RU2773750C2 - Methods for treatment of lung disorders - Google Patents

Abstract

FIELD: medicine; oncology.

SUBSTANCE: group of inventions relates to medicine, namely to oncology, in particular to a method for the treatment of lung tumor using taxan particles. The method includes intrapulmonary administration to a subject with lung cancer by means of nebulizer of the effective amount of a composition in the form of a suspension, containing taxan particles and a pharmaceutically acceptable carrier. Taxan includes paclitaxel or its pharmaceutically acceptable salt. Taxan particles contain at least 95% of taxan, have an average size (quantitative) from 0.4 mcm to 3 mcm, specific surface area (SSA) from 27 m²/g to 60 m²/g. The suspension is converted to aerosol for the administration. Formed aerosol drops have a mass median aerodynamic diameter (MMAD) from 0.5 mcm to 6 mcm. The use of a composition containing taxan particles for the treatment of lung tumor is also proposed.

EFFECT: inventions lead to significantly more long-lasting retention of taxan in the lungs, in particular long-lasting period of action is at least 4 weeks.

21 cl, 54 dwg, 15 tbl, 4 ex

Description

cross reference

This application claims priority based on Provisional Applications for US Patent No. 62/519257, filed June 14, 2017; 62/628582, filed February 9, 2018; 62/653942, filed April 6, 2018; and 62/678387, filed May 31, 2018, the contents of each of which are incorporated herein in their entirety by reference.

State of the art

Lung cancer is the second most common and one of the most deadly forms of cancer. Traditional therapies, such as surgical resection, radiation and chemotherapy, cannot provide a satisfactory level of long-term survival. Systemic drug delivery, even at high doses, only leads to limited uptake of taxane drugs in lung tumors. Thus, improved methods for the treatment of lung tumors are needed.

Brief description of the invention

In one aspect, the invention provides methods for treating a lung disorder, such as a lung tumor or lung fibrosis, comprising intrapulmonary administering to a subject with a lung disorder an effective amount of a composition comprising taxane particles for treating the lung disorder, wherein the taxane particles comprise at least 95% taxane. and have an average size (quantitative) of 0.1 µm to 5 µm. In one embodiment, intrapulmonary administration may include nebulization, where nebulization results in intrapulmonary delivery of taxane particles or suspensions containing them in the form of aerosol droplets to the subject. In another embodiment, the taxane particles may have an average size (quantitative) of 0.4 µm to 2 µm. In further embodiments, the taxane particles may have an average size (quantitative) of about 0.4 microns to about 1.2 microns, or about 0.6 microns to about 1.0 microns.

In another embodiment, the taxane particles may have a specific surface area (SSA) of at least 10 m ² /g or at least 12 m ² /g, 14 m ² /g, 16 m ² /g, 18 m ² /g, 20 m ² /g, 25 m ² /g, 30 m ² /g, 32 m ² /g, 34 m ² /g or 35 m ² /g; or the taxane particles have an SAR from about 10 m ² /g to about 60 m ² /g. In another embodiment, the taxane particles may be present in a suspension, where the suspension contains:

(a) taxane particles;

(b) a pharmaceutically acceptable carrier; and

(c) a polysorbate, wherein the polysorbate is present in the suspension at a concentration of from about 0.01% (v/v) to about 1.5% (v/v) or from about 0.01% (v/v .) to about 1% (v/v), from about 0.01% (v/v) to about 0.5% (v/v), from about 0.01% (v/v) /v) to about 0.4% (v/v), from about 0.01% (v/v) to about 0.25% (v/v), from about 0.05 % (v/v) to about 0.5% (v/v), from about 0.05% (v/v) to about 0.25% (v/v), from about 0.1% (v/v) to about 0.5% (v/v), from about 0.1% (v/v) to about 0.25% (v/v) .), about 0.1% (v/v), about 0.16 (v/v), or about 0.25% (v/v). In one embodiment, the pharmaceutically acceptable carrier may be a saline solution such as 0.9% sodium chloride solution. In another embodiment, the polysorbate may be polysorbate 80. In a further embodiment, the taxane may be present in the suspension at a concentration of from about 1 mg/mL to about 40 mg/mL, or from about 6 mg/mL to about 20 mg/mL.

In another embodiment, the taxane particles and suspensions containing them may be free of coating and lipids, polymers, proteins such as albumin, polyethoxylated castor oil, and/or polyethylene glycol glycerides composed of mono-, di-, and triglycerides and polyethylene mono- and diesters. nglico la.

In other embodiments, the taxane may contain paclitaxel, docetaxel, cabazitaxel, or pharmaceutically acceptable salts thereof. In one embodiment, the taxane may comprise paclitaxel or a pharmaceutically acceptable salt thereof; in this embodiment, the particles may have one or more of the following characteristics:

(a) an average bulk density (without compaction) from about 0.050 g/cm ³ to about 0.12 g/cm ³ or from about 0.060 g/cm ³ to about 0.11 g/cm ³ ;

(b) ARR of at least 12 ^m2 /g, 15 ^m2 /g, 18 ^m2 /g, 20 ^m2 /g, 25 ^m2 /g, 30 ^m2 /g, 32 ^m2 /g, 34 m ² /g or 35 m ² /g;

(c) SAR from about 22 m ² /g to about 40 m ² /g, from 25 m ² /g to about 40 m ² /g, from 30 m ² /g to about 40 m ² /g, or from about 35 m ² /g to about 40 m ² /g; and/or

(d) wherein at least 40% (w/w) of paclitaxel dissolves in 30 minutes or less in a 50% methanol/50% water (v/v) solution at 37° C. and pH 7.0 in a paddle USP II apparatus at 75 rpm.

In another embodiment, the taxane particles may contain docetaxel or a pharmaceutically acceptable salt thereof; in this embodiment, the particles may have one or more of the following characteristics:

(a) an average bulk density (without compaction) from about 0.050 g/cm ³ to about 0.12 g/cm ³ or from about 0.06 g/cm ³ to about 0.1 g/cm ³ ;

(b) SAR of at least 12 m ² /g, 15 m ² /g, 18 m ² /g, 20 m ² /g, 25 m ² /g, 30 m ² /g, 35 m ² /g, 40 m ² /g or 42 m ² /g;

(c) SAR from about 20 m ² /g to about 50 m ² /g or from about 35 m ² /g to about 46 m ² /g; and/or

(d) where at least 20% (w/w) docetaxel dissolves in 30 minutes or less in a solution of 15% methanol/85% water (v/v) at 37° C. and pH 7.0 in a paddle USP II apparatus at 75 rpm.

In another embodiment, the taxane particles may be in crystalline form. In a further embodiment, the taxane particles or suspensions containing them are sprayed for administration and the aerosol droplets have a mass median aerodynamic diameter (MMAD) of about 0.5 µm to about 6 µm, or about 1 µm to about 3 µm, or about 2 µm to about 3 µm.

In one embodiment, the taxane may remain in a detectable amount in the lung tissues of the subject for at least 4 days following administration.

In some embodiments, the taxane particles are retained at the site of the tumor after administration of the composition, allowing the taxane particles to act on the tumor for an extended period of time sufficient to stimulate the subject's endogenous immune system, resulting in the production of antitumor cells and infiltration of antitumor cells into the tumor in an amount enough to cure the tumor. In some embodiments, stimulation of the endogenous immune system elicits a cellular (cell-mediated) immune response. In other embodiments, stimulation of the endogenous immune system elicits a humoral immune response. In some embodiments, upon stimulation of the endogenous immune system, a tumor vaccine is produced. In some embodiments, metastases are reduced or eliminated.

In one embodiment, the extended time period is at least 4 weeks.

In some embodiments, the antitumor cells include T cells, B cells, or natural killer cells (NKCs), or combinations thereof.

In some embodiments, the composition is administered in two or more separate administration steps.

In some embodiments, two or more separate insertions are performed once a week for at least two weeks.

In other embodiments, two or more separate injection operations are carried out twice a week for at least one week, where two or more separate administration operations are carried out at least one day apart.

In some embodiments, the method of treating a tumor is to kill the tumor.

In the context of the present invention, the following embodiments from 1 to 25 are described:

Embodiment 1 is a method of treating a lung disorder, including but not limited to lung tumor or lung fibrosis, comprising intrapulmonary administering to a subject with a lung disorder an effective amount of a composition comprising taxane particles to treat a lung disorder, wherein the taxane particles comprise at least 95% taxane and have an average size (quantitative) of about 0.1 µm to 5 µm.

Embodiment 2 is the method of Embodiment 1, wherein intrapulmonary administration comprises nebulization, and nebulization results in intrapulmonary delivery of taxane particles or a suspension containing them in the form of aerosol droplets to the subject.

Embodiment 3 is a process according to any one of Embodiments 1-2, wherein said taxane particles have an average size (quantitative) of about 0.4 µm to 2 µm.

Embodiment 4 is a process according to any one of Embodiments 1-3, wherein said taxane particles have an average size (quantitative) of about 0.4 µm to about 1.2 µm, or about 0.6 µm to about 1 .0 µm.

Embodiment 5 is a process according to any one of Embodiments 1-4, wherein said taxane particles have a specific surface area (SSA) of at least 10 m ² /g or at least 12 m ² /g, 14 m ² /g, 16m2/g, ^18m2 /g, ^20m2 /g, ^25m2 /g, ^30m2 /g, ^32m2 /g, ^34m2 / ^g or ^35m2 /g ; or the taxane particles have an SAR from about 10 m ² /g to about 60 m ² /g.

Embodiment 6 is a process according to any one of Embodiments 1-5, wherein said taxane particles are present in a suspension, wherein the suspension contains:

(a) taxane particles;

(b) a pharmaceutically acceptable carrier; and

(c) a polysorbate, wherein the polysorbate is present in the suspension at a concentration of from about 0.01% (v/v) to about 1.5% (v/v) or from about 0.01% (v/v .) to about 1% (v/v), from about 0.01% (v/v) to about 0.5% (v/v), from about 0.01% (v/v) /v) to about 0.4% (v/v), from about 0.01% (v/v) to about 0.25% (v/v), from about 0.05 % (v/v) to about 0.5% (v/v), from about 0.05% (v/v) to about 0.25% (v/v), from about 0.1% (v/v) to about 0.5% (v/v), from about 0.1% (v/v) to about 0.25% (v/v) .), about 0.1% (v/v), about 0.16% (v/v), or about 0.25% (v/v).

Embodiment 7 is the method of Embodiment 6 wherein said pharmaceutically acceptable carrier is a saline solution such as 0.9% sodium chloride solution.

Embodiment 8 is a method according to embodiments 6 or 7, wherein said polysorbate is polysorbate 80.

Embodiment 9 is a method according to any one of embodiments 6-8, wherein said taxane is present in suspension at a concentration of from about 1 mg/mL to about 40 mg/mL, or from about 6 mg/mL to about 20 mg/ ml.

Embodiment 10 is a process according to any one of Embodiments 1-9, characterized in that said particles and suspensions containing them are free of coating and lipids, polymers, proteins such as albumin, polyethoxylated castor oil and/or polyethylene glycol glycerides, consisting of mono-, di- and triglycerides; and polyethylene glycol mono- and diesters.

Embodiment 11 is a method according to any one of Embodiments 1-10, wherein said taxane comprises paclitaxel, docetaxel, cabazitaxel, or pharmaceutically acceptable salts thereof.

Embodiment 12 is the method of Embodiment 11 wherein said taxane comprises paclitaxel or a pharmaceutically acceptable salt thereof.

Embodiment 13 is a process according to Embodiment 12, wherein said particles have one or more of the following characteristics:

(a) an average bulk density (without compaction) from about 0.050 g/cm ³ to about 0.12 g/cm ³ or from about 0.060 g/cm ³ to about 0.11 g/cm ³ ;

(b) ARR of at least 12 ^m2 /g, 15 ^m2 /g, 18 ^m2 /g, 20 ^m2 /g, 25 ^m2 /g, 30 ^m2 /g, 32 ^m2 /g, 34 m ² /g or 35 m ² /g;

(c) SAR from about 22 m ² /g to about 40 m ² /g, from 25 m ² /g to about 40 m ² /g, from 30 m ² /g to about 40 m ² /g, or from about 35 m ² /g to about 40 m ² /g; and/or

(d) wherein at least 40% (w/w) of paclitaxel dissolves in 30 minutes or less in a 50% methanol/50% water (v/v) solution at 37° C. and pH 7.0 in a paddle USP II apparatus at 75 rpm.

Embodiment 14 is the method of Embodiment 11 wherein said taxane contains docetaxel or a pharmaceutically acceptable salt thereof.

Embodiment 15 is a process according to Embodiment 14, wherein said particles have one or more of the following characteristics:

(a) an average bulk density (without compaction) from about 0.050 g/cm ³ to about 0.12 g/cm ³ or from about 0.06 g/cm ³ to about 0.1 g/cm ³ ;

(b) SAR of at least 12 m ² /g, 15 m ² /g, 18 m ² /g, 20 m ² /g, 25 m ² /g, 30 m ² /g, 35 m ² /g, 40 m ² /g or 42 m ² /g;

(c) SAR from about 20 m ² /g to about 50 m ² /g or from about 35 m ² /g to about 46 m ² /g; and/or

(d) where at least 20% (w/w) docetaxel dissolves in 30 minutes or less in a solution of 15% methanol/85% water (v/v) at 37° C. and pH 7.0 in paddle USP at 75 rpm.

Embodiment 16 is a method according to any one of Embodiments 1-15, wherein said taxane can remain in a detectable amount in the lung tissues of the subject for at least 4 days after administration.

Embodiment 17 is a process according to any one of Embodiments 1-16, wherein said taxane particles are in crystalline form.

Embodiment 18 is a method according to any one of Embodiments 1-17, wherein said taxane particles or suspensions containing them are sprayed for administration and the aerosol droplets have a mass median aerodynamic diameter (MMAD) of from about 0.5 µm to about 6 µm, or from about 1 µm to about 3 µm, or from about 2 µm to about 3 µm.

Embodiment 19 is a method according to any one of embodiments 1-18, wherein said lung disorder comprises a lung tumor and the taxane particles are retained at the site of the tumor after administration of the composition, allowing the tumor to be exposed to the taxane particles for an extended period of time sufficient to stimulate the endogenous immune system of the subject, resulting in the production of anti-tumor cells and infiltration of anti-tumor cells into the tumor in an amount sufficient to cure the tumor.

Embodiment 20 is a method according to Embodiment 19, characterized in that said extended period of time is at least 4 weeks.

Embodiment 21 is a method according to any of embodiments 19-20, wherein said antitumor cells include T cells, B cells, or natural killer cells (NKCs), or combinations thereof.

Embodiment 22 is a method according to any one of embodiments 1-21, wherein said composition is administered in two or more separate administration steps.

Embodiment 23 is the method according to Embodiment 22, wherein said two or more separate insertions are performed once a week for at least two weeks.

Embodiment 24 is a method according to Embodiment 22, wherein said two or more separate insertions are carried out twice a week for at least one week, wherein the two or more separate insertions are carried out at least one day apart. .

Embodiment 25 is a method according to any one of Embodiments 1-24, wherein said lung disorder comprises a lung tumor and the method of treating the tumor is to kill the tumor.

Brief description of the drawings

FIG. 1 is a graph of paclitaxel lung tissue and plasma levels versus time in an inhalation study.

FIG. 2 is a plot of aerodynamic diameter change for a 6.0 mg/mL paclitaxel particle formulation in an inhalation study.

FIG. 3 is a plot of the change in aerodynamic diameter for a 20.0 mg/mL paclitaxel particle formulation in an inhalation study.

FIG. 4 is a plot of paclitaxel plasma level versus time in an inhalation study.

FIG. 5 is a graph of paclitaxel lung tissue levels versus time in an inhalation study.

FIG. 6 is a graph of paclitaxel lung tissue and plasma levels versus time in an inhalation study.

FIG. 7 schematically shows a Hospitak compression jet nebulizer.

FIG. 8 is a graph of animal body weight versus time in an orthotopic lung cancer study.

FIG. 9 is a plot of an animal's body weight versus time in an orthotopic lung cancer study.

FIG. 10 is a graph of the change in lung weight of an animal in an orthotopic lung cancer study.

FIG. 11 is a graph of the ratio of lung weight to body weight of an animal in an orthotopic lung cancer study.

FIG. 12 is a graph of the ratio of lung weight to brain weight of an animal in an orthotopic lung cancer study.

FIG. 13 is a graph showing the change in mean tumor area in the orthotopic lung cancer study.

FIG. 14 is a plot of mean tumor area in the orthotopic lung cancer study.

FIG. 15 is a graph of tumor reduction in an orthotopic lung cancer study.

FIG. 16 is a photomicrograph of a slide with tissue affected by orthotopic lung cancer - 1006 (control) adenocarcinoma-3, germ cells-1, tumor regression-0. Primary signs of tumor formation in the lungs. (2x).

FIG. 17 is a photomicrograph of a slide with tissue affected by orthotopic lung cancer-1006, control, adenocarcinoma-3, germ cells-1, tumor regression-0. Primary signs of undifferentiated cells in a tumor formation in the lungs.

FIG. 18 is a photomicrograph of a slide with tissue affected by orthotopic lung cancer - 1006 (control) adenocarcinoma-3, germ cells-1, tumor regression-0. Primary signs of undifferentiated cells in a tumor formation in the lungs.

FIG. 19 is a photomicrograph of a slide with tissue affected by orthotopic lung cancer - 1006 (control) adenocarcinoma-3, germ cells-1, tumor regression-0. Primary signs of undifferentiated cells in a tumor formation in the lungs, formations in the alveolar space are shown. a(20x).

FIG. 20 is a photomicrograph of a slide with tissue affected by orthotopic lung cancer - 1006 (control) adenocarcinoma-3, germ cells-1, tumor regression-0. Primary signs of germ cells in a tumor formation in the lungs. b(10x).

FIG. 21 is a photomicrograph of a slide with tissue affected by orthotopic lung cancer - 1006 (control) adenocarcinoma-3, germ cells-1, tumor regression-0. Primary signs of germ cells in a tumor formation in the lungs. b20x.

FIG. 22 is a photomicrograph of a slide with tissue affected by orthotopic lung cancer - 1006 (control) adenocarcinoma-3, germ cells-1, tumor regression-0. Primary signs of germ cells in a tumor formation in the lungs. b(40x).

FIG. 23 is a photomicrograph of a slide with tissue affected by orthotopic lung cancer 1006 (control) adenocarcinoma-3, germ cells-1, tumor regression-0. Primary signs of germ cells in a tumor formation in the lungs. b(40x).

FIG. 24 is a photomicrograph of a slide with tissue affected by orthotopic lung cancer - 1006 (control) adenocarcinoma-3, germ cells-1, tumor regression-0 in the bronchiole. Primary signs of undifferentiated cells in the bronchiole. c(20x).

FIG. 25 is a photomicrograph of a slide with tissue affected by orthotopic lung cancer 1006 (control) adenocarcinoma-3, germ cells-1, tumor regression-0 in the glands. Primary signs of cell differentiation of acinar glands in a tumor formation in the lungs. d(10x).

FIG. 26 is a photomicrograph of a slide with tissue affected by orthotopic lung cancer 1006 (control) adenocarcinoma-3, germ cells-1, tumor regression-0 in the glands. Primary signs of cell differentiation of acinar glands in a tumor formation in the lungs. d(20x).

FIG. 27 is a photomicrograph of a tissue slide with orthotopic lung cancer 2001 (i.v. ABRAXANE®) adenocarcinoma-2, germ cells-1, tumor regression-0. Primary signs of displacement of the tumor formation in the lungs under the bronchiole, no signs of intravascular invasion. (2x).

