Inhalable Compositions of CDK9 Inhibitors

Abstract

The present disclosure describes formulations for the delivery of CDK9 inhibitors.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 371,721, filed August 17, 2022, the entire contents of which are incorporated herein for all purposes.

[0002] STATEMENT REGARDING RIGHTS TO INVENTIONS RESULTING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with government support from the Department of Defense through the Congressionally Managed Healthcare Program under Grant No. PR200947. The government has certain rights in this invention. [Background technology]

[0003] Pulmonary inflammation is a major cause of disease and even death, including pulmonary fibrosis, lung cancer, and more recently, lung failure due to COVID-19. Therefore, anti-inflammatory drugs represent an important target for medical intervention. Previous studies conducted by the present inventors and others identified the potent anti-inflammatory treatment flavopiridol, a well-known small molecule inhibitor of cyclin-dependent kinase 9 (CDK9). It was originally designated by the U.S. Food and Drug Administration (FDA) as an orphan drug for the treatment of rare leukemias. Our team demonstrated that flavopiridol inhibits the activation of primary response genes, resulting in the suppression of downstream inflammation in cells and tissues. Therefore, flavopiridol is being tested in the treatment of diseases ranging from cancer, viral infections, and post-traumatic osteoarthritis (PTOA). Given its efficacy, flavopiridol has been identified as a potentially promising candidate for treating diseases such as lung cancer or coronavirus disease 2019 (COVID-19). In most cases, anti-inflammatory drugs have typically been administered systemically, such as intravenously (i.v.) or orally. Systemic delivery frequently results in high doses and severe side effects. Therefore, local delivery, such as pulmonary delivery via inhalation, is more desirable. Local delivery of drugs to the lungs has been shown to provide rapid therapeutic effects and improve the proportion of drug available for treatment. Additional advantages of inhalation delivery, such as simple dry powder inhalers (DPIs), include non-invasiveness, high safety, and rapid targeting.

[0004] Inhalable dry particles are typically produced using either jet milling or spray drying techniques. Spray drying involves rapidly drying a liquid formulation into a dry powder, which is then filled into either gelatin capsules or blister packs for use in dry powder inhalers. While capable of producing high throughputs, even small spray drying devices such as the Buchi Mini Dryer B290 require hundreds of milligrams of often expensive drug material to produce a single sample, and these small dryers are also expensive (approximately $35,000). Therefore, using spray drying in a laboratory to conduct early research and development (R&D) efforts, such as formulation and particle size optimization and mechanistic understanding, is difficult and expensive. In this study, we introduce a simple, lab-built, benchtop device that enables the production of inhalable ultrasmall particles loaded with flavopiridol for pulmonary delivery. The particles produced meet the physiochemical properties, inhalability, and release profile requirements necessary for pulmonary delivery. Their anti-inflammatory activity has also been demonstrated in in vitro studies. The device and conditions introduced in this study can be utilized by researchers in their R&D efforts in drug delivery. The generated particles will be tested in vivo for the treatment of pulmonary inflammation and as a new therapeutic approach to treat lung cancer, pulmonary fibrosis, and COVID-19. Summary of the Invention

[0005] In one embodiment, the present invention provides a microparticle composition comprising a plurality of microparticles, each microparticle comprising a hydrophobic amino acid or a hydrophobic peptide, or a combination thereof, a lipid, and a cyclin-dependent kinase 9 (CDK9) inhibitor.

[0006] In another embodiment, the present invention provides a composition for use in treating inflammation or respiratory fibrosis in a subject in need thereof, the composition comprising a cyclin-dependent kinase 9 (CDK9) inhibitor.

[0007] In another embodiment, the present invention provides a method for preparing a plurality of particles of the present invention, comprising: sonicating a first reaction mixture comprising L-isoleucine and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), water, and ethanol, wherein the isoleucine and the DPPC are present in a ratio of about 9:1 (w / w) and the ethanol:water ratio is about 70:30 (v / v), to prepare a 0.3% (w / v) feed mixture; applying the feed mixture to a microfluidic piezo array to form a spray of atomized droplets; and and drying the droplets to obtain the plurality of particles.

[0008] In another embodiment, the present invention provides a liquid composition comprising a citrate buffer having a pH of 2 to 7 and a cyclin-dependent kinase 9 (CDK9) inhibitor.

[0009] In another embodiment, the present invention provides a method for administering a therapeutically effective amount of a CDK9 inhibitor to a subject in need thereof, comprising administering to the subject a microparticle composition of the present invention or a liquid composition of the present invention via respiratory administration.

[0010] In another embodiment, the present invention provides a method of treating inflammation in a subject in need thereof, the method comprising administering a therapeutically effective amount of a microparticle composition of the present invention, or a liquid composition of the present invention via respiratory administration, thereby treating the inflammation.

[0011] In another embodiment, the present invention provides a method of treating respiratory fibrosis in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a microparticle composition of the present invention, or a liquid composition of the present invention, via respiratory administration, thereby treating respiratory fibrosis.

[0012] In another embodiment, the present invention provides a method for treating inflammation or respiratory fibrosis in a subject in need thereof, comprising administering a therapeutically effective amount of a composition comprising a cyclin-dependent kinase 9 (CDK9) inhibitor. [Brief explanation of the drawings]

[0013] [Figure 1] FIG. 1 shows a schematic diagram of a Microfluidic Piezo and Cyclone Apparatus (MPCA) for the formation of inhalable particles. [Figure 2] FTIR spectra of excipient materials (A) L-isoleucine, (B) DPPC shown in combination with ILDF (L-isoleucine, DPPC, flavopiridol) microparticles (C). [Figure 3A] 1 shows SEM images from four experiments underway to identify our formulation, showing that the excipient comprises L-isoleucine:DPPC=90:10 (w:w) in a 70% (v / v) ethanol / water mixture. [Figure 3B] Figure 3B shows SEM images from four experiments underway to identify our formulations. Figure 3B shows the same mixture as Figure 3A, but with L-isoleucine:DPPC:CaCl = 89:10:0.75 in the mixed solvent. [Figure 3C] Figure 3C shows SEM images from four experiments underway to identify our formulations. Figure 3C shows the same mixture as Figure 3A, but with a 10:10:80 L-isoleucine:DPPC:glucose ratio in the mixed solvent. [Figure 3D] SEM images from four experiments underway to identify our formulation are shown. Figure 3D shows L-isoleucine:DPPC:glucose = 10:10:80 in an 80% (v / v) ethanol:water mixture. Scale bar = 20 μm. [Figure 4] 1 shows the release profile of flavopiridol from microparticles. [Figure 5-1]Particle stability data are shown, including at 4°C (Figure 5A), 24°C (Figure 5B), and 37°C (Figure 5C). [Figure 5-2] Particle stability data are shown, including at 4°C (Figure 5A), 24°C (Figure 5B), and 37°C (Figure 5C). [Figure 6] Luminescence readings of samples treated with TNF-α or flavopiridol are shown. [Figure 7A] Testing particle dispersity using the device shows particles (Figure 7A) exhibiting high dispersity (B), while the corresponding glucose particles exhibited little or no dispersity (Figure 7C). [Figure 7B] Testing particle dispersity using the device shows particles (Figure 7A) exhibiting high dispersity (B), while the corresponding glucose particles exhibited little or no dispersity (Figure 7C). [Figure 7C] Testing particle dispersity using the device shows particles (Figure 7A) exhibiting high dispersity (B), while the corresponding glucose particles exhibited little or no dispersity (Figure 7C). [Figure 8A]

[0043] Figure 8 shows the results of administration of flavopiridol in a pulmonary fibrosis model. Administration of flavopiridol showed a statistically significant difference in survival rate compared to the BLM + vehicle control (Figure 8A). [Figure 8B] Figure 8 shows the results of administration of flavopiridol in a pulmonary fibrosis model. Flavopiridol-treated mice generally maintained a heavier body weight than BLM+vehicle controls (Figure 8B). [Figure 8C] Figure 8 shows the results of flavopiridol administration in a pulmonary fibrosis model. Flavopiridol-treated mice generally maintained statistically significantly lower levels of hydroxyproline (Figure 8C). [Figure 8D]

[0033] Figure 8 shows the results of administration of flavopiridol in a pulmonary fibrosis model. Lung tissue sections from treated animals were stained with Masson's Trichrome staining protocol, and exemplary images are shown in Figure 8D. [Figure 8E] The results of administering flavopiridol to a pulmonary fibrosis model are shown in Figure 8E. Quantification of staining is shown in Figure 8E. [Figure 9]Mouse body weights measured on the indicated days are shown (mean ± SE). [Figure 10] Kaplan-Meier analysis of overall survival of mice treated with vehicle and inhaled flavopiridol with a single dose of bleomycin (n=12). [Figure 11] Figure 1 shows the hydroxyproline content in right lung homogenate samples as detected by hydroxyproline ELISA assay. Data are presented as the average of individual values ​​(mean ± SE, *p<0.05 vs. saline + vehicle). [Figure 12] Representative hematoxylin and eosin (H&E) and Masson's trichrome staining of lung tissue from vehicle-treated mice and mice treated by inhalation with flavopiridol (left) following a single dose of bleomycin challenge are shown. Semiquantitative fibrosis assessment of fibrotic lesions in H&E-stained sections of mouse lungs (top) and positive staining in histological Masson's trichrome-stained sections of mouse lungs (right). Fibrosis scores are expressed as the percentage of positively stained area per high-power field. Quantitative analysis of 6-12 high-power fields per lung was performed using ImageJ software (mean ± SE, *p<0.05). Detailed Description of the Invention

[0014] I. Overview The present invention describes cyclin-dependent kinase 9 (CDK9) inhibitors such as L-isoleucine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and flavopiridol.

[0015] II. Definition "Amino acid" refers to naturally occurring amino acids, synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Naturally occurring amino acids include alanine (A), glycine (G), aspartic acid (D), glutamic acid (E), asparagine (N), glutamine (Q), arginine (R), lysine (K), isoleucine (I), leucine (L), methionine (M), valine (V), phenylalanine (F), tyrosine (Y), tryptophan (W), serine (S), threonine (T), and cysteine ​​(C).

[0016] "Amino acid analog" refers to a compound that has the same basic chemical structure as a naturally occurring amino acid, i.e., an alpha carbon bonded to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified backbones, but retain the same basic chemical structure as a naturally occurring amino acid.

[0017] An "unnatural amino acid" is not encoded by the genetic code and may, but need not, have the same basic structure as a naturally occurring amino acid. Unnatural amino acids include, but are not limited to, azetidine carboxylic acid, 2-aminoadipic acid, 3-aminoadipic acid, beta-alanine, aminopropionic acid, 2-aminobutyric acid, 4-aminobutyric acid, 6-aminocaproic acid, 2-aminoheptanoic acid, 2-aminoisobutyric acid, 3-aminoisobutyric acid, 2-aminopimelic acid, tert-butylglycine, 2,4-diaminoisobutyric acid, desmosine, 2,2'-diaminopimelic acid, 2,3-diaminopropionic acid, N-ethylglycine, N-ethylasparagine, homoproline, hydroxylysine, allo-hydroxylysine, 3-hydroxyproline, 4-hydroxyproline, isodesmosine, allo-isoleucine, N-methylalanine, N-methylglycine, N-methylisoleucine, N-methylpentylglycine, N-methylvaline, naphthalanine, norvaline, ornithine, pentylglycine, pipecolic acid, and thioproline.

[0018] "L-isoleucine" refers to the compound (2S,3S)-2-amino-3-methylpentanoic acid, having the CAS number 73-32-5.

[0019] "Peptide" refers to a series of amino acid residues covalently linked to one another. A peptide can include two, three, four, or more amino acid residues of any amino acid. A hydrophobic peptide is a peptide that contains a majority of hydrophobic amino acids. An exemplary hydrophobic peptide is trileucine.

[0020] "Tri-leucine" or "Leu-Leu-Leu" or "LLL" refers to the compound having CAS number 10329-75-6.

[0021] "Lipid" refers to a small molecule with hydrophobic or amphiphilic properties and is useful in preparing vesicles, micelles, and liposomes. Lipids include fats, waxes, fatty acids, cholesterol, phospholipids, monoglycerides, diglycerides, and triglycerides. Lipid moieties can include several fatty acid groups using branched groups such as lysine and other branched amines. Phospholipids or phosphine lipids refer to lipids with the moiety P(O)2(OR)2, where one R group is hydrophilic and one R group is hydrophilic.

