Inhalable liposomal sustained release composition for use in treating pulmonary diseases

Abstract

Provided is a liposomal sustained-release composition for use in treatment of pulmonary disease. The liposomal sustained release composition comprises a liposome that includes a polyethylene glycol (PEG)-modified lipid and encapsulates a tyrosine kinase inhibitor. Tyrosine kinase inhibitor is stably entrapped in the liposome, and the resulting liposomal drug formulation can be aerosolized or nebulized for administration via inhalation. This aerosolized liposomal drug formulation yields consistent pharmacokinetic and pharmacodynamic profiles while achieving desired efficacy and safety.

Claims

1. A liposomal sustained-release composition for use in the treatment of a pulmonary disease via inhalation, comprising liposomes having entrapped nintedanib, wherein each liposome comprises: a lipid bilayer comprising one or more phospholipids, a sterol, and a polyethylene glycol (PEG)-modified lipid; and an aqueous interior encompassed by the lipid bilayer and entrapping the nintedanib; wherein the liposomes have a mean particle diameter between about 50 nm and about 400 nm; and wherein the composition has a ratio of nintedanib-to-phospholipid from about 0.1 mol nintedanib/mol phospholipid to about 2.5 mol nintedanib/mol phospholipid.

2. The liposomal sustained release composition of claim 1, wherein the composition has a nintedanib-to-phospholipid ratio selected from a range of about 100 g nintedanib/mol phospholipid to about 1,000 g nintedanib/mol phospholipid, and about 200 g nintedanib/mol phospholipid to about 1000 g nintedanib/mol phospholipid.

3. The liposomal sustained release composition of claim 1, wherein the composition has a lipid concentration ranging from about 1 mM to about 25 mM.

4. The liposomal sustained release composition of claim 1, wherein the concentration of the nintedanib ranges from about 1 mg/mL to about 15 mg/mL.

5. The liposomal sustained release composition of claim 4, wherein the PEG-modified lipid is present in an amount less than 6 mol % on the basis of the total phospholipids and sterol.

6. The liposomal sustained release composition of claim 1, wherein the pulmonary disease is selected from the group consisting of pulmonary fibrosis, non-small cell lung cancer, and systemic sclerosis.

7. The liposomal sustained release composition of claim 1, wherein the PEG-modified lipid has a PEG moiety with an average molecular weight ranging from about 1,000 g/mol to about 5,000 g/mol.

8. The liposomal sustained release composition of claim 7, wherein the PEG-modified lipid is 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)] (DSPE-PEG).

9. The liposomal sustained release composition of claim 8, wherein the one or more phospholipids is a neutral phospholipid and the DSPE-PEG of the liposome is present in an amount ranging from about 0.001 mol % to about 5 mol % on the basis of total phospholipid and sterol.

10. The liposomal sustained release composition of claim 1, wherein the one or more phospholipids is selected from the group consisting of hydrogenated soy phosphatidylcholine (HSPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), phosphatidylethanolamine lipid, and combinations thereof.

11. The liposomal sustained release composition of claim 1, wherein the molar ratio of the total phospholipids to sterol ranges from about 1:1 to about 3:2.

12. The liposomal sustained release composition of claim 1, wherein the nintedanib is encapsulated in the aqueous interior of the liposome via a transmembrane pH gradient-driven remote loading method using a trapping agent.

13. The liposomal sustained release composition of claim 12, wherein the trapping agent is ammonium sulfate.

14. An aerosolized composition of particles for use in the treatment of a pulmonary disease, comprising the liposomal sustained release composition of claim 1.

15. The aerosolized composition of particles of claim 14, wherein the particles have a mass median aerodynamic diameter of from about 0.5 μm to about 5 μm.

16. The aerosolized composition of particles of claim 14, wherein the liposomes with the entrapped nintedanib have a release rate between about 0.5% and about 25% of total nintedanib per hour in the lung with complete release of the entrapped nintedanib occurring after a minimum of about 12 hours or a minimum of about 24 hours.

17. The aerosolized composition of particles of claim 14, which is administered at an amount of 0.001 mg/kg to 50 mg/kg.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a graph showing the storage stability of 300 mM ammonium sulfate (A.S.) liposomal nintedanib at 4° C.;

(2) FIG. 2 is a graph showing the particle size of 300 mM ammonium sulfate (A.S.) liposomal nintedanib stored at 4° C.;

(3) FIG. 3 is a graph depicting the in vitro release profiles of liposomal TKI formulations in simulated lung fluid (SLF); AS=ammonium sulfate; open circle=liposomal TKI formulation comprising 300 mM AS, 3.74 mg/mL nintedanib, and 0.45 mol % PEG-modified lipid; closed square=liposomal TKI formulation comprising 300 mM AS, 3.74 mg/mL nintedanib, and 1.75 mol % PEG-modified lipid; error bars represent the standard deviation;

(4) FIG. 4 is a graph depicting the retention of nintedanib (Nin) in lung tissue of healthy mice; AS=ammonium sulfate; open circle=3.74 mg/mL free form nintedanib (Nin); open triangle=liposomal TKI formulation comprising 300 mM AS, 3.74 mg/mL nintedanib, and 0.45 mol % PEG-modified lipid; closed triangle=liposomal TKI formulation comprising 300 mM AS, 3.74 mg/mL nintedanib, and 1.75 mol % PEG-modified lipid; error bars represent the standard deviation;

(5) FIG. 5 is a graph depicting the retention of nintedanib (Nin) in mouse lung tissue in an IPF animal model; the figure compares the retention of nintedanib administered intratracheally (IT) in a liposomal TKI formulation comprising 300 mM AS, 3.74 mg/mL nintedanib, and 3 mol % PEG-modified lipid (closed circle) to the retention of nintedanib administered orally (free nintedanib) (open triangle); AS=ammonium sulfate; error bars represent the standard deviation.

