Dry powder pharmaceutical composition for inhalation comprising small molecule LPA1 antagonist and process for preparation thereof

Abstract

Problem: An object of the present invention is to provide a novel approach to the treatment of pulmonary fibrosis including idiopathic pulmonary fibrosis and progressive pulmonary fibrosis. Solution: The present invention provides a dry powder pharmaceutical composition for inhalation, comprising a specific amount of compound represented by Formula (I) or a pharmaceutical acceptable salt thereof, a specific amount of magnesium stearate and a specific amount of lactose, wherein a bulk density, etc. is in a specific range.

Description

DRY POWDER PHARMACEUTICAL COMPOSITION FOR INHALATION COMPRISING SMALL MOLECULE LPA1 ANTAGONIST AND PROCESS FOR PREPARATION THEREOF

[0001] The present invention relates to a new dry powder pharmaceutical composition for inhalation, a process for the preparation of the composition, and a method for the prevention and / or the treatment of pulmonary fibrosis, in particular, idiopathic pulmonary fibrosis, using the composition.

[0002] Idiopathic pulmonary fibrosis (IPF) is a progressive, rare disease of unknown cause for which no fundamental treatment exists. The incidence of IPF is 2.5 persons / 100,000 persons in Japan and 1 person / 10,000 persons worldwide. IPF is a chronic, progressive disease that causes respiratory deterioration due to fibrosis of the lungs, and on average, about half of patients die within about five years of diagnosis.

[0003] Although nintedanib / pirfenidone, for example, is considered as a therapeutic agent for IPF, its mechanism of action, such as inhibition of multikinase and effect on inflammation-inducing factors, has not been fully elucidated, and its therapeutic effect is limited to suppression of lung function decline, not halting progression or restoration of function.

[0004] It was reported that (R)-1-[4’-(5-Chloro-3-{[(1-phenylethoxy)carbonyl]amino}thiophen-2-yl)-2’-methoxy-[1,1’-biphenyl]-4-yl]cyclopropanecarboxylic acid is a small molecule LPA1 antagonist and targets IPF (Patent Literature 1).

[0005] Further, many inhalants that are delivered directly to the lungs have been developed to treat lung diseases, etc. (Patent Literatures 2-6).

[0006] [Patent Literature 1] WO2014 / 104372 [Patent Literature 2] JP5873616 [Patent Literature 3] JP5964939 [Patent Literature 4] JP7305842 [Patent Literature 5] US10,238,601 B2 [Patent Literature 6] US10,946,029 B2

[0007] An object of the present invention is to provide a novel approach to the treatment of IPF with inhalable formulation for nasally or orally inhalation.

[0008] As a result of conducting extensive studies to solve the above problem, the inventors of the present invention found that (R)-1-[4’-(5-Chloro-3-{[(1-phenylethoxy)carbonyl]amino}thiophen-2-yl)-2’-methoxy-[1,1’-biphenyl]-4-yl]cyclopropanecarboxylic acid, which has an LPA1 antagonist, a new mechanism that is different from existing drugs for the treatment of IPF, enables a new approach by the inhalation route of administration, and as a result has completed the present invention.

[0009] That is, the present invention provides: (Item 1) A dry powder pharmaceutical composition for inhalation, comprising 1 to 20 wt% of a compound represented by Formula (I): or a pharmaceutical acceptable salt thereof, 0.01 to 5 wt% of magnesium stearate and 75 to 98.99 wt% of lactose with respect to the weight of the composition. (Item 2) The composition according to Item 1, wherein a bulk density is in the range of 0.3 to 1 g / ml. (Item 3) The composition according to claim 1 or 2, a Hausner index is in the range of 1-2. (Item 4) The composition according to any one of Items 1 to 3, wherein a fine particle fraction (FPF) with less than 5 μm is 10 to 80%. (Item 5) The composition according to any one of Items 1 to 4, wherein a mass median aerodynamic diameter (MMAD) is 0.5 to 19 μm. (Item 6) The composition according to any one of Items 1 to 5, wherein a blend uniformity is in the range of 50 to 150 mg / g. (Item 7) A process for the preparation of the composition according to any one of Items 1 to 6, comprising a step of mixing HL001 with magnesium stearate and lactose using a high-shear mixer wherein a mixing energy is 0.1 to 2 mJ / carrier. (Item 8) The process according to Item 7, wherein the step of mixing comprises the following steps: Step 1 of mixing lactose with magnesium stearate at 400 to 1700 rpm and 0.1 to 1.5 mJ / carrier to coat the lactose; and Step 2 of mixing the coated lactose with HL001 at 400 to 1700 rpm and 0.1 to 2 mJ / carrier. (Item 9) A method for the prevention and / or the treatment of pulmonary fibrosis, wherein a therapeutic effective amount of the composition according to any one of Items 1 to 6 is administered by inhalation to a patient need thereof. (Item 10) The method according to Item 9, wherein the composition is administered at a dose of 0.001 to 60 mg / kg once a day. (Item 11) The method according to Item 9 or 10, wherein the composition is administered in combination with the other agents including the drug for treatment of pulmonary fibrosis. (Item 12) The method according to any one of Items 9 to 11, wherein pulmonary fibrosis includes idiopathic pulmonary fibrosis and progressive pulmonary fibrosis. (Item 13) The composition according to any one of Items 1 to 6 for use in the prevention and / or the treatment of pulmonary fibrosis including idiopathic pulmonary fibrosis and progressive pulmonary fibrosis. (Item 14) Use of HL001 for the preparation of the composition according to any one of Items 1 to 6 for use in the prevention and / or the treatment of pulmonary fibrosis including idiopathic pulmonary fibrosis and progressive pulmonary fibrosis.

[0010] The dry powder pharmaceutical composition for inhalation of the present invention is administered by inhalation, so it can efficiently deliver the therapeutic drug concentration to the target organ, in circumstances the lung or any other organs that develop abnormal fibrosis, and has both high efficacy and tolerability from the viewpoint of reducing exposure to other organs. In addition, the dry powder pharmaceutical composition for inhalation is expected to reduce the toxicity of the formulation itself, which has been a problem with existing drugs. High concentrations of LPA antagonists, HL001 can be delivered to the target organ, the lung, by a route and systemic exposure range where toxicity is not a problem. Further, the dry powder pharmaceutical composition has the advantage of higher yields at scaled-up production scales.