[0022] As used herein, "pharmaceutically acceptable salt" refers to an acid or base salt of a compound used in the method of the present invention. Illustrative examples of pharmaceutically acceptable salts include inorganic acid salts (such as hydrochloric acid, hydrobromic acid, phosphoric acid, etc.), organic acid salts (such as acetic acid, propionic acid, glutamic acid, citric acid, etc.), and quaternary ammonium salts (such as methyl iodide, ethyl iodide, etc.). It is understood that pharmaceutically acceptable salts are non-toxic. Further information regarding suitable pharmaceutically acceptable salts can be found in Remington's Pharmaceutical Sciences, 17, incorporated herein by reference. th ed., Mack Publishing Company, Easton, Pa., 1985.

[0023] Pharmaceutically acceptable salts of the acidic compounds of the present invention include salts formed with bases, i.e., alkali metal and alkaline earth metal salts, such as sodium, lithium, potassium, calcium, magnesium, and the like, as well as cationic salts, such as ammonium, trimethylammonium, diethylammonium, tris(hydroxymethyl)methylammonium salts, and the like.

[0024] Likewise, acid addition salts of mineral acids, organic carboxylic acids and organic sulfonic acids, such as hydrochloric acid, methanesulfonic acid, maleic acid, etc., are possible, provided that a basic group, such as pyridyl, is part of the structure.

[0025] The neutral forms of the compounds can be regenerated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner. The parent form of the compound differs from the various salt forms in certain physical properties, such as solubility in polar solvents, but the salts are otherwise identical to the parent form of the compound for purposes of this invention.

[0026] "CDK" refers to cyclin-dependent kinase. CDK9 is cyclin-dependent kinase 9.

[0027] A "derivative" of a CDK9 inhibitor is an ester, amide, or prodrug of a CDK9 inhibitor, where the ester, amide, or prodrug substituent is cleaved or hydrolyzed after administration to a subject.

[0028] "Microparticles" refers to particles of the present invention having a diameter of about 0.5 μm to about 10 μm.

[0029] "Pharmaceutically acceptable carrier" refers to a typically inert substance used as a diluent or vehicle for a drug, such as a therapeutic agent. The term also encompasses a normally inert substance that imparts viscosity to a composition. Typically, physiologically acceptable carriers are present in liquid form. Examples of liquid carriers include saline, phosphate buffer, normal buffered saline, water, buffered water, saline, glycine, glycoproteins (e.g., albumin, lipoproteins, globulins, etc.) to enhance stability, and the like. Because physiologically acceptable carriers are determined in part by the particular composition being administered as well as the particular method used to administer the composition, there are a wide variety of suitable formulations of pharmaceutical compositions of the present invention, as described below (e.g., Remington's Pharmaceutical Sciences, 17th ed., 1989).

[0030] "Sonication" or "sonicating" refers to the application of sound energy to a solution or mixture.

[0031] "Reaction mixture" refers to a mixture of at least two distinct species.

[0032] "Drying" refers to the removal of water or solvent from a mixture.

[0033] "Power" refers to electrical power.

[0034] "Buffer" or "buffering agent" refers to any inorganic or organic acid or base that resists changes in pH and maintains the pH at or near a desired point. Buffers useful in the present invention include, but are not limited to, sodium hydroxide, dibasic sodium phosphate anhydrous, and mixtures thereof. Those skilled in the art will understand that other buffers are useful in the present invention.

[0035] "Treate," "treating," or "treatment" refers to any indication of successful treatment or amelioration of an injury, condition, state, or symptom (e.g., pain), including relief, remission, attenuation of symptoms or making the symptom, injury, condition, or state more tolerable to the patient, reducing the frequency or duration of the symptom or condition, or, in some circumstances, preventing the onset of the symptom. Treatment or amelioration of symptoms can be based on objective or subjective parameters, including, for example, the results of a physical examination.

[0036] "Administering" refers to administration to a subject by oral administration, breathing, inhalation, nasal administration, suppository, topical contact, parenteral administration, intravenous administration, intraperitoneal administration, intramuscular administration, intralesional administration, intranasal administration, or subcutaneous administration, or intraspinal administration.

[0037] As used herein, "subject" refers to a mammal, which may be a human or a non-human mammal, e.g., a companion animal such as a dog, cat, rat, or a farm animal, such as a horse, donkey, mule, goat, sheep, pig, or cow.

[0038] A "therapeutically effective amount" refers to an amount of the microparticles of the present invention sufficient to suppress unwanted inflammation and eliminate or at least partially prevent symptoms and / or complications. Specifically, a therapeutically effective amount is an amount sufficient to suppress the expression of primary response genes, such as IL-1β and IL-6, to 50%, 40%, 30%, 20%, 10%, 5%, or 1% or less of the gene activity otherwise expected. Amounts effective for this use will depend, for example, on the inhibitory composition, the mode of administration, the stage and severity of the disease being treated, the patient's weight and general health, and the judgment of the prescribing physician. In practice, the amount of CDK9 inhibitor required for therapeutic effect in the methods of the present invention is less than that required for systemic administration due to the localized nature of drug release from the microparticles. The microparticles of the present invention can be administered chronically or acutely to reduce, inhibit, or prevent inflammation, cartilage degeneration, and post-traumatic osteoarthritis.

[0039] "Pulmonary fibrosis," "pulmonary fibrosis," or "respiratory fibrosis" refers to a condition involving scarring of the lungs, accompanied by shortness of breath, coughing, fatigue, weight loss, and swelling of the fingernails.

[0040] "Sustained release" refers to the release of the CDK9 inhibitor over an extended period of time following administration, generally from about 1 hour to about 30-60 days.

[0041] III. CDK9 Composition The present invention provides compositions for treating inflammation. In some embodiments, the present invention provides compositions comprising a cyclin-dependent kinase 9 (CDK9) inhibitor.

[0042] A. Cyclin-dependent kinase 9 (CDK9) inhibitors Provided herein are formulations of therapeutic agents that target Cdk9 kinase activity using existing small molecule inhibitors of CDK9. The formulations provided herein are suitable for flavopiridol, voruciclib, and classes of CDK9 inhibitors structurally related to flavopiridol and voruciclib, such that the inhibitors are delivered to the site of injury, e.g., injured tissue, or cell type, of interest, with appropriate overall release capacity and release kinetics.

[0043] In some embodiments, the CDK9 inhibitor is flavopiridol, or an ester, prodrug, or pharmaceutically acceptable salt thereof. In some embodiments, the CDK9 inhibitor is flavopiridol, SNS-032, or voruciclib, or a derivative thereof, or a pharmaceutically acceptable salt thereof. In some embodiments, the CDK9 inhibitor is flavopiridol, SNS-032, or a pharmaceutically acceptable salt thereof. In some embodiments, the CDK9 inhibitor is flavopiridol, SNS-032, or voruciclib. In some embodiments, the CDK9 inhibitor is flavopiridol or a pharmaceutically acceptable salt thereof. In some embodiments, the CDK9 inhibitor is flavopiridol. In some embodiments, the CDK9 inhibitor is flavopiridol HCl.

[0044] In some embodiments, the CDK9 inhibitor is flavopiridol, or an ester, prodrug, or pharmaceutically acceptable salt thereof. In some embodiments, the CDK9 inhibitor is flavopiridol, or a derivative or salt thereof. In some embodiments, the CDK9 inhibitor is flavopiridol (IUPAC name: 2-(2-chlorophenyl)-5,7-dihydroxy-8-[(3S,4R)-3-hydroxy-1-methyl-4-piperidinyl]-4-chromenone; CAS #146426-40-6); [ka] It has the following structure.

[0045] CDK9 inhibitors, such as flavopiridol, broadly and efficiently suppress the transcriptional activation of primary response genes, including inflammatory genes (e.g., IL-1, TNF, IL-6, iNOS) and matrix metalloproteinases (e.g., MMPs, ADAMTS). However, flavopiridol is rapidly metabolized and degraded, with an in vivo half-life of less than six hours. As a small molecule (approximately 400 Da), it rapidly diffuses from the administration site and is therefore generally administered systemically. Provided herein are formulations of CDK9 inhibitors and PLGA polymers, in which the CDK9 inhibitor is encapsulated within particles of appropriate size and with appropriate release capacity and release kinetics to provide a therapeutically effective amount over a period of time to treat injury, reduce inflammation, ameliorate symptoms, and / or prevent further damage to the injured tissue of a subject.

[0046] In some embodiments, the CDK9 inhibitor is SNS-032, or a prodrug thereof, or a pharmaceutically acceptable salt thereof. In some embodiments, the CDK9 inhibitor is SNS-032, or a salt thereof. In some embodiments, the CDK9 inhibitor is SNS-032, which has the following structure: [ka]

[0047] In some embodiments, the CDK9 inhibitor is voruciclib, or an ester, prodrug, or pharmaceutically acceptable salt thereof. In some embodiments, the CDK9 inhibitor is voruciclib, or a derivative or salt thereof. In some embodiments, the CDK9 inhibitor is voruciclib, which has the following structure: [ka]

[0048] Another CDK9 inhibitor is dinaciclib, which is not effectively encapsulated or adequately released in the microparticles of the present invention: [ka]

[0049] Provided herein is a CDK9 inhibitor, which is SNS-32, voruciclib, or flavopiridol, and a PLGA polymer encapsulated within microparticles of appropriate size and with appropriate release capacity and release kinetics to provide a therapeutically effective amount over a period of time to treat an injury, reduce inflammation, ameliorate symptoms, and / or prevent further damage to injured tissue in a subject.

[0050] Also provided are pharmaceutically acceptable salts, hydrates, solvates, tautomeric forms, polymorphs, and prodrugs of the CDK9 inhibitors described herein. "Pharmaceutically acceptable" or "physiologically acceptable" refers to compounds, salts, compositions, dosage forms, and other materials that are useful in the preparation of pharmaceutical compositions suitable for veterinary or human pharmaceutical use.

[0051] The compounds described herein can be prepared and / or formulated as pharmaceutically acceptable salts, or as free bases, if appropriate. A "pharmaceutically acceptable salt" is a non-toxic salt of the free base form of a compound that retains the desired pharmacological activity of the free base. Such salts are derived from inorganic or organic acids or bases. For example, a compound containing a basic nitrogen can be prepared as a pharmaceutically acceptable salt by contacting the compound with an inorganic or organic acid. Non-limiting examples of pharmaceutically acceptable salts include sulfate, pyrosulfate, bisulfite, bisulfite, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propionate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-1,4-diol, and benzoate. Other suitable pharmaceutically acceptable salts include butyne-1,4-dioates, hexyne-1,6-dioates, benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, hydroxybenzoates, methoxybenzoates, phthalates, sulfonates, naphthalene-2-sulfonates, phenylacetates, phenylpropionates, phenylbutyrates, citrates, lactates, gamma-hydroxybutyrates, glycolates, tartrates, and mandelates. Other suitable pharmaceutically acceptable salts are described in Remington: The Science and Practice of Pharmacy, 21 st Edition, Lippincott Williams and Wilkins, Philadelphia, PA, 2006.

[0052] Examples of pharmaceutically acceptable salts of the compounds disclosed herein include alkali metal (e.g., sodium, potassium), alkaline earth metal (e.g., magnesium), ammonium, and NX4 salts. + Also included are salts derived from appropriate bases, such as: (wherein X is C1-C4 alkyl). Base addition salts, such as sodium or potassium salts, are also included.

[0053] The CDK9 inhibitor may be present in the composition in any suitable amount. For example, the CDK9 inhibitor may be present in an amount of 0.01-10% (w / w), or 0.01-5%, or 0.01-1%, or 0.1-1%, or 0.1-0.5%, or 0.1-0.3% (w / w). The CDK9 inhibitor may be present in the microparticles in an amount of about 0.01% (w / w), or 0.05, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.3, 0.35, 0.4, 0.45, or about 0.5% (w / w).

[0054] The CDK9 inhibitor can be present at a certain ratio relative to the lipid. For example, the lipid can be present at a ratio relative to the CDK9 inhibitor of 1000:1 to 1:1000 (w / w), or 1000:1 to 1:100, or 1000:1 to 1:10, or 1000:1 to 1:1, or 1000:1 to 10:1, or 1000:1 to 10:1, or 90:1 to 10:1, or 80:1 to 10:1, or 70:1 to 10:1, or 60:1 to 10:1, or 50:1 to 10:1, or 40:1 to 10:1, or 30:1 to 10:1 (w / w). The lipid may be present in a ratio of about 50:1 (w / w), or 100:1, 95:1, 90:1, 85:1, 80:1, 75:1, 70:1, 65:1, 60:1, 55:1, 50:1, 45:1, 40:1, 35:1, 30:1, 25:1, 20:1, 15:1, or about 10:1 (w / w) relative to the CDK9 inhibitor.

