JPWO2014098232A1 - PHARMACEUTICAL COMPOSITION CONTAINING PI3 KINASE INHIBITOR, PHARMACEUTICAL COMPOSITION CONTAINING COMPOUND HAVING VITAMIN D RECEPTOR, LYophilized Composition, Method for Producing Lyophilized Composition, and Pharmaceutical Composition for Transpulmonary Administration - Google Patents

Abstract

A pharmaceutical composition comprising a PI3 kinase inhibitor as an active ingredient. A pharmaceutical composition comprising a compound acting on a vitamin D receptor as an active ingredient. It is a lyophilized composition of a composition comprising a poorly water-soluble drug, at least two types of amino acids, a surfactant, an alcohol, and water.

Description

  The present invention relates to a pharmaceutical composition containing a PI3 kinase inhibitor, a pharmaceutical composition containing a compound acting on a vitamin D receptor, a lyophilized composition for producing a pharmaceutical composition for pulmonary administration, and the lyophilized composition And a pharmaceutical composition for pulmonary administration.

Chronic obstructive pulmonary disease (COPD), pulmonary emphysema, chronic bronchitis, acute lung distress syndrome (ARDS), pulmonary fibrosis, lung cancer, stroma Pneumonia, pulmonary tuberculosis sequelae, pneumoconiosis, etc. are respiratory diseases that exhibit irreversible alveolar destruction. At present, there is no curative treatment for irreversible destruction of alveoli, and symptomatic treatment is being used.
As a radical therapeutic agent for these diseases, a therapeutic agent that induces differentiation of alveolar epithelial stem cells into type I and type II alveolar epithelial cells constituting the alveoli and regenerates the alveoli is considered promising. It has been.

  As a compound capable of regenerating alveoli, all-trans retinoic acid, a derivative of vitamin A, has been reported (see, for example, Massaro G.D. et. Al., Nature Med (1997) 3: 675-677.). Therefore, clinical trials of oral drugs containing retinoic acid as an active ingredient have been attempted. However, oral administration is metabolized before reaching the lung tissue, making it difficult to maintain an effective drug concentration in the lung tissue, and alveolar repair. No effect has been observed (see, eg, Roth MD et. Al., Chest (2006) 130: 1334-1345.).

By the way, as an inhalation system for powder medicine for pulmonary administration, there is an inhalation system that particles a freeze-dried composition by air impact (see, for example, US Pat. No. 7,735,485 B2 and US Pat. No. 4,822,709). According to this inhalation system, the freeze-dried composition becomes particles suitable for transpulmonary administration by inhalation of the patient, and inhalation is performed as it is to perform transpulmonary administration.
However, this system uses a lyophilization method suitable for water-soluble drugs, such as proteins and peptides, and is suitable for transpulmonary formulation of drugs that are poorly soluble in water such as retinoic acid. Not. Therefore, in order to apply this system to poorly soluble drugs such as retinoic acid, it is necessary to find a new formulation and manufacturing method.

If a compound that induces differentiation of alveolar epithelial stem cells into alveolar epithelial cells has been found and the compound can be formulated into a pharmaceutical composition for transpulmonary administration, regeneration of alveoli will be possible for diseases exhibiting lung tissue damage. It is possible to provide a treatment method having an action point.
In addition, as a mode of administration of a pharmaceutical composition for pulmonary administration, if the powder drug inhalation system for pulmonary administration disclosed in US Pat. No. 7,735,485B2 or US Pat. No. 4,822,709 can be adopted, patients with the above diseases can be treated. A regenerative therapy for alveoli can be provided simply.

  The present invention has been made under the above circumstances.

Under the circumstances described above, there is a need for a differentiation inducer that induces differentiation of alveolar epithelial stem cells into alveolar epithelial cells.
Under the circumstances described above, there is a need for a pharmaceutical composition that induces alveolar regeneration to treat lung tissue damage.

Under the above circumstances, there is a need for a pharmaceutical composition suitable for transpulmonary administration, which contains a poorly water-soluble drug for treating lung tissue damage.
Under the circumstances described above, there is a need for a lyophilized composition containing a drug that is sparingly soluble in water and that is granulated into particles having a particle size suitable for transpulmonary administration by airflow.

Specific means for achieving the above object are as follows.
(A1) A differentiation inducer that acts on alveolar epithelial stem cells, comprising a PI3 kinase inhibitor as an active ingredient.
(A2) A pharmaceutical composition for inducing alveolar regeneration, comprising a PI3 kinase inhibitor as an active ingredient.
(A3) A pharmaceutical composition for treating lung tissue injury, comprising a PI3 kinase inhibitor as an active ingredient.
(A4) Use of a PI3 kinase inhibitor in the production of a differentiation inducer that acts on alveolar epithelial stem cells.
(A5) Use of a PI3 kinase inhibitor in the manufacture of a pharmaceutical composition that induces alveolar regeneration.
(A6) Use of a PI3 kinase inhibitor in the manufacture of a pharmaceutical composition for treating lung tissue damage.
(A7) PI3 kinase inhibitor for use in the treatment of lung tissue damage.
(A8) A method for treating lung tissue damage, comprising administering a pharmaceutical composition comprising a PI3 kinase inhibitor as an active ingredient to a patient having damage to lung tissue.
(A9) The PI3 kinase inhibitor is an agent that directly inhibits PI3 kinase, an agent that inhibits the PI3K-Akt pathway, an agent that inhibits the PI3K-BTK pathway, an agent that inhibits the PI3K-ITK pathway, PI3K-TAPP1 Agents that inhibit the pathway, agents that inhibit the PI3K-DAPP pathway, agents that inhibit the PI3K-GAB1 / 2 pathway, agents that inhibit the PI3K-Rac pathway, agents that inhibit the PI3K-Rho pathway, and that inhibit the PI3K-ARF pathway Agents that inhibit the PI3K-PDK1 pathway, agents that inhibit the PI3K-EEA1 pathway, agents that inhibit the PI3K-Rabenosyn5 pathway, agents that inhibit the PI3K-Rabip4 pathway, agents that inhibit the PI3K-FAB1 pathway, PI3K- PI3, an agent that inhibits the p40phox pathway Agents that inhibit -SNX3 path is at least one selected from agents that inhibit drug, and PI3K-SGK3 pathway inhibits PI3K-SNX17 pathway, wherein (A1) ~ (A8).

Specific means for achieving the above object are as follows.
(B1) A differentiation inducer that acts on alveolar epithelial stem cells, comprising a compound that acts on a vitamin D receptor as an active ingredient.
(B2) A pharmaceutical composition for inducing alveolar regeneration, comprising a compound that acts on a vitamin D receptor as an active ingredient.
(B3) A pharmaceutical composition for treating lung tissue damage, comprising a compound that acts on a vitamin D receptor as an active ingredient.
(B4) Use of a compound that acts on a vitamin D receptor in the production of a differentiation inducer that acts on alveolar epithelial stem cells.
(B5) Use of a compound that acts on a vitamin D receptor in the manufacture of a pharmaceutical composition that induces alveolar regeneration.
(B6) Use of a compound that acts on a vitamin D receptor in the manufacture of a pharmaceutical composition for treating lung tissue damage.
(B7) Compounds that act on vitamin D receptors for use in the treatment of lung tissue damage.
(B8) A method for treating lung tissue damage, comprising administering to a patient having damage to lung tissue a pharmaceutical composition comprising a compound that acts on a vitamin D receptor as an active ingredient.
(B9) The compound acting on the vitamin D receptor is vitamin D ₂ and derivatives thereof, vitamin D ₃ and derivatives thereof, vitamin D ₄ and derivatives thereof, vitamin D ₅ and derivatives thereof, vitamin D ₆ and derivatives thereof, vitamins is at least one member selected from D ₇ and derivatives thereof, wherein (B1) ~ (B8).

Specific means for achieving the above object are as follows.
(C1) Freeze-drying that contains a poorly water-soluble drug, at least two types of amino acids, and a surfactant, and the proportion of particles having a particle diameter of 5 μm or less becomes 10% or more when formed into particles by air flow Composition.
(C101) contains a poorly water-soluble drug, at least two types of amino acids, and a surfactant, and when formed into particles by an air stream, the proportion of particles having a geometric particle diameter of 5 μm or less is 10% or more. A lyophilized composition.
(C102) contains a poorly water-soluble drug, at least two kinds of amino acids, and a surfactant, and when formed into particles by an air stream, the proportion of particles having an aerodynamic particle diameter of 5 μm or less is 10% or more. A lyophilized composition.

(C2) A lyophilized composition of a composition comprising a drug that is sparingly soluble in water, at least two types of amino acids, a surfactant, an alcohol, and water. A freeze-dried composition in which the proportion of particles having a diameter of 5 μm or less is 10% or more.
(C201) A freeze-dried composition of a composition comprising a poorly water-soluble drug, at least two types of amino acids, a surfactant, an alcohol, and water, A freeze-dried composition in which the proportion of particles having a particle size of 5 μm or less is 10% or more.
(C202) A freeze-dried composition of a composition comprising a poorly water-soluble drug, at least two types of amino acids, a surfactant, an alcohol, and water, A freeze-dried composition in which the proportion of particles having a mechanical particle size of 5 μm or less is 10% or more.

(C3) A freeze-dried composition comprising a poorly water-soluble drug, at least two amino acids selected from the following first group, second group and third group and a plurality of groups, and a surfactant: .
-Group 1: Phenylalanine, tryptophan-Group 2: Isoleucine, leucine, valine-Group 3: Alanine, glycine (C4) Drugs poorly soluble in water, and the following groups 1, 2, 3 A freeze-dried composition of a composition comprising at least two amino acids selected from a plurality of groups, a surfactant, an alcohol, and water.
・ Group 1: phenylalanine, tryptophan ・ Group 2: isoleucine, leucine, valine ・ Group 3: alanine, glycine

(C5) The (C1) to (C4), (C101), (C102), (C201), and (C201), wherein the water-insoluble drug is a compound having a differentiation-inducing action on alveolar epithelial stem cells. The freeze-dried composition according to any one of C202).
(C6) The compound having a differentiation-inducing action on alveolar epithelial stem cells is at least one selected from vitamin A, vitamin A derivatives, provitamin A, compounds acting on vitamin D receptors, and PI3 kinase inhibitors The freeze-dried composition according to (C5), which is a seed.
(C601) The compound acting on the vitamin D receptor is vitamin D ₂ and derivatives thereof, vitamin D ₃ and derivatives thereof, vitamin D ₄ and derivatives thereof, vitamin D ₅ and derivatives thereof, vitamin D ₆ and derivatives thereof, vitamins is at least one member selected from D ₇ and its derivatives, freeze-dried composition according to the (C6).
(C602) The PI3 kinase inhibitor is an agent that directly inhibits PI3 kinase, an agent that inhibits the PI3K-Akt pathway, an agent that inhibits the PI3K-BTK pathway, an agent that inhibits the PI3K-ITK pathway, PI3K-TAPP1 Agents that inhibit the pathway, agents that inhibit the PI3K-DAPP pathway, agents that inhibit the PI3K-GAB1 / 2 pathway, agents that inhibit the PI3K-Rac pathway, agents that inhibit the PI3K-Rho pathway, and that inhibit the PI3K-ARF pathway Agents that inhibit the PI3K-PDK1 pathway, agents that inhibit the PI3K-EEA1 pathway, agents that inhibit the PI3K-Rabenosyn5 pathway, agents that inhibit the PI3K-Rabip4 pathway, agents that inhibit the PI3K-FAB1 pathway, PI3K- an agent that inhibits the p40phox pathway, P Agents that inhibit 3K-SNX3 pathway is at least one selected from agents that inhibit drug, and PI3K-SGK3 pathway inhibits PI3K-SNX17 pathway, freeze-dried composition according to the (C6).

