non-viable bacteria.

91. 91. The method of claim 90, wherein the bacterium belongs to a genus selected from the group consisting of Carnobacterium, Lactiplantibacillus, Lactobacillus, Lacticaseibacillus, Ligilactobacillus, Oenococcus, Leuconostoc, Pedicoccus, Enterococcus, Lactococcus, Staphylococcus, Streptococcus, Streptomyces, Bifidobacterium, Propionibacterium, and Moraxella.

92. 91. The method of claim 90, wherein the bacterium is Lacticaseibacillus rhamnosus, Lactobacillus acidophilus, or Lactiplantibacillus plantarum.

93. 91. The method of claim 90, wherein the bacteria is non-pathogenic.

94. The bacteria are at least 10 １ Colony forming units / gram (CFU / g) at least 10 ２ CFU / g, at least 10 ３ CFU / g, at least 10 ４ CFU / g, at least 10 ５ CFU / g, at least 10 ６ CFU / g, at least 10 ７ CFU / g, at least 10 ８ CFU / g, at least 10 ９ CFU / g, at least 10 １０ CFU / g, at least 10 １１ CFU / g, or at least 10 １２ 91. The method of claim 90, wherein the microbial organism is present at a concentration of CFU / g.

95. The bacteria are at least 10 ６ 91. The method of claim 90, wherein the microbial composition is present at a concentration of colony forming units per gram (CFU / g).

96. The bacteria are at least 10 ８ 91. The method of claim 90, wherein the microbial composition is present at a concentration of colony forming units per gram (CFU / g).

97. 91. The method of claim 90, wherein the excipient is selected from the group consisting of De Man, Rogosa and Sharpe (MRS) growth medium, gelatin, whey isolate, sweet whey, reconstituted skim milk powder, maltodextrin, glucooligosaccharides, lacto-oligosaccharides, fructooligosaccharides, inulin, sodium caseinate, goat milk, cow milk, proline, carnitine, acetylcarnitine, propionylcarnitine, glutamate, glycine betaine, glycogen, trehalose, mannose, xylose, mannitol, sorbitol, maltose, dextrose, starch, lactose, sucrose, glucose, leucine, trileucine, sodium salts, potassium salts, lithium salts, and calcium salts.

98. 65. The method of claim 64, wherein the excipient is leucine and / or trehalose.

99. 65. The method of claim 64, wherein the stabilizer is a polysorbate, a poloxamer, or a polyvinyl alcohol.

100. 65. The method of claim 64, wherein the stabilizer is polysorbate 80.

101. 65. The method of claim 64, wherein the aqueous solution is water.

102. 65. The method of claim 64, wherein the liquid feedstock further comprises at least one additional therapeutic agent.

103. 103. The method of claim 102, wherein the at least one additional therapeutic agent is selected from the group consisting of anti-inflammatory agents, antibacterial agents, antiviral agents, antifungal agents, vasodilators, and bronchodilators.

104. 65. The method of claim 64, wherein the spray nozzle into the drying chamber has a diameter of at least 1.2 um.

105. 65. The method of claim 64, wherein the droplets of liquid feedstock produced by the spray nozzle into the drying chamber have a diameter of at least 1.5 um.

106. 65. The method of claim 64, wherein the droplets of liquid feedstock have a flow rate through the drying chamber of at least 0.5 g / min.

107. 65. The method of claim 64, wherein the droplets of liquid feedstock have a flow rate through the drying chamber of at least 15 g / min.

108. 65. The method of claim 64, wherein the droplets of liquid feedstock have a flow rate through the drying chamber of up to 1000 g / min.

109. 65. The method of claim 64, wherein the heated pressurized gas is heated before being introduced into the drying chamber.

110. 65. The method of claim 64, wherein the heated pressurized gas is introduced into the drying chamber at a temperature of at least 100°C.

111. 65. The method of claim 64, wherein the heated pressurized gas is introduced into the drying chamber at a temperature of up to 195°C.

112. 65. The method of claim 64, wherein the heated pressurized gas is discharged from the drying chamber at a temperature of at least 40°C.

113. 65. The method of claim 64, wherein the heated pressurized gas is discharged from the drying chamber at a temperature of at least 48°C.

114. 65. The method of claim 64, wherein the heated pressurized gas is discharged from the drying chamber at a temperature of up to 85°C.

115. 65. The method of claim 64, wherein the heated pressurized gas is pressurized before being introduced into the drying chamber.

116. 65. The method of claim 64, wherein the heated pressurized gas in the drying chamber has an atomizing gas pressure of at least 10 pounds per square inch gauge (psig).

117. 65. The method of claim 64, wherein the heated pressurized gas in the drying chamber has an atomizing gas pressure of at least 20 pounds per square inch gauge (psig).

118. 65. The method of claim 64, wherein the heated pressurized gas in the drying chamber has an atomizing gas pressure of up to 150 pounds per square inch gauge (psig).

119. 65. The method of claim 64, wherein the heated pressurized gas has a flow rate through the drying chamber of at least 5 kg / hr.

120. 65. The method of claim 64, wherein the heated pressurized gas has a flow rate through the drying chamber of at least 18 kg / hr.

121. 65. The method of claim 64, wherein the heated pressurized gas has a flow rate through the drying chamber of up to 150 kg / hr.

122. 65. The method of claim 64, wherein the heated pressurized gas is discharged through the cyclone chamber.

123. 65. The method of claim 64, wherein exposing the liquid feedstock droplets to heated and pressurized gas in the drying chamber takes up to 8 hours.

124. 65. The method of claim 64, wherein the dried particles isolated in the cyclone chamber have a mass median aerodynamic diameter (MMAD) of at least 1.5 μm and up to 7.5 μm.

125. 65. The method of claim 64, wherein the dried particles isolated in the cyclone chamber have a mass median aerodynamic diameter (MMAD) of at least 4.0 μm.

126. 65. The method of claim 64, wherein the step of isolating dry particles of a predetermined range of diameters in the cyclone chamber is performed continuously.

127. 65. The method of claim 64, wherein the spray-dried biotherapeutic matrix composition comprises at least 5% bacterial viability after the step of isolating the dried particles.

128. A unit dosage form comprising at least 1 mg of a spray-dried biotherapeutic matrix composition comprising a bacterial preparation.

129. 10. A unit dosage form comprising at least 1 mg of the spray-dried biotherapeutic matrix composition of claim 1.

130. 65. A unit dosage form comprising at least 1 mg of the spray-dried biotherapeutic matrix composition prepared by the method of claim 64.

131. At least 10 per unit dose ４ A unit dosage form comprising at least 1 mg of a spray-dried biotherapeutic matrix composition containing CFU of bacteria.

132. 129. The unit dosage form of claim 128, wherein the dosage is at least 30 mg of the spray-dried biotherapeutic matrix composition.

133. Dosage is at least 10 per unit dose ４ 129. The unit dosage form of claim 128, comprising CFU of bacteria.

134. Dosage is at least 10 per unit dose ４ 129. The unit dosage form of claim 128, comprising CFUs of viable bacteria.

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