[0055] In some embodiments, the lipid is present in a ratio of 1000:1 to 1:1 (w / w) relative to the CDK9 inhibitor. In some embodiments, the lipid is present in a ratio of 100:1 to 10:1 (w / w) relative to the CDK9 inhibitor. In some embodiments, the lipid is present in a ratio of about 50:1 (w / w) relative to the CDK9 inhibitor.

[0056] B. Composition The compositions of the present invention can be prepared in a wide variety of oral, parenteral, and topical dosage forms. Oral preparations include tablets, pills, powders, dragees, capsules, liquids, lozenges, cachets, gels, syrups, slurries, suspensions, and the like, suitable for ingestion by a patient. The compositions of the present invention can also be administered by injection, i.e., intravenously, intramuscularly, intradermally, subcutaneously, intraduodenally, or intraperitoneally. The compositions described herein can also be administered by inhalation, for example, intranasally. Additionally, the compositions of the present invention can be administered transdermally. The compositions of the present invention can also be administered by ocular, intravaginal, and intrarectal routes, including suppositories, inhalants, powders, and aerosol formulations (for examples of steroid inhalants, see Rohatagi, J. Clin. Pharmacol. 35:1187-1193, 1995; Tjwa, Ann. Allergy Asthma Immunol. 75:107-111, 1995). Accordingly, the present invention also provides pharmaceutical compositions comprising a pharmaceutically acceptable carrier or excipient and a compound of the present invention.

[0057] When preparing pharmaceutical compositions from the compounds of the present invention, pharmaceutically acceptable carriers can be either solid or liquid.Solid preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules.A solid carrier can be one or more substances that can also act as diluents, flavoring agents, binders, preservatives, tablet disintegrating agents, or encapsulating materials.Details about formulation and administration techniques are fully described in the scientific and patent literature.See, for example, the latest edition of Remington's Pharmaceutical Sciences, Maack Publishing Co, Easton PA ("Remington's").

[0058] In the case of powders, the carrier may be a finely divided solid, which is the finely divided active ingredient. In tablets, the active ingredient is mixed with a carrier having the necessary binding properties in suitable proportions and compressed into the desired shape and size. Powders and tablets preferably contain 5% or 10% to 70% of the compound of the present invention.

[0059] Suitable solid excipients include, but are not limited to, magnesium carbonate, magnesium stearate, talc, pectin, dextrin, starch, tragacanth, low-melting waxes, cocoa butter, carbohydrates, including, but not limited to, sugars including lactose, sucrose, mannitol, or sorbitol, starches derived from corn, wheat, rice, potato, or other plants, celluloses such as methylcellulose, hydroxypropylmethylcellulose, or sodium carboxymethylcellulose, gums including acacia and tragacanth, and proteins including, but not limited to, gelatin and collagen. If desired, disintegrating or solubilizing agents such as cross-linked polyvinylpyrrolidone, agar, alginic acid or a salt thereof, e.g., sodium alginate, may be added.

[0060] Dragee cores are provided with suitable coatings, such as gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and / or titanium dioxide, lacquer solutions, and concentrated sugar solutions also containing suitable organic solvents or solvent mixtures. Dyes or pigments may be added to tablets or dragee coatings for product identification or to characterize the active compound dose (i.e., pharmaceutical dose). The present agents may be administered orally using, for example, push-fit capsules made of gelatin and soft, sealed capsules made of gelatin and coatings such as glycerol or sorbitol. Push-fit capsules may contain the compound of the present invention mixed with fillers or binders, such as lactose or starch, lubricants, such as talc or magnesium stearate, and, optionally, stabilizers. In soft capsules, the compound of the present invention may be dissolved or suspended in a suitable liquid, such as fatty oils, liquid paraffin, or liquid polyethylene glycol, with or without stabilizers.

[0061] For preparing suppositories, a low melting wax such as a mixture of fatty acid glycerides or cocoa butter is first melted and the compound of the present invention is dispersed homogeneously therein, for example by stirring, The molten homogeneous mixture is then poured into convenient sized molds, allowed to cool and thereby solidify.

[0062] Liquid preparations include solutions, suspensions, and emulsions, for example, water or water / propylene glycol solutions.For parenteral injection, liquid preparations can be formulated in solution in aqueous polyethylene glycol solution.

[0063] Aqueous solutions suitable for oral use can be prepared by dissolving the compound of the invention in water and adding suitable colorants, flavors, stabilizing, and thickening agents as desired. Aqueous suspensions suitable for oral use can be made by dispersing the active ingredient, finely divided in water, with a dispersing or wetting agent such as a viscous material, such as a natural or synthetic gum, resin, methylcellulose, sodium carboxymethylcellulose, hydroxypropyl methylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth, gum acacia, or the like, a naturally occurring phosphatide (e.g., lecithin), a condensation product of an alkylene oxide with a fatty acid (e.g., polyoxyethylene stearate), a condensation product of ethylene oxide with a long-chain aliphatic alcohol (e.g., heptadecaethylene oxycetanol), a condensation product of ethylene oxide with a partial ester derived from a fatty acid and a hexitol (e.g., polyoxyethylene sorbitol monooleate), or a condensation product of ethylene oxide with a partial ester derived from a fatty acid and a hexitol anhydride (e.g., polyoxyethylene sorbitan mono-oleate). Aqueous suspensions may also contain one or more preservatives, for example, ethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose, aspartame, or saccharin. The preparation may be adjusted for osmolality.

[0064] Also included are solid form preparations intended to be converted immediately before use into liquid form preparations for oral administration.Such liquid forms include solutions, suspensions, solutions, syrups and emulsions.These preparations may contain, in addition to the active ingredient, colorants, flavors, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizers, etc.

[0065] Oily suspensions can be prepared by suspending the compounds of the present invention in vegetable oils, such as peanut oil, olive oil, sesame oil, or coconut oil, or in mineral oils such as liquid paraffin, or mixtures thereof. Oily suspensions can also contain thickening agents such as beeswax, hard paraffin, or cetyl alcohol. Sweeteners, such as glycerol, sorbitol, or sucrose, can be added to provide a palatable oral preparation. These preparations can be preserved by adding antioxidants such as ascorbic acid. For examples of injectable oil vehicles, see Minto, J. Pharmacol. Exp. Ther. 281:93-102, 1997. The pharmaceutical preparations of the present invention can also be in the form of an oil-in-water emulsion. The oily phase can be the above-mentioned vegetable oil or mineral oil, or a mixture thereof. Suitable emulsifying agents include, for example, naturally occurring gums such as gum acacia and gum tragacanth, naturally occurring phosphatides such as soybean lecithin, esters or partial esters derived from fatty acids and hexitol anhydrides, for example, sorbitan monooleate, and condensation products of these partial esters with ethylene oxide, such as polyoxyethylene sorbitan monooleate. Emulsifying agents may also contain sweeteners and flavoring agents, as in the preparation of syrups and elixirs. Such preparations may also contain demulcents, preservatives, or coloring agents.

[0066] The compositions of the present invention can also be delivered to the body as microspheres for sustained release. For example, microspheres can be formulated for administration via intradermal injection of drug-containing microspheres that slowly release subcutaneously (see Rao, J. Biomater Sci. Polym. Ed. 7:623-645, 1995), as biodegradable and injectable gel formulations (see, e.g., Gao Pharm. Res. 12:857-863, 1995), or as oral microspheres (see, e.g., Eyles, J. Pharm. Pharmacol. 49:669-674, 1997). Both transdermal and intradermal routes allow for constant delivery over weeks or months.

[0067] In another embodiment, the compositions of the present invention can be formulated for parenteral administration, such as intravenous (IV) administration or administration into a body cavity or lumen of an organ. Formulations for administration generally comprise a solution of the compositions of the present invention dissolved in a pharmaceutically acceptable carrier. Acceptable vehicles and solvents that may be employed include water, Ringer's solution, and isotonic saline. Additionally, sterile, fixed oils are conventionally used as solvents or suspending media. For this purpose, any bland fixed oil may be used, including synthetic mono- or diglycerides. Additionally, fatty acids such as oleic acid may similarly be used in the preparation of injectable solutions. These solutions are sterile and generally free of undesirable matter. These formulations can be sterilized by conventional, well-known sterilization techniques. Formulations may contain pharmaceutically acceptable auxiliary substances as needed to approximate physiological conditions (pH adjusting agents, buffers, toxicity adjusting agents, e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc.). The concentration of the composition of the present invention in these preparations can vary widely and is selected mainly based on the volume of body fluids, viscosity, body weight, etc., according to the specific administration method selected and the patient's needs. For IV administration, the preparation can be a sterile injectable preparation, such as a sterile injectable aqueous or oily suspension. This suspension can be formulated according to known techniques using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation can also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol.

[0068] In another embodiment, formulations of the compositions of the present invention can be delivered using liposomes that fuse with or are internalized by cell membranes, i.e., by using ligands attached to the liposomes or directly attached to the oligonucleotides that bind to cell surface membrane protein receptors and induce endocytosis. The use of liposomes can also focus delivery of the compositions of the present invention to target cells in vivo, particularly when the liposome surface carries a ligand specific to the target cells or is otherwise directed preferentially to a particular organ (see, e.g., Al-Muhammed, J. Microencapsul. 13:293-306, 1996; Chonn, Curr. Opin. Biotechnol. 6:698-708, 1995; Ostro, Am. J. Hosp. Pharm. 46:1576-1587, 1989).

[0069] The compositions of the present invention can be delivered by any suitable means, including orally, parenterally, and topically. Transdermal administration methods can be formulated as applicator sticks, solutions, suspensions, emulsions, gels, creams, ointments, pastes, jellies, paints, powders, and aerosols by the topical route.

[0070] Pharmaceutical preparations are preferably in unit dosage form.In such form, the preparation is further divided into unit doses containing appropriate amounts of the compound of the present invention.The unit dosage form can be a packaged preparation, and the package contains discrete amounts of preparations, such as packaged tablets, capsules, and powders in vials or ampoules.Alternatively, the unit dosage form can be a capsule, tablet, cachet, or lozenge itself, or it can be the appropriate number of any of these in packaged form.

[0071] In some embodiments, the composition further comprises an amino acid and a lipid. Suitable amino acids and lipids for use in the compositions of the invention are described herein.

[0072] C. Particulates The present invention also provides microparticles for treating inflammation. In some embodiments, the present invention provides a microparticle composition comprising a plurality of microparticles, each microparticle comprising an amino acid, a lipid, and a cyclin-dependent kinase 9 (CDK9) inhibitor.

[0073] Amino acids and peptides Amino acids useful in the compositions and methods of the present invention include, but are not limited to, hydrophobic amino acids, hydrophilic amino acids, and other amino acids. Representative hydrophobic amino acids include, but are not limited to, alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, tryptophan, and derivatives and analogs thereof. Amino acids can have any suitable stereochemistry. For example, amino acids can be in D-configuration or L-configuration.

[0074] In some embodiments, the present invention provides a microparticle composition comprising a plurality of microparticles, each microparticle comprising a hydrophobic amino acid or a hydrophobic peptide, or a combination thereof, a lipid, and a cyclin-dependent kinase 9 (CDK9) inhibitor. In some embodiments, the present invention provides a microparticle composition comprising a plurality of microparticles, each microparticle comprising a hydrophobic amino acid, a lipid, and a cyclin-dependent kinase 9 (CDK9) inhibitor.

[0075] In some embodiments, hydrophobic amino acids include alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, tryptophan, or a combination thereof. In some embodiments, hydrophobic amino acids include alanine, valine, isoleucine, leucine, or a combination thereof. In some embodiments, hydrophobic amino acids include isoleucine, leucine, or a combination thereof. In some embodiments, hydrophobic amino acids include isoleucine. In some embodiments, hydrophobic amino acids include L-isoleucine.

[0076] In some embodiments, the present invention provides a microparticle composition comprising a plurality of microparticles, each microparticle comprising L-isoleucine, a lipid, and a cyclin-dependent kinase 9 (CDK9) inhibitor.

