Compositions for the treatment of α1-antitrypsin deficiency and their use

Abstract

The present invention relates to pharmaceutical compositions comprising (S)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid and their medical uses, such as for use in the treatment of alpha-1-antitrypsin deficiency (AATD). The inventors have surprisingly found that (S)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid is highly effective both in vitro and in vivo in increasing the levels of Z A1AT, while having no effect on the in vitro secretion of M A1AT or of the Shiiyama variant of A1AT.

Description

[Technical Field]

[0001] The present invention relates to pharmaceutical compositions comprising (S)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid and their medical uses. [Background technology]

[0002] Alpha-1 antitrypsin (A1AT) is a member of the serpine superfamily produced by the liver and secreted into the bloodstream. It inhibits various serine proteases, particularly neutrophil elastase. Low blood levels of A1AT can lead to excessive neutrophil elastase activity, which breaks down lung tissue and causes respiratory complications (e.g., chronic obstructive pulmonary disease (COPD)).

[0003] The normal range for blood A1AT is 0.9–2.3 g / L. Lower levels are typical of alpha-1 antitrypsin deficiency (A1AD or AATD) (a genetic disorder caused by mutations in the SERPINA1 gene that encodes A1AT). The most common cause of AATD is the Z mutation, which is a glutamine-to-lysine substitution at position 366 (which corresponds to position 342 in the mature form) of A1AT (UniProtKB - P01009(A1AT_HUMAN)) (Z A1AT). The Z mutation affects the folding of A1AT, resulting in only a small fraction acquiring a native / active state. The remainder are either removed as misfolded proteins or accumulate in the liver as stable polymers. As a result of misfolding, homozygous carriers of the Z mutation (ZZ) have plasma levels of A1AT that are 10–15% of normal and become predisposed carriers of COPD. Accumulation of Z A1AT polymer in hepatocytes makes individuals predisposed to cirrhosis, liver cancer, and other liver pathologies.

[0004] Current treatments for the pulmonary manifestation of AATD include augmentation therapy using A1AT concentrates prepared from blood donor plasma. The U.S. FDA has approved the use of four A1AT products: Prolastin, Zemaira, Glassia, and Aralast. Administration is via weekly intravenous infusion. Augmentation therapy has been clearly shown to slow the progression of COPD. Hepatic manifestations of AATD (e.g., cirrhosis and cancer) are treated with steroids and liver transplantation. Investigational approaches to improving the treatment of hepatic manifestations include increasing polymer clearance through inhibition of A1AT polymerization and activation of autophagy. Investigational approaches to improving the treatment of both pulmonary and hepatic manifestations concern improving A1AT folding and secretion.

[0005] Elliott et al. (Protein Science, 2000, 9, 1274-1281) described the X-ray crystal structure of A1AT and identified five voids that are potential targets for rational drug design to develop agents that affect A1AT polymerization.

[0006] Parfrey et al. (J. Biol. Chem., 2003, 278, 35, 33060-33066) further defined a single lumen as a potential target for rational drug design to develop agents that affect Z A1AT polymerization.

[0007] Knaupp et al. (J. Mol. Biol., 2010, 396, 375-383) found that bis-ANS(4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonate) is 1:1 stoichiometric and 700 nM K d We showed that it can bind to Z A1AT but does not bind to wild-type A1AT(M).

[0008] Chang et al. (J. Cell. Mol. Med., 2009, 13, 8B, 2304-2316) reported a series of peptides (including Ac-TTAI-NH2) that inhibit Z A1AT polymerization.

[0009] Burrows et al. (Proc. Nat. Acad. Sci., 2000, 97, 4, 1796-1801) showed that a series of non-selective chaperones (including 4-phenylbutyrate, glycerol, and trimethylamine oxide) can increase Z A1AT levels in cell supernatant and mouse models.

[0010] Bouchecareilh et al. (Journal of Biological Chemistry, 2012, 287, 45, 38265-38278) describe the use of histone deacetylase inhibitors, particularly SAHA (suberoylanilide hydroxamic acid), to increase the secretion of both M and Z A1AT from cells.

[0011] Berthelier et al. (PLOS ONE, May 11, 2015) clearly demonstrated that S-(4-nitrobenzyl)-6-thioguanosine can prevent Z A1AT polymerization in vivo.

[0012] Mallya et al. (J. Med. Chem., 2007, 50, 22, 5357-5363) describe that a series of phenols (e.g., N-(4-hydroxy-3,5-dimethylphenyl)-2,5-dimethylthiophene-3-sulfonamide) can block the polymerization of Z A1AT in vitro.

[0013] Huntington (13th International Symposium on Proteinases, Inhibitors and Biological Control, 23 September 2012 and 7 th International Symposium on Serpin Biology, Structure and Function, 1 stIn April 2014, we investigated the cavities of Z A1AT from its X-ray crystal structure, which are potential targets for rational drug design to develop agents that affect Z A1AT polymerization.