FIG. 28 is a photomicrograph of a tissue slide with orthotopic lung cancer 2001 (i.v. ABRAXANE®) adenocarcinoma-2, germ cells-1, tumor regression-0. Primary signs of displacement of the tumor formation in the lungs under the bronchiole, no signs of intravascular invasion. (4x).

FIG. 29 is a photomicrograph of a tissue slide with Orthotopic Lung Cancer 2001 (i.v. ABRAXANE®) adenocarcinoma-2, germ cells-1, tumor regression-0. Primary signs of displacement of the tumor formation in the lungs under the bronchiole. (10x).

FIG. 30 is a photomicrograph of a tissue slide with orthotopic lung cancer - 2003 (i.v. ABRAXANE®) adenocarcinoma-1, germ cells-1, tumor regression-1. Signs of regression of tumor formation in the lungs. (4x).

FIG. 31 is a photomicrograph of a tissue slide with orthotopic lung cancer - 2003 (iv ABRAXANE®) adenocarcinoma-1, germ cells-1, tumor regression-1. Signs of regression of tumor formation in the lungs. (10x).

FIG. 32 is a photomicrograph of a tissue slide with orthotopic lung cancer - 2003 (i.v. ABRAXANE®) adenocarcinoma-1, germ cells-1, tumor regression-1. Signs of regression of tumor formation in the lungs. (20x).

FIG. 33 is a photomicrograph of a tissue slide with orthotopic lung cancer - 2003 (iv ABRAXANE®) adenocarcinoma-1, germ cells-1, tumor regression-1. Signs of regression of tumor formation in the lungs. Note the presence of lymphocytes and macrophages at the border. 1(40x).

FIG. 34 is a photomicrograph of a tissue slide with orthotopic lung cancer - 2003 (iv ABRAXANE®) adenocarcinoma-1, germ cells-1, tumor regression-1. Signs of regression of tumor formation in the lungs. Note the presence of lymphocytes and macrophages at the border. 2(40x).

FIG. 35 is a photomicrograph of a tissue slide with orthotopic lung cancer - 2003 (i.v. ABRAXANE®) adenocarcinoma-1, germ cells-1, tumor regression-1. Signs of regression of tumor formation in the lungs. The presence of large foamy and pigmented macrophages should be noted. 2, 2 x (40 x).

FIG. 36 is a photomicrograph of a tissue slide with orthotopic lung cancer - 2010 (i.v. ABRAXANE®) adenocarcinoma-3, germ cells-1, tumor regression-0. Primary signs of tumor formation in the lungs. (2x).

FIG. 37 is a photomicrograph of a tissue slide with orthotopic lung cancer - 2010 (i.v. ABRAXANE®) adenocarcinoma-3, germ cells-1, tumor regression-0. Primary signs of tumor formation in the lungs. (20x).

FIG. 38 shows a micrograph of a tissue slide with orthotopic lung cancer - 2010 (iv ABRAXANE®) adenocarcinoma-3, germ cells-1, tumor regression-0. Primary signs of tumor formation in the lungs. It should be noted the presence of a small number of macrophages at the border. (40x).

FIG. 39 is a photomicrograph of a tissue slide with orthotopic lung cancer 4009 (ing. paclitaxel particle formulation, 1x, high dose) adenocarcinoma-0, germ cells-0, tumor regression-4. Signs of complete regression of tumor formation in the lungs. (2x).

FIG. 40 is a photomicrograph of a tissue slide of orthotopic lung cancer-4009 (ing. paclitaxel particle formulation, 1x, high dose) adenocarcinoma-0, germ cells-0, tumor regression-4. Signs of complete regression of tumor formation in the lungs. Stroma fibrosis should be noted. (10x).

FIG. 41 is a photomicrograph of a tissue slide with orthotopic lung cancer 4009 (ing. paclitaxel particle formulation, 1x, high dose) adenocarcinoma-0, germ cells-0, tumor regression-4. Signs of complete regression of tumor formation in the lungs. Stroma fibrosis and the presence of lymphocytes and macrophages at the border should be noted. (40x).

FIG. 42 is a photomicrograph of a tissue slide of orthotopic lung cancer-5010 (ing. paclitaxel particle formulation, 2x, low dose) adenocarcinoma-1, germ cells-0, tumor regression-3. Signs of regression of tumor formation in the lungs. (2x).

FIG. 43 is a photomicrograph of a tissue slide of orthotopic lung cancer-5010 (ing. paclitaxel particle formulation, 2x, low dose) adenocarcinoma-1, germ cells-0, tumor regression-3. Signs of regression of tumor formation in the lungs. (10x).

FIG. 44 is a photomicrograph of a tissue slide with orthotopic lung cancer 5010 (ing. paclitaxel particle formulation, 2x, low dose) adenocarcinoma-1, germ cells-0, tumor regression-3. Signs of regression of tumor formation in the lungs. (20x).

FIG. 45 is a photomicrograph of a tissue slide of orthotopic lung cancer-5010 (ing. paclitaxel particle formulation, 2x, low dose) adenocarcinoma-1, germ cells-0, tumor regression-3. Signs of regression of tumor formation in the lungs. (40x).

FIG. 46 is a photomicrograph of a tissue slide of orthotopic lung cancer-6005 (ing. paclitaxel particle formulation, 2x, high dose) adenocarcinoma-1, germ cells-0, tumor regression-4. Signs of regression of tumor formation in the lungs. (2x).

FIG. 47 is a photomicrograph of a tissue slide with orthotopic lung cancer 6005 (ing. paclitaxel particle formulation, 2x, high dose) adenocarcinoma-1, germ cells-0, tumor regression-4. Signs of regression of tumor formation in the lungs. Stroma fibrosis and the presence of lymphocytes and macrophages at the border should be noted. (10x).

FIG. 48 is a photomicrograph of a tissue slide with orthotopic lung cancer 6005 (ing. paclitaxel particle formulation, 2x, high dose) adenocarcinoma-1, germ cells-0, tumor regression-4. Signs of regression of tumor formation in the lungs. Note the presence of lymphocytes and macrophages at the border. (20x).

FIG. 49 is a photomicrograph of a tissue slide with orthotopic lung cancer 6005 (ing. paclitaxel particle formulation, 2x, high dose) adenocarcinoma-1, germ cells-0, tumor regression-4. Note the presence of lymphocytes and macrophages at the border. (40x).

FIG. 50 is a photomicrograph of a tissue slide of orthotopic lung cancer 6005 (ing. paclitaxel particle formulation, 2x, high dose) adenocarcinoma-1, germ cells-0, tumor regression-4. It should be noted the presence of a focus of residual tumor cells in the alveolus. 2(40x).

FIG. 51 is a plot of animal body weight versus time in an inhalation study.

FIG. 52 is a plot of the change in animal body weight over time in an inhalation study.

FIG. 53 is a plot of paclitaxel plasma level versus time in an inhalation study.

FIG. 54 is a graph of paclitaxel lung tissue levels versus time in an inhalation study.

Detailed description of the invention

In this document, the singular forms (corresponding to the English "a", "an" and "the") include references to many objects, unless the context clearly requires otherwise. "And" is used interchangeably with "or" herein, unless explicitly stated otherwise.

In this document, "about" means +/- five percent (5%) of the specified value.

All embodiments of any aspect of the invention may be used in combination unless the context clearly requires otherwise.

Unless the context clearly requires otherwise, in the description and claims, the words "comprise", "comprising", etc., should be considered in an inclusive sense, and not in an exclusive or exhaustive sense; that is, in the meaning of "including, but not limited to". Words used in the singular or plural form include the plural and singular, respectively. In addition, the words "in this document", "above" and "below" and similar words when used in this application refer to this application as a whole, and not to specific fragments of the application. Compositions and methods of their use may "include", "consist essentially of" or "consist of" any of the ingredients or steps disclosed in the description.

The description of embodiments according to the invention is not exhaustive or does not limit the invention to the exact form described. While specific embodiments and examples of the invention are described herein by way of illustration, various equivalent modifications are possible within the scope of the invention, which will be apparent to those skilled in the art.

According to a first aspect, the invention provides methods for treating a lung disorder, comprising intrapulmonary administration to a subject with a lung disorder of an effective amount of a composition containing taxane particles for treating a lung tumor, wherein the taxane particles contain at least 95% taxane and have an average size (quantitative) of 0 .1 µm to 5 µm.

The inventors have surprisingly found that intrapulmonary administration of taxane particles in accordance with the methods of the invention results in significantly longer retention of taxane in the lungs than previously possible with any other taxane formulations. As shown in the following examples, the taxane persists at a detectable level in the lung tissues of the subject for at least 96 hours after administration. In various additional embodiments, the taxane persists at a detectable level in the lung tissues of the subject for at least 108, 120, 132, 144, 156, 168, 180, 192, 204, 216, 228, 240, 252, 264, 276, 288, 300, 312, 324 or 336 hours after administration. Thus, the methods can be used to treat any lung disorder for which taxane particles can provide an effective cure, including, but not limited to, lung tumors, mesothelioma, restrictive lung diseases such as pulmonary fibrosis, and obstructive lung diseases such as chronic obstructive pulmonary disease (COPD).

Another aspect of the invention relates to methods that provide exposure of taxane particles in lung tumors after administration of the composition for an extended period of time sufficient to stimulate the endogenous immune system of the subject, which leads to the production of antitumor cells and infiltration of antitumor cells into the tumor in an amount sufficient to cure tumors. In some embodiments, stimulation of the endogenous immune system elicits a cellular (cell-mediated) immune response. In other embodiments, stimulation of the endogenous immune system elicits a humoral immune response. In some embodiments, upon stimulation of the endogenous immune system, a tumor vaccine is produced. In some embodiments, metastases are reduced or eliminated. Antitumor cells may contain T cells, B cells, or natural killer cells (NKCs), or combinations thereof. In some embodiments, the long exposure period is at least 108, 120, 132, 144, 156, 168, 180, 192, 204, 216, 228, 240, 252, 264, 276, 288, 300, 312, 324, or 336 hours. In various additional embodiments, the long exposure period is at least 3, 4, 5, 6, 7, or 8 weeks. The composition may be administered intrapulmonary in a single single dose operation (cycle) or in two or more separate administration operations (2 or more cycles) of single doses. In some embodiments, the interval between two or more separate administration operations is or is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 14 days. In some embodiments, two or more separate insertions are performed at intervals of 2 to 12, 2-11, 2-10, 2-9, 2-8 2-7, 2-6, 2-5, 2-4, 2 -3, 3-12, 3-11, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-12, 4-11, 4-10 , 4-9, 4-8, 4-7, 4-6, 4-5, 5-12, 5-11, 5-10, 5-9, 5-8, 5-7, 5-6, 6 -12, 6-11, 6-10, 6-9, 6-8, 6-7, 7-12, 7-11, 7-10, 7-9, 7-8, 8-12, 8-11 , 8-10, 8-9, 9-12, 9-11, 9-10, 10-12, 10-11, 11-12, 2, 3, 4, 5, 6, 7, 8, 9, 10 , 11 or 12 weeks. In some embodiments, the composition is administered in 2-5, 2-4, 2-3, 3-5, 3-4, 2, 3, 4, 5 or more separate administration steps. In some embodiments, two or more separate injection operations are carried out with an interval of 2 to 12 weeks. In some embodiments, two to five separate operations for administering the composition are carried out. In some embodiments, two or more separate insertions are performed once a week for at least two weeks. In other embodiments, two or more separate injection operations are carried out twice a week for at least one week, where the interval between two or more separate injection operations is at least one day. In some embodiments, the method of treatment results in the elimination (destruction) of the tumor. In some embodiments, the composition is administered in 1, 2, 3, 4, 5, 6 or more separate administration steps. In other embodiments, the composition is administered in 7 or more separate injection operations.

As used herein, "taxane particles" are particles consisting essentially of taxane (i.e., at least 95%, 96%, 97%, 98%, 99%, or 100% taxane) that have an average size (quantitative ) from 0.1 µm to 5 µm. The taxane particles for use according to the invention are uncoated and are not immersed, contained, enclosed or encapsulated in a solid excipient. The taxane particles according to the invention, however, contain impurities and by-products, as a rule, formed during the production of taxane. But even taking this into account, the taxane particles contain at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% taxane, that is, the taxane particles are or are composed of essentially from essentially pure taxane.

Taxanes are a class of taxadiene-containing diterpenoids that are very poorly soluble in water. The taxane particles of the invention may be any suitable taxane including, but not limited to, paclitaxel, docetaxel, cabazitaxel, taxadiene, baccatin III, taxquinine A, brevifoliol and taxuspin D, combinations thereof, or pharmaceutically acceptable salts. In one embodiment, the taxane is selected from the group consisting of paclitaxel, docetaxel, and cabazitaxel, or pharmaceutically acceptable salts thereof.

The active pharmaceutical ingredients (APIs) paclitaxel and docetaxel are commercially available from Phyton Biotech LLC, Vancouver, Canada. The docetaxel API contains not less than 95% or not less than 97.5% docetaxel, based on the weight of the anhydrous substance without solvent. The paclitaxel API contains at least 95% or at least 97% paclitaxel by weight of the anhydrous substance without solvent. In some embodiments, the paclitaxel API and the docetaxel API are USP and/or EP grade. The paclitaxel API can be produced by a semi-synthetic chemical process or from a natural source, such as fermentation or extraction from plant cells.

A lung tumor is any tumor present in the lungs and may be a primary or metastatic lung tumor. Non-limiting examples of lung tumors include small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). In one embodiment, the lung tumor is SCLC. In another embodiment, the lung tumor is NSCLC. The subject may be any mammal affected by a lung tumor, including, but not limited to, humans and other primates, dogs, cats, horses, cattle, pigs, sheep, goats, and the like.

An "effective amount" of taxane particles can be determined by the attending physician, taking into account all important factors. Administration of the taxane particles may include administration of the taxane alone or administration in conjunction with other therapeutic agents as deemed appropriate under all circumstances by the attending physician. In one embodiment, the methods further comprise treating the subject with a standard of care for the tumor being treated, such as intravenous chemotherapy, radiation therapy, surgical resection, and the like.

As used herein, "treat", "method of treatment" or "treatment" means doing one or more of the following: (a) reducing tumor size or fibrosis; (b) reducing the rate of tumor growth; (c) elimination of tumor or fibrosis; (d) reducing or limiting the development and/or spread of metastases or eliminating metastases. In some embodiments, the method of treatment is the elimination of the tumor or fibrosis.

In one particular embodiment, intrapulmonary administration comprises inhalation of a single dose of taxane particles, such as intranasal, oral inhalation, or both. The taxane particles can be administered in two or more separate administration (dose) operations. In this embodiment, the implementation of the taxane particles can be obtained in the form of an aerosol (ie, liquid droplets of a stable dispersion or suspension of antitumor particles in a gaseous environment). Taxane particles delivered as an aerosol may be deposited in the respiratory tract by gravity sedimentation, inertial collision and/or diffusion. Any suitable aerosol device may be used, including, but not limited to, pressurized metered dose inhalers (pMDIs), nebulizers, dry powder inhalers (DPIs), and liquid inhalers.

In one specific embodiment, the methods include inhalation of taxane particles, which are aerosolized by spraying. Nebulizers typically use compressed air or ultrasound to produce aerosol droplets of taxane particles or inhalable suspensions containing them. In this embodiment, the nebulization results in intrapulmonary delivery to the subject of aerosol droplets of taxane particles or a suspension containing them.

In another embodiment, the methods include inhalation of taxane particles aerosolized using pMDI, wherein the taxane particles or suspensions containing them are suspended in a suitable propellant system (including but not limited to hydrofluoroalkanes (HFA)) containing at least one liquefied gas in pressurized container closed with a gauge valve. Activation of the valve results in the delivery of a metered dose of the nebulized aerosol of taxane particles or suspensions containing them.

In other embodiments, the taxane particles have an average size (quantitative) greater than 0.2 µm or 0.3 µm. In another embodiment, the taxane particles have an average size (quantitative) of at least 0.4 microns. In additional embodiments, the taxane particles have an average size (quantitative) of 0.4 µm to 2 µm, or 0.5 µm to 1.5 µm, or 0.2 µm to 1 µm, or 0.2 µm to less than 1 µm.

In additional embodiments, the taxane particles may have a quantitative size ranging from about 0.4 µm to about 5 µm, from about 0.4 µm to about 3 µm, from about 0.5 µm to about 1.4 µm, from about 0 .4 µm to about 0.8 µm, from about 0.4 µm to about 0.7 µm, or from about 0.5 µm to about 0.7 µm. In a further embodiment, the taxane particles have a quantitative size of from about 0.4 microns to about 1.2 microns, or from about 0.6 microns to about 1.0 microns. In another embodiment, the taxane particles have a quantitative size of 0.6 µm to 0.861 µm, or about 0.5 µm to about 0.7 µm, or about 0.2 µm to about 1 µm, or about 0.2 µm to less less than 1 µm, or from about 0.3 µm to about 1 µm, or from about 0.3 µm to less than 1 µm, or from about 0.4 µm to about 1 µm, or from about 0.4 µm to less than 1 µm.

The particle size of the taxane can be determined in a particle size analyzer and the measured value can be expressed as an average diameter calculated from the distribution of the number of particles (quantitative). A suitable particle size analyzer is an instrument that uses a chiaroscuro technique, also referred to as optical photozone or single particle sensing (SPOS), for analysis. A suitable chiaroscuro particle size analyzer is an ACCUSIZER such as the ACCUSIZER 780 SIS available from Particle Sizing Systems, Port Richey, Florida. Another suitable particle size analyzer is a laser diffraction instrument such as the Shimadzu SALD-7101.

In those embodiments where the taxane particles are aerosolized for administration, the mass median aerodynamic diameter (MMAD) of the aerosol droplets of the taxane particles or suspensions containing them can be any diameter suitable for use in the invention. In one embodiment, the aerosol droplets have an MMAD from about 0.5 µm to about 6 µm. In various further embodiments, the aerosol droplets have an MMA from about 0.5 µm to about 5.5 µm, from about 0.5 µm to about 5 µm, from about 0.5 µm to about 4.5 µm, from about 0. 5 µm to about 4 µm, about 0.5 µm to about 3.5 µm, about 0.5 µm to about 3 µm, about 0.5 µm to about 2.5 µm, from about 0.5 µm to about 2 µm, from about 1 µm to about 5.5 µm, from about 1 µm to about 5 µm, from about 1 µm to about 4.5 µm, from about 1 µm to about 4 µm, from about 1 µm to about 3.5 µm, from about 1 µm to about 3 µm, from about 1 µm to about 2.5 µm, from about 1 µm to about 2 µm, from about 1.5 µm to about 5.5 µm, from about 1.5 µm to about 5 µm, about 1.5 µm to about 4.5 µm, about 1.5 µm to about 4 µm, about 1.5 µm to about 3.5 µm, from about 1, 5 µm to about 3 µm, from about 1.5 µm to about 2.5 µm, from about 1.5 µm to about 2 µm, from about 2 µm to about 5.5 µm, from about 2 µm to about 5 µm, from about 2 µm to about 4.5 µm, from about 2 µm to about 4 µm, from about 2 µm to about 3.5 µm, from about 2 µm to about 3 µm, and from about 2 µm to about 2.5 µm. A suitable instrument for measuring mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD) of aerosol droplets is a seven-stage aerosol sampler such as a Mercer-type cascade impactor.