DETAILED DESCRIPTION OF THE EMBODIMENTS

(6) As employed above and throughout the disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings.

(7) As used herein, the singular forms “a”, “an” and “the” include the plural reference unless the context clearly indicates otherwise.

(8) All numbers herein may be understood as modified by “about,” which, when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±10%, preferably ±5%, more preferably ±1%, and even more preferably ±0.1% from the specified value, as such variations are appropriate to obtain a desired amount of liposomal drug, unless otherwise specified.

(9) The term “treating,” “treated,” or “treatment” as used herein includes preventive (e.g., prophylactic), palliative, and curative uses or results.

(10) The term “subject” includes a vertebrate having cancer or other disease(s) affecting pulmonary function. In some embodiments, the subject is a warm-blooded animal, such as a mammal, including a human.

(11) As used herein, the term drug to lipid ratio (“D/L ratio”) refers to the ratio of tyrosine kinase inhibitor to total phospholipid content. The tyrosine kinase inhibitor content of free and liposomal drug was determined by UV-Vis absorbance measurements. The phospholipid content, or concentration, of liposome and liposomal drug was determined by assaying the phosphorus content of liposome and liposomal drug samples using a phosphorus assay (adapted from G. Rouser et al., Lipids 1970, 5, 494-496). The D/L ratio can be expressed in terms of either g/mol or mol/mol. For example, g/mol of liposomal nintedanib can be converted to mol/mol of liposomal nintedanib by dividing the g/mol value by 539.62 to yield the mol/mol value.

(12) As used herein, the term mol % means the percentage of moles of a given component of a mixture relative to the total moles of that mixture.

(13) Liposome

(14) The term “liposome” as used herein refers to a particle characterized by having an aqueous interior space sequestered from an outer medium by a membrane of one or more bilayer membranes forming a vesicle. Bilayer membranes of liposomes are typically formed by lipids, i.e., amphiphilic molecules of synthetic or natural origin that comprise spatially separated hydrophobic and hydrophilic domains. In certain embodiments of the present disclosure, the term “liposomes” refers to small unilamellar vesicle (SUV) liposomes in which one lipid bilayer forms the membrane.

(15) In general, liposomes comprise a lipid mixture typically including one or more lipids selected from the group consisting of: dialiphatic chain lipids, such as phospholipids, diglycerides, dialiphatic glycolipids, single lipids such as sphingomyelin and glycosphingolipid, steroids such as cholesterol and derivatives thereof, and combinations thereof.

(16) Examples of phospholipids according to the present disclosure include, but are not limited to, 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1-palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine (PSPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), hydrogenated soy phosphatidylcholine (HSPC), 1,2-dimyristoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (sodium salt) (DMPG), 1,2-dipalmitoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (sodium salt) (DPPG), 1-palmitoyl-2-stearoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (sodium salt) (PSPG), 1,2-distearoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (sodium salt) (DSPG), 1,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DOPG), 1,2-dimyristoyl-sn-glycero-3-phospho-L-serine (sodium salt) (DMPS), 1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine (sodium salt) (DPPS), 1,2-distearoyl-sn-glycero-3-phospho-L-serine (sodium salt) (DSPS), 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), 1,2-dimyristoyl-sn-glycero-3-phosphate (sodium salt) (DMPA), 1,2-dipalmitoyl-sn-glycero-3-phosphate (sodium salt) (DPPA), 1,2-distearoyl-sn-glycero-3-phosphate (sodium salt) (DSPA), 1,2-dioleoyl-sn-glycero-3-phosphate (sodium salt) (DOPA), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dipalmitoyl-sn-glycero-3-phospho-(1′-myo-inositol) (ammonium salt) (DPPI), 1,2-distearoyl-sn-glycero-3-phosphoinositol (ammonium salt) (DSPI), 1,2-dioleoyl-sn-glycero-3-phospho-(1′-myo-inositol) (ammonium salt) (DOPI), cardiolipin, L-a-phosphatidylcholine (EPC), and L-α-phosphatidylethanolamine (EPE).

(17) Polyethylene Glycol (PEG)-Modified Lipid

(18) A polyethylene glycol-modified lipid comprises a polyethylene glycol moiety conjugated with a lipid. In some embodiments, the PEG moiety has a molecular weight from about 1,000 to about 20,000 daltons. In some embodiments, the PEG-modified lipid is mixed with the phospholipids to form liposomes with one or more bilayer membranes. In some embodiments, the amount of PEG-modified lipid ranges from 0.0001 mol % to 40 mol %, optionally from 0.001 mol % to 30 mol %, and optionally from 0.01 mol % to 20 mol %, on the basis of the total phospholipids and sterol. In some embodiments, the amount of PEG-modified lipid is no more than 6 mol %, no more than 5 mol %, no more than 3 mol %, or no more than 2 mol %, on the basis of the total phospholipids and sterol. In some embodiments, the PEG-modified lipid has a PEG moiety with an average molecular weight ranging from 1,000 g/mol to 5,000 g/mol. In some embodiments, the PEG-modified lipid is phosphatidylethanolamine linked to a polyethylene glycol group (PEG-PE). In some embodiments, PEG-modified phosphatidylethanolamine is 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)] (DSPE-PEG).

(19) Liposomal Sustained Release Compositions

(20) The terms “liposomal drug formulation” and “liposomal sustained release composition” are interchangeably used in the present disclosure. The liposomal sustained release composition in accordance with the present disclosure includes, but is not limited to, liposomes with entrapped tyrosine kinase inhibitor prepared by entrapping the tyrosine kinase inhibitor in the aqueous interior of the liposome via a transmembrane pH gradient-driven remote loading method. In some embodiments, the transmembrane pH gradient is created by using a trapping agent for remote loading of the tyrosine kinase inhibitor into liposomes. In various embodiments, the trapping agent is selected from the group consisting of ammonium sulfate, ammonium mesylate, ammonium tosylate, triethylammonium sucrose octasulfate, and combinations thereof.

