WO2023229023A1 - Powdered agent and production method for same - Google Patents

Abstract

Provided is a powdered agent that forms lipid particles that give an active ingredient an excellent ability to reach the lungs when inhaled and also give the active ingredient a DDS function at the site of arrival. The powdered agent includes constituent particles that contain: a lipid component that contains a PEG-lipid conjugate; and leucine and/or dileucine.

Description

Powder and its manufacturing method

The present specification relates to a powder, a method for producing the same, and the like.
This application claims priority based on Japanese patent applications No. 2022-85620 and No. 2022-85625, which are Japanese patent applications filed on May 25, 2022. The content of the patent application is incorporated herein by reference.

Inhalers, which can deliver drugs directly and non-invasively to the lungs, are expected to have a rapid onset of action and have the advantage of reducing systemic side effects because they require a smaller dose than oral administration. Therefore, it is attracting attention as an administration route that can be expected not only to be a locally active drug but also to have a systemic effect with low absorption in the gastrointestinal tract (Patent Document 1).

Inhalants are classified into three types: metered-dose inhaler (MDI), inhalation solution, and dry powder inhaler (DPI). Among these, in DPI, the drug powder is generally broken down and dispersed into the air by the patient's inhalation effort and delivered to the respiratory treatment area, so synchronization of powder spray and inhalation is easy, no propellant is required, and the inhalation technique is simple. It has the advantage that it is simple and the inhalation device is relatively small and has excellent portability.

On the other hand, since DPI is a powder, it has to reach the lungs, so there are problems with the patient's inhalation ability and adhesion to the oral cavity, pharynx, etc. Therefore, a DPI formulation has been developed that allows constituent particles to easily and effectively reach the lungs (Patent Document 2).

Special Publication No. 2007-522246 International Publication No. 2020/071448 pamphlet

In order to further enhance the usability of DPI preparations, it is necessary to add drug delivery system (DDS) functions such as improving the retention and intracellular transfer of the active ingredient after it reaches the lungs.

The present specification provides a powder that has excellent delivery of active ingredients to the lungs during inhalation and forms lipid particles that impart DDS functionality to the active ingredients at the site of arrival.

The present inventors further investigated and evaluated various formulations for DPI formulations that achieve excellent pulmonary delivery, and found that in addition to the reachability and cellular penetration of active ingredients such as RNA interference agents, the retention We succeeded in discovering an ingredient that is effective in controlling these conditions. According to this specification, the following means are provided based on this knowledge.

[1] A powder agent,
a lipid component containing a polyethylene glycol (PEG)-lipid conjugate;
Leucine and/or dileucine;
A powder containing constituent particles containing.
[2] The powder agent according to [1], wherein the constituent particles produce a large number of lipid particles in an aqueous suspension of the particles.
[3] The lipid component further includes 1,2-dioleyloxy-3-trimethylammoniumpropane (DOTMA), 1,2-dioleyloxy-3-dimelaminopropane (DODMA), and 1,2-dioleyloxy-3-trimethylammoniumpropane (DOTMA). One or more selected from the group consisting of palmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), egg-derived phosphatidylcholine (EPC), and sphingomyelin (SM), or one or two selected from salts thereof. The powder agent according to [1] or [2], which is a type or more.
[4] The powder agent according to any one of [1] to [3], which contains the lipid component in an amount of 10% by mass or more and 60% by mass or less based on the total amount of the constituent particles.
[5] The powder agent according to any one of [1] to [4], wherein the constituent particles are porous spherical particles.
[6] The powder agent according to [5], wherein the porous spherical particles are three-dimensional porous bodies including partition walls and/or skeletons that define continuous pores.
[7] The powder agent according to any one of [1] to [6], wherein the constituent particles have an average particle diameter of 1 μm or more and 100 μm or less.
[8] The lipid particles have an average particle diameter of 50 nm or more and 300 nm or less, as measured by a dynamic light scattering method for a suspension obtained by suspending the particles in water or a buffer solution, [1] The powder agent according to any one of ~[7].
[9] The constituent particles have an OE (%) = recovery amount from the throat onwards (mg) / total recovery amount (mg) x 100 of 80% or more in a characteristic evaluation using a multi-stage liquid impinger (MSLI). The powder agent according to any one of [1] to [8].
[10] The powder according to any one of [1] to [9], which has an FPF (%) of 10% or more in characteristic evaluation by MSLI.
[11] The powder according to any one of [1] to [10], which contains an RNA interference agent as an active ingredient.
[12] The powder according to any one of [1] to [11], wherein the PEG-lipid conjugate is a PEG-phospholipid conjugate and/or a PEG-diacylglycerol conjugate.
[13] The powder according to any one of [1] to [11], wherein the PEG-lipid conjugate is a PEG-cholesterol conjugate.
[14] The powder preparation according to any one of [1] to [13], which contains only dileucine among leucine and dileucine.
[15] A method for producing a powder, comprising:
preparing a liquid containing an active ingredient, a lipid component containing a PEG-lipid conjugate, and leucine and/or dileucine;
Drying the liquid by a spray drying method or a spray freeze drying method to produce a powder containing the active ingredient, the PEG-lipid conjugate, and the leucine and/or dileucine;
Equipped with
The manufacturing method, wherein the powder is a powder containing constituent particles that form a large number of lipid particles in an aqueous suspension of the powder.

FIG. 2 is a diagram showing the formulation of the powder prepared in Example 1 (spray freeze-drying method) and the physical properties of lipid particles produced after dissolution. However, regarding FIGS. 1 and 2, LNP in the table is an abbreviation for lipid particle. In the table, * indicates the lipid component content (w/w) in the powder particles, and ** indicates the mixing ratio (v/v) of water and organic solvent in the component solution. SFD microparticles and SD microparticles with the same # number in the product name have the same composition. SFD#3, SFD#3', SFD#3'', SD#3, and SD#3' have the same composition, but differ in the concentration of component solutions or the mixing ratio of water and organic solvent. The background of results in which the physical properties of lipid particles exceed the standard values is shown in gray. In addition, in the figure, Leu represents leucine, and diLeu represents dileucine. FIG. 2 is a diagram showing the formulation of the powder prepared in Example 1 (spray drying method) and the physical properties of lipid particles produced after dissolution. FIG. 2 is a diagram showing an example of a SEM image of constituent particles of the powder preparation prepared in Example 1 and a constituent particle addition device. 3A and 3B are diagrams illustrating the evaluation results of the inhalation characteristics of the constituent particles of the powder preparation prepared in Example 1. FIG. FIG. 3 is a diagram showing the cell binding and uptake amount of lipid particles generated after dissolving constituent particles of a powder. FIG. 6 is a diagram showing the formulation of the powder prepared in Example 6 (spray freeze-drying method and spray drying method) and the physical properties of lipid particles generated after dissolution. FIG. 6 is a diagram showing the observation results of the shape of constituent particles of the powder preparation prepared in Example 6. FIG. 3 is a diagram showing evaluation results of gene silencing effect and cytotoxicity of lipid particles loaded with siRNA (RNA interference agent). FIG. 3 is a diagram showing the evaluation results of cell binding/uptake ability of siRNA-loaded lipid particles. FIG. 6 is a diagram showing the inhalation characteristics of the powder prepared in Example 6. FIG. 7 is a diagram showing the evaluation results of the intrapulmonary distribution of siRNA after administration of the powder prepared in Example 6 into the lungs of mice.

The disclosure of this specification relates to a powder agent (hereinafter also simply referred to as the agent) and a method for producing the same. This agent is generally intended for medicinal use. The constituent particles of this drug contain a predetermined lipid component and leucine and/or dileucine, so they have excellent ability to reach the lungs, and dissolve (wet) at the site where they reach, for example, a large number of nano-level particles. Generate lipid particles. Therefore, this drug can cause lipid particles to reach the target site in the lungs or to remain there.

Additionally, this agent can effectively incorporate lipid particles and/or active ingredients into cells by lipid particles generated in the presence of water in vivo. Additionally, the lipid particles can retain the lipid particles and/or the active ingredient at the target site.