[0077] Hydrophobic peptides useful in the microparticles of the invention include peptides having two, three, four or more hydrophobic amino acids, such as alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, tryptophan, or combinations thereof. In some embodiments, the invention provides a microparticle composition comprising a plurality of microparticles, wherein the hydrophobic peptide comprises trileucine.

[0078] The hydrophobic amino acids or hydrophobic peptides can be present in the microparticles in any suitable amount. For example, the hydrophobic amino acids or hydrophobic peptides can be present in an amount of 50-99% (w / w), 55-95%, 60-95%, 65-95%, 70-95%, 75-95%, 80-95%, 85-95%, 86-94%, 87-93%, 88-92%, or 89-91% (w / w). The hydrophobic amino acids or hydrophobic peptides can also be present in the microparticles in an amount of about 50% (w / w), or 55, 60, 65, 70, 75, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or about 95% (w / w). The hydrophobic amino acid or hydrophobic peptide may also be present in the microparticles in an amount of about 89.0% (w / w), or 89.1, 89.2, 89.3, 89.4, 89.5, 89.6, 89.7, 89.8, 89.9, 90.0, 90.1, 90.2, 90.3, 90.4, 90.5, 90.6, 90.7, 90.8, 90.9, or about 91.0% (w / w).

[0079] lipids Lipids useful in the compositions and methods of the present invention can include a variety of lipids. Suitable lipids can include, but are not limited to, fats, fatty acids, waxes, sterols, cholesterol, fat-soluble vitamins, monoglycerides, diglycerides, phospholipids, sphingolipids, glycolipids, derivatized lipids, and the like.

[0080] In some embodiments, the lipid is a fatty acid. The fatty acid may be saturated, monounsaturated, or polyunsaturated. Examples of fatty acids include, but are not limited to, butyric acid (C4), caproic acid (C6), caprylic acid (C8), capric acid (C10), lauric acid (C12), myristic acid (C14), palmitic acid (C16), palmitoleic acid (C16), stearic acid (C18), isostearic acid (C18), oleic acid (C18), succinic acid (C18), linole ... Examples of fatty acids include oleic acid (C18), alpha-linoleic acid (C18), gamma-linolenic acid (C18), arachidic acid (C20), gadoleic acid (C20), arachidonic acid (C20), eicosapentaenoic acid (C20), behenic acid (C22), erucic acid (C22), docosahexaenoic acid (C22), lignoceric acid (C24), and hexacosanoic acid (C26).

[0081] In some embodiments, the lipid is a phosphocholine lipid. Suitable phospholipids include, but are not limited to, phosphatidylcholine (PC), phosphatidic acid (PA), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylserine (PS), and phosphatidylinositol (PI). Non-cationic lipids include dimyristoylphosphatidylcholine (DMPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dimyristoylphosphatidylglycerol (DMPG), distearoylphosphatidylglycerol (DSPG), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dimyristoylphosphatidylserine (DMPS), distearoylphosphatidylserine (DSPS), dioleoylphosphatidylserine (DOPS), dipalmitoylphosphatidylserine (DPPS), dioleoylphosphatidylamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyl ...amine (DPPG), palmitoyloleoylphosphatidylamine (DPPG), palmitoyloleoylphosphatidylamine (DPPG), palmitoyloleoylphosphatidylamine (DPPG), palmitoyloleoylphosphatidylamine These include, but are not limited to, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (POPE) and dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidiethanolamine (SOPE), 1,2-dielaidoyl-sn-glycero-3-phosphoethanolamine (transDOPE), and cardiolipin.

[0082] In some embodiments, the lipid is 1,2-diheptanoyl-sn-glycero-3-phosphocholine (DHPC), 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), or a combination thereof. In some embodiments, the lipid is dipalmitoylphosphatidylcholine (DPPC).

[0083] Lipids can also include derivatized lipids, such as PEGylated lipids. PEGylated lipids generally contain a lipid moiety described herein covalently conjugated to one or more PEG chains. PEG can be linear or branched, and branched PEG molecules can have additional PEG molecules emanating from a central core, and / or multiple PEG molecules can be grafted onto the polymer backbone. PEG can include low- or high-molecular-weight PEGs, such as PEG500, PEG2000, PEG3400, PEG5000, PEG6000, PEG9000, PEG10000, PEG20000, or PEG50000, where the number 500 indicates the average molecular weight. Derivatized lipids can include, for example, DSPE-PEG2000, cholesterol-PEG2000, DSPE-polyglycerol, or other derivatives commonly known in the art.

[0084] The lipids can be present in the microparticles in any suitable amount. For example, the lipids can be present in an amount of 1-50% (w / w), 1-45%, 1-40%, 1-35%, 1-30%, 1-25%, 1-20%, 1-15%, 5-15%, 6-14%, 7-13%, 8-12%, or 9-11% (w / w). The lipids can be present in the microparticles in an amount of about 1% (w / w), or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or about 50% (w / w).

[0085] The lipids may also be present in any suitable ratio relative to the L-isoleucine, for example, a ratio of 1000:1 to 1:1000 (w / w), or 1000:1 to 1:100, or 1000:1 to 1:10, or 1000:1 to 1:1, or 1000:1 to 1:1, or 75:1 to 1:1, or 50:1 to 1:1, or 40:1 to 1:1, or 30:1 to 1:1, or 20:1 to 1:1, or 15:1 to 5:1, or 12:1 to 6:1 (w / w) L-isoleucine to lipid. The L-isoleucine can be present in a ratio of about 50:1 (w / w) to lipid, or 45:1, 40:1, 35:1, 30:1, 25:1, 20:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, or about 1:1 (w / w).

[0086] In some embodiments, L-isoleucine is present in a ratio of 1000:1 to 1:10 (w / w) to lipid. In some embodiments, L-isoleucine is present in a ratio of 100:1 to 1:1 (w / w) to lipid. In some embodiments, L-isoleucine is present in a ratio of 9:1 (w / w) to lipid.

[0087] fine particles In some embodiments, the microparticles comprise L-isoleucine, DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), and flavopiridol, in a ratio of L-isoleucine:DPPC:flavopiridol of about 89.8:10.0:0.2 (w / w).

[0088] The microparticles can be of any suitable size. For example, the microparticles can have an average geometric diameter of 0.1 to 100 microns, or 0.1 to 50 microns, or 0.1 to 25 microns, or 0.1 to 10 microns, or 1 to 10 microns, or 1 to 7.5 microns, or 1 to 5 microns, or 1 to 2.5 microns, or 3 to 7 microns. The microparticles can have an average geometric diameter of about 1 micron, or 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 microns. In some embodiments, the microparticles have an average geometric diameter of 1 to 10 microns.

[0089] In some embodiments, the microparticles of the present invention do not contain one or more components, such as sugars or inorganic salts. The microparticles of the present invention exclude one or more sugars, such as, but not limited to, glucose, sucrose, fructose, galactose, mannose, ribose, lactose, trehalose, mannitol, sorbitol, and xylitol. The microparticles of the present invention exclude one or more inorganic salts, such as, but not limited to, sodium chloride (NaCl), potassium chloride (KCl), calcium chloride (CaCl), and magnesium chloride (MgCl).

[0090] In some embodiments, the microparticles do not include CaCl. In some embodiments, the microparticles do not include glucose. In some embodiments, the microparticles do not include CaCl or glucose. In some embodiments, the microparticles do not include CaCl and glucose.

[0091] In some embodiments, the microparticles consist essentially of L-isoleucine, DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), and flavopiridol, wherein the ratio of L-isoleucine:DPPC:flavopiridol is about 89.8:10.0:0.2 (w / w).

[0092] In some embodiments, the microparticles consist essentially of L-isoleucine, DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), and flavopiridol, wherein the ratio of L-isoleucine:DPPC:flavopiridol is about 89.8:10.0:0.2 (w / w).

[0093] In some embodiments, the microparticles release the CDK9 inhibitor at a nearly constant rate over the treatment period. In some embodiments, the microparticles release the CDK9 inhibitor at a constant rate after an initial release of about 3% to about 10% of the encapsulated CDK9 inhibitor. In some embodiments, the initial release occurs within about 24 hours. In some embodiments, the initial release occurs within about 12 hours. In some embodiments, the initial release occurs within about 8 hours. In some embodiments, the initial release occurs within about 1 hour. In some embodiments, the microparticles release about 3% to about 30%, about 3% to about 20%, about 3% to about 10%, about 5% to about 30%, about 5% to about 20%, or about 5% to about 10% of the CDK9 inhibitor over 24 hours. In some embodiments, the microparticles release about 5% to about 40%, about 5% to about 30%, about 5% to about 20%, about 10% to about 40%, about 10% to about 30%, about 10% to about 20%, or about 10% to about 15% of the CDK9 inhibitor over a 2-day period. In some embodiments, the microparticles release about 10% to about 50%, about 10% to about 40%, about 10% to about 30%, about 15% to about 40%, about 15% to about 30%, or about 15% to about 25% of the CDK9 inhibitor over a 5-day period. In some embodiments, the microparticles release about 20% to about 70%, about 20% to about 60%, about 20% to about 50%, about 20% to about 40%, or about 25% to about 35% of the CDK9 inhibitor over an 8-day period. In some embodiments, the microparticles release about 30% to about 70%, about 30% to about 60%, about 30% to about 50%, about 40% to about 70%, about 40% to about 60%, or about 40% to about 50% of the CDK9 inhibitor over a 12-day period. In some embodiments, the particles release about 40% to about 80%, about 40% to about 70%, about 50% to about 70%, or about 55% to about 65% of the CDK9 inhibitor over a 15-day period. In some embodiments, the microparticles release about 40% to about 80%, about 50% to about 80%, about 60% to about 80%, or about 65% to about 75% of the CDK9 inhibitor over a 19-day period. In some embodiments, the microparticles release about 40% to about 90%, about 50% to about 90%, about 60% to about 90%, about 70% to about 90%, or about 75% to about 85% of the CDK9 inhibitor over a 22 day period.In some embodiments, the microparticles release about 50% to about 90%, about 60% to about 90%, about 70% to about 90%, or about 80% to about 90% of the CDK9 inhibitor over 26 days, hi some embodiments, the microparticles release about 60% to about 95%, about 70% to about 95%, about 80% to about 95%, or about 85% to about 95% of the CDK9 inhibitor over 30 days.

[0094] In some embodiments, at least about 80% of the encapsulated CDK9 inhibitor is released by the end of the treatment period. In some embodiments, the amount of encapsulated CDK9 inhibitor released is at least about 85%, at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.5%. In some embodiments, the treatment period is at least about 24 hours, at least about 2 days, at least about 5 days, at least about 7 days, at least about 10 days, at least about 14 days, at least about 20 days, at least about 21 days, at least about 28 days, at least about 30 days, at least about 31 days, at least about 40 days, at least about 42 days, at least about 45 days, at least about 48 days, at least about 50 days, or at least about 60 days. In some embodiments, the treatment period is less than about 60 days, less than about 55 days, less than about 50 days, less than about 45 days, less than about 40 days, less than about 30 days, less than about 28 days, less than about 25 days, less than about 21 days, less than about 20 days, less than about 14 days, less than about 10 days, less than about 7 days, less than about 5 days, or less than about 2 days.

[0095] In some embodiments, the microparticles release about 3% to about 10% of the CDK9 inhibitor over about 24 hours, about 10% to about 20% of the CDK9 inhibitor over about 2 days, about 15% to about 25% of the CDK9 inhibitor over about 5 days, about 25% to about 35% of the CDK9 inhibitor over about 8 days, about 40% to about 50% of the CDK9 inhibitor over about 12 days, about 55% to about 65% of the CDK9 inhibitor over about 15 days, about 65% to about 75% of the CDK9 inhibitor over about 19 days, about 75% to about 85% of the CDK9 inhibitor over about 22 days, about 80% to about 90% of the CDK9 inhibitor over about 26 days, and / or about 85% to about 95% of the CDK9 inhibitor over about 30 days.