(21) In certain embodiments, the liposome with the entrapped tyrosine kinase inhibitor comprises (a) a lipid bilayer comprising one or more phospholipids, a sterol, and a polyethylene glycol (PEG)-modified lipid, including but not limited to a PEG-modified phosphatidylethanolamine; and (b) an aqueous interior encompassed by the lipid bilayer entrapping a tyrosine kinase inhibitor.

(22) In some embodiments, the one or more phospholipids is a neutral phospholipid. In some embodiments, the PEG-modified lipid is D SPE-PEG and the amount of DSPE-PEG in the liposome ranges from 0.001 mol % to 5 mol % on the basis of the total phospholipid and sterol.

(23) In some embodiments, the liposomes with the entrapped tyrosine kinase inhibitor have a mean particle diameter between 50 nm and 400 nm.

(24) The term “tyrosine kinase inhibitors” (TKIs) refers to one or more groups of substances inhibiting tyrosine kinases, enzymes responsible for the activation of many proteins by adding a phosphate group to the protein (phosphorylation). In some embodiments, the term TKI includes but is not limited to indoline compound. In some embodiments, the TKI is a substituted indoline compound such as nintedanib or pharmaceutically acceptable salts thereof.

(25) In some embodiments, the tyrosine kinase inhibitor in accordance with the present disclosure is selected from the group consisting of nintedanib, saracatinib, axitinib, cabozantinib, pazopanib, vandetanib, regorafenib, sorafenib, sunitinib, imatinib, bosutinib, dasatinib, nilotinib, ponatinib, afatinib, erlotinib, gefitinib, lapatinib, crizotinib and ruxolitinib.

(26) In some embodiments, the tyrosine kinase inhibitor in accordance with the present disclosure is nintedanib, wherein 180.6 mg of nintedanib esylate is equivalent to 150 mg of nintedanib base.

(27) In some embodiments, the tyrosine kinase inhibitor in accordance with the present disclosure is a substituted indoline compound, referring to an indole compound with one or more substituted groups, which target vascular endothelial growth factor receptor (VEGFR), fibroblast growth factor receptor (FGFR), and platelet derived growth factor receptor (PDGFR).

(28) In some embodiments, the substituted indoline compound is selected from the group consisting of:

(29) (a) 3-Z-[1-(4-(piperidin-1-yl-methyl)-anilino)-1-phenyl-methylene]-6-ethoxycarbonyl-2-indolinone,

(30) (b) 3-Z-[(1-(4-(piperidin-1-yl-methyl)-anilino)-1-phenyl-methylene]-6-carbamoyl-2-indolinone,

(31) (c) 3-Z-[1-(4-(piperidin-1-yl-methyl)-anilino)-1-phenyl-methylene]-6-methoxycarbonyl-2-indolinone,

(32) (d) 3-Z-[1-(4-(dimethylaminomethyl)-anilino)-1-phenyl-methylene]-6-ethoxycarbonyl-2-indolinone,

(33) (e) 3-Z-[1-(4-((2,6-dimethyl-piperidin-1-yl)-methyl)-anilino)-1-phenyl-methylene]-6-ethoxycarbonyl-2-indolinone,

(34) (f) 3-Z-[1-(4-(N-(2-dimethylamino-ethyl)-N-acetyl-amino)-anilino)-1-phenyl-methylene]-6-ethoxycarbonyl-2-indolinone,

(35) (g) 3-Z-[1-(4-(N-(3-dimethylamino-propyl)-N-acetyl-amino)-anilino)-1-phenyl-methylene]-6-ethoxycarbonyl-2-indolinone,

(36) (h) 3-Z-[1-(4-(N-(2-dimethylamino-ethyl)-N-methylsulphonyl-amino)-anilino)-1-phenyl-methylene]-6-ethoxycarbonyl-2-indolinone,

(37) (i) 3-Z-[1-(4-(dimethylaminomethyl)-anilino)-1-phenyl-methylene]-6-methoxycarbonyl-2-indolinone,

(38) (j) 3-Z-[1-(4-(N-acetyl-N-dimethylaminocarbonylmethyl-amino)-anilino)-1-phenyl-methylene]-6-methoxycarbonyl-2-indolinone,

(39) (k) 3-Z-[1-(4-ethylaminomethyl-anilino)-1-phenyl-methylene]-6-methoxycarbonyl-2-indolinone,

(40) (l) 3-Z-[1-(4-(1-methyl-imidazol-2-yl)-anilino)-1-phenyl-methylene]-6-methoxycarbonyl-2-indolinone,

(41) (m) 3-Z-[1-(4-(N-dimethylaminomethylcarbonyl-N-methyl-amino)-anilino)-1-phenyl-methylene]-6-methoxycarbonyl-2-indolinone,

(42) (n) 3-Z-[1-(4-(N-(2-dimethylamino-ethyl)-N-methylsulphonyl-amino)-anilino)-1-phenyl-methylene]-6-methoxycarbonyl-2-indolinone,

(43) (o) 3-Z-[1-(4-(N-(3-dimethylamino-propyl)-N-methylsulphonyl-amino)-anilino)-1-phenyl-methylene]-6-methoxycarbonyl-2-indolinone,

(44) (p) 3-Z-[1-(4-(N-dimethylaminocarbonylmethyl-N-methylsulphonyl-amino)-anilino)-1-phenyl-methylene]-6-methoxycarbonyl-2-indolinone,

(45) (q) 3-Z-[1-(4-(N4(2-dimethylamino-ethyl)-carbonyl)-N-methyl-amino)-anilino)-1-phenyl-methylene]-6-methoxycarbonyl-2-indolinone,

(46) (r) 3-Z-[1-(4-(N-(2-dimethylamino-ethyl)-N-acetyl-amino)-anilino)-1-phenyl-methylene]-6-methoxycarbonyl-2-indolinone,

(47) (s) 3-Z-[1-(4-methylaminomethyl-anilino)-1-phenyl-methylene]-6-methoxycarbonyl-2-indolinone,

(48) (t) 3-Z-[1-(4-(N-((4-methyl-piperazin-1-yl)-methylcarbonyl)-N-methyl-amino)-anilino)-1-phenyl-methylene]-6-methoxycarbonyl-2-indolinone, and

(49) (u) methyl (3Z)-3-[[4-[methyl-[2-(4-methylpiperazin-1-yl)acetyl]amino]anilino]-phenylmethylidene]-2-oxo-1H-indole-6-carboxylate.

