Dry powder inhalation formulation for preventing or treating idiopathic pulmonary fibrosis, and preparation method therefor

Abstract

The present invention relates to a method for preparing a dry powder inhalation formulation for preventing or treating idiopathic pulmonary fibrosis, the formulation being used in an inhalable-administration form through a dry powder inhaler, and the method comprising: step A of mixing polypeptides prepared as active pharmaceutical ingredients (APIs) and mannitol in distilled water; step B of freeze-drying the mixture mixed through step A; and step C of milling the mixture freeze-dried through step B at least once, thereby powderizing same, wherein the polypeptides prepared as active pharmaceutical ingredients comprise the amino acid sequence consisting of arginine (R, Arg)-glycine (G, Gly)-aspartic acid (D, Asp)-valine (V, Val)-phenylalanine (F, Phe)-proline (P, Pro)-serine (S, Ser)-tyrosine (Y, Tyr)-threonine (T, Thr)-lysine (K, Lys).

Description

Dry powder inhalation formulation for preventing or treating idiopathic pulmonary fibrosis and method for preparing the same

[0001] The present invention relates to a dry powder inhalation formulation for preventing or treating idiopathic pulmonary fibrosis and a method for producing the same.

[0002] Idiopathic pulmonary fibrosis is a fatal disease in which inflammation of unknown cause occurs repeatedly in the lung interstitial tissue, causing permanent scarring and tissue fibrosis, which induces structural changes in the lung tissue, leading to a decline in lung function and death.

[0003] This idiopathic pulmonary fibrosis is a rare disease with an unknown cause. The main symptom is shortness of breath during exercise, and as the symptoms progress, the degree of shortness of breath worsens. Inflammation and fibrosis in the lungs irritate the airways and lungs, causing frequent dry coughs.

[0004] Eventually, if the degree of shortness of breath becomes severe, not only can hypoxia occur, but clubbing, a phenomenon in which the tips of the fingers become rounded, can also occur.

[0005] Currently, there are no drugs that have been proven to be effective and are approved as direct treatments for idiopathic pulmonary fibrosis, but drugs called pirfenidone and nintedanib are being used. These drugs have been proven to be effective in delaying the progression of the disease, but they have the limitation of not being able to achieve a complete cure.

[0006] Accordingly, the applicant has developed a technology for providing a polypeptide for the prevention or treatment of idiopathic pulmonary fibrosis, which can fundamentally treat lung tissues that have progressed to fibrosis due to idiopathic pulmonary fibrosis and restore lung function to a normal level, and further delay or prevent the onset of idiopathic pulmonary fibrosis, through Korean Patent Publication No. 10-2023-0001168 entitled “Polypeptide for the prevention or treatment of idiopathic pulmonary fibrosis and pharmaceutical composition comprising the same” (hereinafter referred to as “prior application patent”).

[0007] Furthermore, the applicant has also developed a technology for a dry powder inhalation formulation for the prevention or treatment of idiopathic pulmonary fibrosis and a method for manufacturing the same, which is used in a form that can be inhaled using a dry powder inhaler, by utilizing an example of a polypeptide for the prevention or treatment of idiopathic pulmonary fibrosis presented in a prior application patent as an active pharmaceutical ingredient (API).

[0008] The present invention was created to solve the above problems, and the purpose of the present invention is to provide a technology for manufacturing a dry powder inhalation formulation for preventing or treating idiopathic pulmonary fibrosis, which is used in a form that can be inhaled using a dry powder inhaler, by utilizing a polypeptide consisting of an amino acid sequence consisting of arginine (R, Arg, Arginine) - glycine (G, Gly, Glycine) - aspartic acid (D, Asp, Aspartic acid) - valine (V, Val, Valine) - phenylalanine (F, Phe, Phenylalanine) - proline (P, Pro, Proline) - serine (S, Ser, Serine) - tyrosine (Y, Tyr, Tyrosine) - threonine (T, Thr, Threonine) - lysine (K, Lys, Lysine) as an active pharmaceutical ingredient (API), as an example of a polypeptide for preventing or treating idiopathic pulmonary fibrosis suggested through a prior application patent.

[0009] In order to achieve the above purpose, the present invention provides a method for manufacturing a dry powder inhalation formulation for preventing or treating idiopathic pulmonary fibrosis, which is used in a form that can be inhaled using a dry powder inhaler, comprising: a step A of mixing a polypeptide and (Mannitol) prepared as active pharmaceutical ingredients (API) with distilled water; a step B of freeze-drying the mixture mixed through the step A; And a C step of grinding the mixture freeze-dried through the B step at least once to powder it; wherein the polypeptide prepared as the active pharmaceutical ingredient is composed of an amino acid sequence consisting of arginine (R, Arg, Arginine) - glycine (G, Gly, Glycine) - aspartic acid (D, Asp, Aspartic acid) - valine (V, Val, Valine) - phenylalanine (F, Phe, Phenylalanine) - proline (P, Pro, Proline) - serine (S, Ser, Serine) - tyrosine (Y, Tyr, Tyrosine) - threonine (T, Thr, Threonine) - lysine (K, Lys, Lysine).