Furthermore, according to the present agent containing an RNA interference agent, by adding a PEG-lipid conjugate, RNA interference agent-loaded lipid particles with a small and uniform particle size can be formed. Furthermore, by employing a PEG-cholesterol conjugate as the PEG-lipid conjugate, RNA interference agent-loaded lipid particles with excellent gene silencing effects and cell binding/uptake abilities can be formed. Furthermore, according to this drug containing an RNA interference agent, when inhaled into the lungs, it dissolves and deposits in the lungs to form lipid particles loaded with the RNA interference agent, improving the stability and retention of the RNA interference agent. do. Furthermore, according to the present agent containing an RNA interference agent and dileucine, even if the contents of the RNA interference agent and lipid components are high, RNA interference agent-loaded lipid particles with a smaller particle size and uniformity can be formed. Furthermore, by employing the SFD method, it is possible to obtain a powder that has excellent lung delivery properties even with a high content of RNA interference agent/lipid components.

From the above, the particles included in this drug can potentially contain lipid particles, and the lipid particles can be used to achieve an effective DDS function by inhaling air into the lungs. Moreover, when this agent is introduced into a living body, the reachability, intracellular transfer, and retention of the active ingredient can be controlled by lipid particles generated in the presence of water.

Furthermore, according to the method for manufacturing a powder disclosed in this specification, an inhalable powder that has excellent ability to reach the lungs and has particles that dissolve at the site of arrival to produce a large number of lipid particles can be easily produced. can do.

(powder)
The powder agent disclosed herein contains particles (constituent particles). The constituent particles can take the form of a powder or the like as a whole. This agent can contain an active ingredient in some of these constituent particles. The constituent components, various properties, etc. of this agent will be explained below, and then the manufacturing method of this agent will be explained.

(Composition of this drug)
The agent is suspended in an aqueous medium such as water or a buffer (eg, a biocompatible phosphate buffered saline, a HEPES buffer (pH 7.4), etc.) to produce lipid particles. From this point of view, the present agent can contain, for example, a lipid component capable of functioning as a lipid particle called a liposome or the like, and a pharmaceutically acceptable excipient.

(lipid component)
As the lipid component contained in this agent, one or more types selected from cationic lipids and non-cationic lipids may be used.

(cationic lipid)
Cationic lipids are advantageous in that they can facilitate uptake of lipid particles into cells. As the cationic lipid, known cationic lipids can be used, such as 1,2-dioleyloxy-3-trimethylammoniumpropane (DOTMA), 1,2-dioleyloxy-3-dimelamino Propane (DODMA), 1,2-dioleyl-3-trimethylammoniumpropane (DOTAP), 1,2-dioleyl-3-dimethylammoniumpropane (DODAP), 3β-(N-(N',N'-dimethylaminoethane) ) carbamoyl) cholesterol (DC-Chol), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl -N-Hydroxyethylammonium bromide (DMRI), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), diheptadecylamidoglycylspermidine (DOGS), N-(1-(2,3-diolyl) and combinations thereof. One type or a combination of two or more types of these can be used. When these cationic lipids are combined with a PEG-lipid conjugate and are formed into lipid particles in vivo, they may be able to quickly contribute to the cellular uptake of the lipid particles and/or the active ingredient within the lipid particles. be. Among these, for example, either or both of DOTMA and DODMA can be used. In some cases, it may be preferable to use DOTMA alone. Note that the above cationic lipids and other cationic lipids may be salts with acid groups such as chlorine ions. In this agent, for example, a cationic lipid can be used as a predominant lipid component among all lipid components.

The content of the cationic lipid is not particularly limited, but may be, for example, 30% by mass or more and 70% by mass or less based on the total amount of lipid components. Within this range, the above effects of cationic lipids are effective. The cationic lipid is, for example, 40% by mass or more and 70% by mass or less, for example, 40% by mass or more and 65% by mass or less, and for example, 40% by mass or more and 60% by mass or less. In addition, the content of cationic lipid is, for example, 3% by mass or more and 30% by mass or less, for example, 4% by mass or more and 25% by mass or less, and for example, 4% by mass or less, based on the total amount of the drug. % or more and 20% by mass or less.

(Non-cationic lipid)
As the non-cationic lipid, known non-cationic lipids such as zwitterionic lipids can be used, such as 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), egg-derived phosphatidylcholine, etc. (EPC), phosphatidyl cholinelipids such as 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), etc. Examples include sphingolipids such as ethanolamine lipids and sphingomyelin (SM). One type or a combination of two or more types of these can be used. When combined with a PEG-lipid conjugate, these non-cationic lipids have a DDS function that improves the sustained release or retention of active ingredients when they are formed into lipid particles in vivo, such as on airway epithelium. You may be able to contribute. In this agent, for example, non-cationic lipids may be used as the predominant lipid component among the total lipid components.

The content of the non-cationic lipid is not particularly limited, but can be, for example, 30% by mass or more and 70% by mass or less based on the total amount of lipid components. Within this range, the above effects of cationic lipids are effective. The non-cationic lipid is, for example, 40% by mass or more and 70% by mass or less, for example, 40% by mass or more and 65% by mass or less, and for example, 40% by mass or more and 60% by mass or less. In addition, the content of non-cationic lipids is, for example, 3% by mass or more and 30% by mass or less, for example, 4% by mass or more and 25% by mass or less, and for example, It can be set to 20% by mass or more and 20% by mass or less.

(PEG-lipid conjugate)
This agent can use known PEG-lipid conjugates as the complex lipid, but for example, in addition to various PEG-cholesterol conjugates, PEG-phospholipid conjugates such as PEG-DSPE (especially non-cationic PEG-diacylglycerol conjugates such as PEG-DMG (dimyristoylglycerol) can be used.

Among PEG-lipid conjugates, PEG-cholesterol conjugates include those in which PEG and lipid are directly bonded. The molecular weight of PEG is also not particularly limited, but can be appropriately selected within the range of, for example, 500 to 10,000. Also, for example, it may be advantageous to use a conjugate of PEG with a number average molecular weight of 1500 to 5500, and for example a conjugate of PEG with a number average molecular weight of 2000 to 5000 can be used. For example, a PEG conjugate with a number average molecular weight of 1,500 to 2,500 may be effective for the intracellular transfer of lipid particles and/or active ingredients. Furthermore, for example, even a PEG conjugate having a number average molecular weight of 4,000 to 6,000 may be effective for the intracellular transfer of lipid particles and/or active ingredients.

PEG-lipid conjugates such as PEG-DSPE and PEG-DMG may have, for example, a glycerol skeleton. It can contribute to the retention effect of the active ingredient.

The content of such a PEG-lipid conjugate is not particularly limited, but may be, for example, 10% by mass or more and 50% by mass or less based on the total amount of lipid components. This is because within this range, various conjugates are effective in exerting their respective effects. Further, for example, the content is 15% by mass or more and 45% by mass or less, and for example, 20% by mass or more and 40% by mass or less. In addition, the content of the PEG-lipid conjugate is, for example, 2% by mass or more and 20% by mass or less, and, for example, 2.5% by mass or more and 15% by mass or less, based on the total amount of the drug. For example, it is 3% by mass or more and 13% by mass or less.

(Other lipid components)
As other lipid components, various known lipids that contribute to the formation of lipid particles such as liposomes can be used. For example, cholesterol, dioleoylphosphatidylethanolamine (DOPE), etc. can be used. These other lipid components can contribute to in-vivo stability and endosomal escape of lipid particles and the like.

The content of other lipids is not particularly limited, but may be, for example, 5% by mass or more and 30% by mass or less based on the total amount of lipid components. This is because within this range, it can contribute to the above-mentioned effects. Further, for example, the content is 10% by mass or more and 30% by mass or less, and for example, 15% by mass or more and 25% by mass or less. In addition, the content of other lipids is, for example, 1% by mass or more and 10% by mass or less, and for example, 1% by mass or more and 5% by mass, based on the total amount of this drug. % or less, and for example, from 1% by mass to 3% by mass.

The total amount of lipid components in this drug is not particularly limited, but is, for example, 5% by mass or more and 60% by mass or less, and, for example, 7% by mass or more and 50% by mass, based on the total amount of this drug. For example, it is 8% by mass or more and 45% by mass or less.

(Excipient)
The agent may further contain an excipient. Although the excipient is not particularly limited, for example, leucine and dileucine, which is a dipeptide, can be used. By using leucine or dileucine, even if the amount of the lipid component is increased, it is possible to suppress the increase in the average particle diameter of the lipid particles when the spherical particles are dissolved in water, and maintain the range of, for example, 50 nm to 250 nm. , PdI can produce lipid particles with a uniform particle size and a low value. Furthermore, by using leucine and/or dileucine, it may be possible to enhance the cell binding/uptake properties of lipid particles.

