DRUG REACTIONS

FIELD OF THE INVENTION

The present invention relates to methods for diagnosis and prognostic of cutaneous adverse drug reactions (cADRs). More specifically present invention relates to methods for diagnosis and monitoring of cutaneous adverse drug reactions (cADRs) through detection of a specific population of microRNAs.

BACKGROUND OF THE INVENTION

Exanthematous drug eruptions also called maculopapular exanthema (MPE) are the most frequent cutaneous adverse drug reactions (cADRs) (50 to 90% of cADR), and occurs in 1 to 5% of first-time users of most drugs ¹ . Although rarer, the Drug Reaction with Eosinophilia and Systemic Symptoms (DRESS) has an incidence ranging between 1 in 1000 and 1 in 10 000 ² ’ ³ and can be life threatening. It is responsible for severe and potentially chronic sequelae ^4-6 . MPE and DRESS pathogenesis depends on a complex interaction between drugs, viruses, genetic susceptibility and the immune system but remains poorly understood ⁷ . The diagnosis of DRESS, especially in early stages, remains challenging due to its heterogeneous clinical presentation and complex natural course, which display different patterns depending on the causal drug ⁸ . Because of classification difficulties, standardized retrospective DRESS clinical, biological and histological diagnosis criteria have been established by the European consortium -RegiSCAR- (RegiSCAR criteria) ⁹ and a Japanese consensus group ¹⁰ . As the resolution of symptoms > 15 days is one of the diagnostic criteria of DRESS ⁹ it is therefore sometimes difficult, at an early stage, to differentiate between MPE and DRESS. Moreover, overlapping features between maculopapular exanthema (MPE) and DRESS, called MPE with systemic symptoms (MPE+SS) have been described, suggesting a continuum spectrum between both diseases, with minor forms of DRESS ¹¹ , also called MP/DR ¹¹ and mini-DRESS ¹² .

To better characterize cADRs, several approaches have been proposed. MicroRNAs (miRNAs) are short non-coding RNA molecules usually composed of 18-25 nucleotides that originate from inter- or intra-genic sequences by the action of RNA pol III and II, respectively ¹³ . A miRNA binding to its target mRNA usually results in degradation or translational inhibition, and hence influences post-transcriptional regulation of target genes ¹⁴ . Multiple studies have explored the potential usefulness of miRNA-expression profiles as highly specific and sensitive noninvasive biomarkers for the diagnosis and prognosis of cancers, as well as inflammatory diseases ^15-18 . Some studies even suggest that miRNAs are involved in many regulatory aspects of inflammation-related functions of epithelial cells, especially in skin with psoriasis or atopic dermatitis ^19-21 or during allergic inflammation ²² .

Serum miR-18a-5p expression had been described as a biomarker of severity ²³ , and disease activity ²⁴ of toxic epidermal necrolysis (TEN), another severe cADR. However, there is no study using miRNA-based approaches to look at the skin of MPE and DRESS patients.

SUMMARY OF THE INVENTION

In the present invention aimed to identify miRNAs differentially expressed in the skin and blood of patients with MPE and DRESS compared with healthy control. The objectives were to assess whether miRNAs expression was able to classify accurately DRESS and MPE patients and to identify gene implicated in the pathogenesis of these diseases. Using real-time qPCR, the expression of 754 miRNAs was quantified in the skin of MPE (n=6), DRESS (n=6) and healthy controls (HC) (n=6). Hierarchical clustering of samples was performed using the Ward’s agglomerative method and correlations between miRNA expressions were performed.

In particular, the inventors have shown that the miRNA expressions analysis of skin tissue samples obtained from cADRs patients is indicative of the status of those patients, (ie discriminate MPE and DRESS pathogenesis) but also monitoring a cutaneous adverse drug reactions (cADRs (moderately- or poorly-differentiated respectively). Among them Let-7c-5p and miR-519e-3p were described in the regulation of the expression of the ATP -binding cassette (ABC) transporter family associated with cell drug efflux. Indeed, inventors found that skin ABCB1 expression was decreased compared to controls during DRESS and MPE. Using a human skin explant model, they confirmed that ABCB1 transporter induction was dependent on Let-7c-5p and miR-519e-3p. Moreover, skin miR-501-3p and miR-520d-5p expression were positively correlated to systemic involvement (respectively eosinophilia and liver abnormalities) during DRESS and MPE.

Thus, in one aspect, the present invention provides a method for diagnosing cutaneous adverse drug reactions (cADRs) in a subject, comprising the steps of : ia) determining in a skin sample obtained from said subject the level of one or more miR nucleic acid selected from a first group of miR nucleic acid consisting of: hsa-miR-1228- 5p; hsa-miR-1294; hsa-miR-548b-5p; hsa-miR-1267; dme-miR-7-5p; hsa-miR-516b-3p; hsa- miR-1253; hsa-miR-520d-5p; hsa-miR-548c-5p; hsa-miR-507 ;hsa-miR-601; hsa-miR-1276; hsa-miR-661; hsa-miR-938; hsa-miR-501-3p; hsa-miR-886-3p; hsa-miR-503-5p; hsa-miR- 519e-3p; hsa-miR-51 l-5p; hsa-miR-939-5p; hsa-miR-1254 ;hsa-miR-223-3p; hsa-miR-1233- 3p; hsa-miR-185-5p and/or ib) determining in a skin sample obtained from said subject the level of one or more miR nucleic acid selected from a second group of miR nucleic acid consisting of: hsa-miR- 411 -5p, hsa-miR-489-3p; hsa-miR-18 la-2-3 p; hsa-let-7c-5p; hsa-miR-27a-3p ; miR-574-3p; hsa-miR-26a-5p; hsa-miR-27b-3p ;hsa-miR-214-3p; hsa-miR-26b-5p, iia) wherein the level of one or more miR nucleic acid of group ia) is positively correlated with the risk of said subject of having or developing a cutaneous adverse drug reactions (cADRs) iib) wherein the level of one or more miR nucleic acid of group ib) is negatively correlated with the risk of said subject of having or developing a cutaneous adverse drug reactions (cADRs).

In particular embodiments, cutaneous adverse drug reactions (cADRs) is selected from the group consisting of maculopapular exanthema (MPE) or Drug Reaction with Eosinophilia and Systemic Symptoms (DRESS).

In another aspect, the present invention provides a method for monitoring a cutaneous adverse drug reactions (cADRs) comprising the steps of ia) determining the level of one or more miR nucleic acid selected from a first group of miR nucleic acid consisting of: hsa-miR-1228-5p; hsa-miR-1294; hsa-miR-548b-5p; hsa- miR-1267; dme-miR-7-5p; hsa-miR-516b-3p ; hsa-miR-1253; hsa-miR-520d-5p; hsa-miR- 548c-5p; hsa-miR-507; hsa-miR-601; hsa-miR-1276; hsa-miR-661; hsa-miR-938; hsa-miR- 501-3p; hsa-miR-886-3p; hsa-miR-503-5p; hsa-miR-519e-3p; hsa-miR-51 l-5p; hsa-miR-939- 5p ; hsa-miR-1254 ;hsa-miR-223-3p; hsa-miR-1233-3p; hsa-miR-185-5p in a skin sample obtained from the subject at a first specific time of the disease and/or ib) determining the level of one or more miR nucleic acid selected from a second group of miR nucleic acid consisting of: hsa-miR-41 l-5p, hsa-miR-489-3p; hsa-miR-18 la-2-3 p; hsa-let-7c-5p; hsa-miR-27a-3p; miR-574-3p; hsa-miR-26a-5p; hsa-miR-27b-3p ;hsa-miR- 214-3p; hsa-miR-26b-5p in a skin sample obtained from the subject at a first specific time of the disease iia) determining the level of one or more miR nucleic acid selected from a first group of miR nucleic acid consisting of:, hsa-miR-1228-5p; hsa-miR-1294; hsa-miR-548b-5p; hsa- miR-1267; dme-miR-7-5p; hsa-miR-516b-3p; hsa-miR-1253; hsa-miR-520d-5p; hsa-miR- 548c-5p; hsa-miR-507 ;hsa-miR-601; hsa-miR-1276; hsa-miR-661 ; hsa-miR-938 ;hsa-miR- 501-3p; hsa-miR-886-3p; hsa-miR-503-5p; hsa-miR-519e-3p; hsa-miR-51 l-5p; hsa-miR-939- 5p; hsa-miR-1254 ;hsa-miR-223-3p; hsa-miR-1233-3p; hsa-miR-185-5p in a skin sample obtained from the subject at a second specific time of the disease, and/or iib) determining the level of one or more miR nucleic acid selected from a second group of miR nucleic acid consisting of: hsa-miR-41 l-5p; hsa-miR-489-3p; hsa-miR-18 la-2-3 p; hsa-let-7c-5p; hsa-miR-27a-3p; miR-574-3p; hsa-miR-26a-5p; hsa-miR-27b-3p ;hsa-miR- 214-3p; hsa-miR-26b-5p in a skin sample obtained from the subject at a second specific time of the disease iii) comparing the level determined at step ia) with the level determined at step iia) and/or comparing the level determined at step ib) with the level determined at step iib) iv) concluding that the disease has evolved in worse manner when the level determined at step iia) is higher than the level determined at step ia) and/or when the level determined at step iib) is lower than the level determined at step ib).

In yet another aspect, the present invention provides a method for monitoring if a treatment with a drug induce a cutaneous adverse drug reactions (cADRs) comprising the steps of : ia) determining the level of one or more miR nucleic acid selected from a first group of miR nucleic acid consisting of: hsa-miR-1228-5p; hsa-miR-1294; hsa-miR-548b-5p; hsa- miR-1267; dme-miR-7-5p; hsa-miR-516b-3p ; hsa-miR-1253; hsa-miR-520d-5p; hsa-miR- 548c-5p; hsa-miR-507 ;hsa-miR-601; hsa-miR-1276; hsa-miR-661 ; hsa-miR-938 ;hsa-miR- 501-3p; hsa-miR-886-3p; hsa-miR-503-5p; hsa-miR-519e-3p; hsa-miR-51 l-5p; hsa-miR-939- 5p ; hsa-miR-1254 ;hsa-miR-223-3p; hsa-miR-1233-3p; hsa-miR-185-5p in a skin sample obtained from the subject before the treatment, and/or ib) determining the level of one or more miR nucleic acid selected from a second group of miR nucleic acid consisting of: hsa-miR-41 l-5p, hsa-miR-489-3p; hsa-miR-18 la-2- 3p; hsa-let-7c-5p; hsa-miR-27a-3p ; miR-574-3p; hsa-miR-26a-5p; hsa-miR-27b-3p ;hsa- miR-214-3p, hsa-miR-26b-5p in a skin sample obtained from the subject before the treatment iia) determining the level of one or more miR nucleic acid selected from a first group of miR nucleic acid consisting of:, hsa-miR-1228-5p; hsa-miR-1294; hsa-miR-548b-5p; hsa- miR-1267; dme-miR-75p; hsa-miR-516b-3p ; hsa-miR-1253; hsa-miR-520d-5p; hsa-miR- 548c-5p; hsa-miR-507 ;hsa-miR-601; hsa-miR-1276; hsa-miR-661 ; hsa-miR-938 ;hsa-miR- 501-3p; hsa-miR-886-3p; hsa-miR-503-5p; hsa-miR-519e-3p; hsa-miR-51 l-5p; hsa-miR-939- 5p ; hsa-miR-1254 ;hsa-miR-223-3p; hsa-miR-1233-3p; hsa-miR-185-5p in a skin sample obtained from the subject after the treatment, and/or iib) determining the level of one or more miR nucleic acid selected from a second group of miR nucleic acid consisting of: hsa-miR-41 l-5p, hsa-miR-489-3p; hsa-miR-181a-2- 3p; hsa-let-7c-5p; hsa-miR-27a-3p; miR-574-3p; hsa-miR-26a-5p; hsa-miR-27b-3p ;hsa-miR- 214;-3p hsa-miR-26b-5p in a skin sample obtained from the subject after the treatment, iii) comparing the level determined at step ia) with the level determined at step iia) and/or comparing the level determined at step ib) with the level determined at step iib) and iv) concluding that the treatment induce a cutaneous adverse drug reactions (cADRs) when the level determined at step iia) is higher than the level determined at step ia) and/or when the level determined at step iib) is lower than the level determined at step ib).

In another aspect, the present invention provides a method for treating cutaneous adverse drug reactions (cADRs) comprising a step of administering to the said individual a compound selected in a group comprising a hsa-let-7c-5p nucleic acid, a compound mimicking a hsa-let-7c-5p nucleic acid, and/or comprising a step of administering to the said individual a compound consisting of a Block-hsa-miR-519e-3p nucleic acid

These and other objects, advantages and features of the present invention will become apparent to those of ordinary skill in the art having read the following detailed description of the preferred embodiments.