[0014] U.S. Patent No. 8,436,013B2 discloses a wide range of structures that can increase the secretion of Z A1AT from cells in the micromolar concentration range.

[0015] Angewandte Chemie International Edition vol 56, no 33, 2017, 9881-9885 discloses 1-((4-chlorophenyl)sulfonyl)piperidine-4-carboxylic acid as a herbicide.

[0016] The Journal of Applicable Chemistry, vol 2, no 6, 2013, pp. 1501-1508 discloses the synthesis of 1-(4-(trifluoromethyl)phenylsulfonyl)piperidine-4-carboxylic acid as an antibacterial agent.

[0017] US2011 / 0065707A1 discloses the use of 1-(2-chlorobenzene-sulfonyl)-piperidine-4-carboxylic acid as a reagent.

[0018] EP0520336A2 discloses 1-(8-quinoyl-sulfonyl)-piperidine-4-carboxylic acid.

[0019] WO2019 / 243841A1 discloses oxoindoline-4-carboxamide compounds as regulators of α-1-antitrypsin and their use in treating diseases associated with α-1-antitrypsin. WO2020 / 081257A1 discloses pyrrolo-indazolyl-propanoate compounds as regulators of α-1-antitrypsin. US2020 / 0361939A1 further discloses pyrrolo-indazolyl-propanoate compounds as regulators of α-1-antitrypsin.

Prior Art Documents

Patent Documents

[0020]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Patent Document 5

Patent Document 6

Non-Patent Documents

[0021]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

[0022] A prior art search based on the structure of 1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid was conducted after the invention of this invention. The closest prior art molecule retrospectively identified by this search was the racemic compound, 1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid (CAS Registry No. 891392-68-0). This compound is listed as being commercially available from Aurora, ChemDiv and the FCH Group, but no publications are recorded. Another close prior art molecule is 1-(1-tosyl-1,2,5,6-tetrahydropyridine-3-yl)ethane-1-one (Example 16 of US9084782B2). This compound is said to inhibit angiogenesis and lower cellular cholesterol levels (however, US9084782B2 does not provide biological data regarding this compound).

[0023] According to a first aspect of the present invention, a compound for use in treating a disease or disorder characterized by low plasma levels of A1AT in a human subject, (S)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid: [ka] A pharmaceutical composition comprising the above is provided, wherein the composition is administered to the human subject at a daily dose of about 1 to 6 mg / kg (i.e., about 1 to 6 mg of the pharmaceutical composition per mass (in kg) of the human subject).

[0024] The above composition may be administered to the above human subjects in a daily dose of approximately 2 to 5 mg / kg, or for example, approximately 1 mg / kg, approximately 1.5 mg / kg, approximately 2 mg / kg, approximately 2.5 mg / kg, approximately 3 mg / kg, approximately 3.5 mg / kg, approximately 4 mg / kg, approximately 4.5 mg / kg, approximately 5 mg / kg, approximately 5.5 mg / kg, or approximately 6 mg / kg.

[0025] In another aspect of the present invention, the composition may be administered to the human subject at different intervals, for example, every two days, every three days, every four days, every five days or every six days, or once a week, in doses equal to the daily dose specified herein. [Modes for carrying out the invention]

[0026] As detailed below, the inventors have found that (S)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid, surprisingly, has no effect on the in vitro secretion of M A1AT or the Siiyama variant of A1AT, while being highly effective in increasing Z A1AT levels both in vitro and in vivo.

[0027] Furthermore, as shown in Examples 7, 8 and 14, (S)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid is its enantiomer (R)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid: [ka] It exhibits pharmacokinetic properties that suggest it is effective for treating AATD at surprisingly low doses.

[0028] The compounds in the pharmaceutical compositions of the present invention may be in pharmaceutically acceptable salt forms.

[0029] The term “pharmaceutically acceptable salt” refers to a pharmaceutically acceptable monoorganic or inorganic salt of the compound of the present invention. This may include those derived from a base such as sodium hydroxide, potassium hydroxide, lithium hydroxide, calcium hydroxide, 1-deoxy-2-(methylamino)-D-glucitol, magnesium hydroxide, zinc hydroxide, aluminum hydroxide, iron(II) or iron(III) hydroxide, or ammonium hydroxide, or an organic amine (e.g., N-methylglucamine, choline, arginine, etc.). For other examples of pharmaceutically acceptable salts, see Gould (1986, Int J Pharm 33: 201-217).

[0030] The pharmaceutical compositions of the present invention may further comprise pharmaceutically or therapeutically acceptable excipients or carriers.

[0031] The term “pharmaceutically or therapeutically acceptable excipient or carrier” refers to a solid or liquid filler, diluent, or encapsulating substance that does not interfere with the efficacy or biological activity of the active ingredient and is non-toxic to the host (which may be a human or an animal to which it may be administered). Depending on the specific route of administration, various pharmaceutically acceptable carriers (e.g., those well known in the art) may be used. Non-limiting examples include sugars, starches, cellulose and their derivatives, malt, gelatin, talc, calcium sulfate, vegetable oils, synthetic oils, polyols, alginic acid, phosphate-buffered salt solutions, emulsifiers, isotonic salt solutions, and pyrogen-free water.

