Glycopeptide Derivatives For Use In The Treatment And/Or Prevention And/Or Attenuation of Fibrosis Diseases

Abstract

The present invention relates to a compound of the following formula (I), as well as to a pharmaceutical composition comprising at least one compound of following formula (I) and at least one pharmaceutically acceptable excipient, for use in the treatment and/or prevention and/or attenuation of fibrosis diseases, in particular excessive scars such as keloids or hypertrophic scars.

##STR00001##

Claims

1-18. (canceled)

19. A method of treating and/or preventing and/or attenuating a fibrosis disease, comprising the administration to a person in need thereof of an effective amount of a compound of following formula (I): ##STR00014## or a salt thereof, a solvate, a tautomer, a stereoisomer or a mixture of stereoisomers in any proportions, in which: n represents an integer from 1 to 6, m represents 0 or 1, p represents 0 or 1 R represents H, F, CH.sub.3, CH.sub.2F, or CH.sub.2OH, R.sub.1, R.sub.2 and R.sub.3 represent, independently from one another, H, F, or OH, R.sub.4 represents a hydrogen, a halogen, or OH, and R.sub.6 and R.sub.7 represent, independently from each other, a hydrogen, a (C.sub.1-C.sub.6)alkyl, an aryl, or an aryl-(C.sub.1-C.sub.6)alkyl.

20. The method according to claim 19, wherein the fibrosis disease is excessive scars.

21. The method according to claim 20, wherein the excessive scars are keloids or hypertrophic scars.

22. The method according to claim 19, wherein the compound of formula (I) has one of the following formulas (Ia), (Ib), (Ic): ##STR00015##

23. The method according to claim 19, wherein n represents an integer from 2 to 6.

24. The method according to claim 19, wherein R represents a CH.sub.2OH group.

25. The method according to claim 19, wherein R.sub.1, R.sub.2 and R.sub.3 each represent a OH group.

26. The method according to claim 19, wherein R.sub.4 represents a OH group.

27. The method according to claim 19, wherein R.sub.6 and R.sub.7 represent, independently from each other, a (C.sub.1-C.sub.6)alkyl, an aryl or an aryl-(C.sub.1-C.sub.6)alkyl.

28. The method according to claim 19, wherein the compound of formula (I) is chosen among the following compounds: ##STR00016## and the salts and/or solvates thereof.

29. A method of treating and/or preventing and/or attenuating a fibrosis disease, comprising the administration to a person in need thereof of an effective amount of a pharmaceutical composition comprising at least one pharmaceutically acceptable excipient and at least one compound of following formula (I): ##STR00017## or a salt thereof, a solvate, a tautomer, a stereoisomer or a mixture of stereoisomers in any proportions, in which: n represents an integer from 1 to 6, m represents 0 or 1, p represents 0 or 1 R represents H, F, CH.sub.3, CH.sub.2F, or CH.sub.2OH, R.sub.1, R.sub.2 and R.sub.3 represent, independently from one another, H, F, or OH, R.sub.4 represents a hydrogen, a halogen, or OH, and R.sub.6 and R.sub.7 represent, independently from each other, a hydrogen, a (C.sub.1-C.sub.6)alkyl, an aryl, or an aryl-(C.sub.1-C.sub.6)alkyl.

30. The method according to claim 29, wherein the fibrosis disease is excessive scars.

31. The method according to claim 30, wherein the excessive scars are keloids or hypertrophic scars.

32. The method according to claim 29, wherein n represents an integer from 2 to 6; R represents a CH.sub.2OH group; R.sub.1, R.sub.2 and R.sub.3 each represent a OH group; R.sub.4 represents a OH group; and R.sub.6 and R.sub.7 represent, independently from each other, a (C.sub.1-C.sub.6)alkyl, an aryl or an aryl-(C.sub.1-C.sub.6)alkyl.

33. The method according to claim 29, wherein the compound of formula (I) is chosen among the following compounds: ##STR00018## and the salts and/or solvates thereof.

34. The method according to claim 29, wherein the pharmaceutical composition is administered topically in combination with or after a laser or surgical treatment.