Note that L-leucine and/or D-leucine can be used as leucine and dileucine. For example, L-leucine and L-leucine dipeptides can be used.

The content of leucine or dileucine is not particularly limited, but is, for example, 5% by mass or more and 95% by mass or less, and, for example, 10% by mass or more and 90% by mass or less, based on the total amount of the drug. , or 10% by mass or more and 80% by mass or less.

This drug can contain active ingredients. The content of the active ingredient is not particularly limited, but may be, for example, about 0.1% by mass or more and 5% by mass or less based on the total amount of the agent.

The active ingredient is not particularly limited as long as it can be used in the spray drying method and spray freeze drying method described below, and is generally an organic compound.

The active ingredient may be, for example, an active ingredient for treatment or prevention of diseases related to the lungs, or an active ingredient for treatment or prevention that is intended to be administered systemically through the bloodstream through the lungs. It may be. This drug reaches the lungs and dissolves on the spot to generate lipid particles, which can be taken into cells or the bloodstream via the lipid particles. Therefore, any active ingredient that can be encapsulated by lipid particles can be used. I can do it.

An example of an active ingredient is a nucleic acid. Nucleic acids include natural nucleic acids that are polymers of naturally occurring deoxyribonucleotides and/or ribonucleotides and non-natural nucleic acids that are polymers that include deoxyribonucleotides and/or ribonucleotides that have at least a non-natural structure. I can do it. Natural deoxyribonucleotides and ribonucleotides contain natural bases. Natural bases are those found in natural DNA and RNA and include adenine, thymine, guanine, cytosine and uracil. In addition, in natural deoxyribonucleotides and/or ribonucleotides, the phosphoric acid at the 5-position of the 2-deoxyribose and/or ribose and the 3' hydroxyl group of the adjacent deoxyribose and/or ribose form a phosphate diester bond. It has a skeleton connected by. In this specification, the natural nucleic acid may be DNA, RNA, or a chimera of deoxyribonucleotides and ribonucleotides (hereinafter also referred to as a DNA/RNA chimera). Moreover, DNA and RNA may each be single-stranded, double-stranded of the same type, or a hybrid of DNA and RNA hybridized. Furthermore, the DNA/RNA chimera may be a hybrid hybridized with DNA, RNA, or a DNA/RNA chimera.

A non-natural nucleic acid refers to a nucleic acid that has a non-natural structure in at least a portion of either the base or the backbone (sugar moiety and phosphate moiety). Various non-natural bases are known as non-natural bases. In addition, various skeletons that can replace the natural ribose phosphate skeleton are also provided. Examples include glycol nucleic acids, peptide nucleic acids, and the like having about 3 carbon atoms instead of a sugar-ribose skeleton. Furthermore, natural nucleic acids are L-DNA or L-RNA, but non-natural nucleic acids include nucleic acids that have at least a portion of the structure of D-DNA and D-RNA. Non-natural nucleic acids also include various forms such as single-stranded, double-stranded, hybrid, and chimeric.

This type of non-natural nucleic acid is generally not a coding strand that encodes a protein or a template strand, but has other functions, such as interacting with a certain type of nucleic acid within a cell to change the function of that nucleic acid. It is used for such things. Typically, it is used to express a function of inhibiting the expression or function of a target protein. Examples include nucleic acids that act directly on in vivo nucleic acids without mediating gene expression, and specific examples include antisense nucleic acids, sense nucleic acids, shRNAs, siRNAs, decoy nucleic acids, aptamers, miRNAs, and the like. This type of non-natural nucleic acid is often an oligonucleotide in which about ten to several dozen nucleotides are polymerized.

As the nucleic acid, for example, when this agent is intended for gene expression, a nucleic acid construct (non-viral vector) using a plasmid can be mentioned. Furthermore, for example, when suppressing gene expression is intended, non-viral vectors such as plasmid DNA encoding shRNA can be used.

The form of the nucleic acid is not particularly limited, and may be linear, circular (closed or open ring), or supercoiled. It can be provided with a form depending on the purpose.

(Particle shape of constituent particles)
The shape of the constituent particles contained in this agent can be observed with a scanning electron microscope (SEM). For observation, for example, after spraying onto a sample stage using a component particle addition device used for dispersing and adding the present agent, platinum coating is applied as necessary to make it suitable for SEM observation, and observation is performed. As the component particle addition device and the spraying method, for example, those used in the examples described later can be employed.

The shape of the constituent particles is not particularly limited, but for example, in consideration of dispersibility, it may be preferable to have a spherical shape. Further, in consideration of dispersibility, swelling property, etc., it may be preferable to be porous.

The constituent particles are, for example, porous spherical particles. Porous spherical particles have, for example, a large number of continuous pores (hollow parts) generated by sublimation of water, and adjacent pores have partition walls and/or mesh-like pores made of components such as lipid components and excipients. It has a three-dimensional structure divided by a skeleton. Such partition walls or skeletons are observed, for example, in the form of pleats or networks on the surface of spherical particles in the SEM. Such constituent particles may be produced, for example, by a spray freeze-drying method when producing the present drug.

Furthermore, the constituent particles can also take the form of spherical particles with generally smooth surfaces. The spherical particles in this case may or may not be porous. Such constituent particles may be produced, for example, by a spray drying method using dileucine when producing the present agent.

Further, the constituent particles can also take the form of wrinkled particles whose surfaces are rich in irregularities. Such constituent particles may be produced, for example, by a spray freeze-drying method when producing the present drug. For example, it may be produced using leucine instead of dileucine.

(Particle diameter of constituent particles)
The average particle diameter of the constituent particles contained in this drug can be determined by, for example, using a scanning electron microscope, setting an observation field that contains 10 to 20 constituent particles, observing their diameters across the field, and determining the average value. I can do it. The average particle diameter of the constituent particles of the present agent is not particularly limited, but in consideration of dispersibility, it can be, for example, 1 μm or more and 100 μm or less, or 1 μm or more and 50 μm or less, or 1 μm or more and 40 μm or less. Further, for example, according to the spray freeze-drying method described below, the thickness can be, for example, 5 μm or more and 40 μm or less, 10 μm or more and 30 μm or less, etc. Further, for example, according to the spray drying method described below, the thickness can be set to, for example, 1 μm or more and 10 μm or less.

(Inhalation characteristics)
This drug is delivered to the respiratory tract by inhalation (gas flow during suction from the oral cavity to the bronchi), and its characteristics in that case (inhalation characteristics), In other words, the dispersibility and delivery properties of this drug can be evaluated. Although dispersibility and deliverability are independent properties, they are interrelated.

(Evaluation method using multi-stage liquid impinger (MSLI) method)
In this specification, the MSLI method refers to the method described in 17th Edition Japanese Pharmacopoeia 1st Supplement General Test Methods 6.15 Aerodynamic Particle Size Measurement Method for Inhalants 5.1 Multi-Stage Liquid Impinger Method (Apparatus 1). Use measuring equipment. A pre-separator can be used as appropriate.

The outline of the measuring device and the measuring method can be based on the above general test method. The evaluation of this drug by the MSLI method can be carried out in accordance with the procedure for measuring inhalation powder in 5.1.2 of the above general test method 5.1 Multi-stage liquid impinger method (apparatus 1).

(Dispersibility and delivery characteristics)
OE (Output Efficiency: %), which is the emission rate from the device, FPF (%) (collection rate of particles equivalent to 5 μm or less), and UPF (%) (Ultrafine Particle Fraction) (collection rate of particles equivalent to 2 μm or less) ) is the above general test method 6.15 Aerodynamic particle size determination method for inhalants 6. Based on calculations, each is calculated using the following formulas (1) to (3).
OE (%) = Recovery amount T/Total recovery amount x 100 (1)
(However, the collected amount T is the collected amount from the throat onward.)
FPF (%) = Collection amount of particles corresponding to 5 μm or less */Total collection x 100 (2)
UPF (%) = Collection amount of particles equivalent to 2 μm or less ** / Total collection amount x 100 (3)

Note that * and ** refer to the above general test method 6.15 Aerodynamic particle size measurement method for inhalants 6. Calculation Table 6.15-6 Applying to the calculation in device 1, calculate the amount of constituent particles by interpolation.

OE is an index of dispersibility, FPF is an index of intrapulmonary delivery, and UPF is an index value of deep lung delivery.

This drug can have, for example, an OE of 80% or more in the inhalation characteristics evaluation using the MSLI method. This is because when it is 80% or more, it can be said that the release rate is good. The OE is, for example, 85% or more, 90% or more, or 95% or more.