[0011] Fig. 1 shows HL001 amount on filters for tested formulations measured by HPLC-UV.Fig. 2 shows the fine particle fraction (FPD<5 μm) plotted against HL001 on filters for tested formulations.Fig. 3 shows the best condition of each Example.Fig. 4 shows the percentage body weight change over time. Data is shown as mean ± SEM.Fig. 5 shows the right lung lobe weights at termination. Data is presented as the individual values and mean ± SEM. A Mann-Whitney test was used to compare bleomycin untreated vs naive control (p: 0.0001). Multiple group comparisons: non-parametric Kruskal Wallis test was used (p: 0.0045), followed by Dunn’s Multiple comparison test for comparisons between groups. Selected comparisons were: HL001 p.o vs bleo untreated, HL001 inh low vs bleo untreated, HL001 inh high vs bleo untreated, BMS p.o low vs bleo untreated, BMS p.o high vs bleo untreated. P<0.05 was considered significant. See Table 17 for mean values and p values.Fig. 6 shows the modified Ashcroft score, from the left lung lobe. Data is presented as individual values and mean ± SEM. A Mann-Whitney test was used to compare bleomycin untreated vs naive control (p: <0.0001). Multiple group comparisons: non-parametric Kruskal Wallis test was used (p: <0.0001), followed by Dunn’s Multiple comparison test for comparisons between groups. Selected comparisons were: HL001 p.o vs bleo untreated, HL001 inh low vs bleo untreated, HL001 inh high vs bleo untreated, BMS p.o low vs bleo untreated, BMS p.o high vs bleo untreated. P<0.05 was considered significant. See Table 18 for mean values and p values.Fig. 7 shows Standard curve for the hydroxyproline assay. Data is presented as mean values ± SD.Fig. 8 shows the hydroxyproline values per right lung lobe 2. A Mann-Whitney test was used to compare bleomycin untreated vs naive control (p: 0.0001). Multiple group comparisons: non-parametric Kruskal Wallis test was used (p: <0.0001), followed by Dunn’s Multiple comparison test for comparisons between groups. Selected comparisons were: HL001 p.o vs bleo untreated, HL001 inh low vs bleo untreated, HL001 inh high vs bleo untreated, BMS p.o low vs bleo untreated, BMS p.o high vs bleo untreated. P<0.05 was considered significant. See Table 19 for mean values and p values.

[0012] 1.　　DRY POWDER PHARMACEUTICAL COMPOSITION FOR INHALATION (1-1) Composition (1-1-1) Active Pharmaceutical Ingredient The dry powder pharmaceutical composition for inhalation of the present invention comprises (R)-1-[4’-(5-Chloro-3-{[(1-phenylethoxy)carbonyl]amino}thiophen-2-yl)-2’-methoxy-[1,1’-biphenyl]-4-yl]cyclopropanecarboxylic acid represented by Formula (I): or a pharmaceutical acceptable salt thereof (hereinafter also collectively referred to as “HL001”) as an active pharmaceutical ingredient. Unless the effect of the present invention is inhibited, the pharmaceutical acceptable salt is not particularly limited, and includes, for example, a metal salt such as a sodium salt, a potassium salt, a calcium salt or a magnesium salt; an inorganic salt such as an ammonium salt; or an organic amine salt such as a triethylamine salt or a guanidine salt, etc. HL001 is available commercially, or can be produced by the known method, for example, by the method disclosed in Patent Literature 1. Further, HL001 can be present as a hydrate or a solvate, and they are also included within the present invention.

[0013] The composition of the present invention comprises 1 to 20 wt%, preferably 5 to 20 wt%, more preferably 8 to 15 wt%, particularly preferably 9 - 12 wt%, and especially preferably 10 wt% of HL001, with respect to the weight of the composition.

[0014] Preferably, HL001 may be micronized by the known method, for example, in a jet mill using 2 bar in milling pressure, and 4 bar of ejector with a feed about 1 g / min. HL001 is preferably micronized to have 0.5 to 19 μm, more preferably 1 to 10μm, particularly preferably 2 to 5μm, especially preferably 2.5 μm of primary particle size.

[0015] (1-1-2) Magnesium Stearate (MgSt) In the composition of the present invention, MgSt is used as a coating agent, and is preferred because it increases the fine particle fraction, the tower efficiency and the filter amounts of the composition. The composition comprises 0.01 to 5 wt%, preferably 0.1 to 5 wt%, more preferably 0.25 to 2.5 wt%, particularly preferably 0.5 to 2 wt%, and especially preferably 1 % of MgSt, with respect to the weight of the composition.

[0016] (1-1-3) Lactose In the composition of the present invention, lactose is used as a carrier. The composition comprises 70 to 99 wt%, more preferably 80 to 95 wt%, particularly preferably 85 to 90 wt%, and especially preferably 89 wt% of lactose, with respect to the weight of the composition. Lactose is preferably lactose hydrate, more preferably lactose monohydrate. Further, the median particle size of lactose is preferably 33 to 214 μm (for example, Respitose SV003, Lactohale 206 or Lactohale 100), more preferably 33 to 154 μm (for example, Respitose SV003 and Lactohale 206), particularly preferably 31 to 95 μm (for example Respitose SV003) and especially preferably 61 μm. Further, the lactose is preferably coated with MgSt.

[0017] (1-2) Parameters In the composition of the present invention, the parameters including a bulk density, a Hausner index, a fine particle fraction (FPF) with less than 5 μm, a mass median aerodynamic diameter (MMAD) and a blend uniformity are preferably in the specific range. They may be measured using know methods, for example, the method provided in Examples. (1-2-1) Bulk Density The bulk density is in the range of for example 0.3 to 1 g / ml, preferably 0.4 to 0.8 g / ml, more preferably 0.45 to 0.7 g / ml and particularly preferably 0.5 to 0.65 g / ml. The bulk density may be increased by higher mixing energy or higher amount of MgSt. (1-2-2) Hausner index The Hausner index is in the range of for example 1-2, preferably 1-1.45, and particular preferably 1-1.34. (1-2-3) FPF with less than 5 μm In the composition of the present invention, the FPF with less than 5 μm is in the range of for example 10 to 80 %, preferably 20 to 60 %, more preferably 30 to 50%, especially preferably 40-45 %, and particularly preferably 23 to 49%. The FPF with less than 5 μm may be increased by higher mixing energy or higher amount of MgSt. (1-2-4) MMAD In the composition of the present invention, the MMAD is in the range of for example 0.5 to 19 μm, preferably 1 to 10 μm, more preferably 1 to 5 μm, and particularly preferably 1.5 to 3 μm. The MMAD may be increased by higher mixing energy or higher amount of MgSt. (1-2-5) Blend uniformity In the composition of the present invention, the blend uniformity is in the range of for example 50 to 150 mg / g, preferably 70 to 130 mg / g, more preferably 80 to 120 mg / g, and particularly preferably 87 to 115 mg / g. The blend uniformity may be decreased by higher amount of MgSt.

[0018] In the composition of the present invention, a delivered dose after high mixing energy is for example 1500 - 3000 μg / dose, preferably 1600 - 2500 μg / dose, more preferably 1800 - 2300 μg / dose and particularly preferably 1900-2200 μg / dose.

[0019] 2.　　PREPARATION OF DRY POWDER PHARMACEUTICAL COMPOSITION FOR INHALATION The dry powder pharmaceutical composition for inhalation of the present invention may be prepared in a process comprising a step of mixing HL001, preferably micronized HL001 with magnesium stearate and lactose using a high-shear mixer wherein a mixing energy is 0.1 to 2 mJ / carrier, preferably 0.2 to 2 mJ / carrier, more preferably 0.2 to 1.7 mJ / carrier, particular preferably 0.3 to 1.5 mJ / carrier, and especially preferably 0.4 to 1.3 mJ / carrier.