[0010] Here, the above step A is a step of mixing 0.5 to 2 parts by weight of the polypeptide prepared as the active pharmaceutical ingredient and 80 to 120 parts by weight of the mannitol in the distilled water, so that the concentration of the polypeptide prepared as the active pharmaceutical ingredient in the distilled water is 200 μg / ml to 300 μg / ml.

[0011] In addition, the step B is a step of cooling the mixture mixed through the step A to a temperature of -45°C to -35°C and then drying it at a temperature of -20°C to 25°C for 8 to 12 days, and the step C is a step of milling the mixture freeze-dried through the step B at least once so that a dry powder having a particle size distribution in which D10, which defines a particle size corresponding to 10% of the cumulative volume concentration (%), corresponds to 0.7 μm to 1.3 μm, D50, which defines a particle size corresponding to 50% of the cumulative volume concentration (%), corresponds to 2.5 μm to 3.3 μm, D90, which defines a particle size corresponding to 90% of the cumulative volume concentration (%), corresponds to 4.2 μm to 8.8 μm, and a span value defined by the following mathematical formula 1 corresponds to 1.1 to 2.6 is prepared.

[0012] Mathematical Equation 1

[0013]

[0014] And the method for manufacturing a dry powder inhalation formulation for preventing or treating idiopathic pulmonary fibrosis further includes a step D of adding 100 to 300 parts by weight of alpha-lactose monohydrate corresponding to a carrier to the dry powder pulverized through the step C and then stirring the mixture at a mixing speed of 40 to 50 rpm for 10 to 30 minutes.

[0015] In addition, when the dry powder pulverized through the above step C is in a capsule-type form that can be inhaled using the above dry powder inhaler, 10 mg to 20 mg is filled per capsule.

[0016] In addition, the dry powder pulverized through the above step C is prepared with a moisture content of 2.5% to 3.7%.

[0017] In addition, the dry powder pulverized through the above step C is prepared in a state in which the emitted dose (ED) measured using a next generation pharmaceutical impinger (NGI) is 70% to 90%, the fine particle fraction (FPF) based on a particle size of 4.4 μm to 4.5 μm is 40% to 70%, the fine particle fraction (FPF) based on a particle size of 2.8 μm to 2.9 μm is 20% to 50%, and the mass median aerodynamic diameter (MMAD) is 3.5 μm to 7.0 μm, and has aerodynamic characteristics.

[0018] Meanwhile, in order to achieve the above purpose, a dry powder inhalation formulation for preventing or treating idiopathic pulmonary fibrosis according to the present invention is prepared through the method for manufacturing a dry powder inhalation formulation for preventing or treating idiopathic pulmonary fibrosis described above.

[0019] According to the present invention, a polypeptide having an amino acid sequence consisting of arginine (R, Arg, Arginine)-glycine (G, Gly, Glycine)-aspartic acid (D, Asp, Aspartic acid)-valine (V, Val, Valine)-phenylalanine (F, Phe, Phenylalanine)-proline (P, Pro, Proline)-serine (S, Ser, Serine)-tyrosine (Y, Tyr, Tyrosine)-threonine (T, Thr, Threonine), which exhibits a reducing effect of fibrosis indicators acting on idiopathic pulmonary fibrosis through a polypeptide for preventing or treating idiopathic pulmonary fibrosis, can be provided as an active pharmaceutical ingredient (API), thereby providing a dry powder inhalation formulation for preventing or treating idiopathic pulmonary fibrosis that can be administered by inhalation using a dry powder inhaler.

[0020] Figure 1 is a flow chart illustrating a method for manufacturing a dry powder inhalation formulation for preventing or treating idiopathic pulmonary fibrosis according to the present invention.

[0021] Figure 2 is a result of observing the particle state before grinding using a scanning electron microscope in Example 1 of the method for manufacturing a dry powder inhalation formulation for preventing or treating idiopathic pulmonary fibrosis of the present invention.

[0022] Figure 3 is a result of observing the particle state before grinding using a scanning electron microscope in Example 2 of the method for manufacturing a dry powder inhalation formulation for preventing or treating idiopathic pulmonary fibrosis of the present invention.

[0023] Figure 4 is a graph showing the results of powder X-ray diffraction analysis of Example 1 related to the method for manufacturing a dry powder inhalation formulation for preventing or treating idiopathic pulmonary fibrosis of the present invention.

[0024] Figure 5 is a graph showing the results of powder X-ray diffraction analysis of Example 2 related to the method for manufacturing a dry powder inhalation formulation for preventing or treating idiopathic pulmonary fibrosis of the present invention.

[0025] Figure 6 is a graph showing the results of comparative analysis of aerodynamic characteristics of Examples 1, 3, and 4 related to the method for manufacturing a dry powder inhalation formulation for preventing or treating idiopathic pulmonary fibrosis of the present invention.

[0026] Figure 7 is a graph showing the results of comparative analysis of aerodynamic characteristics of Examples 1 and 5 related to the method for manufacturing a dry powder inhalation formulation for preventing or treating idiopathic pulmonary fibrosis of the present invention.

[0027] Figure 8 is a graph showing the results of a comparative analysis of aerodynamic characteristics according to the amount of filling in the capsule of Example 1 of the method for manufacturing a dry powder inhalation formulation for preventing or treating idiopathic pulmonary fibrosis of the present invention.