[0096] In some embodiments, the microparticles release the CDK9 inhibitor over a period selected from the group consisting of about 24 hours, about 2 days, about 5 days, about 10 days, about 14 days, about 21 days, about 30 days, about 45 days, and about 60 days. In some embodiments, the microparticles release about 5% to about 40%, about 5% to about 30%, about 5% to about 20%, about 10% to about 40%, about 10% to about 30%, about 10% to about 20%, or about 10% to about 15% of the CDK9 inhibitor over a 2-day period following administration. In some embodiments, the microparticles release about 10% to about 50%, about 10% to about 40%, about 10% to about 30%, about 15% to about 40%, about 15% to about 30%, or about 15% to about 25% of the CDK9 inhibitor over a 5-day period following administration. In some embodiments, the microparticles release about 20% to about 70%, about 20% to about 60%, about 20% to about 50%, about 20% to about 40%, or about 25% to about 35% of the CDK9 inhibitor over 8 days after administration. In some embodiments, the microparticles release about 30% to about 70%, about 30% to about 60%, about 30% to about 50%, about 40% to about 70%, about 40% to about 60%, or about 40% to about 50% of the CDK9 inhibitor over 12 days after administration. In some embodiments, the particles release about 40% to about 80%, about 40% to about 70%, about 50% to about 70%, or about 55% to about 65% of the CDK9 inhibitor over 15 days after administration. In some embodiments, the particles release about 40% to about 80%, about 50% to about 80%, about 60% to about 80%, or about 65% to about 75% of the CDK9 inhibitor over 19 days after administration. In some embodiments, the microparticles release about 40% to about 90%, about 50% to about 90%, about 60% to about 90%, about 70% to about 90%, or about 75% to about 85% of the CDK9 inhibitor over 22 days after administration. In some embodiments, the microparticles release about 50% to about 90%, about 60% to about 90%, about 70% to about 90%, or about 80% to about 90% of the CDK9 inhibitor over 26 days after administration. In some embodiments, the microparticles release about 60% to about 95%, about 70% to about 95%, about 80% to about 95%, or about 85% to about 95% of the CDK9 inhibitor over a 30 day period following administration.

[0097] The pharmaceutical compositions of the present invention comprise the microparticles of the present invention dispersed or suspended in a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" includes any solvent, dispersion medium, coating, surfactant, antioxidant, preservative (e.g., antibacterial, antifungal), isotonicity agent, absorption delaying agent, salt, preservative, drug, drug stabilizer, gel, binder, excipient, disintegrant, lubricant, dye, and combinations thereof, as known to those skilled in the art (see, e.g., Remington's Pharmaceutical Sciences, 18th Ed., Mack Printing Company, 1990, pp. 1289-1329, incorporated herein by reference). Any conventional carrier is contemplated for use in pharmaceutical compositions, provided it is not incompatible with the microparticles of the present invention. While the compositions of the present invention are expected to be administered primarily by injection or other parenteral methods, gels and aerosol compositions may also be used, for example, for application during surgical procedures. Suitable carriers include water, water for injection, saline, phosphate-buffered saline, and the like. The compositions of the present invention may further comprise a propellant, an anti-agglomerating agent, and the additional agents described above.

[0098] In some embodiments, the present invention provides a composition for use in treating inflammation or respiratory fibrosis in a subject in need thereof, the composition comprising a cyclin-dependent kinase 9 (CDK9) inhibitor. The composition for treating inflammation or respiratory fibrosis can be any composition of the present invention. In some embodiments, the present invention provides a composition for use in treating respiratory fibrosis in a subject in need thereof, the composition comprising a cyclin-dependent kinase 9 (CDK9) inhibitor.

[0099] Method for preparing microparticles The present invention also provides methods for preparing microparticles. In some embodiments, the present invention provides a method for preparing a plurality of particles of the present invention, comprising: sonicating a first reaction mixture comprising L-isoleucine and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), water, and ethanol, wherein the L-isoleucine and DPPC are present in a ratio of about 9:1 (w / w) and the ratio of ethanol:water is about 70:30 (v / v), to prepare a 0.3% (w / v) feed mixture; applying the feed mixture to a microfluidic piezoelectric array to form a spray of atomized droplets; and and drying the droplets to obtain the plurality of particles.

[0100] The microfluidic piezo array can be operated at any suitable frequency and power. Typical frequencies can be 0.1 kHz to 1000 kHz, or 1 kHz to 1000 kHz, 10 kHz to 1000 kHz, 10 kHz to 500 kHz, 10 kHz to 250 kHz, 10 kHz to 200 kHz, 50 kHz to 150 kHz, or 75 kHz to 125 kHz. For example, the frequency can be about 10 kHz, or 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or about 200 kHz. The frequency can also be about 110 kHz, or 111, 112, 113, 114, 115, 116, 117, 118, 119, or about 120 kHz.

[0101] Typical power can be 0.1 to 1000 V, or 1 to 900 V, 1 to 500 V, 1 to 250 V, 1 to 100 V, 1 to 75 V, 10 to 50 V, 15 to 45 V, 20 to 40 V, or 25 to 35 V. For example, the power can be about 1 V, or 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or about 100 V.

[0102] In some embodiments, the microfluidic piezo array operates at 113 kHz with 30V of power.

[0103] D. Liquid composition The present invention also provides liquid compositions, in some embodiments, comprising a citrate buffer solution having a pH of 2 to 7 and a cyclin-dependent kinase 9 (CDK9) inhibitor.

[0104] The citrate buffer can include any suitable components. For example, the citrate buffer can include one or more of citric acid, citric acid monohydrate, and trisodium citrate dihydrate. The citrate buffer can have any suitable pH between 2 and 7. For example, the citrate buffer can have a pH between 3 and 6, or between 4 and 5. The citrate buffer can have a pH of about 2, 3, 4, 5, 6, or 7. In some embodiments, the citrate buffer includes citric acid monohydrate and trisodium citrate dihydrate and has a pH between 4 and 5. In some embodiments, the citrate buffer includes citric acid monohydrate and trisodium citrate dihydrate and has a pH between 4 and 5.

[0105] In some embodiments, the CDK9 inhibitor is flavopiridol, SNS-032, or voruciclib, or a derivative thereof, or a pharmaceutically acceptable salt thereof. In some embodiments, the CDK9 inhibitor is flavopiridol, SNS-032, or voruciclib, or a pharmaceutically acceptable salt thereof. In some embodiments, the CDK9 inhibitor is flavopiridol.

[0106] The liquid composition may include various other ingredients, such as, but not limited to, inorganic salts. Exemplary inorganic salts include, but are not limited to, sodium chloride, potassium chloride, magnesium chloride, and calcium chloride. In some embodiments, the liquid composition also includes sodium chloride.

[0107] In some embodiments, the liquid composition comprises a citrate buffer having a pH of 4-5, flavopiridol at a concentration of about 120 μM, and sodium chloride at a concentration of about 70 mM.

[0108] IV. Administration Method The present invention provides methods of administering the microparticle and liquid compositions of the present invention. In some embodiments, the present invention provides a method of administering a therapeutically effective amount of a CDK9 inhibitor to a subject in need thereof, comprising administering to the subject a microparticle composition of the present invention or a liquid composition of the present invention via respiratory administration.

[0109] Suitable dosage ranges for the microparticles and liquid compositions of the present invention include about 0.1 mg to about 100 mg, or about 1 mg to about 1000 mg, or about 1 to about 100 mg, or about 1 to about 50 mg, or about 1 to about 25 mg, or about 1 to about 10 mg, or about 10 mg to about 750 mg, or about 25 mg to about 500 mg, or about 50 mg to about 250 mg. Suitable dosages for the compounds of the invention include about 1 mg, or about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900, or about 1000 mg.

[0110] The microparticles and liquid compositions of the present invention can be administered at any suitable frequency, interval, and duration. For example, the microparticles and liquid compositions of the present invention can be administered hourly, twice, three or more times per hour, once daily, twice, three or more times per day, or once every two, three, four, five, six, or seven days to provide a desired dosage level. When the microparticles and liquid compositions of the present invention are administered more than once per day, typical intervals include 5, 10, 15, 20, 30, 45, and 60 minutes, and 1, 2, 4, 6, 8, 10, 12, 16, 20, and 24 hours. The microparticles and liquid compositions of the present invention can be administered once, twice, or three or more times over a period of 1 hour, 1-6 hours, 1-12 hours, 1-24 hours, 6-12 hours, 12-24 hours, 1 day, 1-7 days, 1 week, 1-4 weeks, 1 month, 1-12 months, 1 year or more, or even indefinitely.

[0111] V. Treatment method Another embodiment of the invention is a method of treating a subject in need thereof, comprising administering a therapeutically effective amount of a plurality of microparticles.

[0112] In another embodiment, the present invention provides a method of treating a disease or disorder in a subject in need thereof, the method comprising administering a therapeutically effective amount of a microparticle composition of the present invention, or a liquid composition of the present invention via respiratory administration, thereby treating inflammation.

[0113] In some embodiments, the disease or disorder is respiratory inflammation, acute pulmonary inflammation, chronic pulmonary distress syndrome (ARDS), chronic pulmonary obstructive disease (COPD), or toxin exposure.

[0114] The following clinical endpoints are non-limiting examples of treatment: reduction in inflammation, reduction in rate of decline in forced vital capacity (FVC) (FVC is the total volume of air exhaled during a pulmonary function test), absolute and relative increase from baseline in FVC, absolute increase from baseline in FVC (% predicted), increase in progression-free survival, reduction from baseline in St. George's Respiratory Questionnaire (SGRQ) total score (SGRQ is a health-related quality of life questionnaire divided into three components: symptoms, activity, and impact; the total score (weighted sum) ranges from 0 to 100, with lower scores indicating better health status), and relative reduction from baseline in high-resolution computed tomography (HRCT) quantitative pulmonary fibrosis (QLF) score (QLF score ranges from 0 to 100%, with higher values ​​indicating greater amounts of pulmonary fibrosis and considered poorer health status).

[0115] In some embodiments, the present invention provides a method of treating inflammation in a subject in need thereof, the method comprising administering a therapeutically effective amount of a microparticle composition of the present invention, or a liquid composition of the present invention via respiratory administration, thereby treating the inflammation.

[0116] In some embodiments, the inflammation is respiratory inflammation.

[0117] In some embodiments, respiratory administration is inhaled administration or intranasal administration.

[0118] Subjects that can be treated with the methods of the present disclosure are humans or non-human mammals, such as companion animals such as dogs, cats, rats, or livestock such as horses, donkeys, mules, goats, sheep, pigs, or cows.

[0119] CDK9 inhibitors exert their effects on inflammatory response pathways. For example, the pharmacological CDK9 inhibitor flavopiridol effectively suppresses the activation of a broad range of key inflammatory response genes in human cell cultures treated with IL-1β for 5 hours (see Figure 1). Of 67 distinct genes induced by IL-1β (out of a total of 84 NFκB target genes tested), 59 were suppressed by flavopiridol cotreatment (including the best-characterized pro-inflammatory cytokines, such as IL-1β, IL-6, and TNF). The average magnitude of suppression was greater than 86% of maximum induction. These data demonstrate that CDK9 inhibition is highly efficient in suppressing the induction of a broad range of primary inflammatory genes. Importantly, housekeeping and non-inducible genes were unaffected by short-term CDK9 inhibition, indicating the potential for reduced side effects.

[0120] Current anti-inflammatory drugs target either various components of upstream inflammatory signaling pathways or downstream effector genes (e.g., IL-1 antagonists, TNF antagonists, antioxidants, etc.). The focus has been on inhibiting specific pathways to prevent transcription of the corresponding response genes, or on inhibiting the function of individual downstream effector genes. None of these existing investigations address the rate-limiting process of transcription elongation controlled by CDK9. These existing drugs may be less effective in addressing diverse physiological pro-inflammatory challenges and may not be able to prevent the activation of a wide range of different downstream inflammatory response genes. Therefore, targeting CDK9, which controls the rate-limiting step in the activation of all inflammatory genes, would be more effective and efficient. CDK9-mediated inhibition of transcription elongation is limited to primary response inflammatory genes, and CDK9 inhibition does not affect the transcription of housekeeping and non-inducible genes within the acute inflammatory phase tested, thus not harming cells or tissues in the short term. One advantage of CDK9 inhibition is that it reduces the transcriptional elongation of inflammatory genes induced by numerous inflammatory stimuli. CDK9 can be specifically and reversibly inhibited with small molecule drugs, including flavopiridol and others disclosed herein, such as SNS-032, voruciclib, and flavopiridol. In conjunction with the formulations and methods herein, CDK9 inhibitors can be delivered locally to the site of inflammation, thereby reducing, alleviating, preventing, or alleviating the inflammatory response and its symptoms.