35. A dressing comprising a pad, compress or sponge impregnated with a pharmaceutical composition comprising at least one pharmaceutically acceptable excipient and at least one compound of following formula (I): ##STR00019## or a salt thereof, a solvate, a tautomer, a stereoisomer or a mixture of stereoisomers in any proportions, in which: n represents an integer from 1 to 6, m represents 0 or 1, p represents 0 or 1 R represents H, F, CH.sub.3, CH.sub.2F, or CH.sub.2OH, R.sub.1, R.sub.2 and R.sub.3 represent, independently from one another, H, F, or OH, R.sub.4 represents a hydrogen, a halogen, or OH, and R.sub.6 and R.sub.7 represent, independently from each other, a hydrogen, a (C.sub.1-C.sub.6)alkyl, an aryl, or an aryl-(C.sub.1-C.sub.6)alkyl.

36. The dressing according to claim 34, wherein n represents an integer from 2 to 6; R represents a CH.sub.2OH group; R.sub.1, R.sub.2 and R.sub.3 each represent a OH group; R.sub.4 represents a OH group; and R.sub.6 and R.sub.7 represent, independently from each other, a (C.sub.1-C.sub.6)alkyl, an aryl or an aryl-(C.sub.1-C.sub.6)alkyl.

37. The dressing according to claim 34, wherein the compound of formula (I) is chosen among the following compounds: ##STR00020## and the salts and/or solvates thereof.

38. A method of treating and/or preventing and/or attenuating a fibrosis disease, comprising the application to a person in need thereof of a dressing according to claim 35.

39. The method according to claim 38, wherein the dressing is applied in combination with or after a laser or surgical treatment.

Description

EXAMPLES

[0102] The following abbreviations have been used in the examples:

ACTA2: Actin, alpha2, smooth muscle, aorta
BCA: Bicinchoninic acid
COL1A: Collagen, type I, alpha 1
COL3A1: Collagen, type III, alpha 1
COL5A1: Collagen, type V, alpha 1
COL8A1: Collagen, type VIII, alpha 1

DCN: Decorin

[0103] DMEM: Dulbecco's modified Eagle's medium

DPT: Dermatopontin

[0104] ECM: Extracellular matrix

ELN: Elastin

FBLN5: Fibulin 5

[0105] FCS: Fetal calf serum
KF: Keloid fibroblasts
LEPR: Leptin receptor
MMP: Matrix metalloproteinase
MMP1: Matrix metallopeptidase 1 (interstitial collagenase)
MMP3: Matrix metallopeptidase 3 (stromelysin, progelatinase)
mRNA: Messenger ribonucleic acid
NF: Normal fibroblasts
PBS: Phosphate buffered saline
PCOLCE: Procollagen C-endopeptidase enhancer

PCR: Polymerase Chain Reaction

[0106] RT-qPCR: Reverse Transcription quantitative PCR
sem: standard error of the mean
TGF-: Transforming growth factor beta
TFPI2: Tissue factor pathway inhibitor 2

TMB: 3,3,5,5-Tetramethylbenzidine

[0107] TP53: Tumor protein p53

[0108] Compounds 1, 2, 3 and 4 used in the examples below were prepared as described in WO2015/140178.

[0109] During excessive scar formation, a dysfunction of the healing process is observed, leading to an excessive matrix synthesis and/or deficient matrix degradation and remodeling.

[0110] In the present invention, the effect of CF.sub.2 glycopeptide of formula I was evaluated on the expression of a panel of genes that are involved in mechanism of reducing excessive scars. The study was performed on normal and aged human fibroblasts at mRNA levels. An additional study was performed on normal and keloid fibroblasts at protein levels.

[0111] In normal human dermal fibroblasts, compound 1 can inhibit the expression of genes implicated in extracellular matrix (ECM) synthesis (COL1A1, COL3A1, ELN, COL5A1, COL8A1, DCN, DPT, FBLN5, PCOLCE) and can stimulate the expression of genes implicated in ECM degradation (MMP1, MMP3 and TFPI2). The results are presented in Table 1.

