Compositions for the treatment of alpha 1-antitrypsin deficiency and their uses

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 serpin superfamily that is produced by the liver and secreted into the blood. It inhibits various serine proteases, especially neutrophil elastase. When blood levels of A1AT are low, excess neutrophil elastase activity can degrade lung tissue, resulting in respiratory complications (e.g., chronic obstructive pulmonary disease (COPD)).

[0003] The reference range for A1AT in blood is 0.9-2.3 g / L. Lower levels are typical for α1-antitrypsin deficiency (A1AD or AATD), a genetic disorder caused by mutations in the SERPINA1 gene that codes for A1AT. The Z mutation, the most common cause of AATD, is a glutamine to lysine substitution (Z A1AT) at position 366 (which corresponds to position 342 in the mature form) of A1AT (UniProtKB - P01009(A1AT_HUMAN)). The Z mutation affects the folding of A1AT, so that only a small portion acquires the native / active state. The remainder is either eliminated as a misfolded protein or accumulates in the liver as a stable polymer. As a result of the misfolding, homozygous carriers of the Z mutation (ZZ) have plasma levels of A1AT that are 10-15% of normal, making them predisposed carriers for COPD. Accumulation of Z A1AT polymers in hepatocytes predisposes the carrier to cirrhosis, liver cancer, and other liver pathologies.

[0004] Current treatments for pulmonary expression of AATD include augmentation therapy using A1AT concentrates prepared from blood donor plasma. The US FDA has approved the use of four A1AT products: Prolastin, Zemaira, Glassia, and Aralast. Administration is via intravenous infusion once a week. Augmentation therapy has been shown to slow the progression of COPD. Hepatic expression of AATD (e.g., cirrhosis and cancer) is treated with steroids and liver transplantation. Investigational approaches to improved treatment of hepatic expression include inhibition of Z A1AT polymerization and increased clearance of the polymer via activation of autophagy. Investigational approaches to improved treatment of both pulmonary and hepatic expression involve improving Z 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 cavities that are potential targets for rational drug design to develop agents that affect Z A1AT polymerization.

[0006] Parfrey et al. (J. Biol. Chem., 2003, 278, 35, 33060-33066) further defined one cavity that is 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) reported that bis-ANS (4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonate) reacts with 1:1 stoichiometry and a K of 700 nM. d We showed that it can bind to Z A1AT at 100 ng / mL, but not 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, could increase Z A1AT levels in cell supernatants and in mouse models.

[0010] Bouchecareilh et al. (Journal of Biological Chemistry, 2012, 287, 45, 38265-38278) describe the use of histone deacetylase inhibitors, in particular SAHA (suberoylanilide hydroxamic acid), to increase 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, such as N-(4-hydroxy-3,5-dimethylphenyl)-2,5-dimethylthiophene-3-sulfonamide, can block the polymerization of Z A1AT in vitro.

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

[0014] US Pat. No. 8,436,013 B2 discloses a wide variety of structures capable of increasing 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] Journal of Applicable Chemistry vol 2, no 6, 2013, 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 modulators of alpha-1-antitrypsin and their use in treating diseases associated with alpha-1-antitrypsin. WO2020 / 081257A1 discloses pyrrolo-indazolyl-propanoic acid compounds as modulators of alpha-1-antitrypsin. US2020 / 0361939A1 further discloses pyrrolo-indazolyl-propanoic acid compounds as modulators of alpha-1-antitrypsin. [Prior art documents] [Patent documents]

[0020] [Patent Document 1] U.S. Pat. No. 8,436,013 [Patent Document 2] US Patent Application Publication No. 2011 / 0065707 [Patent Document 3] European Patent Application Publication No. 0520336 [Patent Document 4] International Publication No. 2019 / 243841 [Patent Document 5] International Publication No. 2020 / 081257 [Patent Document 6] US Patent Application Publication No. 2020 / 0361939 [Non-patent literature]

