WO2011107611A2

WO2011107611A2 - Method for evaluating the irritant potential of a test compound

Info

Publication number: WO2011107611A2
Application number: PCT/EP2011/053390
Authority: WO
Inventors: Hervé GROUX; Françoise Cottrez
Original assignee: Immunosearch
Priority date: 2010-03-05
Filing date: 2011-03-07
Publication date: 2011-09-09
Also published as: FR2957090A1; WO2011107611A3

Abstract

The invention relates to a method for evaluating the irritant potential of a test compound and to a kit for implementing said method.

Description

Method for evaluating the irritating potential of a test compound

The present invention relates to a method for evaluating the irritating potential of a test compound and to a kit for carrying out said method. The perfume, cosmetics and pharmacy industries must remain competitive and efficient and continue to offer new products on a regular basis, with the constraint of meeting the safety standards for humans and their environment attached to their use. Irritation is a reversible inflammation of living tissue by chemical action at the site of contact. It is recognized by edema following an influx of fluids into the tissues, redness, heat and / or pain. In response to a chemical attack, epidermal keratinocytes and fibroblasts of the dermis are stimulated and will release into the skin cytokines IL1, TNF alpha, IL6, IL8, as well as mediators such as prostaglandins (PGE2) which will initiate the inflammatory response.

The deleterious effects caused by a chemical agent such as skin irritation is of major importance in dermatology and may prove to be a barrier to the placing on the market of new products.

The new European constraints now require the use of non-animal methods and it is therefore essential to develop alternative methods for determining whether a new composition or a new product is likely to pose a risk to the animal. man by its irritating properties.

From a histological point of view, sensitizing and irritating contact dermatitis are very similar. However, the clinical consequences are not similar and it is important to have reliable methodologies to distinguish them. An original predictive approach is all the more necessary nowadays no clear correlation has been found between a given molecular structure and its irritant and / or sensitizing power.

Current methods for predicting the irritant potential of a compound are on the one hand, based on the use of animals which is now prohibited, on the other hand are not quantitative and therefore do not allow to evaluate the irritating force of a molecule.

Manufacturers in the perfume and cosmetics industries are looking for methodologies to distinguish weak irritants from strong irritants, which would enable them to carry out marketing strategies for their products with much greater precision.

Surprisingly, the inventors have demonstrated that the response to the action of an irritating substance is directly on the skin without the intervention of immunocompetent cells, and that the degree of irritation of a molecule can be determined through the use of specific biomarkers of irritation.

The inventors have demonstrated that the epidermis is a sufficient model to demonstrate specific biomarkers of sensitization and / or irritation in humans and in mice, and that it is not necessary to add other cell types if the analysis of the identified genes is performed at a given time. The EPISKIN model can thus be the reference tissue for evaluating the sensitizing nature of a test component.

Some genes are known to exhibit differential expression following treatment with irritants. However, this differential expression may vary depending on the irritants, and some markers may exhibit differential expression after contacting a non-irritating compound. Also, the markers of irritation identified to date have not allowed the development of a method for evaluating the irritating potential of a test compound having both good specificity and good sensitivity. The Applicant has identified new genes whose level of expression makes it possible to evaluate the irritating potential of a test compound. These new genes have the advantage of allowing an evaluation of the irritating potential of a compound with increased specificity while maintaining a good sensitivity with respect to the markers known from the prior art.

Thus, the present invention relates to a method for evaluating the irritating potential of a test compound, comprising the steps of:

a) contacting a test compound with a biological sample;

b) determining the level of expression of at least one gene selected from the group consisting of: 4.1 BBL (TNF superfamily member 9), COL7A1 (collagen, type VII, alpha 1), CTSD (cathepsin D), FDXR (ferredoxin reductase), GAA (glucosidase, alpha; acid), HAS1 (hyaluronan synthase 1) and RELT (Member 19 of the TNF superfamily of the receptor).

Preferably, the method according to the present invention is an in vitro method.

Preferably, the method according to the present invention may further comprise a step c) of determining the irritating potential of a test compound.