[0112] Besides keloid scars are characterized by a collection of atypical fibroblasts with excessive deposition of extracellular matrix components. An overproduction and accumulation of collagen (increased type I/III collagen ratio) and elastin, as well as a low level of matrix metalloproteinases MMP1 and MMP3 have been mainly observed in keloids (Histol. Histopathol. 2015, 30, 1033-1057).

[0113] That is why the expression of genes implicated in extracellular matrix generation, as collagen 1, collagen 3 and elastin, and in extracellular matrix degradation, as MMP1, was particularly studied in fibroblasts cultures.

[0114] Compounds 1, 2, 3 and 4 can modulate gene profile in a way to avoid the ECM product deposit by decreasing the expression of COL1A1, COL3A1 and ELN; and also by increasing the expression of MMP1 in aged human dermal fibroblasts. The results are presented in Table 2.

[0115] In addition, it has been showed that expression of transforming growth factor TGF- impact the formation of keloids. The expression of TGF- receptors TGF-R1 and TGF-R2 are higher in keloid fibroblasts compared to normal human dermal fibroblasts (Plast. Reconstr. Surg. 2001, 108, 423-429). Besides, leptin also play a major role in wound healing process and it has been shown that leptin is overexpressed in keloids and hypertrophic scars (Appl. Immunohistochem. Mol. Morphol. 2016, 24, 296-306).

[0116] Thus, LEPR and TGFR2 mRNA expression was measured in normal fibroblasts. The expression of these genes involved in cell proliferation was reduced in the presence of compound 1. The results are presented in Table 3.

[0117] Then, compound 1 also decreases the expression of the ACTA2 gene encoding the alpha smooth muscle actin which has been described to increase in fibrosis/hypertrophic scar or keloid. The results are presented in Table 4.

[0118] Finally, to support the results obtained at mRNA levels, an evaluation of compound 1 on the production of proteins, particularly on collagen I and collagen III, in culture of human normal fibroblasts (NF) and keloid fibroblasts (KF) has been performed.

[0119] In accordance with what is described in the literature an increased collagen I/collagen III ratio was observed in keloids fibroblasts compared to normal fibroblasts control. The results show that, in the presence of compound 1, the collagen I/collagen III ratio in KF decreased up to a value close to NF control. Compound 1 inhibits the synthesis of collagens I and III in fibroblasts obtained from keloid scars. The results are presented in Table 5.

[0120] In conclusion, results obtained at mRNA and proteins levels indicate that compounds of formula I can treat and/or prevent and/or attenuate fibrosis diseases, in particular hypertrophic scars and keloids.

General Experimental Procedure

[0121] 1. Effects of Compounds 1, 2, 3, 4 on Expression of Genes Involved in ECM Synthesis or Degradation, in Normal or Aged Fibroblast Culture (Analysis of mRNA Expression Profile by RT-qPCR)

[0122] In the present study, the transcriptional effects (modulation of gene expression) of compounds 1, 2, 3, 4 was evaluated on aged human dermal fibroblasts (Hayflick model) and normal human dermal fibroblasts (NHDF) in order to assess their potential effect.

[0123] More specifically, the effects of the compound on aged fibroblasts and NHDF were evaluated using RT-qPCR technology. Extracted mRNAs were analyzed using a PCR for the analysis of target genes (including housekeeping genes) selected for their importance in dermal fibroblast physiology.

[0124] Materials and Methods

[0125] a) Biological Model

[0126] Subculturing: Human dermal fibroblasts were grown in culture medium composed of DMEM supplemented with L-glutamine (2 mM), Penicillin (50 U/ml), Streptomycin (50 g/ml) and Fetal calf serum (FCS) 10% in 37 C. and 5% CO.sub.2 incubator.

[0127] Assay: Cells were used at the 7th passage (normal fibroblasts) or the 17th passage (aged fibroblasts). The assay medium was composed of DMEM supplemented with L-glutamine (2 mM), Penicillin (50 U/ml), Streptomycin (50) g/ml and FCS 1%.

[0128] b) Test Compounds

[0129] An intermediate solution of compounds 1, 2, 3, 4 was prepared in assay medium at concentration 100 mg/ml (pH=7.4) to be tested at 94 mM or 34 mM in normal or aged fibroblasts culture.