(C7) A step of preparing a first liquid by dissolving a poorly water-soluble drug in a solvent containing alcohol, the first liquid, at least two kinds of amino acids, a surfactant, water, A method for producing a lyophilized composition comprising the steps of preparing a second liquid by mixing and lyophilizing the second liquid.
(C701) A step of preparing a first liquid by dissolving a poorly water-soluble drug in a solvent containing alcohol, and after mixing the first liquid and the surfactant, at least two kinds of A method for producing a lyophilized composition comprising a step of preparing a second liquid by mixing an amino acid and water, and a step of lyophilizing the second liquid.
(C8) A step of preparing a first liquid by dissolving a poorly water-soluble drug in a solvent containing alcohol, the first liquid, and the following first group, second group, and third group Freezing comprising a step of preparing a second liquid by mixing at least two kinds of amino acids selected over a plurality of groups, a surfactant, and water, and a step of freeze-drying the second liquid A method for producing a dry composition.
-Group 1: Phenylalanine, tryptophan-Group 2: Isoleucine, leucine, valine-Group 3: Alanine, glycine (C801) A first liquid is prepared by dissolving a poorly water-soluble drug in a solvent containing alcohol. After mixing the first liquid and the surfactant, at least two kinds of amino acids selected from the first group, the second group, and the third group described below over a plurality of groups, and A method for producing a lyophilized composition comprising the steps of mixing water to prepare a second liquid, and lyophilizing the second liquid.
・ Group 1: phenylalanine, tryptophan ・ Group 2: isoleucine, leucine, valine ・ Group 3: alanine, glycine

(C9) The drug according to any one of (C7), (C701), (C8) and (C801), wherein the poorly water-soluble drug is a compound having a differentiation-inducing action on alveolar epithelial stem cells. A method for producing a freeze-dried composition.
(C10) The compound having a differentiation-inducing action on alveolar epithelial stem cells is at least one selected from vitamin A, vitamin A derivatives, provitamin A, compounds acting on vitamin D receptors, and PI3 kinase inhibitors The method for producing a lyophilized composition according to (C9), which is a seed.
(C111) The compound acting on the vitamin D receptor is vitamin D ₂ and derivatives thereof, vitamin D ₃ and derivatives thereof, vitamin D ₄ and derivatives thereof, vitamin D ₅ and derivatives thereof, vitamin D ₆ and derivatives thereof, vitamins is at least one member selected from D ₇ and its derivatives, method for producing a freeze-dried composition according to the (C10).
(C112) The PI3 kinase inhibitor is an agent that directly inhibits PI3 kinase, an agent that inhibits the PI3K-Akt pathway, an agent that inhibits the PI3K-BTK pathway, an agent that inhibits the PI3K-ITK pathway, PI3K-TAPP1 Agents that inhibit the pathway, agents that inhibit the PI3K-DAPP pathway, agents that inhibit the PI3K-GAB1 / 2 pathway, agents that inhibit the PI3K-Rac pathway, agents that inhibit the PI3K-Rho pathway, and that inhibit the PI3K-ARF pathway Agents that inhibit the PI3K-PDK1 pathway, agents that inhibit the PI3K-EEA1 pathway, agents that inhibit the PI3K-Rabenosyn5 pathway, agents that inhibit the PI3K-Rabip4 pathway, agents that inhibit the PI3K-FAB1 pathway, PI3K- an agent that inhibits the p40phox pathway, P Production of the lyophilized composition according to (C10) above, which is at least one selected from an agent that inhibits the 3K-SNX3 pathway, an agent that inhibits the PI3K-SNX17 pathway, and an agent that inhibits the PI3K-SGK3 pathway Method.

  (C11) Air flow through the lyophilized composition according to any one of (C1) to (C6), (C101), (C102), (C201), (C202), (C601), and (C602) A pharmaceutical composition for pulmonary administration, which is made into particles by the method.

According to the present invention, a differentiation inducer that acts on alveolar epithelial stem cells is provided.
The present invention also provides a pharmaceutical composition for inducing alveolar regeneration and a pharmaceutical composition for treating lung tissue damage.

According to the present invention, there is provided a freeze-dried composition containing a drug that is sparingly soluble in water, and is formed into particles having a particle size suitable for transpulmonary administration by airflow.
Further, according to the present invention, a freeze-dried composition used for the production of a pharmaceutical composition for inducing regeneration of alveoli, the freeze-dried composition that is granulated into particles having a particle size suitable for transpulmonary administration by air flow Things are provided.
In addition, according to the present invention, there is provided a pharmaceutical composition suitable for pulmonary administration, which contains a drug hardly soluble in water.
The present invention also provides a pharmaceutical composition that induces alveolar regeneration suitable for transpulmonary administration.

It is a graph which shows the HE dyeing | staining image of the section | slice of the lung tissue which carried out the wortmannin treatment in the COPD model mouse | mouth of Example A2, and the average distance between alveolar walls. In COPD model mice of Example B3, a graph showing 1,25-dihydroxyvitamin _{D 3} sections of treating a lung tissue HE stained image, and the average alveolar wall distance.

Embodiments of the present invention will be described below. These descriptions and examples are illustrative of the invention and are not intended to limit the scope of the invention.
In the present specification, a numerical range indicated using “to” indicates a range including the numerical values described before and after “to” as the minimum value and the maximum value, respectively.
In this specification, the term “process” is not limited to an independent process, and is included in this term if the intended purpose of the process is achieved even when it cannot be clearly distinguished from other processes. .
In the present specification, the “treatment” may be any improvement of symptoms, and the term also includes suppression of severity and reduction or alleviation of symptoms.
In this specification, “regeneration” is used in the meaning normally used in this technical field.

  Hereinafter, the compound for which differentiation-inducing action on alveolar epithelial stem cells has been confirmed by the present inventor for the first time and a pharmaceutical composition containing the compound will be described in detail.

[A] Pharmaceutical composition containing PI3 kinase inhibitor PI3 kinase (phosphatidylinositol-3 kinase, PI3K) is an enzyme that phosphorylates the hydroxyl group (-OH group) at the 3-position of the inositol ring of inositol phospholipid. Inositol phospholipids are one of the components of eukaryotic cell membranes, and are catalyzed by kinases (phosphorylating enzymes) such as PI3K, so that phosphatidylinositol 3,4,5-triphosphate (PtdIns (3 , 4, 5) P3), and activation of protein kinase B (PKB) / Akt is associated with the control of cell survival, proliferation, differentiation, and cell death.

  A PI3K inhibitor is an agent that directly inhibits PI3 kinase and an agent that inhibits the downstream signaling pathway of PI3K. The downstream signal pathway of PI3K is, for example, PI3K-Akt pathway, PI3K-BTK pathway, PI3K-ITK pathway, PI3K-TAPP pathway, PI3K-DAPP pathway, PI3K-GAB1 / 2 pathway, PI3K-Rac pathway, PI3K-Rho pathway , PI3K-ARF pathway, PI3K-PDK1 pathway, PI3K-EEA1 pathway, PI3K-Rabenosyn5 pathway, PI3K-Rabip4 pathway, PI3K-FAB1 pathway, PI3K-p40phox pathway, PI3K-SNX3 pathway, PI3K-SNX17 pathway, PI3K-SGK pathway It is. Until now, it has not been reported that PI3K inhibitors have a differentiation-inducing action on alveolar epithelial stem cells. The present inventors have obtained a new finding that PI3K inhibitors induce differentiation of alveolar epithelial stem cells into type I and type II alveolar epithelial cells.

Examples of the PI3K inhibitor include the following compounds.
Direct inhibitors of PI3K: for example, Wortmannin, LY294002, AS605240, ZSTK474, PIK-75 Hydrochloride, IPI-145 (INK1197), GDC-0941, CAL-101 (Idelalizib, GS-1101), BEZ235 NVP-BEZ235, Dactolisib), BKM120 (NVP-BKM120, Buparlisb), GSK2636761, CZC24832, GDC-0032, VS-5684 (SB2343), TG100713, BYL719, CUDC-907, T ), BAY 80-6946 (Copanlist), PF-046 1502, PKI-402, CH5132799, GDC-0980 (RG7422), NU7441 (KU-57788), AS-252424, AS-604850, CAY10505, GSK212645 (GSK458), A66, PF-05212384 (PKI-587), Paloid P529), PIK-294, PIK-293, SAR245409 (XL765), PIK-93, AZD6482, AS-605240, GSK1059615, TG100-115, IC-87114, PIK-75, PIK-90, TGX-221, XL147, PI-103, IC486068, and derivatives thereof PI3K-Akt pathway inhibitors: For example, MK-2206, GSK690693, GDC-0068 , A-745563, CCT128930, and derivatives thereof PI3K-PDK1 pathway inhibitors: for example, BX-912, BX-795, OSU-03012, PHT-427, and derivatives thereof

  Based on such knowledge, the following inventions [1] to [4] are provided.

[1] A differentiation inducer that acts on alveolar epithelial stem cells, comprising a PI3K inhibitor as an active ingredient. The differentiation inducer differentiates alveolar epithelial stem cells into alveolar epithelial cells.
The differentiation inducer contains at least one PI3K inhibitor as an active ingredient in a pharmaceutically acceptable medium.
There are no particular limitations on the type of medium and formulation additive used in the preparation of the differentiation inducer. Examples of the medium include a solid medium (for example, gelatin and lactose) and a liquid medium (for example, alcohol, water, and physiological saline). Examples of the additive for preparation include excipients, disintegrants, binders, lubricants, surfactants, buffers, solubilizers, stabilizers, tonicity agents, and the like.

[2] A pharmaceutical composition for inducing alveolar regeneration comprising a PI3K inhibitor as an active ingredient.
The pharmaceutical composition contains at least one PI3K inhibitor as an active ingredient in a pharmaceutically acceptable medium.
The form of the pharmaceutical composition is not particularly limited, and is suitable for oral administration. Tablet, granule, powder, capsule, suspension, syrup, emulsion, limonade; ampoule for injection, freeze-dried powder for injection Agent; dry powder for transpulmonary administration; and the like.
There are no particular limitations on the type of medium and formulation additives used in the preparation of the pharmaceutical composition. Examples of the medium include a solid medium (for example, gelatin and lactose) and a liquid medium (for example, alcohol, water, and physiological saline). Examples of the additive for preparation include excipients, disintegrants, binders, lubricants, surfactants, buffers, solubilizers, stabilizers, tonicity agents, and the like.

[3] A pharmaceutical composition for treating lung tissue damage, comprising a PI3K inhibitor as an active ingredient.
The pharmaceutical composition contains at least one PI3K inhibitor as an active ingredient in a pharmaceutically acceptable medium.
The form of the pharmaceutical composition is not particularly limited, and is suitable for oral administration. Tablet, granule, powder, capsule, suspension, syrup, emulsion, limonade; ampoule for injection, freeze-dried powder for injection Agent; dry powder for transpulmonary administration; and the like.
There are no particular limitations on the type of medium and formulation additives used in the preparation of the pharmaceutical composition. Examples of the medium include a solid medium (for example, gelatin and lactose) and a liquid medium (for example, alcohol, water, and physiological saline). Examples of the additive for preparation include excipients, disintegrants, binders, lubricants, surfactants, buffers, solubilizers, stabilizers, tonicity agents, and the like.