[0021] [Non-Patent Document 1] Elliott et al. (Protein Science, 2000, 9, 1274-1281) [Non-Patent Document 2] Parfrey et al. (J. Biol. Chem., 2003, 278, 35, 33060-33066) [Non-Patent Document 3] Knaupp et al. (J. Mol. Biol., 2010, 396, 375-383) [Non-Patent Document 4] Chang et al. (J. Cell. Mol. Med., 2009, 13, 8B, 2304-2316) [Non-Patent Document 5] Burrows et al. (Proc. Nat. Acad. Sci., 2000, 97, 4, 1796-1801) [Non-Patent Document 6] Bouchecareilh et al. (Journal of Biological Chemistry, 2012, 287, 45, 38265-38278) [Non-Patent Document 7] Berthelier et al. (PLOS ONE, May 11, 2015) [Non-Patent Document 8] Mallya et al. (J. Med. Chem., 2007, 50, 22, 5357-5363) [Non-Patent Document 9] Huntington(XIIIth International Symposium on Proteinases, Inhibitors and Biological Control, 23 September 2012 [Non-Patent Document 10] Huntington(7th International Symposium on Serpin Biology, Structure and Function, 1st April 2014) [Non-Patent Document 11] Angewandte Chemie International Edition vol 56, no 33, 2017, 9881-9885 [Non-Patent Document 12] Journal of Applicable Chemistry vol 2, no 6, 2013, 1501-1508 Summary of the Invention [Means for solving the problem]

[0022] A prior art search based on the structure of 1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid was conducted after making the present invention. The closest prior art molecule identified retrospectively by the search was the racemic compound, 1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid (CAS Registry Number 891392-68-0). This compound is listed as commercially available from Aurora, ChemDiv and the FCH Group, but no publications of record. Another close prior art molecule is 1-(1-tosyl-1,2,5,6-tetrahydropyridin-3-yl)ethan-1-one (Example 16 of US9084782B2). The compound is stated to inhibit angiogenesis and reduce cellular cholesterol levels (but no biological data is provided for this compound in US9084782B2).

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

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

[0025] In other aspects of the invention, the compositions may be administered to the human subject at different intervals, for example, every 2, 3, 4, 5 or 6 days, or once a week, in doses equivalent to the daily doses specified herein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

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

[0027] Additionally, as shown in Examples 7, 8 and 14, (S)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid can be synthesized from its enantiomer, (R)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid: [ka] The pharmacokinetic properties are shown to be effective for treating AATD at surprisingly lower doses.

[0028] The compounds in the pharmaceutical compositions of the present invention may be in a pharma- ceutically acceptable salt form.

[0029] The term "pharmaceutically acceptable salt" refers to a pharmaceutically acceptable mono-organic or inorganic salt of the compound of the present invention. This may include those derived from bases such as sodium hydroxide, potassium hydroxide, lithium hydroxide, calcium hydroxide, 1-deoxy-2-(methylamino)-D-glucitol, magnesium hydroxide, zinc hydroxide, aluminum hydroxide, iron(II) hydroxide or iron(III) hydroxide, ammonium hydroxide, or organic amines (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 a pharma- ceutically or therapeutically acceptable excipient or carrier.

[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 effectiveness or biological activity of the active ingredient and is not toxic to the host, which may be either a human or an animal to which it may be administered. Depending on the particular route of administration, a variety of pharmaceutically acceptable carriers, such as those well known in the art, may be used. Non-limiting examples include sugars, starches, cellulose and its derivatives, malt, gelatin, talc, calcium sulfate, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffered saline, emulsifiers, isotonic saline, and pyrogen-free water.

[0032] All suitable modes of administration are contemplated according to the present invention.For example, the administration of the composition or medicament can be via oral route, subcutaneous route, direct intravenous route, slow intravenous infusion, continuous intravenous infusion, intravenous or epidural patient-controlled analgesia (PCA and PCEA), intramuscular route, intrathecal route, epidural route, intracisternal route, intraperitoneal route, transdermal route, topical route, transmucosal route, buccal route, sublingual route, transmucosal route, inhalation, intranasal route, intra-atricular route, intranasal route, rectal route or ocular route.The composition or medicament can be formulated in separate dosage units (e.g., as required by the daily dosage regimen of the present invention) and can be prepared by any of the methods well known in the pharmaceutical field.

[0033] The compositions may be administered in particular in oral form, or alternatively, the compositions may be administered intravenously.

[0034] All suitable pharmaceutical dosage forms are contemplated. Medicament administration may be in the form of, for example, oral solutions and suspensions, tablets, capsules, lozenges, effervescent tablets, transmucosal films, suppositories, buccal or oral mucotentive products, topical creams, ointments, gels, films and patches, transdermal patches, abuse deterrent and abuse resistant formulations, sterile solutions suspensions, and depots for parenteral use, and may be administered as immediate release, sustained release, delayed release, controlled release, extended release, and the like.