DETAILED DESCRIPTION OF THE INVENTION

Definitions:

Throughout the specification, several terms are employed and are defined in the following paragraphs.

As used herein, the term “subject” refers to a human or another mammal ( e.g ., primate, dog, cat, goat, horse, pig, mouse, rat, rabbit, and the like), that can be afflicted with cutaneous adverse drug reactions (cADRs). In a preferred embodiment of the present invention, the subject is a human being. In such embodiments, the subject is often referred to as an “individual”.

The term “cutaneous adverse drug reactions” (or “cADRs”) (The terms “cARDs” and “cutaneous adverse drug reactions” are used herein interchangeably) are a group of potentially lethal adverse drug reactions that involve the skin and mucous membranes of various body openings such as the eyes, ears, and inside the nose, mouth, and lips. In more severe cases, cADRs could also involve serious damage to internal organs. cADRs include these different syndromes: Drug reaction with eosinophilia and systemic symptoms (i.e. DRESS syndrome, also termed Drug-induced hypersensitivity syndrome [DIHS]); Stevens-Johnson syndrome (SJS); Toxic epidermal necrolysis (TEN), Stevens- Johnson/toxic epidermal necrolysis overlap syndrome (SJS/TEN); cute generalized exanthematous pustulosis (AGEP), Maculo-Papular Exanthema related to drug (MPE), Fixed Drug Eruption (FDE), Symmetrical Drug Related Intertriginous and Flexural Exanthema (SDRIFE). These disorders have similar pathophysiologies, i.e. disease-causing mechanisms, for which new strategies are in use or development to identify individuals predisposed to develop the cADRs -inducing effects of specific drugs and thereby avoid treatment with them (Adler NR, et al (2017) The British Journal of Dermatology. 177 (5): 1234-1247).

Adverse drug reactions are major therapeutic problems estimated to afflict up to 20% of inpatients and 25% of outpatients. About 90% of these delayed adverse reactions take the form of benign morbilliform rash hypersensitivity drug reactions called maculo-papular exanthema (MPE). cADRS are delayed-hypersentivity reaction called Type IV hypersensitivity reaction of the innate immune system initiated by lymphocytes of the T cell type and mediated by various types of leukocytes and cytokines (Garon SL et al (2017). British Journal of Clinical Pharmacology. 83 (9): 1896-1911). cADRs are here considered as a group focusing on the similarities and differences in their pathophysiologies, clinical presentations, instigating drugs, and recommendations for drug avoidance.

The mains drugs knows to inducing cADRs are for instance but not limited to : anti epileptics (Stern RS; N Engl J Med 2012;366:2492-501), antibiotics (such as Vancomycin , Penicillin , Cephalosporin, Tetracycline , Fluoroquinolone, Sulfonamide, Cotrimoxazole, Carbapenem,...) (Wolfson AR et al Allergy Clin Immunol Pract Month 2018), antiretroviral drugs, Immune Checkpoint inhibitors (ICP) (see Naqash et al. Journal for ImmunoTherapy of Cancer (2019) 7:4), proton pump inhibitors, anticonvulsants (such as phenobarbital, carbamazepine, phenytoin, lamotrigine, and sodium valproate) Allopurinol, (used to decrease high blood uric acid levels) (see also the review V. Descamps, et al Joint Bone Spine 81 (2014) 15-21)

The term “subject suspected of having cADRs” refers to a subject that presents one or more symptoms indicative of cADRs ( e.g ., pain, skins or and mucous membranes lesions associated with drugs administration), or that is screened for cADRs (e.g., during a physical examination). Alternatively or additionally, a subject suspected of having cADRs may have one or more risk factors ( e.g ., age, sex, family history,, etc). The term encompasses subjects that have not been tested for cADRs as well as subjects that have received an initial diagnosis.

The terms “normal” and “healthy” are used herein interchangeably. They refer to a subject that has not presented any cADRs symptoms, and that has not been diagnosed with cADRs or with skin injury. Preferably, a normal subject is not on medication for cADRs and has not been diagnosed with any other disease (in particular an skin disease). In certain embodiments, normal subjects may have similar sex, age, and/or body mass index as compared with the subject from which the biological sample to be tested was obtained. The term “normal” is also used herein to qualify a sample obtained from a healthy subject.

In the context of the present invention, the term “control”, when used to characterize a subject, refers to a subject that is healthy or to a patient who has been diagnosed with a specific skin disease other than cADRs. The term “control sample” refers to one, or more than one, sample that has been obtained from a healthy subject or from a patient diagnosed with a disease other than cADRs.

The terms “biomarker” and “marker” are used herein interchangeably. They refer to a substance that is a distinctive indicator of a biological process, biological event, and/or pathologic condition. In the context of the present invention, the term “biomarker of cADRs” encompasses miR-1228-5p; hsa-miR-1294; hsa-miR-548b-5p; hsa-miR-1267; dme-miR-7-5p; hsa-miR-516b-3p; hsa-miR-1253; hsa-miR-520d-5p; hsa-miR-548c-5p; hsa-miR-507 ;hsa- miR-601; hsa-miR-1276; hsa-miR-661; hsa-miR-938 ;hsa-miR-501-3p; hsa-miR-886-3p; hsa- miR-503-5p; hsa-miR-519e-3p; hsa-miR-51 l-5p; hsa-miR-939-5p; hsa-miR-1254 ;hsa-miR- 223-3p; hsa-miR-1233-3p; hsa-miR-185-5p, hsa-miR-41 l-5p, hsa-miR-489-3p; hsa-miR- 181a-2-3p; hsa-let-7c-5p; hsa-miR-27a-3p ; miR-574-3p; hsa-miR-26a-5p; hsa-miR-27b-3p ;hsa-miR-214;-3p hsa-miR-26b-5p as described herein present in a biological sample of a RA patient..

As used herein, the term “indicative of cADRs”, when applied to a process or event, refers to a process or event which is a diagnostic of cADRs, such that the process or event is found significantly more often in subjects with cADRs than in healthy subjects and/or in subjects suffering from a disease other than cADRs.

Detailed Description of Certain Preferred Embodiments The present inventors have shown that a switch in miRNA expression patterns in skin of cADRs inverts the balance between inductive and repressive signals, triggering decreased protein expression (such as ABC transporter). Such miRNAs expression was able to classify accurately DRESS and MPE patients and to identify gene implicated in the pathogenesis of these diseases. Accordingly the specific miRNA expressions of skin tissue samples obtained from cADRs patients is indicative of the status of those patients, (ie discriminate MPE and DRESS pathogenesis) but also monitoring a cutaneous adverse drug reactions (cADRs (moderately- or poorly-differentiated respectively). These data raise the intriguing possibility that miRNA-dependent epigenetic regulation could underlie the pathophysiology of Severe cutaneous adverse reactions” (or “SCARs”) in human when a genetic cause is not evident, and they hold therapeutic potential for cADRs disorders.

In other words, the inventors found that miR nucleic acids of the invention are up- regulated or down-regulated, and therefore may be considered as good biomarkers in the occurrence of certain cADRs disorders.

Some definitions are given hereunder that are relevant as regards the description of the whole embodiments that are encompassed by the present invention.

MicroRNAs (miRs) are small, noncoding RNAs that are emerging as crucial regulators of biological processes.

“MicroRNA”, “miRNA” or “miR” means a non-coding RNA of about 18 to about 25 nucleotides in length. MicroRNAs (miRNAs) are short noncoding RNAs that silence gene expression post-transcriptionally, principally by binding to 3’ untranslated regions (3’UTR) of target mRNAs These miRs could originate from multiple origins including: an individual gene encoding for a miRNA, from introns of protein coding gene, or from poly-cistronic transcript that often encode multiple, closely related microRNAs.

"Stem-loop sequence" means a RNA having a hairpin structure and containing a mature microRNA sequence. Pre-miRNA sequences and stem-loop sequences may overlap. Examples of stem-loop sequences are found in the microRNA database known as miRBase.

"MicroRNA precursor" means a transcript that originates from a genomic DNA and that comprises a non-coding, structured RNA comprising one or more microRNA sequences. For example, in certain embodiments a microRNA precursor is a pre-miRNA. In certain embodiments, a microRNA precursor is a pri-miRNA.

The following specification will follow the standard nomenclature system with the uncapitalized "mir-X" refers to the pre-miRNA, while a capitalized "miR-X" refers to the mature form. When two mature microRNAs originate from opposite arms of the same pre- miRNA, they are denoted with a -3p or -5p suffix. When relative expression levels are known, an asterisk following the name indicates a microRNA expressed at low levels relative to the microRNA in the opposite arm of a hairpin.

In the following specification, unless otherwise specified, the use of the expression miR-X refers to the mature miRNA including both forms -3p and -5p, if any.

For the avoidance of doubt, in the present specification, the expressions microRNA, miRNA and miR designate the same product

As mentioned above, the present invention provides biomarkers in order to detect the miRNA level in skin samples obtained from patients. These biomarkers are hsa-miR-1228- 5p; hsa-miR-1294; hsa-miR-548b-5p; hsa-miR-1267; dme-miR-7-5p; hsa-miR-516b-3p ; hsa- miR-1253; hsa-miR-520d-5p; hsa-miR-548c-5p; hsa-miR-507 ;hsa-miR-601; hsa-miR-1276; hsa-miR-661; hsa-miR-938 ;hsa-miR-501-3p; hsa-miR-886-3p; hsa-miR-503-5p; hsa-miR- 519e-3p; hsa-miR-51 l-5p; hsa-miR-939-5p ; hsa-miR-1254 ;hsa-miR-223-3p; hsa-miR-1233- 3p; hsa-miR-185-5p , hsa-miR-41 l-5p, hsa-miR-489-3p; hsa-miR-18 la-2-3 p; hsa-let-7c-5p; hsa-miR-27a-3p; miR-574-3p; hsa-miR-26a-5p; hsa-miR-27b-3p ;hsa-miR-214;-3p hsa-miR- 26b-5p as described herein present in the skin sample of cADRs patients (see table 1).

Table 1 miRNA used in the present invention: The invention also provides methods for using these biomarkers in the diagnosis /monitoring methods of cADRs.

I. Methods of Diagnosis

As mentioned above, the biomarkers disclosed herein are specifically detected in in the skin sample of cADRs patients, and in a particular embodiment allow to discriminate cADRs patients between maculopapular exanthema (MPE) or Drug Reaction with Eosinophilia and Systemic Symptoms (DRESS).

Accordingly, the present invention provides a method for diagnosing cutaneous adverse drug reactions (cADRs) in a subject, comprising the steps of: ia) determining in a skin sample obtained from said subject the level of one or more miR nucleic acid selected from a first group of miR nucleic acid consisting of:, hsa-miR- 1228-5p (SEQ ID N°l); hsa-miR-1294 (SEQ ID N°2); hsa-miR-548b-5p (SEQ ID N°3); hsa- miR-1267 (SEQ ID N°4); dme-miR-7-5p (SEQ ID N°5); hsa-miR-516b-3p (SEQ ID N°6); hsa-miR-1253 (SEQ ID N°7); hsa-miR-520d-5p (SEQ ID N°8); hsa-miR-548c-5p (SEQ ID N°9); hsa-miR-507 (SEQ ID N°l l); hsa-miR-601 (SEQ ID N°12); hsa-miR-1276 (SEQ ID N°13); hsa-miR-661 (SEQ ID N°14); hsa-miR-938 (SEQ ID N°15);hsa-miR-501-3p (SEQ ID N°16); hsa-miR-886-3p (SEQ ID N°17); hsa-miR-503-5p (SEQ ID N°18); hsa-miR-519e-3p (SEQ ID N°19); hsa-miR-51 l-5p (SEQ ID N°20); hsa-miR-939-5p (SEQ ID N°22); hsa-miR- 1254 (SEQ ID N°23);hsa-miR-223-3p (SEQ ID N°24); hsa-miR-1233-3p (SEQ ID N°27); hsa-miR-185-5p (SEQ ID N°30) and/or ib) determining in a skin sample obtained from said subject the level of one or more miR nucleic acid selected from a second group of miR nucleic acid consisting of: hsa-miR- 411 -5p (SEQ ID N°10), hsa-miR-489-3p (SEQ ID N°21); hsa-miR-18 la-2-3 p (SEQ ID N°25); hsa-let-7c-5p (SEQ ID N°26); hsa-miR-27a-3p (SEQ ID N°28); miR-574-3p (SEQ ID N°29); hsa-miR-26a-5p (SEQ ID N°31); hsa-miR-27b-3p (SEQ ID N°32); hsa-miR-214-3p (SEQ ID N°33);hsa-miR-26b-5p (SEQ ID N°34), iia) wherein the level of one or more miR nucleic acid of group ia) is positively correlated with the risk of said subject of having or developing a cutaneous adverse drug reactions (cADRs) iib) wherein the level of one or more miR nucleic acid of group ib) is negatively correlated with the risk of said subject of having or developing a cutaneous adverse drug reactions (cADRs). In some embodiments, the said method is performed in vitro or ex vivo.