[0020] Further, the step of mixing as above may comprise the following steps: Step 1 of mixing lactose with magnesium stearate to coat the lactose; and Step 2 of mixing the coated lactose with HL001, preferably micronized HL001. Recommended mixing settings for Step 1 is mixing at speed 400 to 1700 rpm and 0.1 to 1.5 mJ / carrier, preferably 500 to 1600 rpm and 0.2 to 1.2 mJ / carrier, more preferably 600 to 1500 rpm and 0.3 to 1 mJ / carrier, and particularly preferably 800 to 1300 rpm and 0.4 to 0.9 mJ / carrier. Preferred mixing settings for Step 2 is mixing at mixing speed 400 to 1700 rpm and 0.1 to 2 mJ / carrier, more preferably 450 to 1600 rpm and 0.2-1.7 mJ / carrier, particularly 500 to 1500 rpm and 0.3 to 1.5 mJ / carrier, and especially 550 rpm to 1300 rpm and 0.4 to 1.3 mJ / carrier.

[0021] Preferably, MgSt is mixed at the high concentration (1 wt%) at the low mixing speed (800 to 900 rpm) to obtain the composition having the high fine particle fraction.

[0022] 3.　　USE OF DRY POWDER PHARMACEUTICAL COMPOSITION FOR INHALATION The dry powder pharmaceutical composition for inhalation of the present invention may be used by inhalation, for example, nasally or orally inhalation, preferably nasally inhalation in a method for the prevention and / or the treatment of pulmonary fibrosis including idiopathic pulmonary fibrosis and progressive pulmonary fibrosis. Here, a therapeutic effective amount of the composition may be administered by inhalation, for example, nasally or orally inhalation, preferably nasally inhalation to a patient need thereof. The above term “therapeutic effective amount” means the amount of HL001 that produces the desired pharmaceutical effect against pulmonary fibrosis, preferably idiopathic pulmonary fibrosis, when delivered to the lungs by the composition of the present invention. The therapeutic effective amount may be 0.001 to 60 mg / kg, preferably 0.01 to 30 mg / kg, more preferably 0.1 to 10 mg / kg, especially preferably 0.3 to 1 mg / kg. Further, the composition may be administered by inhalation once a day.

[0023] The composition of the present invention may be administered in combination or association, simultaneously, separately or sequentially with other therapeutic agents including the drug for treatment of pulmonary fibrosis, can be consistent with the composition may be administrated as 2ndline therapy.

[0024] The composition of the present invention may comprise only three ingredients of HL001, MgSt and lactose, or unless the effect of the present invention is inhibited, may comprise an additional ingredient other than these three ingredients. Such an additional ingredient may include any therapeutic agent including drug for the prevention and / or the treatment of pulmonary fibrosis, such as Pirfenidone, Nintedanib, Pamrevlumab, Tyvaso, BMS-986278 and Saracatinib; and pharmaceutical acceptable excipients such as disintegrators, binders, fillers, tonicity agents, stabilizers, antioxidants, surfactants, carriers, diluents or lubricants used in formulating pharmaceutical products. Here, BMS-986278 is represented by the following formula:

[0025] 4.　　EXAMPLESExample 1: Micronisation of HL001 About 12 g of HL001 was fed into the milling chamber of 2.5” (radius) Titanium nitride coated jet mill (Airfilco Jet Mill) using 2bar in milling pressure, and 4bar of ejector with a feed about 1g / min. Out of the 12g, almost 11g of micronized HL001 were collected, resulting in a yield of about 89%.

[0026] Example 2: Preparation and Assessment of Dry Powder Formulation (1) HL001 micronized as above was mixed with lactose and optional magnesium stearate (MgSt) to prepare the dry powder formulations: D1-1 to D1-6. The six formulations prepared as above were batched with a batch size of 200 g. The 12 formulation samples were filled in capsules and packaged in aluminium pouch prior to testing with NGI and in some cases delivered dose (DD). ICO cap has been selected for used as a capsule inhaler. High shear mixing and filling were performed at room temperature and at a humidity of ≦35%RH. The mixing was performed using a high shear mixer, Diosna, equipped with a 1 liter jacket bowl (not tempered or cooled). The batch size was 200g. Further, the mixing process includes a Mixing Method A or B.

[0027] (1) Mixing Method A, without MgSt The mixing was performed in one step. Lactose carrier (D1-1: Lactohale 206, D1-3: Respitose SV003, D1-5: Lactohale 100) and the micronized HL001 were added to the mixing vessel in the following order: half the amount lactose carrier, the micronized HL001 and half the amount of lactose carrier. Mixing: 200 rpm for 1 min, then mixing speed and mixing time according to Table 1. The mixing was stopped two times. The temperature of the powder bed was measured after each stop. After stop 1, blend uniformity samples and a 6 g sample were withdrawn. The formulations were then sieved with a 1 mm sieve. Bulk density was measured in duplicates. The formulations were filled into 30 ml and 300 ml container with desiccant caps. Samples of 10 g and 90 g were withdrawn.

[0028] (2) Mixing Method B, with MgSt The mixing was performed in two steps. The lactose carrier was coated with MgSt in the following Mixing Step 1. The micronized HL001 was mixed with the coated lactose carrier in the following Mixing Step 2. (2-1) Mixing Step 1, coating Lactose carrier (D1-2: Lactohale 206, D1-4: Respitose SV003, D1-6: Lactohale 100) and MgSt were added to the mixing vessel in the following order: half the amount lactose carrier, MgSt and half the amount of lactose carrier. Mixing: 200 rpm for 1 min, then mixing speed and mixing time 1 according to Table 1. The walls of the Diosna bowl were scraped. (2-2) Mixing Step 2 Half the amount of the coated lactose carrier was removed from the bowl with a spoon. The micronized HL001 was added to the mixing bowl followed by the remaining amount coated carrier. Mixing: 200 rpm 1 min, then mixing speed and mixing time according to Table 1.

[0029] The mixing was stopped two times. The temperature of the powder bed was measured after each stop. After stop 1, blend uniformity samples and a 6 g sample were withdrawn. Formulation was then sieved with a 1 mm sieve. Bulk density was measured in duplicates. The formulation was filled into 30 ml and 300 ml container with desiccant caps. Samples of 10 g and 90 g were withdrawn.

[0030] The design was presented in Table 1. The quality of lactose carrier, amount magnesium stearate and mixing energy in step 2 were varied. The Mixing Step 2 obtained a mixing energy after stop 1 and 2 of 0.44-0.70 mJ / carrier and 0.87-0.91 mJ / carrier, respectively. Since the three lactose carrier qualities have three particle sizes, the mixing speed and mixing time were adjusted to obtain same mixing energy for the three lactose carriers at one mixing stop of Mixing Step 2. The mixing energy for the experiments is provided in Table 3. The complete composition was summarized in Table 2. All batches were sampled at two time points, which means that the design resulted in 12 samples.