In addition, this drug can have an FPF of 10% or more, for example, 15% or more, or 20% or more, or 25% or more, in the inhalation characteristics evaluation using the MSLI method. , for example, 30% or more, or, for example, 40% or more. For example, it can be said that the larger the value, the better the lung delivery rate. Note that, depending on the active ingredients and uses of the inhalable powder, an FPF of 10% or more may be sufficient.

In addition, this drug has a UPF of, for example, 10% or more, 15% or more, 20% or more, 25% or more, or 30% or more in the inhalation property evaluation using the MSLI method. % or more. This is because it can be said that the larger the value, the better the deep lung delivery rate. Note that depending on the active ingredients and uses of the inhalable powder, a UPF of 10% or less, for example, 5% or less, or, for example, 1% or less may be sufficient.

(Ability to form lipid particles)
Also, when this agent is suspended in water, the water-insoluble lipid component can form lipid particles that are finer than the constituent particles contained in the agent. Specifically, for example, when measuring the particle size distribution of a suspension of the present agent (powder) by dynamic light scattering, an average particle size of 400 nm or less can be obtained. Further, a polydispersity index (PdI) of 0.420 or less can be obtained. The particle size distribution of lipid particles determined by dynamic light scattering was determined using Zetasizer Nano manufactured by Malvern after stirring and standing for 30 minutes at a suspension concentration of 0.2 mg/mL as the concentration of lipid components in the powder. It can be obtained by measuring using ZS. Note that the average particle size and particle size distribution by the dynamic light scattering method can be obtained based on the diffusion coefficient using an autocorrelation function obtained by software attached to this device.

The average particle diameter is, for example, 50 nm or more, 60 nm or more, 70 nm or more, 80 nm or more, and 90 nm or more. The average particle size is also, for example, 300 nm or less, such as 280 nm or less, such as 260 nm or less, such as 240 nm or less, such as 220 nm or less, and such as 200 nm or less. For example, it is 180 nm or less, for example, 170 nm or less, for example, 160 nm or less, and for example, 140 nm or less. The range of the average particle diameter can be appropriately selected and set from the above lower and upper limits, and is, for example, 60 nm or more and 280 nm or less, and also, for example, 60 nm or more and 260 nm or less, and for example, 60 nm or more and 240 nm or less. etc.

PdI is also, for example, 0.400 or less, for example, 0.390 or less, for example, 0.380 or less, and for example, 0.370 or less.

(Manufacturing method of this drug)
This agent can be manufactured by a spray drying method or a spray freeze drying method. By employing such a manufacturing method, it is possible to easily obtain this drug which has excellent lung reach and contains particles that form lipid particles when dissolved in water or when wet. From the viewpoints of formation of spherical porous particles, cell membrane binding/intracellular uptake, and high content of lipid components, it may be advantageous to produce this agent by spray freeze-drying.

The manufacturing method of this drug includes, for example, a step of preparing a liquid containing an active ingredient, a lipid component containing a PEG-lipid conjugate, and leucine and/or dileucine, and spray-drying or spray-freezing this liquid. The method may include a step of producing a powder containing an active ingredient, a component containing a PEG-lipid conjugate, and leucine and/or dileucine by drying by a drying method. Here, when a powder is obtained by a spray drying method or a spray freeze drying method, the powder is a powder containing constituent particles that form a large number of lipid particles in an aqueous suspension of the powder.

In the liquid preparation step, the liquid to be subjected to the spray drying method or the spray freeze drying method contains an active ingredient, a lipid component containing a PEG-lipid conjugate, and leucine and/or dileucine, and these are uniformly dispersed or It is sufficient if it is dissolved. For example, the lipid component may be finely and uniformly dispersed even if it is not dissolved.

In the liquid preparation step, for example, a lipid component is mixed with a liquid dissolved in a solvent that dissolves the lipid component and a liquid dissolved in a solvent that dissolves an excipient such as leucine and/or dileucine, and then sprayed. It may also include making it into a liquid for use. In this way, a homogeneous liquid for spraying can be prepared.

The solvent for dissolving the lipid component is preferably an organic solvent that is miscible with water. This is because when leucine and/or dileucine is dissolved in water, a uniform mixture with the leucine and/or dileucine solution can be prepared. Examples of such organic solvents include alcohols such as methanol, ethanol, n-propanol, 2-propanol, and tert-butyl alcohol, and acetonitrile.

As the solvent for dissolving dileucine, it may be preferable to use water or a mixture of water and an organic solvent that is miscible with water. Such an organic solvent has the same meaning as an organic solvent in a solvent for dissolving a lipid component.

Depending on its solubility, the active ingredient can be dissolved in a solution of either the lipid component and leucine and/or dileucine. In addition, independently of these solutions, they can be dissolved in the same or other solvents that are miscible with the solvents used for these solutions, and mixed with solutions of other components.

The mixing ratio of water and organic solvent in the final spraying liquid is, for example, 9:1 to 1:9, or 9:1 to 3:7, or 9:1 to 3:7. 4:6, or, for example, can be appropriately set in the range of 9:1 to 5:5.

The compositions of the lipid components and leucine and/or dileucine in each solution are not particularly limited, and may be within the composition ranges described above. The concentrations of these components are also adjusted to a final concentration suitable for use in spray drying or spray freeze drying. For example, the total amount of the active ingredient, lipid component, and excipient such as leucine and/or dileucine is, for example, 5 to 100 mg/mL, depending on the content of the lipid component and excipient, and the type of solvent. It can be appropriately set in the range of 5 to 80 mg/mL, for example 5 to 50 mg/mL, further for example 5 to 30 mg/mL, further for example 5 to 25 mg/mL.

The drying step of pulverizing this liquid by a spray drying method or a spray freeze drying method can be carried out according to a conventionally known spray drying method or a spray freeze drying method. In the manufacturing method of this drug, prescribed lipid components and dileucine are used, so by powdering it according to the conventional method, this drug has excellent lung delivery and contains particles that form a large number of lipid particles when dissolved in water. can be easily obtained.

(Applications of this drug)
This agent can be used for various purposes such as medical use depending on the active ingredient. In addition, this drug can be applied to organs that can be accessed from the outside using a catheter or the like non-invasively or almost non-invasively in animals including humans, such as the nasal cavity, eyes, oral cavity, respiratory tract, lungs, stomach, duodenum, etc. For the inner surfaces (mucosal membranes) of the small intestine, large intestine, rectum, bladder, vagina, uterus, heart, blood vessels, etc., the active ingredient can be delivered to the target area by injecting this drug through an appropriate gas. can. For example, the supply of a powder to the lung mucosa or nasal mucosa is well known as an inhalation method. Alternatively, the agent may be directly supplied to the inside of the animal, for example, subcutaneously, into the muscle, into the abdominal cavity, into a lesion such as a tumor, etc., through laparotomy or incision. In addition, when applying this agent, it is also possible to adopt methods such as transplanting it inside the target tissue, on its surface, or in its vicinity. The agent can also be supported on the surface of a gel-like material, a porous body such as a sponge, a nonwoven fabric, and the like.

This agent exhibits sufficient effects even if it is dissolved before use. For example, at the time of use, the agent can be suspended or dissolved in an aqueous medium such as water, physiological saline, a buffer, a glucose solution, or a culture medium to prepare a redissolved product and then applied. For redissolution, the agent is suspended or diluted using an aqueous medium such as water. Since a different amount and type of solvent can be used than before freeze-drying, relatively high concentration suspensions and solutions, which have been difficult to prepare in the past, can be easily prepared.

As described above, the present agent dissolved or suspended in an appropriate liquid medium can be prepared using any method commonly used for introducing nucleic acids or derivatives thereof into living cells.

