Competitive PPAR-gamma antagonists

Abstract

The present invention pertains to a new compound E)-2-(5-((4-methoxy-2-(trifluoromethyl)quinolin-6-yl)methoxy)-2-((4-(trifluoromethyl) benzyl)oxy)-benzylidene) hexanoic acid (MTTB), and its derivatives. The compounds of the invention are useful as selective peroxisome proliferator-activated receptor gamma (PPAR) antagonists and are indicated for the use in the treatment of immune related diseases such as systemic inflammation, sepsis and septic shock.

Claims

1. A compound of the formula (I), or a stereoisomer, or salt thereof: ##STR00014## wherein R.sub.1 is selected from a non-substituted, monosubstituted or polysubstituted aryl or heteroaryl residue, R.sub.2 is a radical of the formula (III), ##STR00015## wherein X and Z are independently selected from C or N, R.sub.9 to R.sub.12 are independently selected from H, hydroxyl, a non-substituted, monosubstituted or polysubstituted C.sub.1-C.sub.18-alkyl, wherein the alkyl can be straight, branched or cyclic, alkenyl, trifluormethyl, a non-substituted, monosubstituted or polysubstituted aryl or heteroaryl residue, a non-substituted, monosubstituted or polysubstituted benzyl group, an acyl group, or a branched or heteroatom- or aryl-substituted acyl group, an alkoxy substituent, the alkyl group thereof is branched, non-branched or cyclic, an alkyl group bound through a sulfur atom, or a sulfonyl group, or a nitrogen substituent, or fluoro, chloro, bromo, iodo, CN or a hetero substituent, if X is N then R.sub.11 is absent, and if Z is N then R.sub.12 is absent; and R.sub.1 and R.sub.2 are different, and R.sub.3 is a non-substituted, monosubstituted or polysubstituted C.sub.1-C.sub.10-alkyl, wherein the alkyl can be straight, branched or cyclic.

2. The compound according to claim 1, wherein R.sub.3 is non-substituted C.sub.1-C.sub.8 alkyl.

3. The compound according to claim 1, wherein R.sub.1 is a radical with the formula (II), ##STR00016## wherein R.sub.4 to R.sub.8 are independently selected from H, OH, SH, a non-substituted, monosubstituted or polysubstituted C.sub.1-C.sub.18-alkyl, wherein the alkyl can be straight, branched or cyclic, alkenyl, trifluormethyl, a non-substituted, monosubstituted or polysubstituted aryl or heteroaryl residue, a non-substituted, monosubstituted or polysubstituted benzyl group, an acyl group, or a branched or heteroatom- or aryl-substituted acyl group, an alkoxy substituent, the alkyl group thereof is branched, non-branched or cyclic, an alkyl group bound through a sulfur atom, or a sulfonyl group, or a nitrogen substituent, or fluoro, chloro, bromo, iodo, CN or a hetero substituent.

4. The compound according to claim 3, wherein R.sub.4, R.sub.5, R.sub.7 and R.sub.8 are H, and R.sub.6 is trifluormethyl (CF.sub.3).

5. The compound according to claim 1, wherein Z is N, and X is C.

6. The compound according to claim 5, wherein R.sub.10 is H, R.sub.12 is absent, R.sub.9 is OMet (OCH.sub.3), and R.sub.11 is trifluormethyl (CF.sub.3).

7. A method for treating an immune disease in a subject in need thereof, the method comprising the step of administering to the subject a therapeutically effective amount of a compound according to claim 1.

8. A combination comprising (a) a compound according to claim 1, and (b) a second compound that is effective in the treatment of sepsis or systemic inflammation.

9. The combination according to claim 8, wherein the second compound (b) that is effective in the treatment of sepsis or systemic inflammation is selected from another PPAR antagonist, an immune suppressive agent, an antibiotic, a vasopressor, a corticosteroids, or activated protein C.

10. A method for treating an immune disease in a subject in need thereof, the method comprising the step of administering to the subject a therapeutically effective amount of the combination according to claim 8.

11. A pharmaceutical composition, comprising a compound according to claim 1, or a combination according to claim 10, together with a pharmaceutically acceptable carrier, adjuvant, diluent and/or excipient.