[4] A method for treating lung tissue damage, comprising administering a pharmaceutical composition comprising a PI3K inhibitor as an active ingredient to a patient having damage to lung tissue. The pharmaceutical composition used for the treatment method is the pharmaceutical composition of [2] or the pharmaceutical composition of [3].
Examples of patients having damage to lung tissue include chronic obstructive pulmonary disease (COPD), emphysema, chronic bronchitis, acute lung distress syndrome (ARDS), pulmonary fibrosis, lung cancer, Examples include interstitial pneumonia, pulmonary tuberculosis sequelae, and pneumoconiosis.
In the treatment method, the administration mode is not particularly limited, and various administration routes such as pulmonary administration, intravenous administration, and oral administration can be selected, and preferably pulmonary administration.
The dose is selected depending on the type of disease; patient symptom, body weight, age; mode of administration; type of PI3K inhibitor;

  Furthermore, the following inventions [5] to [8] and [5 '] to [8'] are provided. [5] to [8] and [5 ′] to [8 ′] correspond to [freeze-dried composition], [production method of freeze-dried composition], and [pharmaceutical composition for transpulmonary administration] described later. And a PI3K inhibitor as an active ingredient. The details of [5] to [8] and [5 ′] to [8 ′] will be described later in [Freeze-dried composition], [Production method of freeze-dried composition], and [Pharmaceutical composition for transpulmonary administration]. It is the same.

[5] A lyophilized composition comprising a PI3K inhibitor, at least two types of amino acids, and a surfactant.

[5 ′] A lyophilized composition comprising a PI3K inhibitor, at least two kinds of amino acids selected from the following first group, second group and third group and a plurality of groups, and a surfactant.
・ Group 1: phenylalanine, tryptophan ・ Group 2: isoleucine, leucine, valine ・ Group 3: alanine, glycine

[6] A lyophilized composition comprising a PI3K inhibitor, at least two amino acids, a surfactant, an alcohol, and water.

[6 ′] a PI3K inhibitor, at least two amino acids selected from the first group, the second group, and the third group described below across a plurality of groups, a surfactant, an alcohol, and water A lyophilized composition of the composition.
・ Group 1: phenylalanine, tryptophan ・ Group 2: isoleucine, leucine, valine ・ Group 3: alanine, glycine

[7] A step of preparing a first liquid by dissolving a PI3K inhibitor in a solvent containing alcohol, mixing the first liquid, at least two kinds of amino acids, a surfactant, and water. A method for producing a lyophilized composition comprising the steps of preparing a second liquid and lyophilizing the second liquid.

[7 ′] A step of preparing a first liquid by dissolving a PI3K inhibitor in a solvent containing alcohol; the first liquid; a plurality of groups from the following first group, second group, and third group: A step of preparing a second liquid by mixing at least two kinds of amino acids selected over the two, a surfactant and water, and a step of freeze-drying the second liquid. A method for producing the composition.
・ Group 1: phenylalanine, tryptophan ・ Group 2: isoleucine, leucine, valine ・ Group 3: alanine, glycine

[8] A pharmaceutical composition for transpulmonary administration, wherein the freeze-dried composition according to [5] or [6] is granulated by airflow.

[8 '] A pharmaceutical composition for transpulmonary administration, wherein the freeze-dried composition according to [5'] or [6 '] is granulated by an air stream.

  Further, the invention of [5], [6], [5 ′] or [6 ′] is filled into a transpulmonary administration device disclosed in, for example, US Pat. No. 7,735,485 B2 or Japanese Patent No. 4822709. The following [9] or [9 ′] invention is provided.

[9] When the patient inhales the pharmaceutical composition for pulmonary administration according to [8], the freeze-dried composition according to [5] or [6] A method for transpulmonary administration of a pharmaceutical composition for pulmonary administration, comprising the step of preparing the pharmaceutical composition for pulmonary administration.

[9 ′] When the patient inhales the pharmaceutical composition for transpulmonary administration described in [8 ′], the freeze-dried composition described in [5 ′] or [6 ′] is generated by inhalation of the patient. A method for transpulmonary administration of a pharmaceutical composition for pulmonary administration, comprising the step of preparing the pharmaceutical composition for pulmonary administration by granulating with air impact.

  According to the above [1] to [9] and [5 ′] to [9 ′], a differentiation inducer that acts on alveolar epithelial stem cells, comprising a PI3K inhibitor as an active ingredient; a pharmaceutical composition that induces alveolar regeneration A pharmaceutical composition for treating lung tissue damage; a method for treating lung tissue damage; a lyophilized composition used in the manufacture of a pharmaceutical composition for treating lung tissue damage; suitable for transpulmonary administration by airflow; A freeze-dried composition that is granulated into particles having different particle sizes; a method for producing the freeze-dried composition; a pharmaceutical composition for treating lung tissue damage suitable for transpulmonary administration; a pharmaceutical composition for treating lung tissue damage A method of transpulmonary administration of the product.

  Hereinafter, the compound for which differentiation-inducing action on alveolar epithelial stem cells has been confirmed by the present inventor for the first time and a pharmaceutical composition containing the compound will be described in detail.

[B] Pharmaceutical composition containing a compound that acts on vitamin D receptor 1,25-dihydroxyvitamin D ₃ (calcitriol) is expressed in lung tissue of patients with chronic obstructive pulmonary disease (COPD) (Sundar IK. Et. Al., Biochem Biophys Res Commun. (2011) 406: 127-133.), But it has been reported to have differentiation-inducing action on alveolar epithelial stem cells. Absent.
The present inventors have found that 1,25-dihydroxyvitamin D ₃ is a lung epithelial stem cells to obtain a new finding that induce differentiation into type I and type II alveolar epithelial cells.
1,25-dihydroxyvitamin _{D 3} is (referred to hereinafter as "VDR active compound".) Because it is a ligand for the vitamin D receptor (vitamin D receptor, VDR), compounds that act on the VDR, on lung epithelial stem cells It was considered to have a differentiation-inducing action.
VDR acting compounds include compounds that bind directly to VDR, eg, VDR ligands, VDR agonists. VDR acting compounds include compounds that do not directly bind to VDR, such as anti-VDR ligand antibodies, compounds that promote signal transduction downstream of VDR, compounds that act on proteins constituting signal transduction pathways downstream of VDR, VDR Compounds that act as activators, compounds that inhibit VDR activator inhibitors are included.

Among VDR acting compounds, VDR ligands and agonists include, for example, vitamin D and derivatives thereof, and specifically include the following compounds.
Vitamin D ₂ and its derivatives: eg ergosterol, ergocalciferol Vitamin D ₃ and its derivatives: eg 7-dehydrocholesterol, previtamin D ₃ , cholecalciferol, 25-hydroxycholecalciferol, 1,25 - dihydroxy cholecalciferol (calcidiol), 1,25-dihydroxyvitamin _{D 3} (calcitriol), calcitroic acid-vitamin _{D 4} and its derivatives: e.g., dihydro ergocalciferol, vitamin _{D 5} and its derivatives, vitamin _{D 6} And its derivatives Vitamin D ₇ and its derivatives Vitamin D analogs: for example, dihydrotaxosterol, calcipotriol, tacalcitol, paricalcitol

Among VDR acting compounds, compounds acting on proteins constituting a signal transduction pathway downstream of VDR include, for example, RANKL (Receptor activator of NF-kappa B ligand) inhibitor and NF-kB (Nuclear factor-kappa B) inhibition. Examples of the material include the following compounds.
-RANKL inhibitor: For example, WP9QY, Osteoprotegerin (OPG), anti-RANKL antibody (denosumab), RANKL siRNA, RANKL shRNA
-NF-kB inhibitors: For example, (5Z) -7-Oxozeaenol, (5Z) -Zeaenol, Andrographolide, Aurothiomalate, Capsaicin, (E) -Capsaicin, Evodiamine, anti-NF-kB antibody, NF-RNABsRNA

The VDR active compound, preferably 1,25-dihydroxyvitamin _{D 3.} 1,25-dihydroxyvitamin _{D 3} shows the differentiation inducing effect on significantly alveolar epithelial stem cells at lower concentrations than ATRA.

  Based on such knowledge, the following inventions [11] to [14] are provided.

[11] A differentiation inducer that acts on alveolar epithelial stem cells, comprising a VDR acting compound as an active ingredient. The differentiation inducer differentiates alveolar epithelial stem cells into alveolar epithelial cells.
The differentiation inducer contains at least one VDR acting compound as an active ingredient in a pharmaceutically acceptable medium.
There are no particular limitations on the type of medium and formulation additive used in the preparation of the differentiation inducer. Examples of the medium include a solid medium (for example, gelatin and lactose) and a liquid medium (for example, alcohol, water, and physiological saline). Examples of the additive for preparation include excipients, disintegrants, binders, lubricants, surfactants, buffers, solubilizers, stabilizers, tonicity agents, and the like.

[12] A pharmaceutical composition for inducing alveolar regeneration comprising a VDR acting compound as an active ingredient.
The pharmaceutical composition contains at least one VDR acting compound as an active ingredient in a pharmaceutically acceptable medium.
The form of the pharmaceutical composition is not particularly limited, and is suitable for oral administration. Tablet, granule, powder, capsule, suspension, syrup, emulsion, limonade; ampoule for injection, freeze-dried powder for injection Agent; dry powder for transpulmonary administration; and the like.
There are no particular limitations on the type of medium and formulation additives used in the preparation of the pharmaceutical composition. Examples of the medium include a solid medium (for example, gelatin and lactose) and a liquid medium (for example, alcohol, water, and physiological saline). Examples of the additive for preparation include excipients, disintegrants, binders, lubricants, surfactants, buffers, solubilizers, stabilizers, tonicity agents, and the like.

[13] A pharmaceutical composition for treating lung tissue damage, comprising a VDR acting compound as an active ingredient.
The pharmaceutical composition contains at least one VDR acting compound as an active ingredient in a pharmaceutically acceptable medium.
The form of the pharmaceutical composition is not particularly limited, and is suitable for oral administration. Tablet, granule, powder, capsule, suspension, syrup, emulsion, limonade; ampoule for injection, freeze-dried powder for injection Agent; dry powder for transpulmonary administration; and the like.
There are no particular limitations on the type of medium and formulation additives used in the preparation of the pharmaceutical composition. Examples of the medium include a solid medium (for example, gelatin and lactose) and a liquid medium (for example, alcohol, water, and physiological saline). Examples of the additive for preparation include excipients, disintegrants, binders, lubricants, surfactants, buffers, solubilizers, stabilizers, tonicity agents, and the like.