This specification can include the following configurations.
[1] A powder agent,
a lipid component containing a PEG-lipid conjugate;
Leucine and/or dileucine;
A powder containing constituent particles containing.
[2] The powder agent according to [1], wherein the constituent particles produce a large number of lipid particles in an aqueous suspension of the particles.
[3] The lipid component is further one or more selected from the group consisting of DOTMA, DODMA, DPPC, EPC, and SM, or one or more selected from salts thereof; [ 1] or the powder according to [2].
[4] The powder agent according to any one of [1] to [3], which contains the lipid component in an amount of 10% by mass or more and 60% by mass or less based on the total amount of the constituent particles.
[5] The powder agent according to any one of [1] to [4], wherein the constituent particles are porous spherical particles.
[6] The powder agent according to [5], wherein the porous spherical particles are three-dimensional porous bodies including partition walls and/or skeletons that define continuous pores.
[7] The powder agent according to any one of [1] to [6], wherein the constituent particles have an average particle diameter of 1 μm or more and 100 μm or less.
[8] The lipid particles have an average particle diameter of 50 nm or more and 300 nm or less, as measured by a dynamic light scattering method for a suspension obtained by suspending the particles in water or a buffer solution, [1] The powder agent according to any one of ~[7].
[9] The constituent particles have an OE (%) = recovery amount from the throat onwards (mg) / total recovery amount (mg) x 100 of 80% or more in a characteristic evaluation using a multi-stage liquid impinger (MSLI). The powder agent according to any one of [1] to [8].
[10] The powder according to any one of [1] to [9], which has an FPF (%) of 10% or more in characteristic evaluation by MSLI.
[11] A method for producing a powder, comprising:
preparing a liquid containing an active ingredient, a lipid component containing a PEG-lipid conjugate, and leucine and/or dileucine;
Drying the liquid by a spray drying method or a spray freeze drying method to produce a powder containing the active ingredient, the PEG-lipid conjugate, and the leucine and/or dileucine;
Equipped with
The manufacturing method, wherein the powder is a powder containing constituent particles that form a large number of lipid particles in an aqueous suspension of the powder.

This specification can include the following configurations.
[1] A powder agent,
an RNA interference agent;
a lipid component containing a PEG-lipid conjugate;
Leucine and/or dileucine;
A powder containing constituent particles containing.
[2] The powder according to [1], wherein the PEG-lipid conjugate is a PEG-cholesterol conjugate.
[3] The powder according to [1] or [2], which contains only dileucine among leucine and dileucine.
[4] The powder agent according to any one of [1] to [3], wherein the constituent particles produce a large number of lipid particles in an aqueous suspension of the particles.
[5] The powder agent according to any one of [1] to [4], which contains the lipid component in an amount of 10% by mass or more and 60% by mass or less based on the total amount of the constituent particles.
[6] The powder agent according to any one of [1] to [5], wherein the constituent particles are porous spherical particles.
[7] The powder agent according to [6], wherein the porous spherical particles are three-dimensional porous bodies including partition walls and/or skeletons that define continuous pores.
[8] The powder agent according to any one of [1] to [7], wherein the constituent particles have an average particle diameter of 1 μm or more and 100 μm or less.
[9] The lipid particles have an average particle diameter of 50 nm or more and 300 nm or less, as measured by a dynamic light scattering method for a suspension obtained by suspending the particles in water or a buffer solution, [1] The powder agent according to any one of ~[8].
[10] The constituent particles have an OE (%) = recovery amount from the throat and beyond (mg) / total recovery amount (mg) x 100 of 80% or more in a characteristic evaluation using a multi-stage liquid impinger (MSLI). The powder agent according to any one of [1] to [9].
[11] The powder according to any one of [1] to [10], which has an FPF (%) of 10% or more in characteristic evaluation by MSLI.
[12] The powder preparation according to any one of [1] to [11], wherein the interfering agent is siRNA.

Hereinafter, examples will be described as specific examples to more specifically explain the disclosure of this specification. The following examples are intended to illustrate the disclosure herein and are not intended to limit its scope.

(Preparation of inhalation powder)
In this example, an inhalable powder was manufactured by spray freeze-drying (SFD) and spray drying (FD) using the lipid components and excipients shown in FIGS. 1 and 2. Note that a fluorescent lipid, nitrobenzoxadiazolated phospholipid (NBD-DPPE), was used as a labeling reagent for nanosized lipid particles, and dexamethasone palmitate (PDEX) was used as a model drug.

The usage amounts of various components shown in Figures 1 and 2 were as follows. In addition, in the SFD method and the SD method, the total amount was set to 50 mg and 100 mg, respectively. In addition, the spraying liquid was prepared as follows. As the PEG derivative, the following PEG-lipid conjugate was used. As for the PEG derivatives, conjugates with a PEG Mn of 2000 were used, except for SFD #12, in which a conjugate with a PEG Mn of 5000 was used.

[1] SFD#1-3
A solution in which the entire amount of the lipid component was dissolved in 0.333 mL of tert-butyl alcohol and a solution in which the entire amount of the excipient was dissolved in 2.667 mL of water were thoroughly mixed to prepare a solution for spraying.

[2] SFD#3'-6
A solution in which the entire amount of the lipid component was dissolved in 2 mL of tert-butyl alcohol and a solution in which the entire amount of the excipient was dissolved in 2 mL of water were thoroughly mixed to prepare a solution for spraying.

[3] SFD#3''~11
A solution in which the entire amount of the lipid component was dissolved in 0.444 mL of tert-butyl alcohol and a solution in which the entire amount of the excipient was dissolved in 3.556 mL of water were thoroughly mixed to prepare a solution for spraying.

[4] SFD#12
A solution in which the entire amount of model drugs, etc. and lipid components were dissolved in 0.278 mL of tert-butyl alcohol, and a solution in which the entire amount of excipients was dissolved in 2.222 mL of water were mixed well to prepare a solution for spraying. .

[5] SD#1-3
A solution in which the entire amount of the lipid component was dissolved in 2.22 mL of ethanol and a solution in which the entire amount of the excipient was dissolved in 17.78 mL of water were thoroughly mixed to prepare a solution for spraying.

[6] SD#3'-11
A solution in which the entire amount of the lipid component was dissolved in 10 mL of ethanol and a solution in which the entire amount of the excipient was dissolved in 10 mL of water were thoroughly mixed to prepare a solution for spraying.

The SFD method was performed as follows. That is, the SFD method consists of two steps: a spraying step and a freeze-drying step. First, using a two-fluid spray nozzle attached to a spray dryer (SD-1000, Tokyo Rikakikai Co., Ltd.), the sample solution was rapidly sprayed at 150 kPa into liquid nitrogen (500 mL) 15 cm below the nozzle tip. Frozen. The sample solution was fed at a rate of 5 mL/min, and spraying was continued for 1.5 min. The obtained ice droplets were placed in a square dry chamber (DRC-1000 Tokyo Rikakikai Co., Ltd.) connected to a freeze dryer (FDU-210 Tokyo Rikakikai Co., Ltd.) and dried at -40°C for 24 hours or more under vacuum conditions, then The desired formulation was obtained by drying at 25° C. for 12 hours or more.

The SD method was performed as follows. That is, the SD method consists of two steps: a spraying step and a drying step. First, a nebulizer (Medium) was attached to the spray head attached to a spray dryer (B-90HP, Nihon Buchi), and the inlet temperature was set to 90°C and the gas flow rate was set to 120 L/min. .

(Evaluation of physical properties of lipid particles after dissolving powder)
The various powders prepared in Example 1 were dissolved in water to a lipid concentration of 0.2 mg/mL, and after standing for 30 minutes, the particle size distribution and zeta potential of the particles in the liquid were measured using dynamic and electrical methods. It was measured by electrophoretic light scattering method (Zetasizer Nano ZS manufactured by Malvern). Note that the average particle size and particle size distribution obtained by the dynamic light scattering method can be obtained based on the diffusion coefficient using the autocorrelation function obtained from the measurement results using software attached to this apparatus. The standard values were 300 nm as the average particle diameter and 0.42 as the polydispersity index (PdI), and when both analytical values were below the standard values, it was determined that the sample had "good ability to form lipid particles." The results are also shown in FIGS. 1 and 2.

As shown in Figures 1 and 2, the average particle diameter is 80 to 150 nm and the PdI is 0.170 to 0.400, which are generally below the standard values, and regardless of the type of main lipid, it is a good product. Lipid particle forming ability was obtained. Furthermore, there were no clear differences in the average particle diameter and PdI with respect to the type of PEG derivative (PEG-lipid conjugate), the concentration of the component solution, and the solvent composition. When comparing SFD constituent particles and SD constituent particles of the same composition, the average particle diameter and PdI of the lipid particles formed were similar. On the other hand, the zeta potential showed -35 to +60 mV corresponding to the charge of the main lipid, confirming that various lipid particles with different surface charges could be formed.