In another embodiment, the taxane particles may have a specific surface area (SSA) of at least 10 m ² /g or at least 12 m ² /g, 14 m ² /g, 16 m ² /g, 18 m ² /g, 20 m ² /g, 25 m ² /g, 30 m ² /g, 32 m ² /g, 34 m ² /g or 35 m ² /g; or the taxane particles have an SAR from about 10 m ² /g to about 60 m ² /g.

In various embodiments, taxane particles are produced by “compressed anti-solvent precipitation” (PCA) methods such as those described in US Pat. published US patent applications US 2015/0375153, US 2016/0354336, US 2016/0374953; and international patent publications WO 2016/197091, WO 2016/197100 and WO 2016/197101; the contents of all of which are incorporated into this application by reference.

Supercritical carbon dioxide particle size reduction processes use supercritical carbon dioxide (anti-solvent) and a solvent such as acetone or ethanol to produce uncoated taxane particles ranging in size from 0.1 to 5 microns with a well-characterized particle size distribution. Carbon dioxide and solvent are removed during processing (up to 0.5% residual solvent may be retained), leaving taxane particles in powder form. Stability studies have shown that paclitaxel powder particles are stable as a dosage form when stored in vials at room temperature for up to 59 months and under accelerated aging conditions (40°C/75% RH) for up to six months. .

Taxane particles produced by various particle size reduction processes using supercritical carbon dioxide may have unique physical characteristics compared to taxane particles produced by traditional particle size reduction methods using mechanical action or grinding, such as wet or dry grinding, grinding, crushing, trituration, microfluidization or spraying. As described in U.S. Published Application US 2016/0374953, the contents of which are incorporated herein by reference, these unique characteristics include bulk density (the mass of the aggregate of particles in a composition divided by the total volume they occupy when poured into a graduated cylinder without compaction). measuring cylinder, where the total volume includes the volume of the particles, the empty volume between the particles and the volume of internal pores) from 0.05 g/cm ³ to 0.15 g/cm ³ and a specific surface area (SAR) of at least 18 m ² /g taxane particles (eg, paclitaxel and docetaxel), which are obtained by grinding methods using supercritical carbon dioxide, described in published application for US patent US 2016/0374953 and below. This range of bulk density is generally lower than the bulk density of taxane particles produced by conventional methods, and the SAR is generally higher than the SAR of taxane particles produced by conventional methods. These unique characteristics lead to a significant increase in the dissolution rate in water/methanol based media compared to taxanes produced by traditional methods. In this document, "Specific Surface Area" (SSA) is the total surface area of taxane particles per unit mass of taxane as measured by the Brunauer-Emmett-Teller ("BET") isotherm in the following way: the measured mass of the analyzed sample from 200 to 300 mg is added to 30 ml sample tube. The sample tube is then placed in a ^{SORPTOMETER®} model BET-202A manufactured by Porous Materials Inc. An automated test is then carried out using the BETWIN ^® software package, after which the surface area of each sample is calculated. Those skilled in the art will appreciate that "taxane particles" may include agglomerated taxane particles and non-agglomerated taxane particles; and since RAA is defined in terms of weight in grams, it takes into account both agglomerated and non-agglomerated taxane particles in the composition. The BET specific surface area test procedure is a compendial method included in the US Pharmacopoeia and the European Pharmacopoeia. Bulk density can be determined by pouring the taxane particles into a graduated cylinder without compaction at room temperature, measuring the mass and volume, and calculating the bulk density.

As described in published US patent application US 2016/0374953, studies have shown an SAR of 15.0 m ² /g and a bulk density of 0.31 g/cm ³ for paclitaxel particles obtained by grinding paclitaxel in a Deco-PBM- V-0.41 using 5 mm balls at 600 rpm for 60 minutes at room temperature. Also, US Published Application US 2016/0374953 describes that one batch of paclitaxel particles had an SAR of 37.7 m ² /g and a bulk density of 0.085 g/cm ³ when prepared in the following way using supercritical carbon dioxide: 65 mg/ml was obtained solution of paclitaxel in acetone. A BETE ^MicroWhirl® fogging nozzle (BETE Fog Nozzle, Inc.) and an ultrasonic probe (Qsonica, model number Q700) were placed in the crystallization chamber at a distance of about 8 mm from each other. A stainless steel wire filter with a mesh size of approximately 100 nm was attached to a crystallization chamber to collect the precipitated paclitaxel particles. Supercritical carbon dioxide was injected into the crystallization chamber using production equipment and brought to a pressure of about 1200 psi (8.3 MPa) at about 38° C. at a rate of 24 kg/hour. The ultrasonic probe was tuned to 60% of full output power at a frequency of 20 kHz. The acetone solution containing paclitaxel was pumped through the nozzle at a rate of 4.5 ml/minute for about 36 hours. Additional batches of paclitaxel particles produced by the supercritical carbon dioxide process described above had SCP values: 22.27 m ² /g, 23.90 m ² /g, 26.19 m ² /g, 30.02 m ² / g, 31.16 m ² /g, 31.70 m ² /g, 32.59 m ² /g, 33.82 m ² /g, 35.90 m ² /g, 38.22 m ² /g and 38.52 m ² /g.

As described in US Published Application US 2016/0374953, studies have shown an SAR of 15.2 m ² /g and a bulk density of 0.44 g/cm ³ for docetaxel particles obtained by grinding docetaxel in a Deco-PBM- V-0.41 using 5 mm balls at 600 rpm for 60 minutes at room temperature. Also, US Published Application US 2016/0374953 discloses that docetaxel particles had an SAR of 44.2 m ² /g and a bulk density of 0.079 g/cm ³ when prepared in the following manner using supercritical carbon dioxide: 79.32 mg/mL was obtained docetaxel solution in ethanol. The nozzle and ultrasonic probe were placed in a pressure-resistant chamber at a distance of about 9 mm from each other. A stainless steel wire filter with a mesh size of approximately 100 nm was attached to a pressure-resistant chamber to collect the precipitated docetaxel particles. Supercritical carbon dioxide was injected into the crystallization chamber using production equipment and pressurized to about 1200 psi (8.3 MPa) at about 38° C. at a flow rate of 68 scl/min. The ultrasonic probe was tuned to 60% of full output power at a frequency of 20 kHz. An ethanol solution containing docetaxel was pumped through the nozzle at a rate of 2 ml/minute for about 95 minutes. The precipitated agglomerates and supercritical carbon dioxide docetaxel particles were then collected as the mixture was pumped through a stainless steel wire filter. The filter containing the docetaxel particles was opened and the resulting product was collected from the filter.

As described in U.S. Published Application 2016/0374953, dissolution studies have shown an increased dissolution rate in methanol/water based media for paclitaxel and docetaxel particles prepared by the supercritical carbon dioxide methods described in U.S. Published Application 2016/0374953 compared to paclitaxel and docetaxel particles obtained by grinding paclitaxel and docetaxel in a Deco-PBM-V-0.41 ball mill using 5 mm balls at 600 rpm for 60 minutes at room temperature. Methods for determining the rate of dissolution are described below. In the case of paclitaxel, about 50 mg of the material was coated on about 1.5 grams of 1 mm glass beads by mixing the material and the shot in a test tube for about 1 hour. The shot was transferred to a stainless steel wire container and placed in a dissolution bath containing methanol/water 50/50 (v/v) at 37° C., pH 7 and a USP II apparatus (paddle) operating at 75 vol. /min A 5 ml aliquot was taken at 10, 20, 30, 60 and 90 minutes, filtered through a 0.22 µm filter and analyzed on a UV/visible spectrophotometer at 227 nm. The absorbance values of the samples were compared with the values for standard solutions prepared in the dissolution medium to determine the amount of material dissolved. In the case of docetaxel, approximately 50 mg of material was placed directly into a dissolution bath containing methanol/water 15/85 (v/v) at 37° C., pH 7, and a USP II apparatus (paddle) operating at 75 v/v. /min A 5 ml aliquot was taken at 5, 15, 30, 60, 120 and 225 minutes, filtered through a 0.22 µm filter and analyzed on a spectrophotometer at 232 nm. The absorbance values of the samples were compared with the values for standard solutions prepared in the dissolution medium to determine the amount of material dissolved. In the case of paclitaxel, the dissolution rate was 47% dissolved material in 30 minutes for particles produced by the supercritical carbon dioxide process and 32% dissolved material in 30 minutes for particles obtained by milling. In the case of docetaxel, the dissolution rate was 27% dissolved material in 30 minutes for particles produced by the supercritical carbon dioxide process and 9% dissolved material in 30 minutes for particles obtained by milling.

In some embodiments, the taxane particles have an SAR of at least 10, at least 12, at least 14, at least 16, at least 18, at least 19, at least 20, at least 21, at least at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least at least 32, at least 33, at least 34 or at least 35 m ² /g. In one embodiment, the antitumor particles have an ALR from about 10 m ² /g to about 50 m ² /g. In some embodiments, the antitumor particles have a bulk density (uncompacted) of about 0.050 g/cm ³ to about 0.20 g/cm ³ .

In additional embodiments, the anticancer particles have an RAA of:

(a) from 16 m ² /g to 31 m ² /g or from 32 m ² /g to 40 m ² /g;

(b) from 16 m ² /g to 30 m ² /g or from 32 m ² /g to 40 m ² /g;

(c) 16 m ² /g to 29 m ² /g or 32 m ² /g to 40 m ² /g;

(d) from 17 m ² /g to 31 m ² /g or from 32 m ² /g to 40 m ² /g;

(e) 17 m ² /g to 30 m ² /g or 32 m ² /g to 40 m ² /g;

(f) 17 m ² /g to 29 m ² /g or 32 m ² /g to 40 m ² /g;

(g) 16 m ² /g to 31 m ² /g or 33 m ² /g to 40 m ² /g;

(h) 16 m ² /g to 30 m ² /g or 33 m ² /g to 40 m ² /g;

(i) from 16 m ² /g to 29 m ² /g or from 33 m ² /g to 40 m ² /g;

(j) from 17 m ² /g to 31 m ² /g or from 33 m ² /g to 40 m ² /g;

(k) 17 m ² /g to 30 m ² /g or 33 m ² /g to 40 m ² /g;

(l) 17 m ² /g to 29 m ² /g or 33 m ² /g to 40 m ² /g;

(m) 16 m ² /g to 31 m ² /g or ≥ 32 m ² /g;

(h) 17 m ² /g to 31 m ² /g or ≥ 32 m ² /g;

(i) 16 m ² /g to 30 m ² /g or ≥ 32 m ² /g;

(j) from 17 m ² /g to 30 m ² /g or ≥ 32 m ² /g;

(k) 16 m ² /g to 29 m ² /g or ≥ 32 m ² /g;

(l) from 17 m ² /g to 29 m ² /g or ≥ 32 m ² /g;

(m) 16 m ² /g to 31 m ² /g or ≥ 33 m ² /g;

(n) 17 m ² /g to 31 m ² /g or ≥ 33 m ² /g;

(o) from 16 m ² /g to 30 m ² /g or ≥ 33 m ² /g;

(p) from 17 m ² /g to 30 m ² /g or ≥ 33 m ² /g;

(q) 16 m ² /g to 29 m ² /g or ≥ 33 m ² /g; or

(r) 17 ^m2 /g to 29 ^m2 /g or ≥ 33 ^m2 /g.

In some embodiments, the taxane particles are paclitaxel particles and have an SAR of at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, or at least measure 35 m ² /g. In other embodiments, the paclitaxel particles have an SLR of 18 m ² /g to 50 m ² /g, or 20 m ² /g to 50 m ² /g, or 22 m ² /g to 50 m ² /g, or 25 m ² /g to 50 ^m2 /g or from 26 ^m2 /g to 50 ^m2 /g or from 30 ^m2 /g to 50 ^m2 /g or from 35 ^m2 /g to 50 ^m2 /g or from 18 ^m2 /g to 45 ^m2 /g or from 20 ^m2 /g to 45 ^m2 /g or from 22 ^m2 /g to 45 ^m2 /g or from 25 ^m2 /g to 45 ^m2 /g or 26 ^m2 /g to 45 ^m2 /g or 30 ^m2 /g to 45 ^m2 /g or 35 ^m2 /g to 45 ^m2 /g or 18 ^m2 /g to 40 ^m2 /g or from 20 m ² /g to 40 m ² /g or from 22 m ² /g to 40 m ² /g or from 25 m ² /g to 40 m ² /g or from 26 m ² /g to 40 m ² /g or from 30 m ² /g to 40 m ² /g or from 35 m ² /g to 40 m ² /g.

In some embodiments, the paclitaxel particles have a bulk density (uncompacted) of 0.05 g/cm ³ to 0.15 g/cm ³ or 0.05 g/cm ³ to 0.20 g/cm ³ .

In some embodiments, the paclitaxel particles have a dissolution rate of at least 40% (w/w) dissolved material in 30 minutes or less in a 50% methanol/50% water (v/v) solution in a USP II paddle apparatus, operating at 75 rpm, 37°C and pH 7.

In another embodiment, the paclitaxel particles have one or more of the following characteristics:

(a) an average bulk density (without compaction) from about 0.050 g/cm ³ to about 0.12 g/cm ³ or from about 0.060 g/cm ³ to about 0.11 g/cm ³ ;

(b) ARR of at least 12 ^m2 /g, 15 ^m2 /g, 18 ^m2 /g, 20 ^m2 /g, 25 ^m2 /g, 30 ^m2 /g, 32 ^m2 /g, 34 m ² /g or 35 m ² /g;

(c) SAR from about 22 m ² /g to about 40 m ² /g, from 25 m ² /g to about 40 m ² /g, from 30 m ² /g to about 40 m ² /g, or from about 35 m ² /g to about 40 m ² /g; and/or

(d) wherein at least 40% (w/w) of paclitaxel dissolves in 30 minutes or less in a 50% methanol/50% water (v/v) solution at 37° C. and pH 7.0 in a paddle USP II apparatus operating at 75 rpm.

In one embodiment, the paclitaxel particles have an average bulk density (uncompacted) of from about 0.050 g/cm ³ to about 0.12 g/cm ³ and an SAR of at least 30 m ² /g. In another embodiment, the paclitaxel particles have an average bulk density (uncompacted) of about 0.050 g/cm ³ to about 0.12 g/cm ³ and an SAR of at least 35 m ² /g. In one embodiment, the paclitaxel particles have an average bulk density (uncompacted) of from about 0.050 g/cm ³ to about 0.12 g/cm ³ and an SAR from about 30 m ² /g to about 40 m ² /g. In another embodiment, the paclitaxel particles have an average bulk density (uncompacted) of from about 0.060 g/cm ³ to about 0.11 g/cm ³ and an SAR from about 30 m ² /g to about 40 m ² /g. In another embodiment, the paclitaxel particles have an average bulk density (uncompacted) of from about 0.060 g/cm ³ to about 0.11 g/cm ³ and an SAR of at least 30 m ² /g. In a further embodiment, the paclitaxel particles have an average bulk density (uncompacted) of from about 0.060 g/cm ³ to about 0.11 g/cm ³ and an SAR of at least 35 m ² /g.

In another embodiment, at least 40% (w/w) of the paclitaxel particles of paclitaxel in the composition is dissolved in 30 minutes or less in a 50% methanol/50% water (v/v) solution in a USP II paddle apparatus at 75 rpm Used pH 7, pH does not affect the solubility of taxanes. In another embodiment, dissolution studies are conducted at 37°C.

In some embodiments, the taxane particles are docetaxel particles and have an SAR of at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41 or at least 42 m ² /g. In other embodiments, the docetaxel particles have an SLR of 18 m ² /g to 60 m ² /g, or 22 m ² /g to 60 m ² /g, or 25 m ² /g to 60 m ² /g, or 30 m ² /g to 60 ^m2 /g or from 40 ^m2 /g to 60 ^m2 /g or from 18 ^m2 /g to 50 ^m2 /g or from 22 ^m2 /g to 50 ^m2 /g or from 25 ^m2 /g to 50 ^m2 /g or from 26 ^m2 /g to 50 ^m2 /g or from 30 ^m2 /g to 50 ^m2 /g or from 35 ^m2 /g to 50 ^m2 /g or from 40 m ² /g to 50 m ² /g.

In some embodiments, the docetaxel particles have a bulk density (uncompacted) of 0.05 g/cm ³ to 0.15 g/cm ³ .

In some embodiments, the docetaxel particles have a dissolution rate of at least 20% (w/w) dissolved material in 30 minutes or less in a 15% methanol/85% water (v/v) solution in a USP II paddle apparatus, operating at 75 rpm, 37°C and pH 7.

In another embodiment, the docetaxel particles have one or more of the following characteristics:

(a) an average bulk density (without compaction) from about 0.050 g/cm ³ to about 0.12 g/cm ³ or from about 0.06 g/cm ³ to about 0.1 g/cm ³ ;

(b) SAR of at least 12 m ² /g, 15 m ² /g, 18 m ² /g, 20 m ² /g, 25 m ² /g, 30 m ² /g, 35 m ² /g, 40 m ² /g or 42 m ² /g;

(c) SAR from about 20 m ² /g to about 50 m ² /g or from about 35 m ² /g to about 46 m ² /g; and/or

(d) where at least 20% (w/w) docetaxel dissolves in 30 minutes or less in a solution of 15% methanol/85% water (v/v) at 37° C. and pH 7.0 in a paddle USP II apparatus at 75 rpm.

In one embodiment, the docetaxel particles have an average bulk density (uncompacted) of about 0.050 g/cm ³ to about 0.12 g/cm ³ and an SAR of at least 30 m ² /g. In another embodiment, the docetaxel particles have an average bulk density (uncompacted) of from about 0.050 g/cm ³ to about 0.12 g/cm ³ and an SAR of at least 35 m ² /g. In a further embodiment, the docetaxel particles have an average bulk density (uncompacted) of from about 0.050 g/cm ³ to about 0.12 g/cm ³ and an SAR of at least 40 m ² /g. In one embodiment, the docetaxel particles have an average bulk density (unconsolidated) of about 0.050 g/cm ³ to about 0.12 g/cm ³ and an SAR of about 20 m ² /g to about 50 m ² /g. In another embodiment, the mean bulk density (uncompacted) of the docetaxel particles is from about 0.06 g/cm ³ to about 0.1 g/cm ³ and the SRL is from about 30 m ² /g to about 50 m ² /g . In another embodiment, the average bulk density (uncompacted) of the docetaxel particles is from about 0.06 g/cm ³ to about 0.1 g/cm ³ and the SLR is from about 35 m ² /g to about 50 m ² /g . In another embodiment, the average bulk density (uncompacted) of the docetaxel particles is from about 0.06 g/cm ³ to about 0.1 g/cm ³ and the SRL is from about 35 m ² /g to about 45 m ² /g .

In another embodiment, at least 20% (w/w) of docetaxel dissolves in 30 minutes or less in a 15% methanol/85% water (v/v) solution in a USP II paddle apparatus at 75 rpm. . Used neutral pH, and the pH does not affect the solubility of taxanes. In another embodiment, dissolution studies are conducted at 37°C.

In any of these various embodiments, the taxane particles may include at least 4.16 x 10 ^-13 grams of taxane or a pharmaceutically acceptable salt thereof per particle. In some embodiments, the taxane particles are non-agglomerated individual particles and are not clusters of multiple taxane particles.

