FR2847167A1 - Method for treating disorders associated with TGF-beta signal deficiency, e.g. cicatrization disorders, ulcers, cancers or graft rejection, comprises administration of retinoic acid receptor-Gamma antagonists - Google Patents

Abstract

The use of RARgamma (retinoic acid receptor-gamma) antagonists (I) in the production of medicaments for treating disorders associated with TGFbeta (transforming growth factor-beta) signal deficiency or requiring amplification of this signal. An Independent claim is also included for pharmaceutical compositions containing (I) for use as above.

Description

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traitement ou à la prévention des pathologies liées à une déficience du signal TGF|3 ou nécessitant une amplification de ce signal. The present invention relates to the use of at least one agent potentiating the action of TGFss for the preparation of a pharmaceutical composition intended for the

treatment or prevention of pathologies linked to a deficiency in the TGF | 3 signal or requiring an amplification of this signal.

TGFss (Transforming Growth Factor-p) is a cytokine which plays an important role in regulating cell growth and differentiation. The Smads proteins translate the signal from the TGFss receptors by regulating the transcription of target genes, either directly or in combination with other specific sequence transcription factors.

The intracellular signaling cascade of members of the TGF superfamily is initiated by the binding of the cytokine to a membrane receptor complex composed of two types of receptors with serine / threonine kinase activity, type I receptors and type II receptors. . The binding of the cytokine to the type II receptor induces the recruitment and phosphorylation of the type I receptor. The signal transduction from the membrane to the nucleus is then carried out thanks to a family of proteins, the Smads.

According to structural and functional criteria, the family of Smads can be divided into three sub-families. R-Smads (Receptor-regulated Smad), which are specifically activated by type I receptors, include Smadl, Smad5 and Smad8 specific for the BMPs pathway and, Smad2 and Smad3 specific for the TGF / activin pathway. Phosphorylation by type I receptors of R-Smads takes place at the level of serine residues located at the C-terminal end of the protein (motif SSXS). The activation of these RSmads allows the formation of a heterocomplex with another member of the Smads family, common to the various R-Smads, Smad4. This protein also called coSmad (common-partner Smad) lacks the SSXS sequence and allows the translocation of the R-Smad / Smad4 complex in the nucleus which can then act as a transcription factor. The third sub-family consists of Smad6 and Smad7 called I-Smads (Inhibitory-Smads), which prevent the phosphorylation of R-Smads and thus their association with Smad4 and / or the translocation of the R-Smads / Smad4 complex in the nucleus. interacting directly with Smad4.

TGFss is also involved in other signaling pathways such as that of the MAP / SAP kinase or the Ras pathway.

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Il existe à l'heure actuelle une série de pathologies liées au TGFJ3 pour lesquelles des solutions thérapeutiques sont nécessaires.

There are currently a series of pathologies linked to TGFJ3 for which therapeutic solutions are necessary.

5:137-52). De plus, TGFJ3 joue un rôle important dans les processus d'immunomodulation, en agissant sur la différenciation des lymphocytes (Kehrl et al, 1986, J Immunol, 137,3855-3860, Gorelik et Flavell, 2002, Nature, 2,46-53). Indeed, TGFss is a key cytokine in the control of connective tissue homeostasis and plays a major role in profibrotic activity by increasing the expression of genes specific to connective tissues (Mauviel A and Uitto J. (1993) Wounds

5: 137-52). In addition, TGFJ3 plays an important role in the immunomodulation processes, by acting on the differentiation of lymphocytes (Kehrl et al, 1986, J Immunol, 137,3855-3860, Gorelik and Flavell, 2002, Nature, 2,46- 53).

The Applicant has surprisingly found that the use of a RAR receptor antagonist has the effect of potentiating the action of TGF. Thus, the present invention provides a solution for treating pathologies linked to a deficiency in the TGFss signal or requiring an amplification of this signal.

This use is in no way obvious in the light of the prior art because it does not allow those skilled in the art to identify any interest in the use of RAR receptor antagonists in the treatment of pathologies linked to a TGF signal deficiency or requiring amplification of this signal.

Indeed, the prior art describes that retinoids and TGFJ3 follow complex signaling pathways, all the more complex as they differ according to the activated receptor subtype, for example for retinoids, the signaling pathway depends on the RARs and RXRs receptors and on the subtype concerned ([alpha], ss or y). Finally, it should be noted that these signaling channels are independent. Thus, it was not envisioned that a retinoid could have any effect on the TGFss signaling pathway.