Preferentially, said step c) may consist of a step of selecting said compound as having an irritating potential if the level of expression of said gene is greater than a threshold value. Advantageously, said step c) makes it possible to evaluate the irritating force of said test compound.

As used herein, the term "biological sample" refers to any solid or liquid sample from a subject.

Preferably, said biological sample is a skin sample. In a particularly preferred manner, the skin sample is a skin sample reconstructed in vitro, such as for example the EpiSkin model (EPISKIN, Lyon France), EpiDerm ™ (MATEK Corporation, Ashland, MA) or SkinEthic ™ RHE (SKINETHIC, Nice , France). Preferably, said skin sample reconstructed in vitro further comprises a keratin layer.

More preferably, the skin sample comprises only the epidemic and does not include the addition of other additional cell types, and still preferably no additional Langerhans cells.

The test compound may be a compound of varied nature, structure and origin, including a biological compound, a chemical compound, synthetic compound, etc.

The test compound can be any product that is in an isolated form or in admixture with other products. The test compound can be defined in terms of structure and / or composition or be defined functionally. The test compound may, for example, be an isolated and structurally defined product, an isolated product of indefinite structure, a mixture of known and characterized products or a composition comprising one or more products. One or more compounds can thus be tested, in admixture or separately.

Such compositions may be, for example, samples of a cosmetic or dermatological product.

Preferably, said test compound is suitable for use on the skin and can be used in a cosmetic or dermatological composition. By "irritating potential" is meant the risk for the test compound to cause reversible inflammation of living tissue by chemical action at the site of contact.

Preferably, said at least one gene is selected from the group consisting of: 4- 1BBL, COL7A1, HAS1 and RELT.

The present invention is particularly suitable for the identification of a large number of compounds. This simple and effective screening can be accomplished in one lapse very short time. In particular, the described methods can be partially automated, thus enabling efficient and simultaneous screening of various and diverse compounds, either as a mixture or in separate form. Preferably, in the method according to the present invention, the level of expression of said gene is determined by measuring the level of expression of the polypeptide encoded by said gene or a fragment thereof, or by measuring the level of expression of the gene. mRNA of said gene or fragment thereof. In a preferred embodiment, the level of expression of said at least one gene is performed by analyzing the expression of mRNA transcripts or mRNA precursors, such as native RNA, of said gene. Said assay can be performed by preparing the mRNA / cDNA of cells from a biological sample of a patient, and hybridizing the mRNA / cDNA with a reference polynucleotide. The mRNA / cDNA prepared can be used in a hybridization or amplification assay which includes, but is not limited to, Southern and Northen analyzes, polymerase chain reaction (PCR) assays, such as quantitative PCR (Taqman ) and the use of "probes arrays" such as GeneChip® DNA templates (AFFYMETRIX).

Advantageously, the analysis of the level of expression of transcribed mRNA of the said at least one gene involves a nucleic acid amplification process, such as, for example, RT-PCR (experimental embodiment described in US Pat. No. 4,683,202), the ligase chain reaction (BARANY, Proc Natl Acad Sci USA, vol.88, p: 189-193, 1991), self sustained sequence replication (GUATELLI and al., Proc Natl Acad Sci USA, vol.87, p: 1874-1878, 1990), the transcriptional amplification system. (KWOH et al., Proc Natl Acad Sci USA, vol.86, p: 1173-1177, 1989), "Q-Beta Replicase" (LIZARDI et al., Biol Technology, vol.6, p: 1197, 1988), "rolling circle replication" (US Pat. No. 5,854,033) or any other nucleic acid amplification method, followed by a step of detecting amplified molecules by techniques well known in the art. skilled person. These modes of detection are particularly useful for the detection of nucleic acid molecules in very small amounts.

Thus, according to a preferred embodiment, the method according to the present invention comprises a further step of amplification of the mRNA or cDNA of said gene, of the sequence complementary thereto or of a fragment thereof. this.