In particular embodiments the diagnostic method of the invention comprising the step of comparing said level of one or more miR nucleic acid of group ia) to a control reference value and/or said level of one or more miR nucleic acid of group ib) to a control reference value wherein:

- a high level of one or more miR nucleic acid of group ia) compared to said control reference value is predictive of a high risk of having or developing a cutaneous adverse drug reactions (cADRs) and

- a low level of one or more miR nucleic acid of group ib) compared to said control reference value is predictive of a high risk of having or developing a cutaneous adverse drug reactions (cADRs)

In preferred embodiments, a plurality of miRNAs biomarkers (miR nucleic acid biomarkers) is used in the method of diagnosis. In other words, the method of the invention may comprise steps of: detecting the skin sample the level of 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 19, 20,21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33 or 34 biomarker present in the biological sample; and detecting any miRNAs biomarker.

In particular embodiments, the method of diagnosis is performed using 34 different biomarkers including the hsa-miR-1228-5p (SEQ ID N°l); hsa-miR-1294 (SEQ ID N°2); hsa- miR-548b-5p (SEQ ID N°3); hsa-miR-1267 (SEQ ID N°4); dme-miR-75p (SEQ ID N°5); hsa-miR-516b-3p (SEQ ID N°6); hsa-miR-1253 (SEQ ID N°7); hsa-miR-520d-5p (SEQ ID N°8); hsa-miR-548c-5p (SEQ ID N°9); hsa-miR-507 (SEQ ID N°l l); hsa-miR-601 (SEQ ID N°12); hsa-miR-1276 (SEQ ID N°13); hsa-miR-661 (SEQ ID N°14); hsa-miR-938 (SEQ ID N°15); hsa-miR-501-3p (SEQ ID N°16); hsa-miR-886-3p (SEQ ID N°17); hsa-miR-503-5p (SEQ ID N°18); hsa-miR-519e-3p (SEQ ID N°19); hsa-miR-51 l-5p (SEQ ID N°20); hsa- miR-939-5p (SEQ ID N°22); hsa-miR-1254 (SEQ ID N°23); hsa-miR-223-3p (SEQ ID N°24); hsa-miR-1233-3p (SEQ ID N°27); hsa-miR-185-5p (SEQ ID N°30), hsa-miR-41 l-5p (SEQ ID N°10), hsa-miR-489-3p (SEQ ID N°21); hsa-miR-18 la-2-3 p (SEQ ID N°25); hsa- let-7c-5p (SEQ ID N°26); hsa-miR-27a-3p (SEQ ID N°28); miR-574-3p (SEQ ID N°29); hsa- miR-26a-5p (SEQ ID N°31); hsa-miR-27b-3p (SEQ ID N°32); hsa-miR-214;-3p (SEQ ID N°33); hsa-miR-26b-5p (SEQ ID N°34) as described herein.

In particular embodiments, the method of the invention is performed to detect cADRs patient at an early stage.

Accordingly, the method of diagnostic of the invention is consequently useful for the diagnosis of early stage cADRs from a skin sample. As used herein, the term “early stage cADRs” refers to the stage of the disease with the onset of clinical symptoms, such as pain, skin or and mucous membrane lesions associated with drugs administration.

In particular embodiments, cutaneous adverse drug reactions (cADRs) is selected from the group consisting of maculopapular exanthema (MPE) or Drug Reaction with Eosinophilia and Systemic Symptoms (DRESS).

In particular embodiments wherein the cutaneous adverse drug reactions (cADRs) is maculopapular exanthema (MPE) the method of diagnosis of the invention comprising the steps of : i) determining in a skin sample obtained from said subject the level of one or more miR nucleic acid selected from a group of miR nucleic acid consisting of:, hsa-miR-1267 (SEQ ID N°4); hsa-miR-516b-3p (SEQ ID N°6); hsa-miR-223-3p (SEQ ID N°24); hsa-miR- 1233-3p (SEQ ID N°27), ii) wherein the level of one or more miR nucleic acid of group i) is positively correlated with the risk of said subject of having or developing a maculopapular exanthema (MPE).

In a particular embodiment, the level of hsa-miR-1267 (SEQ ID N°4); hsa-miR-516b- 3p (SEQ ID N°6); hsa-miR-223-3p (SEQ ID N°24); and hsa-miR-1233-3p (SEQ ID N°27) are determined in step i).

In this particular embodiments the diagnostic method of the invention comprising the step of comparing said level of the level of one or more miR nucleic acid of group i) to a control reference value wherein:

- a high level of one or more miR nucleic acid of group i) compared to said control reference value is predictive of a high risk of having or developing a maculopapular exanthema (MPE).

Another aspect of the present invention concern an method to distinguish between maculopapular exanthema (MPE) and Drug Reaction with Eosinophilia and Systemic Symptoms (DRESS) in a subject having a cutaneous adverse drug reactions (cADRs), comprising the steps of : ia) determining in a skin sample obtained from said subject the level of one or more miR nucleic acid selected from a first group of miR nucleic acid consisting of:, hsa-miR- 181a-2-3p (SEQ ID N°25); hsa-miR-26b-5p (SEQ ID N°34); hsa-miR-214-3p (SEQ ID N°33); hsa-let-7c-5p (SEQ ID N°26) and/or ib) determining in a skin sample obtained from said subject the level of one or more miR nucleic acid selected from a second group of miR nucleic acid consisting of: hsa-miR- 520d-5p (SEQ ID N°8); hsa-miR-501-3p (SEQ ID N°16); hsa-miR-519e-3p (SEQ ID N°19); iia) wherein the level of one or more miR nucleic acid of group ia) is positively correlated with the risk of said subject of having a maculopapular exanthema (MPE); iib) wherein the level of one or more miR nucleic acid of group ib) is negatively correlated with the risk of said subject of having a Drug Reaction with Eosinophilia and Systemic Symptoms (DRESS).

In some embodiments, the said method is performed in vitro or ex vivo.

In preferred embodiments, a plurality of miRNAs biomarkers (miR nucleic acid biomarkers) is used in the method to distinguish between MPE and DRESS. In other words, the method of the invention may comprise steps of: detecting the skin sample the level of 1, 2, 3, 4, 5, 6 or 7 biomarker present in the biological sample; and detecting any miRNA biomarker.

In particular embodiments, the method of diagnosis is performed using the 7 different biomarkers including the hsa-miR-18 la-2-3 p (SEQ ID N°25); hsa-miR-26b-5p (SEQ ID N°34); hsa-miR-214-3p (SEQ ID N°33); hsa-let-7c-5p (SEQ ID N°26), hsa-miR-520d-5p (SEQ ID N°8); hsa-miR-501-3p (SEQ ID N°16); hsa-miR-519e-3p (SEQ ID N°19) as described herein.

In particular embodiment the method to distinguish between MPE and DRESS comprising the steps of: ia) determining in a skin sample obtained from said subject the level of one or more miR nucleic acid selected from a first group of miR nucleic acid consisting of:, hsa-miR- 181a-2-3p (SEQ ID N°25); hsa-let-7c-5p (SEQ ID N°26) and/or ib) determining in a skin sample obtained from said subject the level of one or more miR nucleic acid selected from a second group of miR nucleic acid consisting of: hsa-miR- 520d-5p (SEQ ID N°8); hsa-miR-519e-3p (SEQ ID N°19); iia) wherein the level of one or more miR nucleic acid of group ia) is positively correlated with the risk of said subject of having a maculopapular exanthema (MPE); iib) wherein the level of one or more miR nucleic acid of group ib) is negatively correlated with the risk of said subject of having a Drug Reaction with Eosinophilia and Systemic Symptoms (DRESS).

In particular embodiments the method to distinguish between MPE and DRESS comprising the step of comparing said level of one or more miR nucleic acid of group ia) to a control reference value and/or said level of one or more miR nucleic acid of group ib) to a control reference value wherein:

- a high level of one or more miR nucleic acid of group ia) compared to said control reference value is predictive of a high risk of having or developing a maculopapular exanthema (MPE) and

- a low level of one or more miR nucleic acid of group ib) compared to said control reference value is predictive of a high risk of having or developing Drug Reaction with Eosinophilia and Systemic Symptoms (DRESS)

Control reference values are easily determinable by the one skilled in the art, by using the same techniques as for determining the level of miRNA biomarker in skin samples previously collected from the patient under testing.

A “reference value” can be a “threshold value” or a “cut-off value”. Typically, a "threshold value" or "cut-off value" can be determined experimentally, empirically, or theoretically. A threshold value can also be arbitrarily selected based upon the existing experimental and/or clinical conditions, as would be recognized by a person of ordinary skilled in the art. The threshold value has to be determined in order to obtain the optimal sensitivity and specificity according to the function of the test and the benefit/risk balance (clinical consequences of false positive and false negative). Typically, the optimal sensitivity and specificity (and so the threshold value) can be determined using a Receiver Operating Characteristic (ROC) curve based on experimental data. Preferably, the person skilled in the art may compare the level of miRNA biomarkers of the present invention (“see table 1) with a defined threshold value. In one embodiment of the present invention, the threshold value is derived from the miRNA level (or ratio, or score) determined in a skin sample derived from one or more subjects who are responders (to the method according to the invention). In one embodiment of the present invention, the threshold value may also be derived from miRNA level (or ratio, or score) determined in a skin sample derived from one or more subjects or who are non-responders. Furthermore, retrospective measurement of the miRNA level (or ratio, or scores) in properly banked historical subject samples may be used in establishing these threshold values.

For example, after determining the expression level of the selected miRNA in a group of reference, one can use algorithmic analysis for the statistic treatment of the expression levels determined in samples to be tested, and thus obtain a classification standard having significance for sample classification. The full name of ROC curve is receiver operator characteristic curve, which is also known as receiver operation characteristic curve. It is mainly used for clinical biochemical diagnostic tests. ROC curve is a comprehensive indicator that reflects the continuous variables of true positive rate (sensitivity) and false positive rate (1-specificity). It reveals the relationship between sensitivity and specificity with the image composition method. A series of different cut-off values (thresholds or critical values, boundary values between normal and abnormal results of diagnostic test) are set as continuous variables to calculate a series of sensitivity and specificity values. Then sensitivity is used as the vertical coordinate and specificity is used as the horizontal coordinate to draw a curve. The higher the area under the curve (AUC), the higher the accuracy of diagnosis. On the ROC curve, the point closest to the far upper left of the coordinate diagram is a critical point having both high sensitivity and high specificity values. The AUC value of the ROC curve is between 1.0 and 0.5. When AUC>0.5, the diagnostic result gets better and better as AUC approaches 1. When AUC is between 0.5 and 0.7, the accuracy is low. When AUC is between 0.7 and 0.9, the accuracy is moderate. When AUC is higher than 0.9, the accuracy is high. This algorithmic method is preferably done with a computer. Existing software or systems in the art may be used for the drawing of the ROC curve, such as: MedCalc 9.2.0.1 medical statistical software, SPSS 9.0, ROCPOWER.SAS, DESIGNROC.FOR, MULTIREADER POWER.SAS, CREATE-ROC.SAS, GB STAT VIO.O (Dynamic Microsystems, Inc. Silver Spring, Md., USA), etc.