[14] A method for treating lung tissue damage, comprising administering a pharmaceutical composition comprising a VDR acting compound to a patient having damage to lung tissue. The pharmaceutical composition used for the treatment method is the pharmaceutical composition of [12] or the pharmaceutical composition of [13].
Examples of patients having damage to lung tissue include chronic obstructive pulmonary disease (COPD), emphysema, chronic bronchitis, acute lung distress syndrome (ARDS), pulmonary fibrosis, lung cancer, Examples include interstitial pneumonia, pulmonary tuberculosis sequelae, and pneumoconiosis.
In the treatment method, the administration mode is not particularly limited, and various administration routes such as pulmonary administration, intravenous administration, and oral administration can be selected, and preferably pulmonary administration.
The dosage is selected according to the type of disease; patient symptom, body weight, age; mode of administration; type of VDR acting compound;

  Furthermore, the following inventions [15] to [18] and [15 '] to [18'] are provided. [15] to [18] and [15 ′] to [18 ′] correspond to [freeze-dried composition], [production method of freeze-dried composition], and [pharmaceutical composition for transpulmonary administration] described later. And a VDR acting compound as an active ingredient. The details of [15] to [18] and [15 ′] to [18 ′] are described later in [Freeze-dried composition], [Method of producing freeze-dried composition], and [Pharmaceutical composition for transpulmonary administration]. It is the same.

[15] A lyophilized composition comprising a VDR acting compound, at least two types of amino acids, and a surfactant.

[15 ′] A lyophilized composition comprising a VDR acting compound, at least two amino acids selected from the following first group, second group and third group and a plurality of amino acids, and a surfactant.
・ Group 1: phenylalanine, tryptophan ・ Group 2: isoleucine, leucine, valine ・ Group 3: alanine, glycine

[16] A lyophilized composition of a composition comprising a VDR acting compound, at least two types of amino acids, a surfactant, an alcohol, and water.

[16 ′] a VDR acting compound, at least two amino acids selected from the following first group, second group, and third group, and a plurality of groups, a surfactant, an alcohol, and water A lyophilized composition of the composition.
・ Group 1: phenylalanine, tryptophan ・ Group 2: isoleucine, leucine, valine ・ Group 3: alanine, glycine

[17] A step of preparing a first liquid by dissolving a VDR acting compound in a solvent containing alcohol, mixing the first liquid, at least two kinds of amino acids, a surfactant, and water. A method for producing a lyophilized composition comprising the steps of preparing a second liquid and lyophilizing the second liquid.

[17 ′] A step of preparing a first liquid by dissolving a VDR acting compound in a solvent containing alcohol, the first liquid, and a plurality of groups from the following first group, second group, and third group A step of preparing a second liquid by mixing at least two kinds of amino acids selected over the two, a surfactant and water, and a step of freeze-drying the second liquid. A method for producing the composition.
・ Group 1: phenylalanine, tryptophan ・ Group 2: isoleucine, leucine, valine ・ Group 3: alanine, glycine

[18] A pharmaceutical composition for transpulmonary administration, wherein the freeze-dried composition according to [15] or [16] is granulated by air flow.

[18 '] A pharmaceutical composition for transpulmonary administration, wherein the freeze-dried composition according to [15'] or [16 '] is granulated by air flow.

  Further, the invention of [15], [16], [15 ′] or [16 ′] is filled into a transpulmonary administration device disclosed in, for example, US Pat. No. 7,735,485 B2 or US Pat. No. 4,822,709. The following [19] or [19 ′] invention is provided.

[19] When the patient inhales the pharmaceutical composition for pulmonary administration according to [18], the freeze-dried composition according to [15] or [16] A method for transpulmonary administration of a pharmaceutical composition for pulmonary administration, comprising the step of preparing the pharmaceutical composition for pulmonary administration.

[19 ′] When the patient inhales the pharmaceutical composition for pulmonary administration described in [18 ′], the freeze-dried composition described in [15 ′] or [16 ′] is generated by inhalation of the patient. A method for transpulmonary administration of a pharmaceutical composition for pulmonary administration, comprising the step of preparing the pharmaceutical composition for pulmonary administration by granulating with air impact.

  According to the above [11] to [19] and [15 ′] to [19 ′], a differentiation inducer that acts on alveolar epithelial stem cells, comprising a VDR acting compound as an active ingredient; a pharmaceutical composition that induces alveolar regeneration A pharmaceutical composition for treating lung tissue damage; a method for treating lung tissue damage; a lyophilized composition used in the manufacture of a pharmaceutical composition for treating lung tissue damage; suitable for transpulmonary administration by airflow; A freeze-dried composition that is granulated into particles having different particle sizes; a method for producing the freeze-dried composition; a pharmaceutical composition for treating lung tissue damage suitable for transpulmonary administration; a pharmaceutical composition for treating lung tissue damage A method of transpulmonary administration of the product.

[C] Lyophilized composition, method for producing lyophilized composition, and pharmaceutical composition for pulmonary administration [lyophilized composition]
The lyophilized composition of the present invention is a lyophilized composition containing at least the following components.
・ Drugs poorly soluble in water (hereinafter sometimes referred to as “active ingredients”)
-At least 2 types of amino acids-Surfactant The lyophilized composition is a lyophilized composition of a composition (dispersion or lysate) containing at least the following components.
・ Slightly soluble drug ・ At least two kinds of amino acids ・ Surfactant ・ Alcohol ・ Water And when the freeze-dried composition is made into particles by air flow, the proportion of particles having a particle size of 5 μm or less is 10%. This is the lyophilized composition.
When at least two kinds of amino acids are not used, a freeze-dried composition containing a drug hardly soluble in water cannot be prepared, or it becomes a freeze-dried composition that is difficult to form particles even when subjected to airflow. Or a lyophilized composition that particles into unsuitable large particles.

For the lyophilized composition, the particle size is a geometric particle size or an aerodynamic particle size.
The above-mentioned ratio is the ratio (volume%) of particles having a particle diameter of 5 μm or less with respect to all the particles with respect to the geometric particle diameter.
The above ratio is the ratio (mass%) of the amount of drug contained in particles having a particle diameter of 5 μm or less with respect to the amount of drug contained in all particles (total amount of drug).

The lyophilized composition of the present invention is preferably a lyophilized composition containing at least the following components.
-A poorly water-soluble drug-At least two amino acids selected from the following group 1, group 2 and group 3 across multiple groups-Group 1: phenylalanine, tryptophan-Group 2: isoleucine, Leucine, valine Group 3: Alanine, glycine Surfactant The lyophilized composition is preferably a lyophilized composition of a composition (dispersion or solution) containing at least the following components.
-A poorly water-soluble drug-At least two amino acids selected from the following group 1, group 2 and group 3 across multiple groups-Group 1: phenylalanine, tryptophan-Group 2: isoleucine, Leucine, valine ・ Group 3: alanine, glycine ・ Surfactant ・ Alcohol ・ Water As at least two types of amino acids, a specific type of amino acid selected in the above combination (hereinafter also referred to as “specific amino acid”) is used. By doing so, a freeze-dried composition containing a drug that is sparingly soluble in water becomes a freeze-dried composition that easily undergoes particle formation with a particle size suitable for transpulmonary administration under airflow.

The lyophilized composition of the present invention is a pre-preparation of a pharmaceutical composition for pulmonary administration (a lyophilized composition for the preparation of a pharmaceutical composition for pulmonary administration), and is granulated by air impact by inhalation of a patient, It produces particles with a geometric particle size of 5 μm or less.
The ratio of the particles having a geometric particle diameter of 5 μm or less to the total particles generated from the freeze-dried composition of the present invention is preferably 10% or more, more preferably 15% or more, even more preferably on a volume basis. Is 20% or more, more preferably 25% or more, and particularly preferably 30% or more.
The geometric particle diameter can be measured by, for example, a laser diffraction particle size distribution measuring apparatus.

The lyophilized composition of the present invention is a pre-preparation of a pharmaceutical composition for pulmonary administration (a lyophilized composition for the preparation of a pharmaceutical composition for pulmonary administration). Particles having a particle size of 5 μm or less are generated.
The ratio of the particles having an aerodynamic particle size of 5 μm or less to the total particles generated from the freeze-dried composition of the present invention is preferably 10% or more as the ratio (mass%) of the drug amount to the total drug amount. More preferably, it is 15% or more, more preferably 20% or more, still more preferably 25% or more, and particularly preferably 30% or more.
The aerodynamic particle size can be measured by, for example, Multi-stage Liquid Impinger, Andersen Cascade Impactor, Next Generation Pharmaceutical Impactor and the like.

  In general, it is said that in order for particles administered transpulmonarily to reach and deposit in the lung, the particles must have an aerodynamic particle size of 5 μm or less. Since the aerodynamic particle size of the particles derived from the porous lyophilized composition is usually smaller than the geometric particle size, if the proportion of particles having a geometric particle size of 5 μm or less is within the above range, A sufficient proportion of particles of aerodynamic particle size suitable for pulmonary administration will be included.

  The lyophilized composition of the present invention is preferably a porous solid in that it is easily atomized by receiving an air stream. The porous solid may be cracked, divided into several lumps, or may be partially broken as long as it can be granulated by an air stream.

  Hereinafter, each component used for preparation of the freeze-dried composition of this invention is explained in full detail.

-Drugs poorly soluble in water-
“Slightly soluble in water” for a drug means a compound that is 10 mg or less soluble in 1 ml of water at 20 ° C., and the amount of the compound is preferably 5 mg or less, more preferably 1 mg or less, More preferably, it is 0.5 mg or less, Most preferably, it is 0.1 mg or less.
Examples of the poorly water-soluble drug include compounds having a differentiation-inducing action on alveolar epithelial stem cells.

-Compound having differentiation-inducing action on alveolar epithelial stem cells-
The compound having a differentiation-inducing action on alveolar epithelial stem cells is not particularly limited as long as it has a pharmacological activity that induces differentiation of alveolar epithelial stem cells (preferably human alveolar epithelial stem cells).

  The fact that a certain compound has a differentiation-inducing action on alveolar epithelial stem cells means that when the compound is brought into contact with alveolar epithelial stem cells, at least one of type I alveolar epithelial cells and type II alveolar epithelial cells This can be confirmed by inducing the cells. Specifically, after culturing alveolar epithelial stem cells in a medium containing the compound, the cells were stained by immunostaining and observed with a fluorescence microscope, and AQP-5 (Aquaporin, which is a marker of type I alveolar epithelial cells). -5, aquaporin 5), and the number of cells expressing at least one of SP-A (Surfactant (apo) protein A, surfactant A), a marker of type II alveolar epithelial cells, It can be confirmed by being.