As used herein, the amplification primers are defined as a pair of nucleic acid molecules that can match the 3 'and 5' regions of a gene, specifically (positive and negative strands, or conversely) and frame a short region of said gene. In general, the amplification primers have a length of 10 to 30 nucleotides and allow the amplification of a region of a length of between 50 and 200 nucleotides. Advantageously, the primers used in the context of the present invention are those listed in Table 1.

In another preferred embodiment, the determination of the level of expression of said at least one gene is performed by measuring the level of expression of the polypeptide encoded by said gene, or a fragment thereof. Said assay can be performed using an antibody (eg, a radiolabeled antibody, labeled with a chromophore, a fluorophore, or an enzyme), an antibody derivative (eg an antibody conjugated to a substrate or a protein or a ligand of a protein of a ligand / protein pair (eg, biotin-streptavidin)) or an antibody fragment (e.g., a single chain antibody, a hypervariable domain of an isolated antibody, etc.) that specifically binds to the polypeptide encoded by said gene. Said assays can be performed by many techniques within the abilities of those skilled in the art including, but not limited to, immunoassays based on the use of enzyme activity ("enzyme immunoassay" EIA), immunoassay based the use of radioactive isotopes (RIA), Western blot analysis and enzyme linked immunoabsorbent assay (ELISA).

For the purposes of the present invention, the term "polypeptide" means a sequence comprising at least two amino acids, and the terms "polypeptide", "peptide" and "protein" may be used interchangeably.

By "mRNA or cDNA fragment" is meant a sequence of at least 50 nucleic acids, for example at least 100 or 150 nucleic acids, preferably at least 200 nucleic acids. as an example of at least 250 or 350 nucleic acids, and particularly preferably a polypeptide of at least 400 nucleic acids. By "fragment of the polypeptide" is meant a sequence of at least 50 amino acids, for example at least 100 or 150 amino acids, preferably at least 200 amino acids, for example at least 250 or 350 amino acids, and particularly preferably a polypeptide of at least 400 amino acids. Preferably, the method according to the present invention further comprises a step of comparing the level of expression of said gene with a reference value. This reference value can serve as a positive and / or negative control. Preferably, the step of comparing the level of expression of said gene with a reference value is carried out after step b).

A positive control can for example be performed by comparing the level of expression of said at least one gene in the presence of the test compound with the level of expression of said at least one gene in the presence of a compound known as an irritant.

A negative control can be carried out in the absence of the test compound or in the presence of a compound known as non-sensitizing and non-irritating such as for example olive oil, glycerol, cetyl trimethylammonium bromide (CTAB) or dipropylene glycol. In the context of the present invention, it will be concluded that a test compound has an irritating potential if overexpression of said gene is observed with respect to its level of expression in the absence of said test compound. By "overexpression" is meant a significantly higher level of expression of said gene relative to its normal expression level. Preferably, overexpression means a level of expression in a biological sample which is greater than at least 20% of the normal level of expression of said gene, preferably greater than at least 50% of the normal level of expression of said gene, and particularly preferably at least 90% higher than the normal level of expression of said gene.

The "level of expression in the absence of said test compound" or "normal level" is the level of expression of said gene in a control sample potentially corresponding to the biological sample of a patient not exhibiting irritation or, preferably, at the average level of expression of said gene in different control samples. In addition, if it is concluded that a test compound is irritating, the determination of the level of expression of said at least one gene in the presence of the test compound will make it possible to evaluate the irritating force of the test compound.

Irritant force is defined as the degree of tissue inflammation at the site of contact. Thus, the greater the irritating force, the greater the degree of inflammation of the tissues at the site of contact.

It is thus possible to perform a quantitative analysis of the irritant potential of a compound. By way of example of a compound known as an irritant, mention may be made of sodium lauryl sulphate (SLS), octanoic acid, cholobenzene, lactic acid, bromobutane, methyl salicylate and butanol.

