Hydroxy-substituted amino and ammonium derivatives and their medical use
11564895 · 2023-01-31
Assignee
Inventors
- Georg Schlechtingen (Dresden, DE)
- Hans-Joachim Knolker (Dresden, DE)
- Tim Friedrichson (Dresden, DE)
- Gary Jennings (Dresden, DE)
- Tobias Braxmeier (Kuppenheim, DE)
Cpc classification
A61K45/06
HUMAN NECESSITIES
A61K31/164
HUMAN NECESSITIES
International classification
A61K45/06
HUMAN NECESSITIES
A61K31/164
HUMAN NECESSITIES
Abstract
The present invention relates to hydroxy-substituted amino and ammonium derivatives, in particular the compounds of formula 1 or 2, and their medical use, including their use in the treatment, prevention or amelioration of an inflammatory, autoimmune and/or allergic disorder, or a proliferative, neoplastic or dysplastic disease or disorder. ##STR00001##
Claims
1. A method of inhibiting mast cell degranulation, Akt kinase, delayed-type hypersensitivity, allergic contact dermatitis inflammatory response, or collagen type II-induced arthritis, the method comprising administering an effective amount to a subject in need of such a treatment, a compound of the following formula 1 ##STR00006## wherein: R.sup.1 is a C.sub.10-20 hydrocarbon group; R.sup.2 is a C.sub.1-4 alkyl group, and R.sup.3 is —H, a C.sub.1-4 alkyl group or R.sup.3 is absent; R.sup.4 is —H or a C.sub.1-4 alkyl group; R.sup.5 is a C.sub.2-8 alkyl group, wherein one or more hydrogen atoms of said C.sub.2-8 alkyl group are replaced by —OH, or R.sup.5 is a C.sub.5-7 cycloalkyl group, wherein one ring carbon atom of said C.sub.5-7 cycloalkyl group is optionally replaced by an oxygen atom and wherein one or more hydrogen atoms of said C.sub.5-7 cycloalkyl group are independently replaced by —OH or —CH.sub.2OH; X is N.sup.+or, if R.sup.3 is absent, X is N; and p is 1, 2 or 3; or a pharmaceutically acceptable salt, or solvate thereof.
2. The method of claim 1, wherein the compound is a compound of the formula 1a, 1b, 1c, or 1d: ##STR00007##
3. The method of claim 1, wherein the compound is administered by an oral route, topical route, or transdermal.
4. The method of claim 1, wherein the subject is a human.
5. The method of claim 1, wherein said subject is a nonhuman mammal.
6. The method of claim 1, wherein the method is inhibiting mast cell degranulation.
7. The method of claim 6, wherein mast cell degranulation is selected from asthma, asthma related diseases, urticarial, mast cell disease, mast cell activation syndrome (MCAS), IgE mediated disorders, and eczema.
8. The method of claim 1, wherein the method is inhibiting Akt kinase.
9. The method of claim 1, wherein the method is inhibiting delayed-type hypersensitivity.
10. The method of claim 1, wherein the method is inhibiting allergic contact dermatitis inflammatory response.
11. The method of claim 1, wherein the method is inhibiting collagen type II-induced arthritis.
12. The method of claim 11, wherein the collagen type II-induced arthritis is rheumatoid arthritis.
Description
(1) The invention is also described by the following illustrative figures. The appended figures show:
(2)
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(7) The invention will now be described by reference to the following examples which are merely illustrative and are not to be construed as a limitation of the scope of the present invention.
EXAMPLES
Example 1; Preparation of N-(2-(Dimethyl(tetradecyl)ammonio)acetyl)-N-methyl-D-glucamine Chloride 1a
(8) A mixture of 2-(dimethyl(tetradecyl)ammonio)acetate (449 mg, 1.5 mmol), HBTU (569 mg, 1.5 mmol) and N,N-diisopropylethylamine (DIEA) (279 μL, 1.6 mmol) in a mixture of dry dimethylformamide (DMF) (10 mL) and dichloromethane (5 mL) is stirred for 5 min at room temperature under an argon atmosphere. Solid N-Methyl-D-glucamine (351 mg, 1.8 mmol) is added and stirring Is continued for another 3 h. The reaction mixture is concentrated under reduced pressure to dryness, taken up in a mixture of tetrahydrofuran and ethanol and purified by preparative HPLC to yield 375 mg (42%) of 1a as trifluoroacetate salt. Then, ion exchange is performed in the batch mode using a (P)-NMe.sub.3.sup.+ type resin in the chloride form and ethanol/water as a solvent. The solvent mixture is removed under reduced pressure and the residue dried in vacuum to provide 1a as the corresponding chloride salt.
(9) ESI-MS, Positive: 477.4 (M).sup.+, 989.2 (2M+Cl).sup.+. Negative: 547.5 (M+2Cl).sup.−1, 1059.2 (2M+3Cl).sup.−, .sup.1H-NMR (600 MHz, DMSO-d.sub.6): δ=0.84 (t, J=7.1, 3H), 1.18-1.30 (m, 22H), 1.64 (m, 2H), 2.87 (s, 2.1H), 3.00 (s, 0.9H), [3.19 (s), 3.19 (d, J=1.7), 3.20 (s), Σ=6H], 3.24 (d/d, J=15.0/3.0, 0.8H), 3.45-3.63 (me, 8.4H), 3.80 (m, 1H), [4.36-4.43 (me), 4.46 (d, J=7.2), 4.49 (s), Σ=3.6H], [4.56 (d), 4.58 (t, J=6.5), Σ=1.7H], 4.63 (d, J=5.5, 0.7H), 4.80 (d, J=5.1, 0.3H), 5.15 (d, J=5.4, 0.7H). (NMR-analysis is complicated by the complex nature of the spectrum and hindered rotation around the amide bond.)
Example 2: Preparation of N-(2-(Methyl(tetradecyl)amino)acetyl)-N-methyl-D-glucamine Hydrotrifluoroacetate 1b
(10) Compound 1b is prepared from 2-(methyl(tetradecyl)ammonio)acetate (130 mg, 0.45 mmol) and N-methyl-D-glucamine (88 mg, 0.45 mmol) using HBTU (133 mg, 0.35 mmol) and N,N diisopropylethylamine (DIEA) (0.40 mmol) in a mixture of 3 mL of dimethylformamide (DMF) and 1 mL of dichloromethane in the same way as described for compound 1c in example 3. Preparative HPLC and drying under vacuum provided 116 mg (58%) of 1b as a colorless solid.