(50) Aerosolized Particles of the Liposomal Sustained Release Composition

(51) The liposomal sustained release composition in accordance with the present disclosure can be adapted for the preparation of an aerosolized composition of particles. In some embodiments, the liposome with the entrapped tyrosine kinase inhibitor comprises (a) a lipid bilayer comprising a phospholipid, a sterol, and a PEG-modified phosphatidylethanolamine; and (b) an aqueous interior encompassed by the lipid bilayer and containing a tyrosine kinase inhibitor, and wherein drug leakage of the tyrosine kinase inhibitor from the liposome after aerosolization is less than 10%.

(52) In some embodiments, the liposomal sustained release composition of tyrosine kinase inhibitor for use according to the present disclosure has a lipid concentration ranging from 1 mM to 25 mM. In certain embodiments, the liposomal sustained release composition of tyrosine kinase inhibitor for use according to the present disclosure has a concentration of the tyrosine kinase inhibitor ranging from 1 mg/mL to 15 mg/mL. In various embodiments, the liposomal sustained release composition of tyrosine kinase inhibitor for use according to the present disclosure has a drug-to-phospholipid ratio ranging from 100 g drug/mol phospholipid to 1,000 g drug/mol phospholipid, optionally 500 g drug/mol phospholipid to 1000 g drug/mol phospholipid, and optionally 0.01 mol drug/mol phospholipid to 2.5 mol drug/mol phospholipid, 0.05 mol drug/mol phospholipid to 2 mol drug/mol phospholipid, 0.1 mol drug/mol phospholipid to 1.5 mol drug/mol phospholipid and 0.5 mol drug/mol phospholipid to 1.5 mol drug/mol phospholipid.

(53) In some embodiments, the free tyrosine kinase inhibitor of the liposomal sustained release composition is present in an amount less than 50%, optionally ranging from 0.5% to 40%, from 1% to 30%, from 2% to 20%, or from 3% to 10% of the total amount (i.e., free plus liposome-encapsulated) of the tyrosine kinase inhibitor of the liposomal sustained release composition.

(54) In some embodiments, an aerosolized composition of particles is generated from the liposomal sustained release composition by using a nebulizer. In certain embodiments, the nebulizer is selected from the group consisting of an air-jet nebulizer, an ultrasonic nebulizer, and a vibrating mesh nebulizer.

(55) In some embodiments, the aerosolized composition of particles has a mass median aerodynamic diameter between 0.5 μm and 5 μm.

(56) In some embodiments, the aerosolized composition of particles is administered at an amount of 0.001 mg/kg to 50 mg/kg, 0.005 mg/kg to 40 mg/kg, 0.01 mg/kg to 30 mg/kg, 0.05 mg/kg to 20 mg/kg, 0.1 mg/kg to 10 mg/kg or 0.5 mg/kg to 5 mg/kg per body weight of a subject by pulmonary delivery to the subject to achieve a release rate between about 0.5% and 25% of the administered tyrosine kinase inhibitor dose per hour, with complete release of the tyrosine kinase inhibitor occurring after a minimum of about 12 hours.

(57) Pulmonary Diseases

(58) Pulmonary diseases in accordance with the present disclosure are embodied in non-infectious pulmonary diseases. The non-infectious pulmonary diseases refer to lung related disorders excluding pulmonary infection caused by gram negative bacterium. In some embodiments, the pulmonary diseases include, but are not limited to: pulmonary fibrosis (such as idiopathic pulmonary fibrosis or radiation therapy-induced fibrosis), lung cancer (such as non-small cell lung cancer), or systemic sclerosis (also known as scleroderma). The term “Idiopathic Pulmonary Fibrosis” (IPF) refers to a type of chronic lung disease characterized by a progressive and irreversible decline in lung function. IPF belongs to a family of lung disorders known as interstitial lung diseases (ILDs) or, more accurately, diffuse parenchymal lung diseases. Within this category of diffuse lung diseases, IPF belongs to the subgroup known as idiopathic interstitial pneumonia (IIP). There are seven distinct IIPs, differentiated by specific clinical features and pathological patterns. IPF is the most common form of IIP. Symptoms of IPF typically include gradual onset of shortness of breath and a dry cough. Other symptoms of IPF may include feeling tired and nail clubbing. Exercise-induced breathlessness and chronic dry cough may be prominent symptoms of IPF as well. Complications of IPF include pulmonary hypertension, heart failure, pneumonia, and pulmonary embolism.

(59) The disclosure will be further described with reference to the following specific, non-limiting examples.

EXAMPLES

(60) The following examples illustrate the preparation and properties of certain embodiments of the present disclosure.