[0032] All appropriate modes of administration are intended in accordance with the present invention. For example, administration of the above compositions or pharmaceuticals may be via oral, subcutaneous, direct intravenous, slow intravenous infusion, continuous intravenous infusion, intravenous or epidural patient-controlled analgesia (PCA and PCEA), intramuscular, intrathecal, epidural, intracisional, intraperitoneal, percutaneous, topical, transmucosal, oral, sublingual, transmucosal, inhalation, intranasal, intra-atricular, rectal, or ocular routes. The above compositions or pharmaceuticals may be formulated in separate dose units (for example, as required by the daily dose regimen of the present invention) and may be prepared by any of the methods well known in the pharmaceutical field.

[0033] The above composition may be administered in oral form, or intravenously.

[0034] All appropriate pharmaceutical dosage forms are intended. Drug administration may take the form of, for example, oral solutions and suspensions, tablets, capsules, lozenges, effervescent tablets, transmucosal films, suppositories, buccal products, or oral mucoretentive products, topical creams, ointments, gels, films and patches, transdermal patches, abuse-deterrent and abuse-resistant formulations, sterile solutions and suspensions, and depots for parenteral use, and may be administered as immediate release, sustained release, delayed release, controlled release, or prolonged release.

[0035] Another aspect of the present invention is the use of (S)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid in the manufacture of a pharmaceutical for the treatment of a disease or disorder characterized by low plasma levels of A1AT in a human subject, wherein the pharmaceutical is administered to the human subject in a daily dose of about 1 to 6 mg / kg (including the dose as described above).

[0036] The present invention also provides a method for treating a disease or disorder characterized by low plasma levels of A1AT in a human subject, the method comprising administering a pharmaceutical composition as defined herein, comprising (S)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid, in a daily dose of about 1 to 6 mg / kg (including the doses described above).

[0037] Diseases or disorders suitable for treatment according to the relevant aspects of the present invention are those characterized by low plasma levels of A1AT (e.g., α1-antitrypsin deficiency (AATD)).

[0038] The use of numerical ranges in this description is expressly intended to include, within the scope of the present invention, all individual integers within the above range, as well as all combinations of upper and lower limit digits within the widest range of a given range.

[0039] As used herein, the terms “comprising” should be read and understood to mean both “containing” and “consisting.” In conclusion, where the present invention relates to a “pharmaceutically acceptable composition containing a compound as an active ingredient,” this terminology is intended to encompass both compositions in which other active ingredients may be present and compositions consisting of only one active ingredient as defined.

[0040] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by ordinary experts in the art to which this invention pertains. Similarly, all publications, patent applications, patents, and all other references referred herein are incorporated herein by reference in their entirety (where legally permitted). [Brief explanation of the drawing]

[0041] Specific non-limiting examples of the present invention are described herein with reference to the following drawings.

[0042] [Figure 1] Figure 1 is a graph showing the dose-dependent effect of (S)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid in an in vitro A1AT cell secretion assay using HEK-EBNA cells containing the Z A1AT plasmid. The vehicle and 10 μM SAHA were tested as controls on each plate. The x-axis represents the various treatments of the cells: vehicle, SAHA, and escalating concentrations of (S)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid, and the y-axis represents the concentration of human A1AT in the cell supernatant (in ng / ml units).

[0043] [Figure 2] Figure 2 is a graph showing the effect of 10 μM (S)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid in an in vitro A1AT cell secretion assay using HEK-EBNA cells containing the M A1AT plasmid. Vehicle and 10 μM SAHA were tested as controls. The x-axis represents the various treatments of the above cells: vehicle, SAHA, and (S)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid, and the y-axis represents the concentration of human A1AT in the cell supernatant (in ng / ml units).

[0044] [Figure 3] Figure 3 is a graph showing the effects of 1 μM and 10 μM (S)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid in an in vitro A1AT cell secretion assay using HEK-EBNA cells containing the Siiyama A1AT plasmid. Vehicle and 10 μM SAHA were tested as controls. The x-axis shows the various treatments of the above cells: vehicle, SAHA, and (S)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid at the two concentrations, and the y-axis shows the concentration of human A1AT in the cell supernatant (in ng / ml units).

[0045] [Figure 4] Figure 4 is a graph showing the effect of (S)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid on Z A1AT levels in mice expressing human Z A1AT (huZ mice). Mice were treated by forced oral administration of vehicle, 5 mg / kg, 15 mg / kg, and 50 mg / kg of (S)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid twice daily for 14 consecutive days. Blood was collected on days -12, -7, and -5, and plasma was prepared to determine the circulating baseline level of human Z A1AT. Plasma samples collected during the last three days of the study (days 12, 13, and 14) were used to determine the effect of (S)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid treatment on circulating human Z A1AT levels, compared to baseline levels. The x-axis represents the treatment dose (mg / kg) of (S)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid; the y-axis represents the mean percentage level of human Z A1AT compared to the baseline level for each treatment group.