(11) ESI-MS, Positive: 463.4 (M+H).sup.+, 947.6 (2M+Na).sup.+. Negative: 461.1 (M−H).sup.−, 575.1 (M+TFA).sup.−, 923.3 (2M−H).sup.−.
(12) .sup.1H-NMR (500 MHz, CDCl.sub.3/CD.sub.3OD 8:2): δ=0.80 (t, J=6.9, 3H), 1.18+1.26 (2s, Σ=22H), 1.64 (br, m, 2H), 2.82+2.85 (2s, 3H), 2.94+2.96 (2s+m, 3.50H), 3.14 (d/d+m, J=15.1/3.2 Σ=2.0H), 3.48 (m, 1.3H), 3.62 (m, 5H), 3.95 (m, 1.2H), 4.08 (br, s+m, 9.3H, OH+H.sub.2O), 4.45 (br, m, 0.75H). (Some signals are doubled due to slow rotation of amide.)
Example 3: Preparation of N-(2-(Methyl(dodecyl)amino)acetyl)-N-methyl-D-glucamine Hydrotrifluoroacetate 1c
(13) 2-(Dodecyl(methyl)ammonio)acetate (130 mg, 0.51 mmol) is dissolved in a mixture of 3 mL of dry dimethylformamide (DMF) and 1.5 mL of dry dichloromethane under argon atmosphere, HBTU (133 mg, 0-35 mmol) and N,N-diisopropylethylamine (DIEA) (70 μL, 0.40 mmol) are added and the mixture is stirred at room temperature for 5 min. N-Methyl-D-glucamine (88 mg, 0.45 mmol) is added and stirring is continued for another 2 h. Then, upon control by analytical HPLC, only minor amounts of residual 2-(dodecyl(methyl)-ammonio)acetate are visible. The solvents are removed under reduced pressure and the residue is dried in vacuo, taken up in 3 mL of ethanol and purified by preparative HPLC to TO yield 106 mg (55%) of 1c as a colorless solid.
(14) ESI-MS, Positive; 435.4 (M+H).sup.+. Negative; 433.1 (M−H).sup.−, 547.0 (M+TFA).sup.−.
(15) .sup.1H-NMR (500 MHz, CDCl.sub.3): δ=0.81 (t, J=6.8, 3H), 1.23+1.28 (2s, Σ=18H), 1.68 (br, m, 2H), 2.90 (s, 2.18H), 2.92 (s, Σ=0.84H), 2.94 (s, 2.30H), 2.99 (s+m, 2=1.50H), 3.47 (m, 1.4H), 3.57 (m, 4H), 3.62 (m, 1H), 3.80-4.05 (m+br, Σ=12H). (Some signals are doubled due to slow rotation of amide.)
Example 4: Preparation of N-(2-(Dimethyl(tetradecyl)ammonio)acetyl)-N-methyl-2,3-dihydroxypropylamine Trifluoroacetate 1d
(16) 2-(Dimethyl(tetradecyl)ammonio)acetate (300 mg, 1.0 mmol) Is suspended in 7 mL of dry dimethylformamide (DMF) under argon atmosphere. The compound is dissolved by addition of 3 mL of dry dichloromethane. Solid HBTU (379 mg, 1.0 mmol) is added, followed by N,N-diisopropylethylamine (DIEA) (182 μL, 1.05 mmol). The mixture turns brownish yellow. After 10 min of activation time, 3-(methylamino)propane-1,2-diol (201 μL, 2.1 mmol) is added as a solution in 1 mL of dichloromethane. The solution turns brighter. The mixture is stirred overnight at room temperature. The reaction mixture is then concentrated under reduced pressure. The crude product is purified by preparative HPLC. Removal of the solvent of the product fraction under reduced pressure provides a semisolid which is triturated with a diethyl ether/petrolether mixture. The supernatant is discarded and the semisolid residue is dried in vacuo to give 113 mg (22%) of 1d as a colorless solid.
(17) ESI-MS, Positive: 387.4 (M).sup.+, 887.7 (2M+TFA).sup.+. Negative: 613.0 (M+2TFA).sup.−.
(18) .sup.1H-NMR (300 MHz, CDCl.sub.3/CD.sub.3OD 8:2); δ=0.81 (t, J=6.5, 3H), 1.18+1.25 (2s, Σ=22H), 1.63 (br, m, 2H), 2.91 (s, 2.4H), 3.03+3.10 (s+d/m, Σ=1.3H), 3.23 (s+m, X™ 5.8H), 3.35-3.69 (m, 4H), 3.77 (m, 1.8H), 4.36 (d, J=16.3, 1.2H), 4.72 (d, J=16.3, 0.75H), 5.63 (br, s, 3H). (Some signals were doubled due to slow rotation of amide.)
Example 5: Inhibition of Mast Cell Degranulation
Introduction
(19) Mast cells are key effector cells involved in allergic and inflammatory diseases, and the Rat Basophilic Leukemia clone 2H3 (RBL-2H3) cell line is a commonly used model of allergen dependent immune modulator release (degranulation) in mast cells. On their surface, they express the high affinity receptor for IgE (FcεRI). Upon binding of antigen-specific IgE to the receptor, cells become sensitized to the IgE specific antigen (allergen). When IgE-sensitized cells then encounter multivalent antigen, the antigen clusters IgE-FcεRI complexes and initiates a signal transduction cascade that leads to degranulation, that is, the release of inflammatory mediators, such as cytokines, eicosanoids, histamine and enzymes. The assay can be used as a screening method to Identify immune-modulating compounds, in particular compounds useful in the medical management of allergic and inflammatory diseases and asthma, β-hexosaminidase was previously shown to be released with the same kinetics as histamine (Schwartz et al., J Immunology; 123:1445-1450 (1979)), thus offering a simple means to monitor degranulation. The RBL-2H3 cell line has been successfully used to identify compounds with anti-allergic activity (Choo et al. Plants Med., 69:518-522 (2003)).