[0031]

[0032]

[0033]

[0034] The dry powder formulations: D1-1 to D1-6 prepared as above were evaluated as follows. The results were shown in Table 6. (1) Blend uniformity Method used for assay of blend uniformity samples were below. 30.00 mg ± 2.50 mg of the formulations were accurately weighed into a 25.0 mL volumetric flask. Thereto were added about 20 mL of diluent (prepared by mixing 600 mL acetonitrile and 400 mL water, and being stable for 2 weeks stored at ambient temperature) and dispersed by shaking (use a vortex apparatus) the flask. The formulation stuck were rinsed down to the flask’s neck before continuing with the sample preparation. The solution of the flask was diluted to volume with the above diluent so as to provide a sample of 0.120 mg / ml. The determination of HL001 was carried out by means of reversed phase HPLC with UV detection at 311 nm. Quantification was done by external calibration and identification by comparison of retention times and UV-spectra. The solutions may be sensitive to light and should be protected. The column was equilibrated for about 60 minutes with mobile phase A (prepared by adding 1.0 mL TFA to 1000 mL water, and being stable for 1 week stored at ambient temperature) and mobile phase B (acetonitrile) or until a stable baseline was obtained. A typical pressure for the system during analysis was about 4200 psi. Standard solution S1 (nominal concentration of HL001: 0.12000 mg / ml) was injected 5 times followed by injection of blank (the above diluent). Single injections of the standard solution and samples were made. The standard solution was injected after every 10th sample injection and at least twice during the analysis. The sequence with injection of standard solution was always ended. (2) Delivered dose and Fine particle assessment with NGI Method used for Fine particle assessment with Next Generation Impactor (NGI) and Delivered Dose were below.

[0035] (2-1) Procedure (2-1-1)Delivered dose measurement After dosing to a filter (Respirgard II, model 303EU, Timik AB or equivalent), the filter was put in a beaker, and thereto added Internal standard solution [prepared by dissolve 200 mg butagin per 1000 mL Ethanol / 0.01M buffer 70 / 30 v / v (to facilitate dissolution, the butagin was dissolved first in 5mL ethanol)] with a calibrated dispenser. The filter was rinsed 5 times.

[0036] (2-1-2) Fine particle assessment with the NGI (i) The connection of the NGI to vacuum source was arranged as specified in EU Pharmacopeia. (ii) Use a Pre-separator and humidifier at the inlet Coat the collection cups with Brij / glycerol solution. (iii) After dosing, 15 mL the Internal Standard solution was added with a calibrated dispenser, to all NGI parts (i.e. Throat+adapter, Pre-separator (if used) NGI-cups and filter) (iv) the samples were dissolved in the throat+adapter, Pre-separator (if used). (v) Filter extraction was performed as for the delivered dose measurement described above but with 15 mL Internal standard solution per NGI- filter. (vi) The samples from the NGI parts and the filter were transferred to HPLC vials. The HPLC instrumentation were below. An HPLC-system comprised of an isocratic pump, a column, an auto injector, a column oven and a variable wavelength detector plus a chromatography data system. The HPLC settings were shown in Table 4. The method was linear between 37-2140 μg / 15mL Internal standard solution (IS).

[0037] Device used: ICOcap, tests performed at 71 L / min (=2kPa).

[0038] (2-2) Data handling and calculations (2-2-1) Delivered dose The amount for each dose in the filters was calculated (μg / dose). The mean value and Relative standard deviation per batch were calculated. (2-2-2) Fine particle assessment with the NGI If requested, the total recovered dose, MMAD, GSD and fine particle dose(s) and fraction(s) were calculated according to the general instructions according to European pharmacopeia 2.9.18 or USP (601). Copley CITDAS application could be used for calculations.

[0039] (3) Bulk density The bulk density was measured using two 20 mL vials. Duplicate measurements were performed in connection with manufacturing.

[0040] (4) Tower Efficiency A nominal dose of 1.0 mg / kg (using an estimated animal weight of 0.4 kg) was tested for each formulation in the inhalation tower. Each formulation was tested in triplicate and each run lasted for 5 minutes. An aerodynamic particle sizer (APS) was used to follow the particle concentration and size distribution during each run in real-time. A total filter, connected to one of the tower ports, was weighed before and after each run. For two formulations, a 7-stage Marple impactor fitted with filters was connected to one of the animal ports to determine the particle size (MMAD). The amount of HL001 on total filters and impactor stage plates were quantified with HPLC-UV. The inhalation tower settings for the runs are shown in Table 5.

[0041]

[0042]

[0043] To assess the manufacturing processes of the dry powder formulations, the excipients, compositions, mixing energies, and mixing steps are assessed as batches.

[0044] The bulk density was in the range 0.545 - 0.629 g / mL. The coated batches obtained a higher bulk density compared to non-coated batches with same carrier. The bulk density tends to increase with particle size of lactose carrier for the formulations with non-coated carrier. The fine particle fraction <5 μm was in the range 24 - 43%. The highest fine particle fraction (42-43%) was reached for batches containing MgSt and the lactose carrier Lactohale 206 (D1-2) or Respitose SV003 (D1-4) at high mixing energy. Batches with MgSt (D1-2, D1-4 and D1-6) obtained a lower MMAD than batches without MgSt (D1-1, D1-3 and D1-5). The delivered dose for D1-1, D1-2 and D1-3 after high mixing energy was about 2000 μg and the dose variation was low (2.8-4.3%). The capsule retention was low (2-3% of total dose), which shows that the emptying process in the capsule is almost complete at 2kPa / 71 L / min.

[0045] The particle generation was partially uneven for D1-1, which may be an indicator for issues with the cake integrity that may cause problems, while it was more even for the other formulations. The presence of magnesium stearate in formulations resulted in increased filter amounts as shown in Table 6. A similar pattern was seen for the measured amount of HL001 on total filters and the tower efficiency. Tower efficiency is calculated as the measured HL001 on total filters divided by the theoretical filter deposition and is given in %. The measured amount of HL001 on total filters and the tower efficiency give an indication on the fine particle fraction in the formulation. Among the formulations that contained magnesium stearate, D1-2, D1-4 and D1-6, we observed that D1-4 generated the highest amount of HL001 on total filters (Fig. 1) and the highest tower efficiency (Table 6). The mean amount of HL001 on total filters was 58 μg for D1-4 and 50 and 51 μg for D1-2 and D1-6, respectively. The mean tower efficiency was 50% for D1-4 and 41 and 42% for D1-2 and D1-6, respectively. The amount of particles measured on total filters is dependent on the fine particle fraction of HL001 in a formulation. The fine particle fraction for the formulations were therefor plotted against HL001 on the total filters (Fig. 2). There was a high degree of correlation between these parameters with a R2-value of 0.95 and D1-4 had the highest fine particle fraction and amount of HL001 on total filters.

[0046] Form the above results, Formulation D1-4 stop 2 was the formulation that was selected to be used for the pre-clinical studies due to the high fine particle fraction and high tower efficiency.

[0047] Example 3: Preparation and Assessment of Dry Powder Formulation for inhalation (1) HL001 micronized as above was mixed with lactose and optional magnesium stearate (MgSt) to prepare the dry powder formulations: D2-1 to D2-12. The 12 formulations prepared as above were batched with a batch size of 200 g. The formulations were filled in capsules and packaged in aluminium pouch prior to testing with NGI and some cases delivered dose (DD) using ICO cap as device. High shear mixing and filling, mixing was performed in the similar manner to in Example 1. Further, the mixing process includes a Mixing Method A or B.