Examples of compounds having differentiation-inducing action on alveolar epithelial stem cells include vitamin A, vitamin A derivatives, provitamin A, compounds acting on vitamin D receptors, and PI3 kinase inhibitors. Include the following compounds.
Vitamin A derivatives: for example, retinoids such as all-trans-retinoic acid (ATRA. Tretinoin), retinol, retinal, and synthetic retinoids (for example, Tamibarotene). Provitamin A: for example α-carotene, β-carotene Vitamin D ₂ and its derivatives: eg ergosterol, ergocalciferol Vitamin D ₃ and its derivatives: eg 7-dehydrocholesterol, previtamin D ₃ , cholecalciferol, 25-hydroxycholecalciferol, 1,25-dihydroxycholecalciferol (calcidiol), 1,25-dihydroxyvitamin _{D 3} (calcitriol), calcitroic acid-vitamin _{D 4} and its derivatives: e.g., dihydro ergocalciferol Vita Emissions _{D 5} and derivatives thereof, vitamin _{D 6} and its derivatives, vitamin _{D 7} and its derivatives, vitamin D analogs: e.g., dihydro Taki sterol, calcipotriol, tacalcitol, direct inhibitor of paricalcitol-PI3K: for example, Wortmannin, LY294002, AS605240, ZSTK474, PIK-75 Hydrochloride, IPI-145 (INK1197), GDC-0941, CAL-101 (Idelalisib, GS-1101), BEZ235 (NVP-BEtK235, NVP-BEZ235MN) -BKM120, Buparisib), GSK2636761, CZC24832, GDC-0032, VS-5484 (SB2343), TG10 713, BYL719, CUDC-907, 3-Methyladeline, YM201636, BGT226 (NVP-BGT226), BAY80-6946 (Copanlist), PF-04691502, PKI-402, CH5132799, GDC-0980 (RG74-8K, RU74) ), AS-252424, AS-604850, CAY10505, GSK2126458 (GSK458), A66, PF-05212384 (PKI-587), Palomid529 (P529), PIK-294, PIK-293, SAR245409 (XL765), PIK-93, AZD6482, AS-605240, GSK1059615, TG100-115, IC-87114, PIK- 5, PIK-90, TGX-221, XL147, PI-103, IC486068, and derivatives thereof PI3K-Akt pathway inhibitors: For example, MK-2206, GSK690693, GDC-0068, A-674563, CCT128930, and these Derivatives of PI3K-PDK1 pathway: For example, BX-912, BX-795, OSU-03012, PHT-427, and derivatives thereof

-Other drugs-
In addition, examples of poorly water-soluble drugs that can be expected to have a pharmacological effect on the lung include steroids (beclomethasone, fluticasone, budesonide, etc.) and herbal turmeric components (curcumin).

  The amount of the active ingredient contained in the lyophilized composition of the present invention is not particularly limited. The amount of the active ingredient contained in the lyophilized composition for a single dose is, for example, 20 mg or less, preferably 10 mg or less, more preferably 5 mg or less, still more preferably 2 mg or less, and particularly preferably 1 mg or less.

-Amino acid-
The present invention uses at least two amino acids in combination.
For example, glycine (Gly), alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), serine (Ser), threonine (Thr), aspartic acid (Asp), glutamic acid (Glu), asparagine ( Asn), glutamine (Gln), lysine (Lys), arginine (Arg), cysteine (Cys), methione (Met), phenylalanine (Phe), tyrosine (Tyr), tryptophan (Trp), histidine (His), and proline At least two types are selected from (Pro).
The 20 amino acids other than glycine may be D-type or L-type, or a mixture of D-type and L-type.

A preferable combination of at least two kinds of amino acids selected from the above amino acid group is that the lyophilized composition is easily formed into particles having a geometric particle diameter of 5 μm or less, and Phe or Trp / Ile, Leu, Val, At least one selected from Ala and Gly; at least one selected from Ile, Leu and Val / at least one selected from Ala and Gly;
Among these, at least one combination selected from Phe or Trp / Ile, Leu and Val is more preferable, and a combination of Phe / Ile, Phe / Leu, Phe / Val and Trp / Ile is particularly preferable.

  The total content of amino acids in the lyophilized composition of the present invention is not particularly limited, but is, for example, 0.001% by mass or more and less than 100% by mass, preferably 0.01% by mass or more and less than 100% by mass, more Preferably they are 0.1 mass% or more and less than 100 mass%, More preferably, they are 1 mass% or more and less than 100 mass%, Especially preferably, they are 10 mass% or more and less than 100% mass.

-Specific amino acid-
In the present invention, it is preferable to use at least two kinds of amino acids selected from the following first group, second group and third group as a plurality of groups as at least two kinds of amino acids. The combination of a plurality of groups is a combination of the first group and the second group, a combination of the first group and the third group, a combination of the second group and the third group, and a combination of the first group, the second group and the third group. Any combination may be used.
First group: phenylalanine (Phe), tryptophan (Trp)
Second group: isoleucine (Ile), leucine (Leu), valine (Val)
Group 3: alanine (Ala), glycine (Gly)
The amino acids other than glycine may be D-type or L-type, or a mixture of D-type and L-type.

At least one of the two types is preferably an amino acid selected from the first group in that the freeze-dried composition is easily granulated into particles having a geometric particle size of 5 μm or less, that is, Phe or Trp Is preferred.
A preferred combination of at least two types of amino acids is at least selected from Phe or Trp / Ile, Leu, Val, Ala and Gly in that the lyophilized composition is easily granulated into particles having a geometric particle size of 5 μm or less. At least one selected from Ile, Leu and Val / at least one selected from Ala and Gly;
Among these, at least one combination selected from Phe or Trp / Ile, Leu and Val is more preferable, and a combination of Phe / Ile, Phe / Leu, Phe / Val and Trp / Ile is particularly preferable.

  The total content of the specific amino acids in the lyophilized composition of the present invention is not particularly limited, but is, for example, 0.001% by mass or more and less than 100% by mass, preferably 0.01% by mass or more and less than 100% by mass, More preferably, they are 0.1 mass% or more and less than 100 mass%, More preferably, they are 1 mass% or more and less than 100 mass%, Especially preferably, they are 10 mass% or more and less than 100% mass.

  The lyophilized composition of the present invention may contain an amino acid other than the specific amino acid when at least two kinds of amino acids are the specific amino acid.

-Alcohol-
When producing the freeze-dried composition of the present invention, a drug that is sparingly soluble in water is used by dissolving it in a solvent containing alcohol.
Since alcohol volatilizes by lyophilization, it is usually not included in lyophilized compositions.
Examples of the alcohol include ethanol, methanol, isopropyl alcohol, ethylene glycol, and the like, and preferably ethanol.
These may be used alone or in combination of two or more.

-Surfactant-
As the surfactant, any anionic surfactant, cationic surfactant, or nonionic surfactant can be used as long as it is a surfactant that is usually used in pharmaceuticals. Preferably, nonionic surfactants such as polyoxyethylene sorbitan fatty acid ester (for example, Tween surfactant) and sorbitan trioleate are exemplified.

Specific examples of the surfactant include sucrose fatty acid ester, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, polyoxyethylene hydrogenated castor oil 140, polyoxyethylene hydrogenated castor oil 50, polyoxyethylene hydrogenated castor oil 60. , Polyoxyethylene (42) polyaxpropylene (67) glycol, polyoxyethylene (54) polyaxpropylene (39) glycol, polyoxyethylene (105) polyaxpropylene (5) glycol, polyoxyethylene (196) poly Axypropylene (67) glycol, polyoxyethylene (160) polyaxpropylene (30) glycol, sorbitan sesquioleate, sorbitan trioleate, sorbitan monostearate, sorbita monopalmitate , Polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, glyceryl monostearate, sodium lauryl sulfate, lauromacrogol, polyoxyethylene sorbitan monolaurate (Tween 20), polyoxyethylene sorbitan oleate (Tween 80), etc. Can be mentioned.
These may be used alone or in combination of two or more.

-Water-
In the production of the freeze-dried composition of the present invention, the composition (dispersion or solution) subjected to freeze-drying is a liquid containing water as a main solvent.
Since all or most of the water is lost by the lyophilization process, the water is not included in the lyophilized composition, or a small amount, if any, is included.

-Other ingredients-
The lyophilized composition of the present invention may contain other components in addition to the above components as long as the final preparation (pharmaceutical composition for pulmonary administration) does not affect the human body.
Other components include stabilization of active ingredients in the composition (dispersion or solution) to be lyophilized, stabilization of active ingredients after lyophilization, prevention of adsorption of active ingredients to containers, etc. Pharmaceutical additives commonly used in the art for the purpose (excipients, disintegrants, binders, lubricants, surfactants, buffers, solubilizers, stabilizers, tonicity agents, etc.) May be used.

The lyophilized composition of the present invention may contain a carrier other than amino acids.
As carriers other than amino acids, monosaccharides such as glucose; disaccharides such as sucrose, maltose, lactose and trehalose; oligosaccharides such as cyclodextrin; polysaccharides such as dextran 40 and pullulan; fatty acid sodium such as sodium caprate; Is mentioned. These may be used alone or in combination of two or more.
The amount of these carriers to be blended is not particularly limited as long as the lyophilized composition is granulated by an air stream. As a guide, it is blended so that the proportion of the lyophilized composition is 0.1 mass% or more and less than 100 mass%.

[Method for producing freeze-dried composition]
The method for producing a freeze-dried composition of the present invention includes freeze-drying a composition (dispersion or solution) containing at least the following components.
・ Slightly soluble drugs ・ At least two amino acids ・ Surfactant ・ Alcohol ・ Water

The method for producing a freeze-dried composition of the present invention preferably includes freeze-drying a composition (dispersion or solution) containing at least the following components.
-A poorly water-soluble drug-At least two amino acids selected from the following group 1, group 2 and group 3 across multiple groups-Group 1: phenylalanine, tryptophan-Group 2: isoleucine, Leucine, valine ・ Group 3: Alanine, glycine ・ Surfactant ・ Alcohol ・ Water

  The method for producing a lyophilized composition of the present invention preferably includes the following steps (A) to (C). According to the production method including the steps (A) to (C), it is possible to prepare a liquid (dispersion or solution) containing water as the main solvent in which the active ingredient is well dispersed or dissolved, and as a result, A porous lyophilized composition can be produced that is granulated into a size suitable for transpulmonary administration by airflow.

Step (A): a step of preparing a first liquid by dissolving a poorly water-soluble drug in a solvent containing alcohol. Step (B): the first liquid and at least two kinds of amino acids, Step of preparing a second liquid by mixing a surfactant and water-Step (C): Step of freeze-drying the second liquid

The step (B) is preferably the following step (B ′).
Step (B ′): the first liquid, at least two kinds of amino acids selected from the following first group, second group, and third group across a plurality of groups, a surfactant, and water To prepare a second liquid by mixing the following:-Group 1: phenylalanine, tryptophan-Group 2: isoleucine, leucine, valine-Group 3: alanine, glycine

-Process (A)-
Step (A) is a step of preparing a first liquid in which a poorly water-soluble drug (active ingredient) is dissolved in a solvent containing alcohol.

The solvent containing alcohol may be a solvent of alcohol alone or an aqueous alcohol solution.
Examples of the alcohol include ethanol, methanol, isopropyl alcohol, ethylene glycol, and the like, and preferably ethanol.
The alcohol concentration of the solvent containing alcohol is not particularly limited as long as the active ingredient can be dissolved. However, since it is desirable that the second liquid to be subjected to freeze-drying has a low alcohol concentration in terms of easy freeze-drying, the alcohol concentration of the solvent containing alcohol may be adjusted from the above viewpoint.

  The solvent containing alcohol may contain an organic solvent other than alcohol as a solubilizing agent as long as the final preparation (pharmaceutical composition for pulmonary administration) does not affect the human body. Examples of the organic solvent include acetone.

-Process (B)-
In step (B) (preferably step (B ′)), the first liquid in which the active ingredient is dissolved, at least two kinds of amino acids (preferably specific amino acids), a surfactant, and water are mixed. This is a step of preparing a second liquid (dispersion or solution).
Since the first liquid usually has an alcohol concentration that is so high that it is difficult to freeze-dry, it is necessary to add water to lower the alcohol concentration to adjust the alcohol concentration to be easily freeze-dried. Step (B) is a step of adjusting the alcohol concentration by adding water while favorably dispersing or dissolving the active ingredient with at least two types of amino acids (preferably specific amino acids) and a surfactant.