(20) Materials and Methods
(21) Materials
(22) Chemicals: Rat anti-DNP IgE monoclonal antibody was acquired from Biozol (BZL06936), dinitrophenyl-conjugated human serum albumin (A6661) and Triton X-100 (T9284) were from Sigma-Aldrich, 4-methylumbelliferyl-N-acetyl-β-D-glucosaminide (474502), Phorbol-12-myristate-13-acetate (524400) and thapsigargin (586005) from Calbiochem. Ionomycin (ALX-450-006) was purchased from Alexis Biochemicals. DMSO was from Merck (1.02950.0500) or Sigma-Aldrich (D2650). Cell culture media and supplements, Minimum Essential Medium (21090-022), Minimum Essential Medium without Phenol Red (51200-046), RPMI 1640 Medium (31870-025), L-Glutamine (25030-024) and 0.05% Trypsin-EDTA (25300-054), were obtained from Invitrogen, Fetal bovine serum (A15-151) was from PAA Laboratories. Other reagents were standard laboratory grade or better.
(23) Buffers and solutions: Phosphate buffered saline (PBS) and 1 M HEPES were provided by the in-house service facility. Tyrode's buffer (TyB) consisted of Minimum Essential Medium without Phenol Red supplemented with 2 mM L-glutamine and 20 mM HEPES. Lysis buffer consisted of 25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA and 0.1% (w/v) Triton X-100. DNP-HSA was dissolved to 1 mg/ml in water. MUG substrate solution consisted of 2.5 mM 4-methylumbelliferyl-N-acetyl-β-D-glucosaminide in 0.05 M citrate, pH 4.5; stop solution was 0.1 M NaHCO.sub.3/0.1 M Na.sub.2CO.sub.3, pH 10.
(24) Consumables and equipment: For small-volume liquid handling procedures, Rainin LTS electronic pipettes were routinely used (Mettler-Toledo), Costar-Corning 24-well plates (3337) were centrifuged in an Eppendorf 5804 R centrifuge. A Heraeus 615 table top Incubator was used for incubations at 37° C. under non-sterile conditions. Fluorescence was measured in black Nunc 96-well plates (237105) using a microplate reader (Tecan Safire) or FlexStation 3 (Molecular Devices) multi-mode plate reader. Cells were maintained in Hera Cell 240 CO.sub.2 incubators (Thermo Scientific). Serological pipettes (4487, 4488 and 4489) and cell culture flasks (431080) were from Corning-Costar, 1.5 and 2 ml microcentrifuge tubes (0030 120.086 and 0030 120.094) were from Eppendorf.
(25) Cell Culture: RBL-2H3 cells obtained from the German Collection of Microorganisms and Cell Cultures (ACC312) (Braunschweig, Germany) were maintained in 70% Minimum Essential Medium with Earle's Salts, 20% RPMI 1640 Medium, 10% FBS and 2 mM L-glutamine in 95% alr/5% CO.sub.2 at 37° C. and routinely checked for mycoplasma contamination. Cells were passaged every 3-4 days; after washing cells once with 35 ml PBS cells were incubated 8 min with 5 ml 0.05% Trypsin-EDTA solution at 37° C., Ceils were removed from the incubator, 15 ml culture medium was added and cells were resuspended by repeated pipetting.
(26) Cell seeding: cells were harvested with Trypsin-EDTA as described and 50-100 μl cell suspension seeded Into Costar CellBind 24 well cluster plates (no. 3337). Plates were kept for 30 min at RT under the sterile hood before being transferred to the incubator. Cells were used within one or two days after seeding.
(27) Measurement of β-Hexosaminidase Release
(28) Experimental Procedures
(29) For sensitization, cells for immediate use were sensitized 6-12 h after plating; cells to be used the following day were sensitized 26-38 h after plating. Culture plates were removed from the incubator and checked for cell growth and contamination. The medium was discarded and cells were sensitized with anti-DNP IgE (0.4 μg/ml) in 0.4 ml culture medium overnight. Following overnight sensitization, ceils were washed with 0.8 ml pre-warmed TyB and 0.38 ml test compound or vehicle control (supplemented or not with 1% FBS) were added to duplicate wells. Samples Were adjusted to contain 1% vehicle for test compounds dissolved in organic solvents. Cells were incubated for 1 h at 37° C. At the end of the incubation period, cells were routinely stimulated with 20 μl DNP-HSA (2 μg/ml; final concentration 0.1 μg/ml) diluted in TyB and cells were incubated for 15 min at 37° C., Alternatively, cells were stimulated with 20 μl 5 μM ionomycin (final concentration 0.25 μM) or 20 μl 5 μM thapsigargin (final concentration 0.25 μM), both in the absence or presence of 20 nM PMA (final concentration).
(30) Plates were removed from the incubator and immediately centrifuged at 4° C. for 5 min at 250×g and transferred to an ice bath. Aliquots of supernatants, 25 μl, were transferred to 96-well plates. Remaining supernatant was aspirated from control wells and cells were lysed in 400 μl lysis buffer for 5 min at RT on an orbital shaker at 450 rpm under non-sterile conditions. After lysis, 25 μl aliquots of lysates were transferred to 96-well plates.
(31) MUG substrate solution, 100 μl, were added to supernatant and lysate samples and plates were incubated 30 min at 37° C. The reaction was terminated by addition of 150 μl stop solution, Fluorescence was measured at 365 nm excitation and 440 nm emission wavelengths.
(32) Test compound preparation: test compounds were prepared in 1.5 or 2 ml microcentrifuge tubes and incubated for 30 min at 37° C. in a Thermomixer Comfort with agitation (750 rpm).
(33) An electronic multichannel pipette was used for rapid transfer of compound dilutions from microcentrifuge tubes to the cells.
(34) Controls: controls used are defined as follows: negative control, supernatant of unstimulated cells was measured for unspecific β-hexosaminidase release; positive control, supernatant of DNP-HSA stimulated cells was measured for specific, antigen-stimulated β-hexosaminidase release; maximum control, lysate of unstimulated cells was measured for total β-hexosaminidase content.