12. The method according to claim 7, wherein the immune disease is systemic inflammation or sepsis.

13. The compound according to claim 1, wherein R.sub.3 is non-substituted C.sub.2-C.sub.6 alkyl.

14. The compound according to claim 1, wherein R.sub.3 is non-substituted straight C.sub.4 alkyl.

15. The compound according to claim 6, wherein the compound is (E)-2-(5-((4-methoxy-2-(trifluoromethyl)quinolin-6-yl)methoxy)-2-((4-(trifluoromethyl) benzyl)oxy)-benzylidene) hexanoic acid (MTTB).

Description

(1) The present invention will now be further described in the following examples with reference to the accompanying figures and sequences, nevertheless, without being limited thereto. For the purposes of the present invention, all references as cited herein are incorporated by reference in their entireties. In the Figures:

(2) FIG. 1: Chemical structure and computational modelling of MTTB. The corresponding ligand incorporates all structural features of MTTB (A): an acidic head group, an adjacent lipophilic moiety and a central aromatic core with aromatic substituents on the opposite sites. The docking protocol converged yielding one single binding mode (B). The carboxylic moiety is engaged in ionic interactions with lysine 265 and hydrogen bond with the backbone amide of serine 323. The aromatic moieties and the alpha alkyl chain occupy the hydrophobic sites, indicating favourable interactions.

(3) FIG. 2: Kinetic characterisation of a PPAR agonist, a PPAR antagonist and SPPARMs in HEK293T cells. A PPAR-dependent transactivation assay was used to verify the agonistic effects of the PPAR agonist rosiglitazone, the antagonist GW9662 and the two SPPARMs FMOC-L-leucine and MCC-555 after 24 h stimulation with different concentrations (0.01 M-10 M) of the compounds (A). To determine the antagonistic effects using 10 M GW9662 and FMOC-L-leucine and MCC-555 in time course of the response to agonist (1 M rosiglitazone) stimulation (B) the PPAR-dependent transactivation assay was used again. Values are meansSEM of four experiments. Each experiment was performed in quadruple.

(4) FIG. 3: Comparison of the antagonistic effects of MTTB with those of GW9662 against increasing concentrations of rosiglitazone in vitro. To verify the agonistic and antagonistic effects of MTTB (A and C) and the reference PPAR antagonist GW9662 (B and D) in HEK293T cells (A and B) and Jurkat T cells (C and D) a PPAR-dependent transactivation assay was used. The cell lines were cultured with increasing doses (0.1 M-10 M) of rosiglitazone, MTTB or GW9662 alone or with the same concentrations of rosiglitazone in the presence of MTTB and GW9662 (1 M or 10 M) for 24 h. To determine the IC50 value for MTTB antagonism of response to rosiglitazone in HEK293T cells, a dose-response curve for MTTB antagonism was constructed using the PPAR-dependent transactivation assay (E). Data show responses to 1 M agonist (rosiglitazone) stimulation for 24 h in the presence of increasing concentrations of MTTB (0.1-50 M). All values are meansSEM of four experiments. Each experiment in HEK293T cells was performed in quadruple and in Jurkat T cells in triplicate. Statistically significant differences from antagonist alone were obtained in response to rosiglitazone (1-10 M) in the presence of MTTB at concentrations of x M and y M (p<0.05) in HEK293T cells and Jurkat T cells.

(5) FIG. 4: Cytotoxicity of the competitive PPAR antagonist MTTB and the PPAR antagonist GW9662 in vitro. Cell viability of HEK293T cells, HuT-78 cells and Jurkat T cells was determined by MTT assay after 24 h stimulation with different concentrations (0.1 M-30 M) of MTTB (A) or the PPAR antagonist GW9662 (B). The cell viability was calculated from the following equation: MTT optical density (OD) value of sample/MTT OD value of control (cells treated with DMSO). Data are shown as percent of solvent DMSO control and presented as meansSEM of three separate experiments. Each experiment was performed in octet.

(6) FIG. 5: Intracellular accumulation of MTTB and GW9662 in vitro. HEK293T cells, HuT-78 cells and Jurkat T cells were stimulated either with 10 M MTTB (A) or 10 M GW9662 (B) for 24 h. At different time-points the cells were harvested and the accumulation expressed as a percentage of the intracellular (I) to extracellular (E) concentration in mol per 510.sup.5 cells determined by LC-MS/MS. Values are meansSEM of three experiments.