[0121] In some embodiments, the formulated CDK9 inhibitor is administered on a chronic basis for such pre-existing conditions, e.g., once or twice daily, weekly, every two weeks, every three weeks, monthly (e.g., four weeks), every five, six, seven, eight, nine, or ten weeks. In some embodiments, the formulated CDK9 inhibitor is administered on a chronic basis for such pre-existing conditions until the symptoms, inflammation, or other signs of the condition or disease are alleviated, ameliorated, suppressed, or otherwise affected by treatment. In some embodiments, the formulated CDK9 inhibitor is administered on a chronic basis for such pre-existing conditions for the lifetime of the subject or from the time of diagnosis or acute exacerbation of the disease or condition.

[0122] In some embodiments, the present invention provides a method of treating fibrosis in a subject in need thereof, the method comprising administering a therapeutically effective amount of a microparticle composition of the present invention, or a liquid composition of the present invention via respiratory administration, thereby treating the fibrosis.

[0123] Fibrosis that can be treated by the methods of the present invention includes, but is not limited to, respiratory fibrosis, pulmonary fibrosis, idiopathic pulmonary fibrosis, bleomycin-induced pulmonary fibrosis, fibroblastic lesions, activated fibroblast proliferation, inflammation, or myofibroblastogenesis. In some embodiments, the fibrosis is respiratory fibrosis.

[0124] Additional types of fibrosis, including but not limited to renal fibrosis, liver fibrosis, skin fibrosis, fibroblastic lesions, activated fibroblast proliferation, inflammation, myofibroblastogenesis, and non-idiopathic forms of fibrosis, can be treated with the compositions and methods of the present invention. Still other types of fibrosis that can be treated using the compositions and methods of the present invention include lung scarring, progressive pulmonary fibrosis, or fibrosis caused by drugs, chest radiation therapy, smoking, or environmental or occupational exposures known to cause pulmonary fibrosis. Pulmonary fibrosis caused by autoimmune disorders such as rheumatoid arthritis, scleroderma, Sjögren's syndrome, viral infections, and gastric reflux disease (GERD), and familial pulmonary fibrosis, can also be treated with the compositions and methods of the present invention. Exposure to environmental substances such as asbestos or silica, or bird or animal feces, can also cause pulmonary fibrosis and can be treated with the compositions and methods of the present invention.

[0125] In some embodiments, the present invention provides a method of treating respiratory fibrosis in a subject in need thereof, comprising administering to the subject via respiratory administration a microparticle composition of the present invention, or a liquid composition of the present invention, thereby treating respiratory fibrosis.

[0126] When the disease or disorder is respiratory fibrosis, the following clinical endpoints are non-limiting examples of treatment: reduction in fibrotic tissue, reduction in inflammation, reduction in fibroblast damage, reduction in proliferation of activated fibroblasts, reduction in myofibroblast production, reduction in rate of decline in forced vital capacity (FVC) (FVC is the total volume of air exhaled during a pulmonary function test), absolute and relative increase from baseline in FVC, absolute increase from baseline in FVC (% predicted), increase in progression-free survival, and St. George's Respiratory Questionnaire (SGRQ) total score. The reduction from baseline in core (SGRQ is a health-related quality of life questionnaire, divided into three components: symptoms, activities, and impact; the total score (weighted sum) ranges from 0 to 100, with lower scores indicating better health status), and the relative reduction from baseline in high-resolution computed tomography (HRCT) quantitative pulmonary fibrosis (QLF) score (QLF score ranges from 0 to 100%, with higher values ​​indicating greater amounts of pulmonary fibrosis and considered poorer health status). Non-limiting examples of clinical endpoints for fibrosis treatments, and tests that can be performed to measure the clinical endpoints, are described in the following clinical trials: NCT03733444 (clinicaltrials.gov / ct2 / splay / NCT03733444) (last accessed January 9, 2019), NCT00287729 (clinicaltrials.gov / ct2 / show / NCT00287729) (last accessed January 9, 2019), NCT00287716 (clinicaltrials.gov / ct2 / show / NCT00287716) (last accessed January 9, 2019), NCT02503657 (clinicaltrials.gov / ct2 / show / NCT02503657) (last accessed January 9, 2019), NCT00047645 (clinicaltrials.gov / ct2 / select / NCT00047645) (last accessed January 9, 2019), NCT02802345 (clinicaltrials.gov / ct2 / show / NCT02802345) (last accessed January 9, 2019), NCTO 1979952 (clinicaltrials.gov / ct2 / shel / NCT01979952) (last accessed January 9, 2019), NCT00650091 (clinicaltrials.gov / ct2 / show / NCT00650091) (last accessed January 9, 2019), NCT01335464 (clinicaltrials.gov / ct2 / show / NCT01335464) (last accessed January 9, 2019), NCT01335477 (clinicaltrials.gov / ct2 / show / NCT01335477) (last accessed January 9, 2019), NCT01366209 (clinicaltrials.gov / ct2 / show / NCT01366209) (last accessed January 9, 2019). Further non-limiting example clinical endpoints for fibrosis treatments and tests that can be performed to measure clinical endpoints are described in King et al, (2014) N Engl J Med. May 29;370(22):2083-92 and Richeldi et al, (2014) N Engl J Med. May 29;370(22):2071-82.

[0127] In some embodiments, the respiratory fibrosis is pulmonary fibrosis.

[0128] In some embodiments, respiratory administration is inhaled administration or intranasal administration.

[0129] In some embodiments, the subject is a human.

[0130] In some embodiments, the method includes administering an antifibrotic drug. The method of the present invention may include administering an antifibrotic drug or medication, such as, but not limited to, pirfenidone, idebenone, nintedanib, ifenprodil, n-acetylcysteine, penetaxin, TD139, and corticosteroids. Additional agents useful as antifibrotic agents or medications include colchicine, D-penicillamine, pirfenidone (5-methyl-1-phenyl-2-[1H]-pyridone), interferon-β1a, relaxin, lovastatin, beractant, N-acetylcysteine, keratinocyte growth factor, captopril, hepatocyte growth factor, Rho kinase inhibitor, thrombomodulin-like protein, bilirubin, PPARγ (peroxisome proliferator-activated receptor gamma) activators, imatinib, and interferon-γ. Additional agents are known from the literature, e.g., JPA No. 8-268906, WO00 / 57913, JPA No. 2002-371006, JPA No. 2003-119138, JPA No. 2005-513031, JPA No. 2005-531628, JPA No. 2006-502153, WO2006 / 068232, and Ann Intern Med. 2001;134(2):136-51. In some embodiments, the antifibrotic agent is pirfenidone, idebenone, nintedanib, ifenprodil, n-acetylcysteine, penetaxin, TD139, a corticosteroid, colchicine, D-penicillamine, pirfenidone (5-methyl-1-phenyl-2-[1H]-pyridone), interferon-β1a, relaxin, lovastatin, beractant, N-acetylcysteine, keratinocyte growth factor, captopril, hepatocyte growth factor, a Rho kinase inhibitor, a thrombomodulin-like protein, bilirubin, a PPARγ (peroxisome proliferator-activated receptor gamma) activator, imatinib, or interferon-γ.

[0131] In some embodiments, the present invention provides a method of treating inflammation or respiratory fibrosis in a subject in need thereof, comprising administering a therapeutically effective amount of a composition comprising a cyclin-dependent kinase 9 (CDK9) inhibitor. Compositions useful in the methods of the present invention include any of the compositions of the present invention. In some embodiments, the present invention provides a method of treating respiratory fibrosis in a subject in need thereof, comprising administering a therapeutically effective amount of a composition comprising a cyclin-dependent kinase 9 (CDK9) inhibitor. [Example]

[0132] VI. Working Examples material 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) was purchased from Avanti Polar Lipids (Birmingham, AL). Flavopiridol was purchased from Cayman Chemical Company (Ann Arbor, MI). L-leucine, L-isoleucine, D-(+)-glucose, calcium chloride (CaCl), Tween-20, and 100% ethanol were purchased from Sigma-Aldrich (St. Louis, MO). Phosphate-buffered saline (PBS) (1X) was purchased from Mediatech (Manassas, VA). Nitrogen gas was purchased from Praxair (Danbury, CT). Poly(tetrafluoroethylene) (PTFE) tubing was purchased from Cole-Parmer (Vernon Hills, IL). 96-well plates, 10 mL and 20 mL glass scintillation vials, and 1.00 mm ID glass capillary tubes were purchased from Fisher Scientific (Hampton, NH). The variable area flow meter was purchased from Dwyer Instruments Inc. (Michigan City, IN). Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), and 2X lysis reagent were purchased from Invitrogen (Waltham, MA). Human embryonic kidney 293 cells (HEK-293) were purchased from ATCC (Manassas, VA). Nuclear factor kappa B (NF-κB) reporter (catalog number H-60650) and One-Step Luciferase Assay System were purchased from BPS Bioscience (San Diego, CA). Tumor necrosis factor alpha (TNF-) was purchased from Peprotech (Cranbury, NJ). Ultrapure water with a resistivity of 18.2 MΩ·cm was generated using a Millipore Milli-Q system (EMD Millipore, Billerica, MA). Gaseous nitrogen and gaseous CO2 were purchased from Praxair (Danbury, CT).

[0133] Example 1. Production of flavopiridol-loaded L-isoleucine / DPPC microparticles A microfluidic piezo-cyclone device (MPCA) was designed and fabricated. It is shown schematically in Figure 1. The MPCA was designed and fabricated using components for spray drying technology and further includes a piezoelectric nebulizer disk with an orifice array and cyclone design. The piezo-actuated orifice array is a commercially available humidifier (Steiner & Martins, Inc.) consisting of a stainless steel disk with an array of 4-11 μm holes, with ceramic piezo rings fixed to the top and bottom of the array. The funnel-and-cyclone separator was designed in Solidworks® CAD software using a cylindrical-on-cone design with a tangential inlet and fabricated from PTFE.

[0134] The L-isoleucine / DPPC (ILD) feed solution was prepared using a similar method previously reported (Eedara et al. (2018) International Journal of Pharmaceutics 542(1-2), 72-81). Briefly, 27 mg of L-isoleucine was added to a 10 mL glass scintillation vial containing 3 mL of Milli-Q water. 3 mg of DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine (Avanti Polar Lipids)) was added to a 10 mL glass scintillation vial containing 7 mL of ethanol. Both vials were tightly closed and sonicated for 15 minutes until completely dissolved. After sonication, the entire contents of the DPPC vial were added to the L-isoleucine vial and sonicated for 15 minutes to create a 0.3% (w / v) ILD feed solution.

[0135] The ILD feed solution was drawn into a 10 mL syringe and loaded onto the syringe pump of the microfluidic piezo and cyclone device (MPCA) shown in Figure 1. The ILD feed solution was pumped into the PTFE tubing at 22 mL / h via the syringe pump. The end of the PTFE tubing contained a 30 AWG blunt needle hovering 1 cm directly above the piezo-actuated orifice array. The ILD solution was then dropped onto the microfluidic piezo array, which was actuated via a custom piezo driver with 30 V of power at 113 kHz. Gravity pulled large droplets from the top of the device into the array, forming a spray of atomized droplets. At the top of the acrylic box housing the entire device, a nitrogen inlet was opened, supplying nitrogen gas at 15 L / min into the enclosure. The nitrogen gas carried the atomized droplets 55 cm until they began to dry, and then a vacuum was applied to the custom PTFE funnel leading to the cyclone chamber. Within the cyclone chamber, particles collided with the sidewall, causing a loss of momentum and effectively reaching a screw-top 20 mL glass scintillation collection vial, while gas and evaporated solvent traveled to the vacuum outlet and were removed via an RV5 vacuum pump (Edwards Vacuum). The synthesis was carried out at room temperature (24 °C). The synthesis parameters are listed in Table 1. [Table 1]

[0136] To demonstrate robustness, excipient concentrations of 0.1% and 0.5% (w / v) total were also prepared using the same conditions. Flavopiridol incorporation into the formulation (ILDF:L-isoleucine, DPPC, flavopiridol) was prepared by adding 28.8 μL of 1 mg / mL flavopiridol (Cayman Chemical Company) in ethanol solution to the DPPC vial.