(35) Assessment of Pharmacologic Effect
(36) Degranulation (β-hexosaminidase release): Degranulation was calculated as the percentage of β-hexosaminidase released with respect to maximum control (total β-hexosaminidase) after subtraction of negative control (unspecific release) using the formula;
% Degranulation=100×(test compound−negative control)/(maximum control−negative control).
(37) Inhibition of degranulation (inhibition of β-hexosaminidase release): Inhibition of degranulation was calculated as percent reduction of β-hexosaminidase release with respect to positive control (antigen-stimulated release) after subtraction of negative control (unspecific release) using the formula;
% Inhibition=100×(1−(test compound−negative control)/(positive control−negative control)).
Measurement of Maximum Tolerated Concentration
(38) The maximum tolerated concentration (MTC), i.e. the highest concentration of test compound that does not cause cytotoxicity, as determined by the release of lactate dehydrogenase, was measured over the tested concentration range. A commercially available cytotoxicity test was used (Promega Cytotox-One cat. #67891).
(39) The safety index (SI) of a test compound is the ratio between the maximum tolerated concentration and the IC50 and is used as a measure of the relative safety of the test compound.
(40) Results
(41) Concentration-dependent inhibition of degranulation was determined for all test compounds over a concentration range, as shown in
(42) TABLE-US-00001 TABLE 1 Inhibition of degranulation: IC50, MTC and SI values Compound IC50 (μM) MTC (μM) SI 1a 3.8 150 39.5 1b 4.9 50 10.2 1c 12.3 100 8.1 1d 3.1 75 24.2 Miltefosine 4.2 25 6.0
(43) The MTC of the test compounds was up to 8-40 fold higher than their respective IC50s and hence, the inhibition of degranulation can be ascribed to a pharmacological effect and not to an effect secondary to cytotoxicity.
(44) All substances outlined in Table 1 show IC50 values in the low micromolar range combined with high MTC values when compared to Miltefosine. Thus, the compounds according to the invention and, in particular compounds 1a to 1d, have an advantageously low cytotoxicity. A particularly preferred compound of the present invention is compound 1a which has a very low IC50 and a high MTC value, providing for a high therapeutic window (safety index).
(45) Mast cell degranulation is a key cellular event in allergic and inflammatory reactions, in particular in pathological events involving the release of mediators such as histamine, leukotrienes and prostaglandins as well as proteases. As consequence, the inhibition of mast cell degranulation is a valuable strategy for prevention or treatment of pathological processes involving the aforementioned mediators. Furthermore, the mast cell degranulation assay provides an estimate of the activity of test compounds in other cells that play a key role in the inflammatory response, such as granulocytes, macrophages and thymocytes, which release proinflammatory cytokines and chemokines and tissue eroding proteases.
Example 6: Inhibition of Activation of Akt Kinase
Introduction
(46) The mast cell degranulation assay using the RBL-2H3 cell line (see example 5) was also used to determine the status of the PI3K/Akt axis. Activation of PI3K leads to production of PIP3 on the cytosolic side of the lipid bilayer, Akt is recruited to the PIP3 domain and subsequently activated by phosphorylation on residues Ser473 and Thr308. (Franke et al., Cell 81:727-736, (1995)). Once recruited to the membrane, it is phosphorylated and activated by other kinases (Hemmings, Science 275:628-630 (1997); Hemmings, Science 276:534 (1997); Downward, Science 279:673-674 (1998); Alessi et al., EMBO J. 15:6541-6551 (1996)), Western blotting of the phosphorylated Ser473 residue on Akt (phospho-Akt Ser473) is widely used to assess the level of activation of the PI3K/Akt axis.
(47) Materials and Methods
(48) Materials
(49) All buffers and solutions used for the phosphor-Akt Ser473 assay were from Meso Scale Discovery. Iris Lysis Buffer consisted of 150 mM NaCl, 20 mM Tris, pH 7.5, 1 mM EDTA, 1 mM EGTA and 1% Triton-X-100. Complete Tris Lysis Buffer was prepared prior to use by addition of protease inhibitor, phosphatase inhibitors and PMSF. The 10×Tris Wash Buffer consisted of 500 mM Tris, pH 7.5, 1.5 M NaCl and 0.2% Tween-20, Blocker A was made up of bovine serum albumin in Tris Wash Buffer. Read Buffer T was used according to manufacturer's instructions. The Whole Cell Lysate Kits used were phospho-Akt Ser473 (K11100D, Lot K0011749) and total ERK1/2 (K11107D, Lot K0011698) as a loading control.
(50) Equipment
(51) 12-well multichannel pipettes (30-300 μl) from Eppendorf were used. Assay plates were agitated on a TiMix 5 control (Edmund Buhler), Electrochemiluminescence detection was performed on a SECTOR Imager 6000 (Meso Scale Discovery).
(52) Measurement of phospho-Akt Ser473
(53) Experimental Procedures
(54) Protein assay: protein concentration was determined using the BCA (bicinchoninic acid) Protein Assay kit according to the manufacturer's instructions, Briefly, duplicate 10 μl samples of bovine serum albumin (BSA) standards, blank and lysates were incubated in a 96-well plate with 0.2 ml working reagent for 30 min at 37° C. Plates were cooled to room temperature for 5 min and absorbance at 562 nm was measured in a multi-mode plate reader. Protein concentrations were calculated using FlexStation 3 software (SoftMax Pro version 5.3). Protein concentration of lysates was determined from a standard curve (BSA) using a linear curve fit.
(55) Phosphoprotein assay: protein phosphorylation was determined using the MULTI-SPOT® Assay System (Meso Scale Discovery), providing simultaneous detection of phosphorylated and total proteins. Briefly, capture antibodies against phosphorylated and total protein are patterned on distinct spots in the same well of 96-well plates. Sandwich immunoassay and electrochemiluminescence detection technology are combined to measure intensity of the emitted light from phosphorylated and total protein spots. Analysis of phosphor-Akt Ser473 was performed according to the manufacturer's instructions. The optimal amount of protein was determined at 5 μg lysate per well for ERK1/2 and 10 μg/well for phospho-Akt Ser473. Plates were blocked with 25 μl/well Blocker A for 1 h at room temperature with gentle agitation. During this time, the lysates were thawed and diluted to the desired protein concentration in complete Tris Lysis Buffer. Plates were washed four times in Tris Wash Buffer and 25 μl lysate per well added. Plates were incubated for 1-3 h at room temperature with agitation according to the manufacturer's recommendations. Plates were washed four times with Tris Wash Buffer, followed by addition of 25 μl/well of the respective detection antibody and incubation for 1 h at room temperature, with agitation. After a final four washes with Tris Wash Buffer 150 μl/well, Read Buffer T was added, and plates read on a SECTOR Imager 6000 plate reader.