Preferably, step b) of the method according to the present invention comprises determining the level of expression of at least 2, at least 3, at least 4, at least 5, from minus 6, at least 7, and even more preferably at least 8 genes selected from the group consisting of: 4.1 BBL (member 9 of the superfamily TNF), COL7A1 (collagen, type VII, alpha 1), CTSD (cathepsin D), FDXR (ferredoxin reductase), GAA (glucosidase, alpha; acid), HAS1 (hyaluronan synthase 1), RELT (Member 19 of the superfamily TNF receptor), IL-1A (interleukin 1, alpha), IL-7R (Interleukin 7 receptor), IL-8 (Interleukin 8), JUN (oncogene jun), PLAUR (plasminogen activator, urokinase receptor) and TNF-A (tumor necrosis factor), preferably in the group consisting of: 4.1 BBL (member 9 of the TNF superfamily), COL7A1 (collagen, type VII, alpha 1), CTSD (cathepsin D), FDXR (ferredoxin) reductase), GAA (glucosidase, alpha; acid), HAS1 (hyaluronan synthase 1) and RELT (Member 19 of the TNF superfamily receptor).

Thus, it can be concluded that a test compound has an irritant potential if overexpression of at least 2, at least 3, at least 4, at least 5, at least 6, minus 7, and even more preferably at least 8 genes selected from the group consisting of: 4.1 BBL (TNF superfamily member 9), COL7A1 (collagen, type VII, alpha 1), CTSD (cathepsin D), FDXR ( ferredoxin reductase), GAA (glucosidase, alpha; acid), HAS1 (hyaluronan synthase 1), RELT (Member 19 of the TNF superfamily receptor), IL-1A (interleukin 1, alpha), IL-7R (Receptor of the interleukin 7), IL-8 (Interleukin 8), JUN (oncogene jun), PLAUR (plasminogen activator, urokinase receptor) and TNF-A (tumor necrosis factor) is observed in relation to their level of expression in the absence said test compound.

Preferably, step b) is carried out between 1 and 24 hours after step a), more preferably between 4 and 18 hours after step a), particularly preferably between 5 and 7 hours after step a) and preferably every 6 hours after step a).

According to another aspect, the present invention relates to the use of at least one gene selected from the group consisting of: 4.1 BBL (member 9 of the TNF superfamily), COL7A1 (collagen, type VII, alpha 1), CTSD (cathepsin D), FDXR (ferredoxin reductase), GAA (glucosidase, alpha, acid), HAS1 (hyaluronan) synthase 1) and RELT (Member 19 of the TNF superfamily receptor) for in vitro evaluation of the irritant potential and / or irritant strength of a test compound.

The present invention also relates to a kit for implementing the method according to the present invention, comprising means for determining the level of expression of at least one gene selected from the group consisting of: 4.1 BBL (Member 9 TNF superfamily), COL7A1 (collagen, type VII, alpha 1), CTSD (cathepsin D), FDXR (ferredoxin reductase), GAA (glucosidase, alpha; acid), FIAS1 (hyaluronan synthase 1) and RELT (Member 19 of the superfamily of the TNF receptor).

Preferably, said kit will comprise at least one pair of primers allowing the amplification of at least one gene selected from the group consisting of 4.1 BBL (member 9 of the TNF superfamily), COL7A1 (collagen, type VII, alpha 1 ), CTSD (cathepsin D), FDXR (ferredoxin reductase), GAA (glucosidase, alpha; acid), HAS1 (hyaluronan synthase 1), and RELT (Member 19 of the receiving TNF superfamily). In a particularly preferred manner, said at least one pair of primer is chosen from Table 1.