[0046] [Figure 5] Figure 5 is a graph showing the dose-dependent effect of (R)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid in an in vitro A1AT cell secretion assay using HEK-EBNA cells containing the Z A1AT plasmid. The vehicle and 10 μM SAHA were tested on each plate as controls. The x-axis represents the various treatments of the cells: vehicle, SAHA, and escalating concentrations of (R)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid, and the y-axis represents the concentration of human A1AT in the cell supernatant (in ng / ml units).

[0047] [Figure 6]Figure 6 is a graph showing the effect of 10 μM (R)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid in an in vitro A1AT cell secretion assay using HEK-EBNA cells containing the M A1AT plasmid. Vehicle and 10 μM SAHA were tested as controls. The x-axis represents the various treatments of the cells: vehicle, SAHA, and (R)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid, and the y-axis represents the concentration of human A1AT in the cell supernatant (in ng / ml units).

[0048] [Figure 7] Figure 7 is a graph showing the effects of 1 μM and 10 μM (R)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid in an in vitro A1AT cell secretion assay using HEK-EBNA cells containing the Siiyama A1AT plasmid. Vehicle and 10 μM SAHA were tested as controls. The x-axis shows the various treatments of the above cells: vehicle, SAHA, and the two concentrations of (R)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid, and the y-axis shows the concentration of human A1AT in the cell supernatant (in ng / ml units). [Examples]

[0049] experiment Example 1: (S)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid (S)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid was prepared using the following synthetic procedure. [ka] (S)-piperidine-3-carboxylic acid (1 g, 7.7 mmol), potassium hydroxide (434 mg, 7.7 mmol), and potassium carbonate (2.14 g, 15.4 mmol) were added to water (20 ml) and stirred. 2-(trifluoromethyl)benzenesulfonyl chloride (1.89 g, 7.7 mmol) was added, and the reaction mixture was stirred at room temperature for 3 hours. The reaction mixture was cooled to 0°C and acidified with 2 M hydrochloric acid to obtain a white precipitate. This precipitate was dried and ground with n-pentane to obtain (S)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid.

[0050] Tlc Rf 0.3 70% ethyl acetate in hexane. m / z: 337.98 (calculated value 338.03) 1 H NMR (400 MHz, d6 DMSO) δ 12.33 (1H, s), 8.04 (2H, m), 7.90 (2H, m), 3.69 (1H, dd), 3.50 (1H, dd), 2.93 (1H, m), 2.81 (1H, m), 1.91 (1H, m), 1.72 (1H, m), 1.50 (2H, m).

[0051] Example 2: (R)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid (R)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid was prepared in the same manner as (S)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid, but using (R)-piperidine-3-carboxylic acid. Tlc Rf 0.3 70% ethyl acetate in hexane m / z: 338.03 (calculated value 338.03) 1H NMR (400 MHz, d6 DMSO) δ 12.53 (1H, s), 8.04 (2H, m), 7.90 (2H, m), 3.69 (1H, dd), 3.49 (1H, dd), 2.93 (1H, m), 2.81 (1H, m), 1.90 (1H, m), 1.72 (1H, m), 1.49 (2H, m).

[0052] Example 3: Activity of (S)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid and (R)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid in an A1AT cell secretion assay using HEK-Z cells

[0053] method HEK-Z cells (a human embryonic kidney cell line stably transfected with the human Z A1AT gene) were stored in a humidified atmosphere containing 5% CO2 in a 96-well plate (3.0 × 10⁶). 5 Cells / ml were plated in 200 μl of medium / well and left overnight at 37°C. After incubation, the cells were washed three times with 200 μl of serum-free medium and incubated in a quad-tray at 37°C for 48 hours, with the medium being changed by using serum-free medium containing 10 μM suberanilohydroxamic acid (SAHA), (S)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid or (R)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid (at concentrations of 10 nM, 33 nM, 100 nM, and 333 nM) in a final volume of 200 μl. At the end of the incubation process, the supernatant was removed from the well and centrifuged at 1000 × g at 4°C for 10 minutes. Human A1AT levels were assayed by ELISA (Human Serpin A1 / α1antitrypsin duo set ELISA, R&D Systems, DY1268) according to the manufacturer's instructions.