In some embodiments, the method of the invention comprises the use of a classification algorithm typically selected from Linear Discriminant Analysis (LDA), Topological Data Analysis (TDA), Neural Networks, Support Vector Machine (SVM) algorithm and Random Forests algorithm (RF). In some embodiments, the method of the invention comprises the step of determining the subject response using a classification algorithm. As used herein, the term "classification algorithm" has its general meaning in the art and refers to classification and regression tree methods and multivariate classification well known in the art such as described in US 8,126,690; WO2008/156617. As used herein, the term “support vector machine (SVM)” is a universal learning machine useful for pattern recognition, whose decision surface is parameterized by a set of support vectors and a set of corresponding weights, refers to a method of not separately processing, but simultaneously processing a plurality of variables. Thus, the support vector machine is useful as a statistical tool for classification. The support vector machine non-linearly maps its n-dimensional input space into a high dimensional feature space, and presents an optimal interface (optimal parting plane) between features. The support vector machine comprises two phases: a training phase and a testing phase. In the training phase, support vectors are produced, while estimation is performed according to a specific rule in the testing phase. In general, SVMs provide a model for use in classifying each of n subjects to two or more disease categories based on one k-dimensional vector (called a k-tuple) of biomarker measurements per subject. An SVM first transforms the k-tuples using a kernel function into a space of equal or higher dimension. The kernel function projects the data into a space where the categories can be better separated using hyperplanes than would be possible in the original data space. To determine the hyperplanes with which to discriminate between categories, a set of support vectors, which lie closest to the boundary between the disease categories, may be chosen. A hyperplane is then selected by known SVM techniques such that the distance between the support vectors and the hyperplane is maximal within the bounds of a cost function that penalizes incorrect predictions. This hyperplane is the one which optimally separates the data in terms of prediction (Vapnik, 1998 Statistical Learning Theory. New York: Wiley). Any new observation is then classified as belonging to any one of the categories of interest, based where the observation lies in relation to the hyperplane. When more than two categories are considered, the process is carried out pairwise for all of the categories and those results combined to create a rule to discriminate between all the categories. As used herein, the term "Random Forests algorithm" or "RF" has its general meaning in the art and refers to classification algorithm such as described in US 8,126,690; WO2008/156617. Random Forest is a decision-tree-based classifier that is constructed using an algorithm originally developed by Leo Breiman (Breiman L, "Random forests," Machine Learning 2001, 45:5-32). The classifier uses a large number of individual decision trees and decides the class by choosing the mode of the classes as determined by the individual trees. The individual trees are constructed using the following algorithm: (1) Assume that the number of cases in the training set is N, and that the number of variables in the classifier is M; (2) Select the number of input variables that will be used to determine the decision at a node of the tree; this number, m should be much less than M; (3) Choose a training set by choosing N samples from the training set with replacement; (4) For each node of the tree randomly select m of the M variables on which to base the decision at that node; (5) Calculate the best split based on these m variables in the training set. In some embodiments, the score is generated by a computer program.

In some embodiments, the method of the present invention comprises a) quantifying the level of a plurality of miRNA of Table 1 in the skin sample; b) implementing a classification algorithm on data comprising the quantified plurality of miRNA so as to obtain an algorithm output; c) determining the probability that the subject will develop a cADRs from the algorithm output of step b).

The algorithm used with the method of the present invention can be performed by one or more programmable processors executing one or more computer programs to perform functions by operating on input data and generating output. The algorithm can also be performed by, and apparatus can also be implemented as, special purpose logic circuitry, e.g., an FPGA (field programmable gate array) or an ASIC (application-specific integrated circuit). Processors suitable for the execution of a computer program include, by way of example, both general and special purpose microprocessors, and any one or more processors of any kind of digital computer. Generally, a processor will receive instructions and data from a read-only memory or a random access memory or both. The essential elements of a computer are a processor for performing instructions and one or more memory devices for storing instructions and data. Generally, a computer will also include, or be operatively coupled to receive data from or transfer data to, or both, one or more mass storage devices for storing data, e.g., magnetic, magneto-optical disks, or optical disks. However, a computer need not have such devices. Moreover, a computer can be embedded in another device. Computer- readable media suitable for storing computer program instructions and data include all forms of non-volatile memory, media and memory devices, including by way of example semiconductor memory devices, e.g., EPROM, EEPROM, and flash memory devices; magnetic disks, e.g., internal hard disks or removable disks; magneto-optical disks; and CD- ROM and DVD-ROM disks. The processor and the memory can be supplemented by, or incorporated in, special purpose logic circuitry. To provide for interaction with a user, embodiments of the invention can be implemented on a computer having a display device, e.g., in non-limiting examples, a CRT (cathode ray tube) or LCD (liquid crystal display) monitor, for displaying information to the user and a keyboard and a pointing device, e.g., a mouse or a trackball, by which the user can provide input to the computer. Other kinds of devices can be used to provide for interaction with a user as well; for example, feedback provided to the user can be any form of sensory feedback, e.g., visual feedback, auditory feedback, or tactile feedback; and input from the user can be received in any form, including acoustic, speech, or tactile input. Accordingly, in some embodiments, the algorithm can be implemented in a computing system that includes a back-end component, e.g., as a data server, or that includes a middleware component, e.g., an application server, or that includes a front-end component, e.g., a client computer having a graphical user interface or a Web browser through which a user can interact with an implementation of the invention, or any combination of one or more such back-end, middleware, or front-end components. The components of the system can be interconnected by any form or medium of digital data communication, e.g., a communication network. Examples of communication networks include a local area network (“LAN”) and a wide area network (“WAN”), e.g., the Internet. The computing system can include clients and servers. A client and server are generally remote from each other and typically interact through a communication network. The relationship of client and server arises by virtue of computer programs running on the respective computers and having a client-server relationship to each other.

"Risk" in the context of the present invention, relates to the probability that an event will occur over a specific time period, as in the conversion to cADRs, and can mean a subject's "absolute" risk or "relative" risk. Absolute risk can be measured with reference to either actual observation post-measurement for the relevant time cohort, or with reference to index values developed from statistically valid historical cohorts that have been followed for the relevant time period. Relative risk refers to the ratio of absolute risks of a subject compared either to the absolute risks of low risk cohorts or an average population risk, which can vary by how clinical risk factors are assessed. Odds ratios, the proportion of positive events to negative events for a given test result, are also commonly used (odds are according to the formula p/(l-p) where p is the probability of event and (1- p) is the probability of no event) to no conversion. Alternative continuous measures, which may be assessed in the context of the present invention, include time to cADRs conversion risk reduction ratios.

"Risk evaluation," or "evaluation of risk" in the context of the present invention encompasses making a prediction of the probability, odds, or likelihood that an event or disease state may occur, the rate of occurrence of the event or conversion from one disease state to another, i.e., from a normal condition to a cADRs condition or to one at risk of developing a cADRs. Risk evaluation can also comprise prediction of future clinical parameters, traditional laboratory risk factor values, or other indices of cADRs, such as cellular population determination in peripheral tissues, in serum or other fluid, either in absolute or relative terms in reference to a previously measured population. The methods of the present invention may be used to make continuous or categorical measurements of the risk of conversion to cADRs, thus diagnosing and defining the risk spectrum of a category of subjects defined as being at risk for a cADRs. In the categorical scenario, the invention can be used to discriminate between normal and other subject cohorts at higher risk for cADRs. In other embodiments, the present invention may be used so as to help to discriminate those having cADRs from normal.

In certain embodiments, the levels of miR nucleic acid of the invention are measured from a biological sample by using techniques commonly used in the state of the art. For examples, the whole DNA and RNA content comprised in the biological sample is often harvested and/or isolated in a first step. Harvest and isolation of total RNAs from a sample may be performed by known techniques disclosed in the art and reference may be made to standard DNA and RNA isolation protocols (e.g., Maniatis' Handbook of Molecular Biology.)

In some embodiments, the method does not require that miR nucleic acid be enriched from a standard RNA preparation.

In certain embodiments, miR nucleic acid can be enriched using, for example, an appropriate separating mean. miR nucleic acid levels of the invention may be detected using any appropriate assay known in the art. These assays include, but are not limited to, miRNA arrays (including the commercially available sources from AGILENT® and ILLUMINA®), reverse transcriptase polymerase chain reaction (RT- PCR), quantitative real-time reverse transcriptase PCR (qPCR) using TaqMan microRNA assays (e.g. from APPLIED BIOSYSTEMS®), in situ hybridization, Northern hybridization, hybridization protection assay (HP A) (GENPROBE®), branched DNA (bDNA) assay (CHIRON®), rolling circle amplification (RCA), Invader assay (ThirdWave Technologies), and/or Oligo Ligation Assay (OLA), hybridization, and the like.

In certain embodiments, measuring the levels of miR nucleic acid of the invention may be performed by amplifying all or part of miRNA nucleic acid sequences such as mature miRNAs, precursor miRNAs, and primary miRNAs.

In some embodiments, suitable nucleic acid polymerization and amplification methods are known from a skilled artisan and include, but are not limited to, reverse transcription (RT), polymerase chain reaction (PCR), real-time PCR (quantitative PCR (q- PCR)), nucleic acid sequence-base amplification (NASBA), ligase chain reaction, multiplex ligatable probe amplification, invader technology (Third Wave), rolling circle amplification, in vitro transcription (IVT), strand displacement amplification, transcription-mediated amplification (TMA), RNA (Eberwine) amplification, and the like. One or more amplification methods may be used, such as reverse transcription followed by real time PCR.

In some embodiments, suitable DNA and RNA sequencing techniques methods are known from a skilled artisan and include, but are not limited to, Illumina HiSeq 2500 and mapping of the 450 million 50bp single reads onto the mouse genome (GRCm38) using a splice junction aligner, Tophat v2.0.11.

II Method for Monitoring cADRs

As previously described the specific miRNA expressions of skin tissue samples obtained from cADRs patients is indicative of the status of those patients, (ie discriminate MPE and DRESS pathogenesis) but also monitoring a cutaneous adverse drug reactions (cADRs (moderately- or poorly-differentiated respectively)

Accordingly, another aspect of the present invention provides a method for monitoring a cutaneous adverse drug reactions (cADRs) comprising the steps of ia) determining the level of one or more miR nucleic acid selected from a first group of miR nucleic acid consisting of:, hsa-miR-1228-5p (SEQ ID N°l); hsa-miR-1294 (SEQ ID N°2); hsa-miR-548b-5p (SEQ ID N°3); hsa-miR-1267 (SEQ ID N°4); dme-miR-7-5p (SEQ ID N°5); hsa-miR-516b-3p (SEQ ID N°6); hsa-miR-1253 (SEQ ID N°7); hsa-miR-520d-5p (SEQ ID N°8); hsa-miR-548c-5p (SEQ ID N°9); hsa-miR-507 (SEQ ID N°ll); hsa-miR-601 (SEQ ID N°12); hsa-miR-1276 (SEQ ID N°13); hsa-miR-661 (SEQ ID N°14); hsa-miR-938 (SEQ ID N°15);hsa-miR-501-3p (SEQ ID N°16); hsa-miR-886-3p (SEQ ID N°17); hsa-miR- 503-5p (SEQ ID N°18); hsa-miR-519e-3p (SEQ ID N°19); hsa-miR-51 l-5p (SEQ ID N°20); hsa-miR-939-5p (SEQ ID N°22); hsa-miR-1254 (SEQ ID N°23); hsa-miR-223-3p (SEQ ID N°24); hsa-miR-1233-3p (SEQ ID N°27); hsa-miR-185-5p (SEQ ID N°30) in a skin sample obtained from the subject at a first specific time of the disease and/or ib) determining the level of one or more miR nucleic acid selected from a second group of miR nucleic acid consisting of: hsa-miR-411 5p (SEQ ID N°10), hsa-miR-489-3p (SEQ ID N°21); hsa-miR-18 la-2-3 p (SEQ ID N°25); hsa-let-7c-5p (SEQ ID N°26); hsa-miR- 27a-3p (SEQ ID N°28); miR-574-3p (SEQ ID N°29); hsa-miR-26a-5p (SEQ ID N°31); hsa- miR-27b-3p (SEQ ID N°32) ; hsa-miR-214;-3p (SEQ ID N°33); hsa-miR-26b-5p (SEQ ID N°34), in a skin sample obtained from the subject at a first specific time of the disease iia) determining the level of one or more miR nucleic acid selected from a first group of miR nucleic acid consisting of:, hsa-miR-1228-5p (SEQ ID N°l); hsa-miR-1294 (SEQ ID N°2); hsa-miR-548b-5p (SEQ ID N°3); hsa-miR-1267 (SEQ ID N°4); dme-miR-7-5p (SEQ ID N°5); hsa-miR-516b-3p (SEQ ID N°6); hsa-miR-1253 (SEQ ID N°7); hsa-miR-520d-5p (SEQ ID N°8); hsa-miR-548c-5p (SEQ ID N°9); hsa-miR-507 (SEQ ID N°ll); hsa-miR-601 (SEQ ID N°12); hsa-miR-1276 (SEQ ID N°13); hsa-miR-661 (SEQ ID N°14); hsa-miR-938 (SEQ ID N°15);hsa-miR-501-3p (SEQ ID N°16); hsa-miR-886-3p (SEQ ID N°17); hsa-miR- 503-5p (SEQ ID N°18); hsa-miR-519e-3p (SEQ ID N°19); hsa-miR-51 l-5p (SEQ ID N°20); hsa-miR-939-5p (SEQ ID N°22); hsa-miR-1254 (SEQ ID N°23); hsa-miR-223-3p (SEQ ID N°24); hsa-miR-1233-3p (SEQ ID N°27); hsa-miR-185-5p (SEQ ID N°30) in a skin sample obtained from the subject at a second specific time of the disease, and/or iib) determining the level of one or more miR nucleic acid selected from a second group of miR nucleic acid consisting of: hsa-miR-411 5p (SEQ ID N°10), hsa-miR-489-3p (SEQ ID N°21); hsa-miR-18 la-2-3 p (SEQ ID N°25); hsa-let-7c-5p (SEQ ID N°26); hsa-miR- 27a-3p (SEQ ID N°28); miR-574-3p (SEQ ID N°29); hsa-miR-26a-5p (SEQ ID N°31); hsa- miR-27b-3p (SEQ ID N°32); hsa-miR-214;-3p (SEQ ID N°33); hsa-miR-26b-5p (SEQ ID N°34), in a skin sample obtained from the subject at a second specific time of the disease iii) comparing the level determined at step ia) with the level determined at step iia) and/or comparing the level determined at step ib) with the level determined at step iib) iv) concluding that the disease has evolved in worse manner when the level determined at step iia) is higher than the level determined at step ia) and/or when the level determined at step iib) is lower than the level determined at step ib).