In various embodiments of the present invention, the taxane particles are uncoated (pure) single particles; the taxane particles are not bound or conjugated with any substance; no substance is absorbed or adsorbed on the surface of the taxane particles; the taxane particles are not encapsulated in any substance; the taxane particles do not contain a coating of any substance; the taxane particles are not microemulsions, nanoemulsions, microspheres or taxane liposomes; and/or the taxane particles are not bound, attached, encapsulated or do not contain a monomer, polymer (or biocompatible polymer), protein, surfactant or albumin coating. In some embodiments, the monomer, polymer (or biocompatible polymer), copolymer, protein, surfactant, or albumin is not absorbed or adsorbed onto the surface of the taxane particles. In some embodiments, the compositions are absent/compositions do not include or do not contain a polymer/copolymer or a biocompatible polymer/copolymer. In some embodiments, the compositions are absent/the compositions do not include or contain no protein. According to some aspects of the invention, the compositions are absent/compositions do not include or do not contain albumin. According to some aspects of the invention, the compositions are absent/compositions do not include or do not contain hyaluronic acid. According to some aspects of the invention, the compositions are absent / the compositions do not include or do not contain a conjugate of hyaluronic acid and a taxane. According to some aspects of the invention, the compositions are absent/compositions do not include or do not contain a conjugate of hyaluronic acid and paclitaxel. According to some aspects of the invention, the compositions are absent / compositions do not include or do not contain poloxamers, polyanions, polycations, modified polyanions, modified polycations, chitosan, chitosan derivatives, metal ions, nanovectors, poly-gamma-glutamic acid (PGA), polyacrylic acid (PAA ), alginic acid (ALG), vitamin E-TPGS, dimethylisosorbide (DMI), methoxy-PEG 350, citric acid, anti-VEGF antibody, ethylcellulose, polystyrene, polyanhydrides, polyhydroxyacids, polyphosphazenes, polyorthoesters, polyesters, polyamides, polysaccharides, polyproteins, styrene-isobutylene-styrene (SIBS), polyanhydride copolymer, polycaprolactone, polyethylene glycol (PEG), poly(bis(P-carboxyphenoxy)propane-sebacic acid, poly(d,l-lactic acid) (PLA), poly( d.1-lactic acid-glycolic acid) (PLAGA) and/or poly(D,L-lactic acid-glycolic acid) copolymer (PLGA).In some embodiments, the taxane particles have a crystalline shape. In other embodiments, the taxane particles are in an amorphous form or a combination of crystalline and amorphous forms.

In one embodiment, the taxane particles for administration comprise a suspension dosage form of the taxane (i.e., together with a pharmaceutically acceptable carrier and/or as an aerosol formulation) containing from about 0.1 mg/mL to about 100 mg /ml taxane. In various additional embodiments, the dosage form may contain from about 0.5 mg/ml to about 100 mg/ml, from about 1 mg/ml to about 100 mg/ml, from about 2 mg/ml to about 100 mg/ml, from about 5 mg/ml to about 100 mg/ml, from about 10 mg/ml to about 100 mg/ml, from about 25 mg/ml to about 100 mg/ml, from about 0.1 mg/ml to about 75 mg/ml, from about 0.5 mg/ml to about 75 mg/ml, from about 1 mg/ml to about 75 mg/ml, from about 2 mg/ml to about 75 mg/ml, from about 5 mg/ml ml to about 75 mg/ml, from about 10 mg/ml to about 75 mg/ml, from about 25 mg/ml to about 75 mg/ml, from about 0.1 mg/ml to about 50 mg/ml, from about 0.5 mg/ml to about 50 mg/ml, about 1 mg/ml to about 50 mg/ml, about 2 mg/ml to about 50 mg/ml, about 5 mg/ml to about 50 mg /ml, from about 10 mg/ml to about 50 mg/ml, from about 25 mg/ml to about 50 mg/ml, from about 0.1 mg/ml to about 25 mg/ml, from about 0.5 mg/ml to about 25 mg/ml, about 1 mg/ml to about 40 mg/ml, about 1 mg/ml to about 25 mg/ml, about 2 mg/ml to about 25 mg /ml, from about 5 mg/ml to about 25 mg/ml, from about 10 mg/ml to about 25 mg/ml, from about 0.1 mg/ml to about 15 mg/ml, from about 0.5 mg /ml to about 15 mg/ml, from about 1 mg/ml to about 15 mg/ml, from about 2 mg/mg to about 15 mg/ml, from about 5 mg/ml to about 15 mg/ml, from about 10 mg/mL to about 15 mg/mL, from about 0.1 mg/mL to about 10 mg/mL, from about 0.5 mg/mL to about 10 mg/mL, from about 1 mg/mL to about 10 mg/ml, from about 2 mg/ml to about 10 mg/ml, from about 5 mg/ml to about 10 mg/ml, from about 0.1 mg/ml to about 5 mg/ml, from about 0.5 mg/mL to about 5 mg/mL, from about 1 mg/mL to about 5 mg/mL, from about 2 mg/mL to about 5 mg/mL, from about 0.1 mg/mL to about 2 mg/mL , from about 0.5 mg/ml to about 2 mg/ml, from about 1 mg/ml to about o 2 mg/ml, about 0.1 mg/ml to about 1 mg/ml, about 0.5 mg/ml to about 1 mg/ml, about 0.1 mg/ml to about 0.5 mg /ml, from about 0.1 mg/ml to about 15 mg/ml, from about 0.5 mg/ml to about 15 mg/ml, from about 1 mg/ml to about 15 mg/ml, from about 2 mg /ml to about 15 mg/ml, from about 5 mg/ml to about 15 mg/ml, from about 3 mg/ml to about 8 mg/ml, or from about 4 mg/ml to about 6 mg/ml of a taxane, or at least about 0.1, 0.5, 1, 10, 20, 25, 50, 75, or 100 mg/ml taxane.

In one embodiment, the taxane particles are present in the carrier liquid prior to spraying. Any suitable carrier may be used, such as an aqueous liquid carrier. Any suitable aqueous liquid carrier may be used, including but not limited to 0.9% saline (saline) such as 0.9% sodium chloride for injection USP. In another embodiment, the taxane particles are present in the suspension prior to spraying. In some embodiments, the suspension includes an aqueous carrier. The carrier may contain a buffering agent, an osmotic salt and/or a surfactant in water, and other agents to adjust pH, isotonicity, and viscosity. In one implementation, which uses an aqueous carrier, the concentration of surfactant is less than 1% in terms of wt./mass. or w/v; in other embodiments, less than 0.5%, less than 0.25%, or about 0.1%. In other embodiments, the aqueous vehicle may include surfactants GELUCIRE® (polyethylene glycol glycerides consisting of polyethylene glycol mono-, di- and triglycerides and mono- and diesters of polyethylene glycol) and/or CREMOPHOR® (polyethoxylated castor oil). In some embodiments, the composition or suspension does not include polymers, proteins (such as albumin), polyethoxylated castor oil, and/or polyethylene glycol glycerides consisting of mono-, di-, and triglycerides and polyethylene glycol mono- and diesters.

In some embodiments, the suspension may contain water and optionally one or more excipients selected from the group consisting of a buffer, a tonicity agent, a preservative, an emollient, a viscosity modifier, an osmotic agent, a surfactant, an antioxidant, an alkalizing agent, an acidifying agent, an antifoaming agent and a coloring agent. For example, the suspension may contain taxane particles, water, buffer and salt. It optionally further contains a surfactant. In some embodiments, the suspension consists essentially of or consists of water, paclitaxel particles suspended in water, and a buffer. The suspension may further contain an osmotic salt.

The suspension may contain one or more surfactants. Suitable surfactants include, but are not limited to, polysorbates, lauryl sulfates, acetylated monoglycerides, diacetylated monoglycerides, and poloxamers. Polysorbates are polyoxyethylene sorbitan fatty acid esters which are a group of partial fatty acid esters of sorbitol and its anhydrides copolymerized with about 20, 5 or 4 moles of ethylene oxide for every mole of sorbitol and its anhydrides. Non-limiting examples of polysorbates are polysorbate 20, polysorbate 21, polysorbate 40, polysorbate 60, polysorbate 61, polysorbate 65, polysorbate 80, polysorbate 81, polysorbate 85, and polysorbate 120. Polysorbates containing about 20 moles of ethylene oxide are hydrophilic nonionic surfactants. . Examples of polysorbates containing about 20 moles of ethylene oxide include polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, and polysorbate 120. Polysorbates are commercially available from Croda under the tradename TWEEN™. The polysorbate number corresponds to the TWEEN number, e.g. polysorbate 20 is TWEEN 20, polysorbate 40 is TWEEN 40, polysorbate 60 is TWEEN 60, polysorbate 80 is TWEEN 80, etc. USP/NF polysorbate purity grades include polysorbate 20 NF, polysorbate 40 NF, polysorbate 60 NF, and polysorbate 80 NF. Polysorbates are also available in PhEur (European Pharmacopoeia), BP and JP purity grades. The term "polysorbate" is a generic name. Polysorbate 20 has the chemical name polyoxyethylene-20-sorbitan monolaurate. Polysorbate 40 has the chemical name polyoxyethylene-20-sorbitan monopalmitate. Polysorbate 60 has the chemical name polyoxyethylene-20-sorbitan monostearate. Polysorbate 80 has the chemical name polyoxyethylene-20-sorbitan monooleate. In some embodiments, the suspension may contain mixtures of polysorbates. In some embodiments, the suspension contains polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, and/or polysorbate 120. In other embodiments, the suspension contains polysorbate 20, polysorbate 40, polysorbate 60, and/or polysorbate 80. B in one embodiment, the suspension contains polysorbate 80.

The suspension may contain one or more tonicity agents. Suitable tonicity agents include, but are not limited to, one or more inorganic salts, electrolytes, sodium chloride, potassium chloride, sodium phosphate, potassium phosphate, sodium sulfate, potassium sulfate, sodium and potassium bicarbonate, and alkaline earth metal salts such as inorganic alkaline earth metal salts, for example calcium salts and magnesium salts, mannitol, dextrose, glycerol, propylene glycol and mixtures thereof.

The suspension may contain one or more buffering agents. Suitable buffering agents include, but are not limited to, dibasic sodium phosphate, monobasic sodium phosphate, citric acid, sodium citrate, hydrochloric acid, sodium hydroxide, tris(hydroxymethyl)aminomethane, bis(2-hydroxyethyl)imino-tris-(hydroxymethyl)methane, and sodium bicarbonate; and other agents known to those skilled in the art. Buffers are widely used to adjust the pH to the desired range for intraperitoneal use. Generally, a pH of about 5 to 9, 5 to 8, 6 to 7.4, 6.5 to 7.5, or 6.9 to 7.4 is desirable.

The suspension may contain one or more emollients. An emollient is an agent that forms a soothing film over a mucous membrane, such as membranes lining the abdominal cavity or organs contained therein. An emollient agent can relieve mild pain and inflammation and is sometimes referred to as a mucoprotective agent. Suitable softening agents include cellulose derivatives in an amount of from about 0.2 to about 2.5%, such as sodium carboxymethylcellulose, hydroxyethylcellulose, hydroxypropylmethylcellulose and methylcellulose a; gelatin in an amount of about 0.01%; polyols in an amount of from about 0.05 to about 1% also include from about 0.05 to about 1% of agents such as glycerol, polyethylene glycol 300, polyethylene glycol 400 and propylene glycol; polyvinyl alcohol from about 0.1 to about 4%; povidone in an amount of from about 0.1% to about 2%; and dextran 70 in an amount of from about 0.1% when used in conjunction with another polymeric emollient described herein.

The suspension may contain one or more alkalizing agents to adjust the pH. As used herein, the term "alkalinizing agent" refers to a compound used to provide an alkaline environment. These compounds include, but are not limited to, ammonia solution, ammonium carbonate, potassium hydroxide, sodium carbonate, sodium bicarbonate and sodium hydroxide, and other compounds known to those skilled in the art.

The suspension may contain one or more acidifying agents to adjust the pH. In this document, the term "acidifying agent" means a compound used to provide an acidic environment. These compounds include, but are not limited to, acetic acid, amino acid, citric acid, nitric acid, fumaric acid and other alpha hydroxy acids, hydrochloric acid, ascorbic acid and nitric acid, and other compounds known to those skilled in the art.

The suspension may contain one or more antifoam agents. As used herein, "anti-foaming agent" means a compound or compounds that prevents(-s) foaming or reduces(-s) the amount of foam formed on the surface of the filling composition. Suitable antifoam agents include, but are not limited to, dimethicone, SIMETHICONE, octoxynol, and other agents known to those skilled in the art.

The slurry may contain one or more viscosity modifiers that increase or decrease the viscosity of the slurry. Suitable viscosity modifiers include methylcellulose, hydroxypropylmethylcellulose, mannitol, and polyvinylpyrrolidone.

In some embodiments, the taxane particle is present in the suspension further containing the polysorbate. In one particular embodiment, the taxane particle is present in a suspension further containing a polysorbate, wherein the polysorbate is polysorbate 80. In other embodiments, the polysorbate or polysorbate 80 is present in the suspension at a concentration of from about 0.01% (v/v) to about 1.5% (v/v). The inventors surprisingly found that these very small amounts of polysorbate 80 reduce the surface tension at the interface between the taxane particles and an aqueous carrier in suspension (such as saline). In some embodiments, the particles may be coated with polysorbate or polysorbate 80, in other embodiments, the particles are not coated with polysorbate or polysorbate 80. In various other embodiments, polysorbate or polysorbate 80 is present in the suspension at a concentration of from about 0.01% (v/v). about 1% (v/v), from about 0.01% (v/v) to about 0.5% (v/v), from about 0.01% (v/v) ./vol.) to about 0.4% (v/v), from about 0.01% (v/v) to about 0.25% (v/v), from about 0, 05% (v/v) to about 0.5% (v/v), from about 0.05% (v/v) to about 0.25% (v/v), from about 0.1% (v/v) to about 0.5% (v/v), from about 0.1% (v/v) to about 0.25% (v/v) vol.), about 0.1% (v/v), about 0.16 (v/v), or about 0.25% (v/v). In further embodiments, the taxane, such as paclitaxel, is present in the suspension at a concentration of from about 1 mg/mL to about 40 mg/mL, or from about 6 mg/mL to about 20 mg/mL. In various additional embodiments, the taxane is present in the suspension at a concentration of about 6 mg/mL to about 15 mg/mL, about 6 mg/mL to about 10 mg/mL, about 10 mg/mL to about 20 mg/mL, about 10 mg/ml to about 15 mg/ml, about 6 mg/ml, about 10 mg/ml, or about 15 mg/ml. In various additional embodiments, the aqueous carrier in the composition may be a saline solution, such as about 0.9% sodium chloride.

Example 1 paclitaxel particles (i.e. the paclitaxel particles described herein, approximately 98% paclitaxel with an average particle size (number average) of 0.83 microns, an SAR of 27.9 m ² /g and a bulk density (without compaction) of 0, 0805 g/cm ³ that were used in examples 1, 2, 3 and 4) in suspension - Safety and Efficacy Research Program - a pilot study of pharmacokinetics in Sprague-Dawley rats.

Study number: FY17-008A

EXPLANATORY NOTE

The objective of this pilot study was to determine the timing of sampling in a complete pharmacokinetic (PK) study of the suspension formulation of paclitaxel particles. Because the paclitaxel particle formulation had the potential to increase pulmonary retention, nine time points from 0.5 to 168 hours were evaluated to determine the appropriate sampling strategy in the full pharmacokinetic study.

Sixteen (16) Sprague-Dawley rats were exposed to a single inhalation of the paclitaxel particle formulation (target dose 0.37 mg/kg) through the nose only. Two animals (n=2) were sacrificed at the indicated time points at 0.5, 6, 12, 24, 48, 72, 120 and 168 hours after exposure to the formulation. Blood (plasma) and lung tissue samples were collected.

On the day of exposure, the formulation of paclitaxel particles (6 mg/ml) was prepared according to the instructions provided by the sponsor.

The total duration of aerosol exposure was 63 minutes for all animals. Aerosol concentration was monitored throughout the 63 minute period of exposure to the aerosol formulation of paclitaxel particles by measuring the amount of formulation that accumulated on 47 mm GF/A filters placed in the respiratory zone of the exposure chamber by nasal administration only. Aerosol particle size (droplet size) was measured using a Mercer-type cascade impactor in the animal respiratory zone in the exposure chamber.

The suspension composition of paclitaxel particles was nebulized using two Hospitak compression jet nebulizers (average concentration of paclitaxel in aerosol: target 82.65 μg/l). The total mean measured aerosol concentration on the GF/A filters was 0.24 mg/l and the mean aerosol concentration of paclitaxel was 73.5 μg/ml. When measuring the particle size distribution, MMAD was 2.0 μm at a GSD of 2.2. The measured mean paclitaxel aerosol concentration of 73.5 µg/L was ~11% lower than the target mean paclitaxel aerosol concentration of 82.65 µg/L (within ±15% analytical test accuracy/detection performance criteria). Monitored the level of oxygen and temperature during exposure to the aerosol composition of particles of paclitaxel. The measured oxygen level and temperature ranges were 19.7%-20.9% and 20.4°C-20.8°C, respectively.

The dose of paclitaxel deposited in the lungs was calculated assuming that the mean concentration of paclitaxel in the aerosol was 73.5 μg/l, the mean body weight of the rodent was 326 g, the estimated deposited fraction was 10%, and the duration of exposure was 63 minutes. The average deposited dose for rodents was determined to be 0.33 mg/kg. The mean deposited dose was ~11% lower than the target deposited dose of 0.37 mg/kg, but was within the expected error (±15% of target) for spray exposure.

All animals survived to their indicated necropsy time. During autopsy, minimal red staining of the lungs was observed in several animals. No other macroscopic abnormalities were noted at the time of autopsy. According to the results of body and lung weights obtained at the time of autopsy, the mean final body weight of the animals for all time points (standard deviation) was 346.26 g (24.01); and the mean lung mass (standard deviation) was 1.60 g (0.13).

The systemic blood flow (for plasma in K ₂ -EDTA tubes) was examined by liquid chromatography-mass spectrometry (LCMS) and the lungs were examined, as briefly described in section 4.6 (bioanalytical study), to evaluate the dependence of the amount of paclitaxel on time. In lung tissue analysis, the amount of paclitaxel in the lungs was detectable up to 168 hours. In the systemic circulation, the amount of paclitaxel could not be detected (less than 1 ng / ml) after 24 hours. Based on the data obtained, the following sampling times were proposed for the study of RK: after 0.5 (±10 minutes), 6 (±10 minutes), 12 (±10 minutes), 24 (±30 minutes), 48 (±30 minutes ), 72 (±30 minutes), 120 (±30 minutes) 168 (±30 minutes), 240 (±30 minutes) and 336 (±30 minutes) hours after inhalation.

Tasks

The objective of this pilot study was to determine the timing of sampling in a complete pharmacokinetic (PK) study of paclitaxel particle composition. Preliminary data for the particle composition of paclitaxel administered by intraperitoneal (i.p.) injection indicate a significant retention time in the intra-abdominal cavity. Because the paclitaxel particle formulation had the potential to increase pulmonary retention, time points up to 168 hours were evaluated to determine an appropriate sampling strategy in the full pharmacokinetic study.

MATERIALS AND METHODS

INVESTIGATED SYSTEM

Kind/line: Sprague Dawley Rats Animal age at start 8-10 weeks research: Body weight range at start 308-353 g research: Number of subjects 18 males (16 test animals and 2 spares) animals/gender: Source: Charles River Laboratories (Kingston, NY) Identification: Permanent marker on tail Study and Control Composition and Introduction

The suspension composition of paclitaxel particles (6 mg/ml) was prepared according to the instructions provided by the sponsor. Briefly, 5.0 ml of 1% polysorbate 80 was added to a tube containing paclitaxel particles (306 mg). The suspension of paclitaxel particles was vigorously shaken and inverted to wet all the particles contained in the tube. Immediately after shaking, 46 ml of 0.9% sodium chloride was added to the tube and the tube was shaken for at least 1 minute to ensure sufficient mixing and a suitable dispersion of the suspension. The resulting composition was left for at least 5 minutes to reduce the level of air/foam in the tube, after which it was placed in a nebulizer for spraying. The final formulation was kept at room temperature and used within 3 hours of reconstitution.