[0054] In short, a 96-well plate was coated overnight at room temperature with human A1AT capture antibody (1:180 dilution from stock, 100 μl final volume / well). The capture antibody was then removed, and the wells were washed three times with 300 μl of wash buffer (0.05% Tween® 20 in PBS). Then, 200 μl of reagent dilution (25% Tween® 20 in PBS) was incubated in each well at room temperature for 1 hour. Diluted samples, standards (A1AT at 125 pg / ml, 250 pg / ml, 500 pg / ml, 1000 pg / ml, 2000 pg / ml, 4000 pg / ml, and 8000 pg / ml) or blanks were added in double strips to each well, the plate was covered with a plate sealer, and left at room temperature for 2 hours. At the end of the sample incubation process, the samples were removed, all wells were washed as described above, and 100 μl of detection antibody (1:180 dilution from stock) was added to each well. The mixture was incubated at room temperature for a further 2 hours. After incubation with the detection antibody, the supernatant was removed, the wells were washed as described above, and 100 μl of streptavidin-HRP solution (1:200 dilution from stock) was added to each well in the dark for 20 minutes. Subsequently, 50 μl of stop solution (2 M H2SO4) was added, and the optical density (OD) of each well was read at 450 nm by subtracting the 570 nm blank from each well using a microplate reader. A 4-parameter logistic curve was constructed using GraphPad Prism 7, and the A1AT concentration was determined for each sample by interpolation from the standard curve and multiplication by the appropriate dilution ratio.

[0055] result The amount of human A1AT secreted from transfected HEK-EBNA cells into the culture medium was measured by ELISA. 10 μM SAHA was used as a positive control for all in vitro A1AT secretion experiments.

[0056] The data in Figure 1 show that (S)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid stimulates the secretion of human Z A1AT in a dose-dependent manner, as measured by ELISA.

[0057] The data in Figure 5 show that (R)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid stimulates the secretion of human Z A1AT in a dose-dependent manner, as measured by ELISA.

[0058] Example 4: Activity of (S)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid and (R)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid in an A1AT cell secretion assay using HEK-M cells

[0059] method HEK-M cells (human embryonic kidney cell line stably transfected with M A1AT) were stored in a humidified atmosphere containing 5% CO2 in a 96-well plate (3.0 × 10⁶). 5 Cells / ml were plated into 200 μl of medium / well and left overnight at 37°C. After incubation, the cells were washed three times with 200 μl of serum-free medium and incubated in a hexagonal 37°C incubator for 48 hours. The final volume of the medium was changed to 200 μl with serum-free medium containing either the vehicle, 10 μM suberoylanilide hydroxamic acid (SAHA), (S)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid (at 10 μM) or (R)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid (at 10 μM). At the end of the incubation process, the supernatant was removed from the well and centrifuged at 1000 × g at 4°C for 10 minutes. Human A1AT levels were assayed by ELISA (Human Serpin A1 / α1antitrypsin duo set ELISA, R&D Systems, DY1268) according to the manufacturer's instructions.

[0060] In short, a 96-well plate was coated overnight at room temperature with human A1AT capture antibody (1:180 dilution from stock, 100 μl final volume / well). The capture antibody was then removed, and the wells were washed three times with 300 μl of wash buffer (0.05% Tween® 20 in PBS). Then, 200 μl of reagent dilution (25% Tween® 20 in PBS) was incubated in each well at room temperature for 1 hour. Diluted samples, standards (A1AT at 125 pg / ml, 250 pg / ml, 500 pg / ml, 1000 pg / ml, 2000 pg / ml, 4000 pg / ml, and 8000 pg / ml) or blanks were added in double strips to each well, the plate was covered with a plate sealer, and left at room temperature for 2 hours. At the end of the sample incubation process, the samples were removed, all wells were washed as described above, and 100 μl of detection antibody (1:180 dilution from stock) was added to each well. The mixture was incubated at room temperature for a further 2 hours. After incubation with the detection antibody, the supernatant was removed, the wells were washed as described above, and 100 μl of streptavidin-HRP solution (1:200 dilution from stock) was added to each well in the dark for 20 minutes. Subsequently, 50 μl of stop solution (2 M H2SO4) was added, and the optical density (OD) of each well was read at 450 nm by subtracting the 570 nm blank from each well using a microplate reader. A 4-parameter logistic curve was constructed using GraphPad Prism 7, and the A1AT concentration was determined for each sample by interpolation from the standard curve and multiplication by the appropriate dilution ratio.

[0061] result The amount of human A1AT secreted from transfected HEK-EBNA cells into the culture medium was measured by ELISA. 10 μM SAHA was used as a positive control for all in vitro A1AT secretion experiments.

[0062] The data in Figure 2 show that (S)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid does not stimulate the secretion of human M A1AT at 10 μM, as measured by ELISA. In contrast, the positive control, 10 μM SAHA, stimulates increased M A1AT secretion.

[0063] The data in Figure 6 show that (R)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid does not stimulate the secretion of human M A1AT at 10 μM, as measured by ELISA. In contrast, the positive control, 10 μM SAHA, stimulates increased M A1AT secretion.

[0064] Example 5: Activity of (S)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid and (R)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid in an A1AT cell secretion assay using HEK-Siiyama cells

[0065] A rare Siiyama mutation (from Ser 53 to Phe, based on mature A1AT numbering) was identified in Japanese men with AATD (Seyama et al. (J Biol Chem (1991) 266:12627-32)). Ser 53 is a conserved serpine residue and is thought to be important for the organization of the internal core of the A1AT molecule. The change from an uncharged polar amino acid to a large nonpolar amino acid on the protein's conserved backbone affects folding and intracellular processing of Siiyama A1AT.