[0048] (1) Mixing method A, with MgSt The mixing was performed in two steps. The Lactose carrier was coated with MgSt in the following Mixing Step 1. The micronized HL001 was mixed with the coated carrier in the following Mixing Step 2. (1-1) Mixing Step 1, coating Lactose carrier and MgSt were added to the mixing vessel in the following order: half the amount lactose carrier, MgSt and half the amount of lactose carrier. Mixing: 200 rpm for 1 min, then mixing speed and mixing time 1 according to Table 8. The walls of the Diosna bowl were scraped. (1-2) Mixing Step 2 Half the amount of the coated carrier was removed from the bowl with a spoon. The micronized HL001 was added to the mixing bowl followed by the remaining amount coated carrier. Mixing: 200 rpm 1 min, then mixing speed and mixing time according to Table 8.

[0049] The mixing was stopped one and two times for 7 and 5 batches, respectively. The temperature of the powder bed was measured after each stop. After stop 1, a 6 g sample were withdrawn. Blend uniformity was withdrawn after stop 1 for 3 batches and after final stop for 2 batches. Formulations were then sieved with a 1 mm sieve. Bulk density was measured in duplicates. The formulations were filled into 300 ml container with desiccant caps.

[0050] (2) Mixing Method B, without MgSt The mixing was performed in one step. Lactose carrier and the micronized HL001 were added to the mixing vessel in the following order: half the amount lactose carrier, the micronized HL001 and half the amount of lactose carrier. Mixing: 200 rpm for 1 min, then mixing speed and mixing time according to Table 8. The temperature of the powder bed was measured after mixing. Formulation was then sieved with a 1 mm sieve. Bulk density was measured in duplicates. The formulation was filled into 300 ml container with desiccant caps.

[0051] Twelve batches with a batch size of 200 g were manufactured according to the design presented in Tables 7 and 8. The latter table displays the mixing time and mixing speed used to obtain the desired mixing energy in Table 7. The five first batches (D2-1 to D2-5) generated 9 samples, which were part of a full factorial design with one center point and no replicates. The factors varied are mixing energy and mixing speed for the Mixing Step 2 and amount MgSt. Mixing energy during coating and amount HL001 were varied for D2-7 to D2-11. D2-6 was mixed to two stops and these samples display together with the samples from D1-4 how the mixing energy affects fine particle fraction. D2-12, which contained no MgSt, was mixed with same mixing energy as D1-3 stop 1. D2-12 and D1-3 have different mixing speed and mixing time. Seven and five batches were mixed to one and two stops, respectively. A sample of 6 g was withdrawn after mixing stop 1 for the two stops batches. The design resulted in 17 samples. The samples were filled in capsules with a fill weight of 25 mg. D2-11 was also filled in capsules with a fill weight of 30 mg. The complete composition were summarized in Table 9. The samples were evaluated as below.

[0052]

[0053]

[0054]

[0055] The dry powder formulations: D2-1 to D2-12 prepared as above were evaluated as follows. The results were shown in Table 10. (1) Assay and related substances Method used for assay and related substances was below. 30.00 mg ± 2.50 mg of the formulations were accurately weighed into a 25.0 mL volumetric flask. Thereto were about 20 mL of diluent (prepared by mixing 600 mL acetonitrile and 400 mL water, and being stable for 2 weeks stored at ambient temperature) and dispersed by shaking (use a vortex apparatus) the flask. The formulation stuck were rinsed down to the flask’s neck before continuing with the sample preparation. The solution of the flask was diluted to volume with the above diluent so as to provide a sample of 0.120 mg / ml. The determination of assay and related substances of HL001 was carried out by means of reversed phase HPLC with UV detection at 311 nm. Quantification was done by external calibration and identification by comparison of retention times and UV-spectra. The solutions may be sensitive to light and should be protected. The column was equilibrated for about 60 minutes with mobile phase A (prepared by adding 1.0 mL TFA to 1000 mL water, and being stable for 1 week stored at ambient temperature) and mobile phase B (acetonitrile) or until a stable baseline was obtained. A typical pressure for the system during analysis was about 4200 psi. Standard S1 (nominal concentration of HL001: 0.12000 mg / ml) was injected 5 times followed by injection of blank (the above diluent) and Quantitation limit solution (QL solution). The QL solution was prepared as follows. An automatic pipette was used to transfer 100 μL of Standard S1 to a 100.0 mL volumetric flask, which was diluted to volume with the above diluent to prepare the OL solution. It was stored and protected from light. The QL solution must be freshly prepared at each time of analysis. This solution corresponded to 0.1% of HL001 in the formulation. Single injections of standard solution and samples were made. The standard solution was injected after every 10th sample injection and at least twice during the analysis. The sequence with injection of standard solution was always ended.

[0056] (2) Delivered dose and Fine particle assessment with NGI Fine particle assessment with Next Generation Impactor (NGI) and Delivered Dose were performed according to the method provided in the item “(2) Delivered dose and Fine particle assessment with NGI” of Example 2 except Device used: ICOcap, tests performed at 71 L / min (=2kPa) and 51L / min (=1kPa).

[0057] (3) Bulk density The bulk density was measured using two 20 mL vials. Duplicate measurements were performed in connection with manufacturing.

[0058]

[0059] The blend uniformity was measured on 5 batches (D2-1, D2-3, D2-5, D2-9 and D2-10) after first mixing stop. Four of the batches showed good homogeneity (<1% RSD) and one batch (D2-10) obtained an acceptable homogeneity (3.5% RSD). The fine particle fraction <5 μm of the twelve batches was in the range 13% to 46% of delivered dose. The delivered dose for four (D2-3, D2-4, D2-5 and D2-11) of the formulations with 10% added HL001 and two formulations (D2-9 and D2-10) with 12% added HL001 was in the range 1928 to 2082 μg / dose and 2400 to 2518 μg / dose, respectively. The delivered dose RSD was 4-6%. The selected composition (D1-4 and D2-3) after Examples 2 and 3 was 10% HL001, 1% MgSt and 89% Respitose SV003 and selected fill weight is 25 mg. Preferred mixing process in 1 liter bowl for mixing step 2 is mixing at mixing speed 1100 rpm or 900 rpm to 0.9 mJ / carrier. The recommended mixing settings for mixing step 1 is mixing at speed 1100 rpm to 0.46 mJ / carrier.

[0060] Some of the trends displayed in Example 3: (i) The temperature in powder bed after mixing increased with increasing mixing speed, mixing energy and amount MgSt in the formulations; (ii) Formulation containing a high MgSt concentration (1%), which was mixed at a low mixing speed (900 rpm) obtained a high fine particle fraction; and (iii) MMAD is lower for formulation with high MgSt concentration (1%).

[0061] Example 4: Preparation and Assessment of Dry Powder Formulation for inhalation (Scale up) HL001 micronized as above was mixed with lactose and magnesium stearate (MgSt) to prepare the dry powder formulations: PD-1 to PD-3. The three batches prepared above were scaled up to a batch size of 1 kg. These formulations were filled in capsules and packaged in aluminum pouch prior to testing with NGI and some cases delivered dose using ICO cap as device. High shear mixing and filling was performed in the similar manner to in Example 1. The mixing was performed using a high shear mixer, Diosna, equipped with a 4 liter jacket bowl (not tempered or cooled). The batch size was 1 kg.