[0130] c) Cultures and Treatments

[0131] Aged or normal fibroblasts were seeded in 12-well or 24-well plates and cultured for 24 to 48 hours in culture medium. The medium was then removed and replaced by assay medium containing the test compound (treated) or not (control) and the cells were incubated for 24 hours. All experimental conditions were performed in n=3. At the end of incubation, the cells were washed in a phosphate buffered saline (PBS) solution and immediately frozen at 80 C.

[0132] d) Differential Gene Expression Analysis by RT-qPCR Method

[0133] The expression of markers was analyzed using RT-qPCR method on mRNA extracted from the cell monolayers of each experimental condition (before RNA extraction, the replicates of the same experimental condition were pooled). The analysis of transcripts was performed in n=2 using a PCR array.

Reverse Transcription:

[0134] Total RNA was extracted from each sample using TriPure Isolation Reagent or NucleoSpin RNA Plus kit (Macherey-Nagel) according to the supplier's instructions. The amount and quality of RNA were evaluated using electrophoresis (Bioanalyzer 2100, Agilent technologies). Potential contaminant traces of genomic DNA were removed using the DNAfree system (Ambion). The complementary DNA (cDNA) was synthetized by reverse transcription of total RNA in presence of oligo(dT) and Transcriptor Reverse Transcriptase (Roche). Quantification of cDNA was performed using a spectrophotometer (Nanovue, GE Healthcare) and cDNA quantities were adjusted.

Quantitative PCR:

[0135] The PCRs (Polymerase Chain Reactions) were performed using the LightCycler system (Roche Molecular System Inc.) according to the supplier's instructions. The reaction mix (10 l final) was prepared as follows: 2.5 l of cDNA, primers (forward and reverse), reagent mix containing taq DNA polymerase, SYBR Green I and MgCl.sub.2.

Data Management of Quantitative PCR:

[0136] Raw data were analyzed using Microsoft Excel software. The incorporation of fluorescence in amplified DNA was continuously measured during the PCR cycles. This resulted in a fluorescence intensity versus PCR cycle plot allowing the evaluation of a relative expression (RE) value for each marker.

[0137] The value selected for RE calculations is the output point (Ct) of the fluorescence curve. For a considered marker, the highest is the cycle number, the lowest is the mRNA quantity.

[0138] The RE value was calculated with the formula: (.sup.number of cycles)10.sup.6.

[0139] Two housekeeping genes RPS28 and GAPDH were used for data normalization since their expression is constitutive and theoretically stable. The mean relative expression of housekeeping genes was calculated for all test conditions. The level of expression of the target markers was compared to the mean expression level of these 2 markers for all test conditions (% control mean HK).

Classification of Effects (Treated Conditions Versus Normal Control or Aged Control):

[0140] For a standardized interpretation, the following table was used:

TABLE-US-00001 Relative expression (% of control) Classification of effects >300% Strong stimulation >200% and 300% Stimulation 30% and <50% Inhibition <30% Strong inhibition

II. Evaluation of the Effects of Compound 1 on Collagen Expression in Human Keloid Fibroblasts Culture

[0141] Materials and Methods

[0142] a) Biological Model

[0143] Primary cultures of keloid fibroblasts (KF) and normal fibroblasts (NF) were obtained from operative wastes (keloid and abdominoplasty). After thawing, cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% of fetal calf serum, 40 mg/l of gentamicin and 2 mg/l of fungizone (DMEMc), in an incubator at 37 C., 5% CO.sub.2. Culture medium was changed twice a week and cells were subcultured by trypsinization when confluence was reached. Cells were used under 10 passages.

[0144] b) Tested Compound

[0145] Compound 1 was directly diluted in culture medium at 5 mg/ml.