(7) FIG. 6: Survival of Mice in a Sepsis Model after Treatment with MTTB.

EXAMPLES

(8) Materials and Methods

(9) Chemicals and Reagents

(10) All chemicals and reagents were of highest grade of purity and if not indicated otherwise, commercially available from AppliChem GmbH (Darmstadt, Germany), Carl Roth GmbH (Karlsruhe, Germany), Alfa Aesar GmbH (Karlsruhe, Germany), Apollo Scientific (Manchester, United Kingdom) and Sigma-Aldrich Chemie GmbH (Schnelldorf, Germany). The PPAR antagonist GW9662, the SPPARMs N-(9-fluorenylmethoxycarbonyl)-L-leucine (FMOC-L-leucine) and netoglitazone (MCC-555) were acquired from Cayman Chemical Company (Ann Arbor, USA) and the PPAR agonist rosiglitazone from Enzo Life Sciences GmbH (Lrrach, Germany). Cell culture media and supplements were purchased from PAA Laboratories GmbH (Clbe, Germany) and Sigma-Aldrich Chemie GmbH.

(11) Cell Culture

(12) Human T cell-78 (HuT-78) cells (Gazdar et al., 1980), Jurkat T cells (Schneider et al., 1977) and human embryonic kidney 293 cells, stably expressing the large T antigen of simian vacuolating virus 40 (HEK293T cells) (Graham et al., 1977) were obtained from LGC Standards GmbH (Wesel, Germany). HuT-78 cells and Jurkat T cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium and HEK293T cells in Dulbecco's Modified Eagle's Medium (DMEM) in a humidified 5% carbon dioxide atmosphere at 37 C.

(13) Both media contained 10% (v/v) heat-inactivated fetal calf serum, 100 units ml-1 penicillin and 100 g ml-1 streptomycin. The media were changed three times a week and the cells passaged before reaching confluency. When using DMSO, in all cases, the final concentration of DMSO did not exceed 0.1% and was not found to be cytotoxic in the cell lines used.

(14) Transient Transfection of Cultured Cells Lines

(15) For the PPAR-dependent transactivation assay, 1104 HEK293T cells per well were seeded in 96-well plates and cultured overnight as described above to allow attachment of the HEK293T cells. The next day, the HEK293T cells were transiently transfected using the JetPRIME transfection reagent (PEQLAB Biotechnologie GmbH, Erlangen, Germany), as described by the manufacturer, with 0.01 g per well pFA-PPAR-LBD-GAL4-DBD, kindly provided by Prof. Dr. Manfred Schubert-Zsilavecz (Institute of Pharmaceutical Chemistry, Department of Biochemistry, Chemistry and Pharmacy, Goethe-University Frankfurt am Main, Germany), 0.09 g per well pFR-Luc (Stratagene, La Jolla, USA) and 0.0005 g per well pRL-CMV (Promega GmbH, Mannheim, Germany). After 4 h, the transfection medium was replaced and the HEK293T cells were cultured in fresh growth medium for another 24 h. For the transient transfection of 1106 HuT-78 cells and Jurkat T cells per well, the SuperFect transfection reagent (QIAGEN GmbH, Hilden, Germany) was used as described by the producer with 0.2 g per well pFA-PPAR-LBD-GAL4-DBD, 0.8 g per well pFR-Luc and 0.05 g per well pRL-CMV. After 24 h, 1 ml fresh growth medium was added and the cells were cultured for another 48 h. The PPAR-dependent transactivation assay in HEK293T cells using a 96-well plate format was performed in quadruple and in HuT-78 cells and Jurkat T cells using a 12-well plate format in triplicate.

(16) PPAR-Dependent Transactivation Assay of Cultured Cells Lines

(17) The PPAR-dependent transactivation assay is based on the vector pFA-PPAR-LBD-GAL4-DBD encoding the hybrid protein PPAR-LBD-GAL4-DBD and the reporter vector pFR-Luc, carrying a GAL4-responsive element in front of the Firefly luciferase gene. These two vectors were co-transfected, as described above, in combination with the control vector pRL-CMV, encoding Renilla luciferase, to normalize Firefly luciferase activity for transfection efficiency. Following transfection, the cells were incubated with MTTB, FMOC-L-leucine, MCC-555, GW9662 and rosiglitazone for varying times (2-48 h) and at increasing concentrations (0.01-10 M), with or without rosiglitazone (0.01-10 M). Transactivation was analysed using a 96-well plate format in a Mithras LB940 multimode reader (Berthold Technologies, Bad Wildbad, Germany). The IC50 value for MTTB was determined by fitting the data to a sigmoidal dose-response curve using the GraphPad Software Prism (La Jolla, USA).