[0062] The mixing was performed in two steps. The lactose carrier was coated with MgSt in the following Mixing Step 1. The micronized HL001 was mixed with the coated lactose carrier in the following Mixing Step 2.

[0063] (1) Mixing Step 1, coating Lactose carrier and MgSt were added to the mixing vessel in the following order: half the amount lactose carrier, MgSt and half the amount of lactose carrier. Mixing: 200 rpm for 1 min, then mixing speed and mixing time 1 according to Table 11. The walls of the Diosna bowl were scraped. (2) Mixing Step 2 Half the amount of the coated carrier was removed from the bowl with a spoon. The micronized HL001 was added to the mixing bowl followed by the remaining amount coated carrier. Mixing: 200 rpm 1 min, then mixing speed and mixing time according to Table 11.

[0064] The mixing was stopped one and four times for 2 and 1 batches, respectively. The temperature of the powder bed was measured after each stop. After stop 1, stop 2 and stop 3 for formulation PD-3, a 6 g sample was withdrawn. A 1 g sample was withdrawn after each stop of PD-3. Blend uniformity was withdrawn after stop 1 for 1 batch and after final stop for 2 batches. The formulations were then sieved with a 1 mm sieve. Bulk density was measured in duplicates. The formulations were filled into 300 ml container with desiccant caps. The samples were filled in capsules with a fill weight of 25 mg. The complete composition and the batch formulas are summarized in Tables 12 and 13.

[0065]

[0066]

[0067]

[0068] The dry powder formulations: PD-1 to PD-3 prepared as above were evaluated as follows. The results were shown in Table 14. (1) Blend uniformity, wall samples and assay Blend uniformity, wall samples and assay were performed according to the method provided in the item “(1) Assay and related substances of Example 3. (2) Delivered dose and Fine particle assessment with NGI Fine particle assessment with Next Generation Impactor (NGI) and Delivered Dose were performed according to the method provided in the item “(2) Delivered dose and Fine particle assessment with NGI” of Example 2. Device used: ICOcap, tests performed at 71 L / min (=2 kPa), 51L / min (=1 kPa) and 90 L / min (=2.9 kPa) (3) Bulk density The bulk density was measured using two 20 mL vials. Duplicate measurements were performed in connection with manufacturing. (4) Tapped density and hausner index The tapped density was measured with a Tapped density tester using a 100 mL measuring cylinder. Powder (90-100 mL) was added gently to the measuring cylinder and the weight and volume were measured and poured bulk density was calculated. The volume was also measured after 10, 500 and 1250 taps and tapped density was calculated after 1250 taps. Haunser index (HI) is the ratio between tapped density and bulk density.

[0069]

[0070] The blend uniformity measurement displayed that the three 1 kg batches obtained 97.2 to 99.6 mg HL001 / g powder, which is close to the nominal content of 100 mg HL001 / g powder. The hausner index of the three batches was 1.30 to 1.35, which means that the flowability is passable to poor. This is expected from this type of formulation and should therefore be suitable for commercial capsule filling. The fine particle fraction <5 μm of PD-1, PD-2 and PD-3 (4 stops) was 45%, 62% and 33-52% of delivered dose, respectively. The delivered dose was 2031 μg / dose to 2154 μg / dose for the four samples. The NGI measurements of PD-1 were performed at three flows (51 L / min, 71 L / min and 90 L / min). The fine particle fraction at low flow 51 L / min was about 6% lower than at 71 L / min, while the fine particle fraction obtained a similar level at both 71 L / min and high flow 90 L / min. All of the PD-1, 2, 3 were proper for the manufacturing condition, but both PD-2 and PD-3 indicated that there was an opportunity to increase and optimize the fine particle dose even more. The robustness of PD-1 was considered high due to the supporting data from PD-3 and D2-11. The robustness of the mixing process of PD-2 should be investigated further if this mixing process is used for the product.

[0071] Best conditions of each of Examples 2-3 are summarized in Fig. 3. D1-4 was used in Example 5.

[0072] Example 5: PK assessments (1) Inhalation administration Assessment of the pharmacokinetic profile of HL001, after a three-day repeated administration, once daily, via nose-only dry powder inhalation, in plasma and lung tissue of Sprague Dawley rats

[0073] (1-1) Preparation of Formulation A cake, composed of HL001 blended with Respitose SV003 and Magnesium Stearate (10 % HL001, 89% Respitose SV003 and 1 % Magnesium Stearate (w / w)), was packed in mid-size dust cups. The dust cup was fitted on the Wright dust-feeder (WDF).

[0074] (1-2) Methods Briefly, the rats received HL001, via nose-only dry powder inhalation, once daily, for three consecutive days. Acclimatization to the inhalation holders occurred once before the start of the study. The start of the first inhalation was considered time 0 (zero). Blood samples were collected at specific time points. All times related to HL001 administration, blood sampling and termination were recorded. All times related to HL001 administration, blood sampling and termination were recorded. At termination, all rats were anaesthetised with isoflurane. A terminal blood sample was taken from the inferior vena cava. Broncho-alveolar lavage was performed. The chest cavity was opened, and the lungs were perfused with PBS until the liquid runs clear. The right lung lobes were ligated, excised, weighed and frozen, while the left lung lobe was inflated with 4% PFA.

[0075] Before administration of HL001, the rats were weighed. The rats were placed in animal holders and connected to the inhalation tower, where they were exposed to HL001, in a dpi formulation for 30 min.

[0076] The in-silico prediction of lung tissue pharmacokinetic profile post repeated DPI administration was performed using the NonParametric Superposition method from Phoenix WinNonlin, version 8.4 from Certara. A non-compartmental analysis (NCA) was performed on the bioanalysis results to generate the following parameters: Cmax, Tmax, AUC and T1 / 2, using Phoenix WinNonlin version 8.4 from Certara. The results were shown in Table 15.

[0077] (2) Oral administration Evaluation of the pharmacokinetic profile of HL001, after oral administration, in Sprague-Dawley rats

[0078] (2-1) Preparation of Formulation To 225 mg of HL001, 0.75 ml of Tween 80 (final concentration 1%) was added followed by the addition of 74.25 ml 0.5% Methylcellulose (prepared in water), to obtain a 3 mg / ml suspension. The preparation was sonicated briefly to obtain a homogeneous suspension (dose: 30 mg / kg, administration volume: 10 ml / kg). Values were recalculated before suspension preparation, in accordance with the weight of the rats.

[0079] (2-2) Methods The rats were given 30 mg / kg of HL001, via oral gavage. Blood was collected (200 μl, into EDTA microvettes) at specific time point: pre-dose, 30 min, 1h, 2h, 4h, 8h, and 24h after administration of HL001. The rats were terminated at different time points: 30 min, 2h, 8h and 24h after administration of HL001. A non-compartmental analysis (NCA) was performed on the data to generate the following parameters: Cmax, Tmax, AUC and T1 / 2, using PhoenixWinNonlinversion 8.4 from Certara. The results were shown in Table 15.