[0146] c) Cell Culture and Collect

[0147] Fibroblasts were seeded in 6 wells culture plate at the concentration of 0.08.Math.10.sup.6 cells/well (n=6). After 48 hours of culture, wells were washed with 3 ml of PBS. 2 ml of each solution of tested compound were added per wells. Plates were then incubated at 37 C. with 5% CO.sub.2. After 48 hours of culture, 4200 l of supernatant were collected in Eppendorf vials containing 20 l of protease inhibitor cocktail and stored at 80 C. until collagen synthesis analysis. The monolayer cells were then washed with 3 ml of PBS and 250 l of 0.1N NaOH were added. After 10 minutes supernatants were removed into an Eppendorf and stored at 20 C. until proteins analysis.

[0148] d) Evaluation of Collagen Synthesis

[0149] The synthetized collagen quantity is expressed as the ratio of quantity of collagen vs quantity of total proteins in the sample.

Evaluation of the Quantity of Total Proteins in the Sample:

[0150] The quantity of total protein was determined using a biochemical test, the bicinchoninic acid assay (BCA assay). The total protein concentration is exhibited by a color change of the sample solution from green to purple in proportion to protein concentration, which can then be measured using colorimetric techniques. Bicinchoninic acid (BCA) as soluble sodium salt in aqueous medium reacts in a stably, sensitive and highly specific manner with cuprous ions. Proteins react with cupric ions (issued from copper(II) sulfate) in an alkaline medium to produce cuprous ions. Then, two molecules of BCA with one cuprous ion react to form a purple complex which shows an absorption peak at 562 nm. A standard curve (0-2 mg/ml) is established with a bovine serum albumin solution. 20 l of each tested concentration of the standard or of the sample are placed in wells of a 96 well-plate, in duplicate. 200 l of Pierce reagent (comprising BCA and copper(II) sulfate) were added and the plate is incubated for 30 minutes at 37 C. Absorbance is read immediately at 550 nm (Spectrophotometer Multiscan Ex, Thermo). The standard curve is drawn and the sample protein concentration is determined with this curve. The results are expressed in mg protein/ml.

Evaluation of Collagen I Synthesis:

[0151] Collagen I synthesis was evaluated by Enzyme-linked immunosorbent assay with an ELISA kit (USCN SEA571Hu, Euromedex). The assay was performed according to the supplier's instructions. To summarize, the microtiter plate provided in the kit is pre-coated with an antibody specific to collagen I. Standard or samples are added to the wells with a biotin-conjugated antibody specific to collagen I. Next, avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. After TMB substrate solution is added, only those wells that contain collagen I, biotin-conjugated antibody and enzyme-conjugated avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of sulfuric acid solution and the color change is measured spectrophotometrically at 450 nm. The concentration of collagen I in the samples is then determined by comparing the optical density of the samples to the standard curve. The results were expressed as pg of collagen I by mg of proteins.

Evaluation of Collagen III Synthesis:

[0152] Collagen III synthesis is performed by Enzyme-linked immunosorbent assay with an ELISA kit (USCN SEA576Hu, Euromedex). The assay was performed according to the supplier's instructions. To summarize, the microtiter plate provided in the kit is pre-coated with an antibody specific to collagen III. Standard or samples are then added to the wells with a biotin-conjugated antibody specific to collagen III. Next, avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. After TMB substrate solution is added, only those wells that contain collagen III, biotin-conjugated antibody and enzyme-conjugated avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of sulfuric acid solution and the color change is measured spectrophotometrically at 450 nm. The concentration of collagen III in the samples is then determined by comparing the optical density of the samples to the standard curve. The results were expressed as ng of collagen III by mg of proteins.

Statistical Analysis:

[0153] Data are expressed as mean+/sem. A variance analysis with one factor was performed for cytotoxicity study followed if necessary by a Fisher test. A variance analysis with two factors was performed for synthesis study followed if necessary by a Fisher test. A p value less than 0.05 is considered significant.

Data Management:

[0154] To be able to compare the results, the collagen quantity is expressed as the ratio of quantity of collagen vs quantity of total proteins in the sample.