[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 medicament for the treatment of a disease or disorder characterized by low plasma levels of A1AT in a human subject, wherein the medicament is administered to the human subject at a daily dose of about 1 to 6 mg / kg (including the doses as described above).

[0036] The present invention also includes a method of 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 1-6 mg / kg (including those doses as described above).

[0037] Diseases or disorders suitable for treatment according to related aspects of the invention are those characterized by low plasma levels of A1AT (eg, alpha 1-antitrypsin deficiency (AATD)).

[0038] The use of numerical ranges within this description is expressly intended to include within the scope of the invention all individual integers within the ranges stated, as well as all combinations of upper and lower numerical limits within the broadest range of the stated range.

[0039] As used herein, the term "comprising" should be read and understood to mean both comprising and consisting. In conclusion, when the present invention relates to a pharmaceutical composition that comprises a compound as an active ingredient, this terminology is intended to cover both compositions in which other active ingredients may be present and compositions that consist of only one active ingredient as defined.

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

[0041] Certain non-limiting examples of the present invention will now be described with reference to the following drawings.

[0042] [Figure 1] FIG. 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. Vehicle and 10 μM SAHA were tested as controls on each plate. The x-axis shows the various treatments of the cells: vehicle, SAHA and increasing concentrations of (S)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid, and the y-axis is the concentration of human A1AT in the cell supernatant (in ng / ml).

[0043] [Diagram 2] FIG. 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 shows the various treatments of the cells: vehicle, SAHA and (S)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid, and the y-axis is the concentration of human A1AT in the cell supernatant (in ng / ml).

[0044] [Diagram 3] FIG. 3 is a graph showing the effect 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 cells: vehicle, SAHA and two concentrations of (S)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid, and the y-axis is the concentration of human A1AT in the cell supernatant (in ng / ml).

[0045] [Figure 4] FIG. 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 oral gavage with vehicle, 5 mg / kg, 15 mg / kg, and 50 mg / kg (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 circulating basal levels of human Z A1AT. Plasma samples collected on the last 3 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 basal levels. The x-axis is the treatment dose (in mg / kg) of (S)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid; the y-axis is the mean percentage level of human Z A1AT compared to baseline levels for each treatment group.

[0046] [Diagram 5] FIG. 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. Vehicle and 10 μM SAHA were tested on each plate as controls. The x-axis shows the various treatments of the cells: vehicle, SAHA and increasing concentrations of (R)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid, and the y-axis is the concentration of human A1AT in the cell supernatant (in ng / ml).

[0047] [Figure 6]FIG. 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 shows the various treatments of the cells: vehicle, SAHA and (R)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid, and the y-axis is the concentration of human A1AT in the cell supernatant (in ng / ml).

[0048] [Figure 7] FIG. 7 is a graph showing the effect 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 cells: vehicle, SAHA and two concentrations of (S)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid, and the y-axis is the concentration of human A1AT in the cell supernatant (in ng / ml). 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 was stirred at room temperature for 3 hours. The reaction was cooled to 0° C. and acidified with 2M hydrochloric acid to give a white precipitate. The precipitate was dried and triturated with n-pentane to give (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 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 similarly to (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 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 the 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 cultured in a humidified atmosphere containing 5% CO in a 96-well plate (3.0 × 10 5 Cells were plated in 200 μl of medium / well (cells / ml and 200 μl medium / well) overnight at 37° C. After incubation, cells were washed three times with 200 μl serum-free medium and the medium was replaced in quadruplicate for 48 hours in a 37° C. incubator with treatment using serum-free medium containing vehicle, 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 step, the supernatants were removed from the wells, centrifuged at 1000×g for 10 min at 4° C., and human A1AT levels were assayed by ELISA (Human Serpin A1 / α1 antitrypsin duo set ELISA, R&D Systems, DY1268) according to the manufacturer's instructions.