Examples

1) Demonstration of bio markers according to the Skinethic protocol

The skins, 1.07 cm ² , were purchased from EPISKIN. The different substances were applied either in liquid form (30μ1 at different concentrations) or in solid form (30μ1 PBS or olive oil + 3Cg or less to evaluate different concentrations of powder) on the skins. After an incubation of 15 minutes at room temperature, the skins were washed with PBS (25 ml) and incubated for 3 h, 6 h or 18 h at 37 ° C. in a CO ₂ oven. After incubation the skins were removed with a punch and separated from the collagen support. They were then directly placed in a solution of "Tri Reagent" (Ambion) (1 ml) and immediately mechanically dissociated. Preparation of the cDNA

The tissues were placed in a solution of "Tri Reagent" (Ambion) and milled mechanically. RNA was prepared according to the protocol described by the manufacturer with isopropanol precipitation. For the preparation of the cDNA, the total RNAs are treated with a DNAse to remove contaminating genomic DNAs. For the treatment, 1 to 5 μg of total RNA are used with RNAse free DNAse RNAsin (Ιμΐ) and random primers (3 μg). Superscipt III RT (1.5 μΐ to 200 μ / μl) is then added. The cDNAs are then tested by RT-PCR.

Quantitative PCR

Real-time quantitative PCR was performed using SYBR Green technology from LC480 devices from Roche. The primers were designed to cover the intron-exon junctions to prevent possible traces of amplification of genomic DNA. The amplification gives amplicons between 100 and 150 bp. All primer pairs were qualified by restriction enzyme digestion and analyzed by electrophoresis. The PCR was carried out in 10 μΐ using Roche PCR Sybr green 2X mix PCR mix in PCR plates from Roche. The expression of the target genes was measured after normalization of the RNA by four housekeeping genes, and the values are expressed using the CT method, and expressed in terms of additional expression rate with respect to a theoretical zero (user Bulletin No. 2, Applied Biosystems, December 1997).

Results

For this analysis, an application of 15 minutes followed by a wash and a post-incubation of 6 hours before collection of biopsies and a gene transcription analysis were performed. The results are shown in Table 1.

Table 1

2) Quantitative analysis

The substances in Table 2 were used as irritant controls (SLS, octanoic acid, Cholobenzene, lactic acid, bromo-butane, methyl salicylate, butanol) and non-irritating (glycerol, cetyl trimethylammonium bromide and

Dipropylene Glycol).

( ^' lusse Name (AS Abbreviation

No -

Irritant Cetyl trimethylammonium bromide 57-09-0 CTAB

No -

Irritant Dipropylene Glycol 25265-71-8 LG

No -

Irritant Glycerol 56-81-5 Glyc

Irritant Octanoic Acid / Caprylic Acid 124-07-2 OA

Irritant Lactic Acid 598-82-3 LA

Irritant nButanol- 1 Butanol 71-36-3 Purpose

Irritant Lauryl Sodium Sulphate 151-21-3 SLS

Irritant Chlorobenzene 108-90-7 CBZ

Irritant BromoButane 109-65-9 BrBut

Irritant Methylsalicylate 119-36-8 M

Table 2

Table 3 shows the level of expression of biomarkers of irritation for these different substances. It is clear that irritants strongly induce most biomarkers. It is also noted that in the case of already known biomarkers (TNF-A, IL-1, IL-7, PLAUR, IL-8, JUN), at least one of the non-irritating substances (CTAB, DPG or Glycerol) induces expression of these genes which may lead to the definition of false positive substances if only these markers are used.

In addition, for some already known biomarkers, certain irritating substances do not induce a significant increase in the level of expression. Preferably, if at least 6 genes tested are overexpressed compared to the untreated control and / or the average of the increase in the expression of these genes exceeds by a factor 2 that of the average of the expression of these genes during the untreated control, then the substance is considered irritating in a certain way. expression rate of specific genes of irritation in the model

Table 3

Moreover, for substances that are too corrosive, such as SLS at doses greater than 10% or lactic acid greater than 1%, the tissue degradation is then too great, even with a contact time limited to 15 minutes, the amount the collected RNA is then too weak to be analyzed by RT-PCR. Typically this amount is 2 (g / cm 2, the analysis is not performed if this rate is less than 15μ / αη2 skin.

Furthermore, assays of dose-dependent responses with SLS have shown that the biomarkers mentioned in the present invention could be used as bio markers for irritation, and in particular IL-8, 4-1BBL or col7Al. to quantify the strength of irritation, as shown in Figure 1.