(56) Assessment of Effects of Phospho-Akt Ser473
(57) The mean background signal from each plate was subtracted from averaged raw data. The amount of total protein phosphorylated was expressed as % phosphoprotein according to the manufacturer's (Meso Scale Discovery) instructions.
(58) Results
(59) Levels of phospho-Akt Ser473 were determined in IgE sensitized and antigen stimulated cells after treatment without (positive control) or with 1, 5 and 25 μM test compound and normalized to levels of total Akt. Concentration-dependent inhibition of Akt phosphorylation on Ser473 was demonstrated, as shown in
(60) TABLE-US-00002 TABLE 2 Inhibition of Akt phosphorylation on Ser473 by compound 1a Level of phospho-Akt Ser473 (% positive control) Compound 1 μM 5 μM 25 μM 1a 81.8 ± 42.5 22.6 ± 15.7 6.8 ± 1.8 Percentage of total Akt phosphorylated on Ser473 expressed as percentage of control untreated cells, after induction with IgE and antigen for 15 min.
(61) A dose-dependent decrease in levels of phospho-Akt Ser473 was observed after treatment with compound 1a (Table 2). Thus, the compounds according to the invention can be used to reduce levels of activated Akt and, accordingly, are useful in the medical intervention in indications in which hyperactivated Akt plays a pathogenic role, such as inflammatory and allergic diseases, hyperproliferative diseases and other indications.
Example 7: Inhibition of the Delayed-Type Hypersensitivity (DTH) Reaction in Mice
Introduction
(62) The anti-inflammatory and anti-allergic effects of compound 1a was assessed in a mouse model of skin delayed-type hypersensitivity (DTH) reactions and compared to a vehicle control and to the reference drug, dexamethasone, DTH reactions are antigen-specific cell-mediated immune responses, driven primarily by T helper type 1 (Th1) cells, similar to the tuberculin immunization response. The immune reaction induced by an ovalbumin challenge to animals previously sensitized with ovalbumin in Complete Freund's Adjuvant, is characterized by swelling (edema) at the site of challenge, e.g. the mouse ear. Dexamethasone, an anti-inflammatory steroid, reduces cell-mediated Immune responses and was employed to validate the responsiveness of the assay to pharmacological treatment,
(63) Materials and Methods
(64) Materials
(65) Ovalbumin (fraction V, lyophilized powder), complete Freud's adjuvant (CFA) and methylcellulose were obtained from Sigma-Aldrich, dexamethasone from Pharmaceutical Works Polfa (Pabianice, Poland).
(66) Animals
(67) Female BALB/cJW mice were bred at the University of Lodz, Lodz, Poland and housed in groups of 8 in makrolon cages with a 12 h light-dark cycle. Mice were given free access to food (Agropol S. j., Motycz, Poland) and water.
(68) Antigen Sensitization and Challenge
(69) Group size was n=8 mice unless otherwise stated. Test compound was freshly prepared before administration.
(70) Sensitization: The protein antigen, ovalbumin, was reconstituted in PBS at 4 mg/ml. An ovalbumin-CFA emulsion was prepared by mixing the protein solution with the CFA suspension at a ratio of 1:1, using two Luer-lock syringes. The emulsion was tested by putting a drop of emulsion onto PBS; if the emulsion remained as a tight droplet on the PBS, the emulsion was deemed ready. Mice were sensitized by subcutaneously injecting 25 μL of emulsion into each side of the tail (100 μg ovalbumin per mouse).
(71) Challenge: On the sixth day after sensitization, DTH was elicited by challenging animals subcutaneously (gauge 30 needle, B. Braun Melsungen, Melsungen, Germany) in the left ears with 10 μL of a 1% suspension of heat-aggregated ovalbumin (HOVA) (100 μg ovalbumin per mouse). The right ears were administered subcutaneously with PBS and served to determine the individual differences in ear thicknesses. HOVA was prepared by heating a 5% solution of ovalbumin in saline for 1 h at 80° C. with occasional swirling. After cooling to room temperature and centrifugation (400 g, 10 min at 4° C.), the pellet was washed twice with saline, resuspended at 2% in PBS and aliquots stored at −30° C. Before injection, HOVA was diluted with an equal volume of PBS and sonicated. Ear thickness was measured with a precise spring-loaded caliper (Art. No. 7309, Mitutoyo, Kawasaki, Japan) before challenge, and 24 h after challenge.
(72) Sensitization, challenge and ear thickness measurement were performed under anesthesia (ketamine 80 mg/kg plus xylazine 8 mg/kg, intraperitoneally).
(73) Compound Administration
(74) The anti-inflammatory effects of compound 1a were compared to a vehicle control (0.5% methyl cellulose solution) and to the reference drug, dexamethasone. Test compound was given at 25 or 100 mg/kg orally by gavage (Art. No. 432093, Harvard Apparatus GmbH, March-Hugstetten, Germany) 16 h and 3 h before sensitization and then twice daily with the final dose given 3 h prior to ear challenge (a total of 14 administrations). Dexamethasone was given at 0.1 or 1 mg/kg orally by gavage 3 h before sensitization and once daily with the final dose given 3 h prior to antigen challenge (a total of 7 administrations), All administrations were given in a volume of 10 mL/kg.