(18) Cell Viability Studies

(19) HuT-78 cells, Jurkat T cells and HEK293T cells, were seeded in 96-well plates at a density of 5104 cells per well and cultured overnight as described above to allow attachment of the HEK293T cells. The next day, the medium was replaced with fresh cell-specific growth medium containing varying concentrations of MTTB (1-30 M) and GW9662 (1-30 M). As control, DMSO alone was used. After 24 h stimulation, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) solution (5 mg ml-1 in phosphate buffered saline (PBS)) was added and the cells were incubated for two more hours at 37 C. (Mosmann, 1983). Afterwards, the cells were lysed with MTT lysis buffer. Following cell lysis, cell viability was measured at an absorbance of 560 nm in a Mithras LB940 multimode reader. The cell viability studies using a 96-well plate format were performed in octet.

(20) Determination of the Intracellular MTTB and GW9662 Accumulation

(21) To determine the intracellular accumulation of MTTB and GW9662, HuT-78 cells, Jurkat T cells and HEK293T cells, were seeded in 12-well plates at a density of 5104 cells per well and cultured overnight as described above to allow attachment of the HEK293T cells. The next day the medium was replaced with fresh cell-specific growth medium containing 10 M MTTB and 10 M GW9662. After 24 h treatment with MTTB and GW9662, the cells and the supernatant were harvested and analysed by LC-MS/MS. Accumulation was expressed as a percentage of the ratio of intracellular to extracellular concentration (I/E).

(22) Computational Modeling

(23) Computational docking was performed using the Molecular Operating Environment software suite 2012.10 (Chemical Computing Group Inc., Montreal, Canada). The assignment of hydrogens in the structure was performed using the Protonate3D routine (Labute, 2009).

(24) Statistical Analysis

(25) All data are presented as the meansstandard error of the mean (SEM). Each experiment was performed at least three times. Statistical analysis was done either with one- or two-way-analysis of variance modified with Bonferroni's multiple comparison test or unpaired and paired Student's t-test, respectively. Differences were considered significant when: p<0.05.

Example 1: Synthesis of MTTB

(26) The compounds of the invention, in particular MTTB are synthesized in a four stage process. In the following E refers to a reactant, P refers to a product of a reaction:

(27) Stage 1:

(28) ##STR00010##

(29) In a three-neck reaction vessel with reflux condenser, argon supply, septum and magnet stirrer, E1 (1 eq) and Cs.sub.2CO.sub.3 (1.3 eq) are suspended in dimethylformamide (DMF) under inert gas and slightly heated (about 60 C. for about 15 min). After cooling to room temperature (RT), E2 (1 eq) is quickly added via a septum in few milliliters of DMF and reacted under stirring without heating. After about 1.5 hours the solvent is removed under vacuum until nearly complete dryness. 50 ml of H.sub.2O are added with 50 ml of ethyl acetate to separate phases. The hydrous phase is extracted three times with ethyl acetate, and the organic phase is dried over MgSO.sub.4 and evaporated until complete dryness. The remainings underwent a column chromatography and after isolation the product was recrystallized two times (hexane/ethyl acetate).

(30) Stage 2:

(31) ##STR00011##

(32) In a three-neck reaction vessel with reflux condenser, argon supply, septum and magnet stirrer, P1 (1 eq) and Cs.sub.2CO.sub.3 (1.3 eq) are suspended in dimethylformamide (DMF) under inert gas and slightly heated (about 60 C. for about 15 min). After cooling to room temperature (RT), E3 (1 eq) is quickly added via a septum in few milliliters of DMF and reacted under stirring without heating. After about 1.5 hours the solvent is removed under vacuum until nearly complete dryness, 50 ml of H.sub.2O are added with 50 ml of ethyl acetate to separate phases. The hydrous phase is extracted three times with ethyl acetate, and the organic phase is dried over MgSO.sub.4 and evaporated until complete dryness. The remainings underwent a column chromatography and after isolation the product was recrystallized two times (hexane/ethyl acetate).

