EP2160606A2

EP2160606A2 - Tace inhibitors in the treatment of acne

Info

Publication number: EP2160606A2
Application number: EP08806020A
Authority: EP
Inventors: Jérôme AUBERT; Isabelle Carlavan; Johannes Voegel
Original assignee: Galderma Research and Development SNC
Current assignee: Galderma Research and Development SNC
Priority date: 2007-06-18
Filing date: 2008-06-18
Publication date: 2010-03-10
Also published as: FR2917427A1; CA2690593A1; WO2009004247A3; FR2917427B1; JP2014051500A; US20100168089A1; JP2010532658A; WO2009004247A2

Abstract

The invention relates to an in vitro method of screening for candidate compounds for the preventive or curative treatment of acne, comprising the determination of the ability of a compound to inhibit the expression or the activity of TACE, and also the use of inhibitors of the expression or the activity of this enzyme for treating acne.

Description

TACE inhibitors in the treatment of acne

The invention relates to the identification and use of modulating compounds of the enzyme TACE for the treatment of acne.

Acne is a skin condition that results from the occlusion of the upper extremity as well as the internal part of the pilosebaceous canal by the abnormal multiplication of keratinocytes, and the androgenic hyper-hormonal activity often appearing during puberty. , which causes a significant increase in seborrhea in the sebaceous glands. The obstruction of the pilosebaceous canal causes the formation of comedones or microcysts, accompanied by a proliferation of Propionibacterium acnes bacteria as well as Pytirosporum ovale in the obstructed pilosebaceous follicles. This condition, particularly common in adolescents, is accompanied by an inflammatory reaction of the skin, which may be in the form of papules or pustules usually located in the superficial dermis. In some cases the inflammatory reaction can reach the deep dermis, forming nodules and macrocysts.

Traditional treatments for acne use benzoyl peroxide, erythromycin, isotretinoin and various antiseptics. However, the use of antibiotics such as erythromycin today faces the phenomena of bacterial resistance and changes in flora.

The applicant now proposes to use inhibitors of the enzyme TACE, to prevent and / or improve acne phenomena. Preferably, the TACE inhibitor is used alone, that is to say without other anti-acne active ingredient, or even without any other active principle.

Acne to be treated includes all forms of acne, namely in particular vulgar acne, comedon, polymorphic, nodulocystic acne, conglobata, or secondary acne such as solar acne, drug or professional.

TACE

The enzyme TACE, for "TNF-alpha converting enzyme", is responsible for the production of TNF-alpha. This enzyme is a member of the ADAM family of metalloproteases (Becherer et al, Handb Exp Pharmacol, 2000, 140: 235-258). It quickly converts proTNFα, a 26kDa precursor protein, into protein

TNFα mature 17kDa. Inhibition of TACE (or ADAM 17) leads to inhibition of TNFα synthesis (Nelson et al., Exp Opin. Invest Drugs, 1999, 8: 383-392). In addition, TACE inhibitors have been shown to suppress TNF production and inflammatory response in model animals with collagen-induced arthritis (Newton et al., Ann Rheum, Dis, 2001, 60: iii25-iϋ32). ). It has also been shown that P. acnes stimulates the release of TNFα by keratinocytes and macrophages (Gro et al., Br J Dermatol 2004, 150 (3): 421-428). Overexpression of TNFα has also been observed in acne lesions (Kang et al., Am J Pathol 2005, 166 (6): 1691-9). The transcriptome analysis of an acne lesion at an inflammatory stage allowed the identification of a large panel of genes preferentially expressed in the lesion

(Trivedi et al, JID.2006, May 126 (5): 1071-9.). Without wishing to be bound to a particular mechanism of action, the inventors hypothesize that TACE inhibitors have an impact on the inflammatory phenomena observed in acne, in particular via the modulation by TNFα of several genes involved in the inflammation. Among the markers of inflammatory acne lesion and reported to be induced in response to TNFα exposure are the genes involved in the following "stress response":

SGK (Li X et al., J Biol Chem 2002 11 22777 (47): 45129-40);

SOD2: (Horrevoets et al., Blood 1999 May 15, 93 (10): 3418-31); the gene involved in the following angiogenesis: - ECGF1 (Zhu et al., Oncogene 2002 Dec 5; 21 (55): 8477-85), the genes involved in the following cell migration and remodeling: -MMP1, MMP3 (Reunanen and J Biol Chem 2002 Aug 30, 277 (35): 32360-8),

T1-A-2 (Banno et al., J Biol Chem 2004, Jul 30, 279 (31): 32633-42, Epub 2004 May 15); the genes involved in the following immune response: DEFB4 (Seo SJ et al., J Dermatol Sci 2001 Nov; 27 (3): 183-91),

IL1 F9 (Kumar et al J Biol Chem 2000 Apr 7; 275 (14): 10308-14),

CXCL2 (Banno et al, J Biol Chem 2004, Jul 30, 279 (31): 32633-42, Siwkowski et al, Mol Pharmacol 2004 Sep 01, 66 (3): 572-9),

IL-8: (Brasier et al., J Biol Chem 1998 Feb 6, 273 (6): 3551-61, Terui et al., Blood 1998 Oct. 15; 92 (8): 2672-80),

CCR1 (Li et al., J Biol Chem 2002, 227: 277 (47): 45129-40, Sozzani et al., J. Immunol 1998 Aug. 1, 161 (3): 1083-6),