(75) Quantification of Assay Results
(76) To account for individual variability, the increase in right ear thickness, before and 24 h after administration of PBS, was subtracted from the HOVA-induced increase in left ear thickness. The increase in ear thickness was calculated by the difference between ear thickness before and 24 h after antigen challenge. Percent inhibition of ear swelling was calculated according to the following formula;
% inhibition=100×(IET.sub.vehicle−IET.sub.compound)/IET.sub.vehicle
Where IET=(ET.sub.24 hrs pc−ET.sub.predose).sub.HOVA-treated ears−(ET.sub.24 hrs pc−ET.sub.predose).sub.PBS-treated ears (IET, increase ear thickness; ET, ear thickness; pc, post challenge)
Statistical Evaluation
(77) Mean and standard deviation (SD) were calculated from individual ear edema values. Statistical evaluation was a one-way analysis of variance (ANOVA) with Dunnett's post hoc test or Student's t-test where appropriate.
(78) Results
(79) Suppression of mouse ear swelling by compound 1a as well as dexamethasone, compared to vehicle control is shown in
(80) TABLE-US-00003 TABLE 3 Effect of compound 1a on ear swelling in the DTH response in mice. Inhibition of Compound mouse ear swelling 1a, 25 mg/kg 43* 1a, 100 mg/kg 50* Dexamethasone, 0.1 mg/kg 14 Dexamethasone, 1.0 mg/kg 53* *p < 0.01 vs. vehicle control (Dunnett's post hoc test)
(81) Dexamethasone administered orally at a dose of 1 mg/kg, once daily over the whole sensitization period resulted in a significantly reduced DTH response, with inhibition of 53%, Such high dosing (overdose) is, however, not suitable for treatment of humans due to severe side effects of the corticosteroid and was only used to validate the responsiveness of the model. In addition, in the course of the current study, a significant loss in body weight of 9% (p<0.01 vs. vehicle control with the paired Student's t-test) was seen in the high dose dexamethasone group, A more clinically representative dose of dexamethasone in the mouse is 0.1 mg/kg, but at this dose inhibition was very low (14%) and did not reach significance indicating that only steroid doses which result in significant body weight loss upon repeated administration are active in this model.
(82) Compound 1a, administered orally twice dally over the whole sensitization period at two dosing regimens, 25 mg/kg or 100 mg/kg, reduced the DTH response by 43% and 50%, respectively. Hence, compound 1a was able to produce an inhibition almost equivalent to that of the high dose of dexamethasone (up to 94%). In contrast to dexamethasone, no significant toxic side-effects of compounds 1a were observed during the course of the study.
(83) The reduction of DTH response obtained by treatment with compound 1a demonstrates that the compounds according to the invention and, in particular compound 1a, are effective in the pharmaceutical intervention in allergic and inflammatory diseases involving antigen-specific cell-mediated immune responses. Even at the low dose, compound 1a provided for the similarly high inhibition of the DTH response as obtained by an overdose of dexamethasone, and thus represents a particularly preferred compound of the present invention.
Example 8: Inhibition of the Allergic Contact Dermatitis Inflammatory Response in Mice
Introduction
(84) The anti-inflammatory and anti-allergic effects of compound 1a were assessed in a mouse model of allergic contact dermatitis, a response driven primarily by T helper type 2 (Th2) cells. It has been demonstrated that BALB/c mice are susceptible to the allergen toluene-2,4-diisocyanate (TDI), producing an inflammatory condition of the skin with similar aspects to that of human atopic dermatitis (Baumer et al., J Pharm Pharmacol, 55:1107-1114 (2003); Baumer et al., Br J Dermatol. 151:823-830 (2004); Ehinger et al., Eur J Pharmacol. 392:93-99 (2000)). In this model, an allergic dermatitis response is obtained by sensitizing mice to TDI and subsequently challenging them with antigen by topical administration onto the ears. A quantitative assessment of anti-inflammatory and anti-allergic effects of topically or orally administered test compounds is possible by measuring the resulting ear swelling.
(85) The advantages of the allergic contact dermatitis model (Zöllner et al., Bioessays 26:693-6 (2004)) are reproducibility and reliability (>90% of BALB/c mice respond to sensitization), a short induction protocol, quantitative assessment by measuring ear thickness, atopic dermatitis-like skin lesions can be induced, and clinically relevant pharmaceuticals, such as corticosteroids, calcineurin-inhibitors and PDE4-inhibitors, are effective in this model.
(86) Materials and Methods
(87) Materials
(88) Dexamethasone dihydrogenphosphate (Dexa-Inject) was obtained from Mibe GmbH, Jena, Germany and diflorasone diacetate from Basotherm, Biberach an der Riss, Germany
(89) Animals
(90) Female BALB/c-mice were obtained from Charles River (Sulzfeld, Germany) at age 8 weeks. All animals were housed in groups of eight per cage at 22° C. with a 12 h light/dark-cycle. Water and a standard diet (Altromin, Lage/Lippe, Germany) were available ad libitum. All animals were acclimatized for one week before experimental procedures were commenced.
(91) TDI Sensitization, Allergen Challenge and Mouse Ear Swelling Test
(92) Experimental procedures for BALB/c mice housing, TDI sensitization and challenge, and measurement of ear thickness were performed as previously described (Baumer et al., J Pharm Pharmacol. 55:1107-1114 (2003)) with the following modifications. For active sensitization, 100 μL of 5% (w/v) TDI was administered to the shaved and stripped abdominal epidermis on day one, and for the next three consecutive days, 50 μL of 5% (w/v) TDI was applied. The allergic reaction was boosted 21 days later by application of 50 μL of 0.5% (w/v) TDI. For the examination of test compound effects, the left ears were used for the TDI challenge (20 μL of 0.5% in acetone) and ear thickness measured 3 h before and 24 h after challenge.
(93) Compound Administration for Systemic Treatment
(94) Group size was n=7 mice unless otherwise stated. Test compound was freshly prepared before administration.
(95) Administration time: to determine optimal time for administration treatment groups were treated orally by gavage with 100 mg/kg of compound 1a (suspended in 0.5% tylose, 10 mL/kg) 1, 4 or 16 h before topical TDI challenge. One group was treated with 100 mg/kg miltefosine orally, 16 h before challenge (based on available data for optimal administration time for miltefosine) and vehicle treated mice received tylose (10 mL/kg) orally, 4 h before challenge.