Experiment Design

Sixteen (16) Sprague-Dawley rats were exposed to a single inhalation of a suspension formulation of paclitaxel particles (target dose 0.37 mg/kg) through the nose only. Two animals (n=2) were sacrificed after 0.5 (±10 minutes), 6 (±10 minutes), 12 (±10 minutes), 24 (±30 minutes), 48 (±30 minutes), 72 (± 30 minutes), 120 (±30 minutes) and 168 (±30 minutes) hours after exposure and collected blood (plasma) and lung tissue. Specific modeling of PK was not performed; on the contrary, based on the data obtained, it is planned to determine the post-exposure period during which the amount of paclitaxel is detectable in the PK study.

Conditions of detention, quarantine and distribution to the study

Male Sprague-Dawley rats (6-8 weeks old) were obtained from Charles River Laboratories (Kingston, NY) and kept in quarantine for 14 days. After quarantine was completed, animals were weighed and then randomly distributed by weight to participate in the study. Animals were identified by tail markings and cage cards. Water consumption, lighting, humidity and temperature were controlled and monitored by standard methods. The rats were given access to standard rodent chow ad libitum during non-treatment periods.

Body weight and daily observations

Body weight was determined at the time of randomization, daily during the study, and after sacrifice. Comparative Medicine Animal Resources (CMAR) staff observed each animal twice daily during the study for clinical signs of abnormality, agony, or death.

Aerosol exposure by nasal administration only

Training

The preparation of animals for exposure tubes by introduction only through the nose was carried out for a period of up to 70 minutes by standard methods. Three preparation sessions were performed over the three days prior to exposure, the first session lasting 30 minutes, the second 60 minutes, and the third 70 minutes. The animals were carefully examined during the preparation and exposure periods to ensure that they did not experience anything more than a momentary breakdown.

Impact system

The inhalation exposure system consisted of two compression jet nebulizers (Hospitak) and a rodent exposure chamber by nasal inhalation only. During exposure, the oxygen level (%) was monitored. The paclitaxel particle suspension formulation aerosol was prepared in a two-compression jet nebulizer set (used up to 40 (±1) minutes, then changed to a second two-compression jet nebulizer set for the remainder of exposure) with an inlet pressure of 20 psi (140 kPa). The aerosol was directed through a 24 inch stainless steel aerosol delivery line (1.53 cm diameter) into the treatment chamber by nasal administration only.

Concentration Tracking

Aerosol concentration was monitored by collecting the aerosol on pre-weighed GF/A 47 mm filters. Filters were collected from the rodent respiratory zones in the exposure chamber by nasal insertion only during the rodent exposure period. The flow rate for sampling the aerosol through the GF/A filters was maintained at 1.0±0.5 l/min. A total of six GF/A filters were collected, one every 10 minutes during exposure, except for the last filter, which was collected 13 minutes after the previous one. After sampling, the filters were weighed to determine the total aerosol concentration in the exposure system. The contents of the filters were extracted and analyzed by high performance liquid chromatography (HPLC) to evaluate the amount of paclitaxel collected on each filter. The total aerosol concentration and paclitaxel aerosol concentration for each filter were calculated by dividing the total amount of aerosol and paclitaxel in the collected aerosol by the total volume of air passed through the filter. The average concentration of paclitaxel in the aerosol was used to calculate the average deposited dose of paclitaxel in the lungs of rodents using Equation 1 shown below.

Particle Sizing

Aerosol particle size distribution in the respiratory zone for exposure chamber rodents was determined by nasal only administration using a seven-stage Mercer-type cascade impactor (Intox Products, Inc., Albuquerque, NM). The particle size distribution was determined using mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD). Samples in the cascade impactor were collected at a flow rate of 2.0±0.1 l/min.

Dose Determination

The deposited dose was calculated using Equation 1. For this calculation, the mean aerosol concentration measured during exposure was used as well as the mean body weight in the group of rats. Thus, the estimated amount of paclitaxel deposited in the lungs of rats was calculated using the measured concentration of paclitaxel in the aerosol.

where:

Precipitated dose = (OD) mcg/kg

² Minute respiratory volume (MOD) = 0.608 x MT ^0.852

Exposure Aerosol Concentration (CA) = Aerosol Concentration of Paclitaxel (µg/L)

Settled Fraction (RP) = Expected Settled Fraction of 10%

BW = mean body weight (at the time of randomization; day -1) of study animals (kg)

Mortification and autopsy

Animals were sacrificed at the appropriate time points by i.p. injection of killing solution. During autopsy, blood was collected (for plasma isolation) by cardiac puncture into a K ₂ -EDTA tube, lungs were weighed, lung tissue samples were collected and flash frozen in liquid nitrogen for bioanalytical studies. In addition, qualified personnel performing the autopsy performed a full macroscopic evaluation. External surfaces of the body, sinuses, and contents of the cranial, thoracic, and abdominal cavities were assessed. Lesions were described and their morphology, number, shape, color, structure, and severity were indicated using dictionary terms.

Bioanalytical research

Systemic blood flow (plasma in K ₂ -EDTA tubes) and lung tissue were studied by liquid chromatography-mass spectrometry (LCMS) to assess the time dependence of the amount of paclitaxel. Briefly, the study uses ultra high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) analysis to quantify paclitaxel levels. Extracted plasma samples by the method of protein precipitation, carried out the separation by reversed-phase chromatography. Homogenized lung samples in water at a ratio of 4:1 (water : lung tissue). The homogenate was then treated in a manner similar to protein precipitation prior to LCMS analysis. Quantification was carried out using the calibration curves obtained for the matrix solutions.

Pharmacokinetic modeling of the data obtained was not performed, however, the concentration at which the level of paclitaxel falls below the detection limits of the assay (1 ng/ml) was used to determine the points for sampling in the main PK study.

RESULTS

Clinical abnormalities and survival

All animals survived to the point of autopsy indicated for them and gained weight. No clinical abnormalities were noted during the study.

Exposure to paclitaxel particles

Aerosol concentration

Table 1 shows the total aerosol concentration and paclitaxel aerosol concentration measured after sampling on each GF/A filter during exposure. The mean paclitaxel aerosol concentration during the inhalation exposure of 73.5 µg/L was ~11% lower compared to the target mean paclitaxel aerosol concentration of 82.65 µg/L. The mean aerosol concentration during exposure was within ±15% of the target aerosol concentration, which would be expected for inhalation exposure by nebulization.

Oxygen level and temperature

The measured oxygen level and temperature ranges were 19.7%-20.9% and 20.4°C-20.8°C, respectively.

Particle size

Particle size distribution was determined using MMAD (GSD), which for a 6.0 mg/ml aerosol of paclitaxel particle formulation using a cascade impactor was 2.0 (2.2) µm.

deposited dose

Given that the mean concentration of paclitaxel in the aerosol was 73.5 μg/l, the mean body weight of the rodents on day -1 (randomization) was 326 g, the estimated precipitated fraction was 10%, and the duration of exposure was 63 minutes; it was determined that the average deposited dose for rodents was 0.33 mg/kg. The mean deposited dose was ∼11% lower than the target deposited dose of 0.37 mg/kg, given the expected error (±15% of target) in mean aerosol concentration during exposure.

Opening

All animals survived to their indicated necropsy time. During autopsy, minimal red staining of the lungs was observed in several animals. No other macroscopic abnormalities were noted at the time of autopsy. Individual and mean values of lung weight, body weight and ratio were determined. The mean body weight at the end of the study (standard deviation) was 346.26 g (24.01). The mean lung weight (SD) was 1.60 g (0.13). Lung weight and the ratio of lung weight to body weight are parameters that are widely used to assess possible toxicological responses to inhaled materials. Overall, the data were consistent with historical values and indicated no response according to any of the specified evaluation criteria.

Bioanalysis

The results are shown in Table 2 below and in Figure 1. The mean plasma concentration of paclitaxel was 16.705 ng/mL at 0.5 hours post-exposure and then gradually decreased until the 24 hour point and was below the lower detection limit (1 ng/mL) for all subsequent time points. The mean concentration of paclitaxel in lung tissue was 21940 ng/g at 0.5 hours post-exposure and gradually decreased to 419.6 ng/g at 168 hours. This indicates significant retention of paclitaxel particles in the lungs with minimal systemic exposure.

CONCLUSIONS

Sixteen (16) male Sprague-Dawley rats were exposed to a single aerosol inhalation of paclitaxel particulate formulation (target dose 0.37 mg/kg) through the nose only. Two animals each (n=2) were sacrificed at 0.5, 6, 12, 24, 48, 72, 120 and 168 hours post-exposure to collect blood (plasma) and lung tissue samples.

The mean paclitaxel aerosol concentration of 73.5 µg/L during the 63-minute inhalation exposure was ~11% lower than the target mean paclitaxel aerosol concentration of 82.65 µg/L. The mean aerosol concentration during exposure was within ±15% of the target aerosol concentration, which would be expected for inhalation exposure by nebulization. The particle size distribution was determined using MMAD (GSD), which for the 6.0 mg/ml aerosol of the paclitaxel particle composition in the cascade impactor was 2.0 (2.2) μm. The measured oxygen level and temperature ranges were 19.7%-20.9% and 20.4°C-20.8°C, respectively.

The precipitated dose of paclitaxel was calculated assuming that the mean concentration of paclitaxel in the aerosol was 73.5 μg/l, the mean body weight of the rodents was 326 g, the estimated precipitated fraction was 10%, and the duration of exposure was 63 minutes. The average deposited dose for rodents was determined to be 0.33 mg/kg. The mean deposited dose was ~11% lower than the target deposited dose of 0.37 mg/kg, given the expected error (±15% of target).

All animals survived to their indicated necropsy time. During autopsy, minimal red staining of the lungs was observed in several animals. No other macroscopic abnormalities were noted at the time of autopsy. According to the results of body weight and lung weight obtained at the time of autopsy, the mean body weight at the end of the study (SD) was 346.26 g (24.01) and the mean lung weight (SD) was 1.60 g (0. 13). Lung weight and the ratio of lung weight to body weight are parameters that are widely used to evaluate possible toxicological responses to inhaled materials. Overall, the data indicated no response according to any of the specified evaluation criteria.

The mean plasma concentration of paclitaxel was 16.705 ng/mL at 0.5 hours post-exposure, then decreased gradually up to the 24 hour point and was below the lower detection limit for all time points after 24 hours. The mean concentration of paclitaxel in lung tissue was 21940 ng/g at 0.5 hours post-exposure and gradually decreased to 419.6 ng/g by 168 hours. This indicates significant retention of paclitaxel particles in the lungs with minimal systemic exposure. The following sampling times were suggested for the PK study: 0.5 (±10 minutes), 6 (±10 minutes), 12 (±10 minutes), 24 (±30 minutes), 48 (±30 minutes), 72 ( ±30 minutes), 120 (±30 minutes) 168 (±30 minutes), 240 (±30 minutes) and 336 (±30 minutes) hours after exposure.

Example 2: FY17-008B Study - Paclitaxel Particulate Aerosol Inhalation Exposure Study

EXPLANATORY NOTE

The main objective of this work was to provide exposure to male rats by inhalation only through 6.0 mg/ml and 20.0 mg/ml suspension formulations of paclitaxel particles. Each inhalation exposure for rats was carried out for 65 minutes.

The 6.0 mg/ml and 20.0 mg/ml suspension formulations of paclitaxel particles were prepared according to the instructions provided by the sponsor. Simultaneously, two Hospitak compression jet nebulizers at 20 psi (140 kPa) pressure were used to spray the paclitaxel particle formulation into the rodent inhalation chamber. During each exposure, the aerosol concentration in the respiratory zone for the animals was measured by sampling on 47 mm GF/A filters at a flow rate of 1.0±0.5 l/min. The particle size was determined by taking samples of aerosols from the respiratory zone for animals using a Mercer-type cascade impactor with a flow rate of 2.0±0.1 l/min. Gravimetric analysis of the filters was performed to determine the total aerosol concentration of paclitaxel particles and high performance liquid chromatography (HPLC) to determine the concentration of paclitaxel in the aerosol for each exposure. Monitored the level of oxygen and temperature and indicated during inhalation exposure.

The mean total aerosol concentration of paclitaxel particles and the paclitaxel aerosol concentration were determined to be 0.25 mg/L at 7.43% RSD and 85.64 µg/L at 10.23% RSD, respectively, for an inhalation exposure of 6.0 mg/ml composition of paclitaxel particles. The mass median aerodynamic diameter (geometric standard deviation) measured with the cascade impactor was 1.8 (2.0) µm for the 6.0 mg/mL aerosol of paclitaxel particle formulation. The mean total paclitaxel particle aerosol concentration and paclitaxel aerosol concentration were determined to be 0.46 mg/L at 10.95% RSD and 262.27 µg/L at 11.99% RSD, respectively, for an inhalation exposure of 20.0 mg/ml composition of paclitaxel particles. The mass median aerodynamic diameter (geometric standard deviation) measured with the cascade impactor was 2.3 (1.9) µm for the aerosol 20.0 mg/mL paclitaxel particle formulation.

The mean deposited dose of paclitaxel calculated using Equation 1 for a 65 minute exposure was 0.38 mg/kg and 1.18 mg/kg for the 6.0 mg/mL and 20.0 mg/mL paclitaxel particle formulations, respectively.

COMPOSITION AND INHALATION EFFECT

Getting the composition

MATERIALS

Test item

The test article used for inhalation exposure is shown below.

Paclitaxel particles

The form: Paclitaxel particles (sterile) Description: New dry powder formulation of paclitaxel delivered at 306 mg/vial Provider: US Biotest Manufacturer: Crititech Storage conditions: Environmental conditions

Carrier

The carriers used to prepare the paclitaxel particle formulations are shown below.

Polysorbate 80

The form: Sterile 1% polysorbate 80 in 0.9% sodium chloride for injection Description: clear liquid Provider: US Biotest Manufacturer: Crititech Storage conditions: Environmental conditions

Saline

The form: Sterile 0.9% sodium chloride for injection, USP Description: clear liquid Manufacturer: Hospira, Inc., Ill. Storage conditions: Environmental conditions

COMPOSITION AND INHALATION EFFECT

Getting the composition

A 6.0 mg/ml formulation of paclitaxel particles was prepared as follows: Briefly, 5.0 ml of 1% polysorbate 80 was added to the tube containing paclitaxel particles (306 mg). The tube was vigorously shaken and inverted to wet all particles present in the tube. Immediately after shaking, 46 ml of 0.9% sodium chloride solution was added to the tube and the tube was shaken for at least 1 minute to ensure sufficient mixing and a suitable dispersion of the suspension.

The method for preparing the paclitaxel particle formulation described above for the 6.0 mg/mL formulation was used to prepare the 20.0 mg/mL paclitaxel particle formulation, with the exception that 10.3 mL of 0.9% sodium chloride solution was added to the tube, and not the 46 ml used for the 6.0 mg/ml formulation.

The resulting compositions were left for at least 5 minutes to reduce the level of air/foam in the tube, after which it was placed in a nebulizer for spraying. The final 6.0 mg/ml formulation was kept at room temperature and sprayed within 2 hours of reconstitution. The final 20.0 mg/ml formulation was kept at room temperature and sprayed within 30 minutes of reconstitution.

Experiment Design

Thirty (30) Sprague-Dawley rats were treated with a single intravenous dose of "Clinical Standard ^ABRAXANE® (paclitaxel: target dose 5.0 mg/kg), thirty (30) Sprague-Dawley rats were treated with paclitaxel particle formulations described herein (target dose 0.37 mg/kg) and thirty (30) Sprague-Dawley rats were exposed to single inhalation formulations of paclitaxel particles (target dose 1.0 mg/kg) through the nose only. Three animals (n=3) were sacrificed after 0.5 (±10 minutes), 6 (±10 minutes), 12 (±10 minutes), 24 (±30 minutes), 48 (±30 minutes), 72 (± 30 minutes), 120 (±30 minutes), 168 (±30 minutes), 240 (±30 minutes) and 336 (±30 minutes) hours after exposure to collect blood (plasma) and lung tissues. Conducted non-compartmental analysis of plasma and lung tissue to determine the period after exposure, during which the amount of paclitaxel was detectable for each group.

Exposure system setup/Aerosol production: As in example 1

Aerosol concentration tracking: As in example 1

Particle size distribution: As in example 1

Calculation of the deposited dose: As in example 1

RESULTS

Impact results

Aerosol concentration and particle size

The aerosol concentration was monitored during each exposure by administering an aerosol formulation of paclitaxel particles using 47 mm GF/A filters in the animal respiratory zone of the exposure chamber by nasal administration only. During each exposure, samples were taken on seven 47 mm GF/A filters. Samples in filters FS-1 to FS-6 were collected for 10 minutes and in filter FS-7 for 5 minutes for each of the low and high dose groups. Particle size was measured using a Mercer-type cascade impactor in each animal respiratory zone of the exposure chamber. Tables 3 and 4 show the total aerosol concentration and paclitaxel aerosol concentration measured after sampling on GF/A filters during low dose and high dose exposure, respectively.

Particle size distribution (aerosol droplet size) was determined using MMAD (median aerodynamic particle mass diameter distribution) (GSD; used in MMAD measurement to describe the variability in particle size distribution) for each paclitaxel particle composition aerosol using a cascade impactor. For aerosols of 6.0 mg/mL and 20.0 mg/mL paclitaxel particle formulations, MMAD (GSD) was determined to be 1.8 (2.0) µm and 2.3 (1.9) µm, respectively. Figures 2 and 3 show the particle size distribution for 6.0 mg/mL and 20.0 mg/mL aerosol formulations of paclitaxel particles, respectively.

deposited dose

The deposited dose of paclitaxel was calculated from the mean paclitaxel aerosol concentration, mean body weight of rats, estimated precipitated fraction of 10%, and exposure time of 65 minutes for low dose and high dose paclitaxel particle formulations using Equation 1. Table 5 shows the mean concentration of paclitaxel in aerosol, average body weight of rats, duration of exposure and deposited dose for each exposure. The mean deposited dose in rodents was determined to be 0.38 mg/kg and 1.18 mg/kg for exposure to 6.0 mg/ml and 20.0 mg/ml paclitaxel particle formulations, respectively.

Oxygen level and temperature

Oxygen levels and temperature were monitored during exposure to paclitaxel particulate formulations in aerosol form. The resulting oxygen level and temperature ranges were 19.8%-20.9% and 20.7°C-20.8°C, respectively, for exposure to 6.0 mg/ml paclitaxel particle formulation. For exposure to 20.0 mg/ml paclitaxel particle formulation, the oxygen level was 19.8% at the time of exposure and the temperature range was 20.7°C-20.8°C.

Preliminary data are shown in figures 4-6.

Example 3 Efficacy Efficacy of Inhaled Paclitaxel Particle Formulations in Nude Rat Orthotopic Lung Cancer Model - Study FY17-095

EXPLANATORY NOTE

One hundred twenty-seven (127) athymic NIH-rnu rats were irradiated with X-rays to suppress the immune system on day -1. On day 0, the animals were injected with Calu3 tumor cells by intratracheal (i.t.) insertion. Animals were kept for a three week growth period. At the third week, the animals were randomly distributed by stratification by body weight into 5 study groups. Beginning at week 4, animals in Group 2 received a dose of ^ABRAXANE® (5 mg/kg) intravenously (iv) once a week on days 22, 29 and 36. Animals from groups 3 and 4 were administered by inhalation (ing.) once a week (on Mondays) low (0.5 mg/kg) and high (1.0 mg/kg) target dose, respectively, of paclitaxel particle formulations. Groups 5 and 6 were inhaled twice a week (Mondays and Thursdays) with low (0.50 mg/kg) and high (up to 1.0 mg/kg) target dose, respectively, of paclitaxel particle formulations. Animals from group 1 were not treated, they were used as a control for the normal growth of tumor cells. All animals were sacrificed at 8 weeks.