[0054] Briefly, 96-well plates were coated with human A1AT capture antibody overnight at room temperature (1:180 dilution from stock, 100 μl final volume / well). The capture antibody was then removed, wells were washed 3 times with 300 μl wash buffer (0.05% Tween® 20 in PBS), and then 200 μl reagent diluent (25% Tween® 20 in PBS) was incubated in each well for 1 hour at room temperature. Diluted samples, standards (125 pg / ml, 250 pg / ml, 500 pg / ml, 1000 pg / ml, 2000 pg / ml, 4000 pg / ml and 8000 pg / ml of A1AT) or blanks were then added in duplicate to each well, and the plate was covered with a plate sealer and left at room temperature for 2 hours. At the end of the sample incubation step, samples were removed, all wells were washed as before, and 100 μl detection antibody (diluted 1:180 from stock) was added to each well and incubated for a further 2 hours at room temperature. After incubation with detection antibody, the supernatant was removed, wells were washed as before, and 100 μl streptavidin-HRP solution (diluted 1:200 from stock) was added to each well for 20 minutes, protected from light. Then, 50 μl stop solution (2M H2SO4) was added, and the optical density (OD) of each well was read at 450 nm using a microplate reader, with the 570 nm blank subtracted from each well. A four-parameter logistic curve was constructed using GraphPad Prism 7, and the A1AT concentration was determined in each sample by interpolation from the standard curve and multiplication by the appropriate dilution factor.

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

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

[0057] The data in FIG. 5 show that (R)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid stimulates 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 the A1AT cell secretion assay using HEK-M cells

[0059] method HEK-M cells (a human embryonic kidney cell line stably transfected with M A1AT) were cultured in a humidified atmosphere containing 5% CO in a 96-well plate (3.0 × 10 5 Cells were plated at 1000 x 1000 cells / ml in 200 μl medium / well overnight at 37° C. After incubation, cells were washed three times with 200 μl serum-free medium and the medium was replaced with serum-free medium containing either 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) in a final volume of 200 μl in a 37° C. incubator in sextuplicate for 48 hours. At the end of the incubation step, the supernatants were removed from the wells, centrifuged at 1000×g for 10 min at 4° C., and human A1AT levels were assayed by ELISA (Human Serpin A1 / α1 antitrypsin duo set ELISA, R&D Systems, DY1268) according to the manufacturer's instructions.

[0060] Briefly, 96-well plates were coated with human A1AT capture antibody overnight at room temperature (1:180 dilution from stock, 100 μl final volume / well). The capture antibody was then removed, wells were washed 3 times with 300 μl wash buffer (0.05% Tween® 20 in PBS), and then 200 μl reagent diluent (25% Tween® 20 in PBS) was incubated in each well for 1 hour at room temperature. Diluted samples, standards (125 pg / ml, 250 pg / ml, 500 pg / ml, 1000 pg / ml, 2000 pg / ml, 4000 pg / ml and 8000 pg / ml of A1AT) or blanks were then added in duplicate to each well, and the plate was covered with a plate sealer and left at room temperature for 2 hours. At the end of the sample incubation step, samples were removed, all wells were washed as before, and 100 μl detection antibody (diluted 1:180 from stock) was added to each well and incubated for a further 2 hours at room temperature. After incubation with detection antibody, the supernatant was removed, wells were washed as before, and 100 μl streptavidin-HRP solution (diluted 1:200 from stock) was added to each well for 20 minutes, protected from light. Then, 50 μl stop solution (2M H2SO4) was added, and the optical density (OD) of each well was read at 450 nm using a microplate reader, with the 570 nm blank subtracted from each well. A four-parameter logistic curve was constructed using GraphPad Prism 7, and the A1AT concentration was determined in each sample by interpolation from the standard curve and multiplication by the appropriate dilution factor.

[0061] result The amount of human A1AT secreted into the medium from transfected HEK-EBNA cells 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 secretion of human M A1AT at 10 μM as measured by ELISA. In contrast, the positive control, 10 μM SAHA, stimulates an increase in M ​​A1AT secretion.

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

[0065] The rare Siiyama mutation (Ser 53 to Phe, according to mature A1AT numbering) was identified in a Japanese male with AATD (Seyama et al. (J Biol Chem (1991) 266:12627-32). Ser53 is a conserved serpin residue that is thought to be important for organization of the inner core of the A1AT molecule. The change from an uncharged polar amino acid to a large nonpolar amino acid on the conserved backbone of the protein affects the 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 cultured in a humidified atmosphere containing 5% CO in a 96-well plate (3.0 × 10 5Cells were plated at 1000 x 1000 cells / ml in 200 μl medium / well overnight at 37° C. After incubation, cells were washed three times with 200 μl serum-free medium and the medium was replaced with serum-free medium containing 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 for 48 hours in a 37° C. incubator in eight replicates. At the end of the incubation step, the supernatants were removed from the wells, centrifuged at 1000×g for 10 min at 4° C., and human A1AT levels were assayed by ELISA (Human Serpin A1 / α1 antitrypsin duo set ELISA, R&D Systems, DY1268) according to the manufacturer's instructions.