[0137] Example 2. Determination of the loading capacity and efficacy of flavopiridol in L-isoleucine / DPPC microparticles The loading of flavopiridol in ILDF (L-isoleucine, DPPC, flavopiridol) formulated microparticles was determined by measuring the loading of approximately 4.8 mg of particles (mp The flavopiridol concentration was determined by dispersing 1 mL of flavopiridol in 1 mL of 70% (v / v) aqueous ethanol. The dispersion was sonicated for 15 minutes until the particles were completely dissolved. The dissolved solution was loaded into a 1 mL syringe and filtered into a quartz cuvette using a 0.2 μm PTFE syringe filter (Sigma Aldrich). The filtered solution was subjected to UV-Vis spectroscopy using a Denovix DS-11 spectrometer to measure the absorbance (A) at 358 nm, a characteristic of flavopiridol. The molar extinction coefficient (ε) of flavopiridol at 358 nm was measured using a standard concentration range of 3 to 100 μM, and the concentration (c) of flavopiridol was calculated using Beer's law. The flavopiridol (m) concentration in the ILDF particles was calculated using Beer's law. f The encapsulated mass of flavopiridol was determined by multiplying c by the volume (1.00 mL) and molar mass of flavopiridol. The loading capacity (LC) of flavopiridol was calculated using the formula (1)

number

[0138] The encapsulation efficiency (EE) of flavopiridol was quantified by dividing the LC by the desired loading capacity of flavopiridol in the formulation using equation (2). (LC 所望の =0.192%(w / w)).

number

[0139] Flavopiridol-loaded ILDF particles were produced using the formulations in Table 2 using MPCA with the parameters listed in Table 1 . [Table 2]

[0140] The loading amount of flavopiridol was 0.19% as determined using UV-Vis spectroscopy. As a negative control, unloaded ILD particles were 最大= 358 nm. The encapsulation efficiency (EE) in this synthesis was quantified using equation (2) and reached 99 ± 2%.

[0141] The composition was confirmed using FTIR spectroscopy as shown in Figure 2. Briefly, <1 mg of solid L-isoleucine, DPPC, and flavopiridol starting material and the resulting ILDF formulation particles were placed on an ATR diamond crystal and compressed. FTIR spectra were recorded from 4000 to 400 cm using a Bruker Tensor 27 FTIR (Bruker Corporation). -1 4 cm over a wavenumber range of -1 The spectrum of the ILDF formulation (C) was scanned 30 times at a resolution of 1514 cm. -1 It is most similar to L-isoleucine (A), which has a star-shaped structure, with a slight presence of the characteristic C=O extension (star-shaped) of DPPC (B) described above. Based on the spectrum, L-isoleucine is the major component of the formulation, while DPPC plays a more minor role in the composition.

[0142] Example 3. Scanning electron microscope imaging of generated particles The shape and size of the microparticles were determined using a field emission scanning electron microscope (SEM) (S-4100T, Hitachi High Technologies America). The generated particles, attached to double-sided carbon tape attached to an Al stub, were coated with 10 nm of gold using a sputter coater (Ted Pella Inc.) and then transferred to the SEM vacuum chamber. The typical acceleration voltage and emission current were 2 kV and 10 μA, respectively. The SEM images were loaded into Image J, where the geometric Feret diameter (d g ) was used to determine the size of the microparticles. The aerodynamic diameter (d g ) was calculated using equation (3), where tap density was measured using an established protocol, where the measured tap density (σ) was determined over 1000 taps, and the reference density (ρ) is 1 mg / mL.

number

[0143] SEM images are shown in Figure 3. Figure 3A shows the particle morphology with (90:10) L-isoleucine / DPPC. The addition of phospholipids to the L-isoleucine formulation resulted in particle morphology composed of outwardly folded sheets resembling a "rosebud" morphology. Figure 3B ((89.25:10:0.75) L-isoleucine / DPPC / CaCl2) shows that the addition of CaCl2 to the formulation resulted in shape heterogeneity with a flat, heterogeneous morphology. Based on this result, CaCl2 was omitted from the formulation. The addition of glucose to the formulations shown in Figure 3C ((10:10:80) L-isoleucine / DPPC / glucose in 70% (v / v) ethanol:water) and Figure 3D ((10:10:80) L-isoleucine / DPPC / glucose in 80% (v / v) ethanol:water) results in solid particles with a low surface to volume ratio, characteristics that are undesirable for pulmonary delivery because they may inhibit particle dispersibility during inhalation.

[0144] Example 4. In vitro release of flavopiridol from L-isoleucine / DPPC microparticles In vitro release experiments were conducted utilizing the validated release protocol to characterize the release of flavopiridol from ILDF particles. Briefly, 5 mg of ILDF particles were placed in six 1.5 mL Eppendorf tubes containing 0.5 mL of 1X PBS / 0.2% Tween-20 aqueous solution at 37°C. To establish sink conditions, Tween-20 was added to ensure dissolution of flavopiridol in the release medium. At each selected time point, one vial was removed and centrifuged at 16,000 rpm for 10 minutes. The supernatant was collected and filtered using a 0.2 μm PTFE syringe filter. The supernatant was analyzed at 358 nm using UV-Vis spectroscopy to determine flavopiridol concentration. After all vials were analyzed for flavopiridol release, the total amount of flavopiridol released by a given time t was determined, followed by the amount of loaded flavopiridol (m fThe cumulative rate of flavopiridol was calculated using equation (4) by normalizing with .

number

[0145] The in vitro release profiles described above were quantified using a binomial model similar to that previously described, incorporating an initial burst and a Fickian diffusion release, as shown in Equation (5).

number

[0146] The first term represents the process of "burst emission" and k b is the burst constant, Θ b was expressed as the contribution of burst release. This burst process was mainly explained as interfacial diffusion, and the drug located near or on the particle surface rapidly dissolved. 19 The second term describes the diffusion of the drug from the spherical matrix under Fickian diffusion, where: [ka] is the effective diffusion coefficient, [ka] is the geometric mean particle radius, [ka] is the contribution of the diffuse emission. b +Θ d = 1 was used for mathematical completeness, representing the individual contributions to the release mechanism. The in vitro release profiles were subjected to regression analysis using a nonlinear least squares algorithm in MATLAB (Math Works, USA).

[0147] The overall release profile is shown in Figure 4. By 3 hours, 99.4% of the drug had been released. At each measurement time point, results are shown as the mean ± SD from n = 3 independent batches of ILDF microparticles. A nonlinear least-squares fit of the release profile using Equation (5) is shown in Figure 4 as the solid black line.

[0148] The quantitative kinetics were fitted using the kinetic model described above and in equation (5), and the extracted parameters are shown in Table 3. The in vitro release profile of flavopidol was consistent with the desired release kinetics for pulmonary delivery. [Table 3]

[0149] Example 5. Determining the stability of ILDF particles The stability of ILDF particles (Table 2) was measured. 1–2 mg of the freshly prepared particles described in Example 1 were evenly distributed in four different environments within 24 × 1.5 mL Eppendorf tubes: 24°C light and dark, 4°C refrigerator, and 37°C incubator. At each time point, 0.5 mL of 70% ethanol was dispensed into the vial, and the contents were sonicated for 15 minutes until complete dissolution was achieved. The solution was filtered through a 0.2 μm PTFE syringe filter into a quartz cuvette, and UV-Vis spectra were measured from 220 to 750 nm for each sample. The measured spectra were qualitatively compared to a freshly produced ILDF formulation to determine the stability of the formulation. As shown in Figure 5A, stability at 4°C mimicked storage under home refrigeration, and the spectral appearance of the ILDF particles after 4 weeks closely resembled that of the freshly produced particles. At 24°C and in the presence of room light, simulating indoor storage, stability was maintained throughout the 4-week study period, as shown in Figure 5B. Stability at 37°C is also shown in Figure 5C.

[0150] Example 6. Attenuated Total Reflectance-Fourier Transform Infrared Spectroscopy Attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR) was used to characterize particle formulations to identify components, including excipients and drug. Briefly, <1 mg of freshly prepared formulation particles were placed on an ATR diamond crystal and compressed. FTIR spectra were acquired using a Bruker Tensor 27 FTIR (Bruker Corporation, Billerica, MA). Spectra were collected over the wavenumber range 4000–400 cm. -1 The spectra were obtained over a 4 cm -1 The images were averaged over 30 scans at a wavenumber resolution of 100 Hz. Individual components of the formulation particles, such as flavopiridol, DPPC, and L-isoleucine, were acquired and compared as controls.

[0151] Example 7. In vitro bioactivity assay The bioactivity of flavopiridol released from ILDF particles was measured by luciferase reporter assay as previously described. ILDF particles (18.94 mg) containing 0.190% (w / w) flavopiridol were dissolved in DMEM containing 10% FBS to obtain 8.9 × 10 4 Flavopiridol was prepared as a 100 nM stock solution. The bioactivity of flavopiridol was measured by its ability to suppress TNF-stimulated luciferase reporter expression driven by an NF-kB-responsive promoter. Human embryonic kidney 293 (HEK293) cells (ATCC) harboring an NF-kB-driven luciferase reporter were seeded in 96-well plates (10,000 cells / well, triplicate) 24 h prior to the experiment. Cells were then treated with 100 μL of medium containing 0.6 nM recombinant human TNF-α (Peprotech) in the presence or absence of various amounts of dissolved flavopiridol formulations or blank formulation equivalents. After 24 h, 100 μL of 2X lysis buffer (Invitrogen) containing luciferase substrate was added directly to each well. Luminescence was measured using a plate reader (SpectraMax iD3, Molecular Devices). Experiments were performed in triplicate (N=3) from one batch of particles, and results were reported as mean ± SD.

[0152] The results are shown in Figure 6. Luminescence readings from samples treated with TNF-α alone were arbitrarily set to 100% and then compared to the flavopiridol-treated samples. The expected maximum luciferase activity was detected in cells treated with TNF-α alone (black bars). Blank ILDF particles (gray bars) showed no statistical difference from the TNF-α-only sample. Luciferase activation was inhibited in the presence of 300 nM soluble, unformulated flavopiridol. Cells treated with 300 nM flavopiridol in ILDF particles (red bars) also inhibited luciferase activity, showing no statistical difference from soluble flavopiridol. This result indicates that the biological activity of flavopiridol in ILDF particles is retained.

[0153] Example 8. Dispersion and aerodynamic properties of ILDF particles The dispersibility of ILDF formulation particles (Table 2) was evaluated in a benchtop assay mimicking the pulmonary inhalation process. Briefly, <1 mg of powder formulation was inserted into a 1.00 mm ID glass capillary tube and attached vertically via a three-finger clamp. A syringe-needle port was custom-fitted to the end of the capillary tube, which was inserted into the PTFE tubing by attaching the end of the PTFE tubing directly to a variable area flow meter attached to a nitrogen gas tank (see schematic Figure 7A). The nitrogen flow rate was set at 12 L / min, a rate comparable to human breathing at sea level. Following particle nebulization, samples were prepared for SEM imaging as described in the experimental section. As shown in Figure 7B, the mean interparticle distance (x) was clearly resolved between particles. - ILDF particles exhibit high dispersibility, with a particle size distribution of 7.3 ± 5.5 μm. The physical diameter of the ILDF particles was 5.5 ± 1.3 μm, as determined from SEM images. The mean aerodynamic diameter, measured according to Minne's method (Minne et al. (2008) European Journal of Pharmaceutics and Biopharmaceutics 70(3), 839-844), was determined to be 2.5 ± 0.6 μm, which corresponds to the desired aerodynamic diameter for pulmonary delivery of 1 to 5 μm. A summary of the properties is shown in Table 4. [Table 4]

[0154] To demonstrate the robustness of the benchtop dispersion method, ILDG particles were synthesized using MPCA and the parameters listed in Table 1, with a composition of 80% D-α-glucose and 10% each of DPPC and L-isoleucine, to produce sticky and cohesive particles. As shown in Figure 7C, the ILDG particles aggregated into aggregates of at least 20 particles, demonstrating near-non-dispersibility.

[0155] Example 9. Liquid Formulations for Inhalable Delivery Flavopiridol was prepared as a liquid formulation for inhalable delivery to the lung. A 15 mM citrate buffer solution was made by mixing 15 mM citric acid monohydrate (C6H8O7·H2O, formula weight (FW) 210.14) and 15 mM trisodium citrate dihydrate (C6HO7Na3·2H2O, FW 294.12) to achieve a pH of 4-5. Flavopiridol free base (C6HO7Na3·2H2O) in powder form was also prepared. 21 H 20 ClNO5, FW 401.8) was weighed and dissolved in the buffer to reach a final concentration of 120 μM. The solution was measured by UV-Vis at different time points to assess stability according to the method in Example 2. The formulated flavopidol showed long-term (>3 months) stability.