  In the step (B), the order of mixing the first liquid, the amino acid and the surfactant is not limited, but the dispersibility or solubility of the active ingredient in the second liquid is better. And the aspect which mixes a 1st liquid and surfactant and mixes an amino acid after that is preferable. Specifically, a surfactant is added to the first liquid to prepare a solution containing the active ingredient and the surfactant, and this solution is mixed with an amino acid-containing solution in which an amino acid is dissolved or dispersed in water. Embodiments are preferred.

  The concentration of the active ingredient in the second liquid (the total concentration when multiple types are used) is, for example, 0.001 mg / mL to 1000 mg / mL, preferably 0.001 mg / mL to 100 mg / mL, more preferably 0.001 mg / mL to 10 mg / mL, more preferably 0.001 mg / mL to 1 mg / mL, and particularly preferably 0.001 mg / mL to 0.1 mg / mL.

  In the second liquid, the total amount of amino acids relative to the amount of the active ingredient is, for example, 50 parts by weight to 80000 parts by weight, preferably 100 parts by weight to 40000 parts by weight, when the amount of the active ingredient is 100 parts by weight. Preferably they are 200 mass parts-20000 mass parts, More preferably, they are 400 mass parts-10000 mass parts, Most preferably, they are 800 mass parts-8000 mass parts.

The total concentration of amino acids in the second liquid is not particularly limited. For example, what is necessary is just to adjust according to the quantity and kind of active ingredient, for example, 0.001 mg / mL or more, 0.01 mg / mL or more, and 0.1 mg / mL or more.
The total concentration of the amino acids in the second liquid is preferably 10 mg / mL or less, more preferably 8 mg / mL or less, and even more preferably 4 mg in that the lyophilized composition is well porous and easily atomized by airflow. / ML or less, particularly preferably 2 mg / mL or less.

  The mass ratio of amino acids contained in the second liquid (the mass ratio between any two types when 3 or more types are contained) is, for example, in the range of 10:90 to 90:10, preferably 20:80. It is -80: 20, More preferably, it is 30: 70-70: 30, More preferably, it is 40: 60-60: 40.

  The concentration of the surfactant in the second liquid (when multiple types are used, the total concentration thereof) is, for example, 0.0001% by mass to 1% by mass, preferably 0.001% by mass to 0.5% by mass, More preferably, it is 0.005 mass%-0.5 mass%, More preferably, it is 0.005 mass%-0.3 mass%, Most preferably, it is 0.005 mass%-0.1 mass%.

  The alcohol concentration of the second liquid is preferably 0.1% by mass to 20% by mass, more preferably 0.1% by mass to 10% by mass, still more preferably 0.5% by mass to 10% by mass, particularly preferably. It is 0.5 mass%-5 mass%.

  In step (B), formulation additives (excipients, disintegrants, binders, lubricants, surfactants, buffers, solubilizers, stabilizers, tonicity agents, etc.) are further mixed. May be. A carrier other than amino acids may be mixed.

-Process (C)-
Step (C) is a step of freeze-drying the second liquid obtained in step (B). The method for freeze-drying the second liquid is not particularly limited, and may be, for example, a freeze-drying method that is conventionally applied to the production of a conventionally known freeze-dried drug (for example, an injection that is dissolved at the time of use).

  In the step (C), a container capable of charging a device for transpulmonary administration with a second liquid containing an active ingredient in an effective dose for single dose or several doses (preferably effective dose for single dose) It is preferable to fill in and freeze-dry as it is.

[Pharmaceutical composition for transpulmonary administration]
The pharmaceutical composition for transpulmonary administration of the present invention is a powdery pharmaceutical composition obtained by granulating the lyophilized composition of the present invention into an air stream.
One aspect of the pharmaceutical composition for pulmonary administration of the present invention comprises a compound having an action of inducing differentiation of alveolar epithelial stem cells (preferably human alveolar epithelial stem cells) as an active ingredient.

When the pharmaceutical composition for pulmonary administration of the present invention contains a compound having a differentiation-inducing action on alveolar epithelial stem cells, it can be used as a therapeutic agent for a disease exhibiting lung tissue damage. Examples of the disease include chronic obstructive pulmonary disease (COPD), emphysema, chronic bronchitis, acute lung distress syndrome (ARDS), pulmonary fibrosis, lung cancer, interstitial pneumonia, pulmonary tuberculosis. Examples include sequelae and pneumoconiosis.
Therefore, according to the pharmaceutical composition for transpulmonary administration of the present invention, a method for treating a disease exhibiting lung tissue damage is provided. The therapeutic method is a method for treating pulmonary tissue damage, comprising pulmonary administration of the pharmaceutical composition for pulmonary administration of the present invention to a patient having damage to pulmonary tissue (for example, a patient having the above-mentioned diseases). .
The dosage of the pharmaceutical composition for pulmonary administration of the present invention is selected according to the type of disease; patient symptom, body weight, age; type of compound contained as an active ingredient, and the like.

The pharmaceutical composition for transpulmonary administration of the present invention is prepared by pulverizing a freeze-dried composition for preparation with an air stream.
The flow rate of the airflow is 5 L / min to 300 L / min, more preferably 10 L / min to 200 L / min, still more preferably 10 L / min to 150 L / min, and particularly preferably 10 L / min to 100 L / min.
The flow rate of the airflow is 1 m / sec to 300 m / sec, more preferably 2 m / sec to 250 m / sec, still more preferably 5 m / sec to 200 m / sec, and particularly preferably 10 m / sec to 150 m / sec.

The pharmaceutical composition for pulmonary administration of the present invention can be formed into particles by the lyophilized composition for its preparation by a relatively low velocity and flow rate of air flow, and therefore, as an administration mode, for example, US Pat. No. 7,735,485 B2 or Patent The transpulmonary administration system disclosed in Japanese Patent No. 4822709 can be employed.
That is, if the freeze-dried composition is provided together with the device for transpulmonary administration disclosed in the above document, the user of the device attaches a vial of the freeze-dried composition to the device at the time of use, and the freeze-dried composition is inhaled by the patient's inspiration. The product becomes particles suitable for transpulmonary administration and can be taken as it is by inhalation.
Therefore, if the lyophilized composition of the present invention is provided together with the above-mentioned device for pulmonary administration, for example, one embodiment of a method for pulmonary administration of the pharmaceutical composition for pulmonary administration is provided. In the pulmonary administration method, when the patient inhales the pharmaceutical composition for pulmonary administration of the present invention, the patient applies airflow to the freeze-dried composition of the present invention to form particles that are suitable for pulmonary administration. The process of including.

In the pharmaceutical composition for pulmonary administration of the present invention, the proportion of particles having a particle size of 5 μm or less in the total particles is preferably 10% or more, more preferably 15% or more, still more preferably 20% or more, More preferably, it is 25% or more, and particularly preferably 30% or more.
Here, the particle diameter means a geometric particle diameter or an aerodynamic particle diameter. The above-mentioned ratio is the ratio (volume%) of particles having a particle diameter of 5 μm or less with respect to all the particles with respect to the geometric particle diameter. The above ratio is the ratio (mass%) of the amount of drug contained in particles having a particle diameter of 5 μm or less with respect to the amount of drug contained in all particles (total amount of drug).

In the pulmonary administration pharmaceutical composition of the present invention, the proportion (volume%) of particles having a geometric particle size of 5 μm or less in the total particles is preferably 10% or more, more preferably 15% or more, and further Preferably it is 20% or more, More preferably, it is 25% or more, Most preferably, it is 30% or more.
In the pulmonary administration pharmaceutical composition of the present invention, the ratio (mass%) of the drug amount contained in particles having an aerodynamic particle size of 5 μm or less with respect to the total drug amount is preferably 10% or more, more preferably. It is 15% or more, more preferably 20% or more, further preferably 25% or more, and particularly preferably 30% or more.
In general, it is said that in order for particles administered transpulmonarily to reach and deposit in the lung, the particles must have an aerodynamic particle size of 5 μm or less. Since the aerodynamic particle size of the particles derived from the porous lyophilized composition is usually smaller than the geometric particle size, if the proportion of particles having a geometric particle size of 5 μm or less is within the above range, A sufficient proportion of particles of aerodynamic particle size suitable for pulmonary administration will be included.

  The present invention will be described more specifically with reference to the following examples. The materials, amounts used, ratios, processing procedures, and the like shown in the following examples can be changed as appropriate without departing from the spirit of the present invention. Therefore, the scope of the present invention should not be construed as being limited by the specific examples shown below.

  CD90 is a marker for alveolar epithelial stem cells, AQP-5 is a marker for type I alveolar epithelial cells, and SP-A is a marker for type II alveolar epithelial cells.

[Reference Example 1: Verification of differentiation-inducing action of ATRA on human alveolar epithelial stem cells]
Human alveolar epithelial stem cells used in Reference Example 1 were derived from human lung alveolar epithelial stem cells (Fujino N, at. Al. Am. J) derived from patients with lung disease (biopsy in lung cancer or COPD or surgical tissue removed during surgery). Respir. Cell Mol. Biol., 46, 422-430 (2012)). The human alveolar epithelial stem cells are obtained by sterilizing the culture supernatant of mouse fetal fibroblasts cultured in DMEM medium containing 10% bovine serum (FBS) and 1% 50-fold diluted MEM (Amino Acids Solution). Using the prepared culture solution, it was cultured and maintained in a 37 ° C./5% CO ₂ environment.

All-trans retinoic acid (ATRA) is exposed to human alveolar epithelial stem cells (1 × 10 ^1-4 cells / cm ² ) in the culture at a concentration of 0 μM, 0.01 μM, 0.1 μM, 1 μM, or 10 μM. The culture was stationary for 5 days in a 37 ° C./5% CO ₂ environment.

  The cultured cells were washed with PBS (Phosphate Buffered Saline) and fixed with a 4% paraformaldehyde-PBS solution. A goat anti-human CD90 antibody (manufactured by Santa Cruz), a goat anti-human AQP-5 antibody (manufactured by Santa Cruz), and a goat anti-human SP-A antibody (manufactured by Santa Cruz) are added to the fixed cells and allowed to react. Anti-goat antibody (Santa Cruz) was added and allowed to react. And the number of positive cells was measured using the fluorescence microscope (BZ-9000, the Keyence company make), and the ratio of the positive cell was computed about each marker. The results are shown in Table 1.

  As is apparent from Table 1, when ATRA was brought into contact with human alveolar epithelial stem cells, the proportion of CD90 positive cells decreased and the proportion of AQP-5 positive cells and the proportion of SP-A positive cells increased in a concentration-dependent manner. did. This result indicates that ATRA has a differentiation-inducing action on human alveolar epithelial stem cells.

[A] Pharmaceutical composition containing PI3 kinase inhibitor [Example A1: Verification of differentiation-inducing effect of PI3K inhibitor on human alveolar epithelial stem cells]
The human alveolar epithelial stem cells used in Example A1 are human alveolar epithelial stem cells (Fujino N, at. Al. Am. J) derived from patients with lung diseases (biopsy in lung cancer or COPD, or excised tissues at the time of surgery). Respir. Cell Mol. Biol., 46, 422-430 (2012)). The human alveolar epithelial stem cells are obtained by sterilizing the culture supernatant of mouse fetal fibroblasts cultured in DMEM medium containing 10% bovine serum (FBS) and 1% 50-fold diluted MEM (Amino Acids Solution). Using the prepared culture solution, it was cultured and maintained in a 37 ° C./5% CO ₂ environment.