Two classes of nuclear receptors, the RARs and RXRs receptors are involved in the transcriptional response to retinoic acid (RA).

The classes of retinoic acid receptors (RARs) and retinoic receptors X (RXRs) are respectively subdivided into subtypes [alpha], ss and y with more than one isoform for each subtype (Chambon, 1996). The RARs, associated with the RXRs to form the RAR-RXR heterodimers, bind the target DNA sequence, ie the elements of response to retinoic acid (RAREs), consisting of a direct repetition of the consensus sequence 5'-PuG (G / A) (T / A) CA-3 ', separated by two or five nucleotides. Family

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 RAR recognizes two natural stereoisomers of RA, RA all-trans and RA 9-cis, while the RXR family recognizes RA 9-cis exclusively.

Retinoids are a family of compounds with a wide range of therapeutic activities, ranging from anticancer activity to repairing the skin (Altucci L and Gronemeyer H 2001 Nature Review 181-193, Sun SY and Lotan R (2002) Oncology Hematology 41: 41-55).

Thus, the present invention relates to the use of at least one RAR receptor antagonist for the preparation of a pharmaceutical composition intended for the treatment of pathologies linked to a deficiency of the TGF signal or requiring an amplification of this signal.

By RAR receptor antagonist is meant a synthetic or natural structural analog of retinoic acid capable of binding and specifically activating RAR receptors.

Synthetic antagonists are preferred, preferably 4- (5,5-dimethyl-8-p-tolyl-5,6-dihydro-naphthalen-2-ylethynyl) -benzoic acid.

The term “potentiating agent” is intended to mean a compound potentiating the transcription induced by the Smad factors via the TGFss signaling.

Thus, the composition used according to the present invention will be intended for the treatment of pathologies linked to a transcription disorder induced by the Smad factors via the TGFss signaling.

By pathologies linked to a deficiency of the TGF signal or requiring an amplification of this signal, is meant: - scarring disorders, such as ulcers, in particular, venous, diabetic ulcers and bedsores; - improving normal healing, such as post-operative healing, especially in accelerating healing (Bernstein E, Harisiadis L, Salomon G et al., Transforming Growth Factor-b is improves healing of radiation-impaired wounds J Invest Dermatol 1991; 97: 430-434; Mauviel A and Uitto J. The extracellular Matrix in Wound Healing: Role of the cytokine Network. Wounds 1993; (3): 137-152);

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 - certain cancers, in particular those linked to a loss of control of TGFss over cell proliferation (Engle SJ et al., 1999 Transforming growth factor beta1supresses nonmetastatic colon cancer at an early stage of tumorigenesis - Cancer Res. 59,3379-3386; Tang B et al., 1998); - rejection of transplants, in particular by promoting immune tolerance (Eikmans M et al., 2002 High transforming growth factor-beta and extracellular matrix mRNA response in renal allografts during early acute rejection is associated with absence of chronic rejection - Transplantation. 73, 4, 573-579); - pathologies involving collagen type VII (COL7A1), in particular genetic pathologies in which only one allele is affected such as bullous epidermolysis or certain cases of scarring which can be improved by stimulating the synthesis of collagen type VII favoring epithelialization (Christiano AM, Greenspan DS, Hoffman GG, Zhang X, Tamai Y, Lin AN, Dietz HC, Hovnanian A, Uitto J. A missense mutation in type VII collagen in two affected siblings with recessive dystrophic epidermolysis bullosa. Nat Genet. 1993 May; 4 (1): 62-6.

In particular, the use according to the present invention is suitable for the treatment of pathologies linked to type VII collagen (COL7A1).

Finally, the present invention relates to pharmaceutical compositions comprising at least one RAR receptor antagonist intended for the treatment of pathologies linked to a deficiency of the TGFss signal or requiring an amplification of this signal.

Example 1: Measurement of the potentiation of the action of RARS on the transactivation of a Smad3-dependent promoter by RAR antagonists A first approach to examine the potentiation of RAR signaling and that of TGFp / Smad, is to determine the effect of the various RAR agonists and antagonists on the dependent Smad transactivation, induced by TGFss. of the reporter gene luciferase (lux) in construct-transfected WI-26 lung fibroblasts (CAGA) 9lux. The latter is composed of nine copies of the nucleotide motif CAGA, known to link Smad3 and Smad4 with high affinity, cloned upstream of the major adenovirus promoter (MLP) controlling the expression of the reporter gene lux.