[0067] Briefly, 96-well plates were coated with human A1AT capture antibody overnight at room temperature (1:180 dilution from stock, 100 μl final volume / well). The capture antibody was then removed, wells were washed 3 times with 300 μl wash buffer (0.05% Tween® 20 in PBS), and then 200 μl reagent diluent (25% Tween® 20 in PBS) was incubated in each well for 1 hour at room temperature. Diluted samples, standards (125 pg / ml, 250 pg / ml, 500 pg / ml, 1000 pg / ml, 2000 pg / ml, 4000 pg / ml and 8000 pg / ml of A1AT) or blanks were then added in duplicate to each well, and the plate was covered with a plate sealer and left at room temperature for 2 hours. At the end of the sample incubation step, samples were removed, all wells were washed as before, and 100 μl detection antibody (diluted 1:180 from stock) was added to each well and incubated for a further 2 hours at room temperature. After incubation with detection antibody, the supernatant was removed, wells were washed as before, and 100 μl streptavidin-HRP solution (diluted 1:200 from stock) was added to each well for 20 minutes, protected from light. Then, 50 μl stop solution (2M H2SO4) was added, and the optical density (OD) of each well was read at 450 nm using a microplate reader, with the 570 nm blank subtracted from each well. A four-parameter logistic curve was constructed using GraphPad Prism 7, and the A1AT concentration was determined in each sample by interpolation from the standard curve and multiplication by the appropriate dilution factor.

[0068] result The amount of human A1AT secreted into the medium from transfected HEK-EBNA cells 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 secretion of human Siiyama A1AT at 1 μM or 10 μM as measured by ELISA. In contrast, the positive control, 10 μM SAHA, stimulates an increase in Siiyama A1AT secretion.

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

[0071] Example 6: Activity of (S)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid in huZ mice The huZ mouse (also called PiZZ mouse) is 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 are on a C57Bl / 6 background and express human Z A1AT protein in liver tissue. The mice used in this study are derived from the descendants of Carlson and colleagues (transgenic strain Z11.03). HuZ mice are used as a tool to evaluate the effect of compounds on either increasing circulating levels of Z A1AT in plasma or on 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 200-600 μg / ml were treated by oral gavage twice daily for 14 consecutive days with either vehicle or 5 mg / kg, 15 mg / kg or 50 mg / kg (S)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid. Mice had free access to food (standard mouse chow, SAFE chow) and water. On study day 14, each mouse was dosed 1 hour before the last procedure. Blood was collected from the tail vein of each mouse on days -12, -7 and -5 before dosing, and on days 12, 13 and 14 during dosing. Blood was collected into a microvette containing EDTA, and plasma was prepared by centrifugation at 2700×g for 10 min at 4°C. The plasma was divided into aliquots and stored at -80°C for bioanalysis. Plasma samples from days -12, -7 and -5 before dosing were used to determine the average basal level of human Z A1AT for each mouse. Plasma samples collected on the last three dosing 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 on human Z A1AT secretion by measuring human Z A1AT levels and comparing them to the 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] Briefly, 96-well plates were coated with human A1AT capture antibody overnight at room temperature (1:180 dilution from stock, 100 μl final volume / well). The capture antibody was then removed, wells were washed 3 times with 300 μl wash buffer (0.05% Tween® 20 in PBS), and then 200 μl reagent diluent (25% Tween® 20 in PBS) was incubated in each well for 1 hour at room temperature. Diluted samples, standards (125 pg / ml, 250 pg / ml, 500 pg / ml, 1000 pg / ml, 2000 pg / ml, 4000 pg / ml and 8000 pg / ml of A1AT) or blanks were then added in duplicate to each well, and the plate was covered with a plate sealer and left at room temperature for 2 hours. At the end of the sample incubation step, samples were removed, all wells were washed as before, and 100 μl detection antibody (diluted 1:180 from stock) was added to each well and incubated for a further 2 hours at room temperature. After incubation with detection antibody, the supernatant was removed, wells were washed as before, and 100 μl streptavidin-HRP solution (diluted 1:200 from stock) was added to each well for 20 minutes, protected from light. Then, 50 μl stop solution (2M H2SO4) was added, and the optical density (OD) of each well was read at 450 nm using a microplate reader, with the 570 nm blank subtracted from each well. A four-parameter logistic curve was constructed using GraphPad Prism 7, and the A1AT concentration was determined in each sample by interpolation from the standard curve and multiplication by the appropriate dilution factor.