Subsequently, powders (SFD#3'', 7-11; SD#3', 7-11) were manufactured by changing the type of excipient and lipid component content while fixing the lipid component, and forming after dissolution. The physical properties of lipid particles were compared. Regarding the results of SFD-derived particles, when leucine was used as an excipient, both the average particle diameter and PdI were below the standard values up to a lipid component content of 20%, but when the lipid component content reached 40%, the average particle size and PdI decreased. Both the particle diameter and PdI exceeded the standard values, and it was determined that the ability to form lipid particles had been lost. On the other hand, when dileucine was used as an excipient, even when the lipid component content was 40%, both the average particle diameter and PdI were below the reference value, and good lipid particle forming ability was obtained. Next, regarding the results of SD constituent particles, when leucine was used as an excipient, the lipid component content was 20% or more, and when dileucine was used as an excipient, the lipid component content was 40%, the average particle diameter, PdI One or both of them exceeded the standard value, and it was observed that the lipid particle forming ability was more likely to be lost compared to SFD constituent particles of the same composition. From these results, it became clear that manufacturing by the SFD method using dileucine as an excipient has the advantage of obtaining good lipid particle forming ability even with a high content of lipid components.

Also, when PDEX was applied as a drug (SFD #12), good lipid particle forming ability was obtained.

(Evaluation of particle shape by scanning electron microscope of powder agent)
The particle shape of the powder particles prepared in Example 1 was observed using a scanning electron microscope (SEM: JSM-IT100LA, JEOL Ltd.). Spraying was performed using the component particle addition device for dispersion addition shown in FIG. As for the spraying method, 0.25 mL of air was compressed in a 1 mL syringe (TERUMO) connected via a three-way connection to a 100 μL tip filled with a small amount of the prepared powder, and the three-way stopcock was opened. After dispersing and adding the constituent particles onto a sample stand attached with black double-sided tape, the sample was coated with platinum (EC-3000FC, JEOL Ltd.) under the conditions of 30 mV and 90 seconds, and observed with SEM. The results are shown in Figure 3.

As shown in FIG. 3, in SEM observation, a clear difference in particle structure was confirmed between the spongy SFD-derived particles with a diameter of about 10 to 20 μm and the smaller SD-derived particles with a diameter of about 1 to 2 μm. On the other hand, no clear difference in particle structure was observed depending on the composition and content of lipid components in both particles. An interesting finding regarding the effect of excipients is that no clear difference in particle structure was observed between SFD-derived particles containing leucine and dileucine, whereas the surface of SD-derived particles became uneven when leucine was included. A clear difference was seen in that the particles had a rich and distorted particle structure, while those containing dileucine had a smooth surface and a highly spherical particle structure.

(Evaluation of inhalation characteristics of powder by MSLI)
Among the constituent particles prepared in Example 1, performance evaluation was performed on SFD #7, #10, and #11, which were able to form lipid particles exhibiting good physical properties even with a relatively high content of lipid components. In order to obtain detailed data on inhalation properties, inhalation properties were evaluated using MSLI (Multi-Stage Liquid Impinger, Copley Scientific).

In the evaluation method, approximately 1.0 mg of the sample was filled into a No. 2 HPMC capsule (Qualicaps Co., Ltd.), and suction was performed at a flow rate (60 L/min) using a vacuum pump (KRF40A-V01B, Orion Kikai Co., Ltd.). The suction time was 4 seconds. A jethaler (Jethaler (registered trademark) Standard, Tokico System Solutions Co., Ltd.) was used as an inhalation device.

(Calculation of powder recovery rate)
After dissolving the sample deposited on the device, capsule, throat, filter, and each stage in 10 mL or 20 mL of phosphate buffer (PBS), the concentration of FlNa (sodium fluorescein, contained in the powder at 1 wt% for quantitative determination) was multiplied. Quantification was performed using a mode plate reader (EnSpire, PerkinElmer Japan Co., Ltd.) (excitation wavelength: 490 nm, fluorescence wavelength: 515 nm), and the recovery amount and recovery rate for each part were calculated. Dilution and quantification were performed as necessary. In addition, from the results of this experiment, OE, FPF (pulmonary therapeutic range reachability index value), and UPF (deep lung reachability index value) were calculated using the previously described formulas (1) to (3) and the 17th revised Japanese Pharmacopoeia No. 1. Supplementary General Test Method 6.15 Aerodynamic Particle Size Determination of Inhalants 6. Calculated based on calculations. The results are shown in Figure 4.

As shown in Figure 4, the amount of deposition from the capsule to the throat was relatively small for all the constituent particles, and the index value of OE was 90% or higher, indicating good release properties. On the other hand, in SFD #7 containing leucine as an excipient, most of the deposits occurred at stage 1, whereas in SFD #10 and SFD #11 containing dileucine, the amount of deposition tended to be larger on the higher stage side. Admitted. The index values of FPF/UPF were approximately 12%/4% for SFD#7, approximately 45%/21% for SFD#10, and approximately 33%/13% for SFD#11. As a result, it was revealed that drugs containing dileucine have better lung delivery properties than leucine. Comparison of SFD #10 and SFD #11 revealed a tendency for lung delivery performance to decrease as the lipid component content increased, but both component particles had better lung delivery performance than commercially available inhalation powders (FPF: 20-40%). The inhalation characteristics were the same or better than those of the previous one, and the inhalation characteristics were sufficiently promising for practical use.

(Evaluation of cell binding/uptake ability of lipid particles formed after particle dissolution)
A dispersion containing lipid particles formed by dissolving constituent particles of the powder in Hanks' balanced salt solution was added to human lung cancer (NCI-H441) cells seeded in a microplate. Based on the fluorescence intensity measurement of the cell lysate collected 2 hours later, the amount of lipid particles bound/uptaked by the cells was calculated. Among the powder particles produced, we selected those that were found to have good ability to form lipid particles, and evaluated and compared the cell binding/uptake ability of the formed lipid particles. The results are shown in Figure 5.

As shown in Figure 5, when comparing constituent particles manufactured with different main lipids (SFD #1 to 6; SD #1 to 6), when DOTMA was included, cell binding was more than 15% of the added amount. / uptake amount, and when other main lipids were included, the cell binding/uptake amount was less than 2% of the added amount. When DOTMA is included, the surface of the lipid particles formed is positively charged at physiological pH, which increases cell binding/uptake ability through electrostatic interaction with negatively charged cell membranes. thinking. Furthermore, we have found that when PEG-Chol is used as a PEG derivative compared to PEG-DSPE, the cell binding/uptake ability of lipid particles containing DOTMA can be further enhanced.

By changing the temperature during the experiment from 37°C to 4°C, the cell binding/uptake ability was significantly reduced (difference between #3' and #3' (4°C)), indicating that this lipid particle was endocytosed. It was suggested that the protein may be taken up into cells partially through tosis.

A comparison of constituent particles (SFD#3'', 7, 9-11; SD#3', 9) manufactured with different types of excipients and lipid component contents showed that when leucine was used as an excipient There was a tendency for the cell binding/uptake ability to decrease when the lipid component content increased from 10% to 20%. On the other hand, when dileucine was used as an excipient, the cell binding/uptake ability was higher than when leucine was used, and even when the lipid component content was 40%, the cell binding/uptake ability was relatively high. Although the reason for this effect of promoting intracellular binding/uptake of lipid particles by dileucine is unknown, it was found that it contributes to improving the functionality of lipid particles.

On the other hand, in SFD#1, 4-6 and SD#1, 4-6, the cell binding/uptake ability of lipid particles was low, but this is thought to indicate the retention of lipid particles in the relevant site. It was done. In addition, based on the result that the cell binding/uptake ability of lipid particles was lower when containing PEG-DSPE than PEG-Chol (difference between #2 and #3), the lipid components contained in the above powder It was thought that the combination of the non-cationic lipid, DODMA, and PEG-phospholipid conjugate caused the formed lipid particles to stay on the cells, contributing to sustained release and sustained action of the encapsulated drug.

In comparing SFD-derived particles and SD-derived particles of the same composition, no clear difference was observed in the cell binding/uptake ability of the formed lipid particles, and the cell binding/uptake ability of lipid particles is mainly determined by the composition of the constituent particles. It was shown that the influence of the manufacturing method is relatively small.

According to the above examples, it has become clear that a wide variety of lipids can be used as lipid components in both the SFD method and SD method studied when developing an inhalation powder that constructs intrapulmonary lipid particles. . Furthermore, based on the results that the cellular binding/uptake ability of lipid particles varied greatly depending on the charge of the lipid, it was found that the application of cationic lipids increased the intracellular transferability of the encapsulated drug. Furthermore, we have found that by applying PEG-Chol as a PEG derivative and dileucine as an excipient, the cell binding/uptake ability of lipid particles containing cationic lipids can be further improved. In particular, the sponge-like constituent particles produced by the SFD method using dileucine have lung delivery properties that are equivalent to or better than existing commercially available inhalation powders, good lipid particle formation ability, and high cell binding / The usefulness of demonstrating uptake ability was clarified.