Example 1

(61) Preparation of Liposomal Tyrosine Kinase Inhibitor (TKI)

(62) I. Preparation of Empty Liposomes

(63) Liposomes were prepared via the thin-film hydration method or solvent injection method. The process for preparing empty liposomes by thin-film hydration method comprises the following steps: 1. weighing out a lipid mixture of phospholipids and cholesterol at a predetermined molar ratio either in the presence or absence of DSPE-PEG2000 and adding the lipid mixture to 10 mL of chloroform in a round-bottom flask; 2. placing the flask in a rotary evaporator at 60° C. and stirring the flask to dissolve the lipid mixture, followed by putting the flask under vacuum while stirring to evaporate the chloroform to obtain a dried lipid film; 3. preparing a trapping agent solution (e.g., ammonium sulfate (A.S.)) by adding a trapping agent to 5 mL of distilled water and vortexing the solution to dissolve the powder; 4. adding the trapping agent solution to the dried lipid film and stirring it at 60° C. for 30 minutes to form a proliposome solution; 5. freeze-thawing the proliposome solution 5 times with liquid nitrogen and a 60° C. water bath to obtain a liposome sample; 6. extruding the liposome sample 10 times through a 0.2 μm polycarbonate membrane at 60° C., then 10 times through a 0.1 μm polycarbonate membrane at 60° C.; 7. dialyzing the extruded liposome sample to remove free trapping agent, followed by adding the sample to a dialysis bag (MWCO: 25 kDa), sealing the bag, and stirring the dialysis bag in 100× volume of a 9.4% (w/v) sucrose solution; and further replacing the sucrose solution after 1 hour and after 4 hours, and letting the dialysis bag stir overnight; and 8. sterilizing the dialyzed liposome sample by filtering it through a 0.45 μm polytetrafluoroethylene (PTFE) membrane to obtain the empty liposomes.

(64) The various liposome formulations that were prepared for forming the empty liposomes to be used for loading tyrosine kinase inhibitor (e.g. nintedanib) are listed in Table 1 below (all liposome formulations were prepared in a 9.4% (w/v) sucrose solution). For liposome formulations comprising both DSPC and DPPE lipids, steps #4-6 were performed at 70° C. instead of 60° C. due to DPPE having a relatively high T.sub.m of 63° C. The mean particle diameter of these liposomes was approximately 120 nm.

(65) TABLE-US-00001 TABLE 1 Liposome Formulations Molar ratio Formulation Phospholipids Trapping no. HSPC DSPC DPPE DSPE-PEG2000 Cholesterol agent 1 3 0 0 0 2 300 mM 2 3 0 0 0.045 2 A.S. 3 3 0 0 0.1 2 4 3 0 0 0.15 2 5 3 0 0 0.26 2 6 0 3 0 0.045 2 7 0 3 0 0.26 2 8 0 3 3 0.09 4 9 0 3 3 0.526 4
II. Drug Loading of TKI into Liposomes to Obtain Liposomal TKI

(66) The following method is an exemplary protocol for the encapsulation of TKI (i.e. nintedanib) in liposomes by remote loading, which comprises the steps of: 1. preparing solutions of 9.4% (w/v) sucrose and 9.4% (w/v) sucrose buffer containing 31 mg/mL L-histidine (L-His), pH 6.5; 2. preparing a solution of 15 mg/mL nintedanib ethanesulfonate in 9.4% (w/v) sucrose and briefly heating the solution at 60° C. to obtain a stock solution comprising nintedanib (hereafter denoted as Nin stock solution); 3. mixing together in a conical tube: (a) empty liposomes as prepared by the process according to Part I of Example 1 (in some embodiments, with the condition of DSPC:cholesterol:DSPE-PEG2000 at a molar ratio of 3:2:0.045, 300 mM ammonium sulfate (A.S.), and 58.27 mM lipid concentration), (b) a 9.4% (w/v) sucrose solution, (c) a 9.4% (w/v) sucrose buffer containing 31 mg/mL L-His, pH 6.5, and (d) Nin stock solution, to obtain a loading solution. In some embodiments, the loading solution has a D/L ratio of 500 g/mol. In one embodiment, the empty liposomes, sucrose solution, sucrose buffer, and Nin stock solution are mixed together as provided in Table 2, below; 4. shaking the loading solution vigorously in a 60° C. water bath and incubating at 60° C. for 15 minutes to form the liposomal drug sample, followed by placing the liposomal drug sample on ice for a few minutes; 5. dialysis by adding the chilled liposomal drug sample into a dialysis bag (MWCO: 25 kDa), sealing the bag, and stirring the dialysis bag in 100× volume of a 9.4% (w/v) sucrose solution; and replacing the sucrose solution after 1 hour and after 4 hours, and letting the dialysis bag stir overnight; and 6. determining the drug encapsulation (i.e. loading efficiency) of the final sample using size-exclusion column chromatography and HPLC analysis (drug concentrations of all samples, liposomal or total form, were determined by absorbance measurements at 388 nm).

(67) TABLE-US-00002 TABLE 2 Exemplary conditions for remote loading of TKI into liposome Final 9.4% (w/v) target sucrose Final lipid Empty 9.4% buffer with 15 mg/mL pH of concentra- liposome (w/v) 31 mg/mL L- Nin stock loading tion (mM) volume sucrose His, pH 6.5 solution solution 15 772.2 μL 277.8 μL 150 μL 1,800 μL 6.0 to 6.5

Example 2

(68) Entrapment of TKI in Liposome

(69) Embodied TKI, nintedanib, was loaded into empty liposomes with a mean particle diameter of approximately 120 nm and comprising various phospholipids (e.g., HSPC, DSPC, DSPC/DPPE, or combinations thereof), with 300 mM A.S. as the trapping agent, and the indicated content of PEG-modified phospholipid (e.g., 0.9 mol % DSPE-PEG2000) according to the method described in Example 1, section II.

(70) Table 3 summarizes the nintedanib encapsulation results for these active loading experiments. The 300 mM A.S. empty liposomes comprising 0.9 mol % PEG-modified lipid encapsulated a D/L ratio of at least 0.9 mol/mol of nintedanib, regardless of the PC lipids or combination of PC/PE lipids in the empty liposome formulation. Therefore, for liposomes comprising the same trapping agent (ammonium sulfate) and PEG content, there is flexibility in choosing different phospholipids for an inhalable liposomal nintedanib formulation that can achieve high drug encapsulation, desired sustained release, and prolonged drug retention in the pulmonary environment.