[0028] Figures 9 and 10 are graphs showing the results of RT (Relaxation time) pattern analysis on the 8th and 14th days by test group in a pharmacodynamics evaluation related to the method for manufacturing a dry powder inhalation formulation for preventing or treating idiopathic pulmonary fibrosis of the invention.

[0029] Figures 11 and 12 are graphs showing the results of the f (breathing frequency) pattern analysis on the 8th and 14th days by test group in the pharmacodynamics evaluation related to the method for manufacturing a dry powder inhalation formulation for preventing or treating idiopathic pulmonary fibrosis of the invention.

[0030] Figure 13 is a graph showing the results of Ashcroft score analysis for images captured of the upper, lower, middle, and bronchial portions of lung tissue by test group in a pharmacodynamics evaluation related to a method for manufacturing a dry powder inhalation formulation for preventing or treating idiopathic pulmonary fibrosis of the invention.

[0031] A preferred embodiment of the present invention will be described in more detail with reference to the attached drawings, but already well-known technical parts will be omitted or compressed for the sake of brevity.

[0032] 1. Description of a method for manufacturing a dry powder inhalation formulation for preventing or treating idiopathic pulmonary fibrosis

[0033] The method for manufacturing a dry powder inhalation formulation for preventing or treating idiopathic pulmonary fibrosis according to the present invention relates to a method for manufacturing a dry powder inhalation formulation used in a form that can be inhaled using a dry powder inhaler. The specific process of this method is described in detail below with reference to FIG. 1.

[0034]

[0035] (1) Active pharmaceutical ingredient mixing stage<S110, A단계>

[0036] In this step (S110), a process of mixing polypeptides and mannitol, which are prepared as active pharmaceutical ingredients (API), into distilled water is performed.

[0037] First, the polypeptide prepared as an active pharmaceutical ingredient (API) is a polypeptide synthesized by linking nine amino acids, and is a peptide with the structure 'RGDVFPSYTK' consisting of the amino acid sequence of arginine (R, Arg, Arginine) - glycine (G, Gly, Glycine) - aspartic acid (D, Asp, Aspartic acid) - valine (V, Val, Valine) - phenylalanine (F, Phe, Phenylalanine) - proline (P, Pro, Proline) - serine (S, Ser, Serine) - tyrosine (Y, Tyr, Tyrosine) - threonine (T, Thr, Threonine) - lysine (K, Lys, Lysine), and is prepared in the form of a molecular weight of 1168 Da to 1169 Da (most preferably 1168.2 Da).

[0038] Specifically, the polypeptide provided as an active pharmaceutical ingredient has an RGD motif active site based on an amino acid sequence starting from 'RGDVFPSYT', and corresponds to the fifth polypeptide (NPT-0025) presented as one of the preferred embodiments of a polypeptide for preventing or treating idiopathic pulmonary fibrosis in the published patent application '10-2023-0001168' published after the applicant's prior application.

[0039] The polypeptide prepared as such an active pharmaceutical ingredient (hereinafter referred to as 'NPT-0025') has been shown to exhibit sufficiently significant results in relation to the preventive or therapeutic effect of idiopathic pulmonary fibrosis through the patent application '10-2023-0001168' published after the applicant's prior application.

[0040] Here, the polypeptide prepared as an active pharmaceutical ingredient is synthesized according to a set sequence using the Solid Phase Peptide Synthesis method and is prepared to exhibit a purity of 90% or more.

[0041] The polypeptide prepared as an active pharmaceutical ingredient purified to exhibit a purity of 90% or more can be confirmed to have a molecular weight of 1035 to 1200 Da through mass spectrometry.

[0042] Furthermore, the polypeptide prepared as an active pharmaceutical ingredient can have the carboxyl group '-COOH' of the amino acid located at the terminal of the amino acid sequence modified to '-CONH2' to increase the absorption rate.

[0043] For example, a polypeptide prepared as an active pharmaceutical ingredient corresponding to the structure of 'RGDVFPSYTK' in the amino acid sequence can have its absorption rate improved by modifying the carboxyl group '-COOH' of the amino acid corresponding to 'K' to '-CONH2'.

[0044] Next, for mannitol, it is desirable to apply the product example of 'PEARLITOL® 25 C mannitol'.

[0045] The polypeptide prepared as an active pharmaceutical ingredient having such material properties is mixed with 0.5 to 2 parts by weight (most preferably 1 part by weight) of the polypeptide prepared as an active pharmaceutical ingredient and 80 to 120 parts by weight (most preferably 100 parts by weight) of mannitol in distilled water through this step (S110), so that the concentration of the polypeptide prepared as an active pharmaceutical ingredient in the distilled water is 200 μg / ml to 300 μg / ml (most preferably 250 μg / ml).

[0046]

[0047] (2) Freeze drying processing step<S120, B단계>

[0048] In this step (S120), the mixture mixed in the previously performed active pharmaceutical ingredient mixing step (S110) is subjected to freeze drying.