[0066] method HEK-Siiyama cells (a human embryonic kidney cell line stably transfected with the Siiyama A1AT gene) were stored in a humidified atmosphere containing 5% CO2 in a 96-well plate (3.0 × 10⁶). 5Cells / ml were plated into 200 μl of medium / well and left overnight at 37°C. After incubation, the cells were washed three times with 200 μl of serum-free medium and incubated in an octagonal 37°C incubator for 48 hours. The medium was then replaced with serum-free medium containing a vehicle, 10 μM suberoylanilide hydroxamic acid (SAHA), (S)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid (at 1 μM or 10 μM) or (R)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid (at 1 μM or 10 μM) in a final volume of 200 μl. At the end of the incubation process, the supernatant was removed from the well and centrifuged at 1000 × g at 4°C for 10 minutes. Human A1AT levels were assayed by ELISA (Human Serpin A1 / α1antitrypsin duo set ELISA, R&D Systems, DY1268) according to the manufacturer's instructions.

[0067] In short, a 96-well plate was coated overnight at room temperature with human A1AT capture antibody (1:180 dilution from stock, 100 μl final volume / well). The capture antibody was then removed, and the wells were washed three times with 300 μl of wash buffer (0.05% Tween® 20 in PBS). Then, 200 μl of reagent dilution (25% Tween® 20 in PBS) was incubated in each well at room temperature for 1 hour. Diluted samples, standards (A1AT at 125 pg / ml, 250 pg / ml, 500 pg / ml, 1000 pg / ml, 2000 pg / ml, 4000 pg / ml, and 8000 pg / ml) or blanks were added in double strips to each well, the plate was covered with a plate sealer, and left at room temperature for 2 hours. At the end of the sample incubation process, the samples were removed, all wells were washed as described above, and 100 μl of detection antibody (1:180 dilution from stock) was added to each well. The mixture was incubated at room temperature for a further 2 hours. After incubation with the detection antibody, the supernatant was removed, the wells were washed as described above, and 100 μl of streptavidin-HRP solution (1:200 dilution from stock) was added to each well in the dark for 20 minutes. Subsequently, 50 μl of stop solution (2 M H2SO4) was added, and the optical density (OD) of each well was read at 450 nm by subtracting the 570 nm blank from each well using a microplate reader. A 4-parameter logistic curve was constructed using GraphPad Prism 7, and the A1AT concentration was determined for each sample by interpolation from the standard curve and multiplication by the appropriate dilution ratio.

[0068] result The amount of human A1AT secreted from transfected HEK-EBNA cells into the culture medium was measured by ELISA. 10 μM SAHA was used as a positive control for all in vitro A1AT secretion experiments.

[0069] The data in Figure 3 show that (S)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid does not stimulate the secretion of human Siiyama A1AT at 1 μM or 10 μM, as measured by ELISA. In contrast, the positive control, 10 μM SAHA, stimulates increased Siiyama A1AT secretion.

[0070] The data in Figure 7 show that (R)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid does not stimulate the secretion of human Siiyama A1AT at 1 μM or 10 μM, as measured by ELISA. In contrast, the positive control, 10 μM SAHA, stimulates increased Siiyama A1AT secretion.

[0071] Example 6: Activity of (S)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid in huZ mice HuZ mice (also known as PiZZ mice) are a transgenic mouse strain containing multiple copies of the Z variant of the human A1AT gene, developed by two separate groups (Dycaico et al. (Science (1988) 242:1409-12) and Carlson et al. (J. Clin Invest (1989) 83:1183-90)). HuZ mice have a C57Bl / 6 background and express the human Z A1AT protein in liver tissue. The mice used in this study are derived from the offspring of Carlson and colleagues (transgenic strain Z11.03). Using HuZ mice as a tool, we will evaluate either the effect of a compound on increasing circulating levels of Z A1AT in plasma, or the effect of a compound on the accumulation of Z A1AT polymers in the liver and associated liver pathology.

[0072] HuZ mice (n=4 / group; male or female) with basal human A1AT plasma levels between 200 and 600 μg / ml were treated by forced oral administration of either vehicle or (S)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid at 5 mg / kg, 15 mg / kg, or 50 mg / kg twice daily for 14 consecutive days. Mice had free access to feed (standard mouse feed, SAFE feed) and water. On day 14 of the study, each mouse received the final dose one hour prior to the last procedure. Blood was collected from the tail vein of each mouse on days -12, -7, and -5 before administration, and on days 12, 13, and 14 during administration. Blood was collected in a microbed containing EDTA, and plasma was prepared by centrifugation at 2700 × g at 4°C for 10 minutes. Plasma was divided into aliquots and stored at -80°C for in vivo analysis. Mean basal levels of human Z A1AT for each mouse were determined using plasma samples from -12, -7, and -5 days prior to administration. Plasma samples collected during the last three days of administration in the study (days 12, 13, and 14) were used to determine the effect of (S)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid on human Z A1AT secretion by measuring human Z A1AT levels and comparing them to basal levels for each mouse. Human A1AT levels in mouse plasma samples were assayed by ELISA (Human Serpin A1 / α1antitrypsin duo set ELISA, R&D Systems, DY1268) according to the manufacturer's instructions.