(71) TABLE-US-00003 TABLE 3 Nintedanib encapsulation results for ~120 nm liposomes comprising various phospholipids and 0.9 mol % PEG-modified lipid Total drug Total D/L Loading Liposomal Liposome composition (molar concentration ratio efficiency D/L ratio ratio) (mg/mL) (mol/mol) (%) (mol/mol) HSPC:cholesterol:DSPE- 9.22 1.47 66.0 0.97 PEG2000 3:2:0.045 7.35 0.92 106 0.98 DSPC:cholesterol:DSPE- 7.48 0.93 103 0.96 PEG2000 3:2:0.045 DSPC:DPPE:cholesterol:DSPE- 7.80 1.02 98.0 1.00 PEG2000 3:3:4:0.09

Example 3

(72) Effect of Empty Liposome Size on the Stability of Nebulized Liposomal TKI

(73) The stability of liposomal TKI after nebulization was investigated for liposomal drugs of varying particle sizes for formulations of low PEG content with high encapsulation of nintedanib. Different sized liposomes were prepared by extrusion of hydrated lipids through polycarbonate membranes of varying pore sizes (0.1 μm, 0.2 μm, 0.4 μm, and 1 μm) prior to remote loading of nintedanib into said liposomes. The liposome formulation used for the nebulization stability tests had a composition of HSPC:cholesterol:DSPE-PEG2000 at a molar ratio of 3:2:0.045 with 300 mM A.S. as the trapping agent. The protocol for nebulization of the liposomal TKI sample was as follows: 1. add 2 mL of liposomal drug to the medication chamber of a Vib-Mesh Nebulizer HL100 (commercialized by Health and Life Corporation); 2. slide in and connect the medication chamber to the rest of the nebulizer; 3. turn on the nebulizer to begin aerosolization of the sample and nebulize the entire 2 mL sample (total nebulization time was almost 6 minutes); 4. collect the aerosol particles in a 50-mL conical tube that is firmly connected to the nebulizer outlet and sealed with parafilm; and 5. determine the drug loading efficiency of the liposomal drug sample prior to and after nebulization using size-exclusion column chromatography and HPLC analysis.

(74) The results of the nebulization stability tests are shown in Table 4 below. Liposomal drug formulations comprising liposomes with mean particle diameters below 300 nm were very stable with practically no drug leakage (≤1%). Liposomal formulations of drug with mean particle diameters slightly larger than 300 nm had a small amount of drug leakage (about 5%). Overall, aerosolized particles of the liposomal drug formulation in which liposomes had mean particle diameters in the 100 nm to 300 nm range were stable, as drug leakage was minimal and particle size did not change significantly before and after nebulization.

(75) TABLE-US-00004 TABLE 4 Nebulization stability of liposomal nintedanib Membrane pore size used to Nebulized liposomal extrude Liposomal TKI mean TKI drug-to-lipid empty particle diameter (nm) (D/L) ratio (mol/mol) TKI liposome Pre-nebu- Post-nebu- Free + Lipo- leakage (μm) lization lization liposomal somal (%) 0.1 128 128 0.92 0.92 0 0.2 191 189 0.91 0.90 1.1 0.4 244 244 0.93 0.93 0 1 322 311 1.03 0.98 4.9

Example 4

(76) Storage Stability of Liposomal Drug Formulations

(77) The stability of liposomal TKI stored at 4° C. was monitored for two years. Nintedanib was remotely loaded into liposomes comprising HSPC:cholesterol:DSPE-PEG2000 at a molar ratio of 3:2:0.045 (0.9 mol % PEG-modified lipid) with 300 mM A.S. as the trapping agent. After loading the drug into liposomes, the liposomal drug sample was stirred and dialyzed in 100× volume of a 9.4% (w/v) sucrose solution. The sucrose solution was replaced after 1 hour and after 4 hours, and the sample was stirred in solution overnight. The drug encapsulation (D/L ratio) for the liposome was about 0.9 mol/mol. After storage of the dialyzed liposomal drug sample at 4° C. for two years, practically no encapsulated drug (≤3%) had leaked out of the liposome (FIG. 1). In addition, the particle size of the liposomal TKI suspension remained the same (diameter within ±1 nm) throughout the two-year storage period at 4° C. (FIG. 2).

Example 5

(78) In Vitro Drug Release in Simulated Lung Fluid

(79) The drug release profiles of two liposomal TKI formulations, with 0.45 mol % PEG-modified lipid or 1.75 mol % PEG-modified lipid, were assessed in simulated lung fluid (SLF) to demonstrate their sustained release properties. The two liposomal TKI samples were prepared at about the same nintedanib concentration (3.85 to 3.95 mg/mL drug) with almost no free drug present in either sample (Table 5). The protocol for the in vitro release (IVR) experiments was as follows: 1. dilute each liposomal TKI sample 10-fold by mixing 0.5 mL of each sample with 4.5 mL of SLF (pre-warmed at 37° C.) and placing the diluted sample in a 15-mL centrifuge tube; 2. place the centrifuge tubes, with the diluted samples, into the sample wells of an Intelli-mixer rotator, which is incubating at 37° C. and rotating at 20 rpm; 3. sample 1 mL of the diluted liposomal TKI sample at predetermined time points (e.g., 0, 4, and 24 hours); 4. determine the encapsulation efficiency, wherein the analytical method for determining the encapsulation efficiency of each 1 mL sample was as follows:

(80) a. pack and wash a 2 mL of Sepharose® CL-4B column with a 9.4% sucrose solution (less than 5 mL);

(81) b. add 0.1 mL of the sample to the column, then add 0.15 mL of a 9.4% sucrose solution three separate times and wait for the solution to elute from the column;

(82) c. add 1 mL of a 9.4% sucrose solution to the column and collect the eluent (liposomal drug fraction) in a 10-mL volumetric flask; add methanol to the volumetric flask to bring it up to volume and mix it well (this is the liposomal drug form);

(83) d. in a separate 10-mL volumetric flask, add 0.1 mL of the unpurified sample to the flask and add methanol to the volumetric flask to bring it up to volume and mix it well (this is the total drug form);

(84) e. measure the absorbance of the final, diluted samples at 380 nm using a UV-Vis plate reader to determine the drug concentrations of each sample. 5. The encapsulation efficiency (EE) is defined as the liposomal form (LF) of the drug divided by the total form (TF) of the drug: EE(%)=LF/TF*100%.