[0049] Specifically, the freeze drying process (S120) is performed by mixing 1 part by weight of a polypeptide prepared as an active pharmaceutical ingredient and 100 parts by weight of mannitol in distilled water, so that the mixture, in which the polypeptide prepared as an active pharmaceutical ingredient has a concentration of 250 μg / ml in the distilled water, is cooled to a temperature of -45°C to -35°C (most preferably -40°C), and then dried at a temperature of -20°C to 25°C for 8 to 12 days (most preferably 10 days).

[0050] The result of the manufacturing method produced later through this freeze drying process (S120) has a low-density fluffy particle shape, and the polymorphic crystal structure of mannitol has an α+β type, which contributes to the improvement of structural stability and enables a greater improvement in aerodynamic characteristics.

[0051]

[0052] (3) Grinding processing stage<S130, C단계>

[0053] In this step (S130), the mixture freeze-dried through the previously performed freeze-drying step (S120) is subjected to a process of grinding (milling) at least once to powder.

[0054] Through this, a dry powder having a particle size distribution in which D10, which defines a particle size corresponding to 10% of the cumulative volume concentration (%), corresponds to 0.7 μm to 1.3 μm, D50, which defines a particle size corresponding to 50% of the cumulative volume concentration (%), corresponds to 2.5 μm to 3.3 μm, D90, which defines a particle size corresponding to 90% of the cumulative volume concentration (%), corresponds to 4.2 μm to 8.8 μm, and a span value defined by the following mathematical expression 1 corresponds to 1.1 to 2.6, is prepared.

[0055] Mathematical Equation 1

[0056]

[0057] As an example (first grinding method), the grinding (milling) treatment process (S130) can be performed by repeating jet milling twice using AO Jet Mill equipment under the conditions of a grinding air pressure of 0.45 MPa and a pushing air pressure of 0.5 MPa to match a batch size of 2.5 g.

[0058] This is to ensure that the D90 is not too large to be inhaled, but rather has an appropriate size for inhalation and ensures that the particle size distribution is evenly formed.

[0059] In this case, it is preferable to prepare a dry powder having a particle size distribution in which D10 corresponds to 1.1 μm to 1.4 μm, D50 corresponds to 2.5 μm to 2.7 μm, D90 corresponds to 4.1 μm to 4.4 μm, and a span value corresponds to 1.1 to 1.3.

[0060] As another example (second crushing method), the crushing (milling) process (S130) can be performed once using JET-O-Mizer Alzet equipment for scale-up, with a batch size of 40 g.

[0061] In this case, it is preferable to prepare a dry powder having a particle size distribution in which D10 corresponds to 0.6 μm to 0.9 μm, D50 corresponds to 3.0 μm to 3.4 μm, D90 corresponds to 8.5 μm to 9.0 μm, and a span value corresponds to 2.4 to 2.7.

[0062] When the dry powder that has been ground through the milling process (S130) is in a capsule-type form that can be inhaled using the dry powder inhaler, it is preferable to fill 10 to 20 mg per capsule (most preferably 10 mg).

[0063] This is a result of comprehensively considering the effective drug amount, along with the fact that as the amount of dry powder filled in the capsule increases, the emitted dose (ED) is similar in aerodynamic terms while the fine particle fraction (FPF) decreases.

[0064] In addition, it is preferable that the dry powder milled through the milling process (S130) be prepared with a moisture content of 2.5% to 3.7%.

[0065] More specifically, when the dry powder is ground according to the first grinding method described above, the dry powder has a moisture content of 2.8% to 3.6%, and when the dry powder is ground according to the second grinding method described above, the dry powder has a moisture content of 2.5% to 3.7%.

[0066] Lastly, it is preferable that the dry powder milled through the milling process (S130) has an emitted dose (ED) of 70% to 90% measured using a next-generation pharmaceutical impinger (NGI), a fine particle fraction (FPF) of 40% to 70% based on a particle size of 4.4 μm to 4.5 μm, a fine particle fraction (FPF) of 20% to 50% based on a particle size of 2.8 μm to 2.9 μm, and an aerodynamic mass median aerodynamic diameter (MMAD) of 3.5 μm to 7.0 μm, which are aerodynamic characteristics.

[0067] More specifically, when the dry powder is ground according to the first grinding method described above, the emitted dose (ED) is 85% to 90%, the fine particle fraction (FPF) based on a particle size of 4.4 μm to 4.5 μm is 40% to 45%, the fine particle fraction (FPF) based on a particle size of 2.8 μm to 2.9 μm is 20% to 35%, and the mass median aerodynamic diameter (MMAD) is 6.5 μm to 7.0 μm, and the dry powder is prepared with aerodynamic characteristics.

[0068] In addition, when the dry powder is ground according to the second grinding method described above, the emitted dose (ED) is 70% to 85%, the fine particle fraction (FPF) based on a particle size of 4.4 μm to 4.5 μm is 60% to 70%, the fine particle fraction (FPF) based on a particle size of 2.8 μm to 2.9 μm is 40% to 50%, and the mass median aerodynamic diameter (MMAD) is 3.8 μm to 4.0 μm, and the aerodynamic characteristics are provided.

[0069] In this way, the result of the dry powder prepared through this step (S130) can be used by filling it into a capsule as one embodiment of a dry powder inhalation formulation for preventing or treating idiopathic pulmonary fibrosis. In this case, it is preferable to store it in a storage container that can sufficiently prevent moisture by blocking contact with moisture from the outside, such as a glass vial.