[0073] In short, a 96-well plate was coated overnight at room temperature with human A1AT capture antibody (1:180 dilution from stock, 100 μl final volume / well). The capture antibody was then removed, and the wells were washed three times with 300 μl of wash buffer (0.05% Tween® 20 in PBS). Then, 200 μl of reagent dilution (25% Tween® 20 in PBS) was incubated in each well at room temperature for 1 hour. Diluted samples, standards (A1AT at 125 pg / ml, 250 pg / ml, 500 pg / ml, 1000 pg / ml, 2000 pg / ml, 4000 pg / ml, and 8000 pg / ml) or blanks were added in double strips to each well, the plate was covered with a plate sealer, and left at room temperature for 2 hours. At the end of the sample incubation process, the samples were removed, all wells were washed as described above, and 100 μl of detection antibody (1:180 dilution from stock) was added to each well. The mixture was incubated at room temperature for a further 2 hours. After incubation with the detection antibody, the supernatant was removed, the wells were washed as described above, and 100 μl of streptavidin-HRP solution (1:200 dilution from stock) was added to each well in the dark for 20 minutes. Subsequently, 50 μl of stop solution (2 M H2SO4) was added, and the optical density (OD) of each well was read at 450 nm by subtracting the 570 nm blank from each well using a microplate reader. A 4-parameter logistic curve was constructed using GraphPad Prism 7, and the A1AT concentration was determined for each sample by interpolation from the standard curve and multiplication by the appropriate dilution ratio.

[0074] result The effect of (S)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid on the circulating levels of human Z A1AT was evaluated in a huZ mouse model. Mice were treated with forced oral administration of 5 mg / kg, 15 mg / kg, or 50 mg / kg twice daily for 14 consecutive days.

[0075] The data in Figure 4 show that (S)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid stimulates the secretion of human Z A1AT in a dose-dependent manner compared to the baseline level in huZ mice.

[0076] Example 7: Pharmacokinetics of (S)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid in mice

[0077] (S)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid was administered to male C57BI / 6 mice intravenously (2 mg / kg) or by forced oral administration (10 mg / kg). Whole blood diluted with water was prepared from these dosed animals over a time course up to 24 hours after dosing, enabling the measurement of the drug blood concentration by UPLC-MS / MS. The measured drug levels enabled the calculation of the following parameters for (S)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid.

[0078] Plasma half-life (t 1 / 2 ) = 1.2 hours Measured clearance = 4.7 ml / min / kg Volume of distribution (Vz) = 0.49 l / kg Oral C max = 33520 ng / ml AUC all = 54234 ng.h / ml AUC INF = 54372 ng.h / ml

[0079] Example 8: Pharmacokinetics of (R)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid in mice

[0080] (R)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid was administered intravenously (2 mg / kg) or orally (10 mg / kg) to male C57BI / 6 mice. Whole blood diluted with water was prepared from these administered animals over a time course of up to 24 hours after administration, allowing for the measurement of drug blood concentrations by UPLC-MS / MS. Based on the measured drug levels, the following parameters for (R)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid could be calculated.

[0081] Blood half-life (t 1 / 2 )=0.54 hours Actual clearance = 8.2 ml / min / kg Distribution volume (Vz)=0.38 l / kg Oral C max = 15599 ng / ml AUC all = 24158 ng.h / ml AUC INF = 24736 ng.h / ml

[0082] Example 9: Mouse and human hepatocyte stability of (S)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid and (R)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid

[0083] Intrinsic clearance (CL) of (S)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid and (R)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid intThe clotting factor and half-life of both compounds were measured in hepatocyte suspensions of cryopreserved male C57BL6 mouse hepatocytes or mixed hepatocyte suspensions of cryopreserved human hepatocytes. Briefly, the compounds were incubated with hepatocyte suspensions at 37°C for a set period of time, and the compounds remaining at each time point were evaluated by mass spectrometry (UPLC-MS / MS). For both compounds, clotting factor in mouse hepatocytes was measured. int <3 μl / min / 10 6 These are cells, and in human hepatocytes, <3 μl / min / 10 6 The cells were the target cells. For both compounds, the half-life in mouse hepatocytes was >460 minutes, and in human hepatocytes it was also >460 minutes.

[0084] Example 10: Plasma protein binding of (S)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid and (R)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid

[0085] The degree to which (S)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid and (R)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid binds to plasma proteins such as albumin and α-1 acid glycoprotein in human or mouse blood was determined by rapid equilibrium dialysis. The compounds were incubated at 5 μM for 4 hours at 37°C. For (S)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid, the binding rate to plasma proteins was 78.1% in mouse plasma and 91.5% in human plasma. For (R)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid, the binding rate to plasma proteins was 83.8% in mouse plasma and 90.5% in human plasma.