(Manufacture of powder)
According to the composition shown in the table below, in the spray freeze drying (SFD) method, siRNA (siGL3 or fluorescently labeled siGL3 (Cy5.5-siGL3), manufactured by Hokkaido System Science Co., Ltd.) and excipient (dileucine (also called diLeu)) were used. ) or leucine (also referred to as Leu)) in water and lipid components (DOTMA, DODMA, PEG derivatives, cholesterol (Chol)) in tert-butyl alcohol, a mixture thereof was prepared. Using a two-fluid spray nozzle attached to a spray dryer (SD-1000, Tokyo Rikakikai Co., Ltd.), the mixed solution was quickly frozen by spraying it into liquid nitrogen (500 mL) 15 cm below the nozzle tip at 150 kPa. . The sample solution was fed at a rate of 5 mL/min, and spraying was continued for 60 seconds when the volume of the mixed solution was 2.5 mL, and for 90 seconds when the volume was 5 mL. The obtained ice droplets were placed in a rectangular dry chamber (DRC-1000 Tokyo Rika Kikai Co., Ltd.) connected to a freeze dryer (FDU-210 Tokyo Rika Kikai Co., Ltd.) and continuously heated at -40°C for 24 h or more under vacuum conditions. The desired powder was obtained by drying at 25° C. for 12 hours or more. As the PEG derivative, a conjugate of PEG with Mn of 5000 was used.

In the spray drying (SD) method, siRNA (siGL3 or fluorescently labeled siGL3 (Cy5.5-siGL3)) and excipient (dileucine or leucine) are mixed in water and lipid component (DOTMA, DODMA) according to the composition shown in the table below. , PEG derivative, Chol) were dissolved in ethanol, and a mixture thereof was prepared. By attaching a nebulizer (Medium) to the spray head attached to a spray dryer (B-90HP, Nippon Buchi), setting the inlet temperature to 70°C and the gas flow rate to 120 L/min, and spraying the mixed liquid, It was dried in a high-temperature air stream to obtain the desired powder.

The experiment was conducted as follows. Each powder was dissolved in 10 mM HEPES buffer (pH 7.4) so that the siRNA/lipid concentration was 0.2 mg/mL, and after standing for 30 minutes, the siRNA-loaded lipid particles (siRNA-LNP) in the solution were dissolved. Particle size distribution and zeta potential were measured by dynamic electrophoretic light scattering (Zetasizer Nano ZS manufactured by Malvern). The standard values were 300 nm as the average particle diameter and 0.4 as the polydispersity index (PdI), and when both analytical values were below the standard values, it was determined that the sample had "good ability to form lipid particles." The results are shown in FIG.

Even when dileucine is used as an excipient like leucine, if a PEG derivative is not included as a lipid component (dileucine/leucine #1), the average particle diameter and PdI value of the formed siRNA-LNP are the standard values. exceeded. On the other hand, when a PEG derivative is included, the average particle diameter and PdI value of the formed siRNA-LNP are all within the standard value range, and the addition of the PEG derivative is essential for efficient lipid particle formation. I was able to confirm that. Overall, PdI tended to show a smaller value in the SFD formulation compared to the SD formulation, indicating that the lipid particle forming ability was superior. No clear difference in the physical properties of siRNA-LNP was observed depending on the presence or absence of Cy5.5 chemical modification to siRNA and the chemical structure of the lipophilic portion of the PEG derivative. Almost similar results were obtained with leucine and dileucine, but under the condition with the highest siRNA/lipid content (dileucine/leucine #4), dileucine had a higher average particle diameter and PdI value of siRNA-LNP. was small, suggesting the superiority of dileucine in preparing siRNA/high lipid content formulations.

(Particle shape of constituent particles)
The particle shape of the constituent particles of the powder prepared in Example 6 was observed using a scanning electron microscope (SEM: JSM-IT100LA, JEOL Ltd.). Using a special powder dispersion/dosing device, a small amount of powder was dispersed and added onto a sample stand attached with conductive carbon double-sided tape, and then plated with platinum coating (JEC-3000FC, JEOL Ltd.) under the conditions of 30 mV and 90 sec. ) and observed by SEM. The results are shown in FIG.

As shown in FIG. 7, in SEM observation, a clear difference in particle structure was confirmed between the sponge-like SFD formulation with a diameter of approximately 10 to 20 μm and the smaller SD formulation with a diameter of approximately 1 to 2 μm. On the other hand, in both formulations, no clear difference in particle structure was observed depending on the composition and content of siRNA/lipid components. An interesting finding regarding the effect of excipients is that in the SFD formulation, no clear difference in particle structure was observed between leucine and dileucine, whereas in the SD formulation, when leucine was included, the surface was uneven and distorted. A clear difference was seen in that the particles had a smooth surface and a highly spherical particle structure when dileucine was included.

(In vitro gene silencing effect and cytotoxicity of siRNA-loaded lipid particles formed by dissolving powder)
Each powder prepared in Example 6 was dissolved in Opti-MEM and added to luciferase-expressing human lung cancer cells (A549-Luc cells) seeded in a microplate. Thereafter, luciferin and Alamar Blue reagent were added at a predetermined time, and the luminescence corresponding to luciferase expression and the fluorescence (excitation wavelength: 465 nm, fluorescence wavelength: 600 nm) due to reaction with the Alamar Blue reagent were measured using an in vivo imaging system ( Detection and intensity analysis were performed using IVIS, Perkinelmer). Based on the obtained luminescence/fluorescence intensity, Gene expression and Cell viability were calculated using the following formulas. The results are shown in FIG.

[1] Gene expression(% of No treatment)＝(LIsample－LIblank)/(LInt－LIblank)
×100
LIsample: Luminescence intensity of cells treated with each sample
LIblank: Luminescence intensity of wells without cell seeding
LInt: Luminescence intensity of untreated cells (only medium added)

[2] Cell viability (% of No treatment) = (FIsample－FIblank)/(FInt－FIblank)
×100
FIsample: Fluorescence intensity of cells treated with each sample
FIblank: Fluorescence intensity of wells without cell seeding
FInt: Fluorescence intensity of untreated cells (only medium added)

As shown in Figure 8, regarding the gene silencing effect and cytotoxicity of the formed siRNA-LNP in cultured cells, (1) when the lipid component does not contain a PEG derivative, the gene silencing effect is significantly impaired (# (Comparison of #1 and #3), (2) As a PEG derivative, the gene silencing effect is higher in the order of "PEG-Chol>PEG-DMG>PEG-DSPE" (comparison of #3, #5, #6), (3) Excellent gene silencing effects can be obtained with both SFD and SD formulations even with relatively high siRNA/lipid component content (comparison of #2, #3, #4, #7, and #8); (4) ) Dileucine and leucine showed no significant decrease in cell viability within the range studied.

(Evaluation of cell binding/uptake ability of siRNA-loaded lipid particles generated by dissolving constituent particles of powder)
Each Cy5.5-siGL3-containing powder prepared in Example 6 was dissolved in Opti-MEM and added to luciferase-expressing human lung cancer cells (NCI-H441-Luc cells) seeded in a microplate. Two hours later, the cell surface was washed with phosphate buffer (PBS, pH 7.4), and Lysis buffer was added to lyse and collect the cells. The collected cell lysate was frozen, thawed, and centrifuged, and then the supernatant was collected. The obtained supernatant was added to a microplate, and fluorescence derived from Cy5.5 was detected and intensity analyzed using a fluorescence image analyzer (Amersham Typhoonscanner 5 system, GE Healthcare). Subsequently, this supernatant was subjected to denaturing polyacrylamide gel electrophoresis (PAGE), and a band corresponding to Cy5.5-siGL3 in the gel was detected using a fluorescent image analyzer in the same manner. The presence of Cy5.5-siGL3 was confirmed. For comparison, a solution of Cy5.5-siGL3 alone (naked siRNA) was also added and evaluated in the same manner. The results are shown in FIG.