(33) Stage 3:

(34) ##STR00012##

(35) In a three-neck reaction vessel with reflux condenser, argon supply, septum and magnet stirrer, NaH (1.3 eq) is first added under argon, and then suspended with tetrahydrofuran (THF). After about 15 min stirring and cooling E4 (1.3 eq) in THF is added drop-wise within few minutes. E4 can be prepared via Arbuzov reaction by refluxing the respective -bromo-ester in triethylphosphite and distillation to yield E4. Then the mix is stirred for 30 to 60 minutes in an ice-bath. Via the septum the aldehyde (1 eq) in THF is added drop-wise. The reaction is observed via TLC. After completing the reaction (about 1 to 3 hours), the solvent is removed and the reaction is subject to a column chromatography. The purified product P3 is recrystallized (hexane/ethyl acetate).

(36) Stage 4:

(37) ##STR00013##

(38) In a reaction vessel with reflux condenser and a heating bath, the ester in THF is combined with LiOH mono hydrate (10 eq) dissolved in H.sub.2O. Methanol is added until the solution has only one phase and until complete hydrolysis heated at 40-60 C. while stirring. After about 12-72 hours THF and methanol are evaporated under vacuum. The acid is precipitated with 2N HCl. After removing the solvent from the precipitate and drying, the final product is obtained by recrystallization.

Example 2: MTTB Docks to the PPAR-LBD

(39) In a previous study it was shown that the similarity of the co-crystallized ligand is crucial for predictive modelling of the receptor-bound ligand conformation due to the pronounced induced fit known for PPAR (Weber et al., 2012). Therefore, the inventors have chosen for comparison an X-ray structure of a potent sulfoncarboxamide antagonist in complex with the PPAR-LBD (PDB code: 2HFP) (Hopkins et al., 2006).

(40) The corresponding ligand incorporates all the structural features of MTTB (FIG. 1A): an acidic head group, an adjacent lipophilic moiety and a central aromatic core with aromatic substituents on the opposite sites. The docking protocol converged, yielding one single binding mode as shown in FIG. 1B. The carboxylic moiety is engaged in ionic interactions with lysine 265 and a hydrogen bond with the backbone amide of serine 342. The aromatic moieties and the alpha alkyl chain occupy the hydrophobic sites, indicating favorable interactions. Pharmacophoric features situated on the moieties of the co-crystallized (vide supra) ligand were used for ligand placement. Water molecules remained in the binding site. The alpha HB score, which was shown to be well suited for docking in PPAR-LBD (Weber et al., 2012), was used for initial scoring, GBVI/WSA dG, a forcefield based function, for refinement.

Example 3: MTTB Inhibits Rosiglitazone-Induced Transactivation of PPAR

(41) To analyse the agonistic and antagonistic effects of the PPAR antagonist GW9662 (Leesnitzer et al., 2002), the SPPARMs FMOC-L-leucine (Rocchi et al., 2001) and MCC-555 (Reginato et al., 1998) and the PPAR agonist rosiglitazone (Willson et al., 1996) at the cellular level, a PPAR-dependent transactivation assay was used in HEK293T cells. Firefly luciferase luminescence values and control Renilla luciferase luminescence values were determined in each sample. The ratios of Firefly to Renilla luciferase luminescence were used to normalize luciferase activity values.

(42) The agonist concentration curves to PPAR receptor interacting agents exhibited differing courses after 24 h stimulation (FIG. 2A). Only rosiglitazone showed high agonistic effects and transactivated PPAR in a dose-dependent manner. Surprisingly, GW9662 as an irreversible full PPAR antagonist, showed agonistic effects and led to a dose-independent, 3-fold higher transactivation of PPAR compared to DMSO control treatment. Higher concentrations (above 1 M) of the SPPARM MCC-555 transactivated PPAR. In all cases, FMOC-L-leucine showed no agonistic effects. As shown in FIG. 2B, only the PPAR antagonist GW9962 inhibited the time-course of the response to agonist (1 M rosiglitazone) stimulation. Neither SPPARM, FMOC-L-leucine nor MCC-555, showed any antagonistic effects in the HEK293T cells. The very weak agonistic and non-existent antagonistic effects of FMOC-L-leucine and MCC-555 prompted us to use GW9662 for the following experiments and analyses as a reference compound.