All animals survived to their indicated necropsy time. Clinical abnormalities associated with the model included reddening of the skin and difficulty in breathing. In all groups, weight gain during the study was approximately at the same level.

The mean concentration of paclitaxel in the aerosol after inhalation exposure in the groups in which the low dose formulation of paclitaxel particles was administered once a week and the low dose twice a week was 270.51 μg/l and 263.56 μg/l, respectively. The mean concentration of paclitaxel in the aerosol after inhalation exposure in the groups in which the high dose formulation of paclitaxel particles was administered once a week and the high dose twice a week was 244.82 μg/l and 245.76 μg/l, respectively.

Doses were determined by taking into account the mean concentration of paclitaxel in the aerosol, the most recent measured body weight in the group, an estimated precipitated fraction of 10%, and an exposure time of 33 (low dose) or 65 (high dose) minutes. During the four-week treatment period, the mean precipitating dose for rodents in the group administered the low dose formulation of paclitaxel particles once a week and the group administered the low dose formulation of paclitaxel particles twice a week were 0.655 mg/kg and 0.640 mg/kg (1.28 mg/kg/week), respectively. The mean precipitating dose of paclitaxel in rodents in the group administered the high dose formulation of paclitaxel particles once a week and the group administered the high dose formulation of paclitaxel particles twice a week were 1.166 mg/kg and 1.176 mg/kg ( 2.352 mg/kg/week), respectively. In the group in which iv was administered. injections of ABRAXANE ^® , the average dose administered on days 22, 29 and 36 was 4.94, 4.64 and 4.46 mg/kg, respectively.

At the time of scheduled necropsy, most animals from each group had pale brown lung nodules and/or red or pale brown lung patches. Other single abnormalities included abdominal hernia in one animal and pericardial nodules in another animal. No other macroscopic abnormalities were noted at the time of autopsy.

In ^ABRAXANE® treated animals, lung weight, lung weight to BW ratio, and lung weight to brain weight ratio were significantly lower compared to untreated controls. In the once weekly high dose paclitaxel particle formulation group, weight values were similar to the ^ABRAXANE® group, and lung weight and lung weight to brain weight ratio were significantly lower compared to the no-treatment control.

According to the results of histological assessment, signs of tumor formations were observed in the lungs of most animals in all groups. The tumor masses were characterized by the presence of small masses of various sizes, capable of growth, randomly scattered throughout the lung parenchyma, and larger overgrown and fused masses that occupied up to 75% of the lung parenchyma, small airways and blood vessels. Larger lesions were distributed mainly in the basal areas or near the central axis of the respiratory tract, while smaller lesions were generally located on the periphery.

Primary morphological cellular characteristics of lung tumors ranged from undifferentiated to relatively well differentiated lung adenocarcinoma. For the main type of tumor cells, the morphology of undifferentiated adenocarcinoma was shown; the cells were pleomorphic, large, anaplastic, gave pale amphophilic staining and had intracytoplasmic vacuoles resembling mucous vesicles, moderate to severe anisokaryosis was noted, cells grew singly or in layers, and there were no clearly defined signs of differentiation into adenocarcinoma cells. However, according to their morphological characteristics, the cells that were observed in other formations or grew in the undifferentiated formations described above were more organized and could be attributed to highly differentiated lung adenocarcinoma due to pronounced differentiation into cells of the acinar glands. These amphophilic staining tumor cells were distributed mainly in nested clusters or in glands which were observed to be connected by alveolar septa. In the population of these tumor cells, mitotic figures were rarely observed. Even more rarely, in these formations, foci of relatively small germinal tumor cells with a primitive structure and slight or moderate pale basophilic staining of the cytoplasm, oval and various vesicle-shaped nuclei, and moderate anisokaryosis were observed. These germline tumor cells were observed to grow randomly and in layers. An increased number of mitotic figures and apoptotic bodies was most often noted in this basophilic population of germline tumor cells. Inflammation characterized by infiltration of various inflammatory cells (mainly eosinophils, lymphocytes, foamy macrophages and sometimes giant cells) accompanied by interstitial fibrosis has been frequently observed. Significant necrosis of the parenchyma was absent in rare cases.

The pathologist considered the presence of irregularities at the boundaries of individual tumor formations, characterized by the gradual disappearance of tumor cells up to the complete disappearance of all tumor cells, and residual fibrosis of the connective tissue of the lung parenchyma, together with the invasion of foamy macrophages, as an indicator of tumor regression.

Compared to the positive control group 1 and the comparative ABRAXANE ^® treatment group 2, the paclitaxel particle formulation groups (groups 3-6) showed a reduction in total tumor burden in the lung, characterized by a decrease in the severity of tumor masses. adenocarcinoma and a decrease in the population of germinal tumor cells, as well as signs of tumor regression. No other treatment-related lesions or signs were observed. Active infiltration of mononuclear cells into the lungs of animals treated with the paclitaxel particle formulation by inhalation was observed. Since there are no T cells in the model used, the cells were probably B cells or NK cells. It was hypothesized that localized exposure of the tumor to higher concentrations of paclitaxel particles led to a change in the environment and infiltration of mononuclear cells into the lungs.

TASKS

The objective of this study was to evaluate the efficacy of inhaled paclitaxel particle formulation versus intravenous dose of clinical standard ^ABRAXANE® in reducing tumor burden in an orthotopic lung cancer model.

MATERIALS AND METHODS

System under study Kind/line: nude rats NIH-rnu Animal age at start 3-5 weeks research: Body weight range at start Approximately 150-200 g research: Number of researched 127 males (120 test animals and 7 spares) animals/gender: Source: Envigo Identification: Permanent marker on tail

Composition of ^ABRAXANE®

As a clinical standard v.v. composition used the drug product ABRAXANE ^® . The drug product was reconstituted to a concentration of 5.0 mg/ml in saline on the day of administration and stored according to the manufacturer's instructions.

Composition of paclitaxel particles

The 20.0 mg/mL formulations of paclitaxel impact particles were prepared according to the sponsor's recommendations. Specifically, the paclitaxel particles were re-dissolved in 1% polysorbate 80. The tube was shaken by hand to wet all the particles. An additional amount of 0.9% sodium chloride for injection (to the target concentration) was added and the tube was shaken by hand for another minute. Continue to shake until the disappearance of large lumps and a suitable dispersion of the suspension.

The resulting compositions were left for at least 5 minutes to reduce the level of air/foam in the tube, after which it was placed in a nebulizer for spraying. The final composition was kept at room temperature and sprayed within 2 hours after reconstitution. The final 20.0 mg/ml formulation was kept at room temperature and sprayed within 30 (+5) minutes after reconstitution.

Experiment Design

One hundred twenty-seven (127) animals were used in the study. Before irradiation with X-rays and the introduction of tumor cells, 7 animals were determined in reserve (reserve animals were not irradiated and did not install the cell line). On day -1, all test animals were irradiated with X-rays to suppress the immune system. On day 0, the animals were injected with Calu3 tumor cells by intratracheal (i.t.) insertion. Animals were kept for a three week growth period. At week 3, animals were randomly ^assigned by body weight stratification to the groups described in Table 6 below. ). Animals from groups 3 and 4 were administered by inhalation (ing.) once a week (on Mondays) low (0.5 mg/kg) and high (1.0 mg/kg) target dose, respectively, of paclitaxel particle formulations. Groups 5 and 6 were inhaled twice a week (Mondays and Thursdays) with a low (0.50 mg/kg) and high (1.0 mg/kg) target dose, respectively, of paclitaxel particle formulations. Animals from group 1 were not treated, they were used as a control for the normal growth of tumor cells. All animals were sacrificed at 8 weeks.

Conditions of detention, quarantine and distribution to the study

After quarantine, all animals were weighed and randomly distributed into groups, with the exception of 7 spare animals, taking into account body weight. Between weeks 1 and 3, animals were identified by cage cards (LC numbers) and tail marks.

Animals were weighed at week 3 before treatment and randomly assigned to the groups listed above using body weight stratification and assigned a study number. From that moment on, the animals were identified by the cards on the cages and the bright markings on the tails.

Suppression of the immune system and radiation

On day -1, total x-ray exposure of the animals ~500 rad was performed (Phillips RT 250 X-ray Therapy Unit, Phillips Medical Systems, Shelton, CT, operating characteristics 250 kVp, 15 mA and source-to-object distance 100 cm). Animals were placed in a pie-shaped chamber ("cutie pie"), 2-3 animals for each "piece of pie". The irradiation process took ~10-15 minutes.

Implantation of tumor cells

On day 0 animals v.t. tumor cells (Calu3) were injected. Briefly, after anesthetizing with 3-5% isoflurane in an induction chamber, the animal was positioned so that the upper incisors were hooked onto an inclined suspended insertion platform. The animal's tongue was carefully fixed, and at that time the stylet was inserted into the trachea immediately behind the larynx. A suspension of cells in EDTA (target dose volume: 500 μl; concentration: about 20×10 ⁶ per 0.5 ml) was delivered to the lungs by intratracheal insertion. After installation, the breathing and movement of the animals were carefully monitored. After implantation of the tumor cells, the animals were kept for a growth period of approximately 3 weeks prior to treatment in order for the tumor cells to engraft and develop lung cancer.

Cultivation and preparation of Calu3

Calu3 cells were grown at 37° C., 5% CO ₂ in cell culture flasks. They were grown in Roswell Park Memorial Institute (RPMI) 1640 medium containing 10% fetal bovine serum (EBS) to 80% confluence. The cages were kept until the day of installation. Before installation, they were collected by washing with PBS, then trypsin was added to remove the cells from the flask. Cells were neutralized in RPMI 1640 medium containing 10% EBS. Then they were centrifuged at 100xg for 5 minutes, the medium was removed and the cells were resuspended to a concentration of 20 million cells in 450 μl of serum-free RPMI. Before installation, 50 μl of 70 μM EDTA was added to the cell suspension to obtain the total volume of wt. doses of 500 μl for each rat.

Body weight and daily observations

Body weights were determined at the time of randomization, weekly up to week 3, twice weekly from week 4 until the end of the study, and at autopsy.

Each animal was observed twice daily during the study for any clinical signs of abnormality, disease, or death. The technical staff observed the animals during the introduction and measurement of body weight.

V.v. administration of ABRAXANE ^® -injection into the tail vein

ABRAXANE ^® (5 mg/ml, maximum dose volume 250 μl) was administered to animals from group 2 by i.v. tail vein injections on days 22, 29 and 36.

Paclitaxel Particulate Formulation Administration - Aerosol Exposure by Nasal Only Administration

Training

The preparation of animals for exposure tubes by introduction only through the nose was carried out for a period of up to 70 minutes by standard methods. Three preparation sessions were performed over the three days prior to exposure, the first session lasting 30 minutes, the second 60 minutes, and the third 70 minutes. The animals were carefully examined during the preparation and exposure periods to ensure that they did not experience anything more than a momentary breakdown.

Impact system

Aerosols were generated using two Hospitak compression jet nebulizers as shown in FIG. 7, at 20 psi (140 kPa) nebulizer pressure. A 20.0 mg/mL suspension formulation of paclitaxel particles was used for low dose and high dose exposure. Aerosols were injected through a delivery line into a 32-hole exposure chamber. Inhalation exposure for rodents was carried out for 33 or 65 minutes. The paclitaxel particle suspension aerosol was prepared in a two Hospitak compression jet nebulizer set (used for up to 40 (±1) minutes), which was then changed to a second set of two Hospitak nebulizers for the remainder of the exposure period. Monitored and measured the level of oxygen and temperature during each inhalation exposure.

Concentration Tracking

Aerosol concentration was monitored by collecting aerosols on pre-weighed 47 mm GF/A filters. Filters were collected from the animal respiratory zones of the exposure chamber by nasal administration only during each inhalation exposure. The aerosol sampling flow rate on the GF/A filters was maintained at 1.0±0.5 L/min. The filters were collected during each exposure every 10 minutes except for the last filter. At a low dose (groups 3 and 5) exposure of 33 minutes, the last filter was collected 13 minutes after the previous one, and at a high dose exposure (groups 4 and 6) of 65 minutes, the last filter was collected after 15 minutes. After sampling, the filters were weighed to determine the total aerosol concentration in the exposure system.

After weighing, each filter was placed in a 7 ml glass tube. The contents of the filters were extracted into glass tubes and analyzed by high performance liquid chromatography (HPLC) to evaluate the amount of paclitaxel collected on the filters. The total aerosol concentration and paclitaxel aerosol concentration for each filter were calculated by dividing the total aerosol and paclitaxel aerosol concentration by the total volume of air passed through the filter. The average concentration of paclitaxel in the aerosol was used to calculate the average deposited dose of paclitaxel in the lungs of rodents using equation 1, as shown below in the section "determining the dose".

Dose Determination

The deposited dose was calculated using Equation 1. In this calculation, the mean aerosol concentration measured at the time of exposure and the mean body weight of the rats per group were used. In this way, the estimated amount of paclitaxel deposited in the lungs of rats was calculated from the measured concentration of paclitaxel in the aerosol.

where:

Precipitated dose = (OD) mcg/kg

² Minute respiratory volume (MOD) = 0.608 x MT ^0.852

Exposure Aerosol Concentration (CA) = Aerosol Concentration of Paclitaxel (µg/L)

Settled Fraction (RP) = Estimated Settled Fraction 10%

BW = mean body weight (at time of randomization; day -1) of study animals (kg)

Mortification and autopsy

At the scheduled necropsy period, the animals were euthanized by intraperitoneal injection of an overdose of a barbiturate-based sedative.

Collection of blood and tissue samples

At the time of autopsy, final body weight and brain weight were determined. During scheduled sacrifice, blood (plasma) was collected after cardiac puncture in K ₂ -EDTA tubes. The lungs were removed and weighed. A tumor tissue section containing a tumor, a tracheobronchial lymph node, was frozen in liquid nitrogen for possible future analysis. The remaining lung fragment was fixed for possible histopathology.

Histopathology

The fixed lobes of the left lung were sliced like a "loaf of bread" and sections were placed in turn in 2 cassettes to obtain 2 preparations of each section for 3 typical sections of the left lung. Tissues were processed in the usual way, embedded in paraffin, cut into ~4 μm sections, fixed and stained with hematoxylin and eosin (H&E) for microscopic examination. Conducted a subjective semi-quantitative evaluation of the results.

Lung sections (1-4/animal) obtained from 60 of 120 athymic rats studied were cut lengthwise and processed to obtain H&E-stained slides for evaluation by light microscopy.

During this analysis, microscopic examination results were recorded and sent to an electronic pathology record system (PDS-Ascentos-1.2.0, version 1.2), which aggregated frequency and severity scores for lung exercise data, pooled results, and acquired data for individual animals. Lungs of 60 athymic rats were histologically assessed: group 1 [1001-1010], group 2 [2001-2010], group 3 [3001-3010], group 4 [4001-4010], group 5 [5001-5010], and group 6 [6001-6010]. To assess the level of tumor burden in these lung samples, the lungs were examined and classified during histopathological examination. To diagnose the cumulative load characteristics of the lungs: 1) adenocarcinoma (undifferentiated and differentiated), 2) germinal tumor cells (poorly differentiated pleomorphic cells), and 3) tumor regression, a semi-quantitative classification of the lungs was performed using the following 4-point classification scale, which indicated the level of spread as a percentage of lung tissue as a whole: 0 = none, 1 = minimal (spread on ~1 - 25% of the total area of lung sections), 2 = low (spread on ~ 25 - 50% of the total area of lung sections), 3 = moderate (spread over ~50-75% of total lung section area) and 4 = severe (spread over ~75-100% of total lung section area).

Histomorphometry

Histomorphometric analysis was performed using fixed lobes of the left lung taken from the first 10 animals from each group. Tissue sections were obtained using a knife for morphometry (cutting "slices of bread"). Briefly, sections were started at a random site at a distance of 2 to 4 mm from the cranial edge of the lung. The thickness of each section of the lung was approximately 4 mm. Odd-numbered sections were placed caudally down in cassette 1 and even-numbered sections were placed in cassette 2. Tissue sections were then processed, embedded in paraffin and cut into 4 µm thick fragments and stained with hematoxylin and eosin (HE) for examination. Both preparations (odd and even sections) were used to determine the average tumor level in each animal.

Morphometric analysis was performed on lung tissues stained with hematoxylin and eosin (HE) taken from designated animals at Lovelace Biomedical. Whole preparations (2 for each animal, which contained transverse sections of the left lung as a whole) were scanned on the Hamamatsu Nanozoomer. Scans were analyzed using Visiopharm Integrator System software (VIS, version 2017.2.5.3857). Statistical analysis of the composition of the tumor site was performed using GraphPad Prism 5 (version 5.04).

Computerized quantitative image analysis, designed to estimate tumor area in each section, was performed on the left lung tissue as a whole using scans of whole preparations. The Visiopharm Lung Metastasis Area Quantification application was used to discriminate between tumor cells and normal lung tissue based on cell density, stain intensity, and stain size and intensity. It should be noted that this quantification, based on simple H&E staining, is not ideal (i.e., it does not fully distinguish between types of tumor tissues, dead and viable tumor tissues, and some normal structures can be attributed to tumor) . The value of using this method for H&E sections is that it provides an objective tumor quantification. The area of the lung as a whole is determined, and then the area occupied by structures classified as metastases is indicated as a percentage of the total area. Minor adjustments to the area under analysis can be made manually to exclude non-lung structures and include the entire lung in the analysis. Other changes are not made in a non-automated manner in order to ensure that conditions are constant for all groups and to eliminate the possibility of the appearance of subjectivity. If possible, the development of specific immunohistochemical stains to identify only tumor tissue could increase the specificity of this assay.

Collection and processing of blood samples

Blood collected at the time of autopsy was processed to obtain plasma by centrifugation at a speed of at least 1300g at 4°C for 10 minutes. Plasma samples were stored at -70 to -90°C until analyzed or sent to the sponsor.

results

Clinical abnormalities, survival and body weight.

All animals survived to their indicated necropsy time. Clinical abnormalities associated with the model included skin rash and difficulty breathing. According to observations, one animal had a hernia in the upper part of the stomach. In accordance with the recommendation of the veterinarian, the animal was transferred to group 1 (control without treatment), in which the inhalation exposure was not carried out, so there was no need to use tube restrictors for exposure.

FIG. 8 shows the average body weight over the course of the study. FIG. 9 shows the percent change in mean body weight from day 0. In all groups, body weight gain occurred at about the same rate over the course of the study.

V.v. ABRAXANE ^® injections into the tail vein

In the group in which iv was administered. ^ABRAXANE® injections, mean dose on days 22, 29, and 36, were 4.94, 4.64, and 4.46 mg/kg, respectively.