(85) TABLE-US-00005 TABLE 5 Encapsulation results for IVR samples Drug Drug concentration Sample name form (mg/mL) EE (%) AS300 mM/Nin3.74 mg/mL/PEG0.45 LF 3.85 98.2 mol % TF 3.92 AS300 mM/Nin3.74 mg/mL/PEG1.75 LF 3.88 98.2 mol % TF 3.95

(86) The IVR profiles of two liposomal TKI formulations are shown in FIG. 3. Liposome formulations retained a significant percentage of the total drug content and exhibited slow drug release over a 24-hour time period. Liposomes with PEG were very stable, as almost no nintedanib was released into SLF in the first four hours and only up to 30% of their total drug content was released into SLF over a 24-hour time period. These IVR results indicate that our liposomal drug formulations can achieve sustained release in vivo in the pulmonary environment.

Example 6

(87) Prolonged Lung Retention and Reduced Drug Toxicity of Liposomal TKI in Healthy Animals

(88) A lung retention study of liposomal TKI in healthy mice was conducted to compare the residence times of free form versus liposomal TKI formulations in the mouse lung. Four compositions were used in the study: 1) blank (saline); 2) free nintedanib (drug dissolved in a 9.4% (w/v) sucrose solution); 3) 300 mM A.S. liposomal nintedanib (0.45 mol % PEG); and 4) 300 mM A.S. liposomal nintedanib (1.75 mol % PEG).
The drug concentration of compositions #2-4 was the same: about 3.74 mg/mL nintedanib.

(89) The protocol for the retention study was as follows: 1. anesthetize 7-week old C57BL/6 mice with isoflurane; 2. intratracheally (IT) administer 50 μL of the composition into anesthetized mice using a micro-sprayer (HRH-MAG4); 3. anesthetize and sacrifice mice at predetermined time points (e.g., 2, 6, and 24 hours); 4. at the same predetermined time points (e.g., 2, 6, and 24 hours), collect lung tissue samples for drug determination; 5. analyze nintedanib in lung tissue by:

(90) a) homogenize each lung tissue sample at 6,000 rpm twice (2 runs/cycle) with 1 mL of methanol;

(91) b) centrifuge each sample at 20,000 g for 10 minutes at 4° C.;

(92) c) transfer 0.5 mL of each supernatant into a 5-mL volumetric flask and bring it up to volume with methanol;

(93) d) measure the absorbance of the final, diluted sample at 380 nm using a UV-Vis plate reader to determine the drug concentration of the sample.

(94) The results from the lung retention study are shown in FIG. 4. For free nintedanib (free drug dissolved in an aqueous solution) administered intratracheally, almost none of the drug was retained in the lung after just two hours. Thus, nearly all of the drug was absorbed and cleared from the lung within that short time period. In contrast, animals administered liposomal nintedanib (comprising either 0.45 mol % or 1.75 mol % PEG-modified lipid) retained about half of the total drug dose in the lung six hours after IT administration. Furthermore, from 16% to over 30% of the total dose was still observed in the lung after 24 hours. Therefore, the residence time of liposomal nintedanib was much longer than that of the free drug. In addition, the liposomal drug formulations appear to be suitable for once-daily or even less frequent dosing. In comparison to free drug, liposomal nintedanib retained up to about 20-fold more nintedanib in the lung over 24 hours.

(95) During the lung retention study, no deaths were observed for mice that were administered either a blank solution or liposomal nintedanib (Table 6). However, mice that were administered free nintedanib (as an aqueous solution) at the same 3.74 mg/mL drug concentration as the liposomal form did not fare as well. Two out of the nine mice administered free nintedanib died, with the seven surviving mice exhibiting significant weakness (Table 6). These results demonstrate that the liposomal form of nintedanib is safer and less toxic than the free form of nintedanib. These results also indicate that it would be possible to increase the nintedanib dose in the liposomal drug form above 3.74 mg/mL to enhance efficacy and prolong sustained release while still maintaining safety.

(96) TABLE-US-00006 TABLE 6 Observations of mice during lung retention study Number of mice sampled at different time points (hours) Test Articles 0 2 6 24 Mortality Blank 3 0 0 0 0/3 3.74 mg/mL Nin (free form) 0 3 3 3 2/9 (the 7 surviving mice were very weak) 300 mM A.S. - 3.74 mg/mL 0 3 3 3 0/9 Nin PEG of 0.45 mol % (liposomal form) 300 mM A.S. - 3.74 mg/mL 0 3 3 3 0/9 Nin PEG of 1.75 mol % (liposomal form)

Example 7

(97) Lung Retention of TKI in an IPF Animal Model

(98) To expand upon the lung retention study of Example 6, retention of nintedanib in mouse lung tissue was also investigated in an IPF model. The study design details are given in Table 7. Briefly, 21 mice were divided into two groups (two different nintedanib compositions) with nine mice in Group #1 and 12 mice in Group #2.

(99) The description of each nintedanib composition is given below: Group #1: AS-Nin 3.74 mg/mL-PEG3% represented nintedanib loaded into 300 mM A.S. liposomes comprising 3 mol % PEG; lipid concentration of 15 mM, nintedanib concentration of 3.74 mg/mL; Group #2: free nintedanib represented 150 mg nintedanib soft capsules (free nintedanib solutions were prepared daily by dissolving the soft capsule content in sesame oil to yield a nintedanib concentration of 7.6 mg/mL for dosing; volume for oral administration was 200 μL/mouse).