Human alveolar epithelial stem cells (1 × 10 ^1-4 cells / cm ² ) in the culture medium are mixed with PI3K inhibitor Wortmannin at a concentration of 0 μM, 0.01 μM, 0.1 μM, 1 μM, or 10 μM. After exposure, the cells were statically cultured in a 37 ° C./5% CO ₂ environment for 5 days.

  The cultured cells were washed with PBS (Phosphate Buffered Saline) and fixed with a 4% paraformaldehyde-PBS solution. A goat anti-human CD90 antibody (manufactured by Santa Cruz), a goat anti-human AQP-5 antibody (manufactured by Santa Cruz), and a goat anti-human SP-A antibody (manufactured by Santa Cruz) are added to the fixed cells and allowed to react. Anti-goat antibody (Santa Cruz) was added and allowed to react. And the number of positive cells was measured using the fluorescence microscope (BZ-9000, the Keyence company make), and the ratio of the positive cell was computed about each marker. The results are shown in Table 2.

  As is apparent from Table 2, when a human alveolar epithelial stem cell was contacted with a PI3K inhibitor, the proportion of CD90 positive cells decreased in a concentration-dependent manner, and the proportion of AQP-5 positive cells and the proportion of SP-A positive cells. increased. This result indicates that the PI3K inhibitor has a differentiation-inducing action on human alveolar epithelial stem cells.

  When Example A1 was compared with Reference Example 1, the PI3K inhibitor showed a strong differentiation-inducing action on alveolar epithelial stem cells at a lower concentration than ATRA. For example, when the concentrations of 0.1 μM are compared, the PI3K inhibitor has a differentiation-inducing action on type I alveolar epithelial cells that is 3 times or more of ATRA, and has a differentiation-inducing action on type II alveolar epithelial cells that is twice or more. showed that.

[Example A2: Verification of effect of PI3K inhibitor on COPD animal model]
Using a COPD model mouse (elastase-induced emphysema mouse), an experiment was conducted to confirm the effect of Wortmannin, a PI3K inhibitor.

  A 6-week-old ICR mouse (male) was subjected to pulmonary administration of elastase (7.5 U, manufactured by Wako) suspended in 50 μL of PBS twice a week with a sonde for one week to prepare an emphysema model. Subsequently, wortmannin was transpulmonary administered twice a week for 3 weeks at a dose of 10 μg / kg each time with a sonde. Twenty-one days after the first administration of wortmannin, both lungs were removed, tissues were fixed with paraformaldehyde, sections were prepared with cryostat, and hematoxylin / eosin staining (HE staining) was performed.

  From a HE-stained slide of a cross section (total 6 cross sections) of 3 fields (left lung and right lung, upper lung field, middle lung field and lower lung field) of both lungs of one mouse, 5 fields of each section were observed with a microscope ( The images were taken with Keyence Biozero and analyzed using image analysis software (ImageJ (National Institutes of Health, USA) and Photoshop (Adobe)). The degree of emphysematous lesions was evaluated by measuring the mean alveolar wall distance (mean linear intercept, Lm) according to the Thulbeck method, which is a method known to those skilled in the art. The method draws a number of random lines, places them on the alveolar image, finds the sum of the lengths of the sections separated by the alveolar walls of each random line, and divides this by the total number of broken lines. It is required by doing. The number of divisions by the alveolar septum per visual field is set to 200 or more, this is measured in 30 visual fields (6 cross sections x 5 visual fields) for each individual, the average is calculated, and the average distance between the alveolar walls of each mouse is obtained, The average of each group (6 individuals) was calculated, and statistical analysis was performed by t-test. The results are shown in Table 3 and FIG.

From the observation of the HE-stained slide, it was confirmed that the alveolar space was remarkably expanded in the control group (without administration of wortmannin) and experimental pathological COPD lesions (remarkable emphysema) were formed by elastase.
Compared to this control group, the mean alveolar wall distance was significantly lower in the wortmannin administration group. The low average alveolar wall distance indicates that alveoli were regenerated, and it was found that wortmannin was regenerated and that wortmannin was effective in treating lung tissue damage. .

[B] Pharmaceutical composition containing a compound that acts on vitamin D receptor Human alveolar epithelial stem cells used in Examples B1 and B2 are patients with lung disease (extracted tissue at the time of biopsy or surgery in lung cancer or COPD) Derived from human alveolar epithelial stem cells (Fujino N, at. Al. Am. J. Respir. Cell Mol. Biol., 46, 422-430 (2012)). The human alveolar epithelial stem cells are obtained by sterilizing the culture supernatant of mouse fetal fibroblasts cultured in DMEM medium containing 10% bovine serum (FBS) and 1% 50-fold diluted MEM (Amino Acids Solution). Using the prepared culture solution, it was cultured and maintained in a 37 ° C./5% CO ₂ environment.

[Example B1: Verification of differentiation-inducing action of 1,25-dihydroxyvitamin D ₃ on human alveolar epithelial stem cells (1)]
1,25-dihydroxyvitamin D ₃ is exposed to human alveolar epithelial stem cells (1 × 10 ^1-4 cells / cm ² ) in the culture medium at a concentration of 0 μM, 0.01 μM, 0.1 μM, 1 μM or 10 μM. Then, static culture was performed in a 37 ° C./5% CO ₂ environment for 5 days.

  The cultured cells were washed with PBS (Phosphate Buffered Saline) and fixed with a 4% paraformaldehyde-PBS solution. A goat anti-human CD90 antibody (manufactured by Santa Cruz), a goat anti-human AQP-5 antibody (manufactured by Santa Cruz), and a goat anti-human SP-A antibody (manufactured by Santa Cruz) are added to the fixed cells and allowed to react. Anti-goat antibody (Santa Cruz) was added and allowed to react. And the number of positive cells was measured using the fluorescence microscope (BZ-9000, the Keyence company make), and the ratio of the positive cell was computed about each marker. The results are shown in Table 4.

As is clear in Table 4, is brought into contact with 1,25-dihydroxyvitamin _{D 3} in the human alveolar epithelial stem cells in a concentration-dependent manner, the proportion of CD90 positive cells is reduced, and the proportion of AQP-5 positive cells SP- The proportion of A positive cells increased. This result shows that 1,25-dihydroxyvitamin D ₃ has a differentiation-inducing action on human alveolar epithelial stem cells.

Example B1, is compared with the reference example 1, 1,25-dihydroxyvitamin D ₃ is at a lower concentration than ATRA, it showed strong differentiation-inducing effect on alveolar epithelial stem cells. For example, when comparing concentrations of 0.1 μM, 1,25-dihydroxyvitamin D ₃ exhibits an action of inducing differentiation into type I alveolar epithelial cells that is 7 times or more that of ATRA, and 3 times or more type II alveolar epithelial cells. Showed differentiation-inducing action.

[Example B2: Verification of differentiation-inducing action of 1,25-dihydroxyvitamin D ₃ on human alveolar epithelial stem cells (2)]
An experiment similar to Example B1 was performed except that the concentration of 1,25-dihydroxyvitamin D ₃ was 10 μM and the culture days were 0 days, 1 day, 2 days, 4 days or 6 days. The results are shown in Table 5.
In the experimental example of the cultivation period day 0, 1,25 and washed with dihydroxyvitamin _{D 3} exposure immediately after PBS, the cells were fixed with 4% paraformaldehyde -PBS solution.

As apparent in Table 5, is brought into contact with 1,25-dihydroxyvitamin _{D 3} in the human alveolar epithelial stem cells, contact time dependent manner, decreased the percentage of CD90 positive cells, the proportion of AQP-5 positive cells with SP -Increased proportion of A positive cells. This result shows that 1,25-dihydroxyvitamin D ₃ has a differentiation-inducing action on human alveolar epithelial stem cells.

Example B3: 1,25- Verification of effect on COPD animal model dihydroxyvitamin _{D 3]}
With the COPD model mice (elastase-induced emphysema mice), an experiment was conducted to confirm the effect of 1,25-dihydroxyvitamin _{D 3.}

A 6-week-old ICR mouse (male) was subjected to pulmonary administration of elastase (7.5 U, manufactured by Wako) suspended in 50 μL of PBS twice a week with a sonde for one week to prepare an emphysema model. Subsequently, 1,25-dihydroxyvitamin _{D 3} twice a week for three weeks, at a dose of each time 10 [mu] g / kg, and transpulmonary administration at sonde. Twenty-one days after the first administration of 1,25-dihydroxyvitamin D ₃ , both lungs were removed, tissues were fixed with paraformaldehyde, sections were prepared with cryostat, and hematoxylin / eosin staining (HE staining) was performed.

  From a HE-stained slide of a cross section (total 6 cross sections) of 3 fields (left lung and right lung, upper lung field, middle lung field and lower lung field) of both lungs of one mouse, 5 fields of each section were observed with a microscope ( The images were taken with Keyence Biozero and analyzed using image analysis software (ImageJ (National Institutes of Health, USA) and Photoshop (Adobe)). The degree of emphysematous lesions was evaluated by measuring the mean alveolar wall distance (mean linear intercept, Lm) according to the Thulbeck method, which is a method known to those skilled in the art. The method draws a number of random lines, places them on the alveolar image, finds the sum of the lengths of the sections separated by the alveolar walls of each random line, and divides this by the total number of broken lines. It is required by doing. The number of divisions by the alveolar septum per visual field is set to 200 or more, this is measured in 30 visual fields (6 cross sections x 5 visual fields) for each individual, the average is calculated, and the average distance between the alveolar walls of each mouse is obtained, The average of each group (6 individuals) was calculated, and statistical analysis was performed by t-test. The results are shown in Table 6 and FIG.

From the observation of the HE-stained slide, the control group (without administration of 1,25-dihydroxyvitamin D ₃ ) has a markedly expanded alveolar space and experimental pathological COPD lesions (significant emphysema) due to elastase. It was confirmed.
Compared to the control group, 1,25-dihydroxyvitamin D ₃ administration group, the distance between the average alveolar wall were significantly lower. Low distances between the average alveolar wall shows that the alveoli is played, the alveoli is reproduced by the administration 1,25-dihydroxyvitamin D _3, the 1,25-dihydroxyvitamin D ₃ It has been found to be effective in treating lung tissue damage.

[C] Lyophilized composition The amino acids used in Examples C1 to C4 are L-type other than glycine.

[Example C1: Preparation of lyophilized composition containing ATRA (1)]
[Preparation of lyophilized composition]
4 mg of ATRA was dissolved in 1 mL of ethanol, and 20 μL of Tween 20 was added as a surfactant to prepare an ATRA solution.
Separately, amino acid aqueous solutions of Phe, Leu, Ile and Val were prepared. The amino acid concentration of the aqueous amino acid solution was 6 mg / mL for Phe, and 2.4 mg / mL for Leu, Ile, and Val.
Then, an aqueous amino acid solution was added to the ATRA solution so that the final amount of amino acid was as shown in Table 7, and the amount of the aqueous solution was adjusted with water to prepare a dispersion in which ATRA was dispersed.
The final concentration of ethanol in each dispersion was 5% by weight. Table 7 shows the final amounts of the active ingredients and amino acids of each dispersion and the final concentration of the surfactant. The liquid volume of 1 vial dispersion is 0.5 mL.