(43) To verify the proposed interactions of MTTB with the PPAR-LBD (FIG. 1B) and to test potential agonistic and antagonistic effects in HEK293T cells and Jurkat T cells, the compound was also tested in the PPAR-dependent transactivation assay. The cells were cultured with different doses of MTTB and rosiglitazone alone or in combination for 24 h. As shown in FIGS. 3A and C, MTTB exhibited no agonistic effect in HEK293T cells and Jurkat T cells. In both cell lines, MTTB led to a dose-dependent inhibition of the rosiglitazone-induced transactivation of PPAR with competitive antagonistic/partial agonistic characteristics. The reference compound GW9662 showed full antagonism and inhibited dose-independently the rosiglitazone-mediated PPAR transactivation in HEK293T cells and Jurkat T cells (FIGS. 3B and D). However, GW9662 alone activated PPAR and showed surprisingly high agonistic effects in the transactivation assay in both cell lines. As indicated in FIG. 3E, MTTB was able to inhibit the agonistic activity of rosiglitazone (1 M) with an IC50 value of 4.3 M after 24 h in HEK293T cells.

Example 4: Effects of MTTB and GW9662 on Cell Survival

(44) To investigate the effects of MTTB and the reference compound GW9662 on survival of HEK293T cells, HuT-78 cells and Jurkat T cells, the cells were subjected to increasing doses (1-30 M) of MTTB or GW9662 for 24 h. Cell viability was analysed by a MTT assay. MTTB was without cytotoxic effects at concentrations up to 10 M (FIG. 4A). MTTB demonstrated comparable loss of viability in all three cell lines. In contrast, GW9662 showed varying cytotoxicity in the used cell lines (FIG. 4B). In HEK293T cells, up to a concentration of 30 M, no loss of viability was detectable. In HuT-78 cells and Jurkat T cells, however, GW9662 was clearly cytotoxic at concentrations above 20 M and 30 M, respectively.

Example 5: MTTB Shows High Intracellular Accumulation

(45) The time-courses of the percentage intracellular accumulation of MTTB (10 M) and the reference PPAR antagonist GW9662 (10 M) were analysed by LC-MS/MS. As shown in FIG. 5A, MTTB showed rapid sustained time-independent intracellular accumulation to approximately 10% after 24 h in HuT-78 cells and Jurkat T cells. In HEK293T cells, a time-dependent increase in the intracellular MTTB concentration was seen, achieving approximately 25% of the extracellular concentration after 24 h. As indicated in FIG. 5B, GW9662 showed 10-fold lower intracellular accumulation compared to MTTB decreasing with time in HEK293T cells. In HuT-78 cells and Jurkat T cells, the intracellular accumulation of GW9662 was about 100-fold less than that of MTTB (FIG. 5A). Especially in HuT-78 cells there was a rapid time-dependent decrease in the intracellular GW9662 accumulation.

Example 6: MTTB Significantly Enhances Survival in a Mouse Model of Sepsis

(46) The activity of MTTB was tested in a previously published mouse model of septic mice (Rittirsch D, Huber-Lang M S, Flierl M A, Ward P A. Immunodesign of experimental sepsis by cecal ligation and puncture. Nat Protoc 2009; 4: 31-36). In brief, the cecum ligation and puncture model (CLP) was performed as described by Rittirsch et al. using a 20-gauge puncture needle or without ligation and puncture for sham mice. MTTB loaded nano-particles were intraperitoneal applied 3 hours after ligation and puncturing. FIG. 6 shows a significant survival rate in-vivo of MTTB-treated septic mice compared to control nano-particles. This is a surprising demonstration of the applicability of the compounds of the invention in the treatment of immune diseases such as sepsis.

DISCUSSION AND CONCLUSIONS

(47) The extensive clarification of PPAR molecular interactions and signal transduction pathways, as well as its activation in molecular, cellular and clinical settings, has provided invaluable insights into the design of therapeutically useful PPAR agonists and SPPARMs (Willson et al., 2000, Sporn et al., 2001, Balint et al., 2006). While PPAR activation by natural and synthetic ligands is well established (Forman et al., 1995, Kliewer et al., 1995), only little is known about PPAR antagonism. However, increased understanding of the biological relevance of PPAR antagonism emphasizes the critical need for the discovery of new therapeutically useful PPAR antagonists, which do not exert known side-effects.