[0070]

[0071] (4) Carrier mixing stage<S140, D단계>

[0072] In this step (S140), 100 to 300 parts by weight of alpha-lactose monohydrate, which serves as a carrier, is added to the dry powder that has been ground in the previously performed grinding step (S130), and then the mixture is stirred at a mixing speed of 40 to 50 rpm for 10 to 30 minutes.

[0073] Here, the carrier is an ingredient added to improve the flowability of the formulation, and it is desirable to apply MEGGLE's 'Inhalac 120' as a product example.

[0074] This is to ensure sufficient improvement in flow and balanced improvement in aerodynamic characteristics by adding 100 to 300 parts by weight of Inhalac 120 as a carrier.

[0075] Through this, a carrier is added to the dry powder pulverized through the previously performed pulverization process step (S130), and it can be used by filling it into a capsule as another embodiment of a dry powder inhalation formulation for preventing or treating idiopathic pulmonary fibrosis.

[0076]

[0077] 2. Description of the comparative review of characteristics and efficacy verification test of dry powder inhalation formulations for the prevention or treatment of idiopathic pulmonary fibrosis.

[0078] In relation to the method for manufacturing a dry powder inhalation formulation for preventing or treating idiopathic pulmonary fibrosis of the present invention, the critical significance of the conditions specified in each major step of the method and the level of the preventive or therapeutic effect of the final result were verified through testing, and the testing used the following experimental methods for the purpose of defining properties, etc. by means obvious to those skilled in the art.

[0079]

[0080] (1) Manufacturing of the example

[0081] - First manufacturing method

[0082] A polypeptide (NPT-0025) prepared as an active pharmaceutical ingredient (API) consisting of an amino acid sequence of RGDVFPSYTK and mannitol are mixed in distilled water, and the mixture in which the polypeptide prepared as an active pharmaceutical ingredient in the distilled water has a concentration of 250 μg / ml is cooled to a temperature of -40°C and then dried at a temperature of -20°C to 25°C for 10 days, and the freeze-dried product is repeatedly jet milled twice using AO Jet Mill equipment under conditions of a grinding air pressure of 0.45 MPa and a pushing air pressure of 0.5 MPa, in a batch size of 2.5 g, to prepare a dry powder inhalation formulation.

[0083] - Second manufacturing method

[0084] A polypeptide (NPT-0025) prepared as an active pharmaceutical ingredient (API) consisting of an amino acid sequence of RGDVFPSYTK and mannitol are mixed in distilled water, and the mixture in which the polypeptide prepared as an active pharmaceutical ingredient in the distilled water has a concentration of 250 μg / ml is cooled to a temperature of -40°C and then dried at a temperature of -20°C to 25°C for 10 days, and the freeze-dried product is jet milled once using a JET-O-Mizer Alzet equipment in a batch size of 40 g to prepare a dry powder inhalation formulation.

[0085] - Third manufacturing method

[0086] A polypeptide (NPT-0025) prepared as an active pharmaceutical ingredient (API) consisting of an amino acid sequence of RGDVFPSYTK and mannitol are mixed in distilled water, and the mixture in which the polypeptide prepared as an active pharmaceutical ingredient in the distilled water has a concentration of 250 μg / ml is cooled to a temperature of -40°C and then dried at a temperature of -20°C to 25°C for 10 days, and the freeze-dried product is jet-milled once using a JET-O-Mizer Alzet equipment in a batch size of 40 g, and a carrier is added to the pulverized product and stirred at a mixing speed of 45 rpm for 20 minutes to prepare a dry powder inhalation formulation.

[0087] - Manufacturing method 4

[0088] The polypeptide (NPT-0025) prepared as an active pharmaceutical ingredient (API) consisting of the amino acid sequence of RGDVFPSYTK and mannitol are mixed with distilled water by stirring at a mixing speed of 45 rpm for 20 minutes to prepare a mixture in which the polypeptide prepared as an active pharmaceutical ingredient in the distilled water has a concentration of 250 μg / ml, and the prepared mixture is repeatedly jet milled twice using AO Jet Mill equipment under conditions of a grinding air pressure of 0.45 MPa and a pushing air pressure of 0.5 MPa, in a batch size of 2.5 g, to prepare a dry powder inhalation formulation.

[0089] Composition of the example (unit: parts by weight)

[0090] NPT-0025Mannitol25CInhalac251Inhalac120Manufacturing methodExample 11100--1Example 21100--4Example 3150--1Example 4110--1Example 51100--2Example 611001003Example 711001003Example 811003003

[0091]

[0092] (2) Comparison of Examples 1 and 2

[0093] - Comparison of particle observations (SEM) by example

[0094] The particle state before the pulverization treatment of Examples 1 and 2 was observed using a scanning electron microscope (SEM) from Ultra Plus, Carl Zeiss, and the results are as shown in Figures 2 and 3.

[0095] As shown in FIGS. 2 and 3, the particle state before the grinding process of Example 2, which did not undergo the freeze-drying process, maintains the characteristics of raw mannitol and maintains a dense columnar shape, whereas the particle state before the grinding process of Example 1, which went through the freeze-drying process, shows a low-density, fluffy shape due to the freeze-drying.