  0.5 mL of this dispersion is put into a glass container (vial) having a barrel diameter of 18 mm and a volume of 2 mL, and is rapidly frozen under acetone-dry ice, and then a shelf-like freeze dryer (Freezone Triad, manufactured by LABCONCO) is used. And lyophilized to obtain a lyophilized composition.

[Evaluation of appearance of freeze-dried composition]
The appearance of the lyophilized composition was evaluated according to the following evaluation criteria. The results are shown in Table 7. As the lyophilized composition, 5 and 4 are preferable.
5: The shape is a lyophilized composition, and there is no bias or crack in its appearance.
4: The shape is a lyophilized composition, and its appearance has minor cracks.
3: The shape is a lyophilized composition, and the appearance is uneven and / or cracked.
2: The shape does not form a lyophilized composition.
1: The freeze-dried composition is not completed or is extremely small.

[Evaluation of particle formation of freeze-dried composition]
A part of the freeze-dried composition is taken out and placed in a sample chamber of a laser diffraction particle size distribution analyzer (LDSA-3500A, manufactured by Tohnichi Computer Applications), and compressed air (speed 60 m / sec, flow rate 750 ml / sec) The lyophilized product was sent to the sample chamber and scattered, and the particle size distribution in air (volume basis) was measured. From the particle size distribution, a particle ratio (%) of 5 μm or less and a particle ratio (%) of 10 μm or less were calculated. The results are shown in Table 7.

  As is apparent from Table 7, the lyophilized compositions of Examples C1-1 to C1-3 were granulated by an air flow to become a powdered drug in which particles having a particle diameter of 5 μm or less accounted for 25% or more. This result shows that a drug suitable for pulmonary administration is prepared from the lyophilized composition of the present invention.

[Example C2: Preparation of lyophilized composition containing ATRA (2)]
[Example C2-1 and Comparative Example C2-1]
1 mg of ATRA was dissolved in 0.25 mL of ethanol, and 2.5 μL of Tween 80 was added as a surfactant to prepare an ATRA solution.
Separately, amino acid aqueous solutions of Phe and Leu were prepared. The amino acid concentration of the aqueous amino acid solution was 4 mg / mL for Phe and 16 mg / mL for Leu.
Then, an aqueous amino acid solution was added to the ATRA solution so that the final amount of amino acid was as shown in Table 8, and the amount of the aqueous solution was adjusted with water to prepare a dispersion in which ATRA was dispersed.

[Example C2-2]
1 mg of ATRA was dissolved in 0.25 mL of ethanol, and 5 μL of Tween 80 was added as a surfactant to prepare an ATRA solution.
Separately, amino acid aqueous solutions of Phe and Leu were prepared. The amino acid concentration of the aqueous amino acid solution was 4 mg / mL for Phe and 16 mg / mL for Leu.
Then, an aqueous amino acid solution was added to the ATRA solution so that the final amount of amino acid was as shown in Table 8, and the amount of the aqueous solution was adjusted with water to prepare a dispersion in which ATRA was dispersed.

[Comparative Example C2-2]
5 mg of ATRA was dissolved in 1.25 mL of ethanol, 25 μL of Tween 80 was added as a surfactant, and serially diluted with water to prepare an ATRA solution.
Separately, an amino acid aqueous solution of Phe (Phe concentration 4 mg / mL) was prepared.
Then, an aqueous amino acid solution was added to the ATRA solution so that the final amount of amino acid was as shown in Table 8, and the amount of the aqueous solution was adjusted with water to prepare a dispersion in which ATRA was dispersed.

[Example C2-3 and Comparative Example C2-3]
5 mg of ATRA was dissolved in 1.25 mL of ethanol, and 25 μL of Tween 80 was added as a surfactant to prepare an ATRA solution.
Separately, amino acid aqueous solutions of Phe and Gly were prepared. The amino acid concentration of the aqueous amino acid solution was 8 mg / mL for Phe and 8 mg / mL for Gly.
Then, an aqueous amino acid solution was added to the ATRA solution so that the final amount of amino acid was as shown in Table 8, and the amount of the aqueous solution was adjusted with water to prepare a dispersion in which ATRA was dispersed.

  The final concentration of ethanol in each dispersion was 5% by weight. Table 8 shows the final amounts of the active ingredients and amino acids of each dispersion and the final concentration of the surfactant. The liquid volume of 1 vial dispersion is 0.5 mL.

  A freeze-dried composition was obtained from this dispersion in the same manner as in Example C1, and the appearance and particle formation were evaluated. The results are shown in Table 8.

  As is apparent from Table 8, the lyophilized compositions of Examples C2-1 to C2-3 were granulated by an air flow to become a powdered drug in which particles having a particle diameter of 5 μm or less accounted for 15% or more. This result shows that a drug suitable for pulmonary administration is prepared from the lyophilized composition of the present invention.

[Example C3: Preparation of lyophilized composition containing ATRA (3)]
5 mg of ATRA was dissolved in 1.25 mL of ethanol, and 25 μL of Tween 80 was added as a surfactant to prepare an ATRA solution.
Separately, amino acid aqueous solutions of amino acids described in Table 9 were prepared. The amino acid concentration of the aqueous amino acid solution was 8 mg / mL.
Then, an aqueous amino acid solution was added to the ATRA solution so that the final amount of amino acid was as shown in Table 9, and the liquid volume was adjusted with water to prepare a dispersion in which ATRA was dispersed.
The final concentration of ethanol in each dispersion was 5% by weight. Table 9 shows the final amounts of the active ingredients and amino acids and the final concentration of the surfactant in each dispersion. The liquid volume of 1 vial dispersion is 0.5 mL.

  A freeze-dried composition was obtained from this dispersion in the same manner as in Example C1, and the appearance and particle formation were evaluated. The results are shown in Table 9.

  As is apparent from Table 9, the freeze-dried compositions of Examples C3-1 to C3-4 were granulated by an air flow to become a powdered drug in which particles having a particle diameter of 5 μm or less accounted for 10% or more. This result shows that a drug suitable for pulmonary administration is prepared from the lyophilized composition of the present invention.

Example C4: 1,25- Preparation of lyophilized compositions that comprise the dihydroxyvitamin _{D 3]}
0.1 mg of 1,25-dihydroxyvitamin D ₃ was dissolved in 0.25 mL of ethanol, and 5 μL of Tween 20 was added as a surfactant and dissolved.
Separately, amino acid aqueous solutions of Phe and Leu were prepared. The amino acid concentration of the aqueous amino acid solution was 4 mg / mL for Phe and 16 mg / mL for Leu.
Then, an aqueous amino acid solution is added to the 1,25-dihydroxyvitamin D ₃ solution so that the final amount of amino acid is as described in Table 10, and the amount is adjusted with water, and 1,25-dihydroxyvitamin D ₃ is added. A solution (5 mL) in which was dissolved was prepared.
The final concentration of ethanol in each dispersion was 5% by weight. Table 10 shows the final amounts of active ingredients and amino acids and the final concentration of the surfactant in each dispersion. The liquid volume of 1 vial dispersion is 0.5 mL.

  A freeze-dried composition was obtained from this dispersion in the same manner as in Example C1, and the appearance and particle formation were evaluated. The results are shown in Table 10.

  As is apparent from Table 10, the lyophilized composition of Example C4 was granulated by an air stream to become a powdered drug in which particles having a particle diameter of 5 μm or less accounted for 20% or more. This result shows that a drug suitable for pulmonary administration is prepared from the lyophilized composition of the present invention.

The disclosure of Japanese Patent Application No. 2012-280070 filed on December 21, 2012 is incorporated herein by reference in its entirety.
All documents, patent applications, and technical standards mentioned in this specification are to the same extent as if each individual document, patent application, and technical standard were specifically and individually stated to be incorporated by reference, Incorporated herein by reference.

Claims (17)

  A differentiation inducer that acts on alveolar epithelial stem cells, comprising a PI3 kinase inhibitor as an active ingredient.   A pharmaceutical composition for inducing alveolar regeneration, comprising a PI3 kinase inhibitor as an active ingredient.   A pharmaceutical composition for treating lung tissue damage, comprising a PI3 kinase inhibitor as an active ingredient.   A differentiation inducer that acts on alveolar epithelial stem cells, comprising a compound that acts on a vitamin D receptor as an active ingredient.   A pharmaceutical composition for inducing alveolar regeneration comprising a compound that acts on a vitamin D receptor as an active ingredient.   A pharmaceutical composition for treating lung tissue damage, comprising a compound that acts on a vitamin D receptor as an active ingredient. Containing a poorly water-soluble drug, at least two amino acids, and a surfactant,
A freeze-dried composition in which the proportion of particles having a particle size of 5 μm or less becomes 10% or more when formed into particles by an air stream. A freeze-dried composition of a composition comprising a poorly water-soluble drug, at least two amino acids, a surfactant, an alcohol, and water;
A freeze-dried composition in which the proportion of particles having a particle size of 5 μm or less becomes 10% or more when formed into particles by an air stream. A freeze-dried composition comprising a poorly water-soluble drug, at least two amino acids selected from the first group, the second group, and the third group described below, and a surfactant, and a surfactant.
・ Group 1: phenylalanine, tryptophan ・ Group 2: isoleucine, leucine, valine ・ Group 3: alanine, glycine A composition comprising a poorly water-soluble drug, at least two amino acids selected from the following first group, second group and third group, a plurality of groups, a surfactant, alcohol, and water Freeze-dried composition.
・ Group 1: phenylalanine, tryptophan ・ Group 2: isoleucine, leucine, valine ・ Group 3: alanine, glycine   The freeze-dried composition according to any one of claims 7 to 10, wherein the water-insoluble drug is a compound having a differentiation-inducing action on alveolar epithelial stem cells.   The compound having differentiation-inducing action on alveolar epithelial stem cells is at least one selected from vitamin A, vitamin A derivatives, provitamin A, compounds acting on vitamin D receptors, and PI3 kinase inhibitors. The lyophilized composition according to claim 11. A step of preparing a first liquid by dissolving a poorly water-soluble drug in a solvent containing alcohol;
Mixing the first liquid, at least two types of amino acids, a surfactant, and water to prepare a second liquid;
A method of producing a freeze-dried composition comprising the step of freeze-drying the second liquid. A step of preparing a first liquid by dissolving a poorly water-soluble drug in a solvent containing alcohol;
The first liquid, at least two kinds of amino acids selected from the following first group, second group and third group, a plurality of groups, a surfactant and water are mixed to form a second Preparing a liquid;
A method of producing a freeze-dried composition comprising the step of freeze-drying the second liquid.
・ Group 1: phenylalanine, tryptophan ・ Group 2: isoleucine, leucine, valine ・ Group 3: alanine, glycine   The method for producing a freeze-dried composition according to claim 13 or 14, wherein the water-insoluble drug is a compound having a differentiation-inducing action on alveolar epithelial stem cells.   The compound having differentiation-inducing action on alveolar epithelial stem cells is at least one selected from vitamin A, vitamin A derivatives, provitamin A, compounds acting on vitamin D receptors, and PI3 kinase inhibitors. The method for producing a lyophilized composition according to claim 15.   The pharmaceutical composition for transpulmonary administration which made the freeze-dried composition of any one of Claims 7-12 the particle | grains with the airflow.