As shown in Figure 9, the fluorescence intensity of the measurement sample (cell lysate) (Figure 9(a)) and the band density in the electrophoretic image (Figure 9(b)) were correlated to some extent. It was determined that the difference in intensity reflects the difference in the dynamics of Cy5.5-siGL3 itself (the influence of fluorescence derived from decomposition products or dissociated Cy5.5 is small). Compared to naked siRNA, the fluorescence intensity was higher in the siRNA-LNP addition group formed from each powder, suggesting that the cell binding/uptake ability of siRNA was increased by lipid particle formation. As a PEG derivative, the fluorescence intensity is higher in the order of "PEG-Chol > PEG-DMG > PEG-DSPE" (comparison of leucine #2', #9', #10'), and as a result of gene silencing effect in cultured cells ( Since this corresponds to Fig. 8), it is considered that the difference in the gene silencing effect was caused by the difference in the cell binding/uptake ability of siRNA-LNP depending on the PEG derivative. It was confirmed that when dileucine was used as an excipient like leucine, siRNA-LNPs with similarly high fluorescence intensity and excellent cell binding/uptake ability were formed. The fluorescence intensity of the dileucine preparation (dileucine #3') was lower than that of the leucine preparation (leucine #3') with the same siRNA/lipid content, but this difference is due to the difference in fluorescence intensity during siRNA-LNP formation (same Cy5. Comparing the fluorescence intensity of the powder solution with 5-siGL3 concentration, it seems that this is due to the fact that dileucine #3 has a lower fluorescence intensity.

(Inhalation characteristics of powder)
The inhalation properties of the powder were evaluated using MSLI (Multi-Stage Liquid Impinger, Copley Scientific). Approximately 1.0 mg of the powder was filled into a No. 2 HPMC capsule (Qualicaps Co., Ltd.), and suction was performed at a flow rate (60 L/min) using a vacuum pump (KRF40A-V01B, Orion Kikai Co., Ltd.). The suction time was 4 seconds. A jethaler (Jethaler (registered trademark) Standard, Tokico System Solutions Co., Ltd.) was used as an inhalation device. After dissolving the powder deposited on the device, capsule, throat, filter, and each stage in 10 mL or 20 mL of PBS, the concentration of fluorescein sodium (FlNa: contained in the powder at 1 wt% for quantification) was determined using a multimode plate reader (EnSpire, PerkinElmer Japan Co., Ltd.) to quantify the amount (excitation wavelength: 490 nm, fluorescence wavelength: 515 nm), and calculate the recovery amount and recovery rate for each part. Dilution and quantification were performed as necessary. In addition, from the results of this experiment, OE, FPF (pulmonary therapeutic range reachability index value), and UPF (deep lung reachability index value) were calculated using the previously described formulas (1) to (3) and the 17th revised Japanese Pharmacopoeia No. 1. Supplementary General Test Method 6.15 Aerodynamic Particle Size Determination of Inhalants 6. Calculated based on calculations. The results are shown in FIG.

As shown in FIG. 10, all of the powders evaluated showed an OE of 75% or more, and the release properties from the inhalation device were relatively good. When leucine was used, both SFD and SD formulations showed excellent lung delivery of over 40% in FPF and over 20% in UPF under conditions with lower siRNA/lipid content (#2, #7). , FPF decreased to 27% or less and UPF decreased to 8% or less under conditions with higher siRNA/lipid content (#3, #8). On the other hand, in the SFD formulations using dileucine (#2, #3), the FPF was as high as 35-40% regardless of the siRNA/lipid content, indicating that lung delivery was possible even at high siRNA/lipid content. The advantage of being able to maintain sex has become clear. However, in the SD formulations using dileucine (#7, #8), a decrease in FPF and UPF was observed as the siRNA/lipid content increased, similar to the case with leucine. The importance of adopting

(Lung distribution/stability of siRNA after intrapulmonary administration in mice)
Each Cy5.5-siGL3-containing powder (dileucine/leucine #3'powder) was intrapulmonarily administered to mice, and 24 hours later, Cy5.5-derived fluorescence images of the whole mouse body were captured using IVIS. Based on the obtained fluorescence image, the lung area (vertical: 1.5 cm, horizontal: 3.5 cm) and non-lung area (vertical: 3.5 cm, horizontal: 3.5 cm) were determined as shown in Figure 11(a). The fluorescence intensity within the area was analyzed. After that, PAGE was performed on lung tissue samples (RNA extracts) prepared from the excised lungs, and a band corresponding to Cy5.5-siGL3 in the gel was detected using a fluorescence image analyzer. The presence of Cy5.5-siGL3 was confirmed. For comparison, Cy5.5-siGL3 alone solution (naked siRNA) and each Cy5.5-siGL3-containing powder solution (dileucine/leucine #3'solution) were similarly administered and evaluated.

Fluorescence intensity in areas other than the lungs analyzed from in vivo fluorescence imaging images (Figure 11(b)) was lower in the group administered with each powder and its solution compared to the group administered with naked siRNA. -LNP formation) to avoid siRNA degradation and systemic translocation. From the electrophoretic image of the lung tissue sample (Figure 11(c)), a band corresponding to Cy5.5-siGL3 could not be detected in the naked siRNA administration group, so the fluorescence detected in the lung region (Figure 11(b) )) was determined to be due to a degradation product of Cy5.5-siGL3 or dissociated Cy5.5. On the other hand, in the groups administered with each powder and its solution, a band corresponding to Cy5.5-siGL3 could be detected, revealing that Cy5.5-siGL3 exists in the lungs while maintaining its structure. From this result, it is considered that the retention/stability of siRNA in the lungs was improved by the formation of lipid particles. In addition, similar results were obtained with a powder containing dileucine (dileucine #3' powder) as well as with its solution (dileucine #3' solution: LNPs were formed in water in advance), so administration was conducted. It is thought that it was possible to demonstrate that the powder was deposited and dissolved in the lungs to form LNPs as expected.

Claims (15)

1. A powder agent,
   a lipid component containing a PEG-lipid conjugate;
   Leucine and/or dileucine;
   A powder containing constituent particles containing.
2. The powder agent according to claim 1, wherein the constituent particles produce a large number of lipid particles in an aqueous suspension of the particles.
3. The lipid component further includes 1,2-dioleyloxy-3-trimethylammoniumpropane (DOTMA), 1,2-dioleyloxy-3-dimelaminopropane (DODMA), 1,2-dipalmitoyl-sn - One or more selected from the group consisting of glycero-3-phosphatidylcholine (DPPC), egg-derived phosphatidylcholine (EPC), and sphingomyelin (SM), or one or more selected from salts thereof. The powder agent according to claim 1, which is.
4. The powder agent according to claim 1, which contains the lipid component in an amount of 10% by mass or more and 60% by mass or less based on the total amount of the constituent particles.
5. The powder agent according to claim 1, wherein the constituent particles are porous spherical particles.
6. The powder agent according to claim 5, wherein the porous spherical particles are three-dimensional porous bodies comprising partition walls and/or skeletons that define continuous pores.
7. The powder agent according to claim 1, wherein the constituent particles have an average particle diameter of 1 μm or more and 100 μm or less.
8. The lipid particles according to claim 1, have an average particle diameter of 50 nm or more and 300 nm or less, as measured by a dynamic light scattering method for a suspension obtained by suspending the particles in water or a buffer solution. Powder.
9. The constituent particles are characterized in that OE (%) = recovery amount from the throat and beyond (mg) / total recovery amount (mg) x 100 is 80% or more in characteristic evaluation using a multi-stage liquid impinger (MSLI). Item 1. Powder preparation according to item 1.
10. The powder agent according to claim 1, which has an FPF (%) of 10% or more in characteristic evaluation by MSLI.
11. The powder according to any one of claims 1 to 10, which contains an RNA interference agent as an active ingredient.
12. The powder according to claim 11, wherein the PEG-lipid conjugate is a PEG-phospholipid conjugate and/or a PEG-diacylglycerol conjugate.
13. The powder according to claim 11, wherein the PEG-lipid conjugate is a PEG-cholesterol conjugate.
14. The powder according to claim 12 or 13, which contains only dileucine among leucine and dileucine.
15. A method for producing a powder, the method comprising:
    preparing a liquid containing an active ingredient, a lipid component containing a PEG-lipid conjugate, and leucine and/or dileucine;
    Drying the liquid by a spray drying method or a spray freeze drying method to produce a powder containing the active ingredient, the PEG-lipid conjugate, and the leucine and/or dileucine;
    Equipped with
    The manufacturing method, wherein the powder is a powder containing constituent particles that form a large number of lipid particles in an aqueous suspension of the powder.

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