[0096]

[0097] - Comparison of crystallinity by example

[0098] The results of powder X-ray diffraction (PXRD) analysis (conditions - Wave length: 1.54 Å, 2θ range: 5-45 degree) for Examples 1 and 2 using the equipment of 'D8 Discover with GADDS, Bruker AXS' are as shown in Figures 4 and 5 below.

[0099] Here, mannitol has a polymorphism in its crystal structure, with stable forms in the order of β, α, and δ, and unstable forms contributing to the stability of peptide or protein structure compared to stable forms.

[0100] As shown in Fig. 4, Example 1 (M2-0 ​​(before grinding treatment) and M2-2 (after grinding treatment) based on Fig. 3) shows that the crystal structure of mannitol changes to the α+β form through freeze-drying treatment.

[0101] In contrast, as shown in Fig. 5, Example 2 (M1-0 (before grinding treatment) and M1-2 (after grinding treatment) based on Fig. 4) shows that the crystal structure of mannitol maintains the β form without change.

[0102]

[0103] (2) Comparison according to the composition ratio of mannitol

[0104] The moisture content and aerodynamic characteristics of Examples 1, 3, and 4 were compared and analyzed, and the results are shown in Tables 2, 3, and Fig. 6 below.

[0105] First, comparing the moisture contents of Examples 1, 3, and 4 as shown in Table 2 below, it can be seen that as the ratio of NPT-0025 to mannitol increases, the moisture content of the formulation itself increases.

[0106] Moisture content (%) Example 13.21±0.37 Example 34.28±1.67 Example 44.64±0.55

[0107] Next, the analysis of the aerodynamic characteristics for Examples 1, 3, and 4 (Condition - Dose: 20 mg in 03 capsule) was performed using a Next Generation Pharmaceutical Impinger (NGI), and the results are shown in Table 3 and Figure 6 below.

[0108] Example 1 Example 3 Example 4 ED (%) 86.82 ± 1.66 85.41 ± 2.84 65.68 ± 7.15 FPF (%) (Cut-off) S2 (4.46 μm) 43.32 ± 0.54 36.21 ± 6.5 27 8.23 ​​± 13.92 S3 (2.82 μm) 28.40 ± 3.42 29.28 ± 5.6 4 65.81 ± 11.12 MMAD (μm) 4.07 ± 0.28 6.86 ± 1.4 4 7.66 GSD 1.57 ± 0.04 3.12 ± 0.5 9 3.09

[0109] As shown in Table 3 and Fig. 6, in the case of Example 3 (M6-2 as shown in Fig. 6), despite the large D90, the FPF decreased by about 7% compared to Example 1 (M2-2 as shown in Fig. 6), and in the case of Example 4 (M7-2 as shown in Fig. 6), the ED decreased significantly to 65.68%, indicating that a significant amount was not released from the device. Accordingly, it can be seen that when the composition ratio of polypeptide and mannitol according to Example 1 (M2-2 as shown in Fig. 6) is satisfied, the aerodynamic characteristics are the best, considering the effective amount of drug and the aerodynamic characteristics.

[0110]

[0111] (3) Analysis of physicochemical and aerodynamic properties of the optimal embodiment

[0112] First, the results of analyzing the particle size distribution and moisture content for Raw NPT-0025, Examples 1 and 5 are as shown in Table 4 below.

[0113] D10(μm)D50(μm)D90(μm)SpanMoisture Content(%)Raw NPT-002513.37±0.9957.43±0.86103.00±2.001.54±0.075.44±0.45Example11.28±0.012.59±0.014.29±0.041.16+0.013.21±0.37Example50.73±0.003.20±0.088.77±0.222.52±0.013.10±0.57

[0114] Next, the analysis of the aerodynamic characteristics for Examples 1 and 5 (Condition - Dose: 20 mg in 03 capsule) was performed using a Next Generation Pharmaceutical Impinger (NGI), and the results are shown in Table 5 and Figure 7 below.

[0115] Example 1 Example 5 ED (%) 86.82 ± 1.66 77.62 ± 3.89 FPF (%) (Cut-off) S2 (4.46 μm) 43.32 ± 0.54 64.55 ± 4.01 S3 (2.82 μm) 28.40 ± 3.42 45.68 ± 2.47 MMAD (μm) 4.07 ± 0.28 3.84 ± 0.06 GSD 1.57 ± 0.04 1.74 ± 0.07

[0116] As shown in Table 4, Table 5 and Figure 7, Examples 1 and 5 are prepared as optimal embodiments with the targeted physicochemical properties and aerodynamic properties.

[0117] (4) Comparison of the effect of improving flowability by carrier

[0118] Using Examples 5 to 8, the difference in flowability depending on whether or not a process of additionally mixing a carrier within an appropriate content range was performed after the freeze-drying and grinding processes was compared and analyzed, and the results are as shown in Table 6 below.

[0119] Specifically, the flowability evaluation for each example was performed using 'PT-TD300, Pharma test' (condition - Cylinder: 10 ml / Tapping number of times: 150 taps (Tap-1), 150 taps (Tap-2), and 150 taps (Tap-3)), and as previously known, the calculation formulas of 'Hausner Raio = ρtapped / ρbulk' and 'Carr's index (CI) = 100 x (ρtapped - ρbulk / ρtapped)' were utilized.