(96) Comparison with dexamethasone: one group of mice was treated orally with compound 1a at 100 mg/kg suspended in 0.5% tylose, 4 h before topical TDI challenge. Vehicle treated mice received 0.5% tylose orally 4 h before challenge. As a positive control, dexamethasone was administered in saline solution at 1 mg/kg or 3 mg/kg, 2 h and 30 min before challenge and 1 h after challenge. The dose and dosing scheme for dexamethasone was based on previous experience showing a maximal effect in this model.
(97) Compound Administration for Topical Treatment
(98) Compound 1a was administered to two groups of mice topically in 20 μl of a 2% or 6% solution in propyleneglycol. The solution was applied, 2 h before topical TDI challenge by administration of 10 μl onto each of the inner and outer surfaces of the left ears. A vehicle group (n-5) was treated with propyleneglycol. As a positive control, diflorasone diacetate was administered at 0.01% (low dose) and 0.05% (high dose) in 20 μl acetone, 2 h before challenge. A basal control group was left untreated.
(99) Determination of Local Lymph Node Weight and Cell Count
(100) Directly after sacrifice, the ear draining lymph node (Ln. auricularis) was prepared and excised. Organ weight was determined by means of an analytical balance (Kern, Balingen, Germany). Single cell suspensions were prepared by means of a glass potter (VWR, Darmstadt, Germany) and cells were counted with a hemocytometer (Neubauer, VWR, Germany).
(101) Statistical Evaluation
(102) Mean and standard error of the mean (SEM) were calculated from individual ear edema values. Statistical evaluation was a one-way analysis of variance (ANOVA) (if the test for normal distribution was passed) or the Kruskal-Wallis one-way ANOVA on Ranks (if the normal distribution test failed). Both were followed by a post-hoc test (Dunnett's method or Dunn's test, respectively). A p<0.05 was considered to be significant.
(103) Results
(104) Suppression of mouse ear swelling by compound 1a after oral administration, compared to vehicle control is shown in
(105) TABLE-US-00004 TABLE 4 Effect of orally administered compound 1a on ear swelling in the allergic contact dermatitis response in mice. Inhibition of Compound mouse ear swelling Administration time (oral) 1a, 100 mg/kg, 1 h 43.5* 1a, 100 mg/kg, 4 h 62.9*** 1a, 100 mg/kg, 16 h 43.5.sup.§ Miltefosine, 100 mg/kg, 16 h 47.3.sup.§ Comparison with dexamethasone (oral) 1a, 100 mg/kg 32.0** Dexamethasone, 1 mg/kg 78.6*** Dexamethasone, 3 mg/kg 87.1*** *p < 0.05, **p < 0.01, ***p < 0.001 vs. vehicle control (Dunnett's post hoc test) compared to vehicle, .sup.§p < 0.05 vs. vehicle control (t-test)
(106) In the administration time study with oral administration, compound 1a reduced ear swelling significantly (63% of vehicle control) when administered 4 h before challenge, as also shown in
(107) In the comparison with dexamethasone, compound 1a administered 4 h before challenge reduced ear swelling significantly (32%) at 100 mg/kg, as shown in
(108) Compound 1a at a concentration of 100 mg/kg had a significant impact on the TDI induced inflammatory reaction. Thus, the compounds according to the invention and, in particular compound 1a, are particularly effective and thus useful for the oral pharmaceutical intervention in inflammatory diseases, in particular in atopic dermatitis.
(109) Suppression of mouse ear swelling by compound 1a after topical administration, compared to vehicle control is shown in
(110) TABLE-US-00005 TABLE 5 Effect of topically administered compound 1a on ear swelling in the allergic contact dermatitis response in mice. Inhibition of Compound mouse ear swelling 1a, 2% 67.7*** 1a, 6% 82.2*** Diflorasone, 0.1% 101.1*** Diflorasone, 0.5% 110.8*** ***p < 0.001 vs. vehicle control (Dunnett's post hoc test) compared to vehicle. Diflorasone treatment reduced ear thickness below that of untreated mice.
(111) Compound 1a topically administered as a solution at 2% or 6% highly significantly reduced ear swelling by 68% and 83%, respectively. The positive control, diflorasone, completely eliminated ear swelling and even reduced ear thickness to below the level of untreated mice. This indicates that the doses of diflorasone used here are not representative of a clinical benchmark, but were used to validate the responsiveness of the model, it must also be stressed, that diflorasone is one of the strongest dermal corticosteroids and is taken for severe eczema.
(112) One of the most undesirable side-effects of corticosteroid administration is immunosuppression, which leads to the inability to effectively address parasitic infection, wound healing and tumor growth. In the current study, the local lymph node reaction after TDI challenge (lymph node weight and cell number) was determined to assess the response of immune organs. Diflorasone produced a highly significant reduction in the local lymph node reaction at both 0.1% and 0.5%, by completely inhibiting the Increase in lymph node weight and cell number (
(113) In view of the strong effect shown in the allergic contact dermatitis model, the compounds of the present invention, including compound 1a, are particularly effective and thus useful for the topical pharmaceutical intervention in inflammatory diseases, in particular in atopic dermatitis. In addition, the compounds of the present invention, including compound 1a, do not show adverse effects typical of topically administered corticosteroids, such as inhibition of the lymph node reaction and loss in body weight.
Example 9: Inhibition of Collagen Type Ll-Induced Arthritis (CIA) in the Mouse
Introduction
(114) The inhibitory effect of compound 1a was assessed for anti-inflammatory and anti-arthritic activity in the type if collagen-induced arthritis (CIA) model in the mouse. CIA has been proposed as a pertinent animal model of rheumatoid arthritis in humans. In this model, a peripheral arthritis is elicited by intradermal injection of homologous or heterologous (e.g. bovine, chicken) type II collagen (CM) in complete Freund's adjuvant (CFA) into rats or mice (Stuart et al., Ann Rev Immunol, 2:199-218 (1984); Marty et al., J Clin Invest. 107:631-640 (2001); Boissier et al., Eur J Immunol. 25:1184-90 (1995)). The central role played by T cells in the development of type II CIA is demonstrated by the T cell proliferative response to mouse CII in immunized mice, the successful adoptive transfer of the disease with immune cells from the spleen, and the resistance of athymic nude mice to the induction of the pathology (Stuart et al., Ann Rev Immunol. 2:199-218 (1984); Marty et al., J Clin Invest. 107:631-640 (2001)). An advantage of this model of arthritis as compared to others is the development of an arthritogenic response toward a well defined antigen (CII), which also permits the study of antigen-induced immunological phenomena and their selective modification by immunopharmacological intervention.