In some embodiments, the said method is performed in vitro or ex vivo.

In particular embodiments, cutaneous adverse drug reactions (cADRs) is selected from the group consisting of maculopapular exanthema (MPE) or Drug Reaction with Eosinophilia and Systemic Symptoms (DRESS).

In particular embodiments wherein the cutaneous adverse drug reactions (cADRs) is maculopapular exanthema (MPE) the method for monitoring cADRs of the invention comprising the steps of : i) determining the level of one or more miR nucleic acid selected from a group of miR nucleic acid consisting of:, hsa-miR-1267 (SEQ ID N°4); hsa-miR-516b-3p (SEQ ID N°6); hsa-miR-223-3p (SEQ ID N°24); hsa-miR-1233 -3 p (SEQ ID N°27) in a skin sample obtained from the subject at a first specific time of the disease, ii) determining the level of one or more miR nucleic acid selected from a group of miR nucleic acid consisting of:, hsa-miR-1267 (SEQ ID N°4); hsa-miR-516b-3p (SEQ ID N°6); hsa-miR-223-3p (SEQ ID N°24); hsa-miR-1233-3p (SEQ ID N°27) in a skin sample obtained from the subject at a second specific time of the disease, iii) comparing the level determined at step i) with the level determined at step ii) iv) concluding that the disease has evolved in worse manner when the level determined at step ii) is higher than the level determined at step i).

In yet another aspect, the present invention provides a method for monitoring if a treatment with a drug induce a cutaneous adverse drug reactions (cADRs) comprising the steps of : ia) determining the level of one or more miR nucleic acid selected from a first group of miR nucleic acid consisting of:, hsa-miR-1228-5p (SEQ ID N°l); hsa-miR-1294 (SEQ ID N°2); hsa-miR-548b-5p (SEQ ID N°3); hsa-miR-1267 (SEQ ID N°4); dme-miR-7-5p (SEQ ID N°5); hsa-miR-516b-3p (SEQ ID N°6); hsa-miR-1253 (SEQ ID N°7); hsa-miR-520d-5p (SEQ ID N°8); hsa-miR-548c-5p (SEQ ID N°9); hsa-miR-507 (SEQ ID N°ll); hsa-miR-601 (SEQ ID N°12); hsa-miR-1276 (SEQ ID N°13); hsa-miR-661 (SEQ ID N°14); hsa-miR-938 (SEQ ID N°15);hsa-miR-501-3p (SEQ ID N°16); hsa-miR-886-3p (SEQ ID N°17); hsa-miR- 503-5p (SEQ ID N°18); hsa-miR-519e-3p (SEQ ID N°19); hsa-miR-51 l-5p (SEQ ID N°20); hsa-miR-939-5p (SEQ ID N°22); hsa-miR-1254 (SEQ ID N°23); hsa-miR-223-3p (SEQ ID N°24); hsa-miR-1233-3p (SEQ ID N°27); hsa-miR-185-5p (SEQ ID N°30) in a skin sample obtained from the subject before the treatment, and/or ib) determining the level of one or more miR nucleic acid selected from a second group of miR nucleic acid consisting of: hsa-miR-411 5p (SEQ ID N°10), hsa-miR-489-3p (SEQ ID N°21); hsa-miR-18 la-2-3 p (SEQ ID N°25); hsa-let-7c-5p (SEQ ID N°26); hsa-miR- 27a-3p (SEQ ID N°28); miR-574-3p (SEQ ID N°29); hsa-miR-26a-5p (SEQ ID N°31); hsa- miR-27b-3p (SEQ ID N°32) ;hsa-miR-214;-3p (SEQ ID N°33); hsa-miR-26b-5p (SEQ ID N°34), in a skin sample obtained from the subject before the treatment iia) determining the level of one or more miR nucleic acid selected from a first group of miR nucleic acid consisting of: hsa-miR-1228-5p (SEQ ID N°l); hsa-miR-1294 (SEQ ID N°2); hsa-miR-548b-5p (SEQ ID N°3); hsa-miR-1267 (SEQ ID N°4); dme-miR-7-5p (SEQ ID N°5); hsa-miR-516b-3p (SEQ ID N°6); hsa-miR-1253 (SEQ ID N°7); hsa-miR-520d-5p (SEQ ID N°8); hsa-miR-548c-5p (SEQ ID N°9); hsa-miR-507 (SEQ ID N°ll); hsa-miR-601 (SEQ ID N°12); hsa-miR-1276 (SEQ ID N°13); hsa-miR-661 (SEQ ID N°14); hsa-miR-938 (SEQ ID N°15); hsa-miR-501-3p (SEQ ID N°16); hsa-miR-886-3p (SEQ ID N°17); hsa-miR- 503-5p (SEQ ID N°18); hsa-miR-519e-3p (SEQ ID N°19); hsa-miR-51 l-5p (SEQ ID N°20); hsa-miR-939-5p (SEQ ID N°22); hsa-miR-1254 (SEQ ID N°23); hsa-miR-223-3p (SEQ ID N°24); hsa-miR-1233-3p (SEQ ID N°27); hsa-miR-185-5p (SEQ ID N°30) in a skin sample obtained from the subject after the treatment, and/or iib) determining the level of one or more miR nucleic acid selected from a second group of miR nucleic acid consisting of: hsa-miR-411 5p (SEQ ID N°10), hsa-miR-489-3p (SEQ ID N°21); hsa-miR-18 la-2-3 p (SEQ ID N°25); hsa-let-7c-5p (SEQ ID N°26); hsa-miR- 27a-3p (SEQ ID N°28); miR-574-3p (SEQ ID N°29); hsa-miR-26a-5p (SEQ ID N°31); hsa- miR-27b-3p (SEQ ID N°32) ;hsa-miR-214;-3p (SEQ ID N°33); hsa-miR-26b-5p (SEQ ID N°34), in a skin sample obtained from the subject after the treatment, iii) comparing the level determined at step ia) with the level determined at step iia) and/or comparing the level determined at step ib) with the level determined at step iib) and iv) concluding that the treatment induce a cutaneous adverse drug reactions (cADRs) when the level determined at step iia) is higher than the level determined at step ia) and/or when the level determined at step iib) is lower than the level determined at step ib).

In some embodiments, the said method is performed in vitro or ex vivo.

In particular embodiments, cutaneous adverse drug reactions (cADRs) is selected from the group consisting of maculopapular exanthema (MPE) or Drug Reaction with Eosinophilia and Systemic Symptoms (DRESS).

In particular embodiments wherein the cutaneous adverse drug reactions (cADRs) is maculopapular exanthema (MPE) the method for monitoring if a treatment with a drug induce MPE comprising the steps of : i) determining the level of one or more miR nucleic acid selected from a group of miR nucleic acid consisting of:, hsa-miR-1267 (SEQ ID N°4); hsa-miR-516b-3p (SEQ ID N°6); hsa-miR-223-3p (SEQ ID N°24); hsa-miR-1233 -3 p (SEQ ID N°27) in a skin sample obtained from the subject before the treatment ii) determining the level of one or more miR nucleic acid selected from a group of miR nucleic acid consisting of:, hsa-miR-1267 (SEQ ID N°4); hsa-miR-516b-3p (SEQ ID N°6); hsa-miR-223-3p (SEQ ID N°24); hsa-miR-1233 -3 p (SEQ ID N°27) in a skin sample obtained from the subject after the treatment, iii) comparing the level determined at step i) with the level determined at step ii) iv) concluding that the treatment induce maculopapular exanthema (MPE) when the level determined at step ii) is higher than the level determined at step i). In preferred embodiments, a plurality of miRNAs biomarkers (miR nucleic acid biomarkers) is used in the method of monitoring a cADRs (or if a treatment with a drug induce a cADRs). In other words, the method of monitoring cADRs (if a treatment with a drug induce a cADRs) according to the invention may comprise steps of: detecting the skin sample the level of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33 or 34 biomarker present in the biological sample; and detecting any miRNAs biomarker.

In particular embodiments, the method of monitoring a cADRs (or if a treatment with a drug induce a cADRs) is performed using 34 different biomarkers including the hsa-miR- 1228-5p (SEQ ID N°l); hsa-miR-1294 (SEQ ID N°2); hsa-miR-548b-5p (SEQ ID N°3); hsa- miR-1267 (SEQ ID N°4); dme-miR-7-5p (SEQ ID N°5); hsa-miR-516b-3p (SEQ ID N°6); hsa-miR-1253 (SEQ ID N°7); hsa-miR-520d-5p (SEQ ID N°8); hsa-miR-548c-5p (SEQ ID N°9); hsa-miR-507 (SEQ ID N°ll); hsa-miR-601 (SEQ ID N°12); hsa-miR-1276 (SEQ ID N°13); hsa-miR-661 (SEQ ID N°14); hsa-miR-938 (SEQ ID N°15); hsa-miR-501-3p (SEQ ID N°16); hsa-miR-886-3p (SEQ ID N°17); hsa-miR-503-5p (SEQ ID N°18); hsa-miR-519e-3p (SEQ ID N°19); hsa-miR-51 l-5p (SEQ ID N°20); hsa-miR-939-5p (SEQ ID N°22); hsa-miR- 1254 (SEQ ID N°23); hsa-miR-223-3p (SEQ ID N°24); hsa-miR-1233-3p (SEQ ID N°27); hsa-miR-185-5p (SEQ ID N°30), hsa-miR-41 l-5p (SEQ ID N°10), hsa-miR-489-3p (SEQ ID N°21); hsa-miR- 181 a-2-3 p (SEQ ID N°25); hsa-let-7c-5p (SEQ ID N°26); hsa-miR-27a-3p (SEQ ID N°28); miR-574-3p (SEQ ID N°29); hsa-miR-26a-5p (SEQ ID N°31); hsa-miR-27b- 3p (SEQ ID N°32); hsa-miR-214;-3p (SEQ ID N°33); hsa-miR-26b-5p (SEQ ID N°34) as described herein.

In a particular embodiments, the method of monitoring a MPE (or if a treatment with a drug induce a MPE) is performed using the 7 different biomarkers including thehsa-miR- 181a-2-3p (SEQ ID N°25); hsa-miR-26b-5p (SEQ ID N°34); hsa-miR-214-3p (SEQ ID N°33); hsa-let-7c-5p (SEQ ID N°26) hsa-miR-520d-5p (SEQ ID N°8); hsa-miR-501-3p (SEQ ID N°16); hsa-miR-519e-3p (SEQ ID N°19) as described herein.

In a particular embodiments, the method of monitoring a MPE (or if a treatment with a drug induce a MPE) is performed using the 4 different biomarkers including the hsa-miR- 1267 (SEQ ID N°4); hsa-miR-516b-3p (SEQ ID N°6); hsa-miR-223-3p (SEQ ID N°24); and hsa-miR- 1233 -3 p (SEQ ID N°27 is determined at step i) and step ii).

III Methods of treatment Among the miRNA involved in cADRs identified by the inventors, Let-7c-5p and miR-519e-3p were described in the regulation of the expression of the ATP -binding cassette (ABC) transporter family associated with cell drug efflux. Indeed, inventors found that skin ABCB1 expression was decreased compared to controls during DRESS and MPE. Using a human skin explant model, they confirmed that ABCB1 transporter induction was dependent on Let-7c-5p and miR-519e-3p.

This two essential components of the switch observed in skin biopsy of cADRs patients, Let-7c-5p and miR-519e-3p, respectively regulate the expression of the ATP -binding cassette (ABC) transporter family associated with cell drug efflux, at the skin level. This alteration in the delicate balance between inductive and repressive signals induces alteration of the normal expression of ATP -binding cassette (ABC) transporter , and disruption in the balance between Let-7c-5p and miR-519e-3p expression during DRESS induce a decrease of ABCB1 expression in the skin during drug intake.