[0080] The 29.3 mg / kg averaged dose of HL001 p.o. shows the plasma Cmax as 83.8 ng / mL and the lung Cmax as78.8 ng / g. Otherwise, the 1.2 mg / kg averaged dose of HL001 inhalation shows the plasma Cmax as 185.8 to 250.8 ng / mL and the lung Cmax as 11042.5 ng / g (Table 15). Thus, the inhalation route was 140-fold efficient for lung delivery with dramatically low systemic exposure.

[0081]

[0082] PK efficacy was 140 fold higher in the inhalation form of HL001 than in oral administration to rat based on the Cmax.

[0083] Example 5: Drug efficacy assessments HL001 and BMS-986278 were assessed in the following formulations. The results were shown in Tables 16-18 and Fig. 4-8. (1) Preparation of Formulations (1-1) Formulation for inhalation comprising HL001: D1-4 prepared in Example 2 was employed. (1-2) Formulation for oral administration comprising HL001: HL001 was wet with Tween 80 (final concentration 1%). To this mixture, 0.5% Methylcellulose (prepared in water) was added to obtain a 3 mg / ml suspension. The preparation was sonicated briefly to obtain a homogeneous suspension (dose: 30 mg / kg, administration volume: 10 ml / kg). Volumes were calculated before suspension administration, in accordance with the weight of the rats. (1-3) Formulation for oral administration comprising BMS-986278 BMS-986278 was used as a benchmark compound. BMS-986278 was prepared in 10% Cremophor-EL, 40% PEG400 and 50% PBS, according to Cheng PTW, Kaltenbach RF, Zhang H, Shi J, Tao S, Li J, et al. Discovery of an Oxycyclohexyl Acid Lysophosphatidic Acid Receptor 1 (LPA. J Med Chem. 2021; 64(21):15549-81.

[0084] (2) Bleomycin administration - i.t. instillation Bleomycin formulation was prepared as follows. Amount of Bleomycin / vial: 15000 IU Dose to administer: 1000 IU Volume to administer: 200 μL One vial of Bleomycin was diluted in 3 mL of 0.9% NaCl. Male Sprague Dawley rats were anaesthetized. The Bleomycin formulation (1000 IU) was administered i.t. (200 μl) followed by a puff of air (100 μl) for better distribution in the lung. The i.t. administration was performed using a syringe connected to a blunt steel cannula (with a small marble at the top to prevent injury to the airways). The cannula passes the larynx, and the administration was made when the rings of cartilage in the trachea were felt. After Bleomycin administration, the rats were housed with cages for one week, due to the excretion of active metabolites.

[0085] (3) Acclimatization All rats were acclimatized to the inhalation holders for 10-30 min, at least one time before treatment started. Acclimatization started 1-3 days before treatment start.

[0086] (4) Allocation to groups Before the start of treatment, the bleomycin-induced rats were randomized and allocated to groups 1-6. The main goal was to have animals with similar body weight between all groups. For that purpose, body weights were collected on day 9 post bleomycin administration.

[0087] (5) Methods The rats were weighed, marked for identification, and received bleomycin by intratracheal instillation, as mentioned above. Ten days after bleomycin administration, the formulations prepared as above were used to treat the rats with each of the following: HL001 dose 1 (0.3 mg / kg, dpi inhalation, QD), HL001 dose 2 (1 mg / kg, dpi inhalation, QD), HL001 30 mg / kg (p.o., BID), BMS-986278 dose 1 (3 mg / kg, p.o., BID), BMS-986278 dose 2 (10 mg / kg, p.o., BID). One group received bleomycin, but no treatment. One group was kept as naive controls. The inhalations were performed using the inhalation tower wherein the rats were exposed nasally for 30 minutes / inhalation session (QD). Inhalation start and end time was registered. At day 28 post bleomycin administration, the rats were terminated as follows: the rats were anaesthetised with isoflurane, and a terminal blood sample was collected from the inferior vena cava. The chest cavity was opened, the right lung lobes were ligated off, weighed and frozen in cold isopentane. The left lung lobe was inflated with 4% PFA and processed for histological assessment. Due to the size and complexity of the study, the study was performed with three starts where one third of the animals were included on three consecutive days until all 96 rats had been included in the study. Each start had the same number of rats per group to limit the risk for introducing bias between groups.

[0088] (6) Experimental Procedures In Vitro One of the right lung lobes was used for collagen analysis. The left lung lobe was processed for histopathological evaluation (Modified Ashcroft score) based on the known method (J Clin Pathol 1988;41:467-470). Rat lungs were perfused with PBS until PBS ran clear. The left lung lobe was inflated with 4% NBF. The NBF was changed after 24h and replaced with 70% ethanol after 48h. After the ethanol treatment, the samples were transferred, dehydrated, and infiltrated with paraffin in a TISSUE-TEK V.I.P. (Miles Scientific) and embedded in paraffin. The paraffin blocks were then trimmed on the microtome to expose the tissue surface to a level where a representative section could be cut. 4 μm paraffin sections were prepared, dried in an oven at 37oC overnight, and stained with Masson’s Trichrome Staining. Fibrosis was assessed on all animals by the modified Ashcroft scoring. The scoring was performed blinded. Briefly, each slide was anonymized and scanned. After scanning, slides were accessed and visualized using QuPath image viewer. Scoring was done using 10x amplification. Several fields of view per slide were scored. The average scoring of all fields of view, per slide, was taken. Scoring parameters and information can be found on the below:

[0089]

[0090] (7) Collagen analysis Collagen (hydroxyproline) analysis was performed on the lung tissue. Hydroxyproline was measured in right lung lobe 2. Briefly, right lung lobe 2 was homogenized in distilled water (100 μL / 10 mg tissue) using a Bead Mill Homogenizer and 2.8 mm ceramic beads. After homogenization, 100 μL of the homogenate was transferred to a new tube and mixed with 100 μL 10M NaOH. Sample was boiled at 120°C for 1h, whereafter 100 μL 10 M HCl was added to neutralize residual NaOH. After centrifugation at 10000g for 5 minutes, 10 μL supernatant was used for hydroxyproline analysis according to kit protocol (Sigma-Aldrich MAK357).

[0091] (8) Results (8-1) Clinical Observations and Mortality The rats that received bleomycin had an expected weight loss during the first 5 days following instillation (Fig. 4). All rats recovered before treatment start. HL001 was well tolerated, regardless of administration route. Some rats in the inhalation groups exhibited stress signs (porphyrin secretion around eyes) at the end of some inhalation sessions, but these symptoms were transient. All animals in the BMS-986278 groups exhibited diarrhoea and stress signs (porphyrin secretion in the fur). In addition, it became increasingly difficult to administer the compound. One rat from the BMS-986278 low dose group was terminated on day 16 due to extensive weight loss.

[0092] (8-2) Body Weight Change Over Time All rats that received bleomycin had an expected weight loss during the first days after bleomycin instillation. This weight loss peaked at about 4 days after bleomycin administration, followed by weight recovery (Fig. 4). The naive control rats arrived one week after the rats in bleomycin groups and were included from day 9. They were ordered to have a starting weight corresponding to a naive control that had been included from day 0. For the percent weight change calculation, the weight for the naive control animals on day 0 is set to the mean weight of all animals in the bleomycin groups on day 0 (before bleomycin instillation).