Indeed, Figure 1 shows a linearity in the level of expression of these 3 genes relative to the amount, thus demonstrating a dose-dependent reaction.

It is therefore possible to use the SLS at different doses to establish a standard curve for each experiment. This is of interest to the extent that the thickness, in particular of the stratum corneum, introduces variations between the experiments, in particular by slowing the rate of penetration, which has an influence on the level of expression observed at 6 h. However, whatever the conditions used, a linearity in the expression as a function of the dose is observed and it is possible to find the same values of irritation force, as shown in the following Table 4.

The value of SLS 5% was arbitrarily set to 1.

Methyl salicylate 10% is not irritating in the test.

Table 4: Quantitative Irritant Analysis

Claims

A method for evaluating the irritant potential of a test compound, comprising the steps of:

a) contacting a test compound with a biological sample;

2. Evaluation method according to claim 1, further comprising a step of comparing the level of expression of said at least one gene with a reference value.

3. Evaluation method according to one of claims 1 or 2, wherein the level of expression of said at least one gene is determined by measuring the level of expression of the polypeptide encoded by said gene or a fragment thereof or by measuring the level of expression of the mRNA of the at least one gene or fragment thereof.

4. Evaluation method according to any one of the preceding claims, comprising a further step of amplifying the mRNA or cDNA of said at least one gene, the complementary sequence thereof or a fragment. of it.

5. Method according to any one of the preceding claims, characterized in that the biological sample is a skin sample, preferably a skin sample reconstructed in vitro.

The method according to any one of the preceding claims, wherein step b) comprises determining the level of expression of at least 2, at least 3, at least 4, at least 5. , at least 6, at least 7, and even more preferably at least 8 genes selected from the group consisting of: 4.1 BBL (TNF superfamily member 9), COL7A1 (collagen, type VII, alpha 1 ), CTSD (cathepsin D), FDXR (ferredoxin reductase), GAA (glucosidase, alpha; acid), HAS1 (hyaluronan synthase 1), RELT (Member 19 of the TNF superfamily receptor), IL-1A (interleukin 1, alpha ), IL-7R (Interleukin 7 receptor), IL-8 (Interleukin 8), JUN (oncogene jun), PLAUR (plasminogen activator, urokinase receptor) and TNF-A (tumor necrosis factor).

The method according to any of the preceding claims, wherein step b) is performed between 2 and 24 hours after step a), more preferably between 4 and 18 hours after step a), particularly preferably between 5 and 7 hours after step a) and preferably between 6 hours after step a).

The method according to any of the preceding claims, comprising a step c) of evaluating the irritating force of said test compound.

9. Use of at least one gene selected from the group consisting of: 4.1 BBL (member 9 of the TNF superfamily), COL7A1 (collagen, type VII, alpha 1), CTSD (cathepsin D), FDXR (ferredoxin reductase) , GAA (glucosidase, alpha; acid), HAS1 (hyaluronan synthase 1) and RELT (Member 19 of the TNF superfamily receptor), for the in vitro evaluation of the irritant potential and / or the irritant strength of a test compound.

10. Kit for implementing the method according to any one of claims 1 to 8, comprising means for determining the level of expression of at least one gene selected from the group consisting of: 4.1 BBL (TNF superfamily member 9), COL7A1 (collagen, type VII, alpha 1), CTSD (cathepsin D), FDXR (ferredoxin reductase), GAA (glucosidase, alpha, acid), HAS1 (hyaluronan synthase 1), and RELT (Member 19 of the TNF receptor superfamily).

11. Kit according to claim 10, comprising at least one pair of primers allowing the amplification of at least one gene selected from the group consisting of: 4.1 BBL (member 9 of the TNF superfamily), COL7A1 (collagen, type VII, alpha 1), CTSD (cathepsin D), FDXR (ferredoxin reductase), GAA (glucosidase, alpha; acid), HAS1 (hyaluronan synthase 1) and RELT (Member 19 of the TNF superfamily of the receptor).