(48) Currently, GW9662 is the best described and most prominent PPAR antagonist (Leesnitzer et al., 2002), but it shows adverse properties including irreversible PPAR antagonism. Therefore, the inventors have identified and characterised MTTB, from a screen of structurally related compounds, as a promising prototype for a new class of competitive PPAR antagonists. Compared to the commercially available PPAR antagonist GW9662, MTTB offers the possibility to regulate inhibition of PPAR and immune responses and may be well suited for controlled therapeutic use in inflammatory conditions.

(49) In line with Leesnitzer and colleagues, the inventors observed non-competitive irreversible antagonism with GW9662 (Leesnitzer et al., 2002) so that the rosiglitazone-mediated PPAR transactivation was completely inhibited. Co-treatment of HEK293T cells and Jurkat T cells with rosiglitazone and GW9662 revealed the expected antagonistic effects. Interestingly, GW9662 alone led to dose-independent, approximately 3-fold PPAR transactivation, which to the inventor's knowledge has not been reported before. These results suggest that GW9662 is not a classical full PPAR antagonist in both HEK293T cells and Jurkat T cells, but rather acts as a SPPARM (Olefsky et al., 2000, Knouff et al., 2004) with strong antagonistic and distinct dose-independent agonistic activity. Because of the irreversible binding and the dose-independent agonistic effects of GW9962 at the PPAR protein, it is not suitable for therapeutic use.

(50) GW9662 has been reported to act as a potent PPAR antagonist with high-affinity and selectivity in both cell-free and cell-based assays (IC50 of 3.3 nM) and in human mammary tumour cell lines by covalently modifying the cysteine 285 residue in the ligand-binding site of PPAR (Leesnitzer et al., 2002, Seargent et al., 2004). As a consequence, GW9662 fully and irreversibly abrogates PPAR activation and signalling. The inventors observed only moderate or weak effects of the SPPARMs, FMOC-L-leucine and MCC-555, while GW9662 and MTTB potently inhibited the action of rosiglitazone in both HEK293T cells and Jurkat T cells. With an IC50 of 4.3 M in HEK293T cells, MTTB exhibited moderate dose-dependent, competitive antagonistic activity at lower concentrations than those reported for antagonism of PPAR by GW9662 in cell lines. This competitive antagonistic potency of MTTB against rosiglitazone mediated PPAR transactivation, was also verified in Jurkat T cells. In contrast to GW9662, MTTB showed less cytotoxicity in both HuT-78 cells and Jurkat T cells, indicating that at the concentrations tested, it is likely to exert pharmacological rather than toxicological effects on inflammatory cells. The inventor's observation of approximately 100-fold higher intracellular accumulation of MTTB, as compared to GW9662, into HuT-78 cells and Jurkat T cells, in contrast to HEK293T cells, in which uptake of MTTB was only 10-fold higher than of GW9662, suggests potential selectivity for lymphocytes.

(51) Interestingly, structural and computational docking analysis of MTTB emphasizes the potential affinity of MTTB for the PPAR-LBD and accentuates differences in the binding characteristics between MTTB, the antagonist GW9662 and the agonist rosiglitazone at PPAR (Chandra et al., 2008).

(52) Therefore MTTB will be used as a lead compound to enhance PPAR binding affinity by modification of its external residues. Taking into consideration that subtle changes in ligand receptor interaction lead to differences in pharmacology as has already been shown for the structurally similar PPAR TZD full agonists rosiglitazone and pioglitazone (Berger et al., 2005, Nissen et al., 2007), structural modifications may further improve the antagonistic effect of MTTB.

(53) Because of the strong antagonistic activity of GW9662, it is widely used as a research tool in cell culture systems and animal models to study the role of PPAR in biological processes. In contrast to the irreversible nature of GW9662 binding, MTTB appears to bindreversibly to the PPAR protein. Taken together, the competitive antagonism, low partial agonism, low cytotoxicity and high intracellular uptake are properties which would allow safe and repeated dosing of MTTB for potential therapeutical use. Further studies in animal models are planned. The results presented here with MTTB indicate that it is a prototype of a new class of competitive PPAR antagonists, and a promising candidate for a broadly applicable therapeutic approach to controlled treatment of inflammatory and immunological disorders.