[0120] ρ bulk (g / ml)ρ tapped (g / ml)Hausner RatioCI (%)Fluterol® 100 / 500.650.851.34225.53Example 50.110.171.56936.27Example 60.030.041.73042.22Example 70.260.411.56636.17Example 80.450.621.37327.17

[0121] As shown in Table 6, the introduction of Lactose 251 according to Example 6 did not improve the flowability, while the introduction of Lactose 120 according to Examples 7 and 8 improved the flowability. In addition, it can be seen that when the composition content ratio of the carrier according to Example 8 increases compared to Example 7, the flowability obtains a value similar to that of the control agent, pluterol.

[0122]

[0123] (5) Comparison of aerodynamic characteristics according to the filling amount in the capsule of the dry powder inhalation formulation

[0124] Based on Example 1, the differences in aerodynamic characteristics were compared and analyzed when 10 mg, 20 mg, and 40 mg were filled in capsules used in a form that can be inhaled using a dry powder inhaler, and the results are as shown in Table 7 and Figure 8 below.

[0125] 10 mg20 mg40 mgED(%)87.82±1.6686.82±1.6688.27±3.66FPF(%)(Cut-off)S2(4.46 μm)52.55±1.0843.32±0.5441.44±3.98S3(2.82 μm)35.33±1.9828.40±3.4225.52±3.47MMAD(μm)4.07±0.285.52±0.146.86±1.44GSD1.57±0.043.14±0.563.12±0.59

[0126] As shown in Table 7 and Figure 8, as the filling amount in the capsule increases, the ED is similar, but the FPF decreases. It can be seen that filling a small amount is advantageous in terms of FPF, considering the effective drug amount and the filling amount.

[0127] (6) Pharmacodynamics evaluation

[0128] In order to perform a pharmacodynamic evaluation of a dry powder inhalation formulation for the prevention or treatment of idiopathic pulmonary fibrosis as a suitable example according to the present invention, first, SD-rats (male, 8 weeks) were acclimatized for one week, and then bleomycin was administered via intratracheal instillation (ITI) at approximately 300 g to induce pulmonary fibrosis.

[0129] Next, as control groups, Negative control (NC), Positive control (PC), and PRF_Oral (daily) group were established, which were administered pirfenidone orally at 30 mg / kg daily from day 7.

[0130] In addition, as a drug administration group, 0.1 mg / kg of Example 1 formulation was administered by inhalation once on day 7, and the details are summarized in Table 8 below.

[0131] GroupNumberDosage formRouteDose / weight(mg / kg)Dose / animal(mg / ea)Formulationdosage(mg)PrescriptionNC6------PC8-Insufflator----PRF_Oral (daily)6Pirfenidone(0.9 ml, solution)Oral30910 mg / mlDailyadministrationM12_0.1 mpk8M12-2 (1:100)Insufflator0.10.033.0 mgSingleadministration

[0132] Accordingly, using the prepared experimental groups (NC, PC, PRF_Oral, M12_0.1 mpk), a lung function test (condition - measurement period: day 8 and day 14 after bleomycin administration / measurement method: analyzing the breathing pattern for 30 minutes by calibrating the rat in the chamber where calibration was performed) was performed using double-chamber plethysmography (DCP) equipment, and the results (* ANOVA, p-value < 0.05 compared with NC, ** ANOVA, p-value < 0.005 compared with NC) are as shown in Figs. 9 to 12. First, the results of the RT (Relaxation time) pattern analysis on the 8th day for each experimental group (NC, PC, PRF_Oral, M12_0.1 mpk) according to Fig. 9 and the results for each experimental group (NC, PC, PRF_Oral, M12_0.1 mpk) according to Fig. 10 Comparing the results of the RT (Relaxation time) pattern analysis on the 14th day, in the case of M12_0.1 mpk, there was no significant difference from PC immediately after drug administration, but 7 days after drug administration, there was no difference from NC, confirming the possibility of an effect in preventing or treating idiopathic pulmonary fibrosis.

[0133] Next, comparing the results of the f(Breathing frequency) pattern analysis on the 8th day by experimental group (NC, PC, PRF_Oral, M12_0.1 mpk) according to Fig. 11 with the results of the f(Breathing frequency) pattern analysis on the 14th day by experimental group (NC, PC, PRF_Oral, M12_0.1 mpk) according to Fig. 12, in the case of M12_0.1 mpk, there is no significant difference from PC immediately after drug administration, but 7 days after drug administration, the difference from NC disappears, confirming the possibility of an effect in preventing or treating idiopathic pulmonary fibrosis.

[0134] And the lungs collected from the prepared experimental groups (NC, PC, PRF_Oral, M12_0.1 mpk) were fixed with formalin, and then H&E or MT staining was performed. For each individual, the upper, lower, middle, and bronchial portions of the lung tissue were captured at a magnification of x10 on each H&E or MT stained lung tissue cross-section slide, and the results are as shown in Table 9 below.

[0135] Accordingly, the captured images were randomly scored by 10 people based on the literature Standardized quantification of pulmonary fibrosis in histological samples, and the results (* ANOVA, p-value < 0.05 compared with NC, # ANOVA, p-value < 0.05 compared with PC, ## ANOVA, p-value < 0.05 compared with PC) are as shown in Fig. 13 below.