(100) First, pulmonary fibrosis was induced by IT instillation of 2.5 mg/kg bleomycin to each mouse. After seven days, either 25 μL of liposomal nintedanib (Group #1) was instilled intratracheally or oral nintedanib (Group #2), one of two FDA-approved drugs for IPF treatment, was administered orally to the fibrotic mice. The dosing regimens are shown in Table 7. Q2D×2 signifies one dose administered every other day for a total of two doses. Blood and lung tissue samples were collected at predetermined time points (Table 7).

(101) TABLE-US-00007 TABLE 7 Study design of lung retention of nintedanib in an IPF animal model Blood/lung tissue Number sampling Group of Dose frequency time points # Composition animals (numbers) (post-dose)* 1 AS-Nin 3.74 mg/mL- 9 Once (N = 6) 24 and 48 h PEG3% Q2Dx2 (N = 3) 48 h (4.68 mg/kg, IT) 2 free nintedanib (60 12 Once (N = 6) 1 and 5 h mg/kg, oral) QD for 5 days 1 and 5 h (N = 6) *3 mice per time point in all groups.

(102) The lung retention results from the IPF animal model study are shown in FIG. 5. The amount of nintedanib in mouse lung tissue was determined and monitored for up to five days. Despite once-daily oral dosing of 60 mg/kg nintedanib, free nintedanib showed very little, if any, drug retention in mouse lung as no nintedanib was detected in lung tissue after just a few hours. In contrast, an IT dose of 4.68 mg/kg liposomal nintedanib administered every other day yielded sustained drug release in mouse lung tissue for up to 48 hours. In addition, more than 12% of the instilled dose of the 300 mM A.S. liposomal nintedanib formulation (˜0.06 mg of drug/g of lung tissue) was still observed in lung tissue after 48 hours. These results demonstrate that our liposomal drug formulation, at a significantly lower dose, was vastly superior to oral drug in prolonging retention of nintedanib in the lung of diseased mice and that infrequent dosing of inhaled liposomal nintedanib is possible.

Example 8

(103) Efficacy of Inhaled Liposomal Nintedanib in an IPF Animal Model

(104) The efficacy of liposomal and oral nintedanib in treating bleomycin-induced pulmonary fibrosis in mice was investigated. The study design details are given in Table 8. Briefly, eight mice were divided into three groups (N=3 each for two different nintedanib compositions and administration routes, N=2 for untreated control).

(105) The description of each nintedanib composition is given below: Group #1: AS-Nin 3.74 mg/mL-PEG3% represented nintedanib loaded into 300 mM A.S. liposomes comprising 3 mol % PEG; lipid concentration of 15 mM, nintedanib concentration of 3.74 mg/mL; Group #2: free nintedanib represented 150 mg nintedanib soft capsules (free nintedanib solutions were prepared daily by dissolving the soft capsule content in sesame oil to yield a nintedanib concentration of 7.6 mg/mL for dosing; volume for oral administration was 200 μL/mouse.)
First, pulmonary fibrosis was induced by IT instillation of bleomycin at 2.5 mg/kg on day 0. After seven days, either 25 μL of liposomal nintedanib (Group #1, N=3) was instilled intratracheally or free nintedanib (Group #2, N=3) was administered orally to the fibrotic mice for the evaluation of therapeutic effect compared to untreated control (Group #3, N=2). The dosing regimens of the nintedanib compositions are shown in Table 8. Q2D×4 signifies one dose administered every other day on day 7, 9, 15, and 17. QD×10 signifies mice received once daily dosing on day 7, 8, 9, 10, 11, 14, 15, 16, 17, and 18. Blood and lung tissue samples were collected on day 21.

(106) TABLE-US-00008 TABLE 8 Study design of nintedanib efficacy study in an IPF animal model Blood/lung tissue Group Dose sampling # Composition Animal No. frequency time points 1 AS-Nin3.74 1 to 3 Q2Dx4 Day 21 mg/mL-PEG3% (4.68 mg/kg, IT) 2 free nintedanib 4 to 6 QDx10 Day 21 (60 mg/kg, oral) 3 Untreated control 7 to 8 N/A Day 21

(107) Efficacy was determined by histopathological evaluation of mouse lung tissue. Table 9 shows the histopathological results after treatment of fibrotic mice with Groups #1-3. Reduction of lung fibrosis was observed in both Group #1 and Group #2 compared with Group #3, as lungs exhibited slight chronic, bronchioloalveolar inflammation and slight interstitial and subpleural fibrosis after treatment with either liposomal or free nintedanib (fibrosis score of ˜2). Because Example 7 demonstrates that IT administration of liposomal nintedanib results in a higher concentration of drug in lung tissue for longer periods of time, our data show that intratracheal instillation of liposomal nintedanib could greatly reduce the required dose and frequency of orally administered nintedanib for the treatment of IPF.

(108) TABLE-US-00009 TABLE 9 Histopathological results of mouse lung tissue in efficacy study (scored by Masson's trichrome staining) Group #1: liposomal Group #2: free Group #3: Histopathological nintedanib nintedanib untreated findings (4.68 mg/kg, IT) (60 mg/kg, oral) control Animal No. 1 2 3 4 5 6 7 8 Interstitial and 2 3 2 2 2 2 5 4 subpleural, 2 2 2 2 2 2 4 3 multifocal 2 2 2 2 1 2 4 3 fibrosis scores 2 2 2 2 1 2 3 2 2 2 1 2 1 2 3 2 Mean score 2.0 2.2 1.8 2.0 1.4 2.0 3.8 2.8
Degree of lesions stained with HE was graded from one to five depending on severity: 1=minimal (<1%); 2=slight (1-25%); 3=moderate (26-50%); 4=moderate/severe (51-75%); 5=severe/high (76-100%).

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