Accordingly in another aspect, the present invention provides a method for treating cutaneous adverse drug reactions (cADRs) in an individual comprising a step of administering to the said individual a compound selected in a group comprising a hsa-let-7c-5p nucleic acid (SEQ ID N°26), a compound mimicking a hsa-let-7c-5p nucleic acid (SEQ ID N°26), and/or comprising a step of administering to the said individual a compound consisting of a Block- hsa-miR-519e-3p nucleic acid

In a still further aspect, the invention relates to a compound selected in a group comprising a hsa-let-7c-5p nucleic acid (SEQ ID N°26), a compound mimicking a hsa-let-7c- 5p nucleic acid (SEQ ID N°26)and/or a compound a compound consisting of a Block-hsa- miR-519e-5p nucleic acid, for use in the prevention or the treatment of cutaneous adverse drug reactions (cADRs) in an individual.

In some embodiments, the individual is an individual in need of such a treatment for treating said cutaneous adverse drug reactions (cADRs).

In a particular embodiments, the individual in need of such a treatment for cutaneous adverse drug reactions (cADRs), was previously diagnosed according the method of the present invention.

Within the scope of the instant invention, the expression “a compound mimicking a miR-X nucleic acid” refers to the properties of said compound to exert the naturally occurring functions of said miR-X, namely bind to its site specific target nucleic acid and upregulate or downregulate the expression said target nucleic acid. In certain embodiments, the said individual has a low expression of hsa-let-7c nucleic acid or a high expression of hsa-miR-519e nucleic acid.

Within the scope of the instant invention, the expression “low expression” encompasses no detectable expression or expression below a reference value reflecting the average expression level measured in a biological sample from a healthy individual or a population of healthy individuals or sequence variants (e.g., mutation) with deficient biological activity.

In some embodiments, a low expression refers to expression levels which are at least 10% or more, for example, 20%, 30%, 40%, or 50%, 60%, 70%, 80%, 90% lower than a reference value.

In certain embodiments, a low expression refers to expression levels which are at least 5.0 fold, 4.0 fold, 3.0 fold, 2.0 fold, 1.8 fold, 1.6 fold, 1.4 fold, 1.2 fold lower than a reference value.

In certain embodiments, a low expression refers to variants in hsa-let-7c nucleic acid sequences (e.g., mutation) resulting in deficient biological activity.

In some embodiments, the therapeutic method may be of use in a mammal, preferably in a human.

Within the scope of the instant invention, the expression “high expression” encompasses expression above a reference value reflecting the average expression level measured in a biological sample from a healthy individual or a population of healthy individuals.

In some embodiments, a higher expression refers to expression levels which are at least 10% or more, for example, 20%, 30%, 40%, or 50%, 60%, 70%, 80%, 90% higher than a reference value.

In certain embodiments, a low expression refers to expression levels which are at least 1.2 fold, 1.4 fold, 1.6 fold, 1.8 fold, 2.0 fold, 3.0 fold, 4.0 fold, 5.0 fold higher than a reference value.

As further developed herein, the use of Block-miRs is also encompassed by the instant invention.

Within the scope of the instant invention, the term “Block-miR” refers to a nucleic acid compound that prevents other molecules from binding to a site specific region of a target nucleic acid, and more particularly prevent the binding of a given miRNA nucleic acid to its target nucleic acid. In certain embodiments, the compound preventing the binding of a hsa-miR-519e-3p nucleic acid to a target nucleic acid consists of a Block- hsa-miR-519e-3p nucleic acid.

In certain embodiments, the Block- hsa-miR-519e-3p nucleic acid is a nucleic acid having at least 22 consecutive nucleotides being 100% complementary to a target nucleic acid consisting to the sequence SEQ ID NO: 19.

Within the scope of the instant invention, the expression “at least 22 consecutive nucleotides” encompasses at least 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,

39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,

64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,

89, 90, 91, 92, 93, 94, 95, 96, 97, 98 and 99 consecutive nucleotides.

In certain embodiments, for the therapeutic purpose, a hsa-let-7c-5p nucleic acid, a compound mimicking a hsa-let-7c-5p nucleic acid, a Block- hsa-miR-519e-3p nucleic acid according to the invention may comprise naturally occurring or non-naturally occurring nucleic acid sequences.

In some embodiments, hsa-let-7c-5p nucleic acids encompass nucleic acids comprising the mature or precursor forms of hsa-let-7c-5p, which optionally further comprise additional flanking nucleotides 5' or 3' to the hsa-let-7c-5p.

Within the scope of the instant invention, the length of the said miRNA nucleic acids may vary provided that they still achieve binding to the target nucleic acid and exert its regulatory function (up-regulation or down-regulation).

In certain embodiments, the miRNA nucleic acids may range from about 23 to about 100 nucleotides in length, which encompasses, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,

36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85,

86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 and 99 nucleotides in length.

In certain embodiments, the said miRNA nucleic acids may consist in sequences having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% identities with respect to the mature or precursor hsa-let-7c-5p sequence.

In certain embodiments, a naturally occurring hsa-let-7c-5p sequence is RNA in nature and comprises a phosphodiester backbone.

In certain embodiments, non-naturally occurring hsa-let-7c-5p sequence may comprise RNA elements, such as naturally occurring ribonucleotides, and non- naturally occurring elements, such as non-naturally occurring ribonucleotides or other nucleotide-like residues or backbone linkages other than phosphodiester linkages including but not limited to phosphorothioate linkages.

In certain embodiments, naturally occurring or non-naturally occurring nucleic acids may be synthesized in vivo, in vitro or ex vivo. Whenever applicable, suitable vectors, such as plasmid or viral vectors may be employed following the acknowledged indications and protocols available in the state of the art.

The current main symptomatic treatment for cARDs is anti-inflammatory agents and potent topical glucocorticoids. In case of severe cADRs systemic steroids and immunosuppressive agent could be used.

The invention also relates to a method for treating cADRs with anti-inflammatory agents and /or glucocorticoids and /or systemic steroids and/or immunosuppressive therapy in a subject wherein the level of miRNA obtained from skin of said subject have been detected by one of diagnostic method of the invention.

Another object of the present invention is a method of treating cADRs in a subject comprising the steps of: i) providing a skin sample from a subject, iia) ia) determining in a skin sample obtained from said subject the level of one or more miR nucleic acid selected from a first group of miR nucleic acid consisting of: hsa-miR- 1228-5p (SEQ ID N°l); hsa-miR-1294 (SEQ ID N°2); hsa-miR-548b-5p (SEQ ID N°3); hsa- miR-1267 (SEQ ID N°4); dme-miR-7-5p (SEQ ID N°5); hsa-miR-516-3p516b-3p (SEQ ID N°6); hsa-miR-1253 (SEQ ID N°7); hsa-miR-520d-5p (SEQ ID N°8); hsa-miR-548c-5p (SEQ ID N°9); hsa-miR-507 (SEQ ID N°11); hsa-miR-601 (SEQ ID N°12); hsa-miR-1276 (SEQ ID N°13); hsa-miR-661 (SEQ ID N°14); hsa-miR-938 (SEQ ID N°15);hsa-miR-501-3p (SEQ ID N°16); hsa-miR-886-3p (SEQ ID N°17); hsa-miR-503-5p (SEQ ID N°18); hsa-miR-519e-3p (SEQ ID N°19); hsa-miR-51 l-5p (SEQ ID N°20); hsa-miR-939-5p (SEQ ID N°22); hsa-miR- 1254 (SEQ ID N°23);hsa-miR-223-3p (SEQ ID N°24); hsa-miR-1233-3p (SEQ ID N°27); hsa-miR-185-5p (SEQ ID N°30) and/or iib) determining in a skin sample obtained from said subject the level of one or more miR nucleic acid selected from a second group of miR nucleic acid consisting of: hsa-miR- 411 -5p (SEQ ID N°10), hsa-miR-489-3p (SEQ ID N°21); hsa-miR-18 la-2-3 p (SEQ ID N°25); hsa-let-7c-5p (SEQ ID N°26); hsa-miR-27a-3p (SEQ ID N°28); miR-574-3p (SEQ ID N°29); hsa-miR-26a-5p (SEQ ID N°31); hsa-miR-27b-3p (SEQ ID N°32); hsa-miR-214;-3p (SEQ ID N°33); hsa-miR-26b-5p (SEQ ID N°34), iii) comparing said level of one or more miR nucleic acid of group iia) to a control reference value and/or said level of one or more miR nucleic acid of group iib) to a control reference value wherein if level of one or more miR nucleic acid of group determined at step iia) is higher than the reference value and if level of one or more miR nucleic acid of group determined at step iib) is higher than the reference value, treating the subject with anti-inflammatory agents and /or glucocorticoids.

In the context of the invention, the term "treating" or "treatment", as used herein, means reversing, alleviating, inhibiting the progress of, or preventing the disorder or condition to which such term applies, or reversing, alleviating, inhibiting the progress of, or preventing one or more symptoms of the disorder or condition to which such term applies.

Example of common anti-inflammatory agents include, without limitation: a) Topical anti-inflammatory agents in the form of creams and sprays with the active ingredients usually belong to Corticosteroids classes such as betamethasone dipropionate, clobetasol propionate, or topical cream, see topical steroid b) Oral antihistamines can also be used: Antihistamines such as and Hydroxyzine c) : or systemic steroids and/or immunosuppressive therapy

• Pharmaceutical compositions

In certain embodiments, the compounds according to the instant invention may be used as an active agent, for therapeutic purposes, in the form of a pharmaceutic composition.

In one aspect, the present invention relates to a pharmaceutic composition comprising (1) a compound selected in a group comprising a hsa-let-7c-5p nucleic acid, a compound mimicking a hsa-let-7c-5p nucleic acid, and/or a a Block- hsa-miR-519e-3p and (2) a pharmaceutically acceptable carrier.

In certain embodiments, the pharmaceutic composition may further comprise one or more salts, one or more buffering agents, one or more preservatives, one or more secondary therapeutic agents.

Within the scope of the instant invention, a “pharmaceutically acceptable carrier” refers to a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting a prophylactically or therapeutically active agent.

In certain embodiments, a suitable pharmaceutically acceptable carrier may be selected in a group including sugars, such as lactose, glucose and sucrose; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; buffering agents, such as magnesium hydroxide and aluminum hydroxide; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solutions; and the like.

In some embodiments, a pharmaceutical composition according to the invention may be in any suitable form encompassed by the state in the art, e.g. in the form of an injectable solution or suspension, a tablet, a coated tablet, a capsule, a syrup, a suppository, a cream, an ointment, a lotion, and the like.

In some embodiments, an effective amount of said compound is administered to said individual in need thereof.

Within the scope of the instant invention, an “effective amount” refers to the amount of said compound that alone stimulates the desired outcome, i.e. alleviates or eradicates the symptoms of the cADRs disorder.

Within the scope of the instant invention, the effective amount of the compound to be administered may be determined by a physician or an authorized person skilled in the art and can be suitably adapted within the time course of the treatment.

In certain embodiments, the effective amount to be administered may depend upon a variety of parameters, including the material selected for administration, whether the administration is in single or multiple doses, and the individual’s parameters including age, physical condition, size, weight, and the severity of the disorder.

In certain embodiments, an effective amount of the active agent may comprise from about 0.001 mg to about 3000 mg, per dosage unit, preferably from about 0.05 mg to about 100 mg, per dosage unit.

Within the scope of the instant invention, from about 0.001 mg to about 3000 mg includes, from about 0.002 mg, 0.003 mg, 0.004 mg, 0.005 mg, 0.006 mg, 0.007 mg, 0.008 mg, 0.009 mg, 0.01 mg, 0.02 mg, 0.03 mg, 0.04 mg, 0.05 mg, 0.06 mg, 0.07 mg, 0.08 mg, 0.09 mg, 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 750 mg, 800 mg, 850 mg, 900 mg, 950 mg, 1000 mg, 1100 mg, 1150 mg, 1200 mg, 1250 mg, 1300 mg, 1350 mg, 1400 mg, 1450 mg, 1500 mg, 1550 mg, 1600 mg, 1650 mg, 1700 mg, 1750 mg, 1800 mg, 1850 mg, 1900 mg, 1950 mg, 2000 mg, 2100 mg, 2150 mg, 2200 mg, 2250 mg, 2300 mg, 2350 mg, 2400 mg, 2450 mg, 2500 mg, 2550 mg, 2600 mg, 2650 mg, 2700 mg, 2750 mg, 2800 mg, 2850 mg, 2900 mg and 2950 mg, per dosage unit.

In certain embodiments, the active agent may be at dosage levels sufficient to deliver from about 0.001 mg/kg to about 100 mg/kg, from about 0.01 mg/kg to about 50 mg/kg, preferably from about 0.1 mg/kg to about 40 mg/kg, preferably from about 0.5 mg/kg to about 30 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, and more preferably from about 1 mg/kg to about 25 mg/kg, of subject body weight per day.

In certain embodiments, each dosage unit may be administered three times a day, two times a day, once a day, every other day, every third day, every week, every two weeks, every three weeks, or every four weeks.

In certain embodiments, the therapeutic treatment encompasses an administration of a plurality of dosage units, including two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more administrations.