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- l'ATRA (all trans retinoic acid) à 10-7M (1 III d'une solution à 10,4M introduit dans du DMSO dans 1 ml de milieu 1 %) ; - contrôle (Ctrl) : 1 l de DMSO. A culture at confluence of WI-26 fibroblasts was co-transfected with (CAGA) 9-lux and an expression vector coding for RARy, in the absence (-) or in the presence (+) of the expression vector expressing Smad3. Three hours after transfection, four compounds are added: - the RAR antagonist: 4- (5,5-dimethyl-8-p-tolyl-5,6-dihydro-naphthalen-2-ylethynyl) benzoic acid at 10 -6M (1 (of a 10-3M solution in DMSO introduced into 1 ml of 1% medium); - the RAR agonist: acid 4 - [(5,5,8,8-Tetramethyl- 5,6,7,8-tetrahydro-naphthalen-2ylamino) -methyl] -benzoic at 10-7M (1 [of a 10-4M solution introduced into DMSO in 1 ml of 1% medium);

- ATRA (all trans retinoic acid) at 10-7M (1 III of a 10.4M solution introduced into DMSO in 1 ml of 1% medium); - control (Ctrl): 1 l of DMSO.

The luciferase activity is determined 24 hours later.

The role of synthetic RARs agonists and antagonists on the functional interactions between Smad3 and RARy has been examined, the results are shown in Figure 1.

Acid 4- (5,5-dimethvl-8-a-tolyl-5.6-dihydro-naphthalen-2-ylethyl -benzoic (RAR antagonist) potentiates the effect of RARy on the Smad3 dependent transcription while acid 4 -i5.5.8.8-Tetramethvl-5.6.7.8-tetrahvdro-naphthalen-2-vlarnino) methvll-benzoique (RAR agonist) abolishes the effect of RARy.

Example 2: Demonstration of the dose-dependent potentiating effect of the RAR antagonists on the specific Smad transcriptional response induced by TGFss.

A confluence culture of WI-26 fibroblasts is transfected with the g-lux (CAGA) gene.

After a shock with glycerol, DMEM containing 1% of FCS (Fetal Calf Serum) is added. Three hours later, increasing concentrations of the synthetic RAR antagonist, 4- (5,5-Dimethyl-8-p-tolyl-5,6-dihydro-naphthalen-2-ylethynyl) benzoic acid are added.

diméthvl-8-p-tolvl-5.6-dihvdro-naphthalen-2-vlethvnvlVbenzoique, potentialise de façon These results (represented in FIG. 2) show that the antagonist, acid 4- (5.5-

dimethvl-8-p-tolvl-5.6-dihvdro-naphthalen-2-vlethvnvlVbenzoique, potentiates in a way

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 dependent dose the transcriptional response Smad dependent induced by TGFss, with a maximum effect (+ 190%) at the concentration of 10-6 M. At a concentration of 10-8 M, the acid 4- (5,5-Dimethyl- 8-p-tolyl-5,6-dihydro-naphthalen-2-ylethynyl) -benzoic doubles the extent of the TGFss-dependent response.

Example 3: Molecular demonstration of the RAR / Smad antagonist interaction by immunoprecipitation (IP) and immunoblottin experiments.

Cos-7 cells are transfected with the Smad3 expression vectors marked with Flag and Gal4BD-RARy-DEF, in the presence or in the absence (see FIG. 3, lines 1-3) of either acid 4 - [(5, 5,8,8-Tetramethyl-5,6,7,8-tetrahydro-naphthalen-2-ylamino) -methyl] benzoic (lines 4-6), i.e. 4- (5,5-dimethyl-8- p-tolyl-5,6-dihydro-naphthalen-2ylethynyl) -benzoic (lines 7-9).

The empty expression vector Gal 4 (Gal4-e) is used as a control.

Three hours after transfection, 10-6 M of 4- (5,5-dimethyl-8-p-tolyl-5,6-dihydronaphthalen-2-ylethynyl) -benzoic acid (RAR antagonist) are added. Forty hours later, the cell lysate is immunoprecipitated by anti-Gal4 antibodies and the coprecipitation of Smad3 is detected by immunoblotting with anti-Flag antibodies (labeling of Smad) (top panel). The expression of RARy and Smad3 is confirmed in Western Blot using anti-Flag antibodies (middle panel) and anti-Gal4 antibodies (bottom panel).

The migration bands at lines 2.5 and 8 obtained with the antiGal4 antibody correspond to the Gal4BD protein not fused to RARy expressed by the empty Gal4BD vector.