[0136] H&EMTNC PC PRF_Oral M12_0.1 mpk

[0137] As shown in Table 9 and Figure 13, in the case of the experimental group of M12_0.1 mpk, there is no difference compared to NC, and by confirming the difference from PC, the possibility of histological therapeutic effect can be confirmed. The embodiments disclosed in the present invention are not intended to limit the technical idea of ​​the present invention, but to explain it, and the scope of the technical idea of ​​the present invention is not limited by these embodiments. The scope of protection should be interpreted by the following claims, and all technical ideas within a scope equivalent thereto should be interpreted as being included in the scope of the rights of the present invention.

Claims

1. A method for manufacturing a dry powder inhalation formulation that can be administered by inhalation using a dry powder inhaler, Step A: Mixing the polypeptide and (Mannitol) prepared as active pharmaceutical ingredients (API) into distilled water; Step B, which involves freeze drying the mixture mixed through the above Step A; and Step C, which comprises grinding the mixture freeze-dried through Step B at least once to powder it; The polypeptide prepared as the above active pharmaceutical ingredient is characterized by being composed of an amino acid sequence consisting of arginine (R, Arg, Arginine) - glycine (G, Gly, Glycine) - aspartic acid (D, Asp, Aspartic acid) - valine (V, Val, Valine) - phenylalanine (F, Phe, Phenylalanine) - proline (P, Pro, Proline) - serine (S, Ser, Serine) - tyrosine (Y, Tyr, Tyrosine) - threonine (T, Thr, Threonine) - lysine (K, Lys, Lysine). Method for preparing a dry powder inhalation formulation for preventing or treating idiopathic pulmonary fibrosis.

2. In paragraph 1, The above step A is characterized in that it is a step of mixing 0.5 to 2 parts by weight of the polypeptide prepared as the active pharmaceutical ingredient and 80 to 120 parts by weight of the mannitol in the distilled water so that the concentration of the polypeptide prepared as the active pharmaceutical ingredient in the distilled water is 200 μg / ml to 300 μg / ml. Method for preparing a dry powder inhalation formulation for preventing or treating idiopathic pulmonary fibrosis.

3. In paragraph 2, The above step B is a step of cooling the mixture mixed through the above step A to a temperature of -45°C to -35°C and then drying it at a temperature of -20°C to 25°C for 8 to 12 days. The above step C is characterized in that the step is a step of milling the mixture freeze-dried through the above step B at least once to prepare a dry powder having a particle size distribution in which D10, which defines a particle size corresponding to 10% of the cumulative volume concentration (%), corresponds to 0.7 μm to 1.3 μm, D50, which defines a particle size corresponding to 50% of the cumulative volume concentration (%), corresponds to 2.5 μm to 3.3 μm, D90, which defines a particle size corresponding to 90% of the cumulative volume concentration (%), corresponds to 4.2 μm to 8.8 μm, and a span value defined by the following mathematical formula 1 corresponds to 1.1 to 2.

6. Method for preparing a dry powder inhalation formulation for preventing or treating idiopathic pulmonary fibrosis.

4. In paragraph 3, The method for manufacturing the above dry powder inhalation formulation for preventing or treating idiopathic pulmonary fibrosis is as follows: It is characterized by further comprising a D step of adding 100 to 300 parts by weight of alpha-lactose monohydrate corresponding to a carrier to the dry powder pulverized through the above C step and then stirring the mixture at a mixing speed of 40 to 50 rpm for 10 to 30 minutes. Method for preparing a dry powder inhalation formulation for preventing or treating idiopathic pulmonary fibrosis.

5. In paragraph 3, The dry powder pulverized through the above step C is characterized in that, when it has a capsule-type usable form that can be inhaled using the dry powder inhaler, it is filled with 10 mg to 20 mg per capsule. Method for preparing a dry powder inhalation formulation for preventing or treating idiopathic pulmonary fibrosis.

6. In paragraph 3, The dry powder pulverized through the above step C is characterized in that it is prepared in a state having a moisture content of 2.5% to 3.7%. Method for preparing a dry powder inhalation formulation for preventing or treating idiopathic pulmonary fibrosis.

7. In paragraph 3, The dry powder pulverized through the above step C is characterized in that it is prepared in a state in which the emitted dose (ED) measured using a next generation pharmaceutical impinger (NGI) corresponds to 70% to 90%, the fine particle fraction (FPF) based on a particle size of 4.4 μm to 4.5 μm corresponds to 40% to 70%, the fine particle fraction (FPF) based on a particle size of 2.8 μm to 2.9 μm corresponds to 20% to 50%, and the mass median aerodynamic diameter (MMAD) corresponds to 3.5 μm to 7.0 μm. Method for preparing a dry powder inhalation formulation for preventing or treating idiopathic pulmonary fibrosis.

8. A dry powder inhalation formulation for the prevention or treatment of idiopathic pulmonary fibrosis manufactured by a method for manufacturing a dry powder inhalation formulation for the prevention or treatment of idiopathic pulmonary fibrosis according to any one of claims 1 to 7.

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