In some embodiments, the active agent, e.g. in the form of a pharmaceutic composition may be administered by any suitable route, including enteral (e.g. , oral), parenteral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, subcutaneous, intraventricular, transdermal, intradermal, rectal, intravaginal, intraperitoneal, topical, mucosal, nasal, buccal, sublingual; by intratracheal instillation, bronchial instillation, and/or inhalation; and/or as an oral spray, nasal spray, and/or aerosol.

In preferred embodiments, the active agent is administered by topical administration, transdermal, intradermal administration.

The invention will be further illustrated by the following example. However, this example should not be interpreted in any way as limiting the scope of the present invention.

FIGURES:

Figure 1: Skin miRNAs expression levels in DRESS, MPE and healthy controls

A: Principal component analysis (PCA) of DRESS, MPE and healthy (HC) controls based on total skin miRNAs expression levels. The first two components correctly separate healthy controls (green circle) from MPE (E1-E6) and DRESS (D1-D6) patients B: PCA of DRESS, MPE and HC based on the 34 skin miRNAs differentially expressed in DRESS and MPE compared with HC. The first two components globally correctly separate DRESS (circle) and MPE patients (square). C: Skin miRNAs with biological functions possibly involved in the pathogenesis of DRESS and MPE based on literature review from www.mirbase.org and MEDLINE/Pubmed with Venn diagram representation.

Figure 2: Skin miRNAs differentially expressed between DRESS and MPE

A: PCA of DRESS and MPE based on the 7 skin miRNAs significantly differentially expressed between DRESS (D1-D6) and MPE (E1-E6). The first two components correctly separate DRESS (circle) and MPE patients (square). B: Skin miRNAs expression level between MPE (E1-E6) and DRESS (D1-D6), patients based on fold change (FC=2 ^{( AACT)} ) regarding miRNAs involved in keratinocyte differentiation/skin inflammation, T cell polarization, Type I interferon pathway/viral replication and ABC transporter. Mann- Whitney test, *: p<0.05, **: p<0.01. C: Skin miRNAs expression level of the 4 miRNAs: miR-181a-2- 3p, -520d-5p, -519e-3p, and Let-7c-5p, in a second cohort of MPE (E7-9) and DRESS (D7- D10) patients, based on fold change (FC=2 ^{( AACT)} ) using TaqMan® MicroRNA Reverse Transcription method. No significant difference had been showed in skin miRNAs expression in the second cohort of patients.

Figure 3: Skin miRNAs as potential markers of systemic involvement during DRESS and MPE

A: Skin miR-501-3p expression level between patients with (Eo+) or without (Eo-) eosinophilia defined by absolute eosinophils count > 700/mm3. Mann- Whitney test, **: p<0.01. B: Receiver Operative Characteristic (ROC) curve of skin miR-501-3p expression levels among patients with or without eosinophilia. C: Skin miR-520d-5p expression level between patients (Liver+) or without (Liver-) liver abnormalities defined by ALT (alanine aminotransferase) and/or AST (aspartate aminotransferase) levels higher than 2 upper limit normal values. Mann- Whitney test **: p<0.01. D: Receiver Operative Characteristic (ROC) curve of skin miR-520d-5p expression levels among patients with or without liver abnormalities. E: Skin Let-7c-5p expression level with (Eo+) or without eosinophilia (Eo-) defined by absolute eosinophils count > 700/mm ³ presence in MPE (E1-E6) and DRESS (Dl- D6) patients based on fold change (FC=2 ^{( AACT)} ).

Figure 4: Skin ABCB1 transporter differentially expressed between DRESS, MPE, and healthy controls

A: Quantitative PCR analysis of ABCBl expression level in lesional skin of MPE (E7- E15) and DRESS (D7-D9) patients compared to healthy control (HC) based on fold change (FC=2 ^{( AACT)} ) using TaqMan® RNA Reverse Transcription method. B: Quantitative PCR analysis of ABCB1 expression level after amoxicillin (AX) 10 and 50 mg/mL 24 hours at 37°C in 2 and 4 human epidermal sheets respectively, based on fold change between skin with and without AX (FC=2 ^{( AACT)} ) using TaqMan® RNA Reverse Transcription method. C: Quantitative PCR analysis of Let-7c-5p and miR-519e-3p expression level after amoxicillin (AX) 10 and 50 mg/mL 24 hours at 37°C in human epidermal sheets, based on fold change between skin with and without AX (FC=2 ^{( AACT)} ) using TaqMan® RNA Reverse Transcription method. D: Schematic representation of the Let-7c-5p and miR-519e3-p expression levels and ABCB1 expression with drug in normal condition in the skin during and during DRESS MPE.

EXAMPLE

Material and Methods:

Skin and blood samples:

Skin samples were obtained from the PEAUTOX collection approved by our local Ethics Committee (CPP- He de France VI and Agence Nationale de Securite du Medicament et des Produits de Sante: N° ID RCB: 2016-A01468-43), a biobank including consecutive patients with skin adverse drug reactions seen in the 4 French dermatological university departments of Assistance Publique-Hopitaux de Paris. In all cases, blood samples were collected and stored in PAXgene™ Blood RNA Tubes (Qiagen ^® ). Clinical characteristics were reported Table 2. Skin explants from healthy controls were collected from plastic surgery..

ABCB1 transporter induction human skin model:

Fresh skin samples were prepared as previously described in order to separate epidermal sheets from the dermis ²⁵ .

Real-time qPCR of skin miRNA: TaqMan Low Density Arrays (TLDA) using TaqMan® MicroRNA Reverse Transcription Kit and the Megaplex™ RT Primers (Applied Biosystems) were used to synthesize single-stranded cDNA from pool A and B allowing quantification of 754 human microRNAs as previously described ²⁶ , for details see Supplemental document 1. The expression levels of 123 miRNAs (32%) were below the detection limit in the skin biopsies. Eighteen (4.7 %) miRNAs from pool A and 105 (27.3 %) miRNAs from pool B with at least 6 samples displaying Ct values above 40 were considered not expressed and were excluded from further analyses.

For the validation cohort, TaqMan® MicroRNA Reverse Transcription Kit (Applied Biosystems) was used with Primers (Let-7c-5p, miRNA- 18 la-2-3 p, -520d-5p and -519e-3p),. Skin miRNA statistical analysis: Using the vooma-limma workflow, the mean-variance relationship of the qPCR data was estimated and linear regression models were performed to assess differences between MPE, DRESS patients, and healthy controls. The empirical Bayes method was used to compute moderated p-values. E-values were then corrected for multiple comparisons using the Benjamini and Hochberg’s false discovery rate (FDR) controlling procedure.

Total blood RNA extraction, filtering, normalization and quality:

Briefly after extraction and purification of total RNA, including miRNA, from whole blood collected into PAXgene blood RNA tubes, (Paxgene Blood miRNA kit, Preanalytix, Qiagen) we applied HTG EdgeSeq technology (HTG Molecular Diagnostics, Inc., Tucson, AZ), a new next-generation sequencing-based miRNA profiling platform to determine the concentration of 2,083 miRNAs ^27,28 .

Total blood miRNA statistical analysis:

Differences between MPE (n=5), DRESS (n=5), and healthy control (n=5) were estimated using the voomWithQualityWeights-limma workflow. To correct for a hidden source of variability an additional parameter estimated by weighted surrogate variable analysis was included in the statistical model. The empirical Bayes method was used to compute moderated p-values. E-values were then corrected for multiple comparisons using the Benjamini and Hochberg’s false discovery rate (FDR) controlling procedure.

Immunostaining of skin ABCB1:

Skin samples were directly frozen in OCT at -80°C (Tissue-Tek, Sakura, Belgium) before cryosectionned (5 pm sections) with a Microm HM550 cryostat, as previously described ²⁹ .

Real-time qPCR of skin ABCB1:

Skin biopsies were frozen in liquid nitrogen and then reduced to powder. Total RNA was extracted using RNAble® (Eurobio Life Science) according to the manufacturer’s instructions. Skin RNA concentration was determined using NanoDrop™ spectrophotometers (Thermo Fischer Scientific) and RNA quality was assessed using bio analyzer RNA chips (Agilent),.

Statistical analysis for miRNA:

Principal Component Analyses (PCA) of sample expression levels were performed with miRNA signals centered but not scaled. A Mann- Whitney test was used to compare non- paired non-normally distributed variables and the Spearman test was used for correlations. Receiver operating characteristic (ROC) curves were calculated to assess Sensitivity and Specificity of potential biomarkers of systemic involvement among cADRs patients. Statistical significance was indicated: *, p< 0.05 **; p< 0.01; ***, p<0.001. Normalizations were performed using the ‘R’ software and other analyses were performed using JMP 13 Stastistical Discovery™ (SAS Institute).

Results

The expression levels of thirty-four skin miRNAs distinguish DRESS and MPE patients from healthy controls.

To identify miRNAs specifically expressed in DRESS and MPE patients, we performed microarray analysis of skin samples collected from 6 patients with MPE, 6 patients with DRESS and 6 healthy matched donors using TaqMan Low Density Arrays (TLDA). PCA of the 631 detectable miRNAs showed that the total skin miRNA profile clearly separated healthy controls (HC) from patients with MPE and DRESS (Figure 1A). MPE and DRESS samples were however not distinguishable using 631 miRNA expression.

We identified 34 miRNAs that were differentially expressed between DRESS patients and HC, with a fold-change < 2 or > 2 and adjusted p-value < 0.05 (data not shown). We next assessed whether the 34-skin miRNAs differentially expressed in DRESS patients were able to accurately classify between patients with MPE, DRESS and HC. the 3 types of samples were classified in two main clusters. We thus used the 34 miRNA-based signatures to perform a new PCA and showed that MPE and DRESS patients were correctly separated (Figure IB). Among them, 24 were over-expressed and 10 under-expressed (Table 2). Conversely, 4 miRNAs (miR-1267, -516b-3p, -1233-3p and -223-3p) included in these 34 miRNAs were significantly over-expressed between MPE patients and HC (Table 3 and Table 1 for target sequence). All miRNAs significantly expressed in MPE compared to HC were also over expressed in DRESS patients. Using non-supervised hierarchical clustering analysis, two MPE patients, E2 and E4, with Herpes viral replication (Table 2), had skin miRNA expression profile very similar to DRESS.

We then searched for biological functions of these miRNAs differentially expressed between MPE, DRESS and HC. Using miRBase (www.mirbase.org) and Pubmed/MEDLINE we searched for putative target genes with published biological functions that may be related to the pathogenesis of DRESS and MPE. The list of additional references is available in the Supplemental document 2. As shown in Figure 1C, we identified 18 miRNAs involved in mechanisms associated with MPE and DRESS pathogenesis as well as ABC transporters (i.e; differentiation of keratinocytes, skin inflammation, T lymphocyte polarization, ABC transporters, type I interferon pathway and/or viral replication). These data suggest that skin miRNAs could discriminate between MPE and DRESS patients.

Seven skin miRNAs distinguish between DRESS and MPE patients.

Among the 34 miRNAs differentially expressed between DRESS patients and healthy controls, we searched for miRNAs differentially expressed with a fold-change < 2 or >2 and adjusted p-value < 0.05, between DRESS and MPE patients. The hierarchical clustering analysis (data not shown) and the PCA (Figure 2A and Table 2) showed that 7 miRNAs correctly classified DRESS and MPE patients. MiR-18 la-2-3 p, miR-26b-5p, miR-214-3p and Let-7c-5p were significantly over-expressed in MPE compared to DRESS patients. Conversely, miR-520d-5p, miR-501-3p and miR-519e-3p were significantly under expressed in MPE compared to DRESS patients (Figure 2B). Using skin biopsies from patients, we observed that 4 out of the seven miRNAs (miR-18 la-2-3 p, miR-520d-5p, miR-519e-3p and Let-7c-5p) were differentially expressed with p-value<0.01 (**) in the skin of MPE compared to DRESS patients. These 7 skin miRNAs that are differentially expressed between DRESS and MPE patients are implicated in biological functions relevant to diseases pathogenesis (Figure 1C).

We further validated the results from this pilot study using biopsies from additional MPE (E7-9) and DRESS (D7-D10) patients and HC. We confirmed 4 miRNAs differentially expressed with p-value <0.01 in lesional skin of MPE and DRESS patients (miR-18 la-2-3 p, miR-520d-5p, Let-7c-5p and miR-519e-3p). As in the first study, skin miR-181a-2-3p and Let-7c-5p were under expressed in lesional skin of DRESS compared to MPE patients whereas miR-520d-5p and -519e-3p were overexpressed in lesional skin of DRESS compared to MPE patients (Figure 2C). However, due to rarity of patient samples, the differences in expression levels of these four miRNAs between DRESS and MPE patients did not reach statistical significance. But this is the first time that skin miRNA screening has been done in DRESS end MPE patients.