After immunoprecipitation with an anti-Gal4BD antibody (top panel) a western blot with anti-flag antibodies reveals the co-immunoprecipitation of Smad3 with RARy at lines 3.

l'acide 4-(5.5-Dimethvl-8-p-tolvl-5.6-dihvdro-naphthalen-2-vlethvnvl)-benzoique. liane 9. The amount of RARy binding Smad3 is significantly increased by the presence of

4- (5.5-Dimethvl-8-p-tolvl-5.6-dihvdro-naphthalen-2-vlethvnvl) -benzoic acid. liana 9.

This indicates potentiation by the RAR antagonist. 4- (5,5-Dimethyl-8-r) tolvl-5.6-dihvdro-naphthalen-2-vlethvnvlVbenzoic acid, from the action of TGF (3, i.e.

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 the increase in transcriptional response induced by TGFss requires an increase in the amount of Smad3-RARy heterocomplex formed.

Example 4: Effects of RARs antagonists on the activity of the promoter of collagen type VII (COL7A1) induced by TGFB.

The effect of the RAR antagonists on the COL7A1 promoters (type VII collagen promoter) is quantified via the transactivation of the reporter gene luciferase (lux).

We observe (FIG. 4), in the presence of the antagonist RAR, 4- (5,5-Dimethyl-8-p-tolyl-5,6-dihydro-naphthalen-2-ylethynyl) -benzoic acid, and TGFss, a dose-dependent increase in the transcription induced by the TGFss of the reporter gene under the control of the type VII collagen promoter (COL7A1).

Example 5 Effect of RAR Antagonists and Agonists on the Modulation of the Response to the Endogenous COL7A1 TGFss Furthermore, FIG. 5 represents a Nothern Blot gel on which, apart from any transfection, the response to the COL7A1 TGFss is observed endogenous is potentiated by the antagonist RAR. This test also makes it possible to confirm all the transfection experiences described above.

This test was carried out according to the following protocol: confluent HaCaT cultures were treated with the agonist, acid 4 - [(5,5,8,8-Tetramethyl-5,6,7,8-tetrahydro-naphthalen -2- ylamino) -methyl] -benzoic at 10-7M, and the antagonist, 4- (5,5-dimethyl-8-p-tolyl-5,6dihydro-naphthalen-2-ylethynyl) -benzoic acid at 10 -6M as described in Example 1, in the presence or absence of TGFss (at 10 ng / ml) for 48 hours. After extraction of the total RNAs, the level of mRNAs of type VII collagen was quantified by Nothern Blot using a specific cDNA probe labeled with P32. A probe characteristic of the mRNAs of the GADPDH protein was used as a deposit control.

The target genes for the action of the antagonist, acid 4- (5,5-Dimethyl-8-p-tolyl-5,6-dihydronaphthalen-2-ylethynyl) -benzoic, are the genes involved in pathologies

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 specific, involving the type VII collagen promoter (COL7A1), such as: certain genetic pathologies such as bullous epidermolysis and certain cases of scarring which can be improved by stimulating the synthesis of type VII collagen promoting epithelialization.

Thus, the potentiation of its activity by these antagonists can prevent or treat such pathologies.

Claims (6)

 CLAIMS 1. Use of at least one RARy receptor antagonist for the preparation of a pharmaceutical composition intended for the treatment of pathologies linked to a deficiency of the TGFss signal or requiring an amplification of this signal. 2. Use according to claim 1, characterized in that the RARs receptor antagonist is 4- (5,5-Dimethyl-8-p-tolyl-5,6-dihydro-naphthalen-2-ylethynyl) acid benzoic acid. 3. Use according to claim 1 or 2, characterized in that the composition is intended for the treatment of pathologies linked to a transcription disorder induced by the Smad factors via the TGFss signaling. 4. Use according to claim 1 or 2, characterized in that the composition is intended for the treatment of: - scarring disorders, such as ulcers, in particular, venous, diabetic ulcers and bedsores; - physiological situations such as post-operative wound healing, in particular in accelerating wound healing; - cancers linked to a loss of control of TGFss over cell proliferation; - transplant rejection, in particular by promoting immune tolerance; - pathologies involving collagen type VII (COL7A1), in particular certain genetic pathologies such as epidermolysis bullosa or certain cases of scarring which can be improved by stimulating the synthesis of collagen type VII promoting epithelialization. 5. Use according to claims 1 or 2 for the preparation of a pharmaceutical composition intended for the treatment of pathologies linked to collagen type VII (COL7A1). 6. Pharmaceutical composition comprising at least one RARy receptor antagonist intended for the treatment of pathologies linked to a deficiency of the TGFss signal or requiring an amplification of this signal.