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

[0075] The data in FIG. 4 show that (S)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid stimulates secretion of human Z A1AT in a dose-dependent manner compared to baseline levels 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 intravenously (2 mg / kg) or by oral gavage (10 mg / kg) to male C57BI / 6 mice. Whole blood diluted with water was prepared from these dosed animals over a time course up to 24 hours post-dose to allow blood concentrations of drug to be measured by UPLC-MS / MS. The measured drug levels allowed the calculation of the following parameters for (S)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid:

[0078] Blood half-life (t 1 / 2 )=1.2 hours Actual clearance = 4.7ml / min / kg Distribution volume (Vz)=0.49 l / kg Oral C max =33520ng / ml AUC all =54234ng.h / ml AUC INF =54372ng.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 by oral gavage (10 mg / kg) to male C57BI / 6 mice. Whole blood diluted with water was prepared from these dosed animals over a time course up to 24 hours post-dose to allow blood fluid concentrations of drug to be measured by UPLC-MS / MS. The measured drug levels allowed the calculation of the following parameters for (R)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid:

[0081] Blood half-life (t 1 / 2 )=0.54 hours Actual clearance = 8.2ml / min / kg Distribution volume (Vz)=0.38 l / kg Oral C max =15599ng / ml AUC all =24158ng.h / ml AUC INF =24736ng.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 int) and half-life 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 time course, and the compound remaining at each time point was assessed by mass spectrometry (UPLC-MS / MS). For both compounds, CL in mouse hepatocytes was int <3μl / min / 10 6 cells, and <3 μl / min / 10 in human hepatocytes. 6 For both compounds, the half-life in mouse hepatocytes was >460 min and in human hepatocytes was >460 min.

[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 extent to which (S)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid and (R)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid are bound to plasma proteins such as albumin and alpha-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 plasma protein bound in mouse plasma was 78.1% and in human plasma was 91.5%. For (R)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid, the plasma protein bound in mouse plasma was 83.8% and in human plasma was 90.5%.

[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 the cardiac potassium (hERG) channel using a Patchliner automated patch clamp. Six-point concentration-response curves were generated using half-log serial dilutions from a maximum final test concentration of 100 μM. IC 50 Values ​​were obtained from a four-parameter logistic fit of the concentration-response data. (S)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid was shown to have an IC50>100 μM with 7% inhibition at 100 μM. Reference compound values ​​were consistent with those presented 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 against 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 were tested at 10 μM using the DiscoverX Safety47 TM When tested against the panel, they displayed a very clean off-target profile: no target was inhibited by more than 25% at this concentration for any compound.

[0093] Example 14: Determination of Human Equivalent Dose

[0094] The highest dose of (S)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid in mice used in efficacy studies to date is 50 mg / kg (data not shown). Converting this to a human equivalent dose using Table 1 from 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 2005 - see https: / / www.fda.gov / media / 72309 / download) indicates a human equivalent dose (HED) of 4 mg / kg.

[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 2.2-fold lower AUC in mice. Expecting clinical efficacy to be driven by either AUC or time above a given concentration, we conclude that the equivalent efficacy of the R enantiomer is at a 2.2-fold higher dose or 8.8 mg / kg compared to the S enantiomer.

Claims

1. Use in treating a disease or disorder characterized by low plasma levels of α １ - antitrypsin (A1AT) in a human subject, a pharmaceutical composition comprising the compound (S)-1-((2-(trifluoromethyl)phenyl)sulfonyl)piperidine-3-carboxylic acid 【Chemical Formula 4】 and wherein said composition is administered to said human subject at a daily dose of about 1 to 6 mg / kg, a pharmaceutical composition.

2. The pharmaceutical composition for use according to claim 1, wherein said composition is administered to said human subject at a daily dose of about 2 to 5 mg / kg, such as about 2 mg / kg, about 3 mg / kg, about 4 mg / kg or about 5 mg / kg.

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

4. The pharmaceutical composition for use according to claim 1, wherein said composition is administered orally or intravenously.

5. Use of a compound as defined in claim 1 in the manufacture of a medicament for the treatment of a disease or disorder characterized by low plasma levels of A1AT in a human subject, wherein said medicament is administered to said human subject at a daily dose of about 1 to 6 mg / kg.

6. The disease or disorder is α １ - antitrypsin deficiency (AATD), a pharmaceutical composition for use according to any of claims 1 to 4 or use according to claim 5.

Images