[0093] (8-3) Lung Lobe Weights at Termination The bleomycin untreated group exhibited significantly increased lung weights compared to the naive control group (p: 0.0001) (Table 17, Fig. 5). None of the treatment groups (HL001 p.o, HL001 inhalation low dose, HL001 inhalation high dose, BMS-986278 low dose, BMS-986278 high dose) had significantly reduced lung weights compared to the bleomycin untreated group. However, the HL001 inhalation low dose group had the lowest p value (0.2822), indicating a trend towards decreased lung weights compared to the bleomycin untreated group.

[0094] (8-4) Modified Ashcroft Score Bleomycin administration resulted in increased lung fibrosis, as shown by the significantly increased Ashcroft score of the bleomycin untreated group compared to the naive control group (p value:<0.0001) (Fig. 6, Table 18). None of the treatment groups demonstrated significant anti-fibrotic effects compared to the untreated bleomycin group. However, rats receiving inhaled HL001 (both doses) and BMS-986278 (3 mg / kg) demonstrated the lowest Ashcroft scores of all bleomycin-induced groups (3.0 ± 0.3 and 2.9 ± 0.3, respectively) compared to the untreated bleomycin group (3.6 ± 0.2). In addition, the HL001 treatment groups and the BMS-986278 low dose group all included at least one rat with an Ashcroft score that was comparable to the naive control group.

[0095] (8-5) Hydroxyproline analysis Hydroxyproline was measured in right lung lobe 2. Briefly, right lung lobe 2 was homogenized in distilled water (100 μL / 10 mg tissue) using a Bead Mill Homogenizer and 2.8 mm ceramic beads. After homogenization, 100 μL of the homogenate was transferred to a new tube and mixed with 100 μL 10M NaOH. Sample was boiled at 120°C for 1h, whereafter 100 μL 10 M HCl was added to neutralize residual NaOH. After centrifugation at 10000g for 5 minutes, 10 μL supernatant was used for hydroxyproline analysis according to kit protocol (Sigma-Aldrich MAK357). Hydroxyproline levels were interpolated from a standard curve (Fig. 7). Unfortunately, levels were low, and all samples fell below the standard curve. However, as the standard curve was linear, we conclude that the interpolated levels could still be informative, but the levels should be interpreted with some caution (Table 19, Fig. 8). Bleomycin administration resulted in increased hydroxyproline levels, as shown by the significantly increased levels compared to the naive control group (p: 0.0001) (Fig. 8, Table 19). The HL001 p.o, HL001 inhalation low dose, HL001 inhalation high dose and BMS-986278 low dose groups all exhibited significantly decreased hydroxyproline levels compared to the bleomycin untreated group.

[0096]

[0097]

[0098]

[0099] Single intra-tracheal administration of bleomycin caused significant lung fibrosis in the rats, as evident by the increased modified Ashcroft score and increased hydroxyproline content, compared to the naive control group. Aerosolized HL001 generated particles of a particle size suitable to inhalation therapy with an average MMAD of 1.54 μm. The delivered dose of the test item was on average 0.29 mg / kg (nominal 0.3 mg / kg) for the lower dose level and 0.89 mg / kg (nominal 1.0 mg / kg) for the higher dose level. Some issues regarding cake integrity were experienced that resulted in lower delivered dose initially, but from day 4 and onwards the delivered doses were close to the nominal dose levels. HL001 administered by inhalation was well tolerated at both doses (0.3 and 10 mg / kg) doses. Rats did not show any signs or symptoms of airway irritation or general toxicity. There were also no evident signs of irritation in the histological analysis. Using the modified Ashcroft scoring system, none of the treated groups (HL001 or BMS-986278) demonstrated significant anti-fibrotic effects as compared to the untreated bleomycin group. However, the mean Ashcroft score was lower in animals receiving inhaled HL001 and low dose BMS-986278 (3.0 ± 0.3 and 2.9 ± 0.3, respectively) compared to untreated animals (3.6 ± 0.2). Similar results were seen for hydroxyproline, with exception for the high dose BMS. Although the mean Ashcroft score is not significant, it is important to note the exposure level difference between the treatment groups. Inhaled HL001 is administered once daily at 0.29 or 0.89 mg / kg, compared to orally administered HL001 (BID at 30 mg / kg) and orally administered BMS-986278 (BID at 3 or 10 mg / kg). Systemic exposure levels will then be significantly reduced for inhaled HL001, while keeping the efficacy on par with higher doses of the orally administered therapies.

[0100] The HL001 inhalation form showed that anti-fibrotic activity was equally or more effective than those in HL001 oral form and BMS-986278, a same-class competitor drug candidate (Bristol Myers Squibb). It was noteworthy that the HL001 inhalation form showed such an anti-fibrotic effect even in lower concentrations and low frequency than the other study group - HL001 Inhalation: 0.3-1 mg / kg, QD - HL001 Oral: 30 mg / kg, BID - BMS-986278 Oral: 3-10 mg / kg, BIDIndustrial Availability

[0101] The dry powder pharmaceutical composition for inhalation of the present invention comprises the LPA1 antagonist and it is useful as a medicament (a medicament for the treatment and / or prevention of pulmonary fibrosis including idiopathic pulmonary fibrosis and progressive pulmonary fibrosis).

Claims

1. A dry powder pharmaceutical composition for inhalation, comprising 1 to 20 wt% of a compound represented by Formula (I): or a pharmaceutical acceptable salt thereof, 0.01 to 5 wt% of magnesium stearate and 75 to 98.99 wt% of lactose with respect to the weight of the composition.

2. The composition according to claim 1, wherein a bulk density is in the range of 0.3 to 1 g / ml.

3. The composition according to claim 1 or 2, a Hausner index is in the range of 1-2.

4. The composition according to any one of claims 1 to 3, wherein a fine particle fraction (FPF) with less than 5 μm is 10 to 80%.

5. The composition according to any one of claims 1 to 4, wherein a mass median aerodynamic diameter (MMAD) is 0.5 to 19 μm.

6. The composition according to any one of claims 1 to 5, wherein a blend uniformity is in the range of 50 to 150 mg / g.

7. A process for the preparation of the composition according to any one of claims 1 to 6, comprising a step of mixing HL001 with magnesium stearate and lactose using a high-shear mixer wherein a mixing energy is 0.1 to 2 mJ / carrier.

8. The process according to claim 7, wherein the step of mixing comprises the following steps: Step 1 of mixing lactose with magnesium stearate at 400 to 1700 rpm and 0.1 to 1.5 mJ / carrier to coat the lactose; and Step 2 of mixing the coated lactose with HL001 at 400 to 1700 rpm and 0.1 to 2 mJ / carrier.

9. A method for the prevention and / or the treatment of pulmonary fibrosis, wherein a therapeutic effective amount of the composition according to any one of claims 1 to 6 is administered by inhalation to a patient need thereof.

10. The method according to claim 9, wherein the composition is administered at a dose of 0.001 to 60 mg / kg once a day.

11. The method according to claim 9 or 10, wherein the composition is administered in combination with the other agents including drug for pulmonary fibrosis.

12. The method according to any one of claims 9 to 11, wherein pulmonary fibrosis including idiopathic pulmonary fibrosis and progressive pulmonary fibrosis.

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