(115) Materials and Methods
(116) Materials
(117) Bovine type II collagen (Chondrex, Redmond Wash., USA) was dissolved at 2 mg/ml in 0.05 M acetic acid by gentle stirring overnight at 4° C. CFA was prepared by adding Mycobacterium tuberculosis H37Ra (Difco, Detroit, Mich.)) at 2 mg/ml to IFA (incomplete Freund's adjuvant, Sigma Aldrich, Milano, Italy). Before injection, CII was emulsified with an equal volume of CFA.
(118) Animals
(119) Eight to 9 week old male DBA/1j mice were purchased from Harlan Laboratories srl (San Pietro al Natisone, Udine, Italy) and kept under standard laboratory conditions with free access to food and water. Mice were allowed to adapt one week to their environment before starting the study.
(120) Induction of Collagen Induced Arthritis (CIA)
(121) Group size was n=11 mice unless otherwise stated. Test compounds were freshly prepared before administration. Mice were injected intradermally at the base of the tail with 100 μL of an emulsion containing 100 μg of CII, IFA and 100 μg of Mycobacterium tuberculosis. On day 21, a boost of CII in IFA was administered.
(122) Prophylactic Treatment
(123) Five groups of mice were treated under a prophylactic regimen from day 0 to 47 and an additional group of sham treated mice was treated only with the CII vehicle, on days 0 and 21. Compound 1a was administered as a suspension in 0.5% carboxymethylcellulose (10 mL/kg) and, as a positive control, dexamethasone was administered at 0.3 mg/kg as detailed below:
(124) Group 1: compound 1a at 25 mg/kg, orally by gavage, twice daily
(125) Group 2: compound 1a at 100 mg/kg, orally by gavage, twice daily
(126) Group 3: dexamethasone 0.3 mg/kg, intraperitoneally, once daily
(127) Group 4: vehicle (carboxymethylcellulose), orally by gavage
(128) Group 5: sham treated mice
(129) Animals were sacrificed on day 47 after immunization.
(130) Therapeutic Treatment
(131) Groups of mice were treated with compound 1a from the onset of arthritic symptoms, defined as first day on which a disease score of 1 or higher was observed and mice expressing the respective disease score were randomly assigned to each experimental group. Treatment was continued for 20 consecutive days.
(132) Group 6: compound 1a at 100 mg/kg, orally by gavage, twice daily
(133) As the treatment was based on the individual expression of arthritis symptoms, the mice were synchronized to the first day of treatment for evaluation of the disease progression. No separate vehicle group was included for the therapeutic treatment regimen hence vehicle group 4 was reanalyzed after synchronization to the first day on which a disease score of 1 or higher was observed. Animals were sacrificed after 20 days of treatment.
(134) Clinical Assessment
(135) Mice were evaluated for arthritis daily by an observer unaware of the treatment regimens according to a macroscopic scoring system: 0=no signs of arthritis; 1=swelling and/or redness of the paw or one digit; 2=involvement of 2 joints; 3=involvement of more than 2 joints; 4=severe arthritis of the entire paw and digits. An arthritis index was calculated for each mouse by summing the scores for individual paws. Clinical severity was also determined by evaluation of paw thickness of both front and hind-paws using a thickness gauge. An index was calculated for each mouse by summing the thickness for individual paws. Body weights were also recorded daily.
(136) Statistical Evaluation
(137) Mean and standard deviation (SD) were calculated from individual score values.
(138) For the arthritis score two different statistical calculations were performed. For each treatment day the arthritis scores of each group were compared to the vehicle control group using the student's-t test and a p<0.05 was considered significant.
(139) Additionally, a cumulative arthritis score was calculated for each treatment group by summing all arthritis scores throughout the study period. The cumulative arthritis scores were compared using the student's-t test and a p<0.05 was considered significant. The cumulative arthritis score requires that all animals are evaluated for the same length of time; in order to determine the cumulative arthritis score of animals which had died during the study, the missing values were substituted with the group mean for the day of the missing value. The substitution of missing values was only performed for the cumulative arthritis score and not used for other calculations.
(140) Results
(141) Effects of the Prophylactic Treatment of Test Compounds on Arthritic Score
(142) As expected, starting 5-6 days after the CII boost, clinical signs of arthritis became observable in vehicle treated control mice, consisting of progressively augmenting arthritic scores, accompanied by increased paw thickness. Significant loss in body weight of vehicle treated animals compared to the sham-treated group was observed after the CII boost.
(143) Relative to vehicle treated control mice, prophylactic treatment at both 25 and 100 mg/kg compound 1a significantly reduced the arthritic score from day 34 and 40, respectively, until the end of the study on day 47, as shown in
(144) Effects of the Therapeutic Treatment of Test Compounds on Arthritic Score
(145) Compound 1a at 100 mg/kg reduced the arthritis score and cumulative arthritis score compared to vehicle treated mice (
(146) Furthermore, a trend to a reduction in the duration of disease compared to vehicle treated mice was observed.
(147) Immune Organ Weights
(148) At sacrifice, thymuses and spleens were collected and weighed to assess the effect on these important immune organs. Compared to the sham-treated mice, animals treated with the vehicle exhibited a significant increase in spleen weight, due to the proliferation of lymphocytes in answer to elicitation by CII injection. As expected, treatment with the positive control drug, dexamethasone, markedly reduced the weights of both spleens and thymuses compared to both vehicle treated and sham groups demonstrating the known immunosuppressive effect of corticosteroids (
(149) The data demonstrate that compound 1a at 25 or 100 mg/kg exerted marked anti-inflammatory effects in murine type II CIA, leading to a significant reduction of clinical parameters associated to development of the disease. At the most clinically effective dose of 25 mg/kg, compound 1a did not show any toxic effects, whereas dexamethasone caused a significant loss in body weight and spleen and thymus weights. These results suggest that the compounds of the present invention, including compound 1a, are particularly effective and thus useful for the medical intervention in rheumatoid arthritis.