Exposure to paclitaxel particles

Aerosol concentration and deposited dose

The total aerosol concentration and the aerosol concentration of paclitaxel were measured by sampling on GF/A filters during each exposure. During inhalation exposure, the mean aerosol concentrations of paclitaxel in the groups administered the low dose formulation of paclitaxel particles once a week and the low dose formulation of paclitaxel particles twice a week were 270.51 µg/L and 263.56 µg/L, respectively. During inhalation exposure, the mean aerosol concentrations of paclitaxel in the groups administered the high dose formulation of paclitaxel particles once a week and the high dose formulation of paclitaxel particles twice a week were 244.82 µg/L and 245.76 µg/L, respectively. Oxygen levels and temperature were monitored during each exposure.

Doses were calculated taking into account the mean concentration of paclitaxel in the aerosol, the most recently measured average body weight in the group, the estimated precipitated fraction of 10%, and the duration of exposure of 33 or 65 minutes. During the four-week treatment period, the mean precipitating dose in the group administered the low dose formulation of paclitaxel particles once a week and the group administered the low dose formulation of paclitaxel particles twice a week were 0.655 mg/kg and 0.640 mg/kg. kg (1.28 mg/kg/week), respectively.

The mean deposited dose in the group administered the high dose formulation of paclitaxel particles once a week and the group administered the high dose formulation of paclitaxel particles twice a week were 1.166 mg/kg and 1.176 mg/kg (2.352 mg/kg). kg/week), respectively.

Particle size (MMAD and GSD)

Particle size distribution was determined by mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD) for each paclitaxel particle composition aerosol using a cascade impactor. For the 20.0 mg/mL aerosol of the paclitaxel particle formulation, the mean MMAD was determined to be 2.01 µm and the GSD 1.87.

Observations during autopsy and organ mass

All animals survived to their indicated necropsy time. At necropsy, animals from each group had pale brown nodules in the lungs and/or red or pale brown spots in the lungs. Other single abnormalities included abdominal hernia in one animal and pericardial nodules in another animal. No other macroscopic abnormalities were noted at the time of autopsy. One animal had no visible tumors (nodules) at the time of autopsy.

Organ weight data from individual animals is graphically shown in FIG. 10 FIG. 11 and FIG. 12. In ^ABRAXANE® treated animals, lung weight, lung weight to BW ratio and lung weight to brain weight ratio were significantly lower compared to untreated controls. In the once weekly high dose paclitaxel particle formulation group, measured weight was similar to the ^ABRAXANE® group, and lung weight and lung weight to brain weight ratio were significantly lower compared to the no-treatment control. The groups given low dose once weekly, low dose paclitaxel particle formulation twice weekly, and high dose paclitaxel particle formulation twice weekly generally had similar mean lung weights and ratios.

Morphometry

In all treatment groups, there was a decrease in the average level of tumor in the lungs compared with the control group; however, there were no statistically significant differences between the groups. There were also no statistically significant differences between v.v. the introduction of ABRAXANE ^® and any mode of administration of the composition of particles of paclitaxel in relation to the area of the tumor in the study of transverse sections of the lung. Animals in all groups showed high variability in tumor area, which is typical for this model. These data should be considered in combination with other measures of lung tumor burden in this model, including the ratio of lung mass to brain mass, and histopathology findings to arrive at the final score. It is important to note that morphometric analysis and histopathological evaluation were performed on fixed lung tissue obtained from the left lobe, while other lung tissue analyzes can be performed on frozen tissue from right lung lobes. The average tumor area is shown in FIG. 13 and FIG. fourteen.

RESULTS OF PATHOLOGY

Based on the results of studying the preparations of the identified neoplastic cell populations, the pathologist determined that: (1) the total tumor burden of adenocarcinoma (undifferentiated and differentiated) in the lungs decreased slightly in all treatment groups (group 2 (1.7), group 3 (1 .8), group 4 (1.7), group 5 (1.6) and group 6 (1.6)), compared with control group 1 without treatment (2.1). (2) the germline tumor cell population decreased as evidenced by the reduction in severity in group 3 (0.3), group 4 (0.3), group 5 (0.2) and group 6 (0.2) compared with the corresponding control group 1 (0.9) and group 2 (1.0), and (3) tumor regression occurred in group 3 (0.6), group 4 (1.0), group 5 (0.8) and group 6 (1.0) compared to the corresponding control group 1 (0.0) and group 2 (0.1). The frequency and severity of signs of tumor burden are shown in table 7 and in FIG. 15. Micrographs of preparations are shown in FIG. 16-50.

HISTOLOGICAL REVIEW OF THE MICROPHOTOGRAPHS IN FIG. 16-50

General observations:

Control: A large number of viable tumors with proliferating cells and no infiltration of immune cells.

ABRAXANE ^® IV: Many viable tumors, little lymphocytic response and little tumor regression.

Composition of paclitaxel particles, once a week, high dose: Disappearance of the tumor in the lungs, a small number of remaining viable tumor cells. The remaining lesions likely include immune cell infiltrate and fibrosis.

Particle composition of paclitaxel, 2 times a week, low dose: A small number of tumor nodules surrounded by an immune cell infiltrate, including macrophages and mononuclear cells.

Composition of paclitaxel particles, 2 times a week, high dose: Several tumor nodules with immune cell infiltrates, stromal fibrosis at the site of the tumor.

A highly active infiltration of mononuclear antitumor cells was observed in the lungs of animals injected with the paclitaxel particle formulation by inhalation. Since there are no T cells in the model used, the cells are likely to be B cells or NK cells, or both of these cell types. B cells are responsible for the production of antibodies and may be involved in the destruction of tumor cells due to antibody-dependent cellular cytotoxicity (antibodies bind to cells expressing Fc receptors and increase the killer activity of these cells). NK cells are innate lymphoid cells that are important for the destruction of tumor cells. In patients with tumors, NK cell activity is reduced, resulting in tumor growth. Along with T cells, NK cells are targets for some checkpoint inhibitors that can increase their activity.

Through the use of a variety of surface receptors that can deliver activating or inhibitory signals, NK cells can track cells in the appropriate microenvironment and determine if a cell is diseased (tumor or virus-infected) and should be killed using the mechanism of cytotoxicity.

Cytotoxicity and chemotaxis of NK cells can be modified by a variety of pathological processes involving tumor cells and metabolic products in these cells. In response to certain signals, their functions increase or increase. In response to certain pathogen-associated molecular patterns (PAMPs) through the use of various toll-like receptors (TLRs); NK cells may increase cytokine production and/or cytolytic activity. Cytokines, including IL-2, IL-15, IL-12, IL-18 and IFN α/β, can also modify the activity of NK cells. NK cells are not simple cells that act only as cytolytic effectors and can destroy various target tumor cells; on the contrary, they are a heterogeneous population that can fine-tune their activity in different environments.

As can be seen, tumor burden is significantly reduced in the lungs of animals after treatment with paclitaxel particle formulation and is lower compared to burden after iv. introduction of ABRAXANE ^® . Thus, localized administration of paclitaxel as a particulate formulation of paclitaxel provides additional activity. This is probably due to the increased duration of exposure to the chemotherapeutic agent and intense cell infiltration into the tumor site. This latter type of response probably depends on dose density (actual dose and frequency of administration).

Observations on specific photomicrographs

FIG. 16: Subject 1006 (control) adenocarcinoma-3, germ cells-1, tumor regression-0. Low magnification (2x) shows the usual distribution of undifferentiated pleomorphic large anaplastic tumor cells located within the alveolar space or lining the alveolar septa. The main part of the cells does not have signs of adenocarcinoma and is present in the layers adjacent to the tumor. Many cells have a basophilic cytoplasm, but others are large and anaplastic and have a pale amphophilic stain. The presence of a previously existing resident population of macrophages and the absence of tumor regression should be noted.

FIG. 30: Subject 2003 (vv ABRAXANE ^® ) adenocarcinoma-1, germ cells-1, regression-1. At low magnification (4x), the usual distribution of tumor formations is shown, mainly along the periphery, as well as several smaller growing tumor formations filling the alveolar space. Tumor cells are pleomorphic, large, anaplastic and have a pale amphophylic staining, various formations corresponding to both undifferentiated and differentiated forms of adenocarcinoma. Signs of tumor regression are observed along the periphery of the tumor and are characterized mainly by macrophage infiltration.

FIG. 36: Subject 2010 (v.v. ABRAXANE ^® ) adenocarcinoma-3, germ cells-1, tumor regression-0. At a low magnification (2x), the usual distribution of a large growing tumor formation is shown, filling most of the alveolar spaces, as well as neoplastic cells in the periphery. The majority of tumor cells are generally undifferentiated, pleomorphic, large, anaplastic, and have a pale amphophilic stain. Germ cells have a smaller oval shape and a cytoplasm with more intense basophilic staining, as well as various vesicular nuclei and moderate to severe anisokaryosis. The infiltrating inflammatory cells are mainly neutrophils and macrophages. This image shows no signs of tumor regression.

FIG. 39: Subject 4009 (ing. paclitaxel particle formulation, 1 time/week, high dose) adenocarcinoma-0, germ cells-0, tumor regression-4. Low magnification (2x) shows the usual distribution of previously colonized tumor masses, the presence of several small areas of fibrous connective tissue, central collagen stroma and fibrocytes are present in the peripheral alveolar spaces, and thickened alveolar septa confirm tumor regression. In addition, most of the alveolar spaces are filled with an infiltrate of macrophages and lymphocytes, which is another confirmation of tumor regression.

FIG. 42: Subject 5010 (ing. paclitaxel particle formulation, 2 times/week, low dose) adenocarcinoma-1, germ cells-0, tumor regression-3. At a low magnification (2x), the usual distribution of previously populated tumor formations is shown. The regressing formations are of variable but small size and are randomly distributed. According to observations, fibrous connective tissue fills/replaces alveolar spaces, which suggests the presence of foci of adenocarcinoma regression. Acute necrosis, a fibrous backbone of the connective tissue, and mixed infiltration of macrophages, giant cells, and lymphocytes in the epithelium and around the stroma are signs of tumor regression.

FIG. 46: Subject 6005 (ing. paclitaxel particle formulation, 2 times/week, high dose) adenocarcinoma-1, germ cells-0, tumor regression-4. At a low magnification (2x), the usual distribution of previously colonized tumor formations in several small areas of fibrous connective tissue that filled/replaced the alveolar spaces is shown, which suggests the presence of foci of early infiltrates in adenocarcinoma cells. Tumor regression is confirmed by fibrosis of previously populated tumor formations, the presence of a central collagen core of the stroma and fibrous connective tissue on the periphery that fills / replaces the alveolar spaces, thickening of the septa, and the presence of fibrocytes filling the alveolar space infiltrated by lymphocytes and macrophages.

CONCLUSIONS

One hundred twenty-seven (127) athymic NIH-rnu rats were irradiated with X-rays to suppress the immune system on day -1. On day 0, the animals were injected with Calu3 tumor cells by intratracheal (i.t.) insertion. Animals were kept for a three week growth period. In the third week, the animals were randomly assigned to groups by stratification by body weight. Beginning at week 4, animals in Group 2 received a once weekly dose of ^ABRAXANE® (5 mg/kg) intravenously (iv) on days 22, 29 and 36. Animals from groups 3 and 4 were administered once a week (Mondays) by inhalation (ing.) low (0.5 mg/kg) and high (1.0 mg/kg) target dose of the paclitaxel particle formulation, respectively. Animals in Groups 5 and 6 were administered twice weekly (Mondays and Thursdays) by inhalation at the low (0.50 mg/kg) and high (1.0 mg/kg) target dose formulation of paclitaxel particles, respectively. Group 1 animals were left untreated as controls for normal tumor cell growth. All animals were sacrificed at 8 weeks.

All animals survived to their indicated necropsy time. Clinical abnormalities associated with the model included skin rash, difficulty breathing. In all groups, body weight gain occurred at approximately the same rate during the study.

During inhalation exposure, the mean aerosol concentrations of paclitaxel in the groups administered the low dose formulation of paclitaxel particles once a week and the low dose formulation of paclitaxel particles twice a week were 270.51 μg/L and 263.56 μg/L, respectively. During inhalation exposure, the mean aerosol concentrations of paclitaxel in the groups administered the high dose formulation of paclitaxel particles once a week and the high dose formulation of paclitaxel particles twice a week were 244.82 µg/L and 245.76 µg/L, respectively.

Doses were calculated taking into account the mean concentration of paclitaxel in the aerosol, the most recently measured average body weight in the group, the estimated precipitated fraction of 10%, and the duration of exposure of 33 or 65 minutes. During the four-week treatment period, the mean precipitating dose for rodents from the group administered the low dose formulation of paclitaxel particles once a week and the group administered the low dose formulation of paclitaxel particles twice a week was 0.655 mg/kg and 0.640 mg /kg (1.28 mg/kg/week), respectively. The mean precipitating dose for rodents from the group administered the high dose formulation of paclitaxel particles once a week and the group administered the high dose formulation of paclitaxel particles twice a week were 1.166 mg/kg and 1.176 mg/kg (2.352 mg /kg/week), respectively. In the group in which IV. ^ABRAXANE® injections, mean dose on days 22, 29, and 36, were 4.94, 4.64, and 4.46 mg/kg, respectively.

At the time of the scheduled autopsy, most animals from each group had pale brown lung nodules and/or red or pale brown lung patches. Other single abnormalities included abdominal hernia in one animal and pericardial nodules in another animal. No other macroscopic abnormalities were noted at the time of autopsy.

In ^ABRAXANE® treated animals, lung weight, lung weight to BW ratio, and lung weight to brain weight ratio were significantly lower compared to untreated controls. In the once-weekly high dose paclitaxel particle formulation group, weight values were similar to the ^ABRAXANE® group and were significantly lower compared to lung weight and lung-to-brain weight ratio for no-treatment controls.

Compared with the positive control group 1 and comparison group 2, which was injected with ABRAXANE ^® , there was a therapeutic effect, defined by a decrease in the ratio of lung mass/brain mass and a decrease in the total tumor burden in the lungs and the absence of obvious adverse events. Histological analysis of tumor burden in the lungs after treatment with the inhaled formulation of paclitaxel particles showed a decrease in tumor formation, a decrease in the germinal tumor cell population, and an increase in tumor regression. Active infiltration of mononuclear cells was observed in the lungs of animals administered the paclitaxel particle formulation by inhalation. Since there are no T cells in the model used, the cells are likely to be B cells or NK cells. It was hypothesized that localized exposure to paclitaxel particles at a presumably higher concentration affected tumors and led to environmental changes and infiltration of mononuclear cells into the lungs.

Example 4: Study FY 17-008B - Study of the pharmacokinetics of paclitaxel particles

EXPLANATORY NOTE

Ninety (90) male Sprague-Dawley rats were treated with the "clinical standard" dose of paclitaxel, ^ABRAXANE® (paclitaxel protein-bound particles for suspension for injection, or nab-paclitaxel), by intravenous (iv) bolus injection. or paclitaxel particle formulation (target dose 0.37 or 1.0 mg/kg) by single inhalation through the nose only. Three animals (n=3) were sacrificed at ten (10) time points from 0.5 to 336 hours post-exposure and blood (plasma) and lung tissue were collected. Conducted non-compartmental analysis (NCA) of plasma and lung tissue to determine the period of time after exposure, during which the amount of paclitaxel is detectable in each group. Animals in which the time point of 336 hours was studied had the right lungs collected from all groups for liquid chromatography-mass spectrometry (LCMS) analysis, while the left lungs were perfused in 10% neutral buffered formalin (NBF) and stored for possible performing histopathology. For comparative histopathology, three spare animals (control animals not previously used in experiments) were sacrificed 336 hours later and lungs were harvested in the same manner. Lungs collected from animals at all other time points were frozen individually for LCMS analysis.

During inhalation exposure, mean paclitaxel aerosol concentrations in the low dose and high dose paclitaxel particle formulation groups were 85.64 µg/L and 262.27 µg/L, respectively. The mean aerosol concentration during exposure was within ±15% of the target aerosol concentration, which was expected for inhalation exposure by nebulization. Particle size distribution was determined by MMAD (GSD) for each paclitaxel particle composition aerosol using a cascade impactor. For the 6.0 mg/mL and 20.0 mg/mL aerosols, the particle composition of paclitaxel MMAD (GSD) was determined to be 1.8 (2.0) µm and 2.3 (1.9) µm, respectively.

The precipitated dose of paclitaxel using the low dose was calculated taking into account the mean concentration of paclitaxel in the aerosol of 85.64 μg/l, the mean body weight in the group per day of 0 420.4 g, the estimated precipitated fraction of 10% and the duration of exposure of 65 minutes; it was determined that the mean precipitating dose in rodents was 0.38 mg/kg in the low dose paclitaxel particle formulation group. In the high-dose paclitaxel particle formulation group, the mean aerosol concentration of paclitaxel was 262.27 μg/L, the mean group body weight on day 0 was 420.5 g, the estimated precipitated fraction was 10%, and the duration of exposure was 65 minutes; it was determined that the average deposited dose for rodents was 1.18 mg/kg. The measured oxygen level and temperature ranges were 19.8%-20.9% and 20.7°C-20.8°C, respectively, for exposure to 6.0 mg/ml paclitaxel particle formulation. In the case of exposure to 20.0 mg/ml paclitaxel particle composition, the measured oxygen level was 19.8% at the time of exposure, and the temperature was in the range of 20.7°C-20.8°C.

In the group in which iv was administered. injections of ABRAXANE ^® , body weight values on day 1 ranged from 386.1 to 472.8 g, giving ABRAXANE ^® doses of 2.6-3.2 mg/kg, and the average dose in the group was 2.9 mg/ kg.

All groups experienced weight gain during the study. No clinical abnormalities were noted during the study. All animals survived to their indicated necropsy time. All animals were sacrificed within the time range assumed for each time point.

At autopsy, about half of the animals in each group had minimal or small pale brown patches in their lungs. These deviations are often associated with inhalation exposure. Other abnormalities seen in individual animals included enlargement of the heart (animal #2016) and enlargement of the tracheobronchial lymph nodes. No other macroscopic abnormalities were noted at the time of autopsy. In histopathology, the lungs and trachea harvested from rats injected with the test product and standard were shown to be normal and indistinguishable in this study from the corresponding organs of rats that had not previously been used in studies. When sacrificed 336 hours post-dose on lung sections of treated animals in this study, macrophage accumulation, which is often observed in inhalation studies as a normal physiological response to exogenous lung deposition, did not occur.

An NCA was developed to quantify exposure (area under the concentration-time curve [AUC]), time to peak concentration (T _max ), maximum concentration (C _max ) and, if possible, apparent terminal half-life (T _{1/ 2} ).

It has been hypothesized that the novel formulation of paclitaxel particles will increase the retention of paclitaxel in lung tissues and reduce systemic exposure. The plasma half-life in the systemic circulation remained unchanged for the studied formulations/doses, and the half-life in lung tissue increased with the paclitaxel particle formulation delivered by inhalation. Lung tissue levels (AUC, dose-adjusted) increased when the paclitaxel particle formulation was delivered by inhalation.

Taken together, the data indicate a significant retention of paclitaxel particles in lung tissues when delivered by inhalation compared to i.v. "clinical standard".

TASKS

The objective of this study was to determine the pharmacokinetics of the composition of paclitaxel particles in comparison with the clinical standard dose of paclitaxel. FY 17-008A pharmacokinetic (PK) pilot study data from Lovelace Biomedical (Example 1 above) using an inhaled paclitaxel particle formulation indicated retention times in lung tissue in excess of 168 hours. In this study, animals were administered one low or high dose formulation of paclitaxel particles by nasal inhalation only or one dose of clinical standard paclitaxel by intravenous (i.v.) tail injection and assessed plasma and lung tissue levels for time points from 0.5 up to 336 hours.