MiR-501-3p and -520d-5p represent potential markers of systemic involvement.

As DRESS but also MPE patients may have systemic involvement including eosinophilia, liver abnormalities or renal failure but usually with milder symptoms, we aimed to identify miRNAs associated with systemic involvement among the 7 miRNAs differentially expressed in MPE and DRESS patients compared to healthy controls.

The expression levels of miR-501-3p were however significantly different among patients with or without eosinophilia (Figure 3A). Using the Receiver Operating Characteristic (ROC) analysis, we found that a fold change of miR-501-3p higher than 11.23 was associated with the presence of eosinophilia with a sensitivity of 100% and a specificity of 87.5% (Figure 3B). Moreover, in the skin, the expression level of miR-520d-5p was significantly correlated with liver abnormalities (Figure 3C). Using the ROC analysis, a fold change of miR-520d-5p higher than 32 was associated with the presence of liver abnormalities with a sensitivity of 100% and a specificity of 87.5% (Figure 3D). When comparing miRNAs expression levels with clinical features of patient skin, Let-7c-5p was under expressed in skin presenting a peripherical eosinophilia compared to skin without eosinophilia, based on fold change (FC=2 ^{( AACT)} ) (Figure 3E). Only one DRESS patient had no peripherical eosinophilia (D3). Among them, Let-7c-5p showed a moderate negative correlation (r=-0.66, p=0.02) (data not shown). Finally, no miRNA was found significantly correlated with renal failure (data not shown).

Skin mRNA ABCB1 transporter expression is reduced in DRESS and MPE patients

We have validated in DRESS and MPE patients Let-7c-5p and miR-519e-3p associated with ABC transporters. The role of ABC transporters in the pathogenesis of both MPE and DRESS has not yet been reported. We hypothesized that ABC transporters could be implicated in DRESS and MPE pathogenesis.

ABCBl transporters had already been reported as expressed in steady-state human skin especially in dermis layers ^30,3 f As we found that skin miRNAs involved in the ABC transporter pathway were differentially expressed between MPE, DRESS patients and HC, we performed an in silico analysis to identify putative target genes.

We then aimed to investigate the presence of the ABCBl transporter in skin biopsies of patients and HC. In the lesional skin of DRESS and MPE patients, we found a decreased expression of ABCBl transporters compared to controls, without significant differences between DRESS and MPE patients (Figure 4A). We then used immunofluorescent staining of the skin dermis to characterize ABCBl expression at the protein level. Skin lesions of MPE patients revealed a reduced expression of ABCBl in the dermal layers compared to HC.

To investigate the link between ABCBl and miRNAs abnormally expressed in MPE skin after drug intake, we developed an in vitro human skin model mimicking the induction of ABCBl transporter expression. As previously described, we exposed human epidermal sheets to increasing concentrations of amoxicillin (AX) ²⁵ , a common inducer of DRESS syndrome as already reported ³² . As expected, we observed a dose-dependent increase of ABCBl mRNA expression in human epidermal sheets after 24 hours of AX treatment (Figure 4B). Using the ABCBl induction human skin model, we investigated skin expression levels of Let- 7c-5p and miR-519e-3p, both implicated in the regulation of ABC transporters. After incubation with the ABCB1 inducer molecule AX, we observed a down-regulation of Let-7c- 5p expression only with the low dose (FC = 0.5). Let-7c-5p expression was increased with high doses of AX, and miR-519e-3p was over-expressed with both doses of AX used (Figure 4C). Interestingly, we observed an under expression of Let-7c-5p and an over expression of miR-519e-3p in skin during DRESS and MPE compared to healthy controls (Figure 2C). We thus suggest that the modulation of the Let-7c-5p and miR-519e3-p expression levels during DRESS and MPE may contribute to a decrease in ABCBl expression in the skin, responsible for drug accumulation in cells, and inducing pathogenesis as schematically represented in Figure 4D.

These data suggested that, during MPE and DRESS, regulatory mechanisms of ABCBl expression in the skin might be deficient, inducing a decreased ABCBl expression in the skin and resulting in drug accumulation within cells.

Table 2. Clinical and biological characteristics of included cutaneous adverse drug reactions patients.

Patients Diagnosis Sex/age Time from onset of skin Systemic Viral RegiSCAR Culprit drug

M: Male, F: Female, DRESS: Drug reaction with eosinophilia and systemic symptoms, MPE: maculopapular exanthema, +SS: systemic symptoms; F: fever, RF: renal failure, LF: liver failure, Eo: eosinophilia, Eln: enlarged lymph nodes, P: pancreatitis, DF: digestive failure,

EBV: Epstein barr virus, CMV: cytomegalovirus, HSV: herpes simplex virus, HHV6: human herpesvirus 6, NA: not available, $ : death attributable to the DRESS (multivisceral failure),

$$ : hypovolemic shock due to DRESS, * with Polymerase Chain Reaction, ** according to Kardaun et al. ⁹ Table 3. Skin miRNAs differentially expressed between MPE and DRESS patients compared with healthy controls (HC) miRNAs Folds change Adjusted p- Folds change Adjusted p- vs HC) value (MPE vs HC) value hsa-mi 0 lisa-mi R- 1204 \ 180 11.1 hsa-ini R-548b-5p lisa-mi K- 12o7 \ 140 x 200 ool I hsa-ini R-50.1 \ 1.1 1.38 hsa-ini R-51 o _L ^« 14.3 lisa-mi R-5 I I \ 8.0 028 hsa-ini R-480 / 7,8 14.3 hsa-ini R-O.lo \ 0.5 14.3 lisa-in i R- 1254 x 0.2 14.3 lisa-mi R-223 0.2 1053 hsa-iniR- 181 0.2 122 hsa-lel-7c 4.0 14.3 hsa-iniR-l 2.1 4.0 14.3 hsa-iniR-27a 4.8 miR-574-.lp 4.0 hsa-ini R- 18 4.4 14.3 hsa-iniR-26a .1.0 14.3 hsa-ini R-27h 1.8 hsa-iniR-214 1.2 hsa-miR-26b / 3 0.043

34 skin miRNAs were differentially expressed between DRESS patients and healthy control (HC), with fold change <2 or >2 (p-value<0.05, Mann- Whitney test); 24 of them were over expressed (x) and 10 under expressed (/). Four skin miRNAs were overexpressed (x) between MPE patients and HC, with fold change <2 or >2 (p-value<0.05, Mann- Whitney test). Discussion:

In the present study, we assessed miRNAs expression of MPE or DRESS patients in lesional skin using real-time qPCR. In skin lesions, 4 miRNAs in MPE patients and 34 miRNAs in DRESS patients were found to be differentially expressed compared to healthy controls. The 4 miRNAs overexpressed in MPE were also overexpressed in DRESS.

Most of MPE patients included in our study exhibited systemic symptoms and a continuum spectrum between MPE and DRESS has already been reported ^{11 12} . We therefore tried to identify miRNAs associated with severity of systemic involvement that could further discriminate between MPE and DRESS patients. Interestingly we found several miRNAs correlated with systemic involvement; eosinophilia and liver abnormalities. Indeed, Let-7c-5p was negatively correlated with eosinophilia. Whereas, miR-501-3p and miR-520d-5p were positively correlated respectively with eosinophilia and with liver abnormalities. A significant decrease in expression of Let-7c-5p has recently been reported in skin lesions of acute graft- versus host disease compared with healthy controls ¹⁷ . In DRESS, only serum biomarkers have already been reported as associated with the severity of the disease, including serum thymus and activation-regulated chemokine (TARC) levels ³³ , CD8 ⁺ T lymphocyte repertoire spreading ³⁴ and HHV6 replication levels ³⁵ . A significant positive correlation between the intensity of lymphocyte infiltration in skin biopsies and the severity of hepatic cytolysis and eosinophilia in DRESS patients has been suggested ³⁶ but no classification markers between MPE and DRESS were reported so far.

Using unsupervised hierarchical clustering analyses, we identified 7 miRNAs capable of discriminating accurately MPE and DRESS patients. These miRNAs are known to be involved in some biological functions correlated with the pathogenesis of cADRs. Indeed, we identified miRNAs including miR-18 la-2-3 p, miR-26b-5p and miR-520d-5p differentially expressed between MPE and DRESS involved in keratinocyte differentiation and skin inflammation. Pro-inflammatory cytokine such as TNF-a and cytotoxic proteins such as serum FasL leading to skin inflammation were found overexpressed in cutaneous lesions of DRESS compared to MPE ³⁷ . Moreover, differences in T cell polarization may partly explain differences observed between DRESS and MPE. Indeed, FoxP3+ regulatory T cells were significantly higher in skin lesions of DRESS compared to MPE in the acute stage of disease ³⁸ with a shift to Thl7 cells during the clinical course of DRESS ³⁹ . Interestingly, miR- 181a and miR-214 were described to be linked with Thl7 polarization ⁴⁰ ’ ⁴¹ . Furthermore, miR-501-3p and Let-7c are involved in type I Interferon pathway and viral replication ⁴² ’ ⁴³ and differences in term of viral replications between DRESS and MPE have been reported ⁴⁴ . Total blood miRNA wasn’t differentially expressed between DRESS, MPE, and healthy controls in this study.

Let-7c and miR-519 ^{45 46} are implicated in the regulation of the ATP -binding cassette (ABC) transporter family. Multiple isoforms of ABC transporters are expressed in human skin ³⁰ ’ ³¹ , and their expression level may be modified by drugs or inflammation. Interestingly, we found that Let-7c-5p and miR-519e-3p were respectively under and over-expressed in lesional skin during DRESS compared to healthy control. Moreover, we showed using a qPCR analysis, a decrease in the expression of ABCB1 transporters in lesional skin isolated from DRESS and MPE patients compared to healthy control. The ABC drug transporter ABCB1 (also known as P-glycoprotein or MDRl) is one of the most-studied mammalian transporters ^47_49 . In skin, ABCB1 is mainly localized in the dermal layer, it is implicated in the efflux of drugs, metabolic products, lipids and sterols, other xenobiotics and especially drugs implicated in MPE and DRESS pathogenesis (i.e; antiepileptics, antibiotics, antidepressants...) ⁴⁹ . Some studies have identified the ABC transporter pathways as potentially implicated in the genetic susceptibility of cADRs ⁵⁰ . ABCB1 polymorphism has been associated with the occurrence of cADRs in patients treated with erlotinib ⁵¹ , and carbamazepine-induced TEN ⁵² . The role of ABC transporters in the pathogenesis of both MPE and DRESS has not yet been reported.

The regulation of ABCBl transporters seems to be dependent of several miRNAs, as already reported in different tissues or cells lineages; among them Let-7c and miR-519 have been already described as capable of inhibiting its expression ⁴⁵ ’ ⁵³ . A decrease of skin ABCBl expression may thus lead to the accumulation of drugs in keratinocytes and could be involved in cADRs pathogenesis. Using Target Scan (Released.2, March 2018) we identified putative miRNA binding sites on ABCBl mRNAs for miR-1253 ⁵⁴ at the 3 ’-untranslated region (3 - UTR) and for miR-27a-3p both at the 3'-UTR ⁵⁵ and indirect modulation expression ⁵⁶ . However, there was no potential binding for Let-7c-5p, miR-503-5p, and miR-519e-3p, thus suggesting an indirect regulation.

Here, we propose that a disruption in the balance between Let-7c-5p and miR-519e-3p expression during DRESS could induce a decrease of ABCBl expression in the skin during drug intake. In addition, ABC transporters not only have a protective function related to their capacity to extrude toxic compounds but also display additional regulatory functions regarding the immune system ^{57 58} particularly antigen-presenting cells functions ⁵⁹ . In addition, we determined whether miRNAs from the blood might represent potential biomarkers for clinical use. Among 2083 miRNAs screened, 995 (47.4 %) were not expressed and were excluded from further analyses. PCA showed that blood miRNA expression patterns were not capable of discriminating between the three types of samples. Only miR-185-3p was significantly higher in MPE blood compared to HC (FC xl.5, adjusted p-value 0.03), and none of the miRNAs were differentially expressed in the blood of DRESS compared to HC. Overall, these results suggest that blood is not an optimal material for miRNA-based biomarkers in MPE and DRESS.

Although our study is limited by the small sample size, to our knowledge, it is the first to address the differential expression of miRNAs in skin lesions of DRESS and MPE in a well-characterized cohort of patients. Our data showed that Let-7c-5p and miRNA-519e-3p might be implicated in the regulation of ABC transporters and deregulated during DRESS and MPE. MiR-501-3p and miR-520d-5p skin expression represent potential markers of systemic involvement (eosinophilia and liver abnormalities) in the acute phases of DRESS and MPE. Overall, we identified several skin miRNA candidates with potential applications as biomarkers that also shed light on DRESS and MPE pathogenesis.

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Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

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