[0086] Example 11: Activity of (S)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid and (R)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid against cytochrome P450

[0087] The inhibition of individual CYPs by (S)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid and (R)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid was evaluated using E. coli CYPEX membranes in combination with specific probe substrates (see Weaver et al., 2003, Drug Metab Dispos 31:7, 955-966).

[0088] [Table 1]

[0089] Example 12: Activity of (S)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid on HERG channels

[0090] (S)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid was tested for inhibition of cardiac potassium (hERG) channels using a Patchliner automated patch clamp. Six-point concentration-response curves were generated using half-log serial dilutions from the maximum final test concentration of 100 μM. IC 50 The values ​​were obtained from four-parameter logistic fitting of concentration-response data. (S)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid showed an IC50 > 100 μM at 100 μM with 7% inhibition. The reference compound value was consistent with that shown in the literature (Elkins et al., 2013 J.Pharm.Tox.Meth. 68:11-122).

[0091] Example 13: Activity of (S)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid and (R)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid on a panel of enzymes, ion channels, and receptors.

[0092] (S)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid and (R)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid are found in DiscoverX Safety47 at 10 μM. TM When tested against a panel, the compounds showed very clean off-target profiles. None of the compounds inhibited any target by more than 25% at these concentrations.

[0093] Example 14: Determination of Human Equivalent Dose

[0094] To date, the highest dose of (S)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid used in efficacy studies in mice is 50 mg / kg (data not shown). Converting this to a human equivalent dose using Table 1 of the US FDA Center for Drug Evaluation and Research (CDER) "Guidance for Industry: Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers" (published in 2005 - see https: / / www.fda.gov / media / 72309 / download), a human equivalent dose (HED) of 4 mg / kg is obtained.

[0095] By comparison, as shown in Examples 7 and 8, the enantiomer (R)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid has a 2.2-fold shorter t1 / 2 and a 2.2-fold lower AUC in mice. Expecting that clinical efficacy is driven by either AUC or time above a given concentration, we conclude that the equivalent efficacy of the R enantiomer lies at a 2.2-fold higher dose or 8.8 mg / kg compared to the S enantiomer. In certain embodiments, for example, the following are provided: (Item 1) α in human subjects 1 - Compound for use in treating diseases or disorders characterized by low plasma levels of antitrypsin (A1AT), (S)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid [ka] A pharmaceutical composition comprising, wherein the composition is administered to a human subject at a daily dose of about 1 to 6 mg / kg. (Item 2) The composition is a pharmaceutical composition for use as described in item 1, administered to the human subject in a daily dose of about 2 to 5 mg / kg, for example, about 2 mg / kg, about 3 mg / kg, about 4 mg / kg, or about 5 mg / kg. (Item 3) A pharmaceutical composition for use as described in either item 1 or 2, further comprising a pharmaceutically or therapeutically acceptable excipient or carrier. (Item 4) The composition is a pharmaceutical composition for use as described in any of the preceding items, administered orally or intravenously. (Item 5) The use of a compound as defined in item 1 in the manufacture of a pharmaceutical product for the treatment of a disease or disorder characterized by low plasma levels of A1AT in a human subject, wherein the pharmaceutical product is administered to the human subject in a daily dose of approximately 1 to 6 mg / kg. (Item 6) A method for treating a disease or disorder characterized by low plasma levels of A1AT in a human subject, the method comprising the step of administering a pharmaceutical composition defined in any of items 1 to 4 at a daily dose of about 1 to 6 mg / kg. (Item 7) The aforementioned disease or disorder is α 1 - Antitrypsin deficiency (AATD), a pharmaceutical composition for use as described in any of items 1-4, a compound for use as described in item 5, or the method described in item 6.

Claims

1. α in human subjects １ - A pharmaceutical composition for use in treating antitrypsin deficiency (AATD), comprising the compound (S)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid 【Chemistry 4】 A pharmaceutical composition comprising, wherein the composition is administered to the human subject at a daily dose of approximately 1 to 6 mg / kg.

2. The pharmaceutically acceptable composition for use according to claim 1, wherein the composition is administered to the human subject in a daily dose of about 2 to 5 mg / kg, for example, about 2 mg / kg, about 3 mg / kg, about 4 mg / kg, or about 5 mg / kg.

3. A pharmaceutical composition for use according to claim 1, further comprising a pharmaceutically or therapeutically acceptable excipient or carrier.

4. The pharmaceutically acceptable composition for use according to claim 1, wherein the composition is administered orally or intravenously.

5. Use of the compound defined in claim 1 in the manufacture of a pharmacopoeia for the treatment of α1-antitrypsin deficiency (AATD) in a human subject, wherein the pharmacopoeia is administered to the human subject in a daily dose of about 1 to 6 mg / kg.

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