MATERIALS AND METHODS

System under study

Species/line: Sprague-Dawley rats

Age of animals at baseline: 8-10 weeks

Body weight range at baseline: 345-447 g

Number of test animals/sex: 95 males (90 test animals and 5 spares)

Source: Charles River Laboratories (Kingston, NY)

Identification: Permanent marker on tail

Composition of ^ABRAXANE®

V.V. was used as a clinical standard. drug product formulation ^ABRAXANE® (Manufacturer: Celgene Corporation, Summit, NJ; Lot No: 6111880). The drug product was reconstituted to 5.0 mg/mL in saline (manufacturer: Baxter Healthcare, Deerfield, 11..; lot no: P357889) on the day of administration and stored according to the manufacturer's instructions.

Composition of paclitaxel particles

The 6.0 mg/mL paclitaxel particle formulation for the low dose groups and the 20.0 mg/mL paclitaxel particle formulation for the high dose groups were prepared as recommended by the sponsor. Specifically, the paclitaxel particles were re-dissolved in 1% polysorbate 80. The tube was shaken by hand to wet all the particles. An additional amount of 0.9% sodium chloride for injection (to the target concentration) was added and the tube was shaken by hand for another minute.

Continue to shake until the disappearance of large lumps and a suitable dispersion of the suspension. The resulting compositions were left for at least 5 minutes to reduce the level of air/foam in the tube, after which it was placed in a nebulizer for spraying. The final 6.0 mg/ml formulation was kept at room temperature and sprayed within 2 hours of reconstitution. The final 20.0 mg/ml formulation was kept at room temperature and sprayed within 30 minutes of reconstitution.

Experiment Design

Group 1 animals, as shown in Table 8, received a single dose of "clinical standard" (formulation concentration: 5 mg/mL, target dose: 5.0 mg/kg body weight; target dose volume: 250 µl or less) of ^ABRAXANE® (paclitaxel protein-bound particles for suspension for injection) by iv. tail vein injections. Animals in Groups 2 and 3, as shown in Table 9, were exposed to paclitaxel particle formulation aerosols (target dose 0.37 or 1.0 mg/kg) by single nasal inhalation only (ing) according to the study design described below. Three animals (n=3) were sacrificed after 0.5 (±10 minutes), 6 (±10 minutes), 12 (±10 minutes), 24 (±30 minutes), 48 (±30 minutes), 72 (± 30 minutes), 120 (±30 minutes), 168 (±30 minutes) 240 (±30 minutes) and 336 (±30 minutes) hours after exposure to collect blood (plasma) and lung tissue. Conducted non-compartmental analysis of plasma and lung tissue to determine the period of time after exposure, during which the amount of paclitaxel is detectable, for each group, which was administered the dose. Animals in which the 336 hour time point was examined had right lungs harvested from all groups and frozen individually for LCMS analysis, while left lungs were perfused in 10% neutral buffered formalin (NBF) and stored until histopathology could be performed. For comparative histopathology, three spare animals (controls, animals not previously used in the experiments) were also sacrificed at the time point corresponding to the time point of 336 hours, and the lungs were harvested in the same way.

Conditions of detention, quarantine and distribution to the study

Male Sprague-Dawley rats (6-8 weeks old) were obtained from Charles River Laboratories (Kingston, NY) and kept in quarantine for 14 days. At the end of quarantine, the animals were weighed and then randomized by weight and assigned to the study. Animals were identified by markings on their tails and cards on the cages. Water flow, lighting, humidity and temperature were monitored and monitored according to the relevant SOPs. Rats were given access to standard rodent chow ad libitum during non-treatment periods.

Body weight and daily observations

Body weight was determined at the time of randomization, daily during the study, and after sacrifice. The staff of the Center for Comparative Medicine and Animal Resources (CMAR) observed the animals twice daily for clinical signs of abnormality, agony, or death.

V.v. introduction of ABRAXANE ^® - Injection into the tail vein

ABRAXANE ^® (concentration: 5 mg/ml, target dose: 5.0 mg/kg body weight; dose volume: not more than 250 μl) was administered to animals from group 1 by a single iv. tail vein injections.

Paclitaxel Particulate Administration - Aerosol Exposure by Nasal Only

Training

The preparation of animals for exposure tubes by nasal only administration was carried out for a period of up to 70 minutes. Three preparation sessions were performed over the three days prior to exposure, the first session lasting 30 minutes, the second 60 minutes, and the third 70 minutes. The animals were carefully examined during the preparation and exposure periods to ensure that they did not experience anything more than a momentary breakdown.

Impact system

Aerosols were generated using two Hospitak compression jet nebulizers as shown in FIG. 7 (see example 3) under 20 psi (140 kPa) nebulizer pressure. The 6.0 mg/mL and 20.0 mg/mL paclitaxel particle suspension formulations were used for low dose and high dose exposure, respectively. Both formulations were sprayed separately and injected through a delivery line into a 32-hole exposure chamber. Inhalation exposure for rodents was carried out for 65 minutes. An aerosol suspension of paclitaxel particles was prepared on a two Hospitak compression jet nebulizer set (used up to 40 (±1) minutes), which was then changed to a second set of two Hospitak nebulizers for the remainder of the exposure period. Monitored the level of oxygen and temperature during each inhalation exposure.

Concentration Tracking

Same as example 1

Particle Sizing

Same as example 1

Dose Determination

Same as example 1

Mortification and autopsy

Animals were sacrificed at the time points indicated above in the study design by intraperitoneal (i.p.) injection of the killing solution.

Time point 336 hours (and spare animals, n=3): At the time of autopsy, blood (plasma) was collected by cardiac puncture in K ₂ -EDTA tubes. The weight of the whole lungs was determined, the left lung was separated and filled with neutral buffered formalin and stored for possible histopathology. The right lung lobes were individually weighed and flash frozen in liquid nitrogen and stored at -70 to -90°C for bioanalytical studies. In addition, qualified personnel performing the autopsy performed a full macroscopic evaluation. External surfaces of the body, sinuses, and contents of the cranial, thoracic, and abdominal cavities were assessed. Lesions were described and their morphology, number, shape, color, structure, and severity were indicated using dictionary terms.

All other time points: At autopsy, blood (plasma) was collected by cardiac puncture into a K ₂ -EDTA tube. The mass of the lungs as a whole was determined, the lung lobes were weighed individually and flash frozen in liquid nitrogen and stored at -70 to -90°C for bioanalytical studies. In addition, qualified personnel performing the autopsy performed a full macroscopic evaluation. External surfaces of the body, sinuses, and contents of the cranial, thoracic, and abdominal cavities were assessed. Lesions were described and their morphology, number, shape, color, structure, and severity were indicated using dictionary terms.

Histopathology

The available fixed tissues were cut. Fixed lobes of the left lung were sliced to obtain typical sections to determine the toxicological pathology, including the respiratory tract. Tissues were processed in the usual way, embedded in paraffin, cut into ~4 μm fragments, fixed and stained with hematoxylin and eosin (H&E) for microscopic examination. One pathologist specializing in toxicological pathology performed a subjective semi-quantitative classification of the results on a scale of 1-5 (1=minimal, 2=low, 3=moderate, 4=severe, 5=severe). Provantis™ computer software/database (Instem LSS Ltd., Staffordshire, England) was used to acquire, describe and analyze histopathology data.

Collection and processing of blood samples

Blood collected at the time of autopsy was processed to obtain plasma by centrifugation at a speed of at least 1300g at 4°C for 10 minutes. Plasma samples were stored at -70 to -90° C. until analysis.

Bioanalytical research

Blood from the systemic circulation (as plasma in K ₂ -EDTA tubes) and lung tissues were examined in a liquid chromatography-mass spectrometry (LCMS) analysis to assess the time dependence of the amount of paclitaxel. Briefly, the study uses ultra high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) analysis to quantify paclitaxel levels.

Samples were extracted by protein precipitation and separated by reverse phase chromatography. Quantification was carried out according to the calibration curve obtained for the matrix solutions.

A non-compartmental data analysis was performed for plasma and lung tissue concentrations. Determined at least C _max , T _max , AUC and apparent terminal half-life. Based on the obtained data, other parameters can be determined.

RESULTS

Clinical abnormalities, survival and body weight

All animals survived to their indicated necropsy time. All animals were sacrificed within the time range assumed for each time point.

No clinical abnormalities were noted during the study.

FIG. 51 and FIG. 52 shows mean body weights over the course of the study and mean percent change over 1 day. In all groups during the study, weight gain occurred at approximately the same rate.

V.v. ABRAXANE ^® injections into the tail vein

In the group in which iv was administered. injections of ABRAXANE ^® , body weight on day 1 ranged from 386.1 to 472.8 g, respectively, the doses of ABRAXANE ^® were 2.6-3.2 mg/kg. The mean dose (standard deviation) was 2.9 (0.16) mg/kg. Individual doses of ^ABRAXANE® are shown in Table 9.

Exposure to paclitaxel particles

Aerosol concentration and particle size

See: Results - Aerosol Concentration and Particle Size in Example 2.

Oxygen level and temperature

See: Results - Oxygen Level and Temperature in Example 2.

deposited dose

See: Results - Deposited Dose in Example 2.

Opening

All animals survived to the points indicated for them at autopsy. At necropsy, animals from each group had minimal or small pale brown patches in their lungs (Table 10). These deviations are often associated with inhalation exposure. Other single abnormalities included enlargement of the heart (animal no. 2016) and enlargement of the tracheobronchial lymph nodes. No other macroscopic abnormalities were noted at the time of autopsy.

Histopathology

There were no significant abnormalities in the trachea and left lung in animals euthanized 336 hours (~14 days) after administration, which were studied in this study. According to the results of microscopic examination, the tissues were indistinguishable from the "reserve" animals, which were used as controls.

Accumulation of macrophages was not observed in lung regions of the treated animals studied in this study. Some elevation of alveolar macrophage levels is often seen in inhalation studies as a normal physiological response to exogenous lung deposition (low levels can also be relatively common in untreated animals). Their actual absence in inhaled dosed animals in this study may be due in part to the relatively late time point (336 hours or ~14 days) post-dose followed by histology.

Bioassay and PK modeling

The results are summarized below in tables 11, 12 and 13 and in FIG. 53 and FIG. 54. Mean paclitaxel plasma concentration versus time data and mean lung tissue paclitaxel concentration versus time data were simulated as shown above, the results are shown in Tables 14 and 15, respectively.

Modeling was performed using WinNonlin for the average concentration in plasma or lung tissue at each time point. An NCA was developed to quantify exposure (area under the concentration-time curve [AUC]), time to peak concentration (T _max ), maximum concentration (C _max ) and, where possible, apparent terminal half-life (T _1/2 ).

The plasma half-life in the systemic circulation remained unchanged for the studied formulations/doses, and the half-life in lung tissues increased when the paclitaxel particle formulation was delivered by inhalation. Lung tissue levels increased (dose-adjusted AUC) upon delivery of the paclitaxel particle formulation by inhalation.

Taken together, these data indicate significant retention of paclitaxel particles in lung tissue upon delivery by inhalation.

CONCLUSIONS

Ninety (90) male Sprague-Dawley rats were exposed to the "clinical standard" dose of paclitaxel, ^ABRAXANE® (paclitaxel protein-bound particles for suspension for injection), by intravenous (i.v.) injection of a bolus or formulation of paclitaxel particles ( target dose of 0.37 or 1.0 mg/kg) by single inhalation through the nose only. Three animals (n=3) were sacrificed at ten (10) time points from 0.5 to 336 hours post-exposure and blood (plasma) and lung tissue were collected. Conducted non-compartmental analysis of plasma and lung tissue to determine the period of time after exposure, during which the amount of paclitaxel was detectable in each group, which was administered the dose. Animals in which the 336 hour time point was studied had their right lungs collected from all groups for liquid chromatography-mass spectrometry (LCMS) analysis, while the left lungs were perfused in 10% neutral buffered formalin (NBF) and stored for possible histopathology . For comparative histopathology, three spare animals (control animals not previously used in the experiments) were also sacrificed after 336 hours and the lungs were harvested in the same way. Animals on which all other time points were studied were harvested lungs and frozen individually for LCMS analysis.

During inhalation exposure, mean paclitaxel aerosol concentrations in the low dose and high dose paclitaxel particle formulation groups were 85.64 µg/L and 262.27 µg/L, respectively. The mean aerosol concentration during exposure was within ±15% of the target aerosol concentration, which would be expected for inhalation exposure by nebulization. Particle size distribution was determined by MMAD (GSD) for each paclitaxel particle composition aerosol using a cascade impactor. For aerosols of 6.0 mg/mL and 20.0 mg/mL paclitaxel particle formulations, MMAD (GSD) was determined to be 1.8 (2.0) µm and 2.3 (1.9) µm, respectively.

The precipitated dose of paclitaxel was calculated assuming that the mean concentration of paclitaxel in the aerosol was 85.64 μg/l, the mean body weight in the group on day 0 was 420.4 g, the estimated precipitated fraction was 10%, and the duration of exposure was 65 minutes; it was determined that the mean precipitating dose in rodents was 0.38 mg/kg in the low dose paclitaxel particle formulation group. It was determined that in the high-dose paclitaxel particle formulation group, in which the mean aerosol concentration of paclitaxel was 262.27 μg/L, the mean body weight in the group on day 0 was 420.5 g, the estimated precipitated fraction was 10 %, and the duration of exposure was 65 minutes; the average deposited dose for rodents was 1.18 mg/kg. The measured oxygen level and temperature ranges were 19.8%-20.9% and 20.7°C-20.8°C, respectively, for exposure to a 6.0 mg/ml paclitaxel particle formulation. For exposure to 20.0 mg/ml paclitaxel particle formulation, the measured oxygen level was 19.8% at the time of exposure and the temperature was in the range of 20.7°C-20.8°C.

In the group in which iv was administered. injections of ABRAXANE ^® , body weight on day 1 ranged from 386.1 to 472.8 g, giving an ABRAXANE ^® dose of 2.6-3.2 mg/kg, and the average dose in the group was 2.9 mg/kg .

All groups experienced weight gain during the study. No clinical abnormalities were noted during the study. All animals survived to their indicated necropsy time. All animals were sacrificed within the time range assumed for each time point.

At necropsy, approximately half of the animals from each group had minimal or slight pale brown staining in the lungs. Such deviations are often associated with inhalation exposure. Other single abnormalities included enlargement of the heart (animal no. 2016) and enlargement of the tracheobronchial lymph nodes. No other macroscopic abnormalities were noted at the time of autopsy. According to the results of histopathology, it was shown that the lungs and trachea of rats, which were injected with the test product and the standard, were within the normal range and were indistinguishable from the organs of rats that had not previously been used in experiments under the conditions of this study.

An NCA was developed to quantify exposure (area under the concentration-time curve [AUC]), time to peak concentration (T _max ), maximum concentration (C _max ) and, where possible, apparent terminal half-life (T _1/2 ).

It has been hypothesized that the novel formulation of paclitaxel particles will increase the retention of paclitaxel in lung tissues and reduce systemic exposure. The plasma half-life in the systemic circulation remained unchanged for the studied formulations/doses, and the half-life in lung tissue increased with the paclitaxel particle formulation delivered by inhalation. Lung tissue levels (AUC, dose-adjusted) increased when the paclitaxel particle formulation was delivered by inhalation.

Taken together, the data indicate a significant retention of paclitaxel particles in lung tissues when delivered by inhalation compared to i.v. "clinical standard".

Claims (21)

1. A method for treating a lung tumor, comprising intrapulmonary administration to a subject with a lung tumor of an effective amount of a composition containing taxane particles for treating a lung tumor, where the taxane includes paclitaxel or a pharmaceutically acceptable salt thereof, where the taxane particles contain at least 95% taxane and have an average size (quantitative) from 0.4 µm to 3 µm, where the taxane particles have a specific surface area (SSA) from 27 m ² /g to 60 m ² /g, where the taxane particles are present in a suspension containing taxane particles and a pharmaceutically acceptable carrier , where the suspension is converted into an aerosol for administration, and the resulting aerosol droplets have a mass median aerodynamic diameter (MMAD) from about 0.5 μm to about 6 μm, where said intrapulmonary administration includes spraying by means of a nebulizer, and said spraying results in intrapulmonary delivery of drops to the subject aerosol suspension of taxane particles. 2. The method according to claim 1, characterized in that said taxane particles have an average size (quantitative) of 0.4 µm to 2 µm. 3. The method according to claim 1, characterized in that said taxane particles have an average size (quantitative) of 0.4 µm to 1.2 µm. 4. The method of claim 1, wherein the resulting aerosol droplets have a mass median aerodynamic diameter (MMAD) of about 1 µm to about 3 µm. 5. The method of claim 1, wherein the suspension further comprises a polysorbate, wherein the polysorbate is present in the suspension at a concentration of from about 0.01% (v/v) to about 1.5% (v/v) . 6. The method of claim 1, wherein said pharmaceutically acceptable carrier is saline. 7. The method according to claim 5, characterized in that said polysorbate is polysorbate 80. 8. The method of claim 1, wherein said taxane is present in the suspension at a concentration of from about 1 mg/mL to about 40 mg/mL. 9. The method according to claim 1, characterized in that these particles and their suspensions do not contain coatings and lipids, polymers, proteins, polyethoxylated castor oil and polyethylene glycol glycerides, consisting of mono-, di- and triglycerides and complex mono- and diesters of polyethylene glycol . 10. The method of claim 4, wherein the suspension further comprises a polysorbate, wherein the polysorbate is present in the suspension at a concentration of from about 0.01% (v/v) to about 1.5% (v/v) . 11. The method according to claim 10, characterized in that said pharmaceutically acceptable carrier contains a saline solution. 12. The method of claim 1, wherein said taxane particles have an average bulk density of from about 0.050 g/cm ³ to about 0.12 g/cm ³ . 13. The method according to claim 1, characterized in that said taxane particles have a crystalline form. 14. The method according to claim 10, characterized in that said taxane particles or suspensions thereof do not contain coatings and lipids, polymers, proteins, polyethoxylated castor oil and polyethylene glycol glycerides consisting of mono-, di- and triglycerides and complex mono- and diesters polyethylene glycol. 15. The method according to claim 1, characterized in that the taxane particles remain in the site of the lung tumor after administration of the composition and provide exposure of the taxane particles to the tumor for an extended period of time sufficient to stimulate the endogenous immune system of the subject, which leads to the production of antitumor cells and infiltration of antitumor cells into the lung tumor in an amount sufficient to cure the tumor. 16. The method according to claim 15, characterized in that said extended period of time is at least 4 weeks. 17. The method of claim 15, wherein said antitumor cells comprise T cells, B cells, or natural killer (NK) cells, or combinations thereof. 18. The method of claim 1, wherein said composition is administered in two or more separate administration steps. 19. The method of claim 18, wherein said composition is administered once a week for at least two weeks. 20. The method of claim 18, wherein said composition is administered twice a week for at least one week, wherein two or more separate administrations are performed at least one day apart. 21. The use of a composition containing taxane particles for the treatment of a lung tumor, where the taxane includes paclitaxel or a pharmaceutically acceptable salt thereof, where the taxane particles contain at least 95% taxane and have an average size (quantitative) from 0.4 μm to 3 μm, wherein the taxane particles have a specific surface area (SSA) of 27 m ² /g to 60 m ² /g, where the taxane particles are present in a suspension containing the taxane particles and a pharmaceutically acceptable carrier, where the suspensions are converted into an aerosol for administration and the resulting aerosol droplets have a mass median aerodynamic diameter (MMAD) of about 0.5 µm to about 6 µm, wherein said intrapulmonary administration comprises nebulization, and said nebulization results in intrapulmonary delivery of an aerosol droplet of a suspension of taxane particles to the subject.

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