TABLE-US-00002 TABLE 1 Effect of compound 1 (at 94 mM) on gene expression involved in ECM synthesis and degradation in normal human dermal fibroblasts. Normal fibroblasts Compound 1 Control % Control Genes Cycles Cycles Mean HK ECM synthesis/ COL1A1 14.25 17.03 17 ECM assembly 14.35 17.13 COL3A1 20.14 22.87 18 20.06 22.72 COL5A1 20.58 22.87 24 20.67 22.87 COL8A1 22.1 24.57 22 22.14 24.51 DCN 17.89 19.94 29 17.91 19.89 DPT 21.86 24.18 24 21.95 24.14 ELN 25.47 27.17 34 25.35 27.16 FBLN5 23.11 24.21 48 22.65 24.06 PCOLCE 20.75 22.02 49 20.84 22.04 ECM degradation MMP1 20.87 16.24 2759 20.77 16.24 MMP3 22.9 20.88 448 22.86 20.97 TFPI2 29.91 26.14 1694 29.99 26.01 Up-regulated genes (arbitrary selection for stimulation): % > 200 Down-regulated genes (arbitrary selection for inhibition): % < 50

TABLE-US-00003 TABLE 2 Effect of compounds 1, 2, 3 and 4 (at 34 mM) on gene expression involved in ECM synthesis and degradation in aged human dermal fibroblasts. Aged fibroblasts Compound 1 Compound 2 % % Compound 3 Compound 4 Control Control Control % Control % Control Genes Cycles Cycles Mean HK Cycles Mean HK Cycles Mean HK Cycles Mean HK ECM synthesis/ COL1A1 14.45 16.23 31 16.60 23 16.29 33 15.67 42 ECM assembly 14.51 16.16 16.70 16.01 15.73 COL3A1 21.63 22.94 39 23.29 32 23.46 33 23.84 21 21.65 23.09 23.44 23.21 23.85 ELN 27.83 29.89 21 29.97 20 28.99 39 29.72 21 27.52 30.08 30.21 29.28 30.04 ECM degradation MMP1 20.82 17.53 962 17.14 1341 18.93 405 18.33 535 20.78 17.60 17.13 18.81 18.35 Up-regulated genes (arbitrary selection for stimulation): %>200 Down-regulated genes (arbitrary selection for inhibition): %<50

TABLE-US-00004 TABLE 3 Effect of compound 1 (at 94 mM) on gene expression involved in cell proliferation in normal human dermal fibroblasts. Normal fibroblasts Compound 1 Control % Control Genes Cycles Cycles Mean HK Cell proliferation LEPR 27.60 29.64 38 28.01 29.16 TGFBR2 25.21 27.51 26 25.24 27.26 Up-regulated genes (arbitrary selection for stimulation): % > 200 Down-regulated genes (arbitrary selection for inhibition): % < 50

TABLE-US-00005 TABLE 4 Effect of compound 1 (at 94 mM) on gene expression involved in cytoskeletal integrity in normal human dermal fibroblasts. Normal fibroblasts Compound 1 Control % Control Genes Cycles Cycles Mean HK Cytoskeletal integrity ACTA2 24.68 26.76 28 24.83 26.87 Up-regulated genes (arbitrary selection for stimulation): % > 200 Down-regulated genes (arbitrary selection for inhibition): % < 50

TABLE-US-00006 TABLE 5 Synthesis of Collagen I and Collagen III proteins in the presence or not of compound 1 (at 9.4 mM) in normal and keloids fibroblasts. Normal fibroblasts Keloids fibroblasts NF Control NF + compound 1 KF Control KF + compound 1 Mean sem Mean sem Mean sem Mean sem Protein Collagen I 103.5 5.61 28.85*** 5.04 149.89*** 12.58 44.68.sup.### 6.03 (pg/mg) Protein Collagen III 11.84 0.82 6.57*** 0.61 6.46*** 0.46 4.42.sup.# 0.25 (ng/mg) Ratio [00001]$\frac{\mathrm{Collagen}.Math..Math.I}{\mathrm{Collagen}.Math..Math.\mathrm{III}}$ 8.74.10.sup.3 4.39.10.sup.3 23.20.10.sup.3 10.11.10.sup.3 ***p < 0.001 versus NF Control .sup.#p < 0.05 versus KF Control .sup.###p < 0.001 versus KF Control