[0156] A second formulation was prepared for nebulization. A 15 mM citrate buffer solution was made by mixing 15 mM citric acid monohydrate (C6H8O7·H2O, FW 210.14) and 15 mM trisodium citrate dihydrate (C6H5O7Na3·2H2O, FW 294.12) solutions in a ratio to achieve a pH of 4.0-5.0. Sodium chloride (NaCl, FW 58.5) was dissolved in the buffer to achieve a NaCl concentration of 70 mM. Flavopiridol free base (C 21 H 20 ClNO5, FW 401.8) was weighed and dissolved in the buffer to reach a final concentration of 120 μM.

[0157] Example 10. Administration of flavopiridol in a pulmonary fibrosis model The effects of flavopiridol on lung inflammation and fibrosis were evaluated in a mouse model of pulmonary fibrosis. C57B / L6 mice were treated with bleomycin (BLM) by microspray. Seven days after BLM treatment, alveolar damage was evident in the treated mice. On day 9, the mice were divided into four groups of eight animals each. Each group received the corresponding treatment every other day, starting on day 9. On day 30, the animals were sacrificed and evaluated. One group received phosphate-buffered saline (PBS) alone. One group received 28 mg / kg nitedanib via intraperitoneal injection (ip). One group received 2.5 mg / kg flavopiridol via intraperitoneal administration, and one group received 28 mg / kg idebenone via intraperitoneal administration. Survival and lung morphology were evaluated on day 30. The results are shown in Figures 8A-8E. Administration of flavopiridol demonstrated a statistically significant difference in survival compared with BLM + vehicle controls (Figure 8A). Flavopiridol-treated mice generally maintained a heavier body weight (Figure 8B) and statistically significantly lower levels of hydroxyproline (Figure 8C) than BLM + vehicle controls. Lung tissue sections from treated animals were stained with Masson's trichrome staining protocol, and exemplary images are shown in Figure 8D. Quantification of staining is shown in Figure 8E. Flavopiridol-treated mice had a statistically significant decrease in the percentage of stained area, indicating reduced pulmonary fibrosis in flavopiridol-treated mice compared with BLM + vehicle controls.

[0158] Example 11. Formulation of Flavopiridol Hydrochloride Solution Preparation of 15 mM citrate buffer. 176.46 mg of sodium citrate dihydrate was weighed and dissolved in 40 mL of Milli Q water to prepare a 15 mM sodium citrate dihydrate solution. 115.272 mg of citric acid was weighed and dissolved in 40 mL of Milli Q water to prepare a 15 mM citric acid solution. 18 mL of the 15 mM sodium citrate dihydrate solution was mixed with 28 mL of the 15 mM citric acid solution to obtain a 15 mM citrate buffer with a pH of 4.1.

[0159] Preparation of 4.9 mM flavopiridol hydrochloride solution. 22.0 mg of flavopiridol hydrochloride was dispersed in 0.922 mL of ethanol. 9.322 mL of 15 mM citrate buffer (pH 4.1) was added to this dispersion. Flavopiridol hydrochloride was dissolved in the above solvent by heating in a 50°C water bath for 15 minutes. The flavopiridol solution was cooled to room temperature and filtered through a 0.22 μm membrane filter. The concentration of the flavopiridol hydrochloride solution was measured using UV-visible spectroscopy, confirming a 4.9 mM flavopiridol hydrochloride solution. [Table 5]

[0160] Example 12. Effervescent formulation of flavopiridol hydrochloride solution Preparation of 15 mM citrate buffer. 176.46 mg of sodium citrate dihydrate was weighed and dissolved in 40 mL of Milli Q water to prepare a 15 mM sodium citrate dihydrate solution. 115.272 mg of citric acid was weighed and dissolved in 40 mL of Milli Q water to prepare a 15 mM citric acid solution. 18 mL of the 15 mM sodium citrate dihydrate solution was mixed with 28 mL of the 15 mM citric acid solution to obtain a 15 mM citrate buffer with a pH of 4.1.

[0161] Preparation of 4.9 mM flavopiridol hydrochloride solution. 22.0 mg of flavopiridol hydrochloride was dissolved in 10.243 mL of 15 mM citrate buffer (pH 4.1) by heating in a 50°C water bath for 15 minutes. The flavopiridol solution was cooled to room temperature and filtered through a 0.22 μm membrane filter. The concentration of the flavopiridol hydrochloride solution was measured using UV-visible spectroscopy, confirming a 4.9 mM flavopiridol hydrochloride solution. [Table 6]

[0162] Example 13. Local delivery for antifibrotic therapy Our experimental model involved inducing pulmonary fibrosis in mice using bleomycin, followed by administration of inhaled flavopiridol during the early fibrotic phase of the model. Eight days after intratracheal instillation of bleomycin, mice were treated every other day with either vehicle or inhaled flavopiridol. After a 14-day treatment course, lung tissues were harvested and subjected to histological and biochemical analysis. The significant weight loss observed in bleomycin-exposed mice served as an indicator of successful fibrosis induction (Figure 9). A higher survival rate was observed in the group of bleomycin-induced mice treated with inhaled flavopiridol compared with the vehicle group (Figure 10). Furthermore, this treatment appeared to suppress the bleomycin-induced increase in hydroxyproline levels, a marker of fibrosis (Figure 11).

[0163] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, those skilled in the art will recognize that certain changes and modifications may be practiced that are within the scope of the appended claims. Furthermore, each reference provided herein is incorporated by reference in its entirety to the same extent as if each reference were individually incorporated by reference. In the event of a conflict between this application and a reference provided herein, this application shall control.

Claims

1. A particulate composition comprising multiple fine particles, wherein each fine particle is Hydrophobic amino acids or hydrophobic peptides, or combinations thereof, Lipids, and The microparticle composition comprising a cyclin-dependent kinase 9 (CDK9) inhibitor.

2. The fine particle composition according to claim 1, wherein the hydrophobic amino acid and / or hydrophobic peptide comprises alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, tryptophan, L-isoleucine, or a combination thereof.

3. The fine particle composition according to claim 1, wherein the hydrophobic peptide comprises trileucine.

4. The fine particle composition according to claim 1, wherein the lipid is a phosphocholine lipid.

5. The fine particle composition according to claim 3, wherein the lipid is a phosphocholine lipid.

6. The fine particle composition according to claim 1 or 4, wherein the lipid is 1,2-diheptanoyl-sn-glycero-3-phosphocholine (DHPC), 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimiristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), or a combination thereof.

7. The fine particle composition according to claim 1 or 5, wherein the lipid is 1,2-difeptanoyl-sn-glycero-3-phosphocholine (DHPC), 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimiristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), or a combination thereof.

8. The fine particle composition according to claim 2, wherein the L-isoleucine is present in a ratio of 1000:1 to 1:10 (w / w), 100:1 to 1:1 (w / w), or about 9:1 (w / w) relative to the lipid.

9. The particulate composition according to claim 7, wherein the L-isoleucine is present in a ratio of 1000:1 to 1:10 (w / w), 100:1 to 1:1 (w / w), or about 9:1 (w / w) relative to the lipid.

10. The particulate composition according to claim 1, wherein the CDK9 inhibitor is flavopyridol, flavopyridol HCl, SNS-032, borcilib, pharmaceutically acceptable salts thereof, or derivatives thereof.

11. The particulate composition according to claim 9, wherein the CDK9 inhibitor is flavopyridol, flavopyridol HCl, SNS-032, or borcilib, a pharmaceutically acceptable salt thereof, or a derivative thereof.

12. The fine particle composition according to claim 1, wherein the lipid is present in a ratio of 1000:1 to 1:1 (w / w), 100:1 to 10:1 (w / w), or 50:1 (w / w) with respect to the CDK9 inhibitor.

13. The fine particle composition according to claim 11, wherein the lipid is present in a ratio of 1000:1 to 1:1 (w / w), 100:1 to 10:1 (w / w), or 50:1 (w / w) with respect to the CDK9 inhibitor.

14. The fine particle composition according to claim 13, wherein the hydrophobic peptide is L-isoleucine, and the L-isoleucine:DPPC:flavopyridol ratio is about 89.8:10.0:0.2 (w / w).

15. The particulate composition according to claim 1, wherein each particulate has an average geometric diameter of 1 to 10 microns.

16. The particulate composition according to claim 14, wherein each particulate has an average geometric diameter of 1 to 10 microns.

17. A liquid composition, A citrate buffer having a pH of 2 to 7, and The liquid composition comprising a cyclin-dependent kinase 9 (CDK9) inhibitor.

18. The citrate buffer solution contains citrate monohydrate and trisodium citrate dihydrate, has a pH of 4 to 5, and The liquid composition according to claim 17, wherein the CDK9 inhibitor is flavopidol, SNS-032, borsicrib, or a derivative thereof, or a pharmaceutically acceptable salt thereof.

19. The liquid composition according to claim 17, further comprising the cyclin-dependent kinase 9 (CDK9) inhibitor at a concentration of about 120 μM, sodium chloride at a concentration of about 70 mM, and the citrate buffer having a pH of 4 to 5.

20. Use of the particulate composition according to claim 1 or the liquid composition according to claim 17 in the manufacture of a pharmaceutical for respiratory administration.

21. Use of the particulate composition according to claim 16 in the manufacture of a pharmaceutical product for respiratory administration.

22. Use of the liquid composition according to claim 19 in the manufacture of a pharmaceutical product for respiratory administration.

23. Use of the particulate composition according to claim 1 or the liquid composition according to claim 17 in the manufacture of a pharmaceutical for treating inflammation in a subject requiring it via respiratory administration.

24. Use of the particulate composition according to claim 16 in the manufacture of a pharmaceutical for treating inflammation in a subject requiring it via respiratory administration.

25. Use of the liquid composition according to claim 19 in the manufacture of a pharmaceutical for treating inflammation in a subject requiring it via respiratory administration.

26. The inflammation is respiratory inflammation, respiratory fibrosis, or pulmonary fibrosis, and The use according to claim 23, wherein the respiratory administration is by inhalation or nasal administration.

27. ​​The inflammation is respiratory inflammation, respiratory fibrosis, or pulmonary fibrosis, and The use according to claim 24, wherein the respiratory administration is by inhalation or nasal administration.

28. The inflammation is respiratory inflammation, respiratory fibrosis, or pulmonary fibrosis, and The use according to claim 25, wherein the respiratory administration is by inhalation or nasal administration.

29. The use according to claim 23, wherein the pharmaceutical agent is to be used in combination with an antifibrotic agent.

30. The use according to claim 24, wherein the pharmaceutical agent is to be used in combination with an antifibrotic agent.

31. The use according to claim 25, wherein the pharmaceutical agent is to be used in combination with an antifibrotic agent.

32. The use according to claim 29, wherein the antifibrotic agent is pirfenidone, idebenone, nintedanib, ifenprodil, n-acetylcysteine, penetaxin, TD139, corticosteroid, colchicine, D-penicillamine, pirfenidone (5-methyl-1-phenyl-2-[1H]-pyridone), interferon-β1a, relaxin, lovastatin, belactant, N-acetylcysteine, keratinocyte growth factor, captopril, hepatocyte growth factor, Rho kinase inhibitor, thrombomodulin-like protein, bilirubin, PPARγ (peroxisome proliferator-activated receptor gamma) activator, imatinib, or interferon-γ.

33. The use according to claim 30, wherein the antifibrotic agent is pirfenidone, idebenone, nintedanib, ifenprodil, n-acetylcysteine, penetaxin, TD139, corticosteroid, colchicine, D-penicillamine, pirfenidone (5-methyl-1-phenyl-2-[1H]-pyridone), interferon-β1a, relaxin, lovastatin, belactant, N-acetylcysteine, keratinocyte growth factor, captopril, hepatocyte growth factor, Rho kinase inhibitor, thrombomodulin-like protein, bilirubin, PPARγ (peroxisome proliferator-activated receptor gamma) activator, imatinib, or interferon-γ.

34. The use according to claim 31, wherein the antifibrotic agent is pirfenidone, idebenone, nintedanib, ifenprodil, n-acetylcysteine, penetaxin, TD139, corticosteroid, colchicine, D-penicillamine, pirfenidone (5-methyl-1-phenyl-2-[1H]-pyridone), interferon-β1a, relaxin, lovastatin, belactant, N-acetylcysteine, keratinocyte growth factor, captopril, hepatocyte growth factor, Rho kinase inhibitor, thrombomodulin-like protein, bilirubin, PPARγ (peroxisome proliferator-activated receptor gamma) activator, imatinib, or interferon-γ.

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