CD14 (Enomoto et al., J. Pharmacol Exp Ther 2003 Sep 01; 306 (3): 846-54),

CD47 (Banno et al., J Biol Chem 2004 JuI 30; 279 (31): 32633-42); FPR1 (Mandai et al., J. Immunol 2005 Nov. 01; 175 (9): 6085-91);

PLSCR1 (Banno et al J Biol Chem 2004 JuI 30; 279 (31): 32633-42), TNC (Scherberich et al., Oncogene 2005 Feb 24; 24 (9): 1525-32; Banno et al., J Biol Chem 2004 JuI 30; 279 (31): 32633-42),

- CYBB (Dusi et al., Eur J Immunol 2001 Mar 1; 31 (3): 929-38).

TNFα, however, inhibits the FRZB gene involved in development (Tian et al., J. Biol Chem 2005 Apr 29; 280 (17): 17435-48).

Finally, TNFα inhibits two genes involved in metabolism:

KYNU (Banno et al., J Biol Chem 2004 JuI 30, 279 (31): 32633-42), AKR1 B10 (Iwata et al., J Biol Chem 1999 Mar 19, 274 (12) -7993-8001).

Several inhibitory molecules of TACE are in development, mainly for the treatment of arthritis. Many patent applications also offer TACE inhibitors, for the treatment of arthritis, cancers, or other diseases such as patent applications US200412201 1 (MASFERRER JL) and WO9942436 (AMERICAN CYANAMID). Indeed, these two applications describe treatment methods or compositions containing TACE inhibitors for the treatment of various pathologies including acne.

In the context of the invention, the term "TACE gene" or "TACE nucleic acid" means the gene or nucleic acid that encodes the TACE enzyme. If the targeted target is preferably the human gene or its expression product, the invention may also use cells expressing a heterologous TACE, by genomic integration or transient expression of an exogenous nucleic acid encoding the enzyme.

SCREENING METHODS An object of the invention is an in vitro method for screening candidate compounds for the preventive and / or curative treatment of acne, comprising determining the ability of a compound to inhibit the expression or the TACE activity or the expression of its gene or the activity of at least one of its promoters, an inhibition of the expression or activity of TACE, or the expression of its gene or activity of at least one of its promoters indicating the utility of the compound for the preventive or curative treatment of acne. By "inhibition" is meant any diminishing effect on the expression or the activity of the enzyme, on the expression of its gene or on the activity of at least one of its promoters. The compounds tested can be of any type. They can be of natural origin or have been produced by chemical synthesis. It can be a library of structurally defined chemical compounds, compounds or uncharacterized substances, or a mixture of compounds. Various techniques can be implemented to test these compounds and to identify compounds of therapeutic interest, modulators of TACE expression or activity.

Preferably, the screening method comprises the following steps: a. Preparation of at least two biological samples or reaction mixtures; b. Contacting one of the samples or reaction mixtures with one or more of the test compounds; vs. Measuring the expression or the activity of the TACE enzyme, the expression of its gene or the activity of at least one of its promoters, in biological samples or reaction mixtures; d. Selection of compounds for which an inhibition of the expression or activity of the TACE enzyme, or an inhibition of the expression of its gene or an inhibition of the activity of at least one of its promoters, is measured in the sample or mixture treated in (b) in relation to the untreated sample or mixture.

Throughout this text, unless otherwise specified, "expression of an enzyme" means the amount of that enzyme.

By "activity of an enzyme" is meant its biological activity.

By "promoter activity" is meant the ability of this promoter to trigger the transcription of the coded DNA sequence downstream of this promoter (and thus indirectly the synthesis of the protein, in this case the corresponding enzyme).

According to a first embodiment, the biological samples or reaction mixtures of step a) are cells transfected with a reporter gene operably linked to all or part of the promoter of the TACE gene. The method is then performed by contacting a compound (step b) with these biological samples or reaction mixtures, then by determining the level of expression of said reporter gene (step c)). The selection of step d) is done by the observation of a difference of expression level, compared to a control carried out in the absence of the compound. This difference indicates the utility of the compound for the preventive or curative treatment of acne. The reporter gene may in particular encode an enzyme which, in the presence of a given substrate, leads to the formation of colored products, such as CAT (chloramphenicol acetyltransferase), GAL (beta galactosidase), or GUS (beta glucuronidase). It may also be the gene for luciferase or GFP (Green Fluorescent Protein). The assay of the protein encoded by the reporter gene, or of its activity, is carried out conventionally, by colorimetric, fluorometric or chemiluminescence techniques, among others.

According to a second embodiment, the biological samples or reaction mixtures of step a) are cells expressing the TACE gene coding for TACE. The screening method then comprises contacting a compound (step b) with the biological samples or reaction mixtures, and then determining the level of expression of said gene (step c)). The selection of step d) is made by the observation of a difference in expression level of the TACE gene, compared to a control carried out in the absence of the compound. This difference indicates the utility of the compound for the preventive or curative treatment of acne. The cell used here can be of any type. It can be a cell expressing the gene

TACE endogenously, such as a liver cell, an ovarian cell, or even better a keratinocyte or a cell of the immune system such as THP1 cells. It may also be a cell transformed with a heterologous nucleic acid, coding for TACE, preferably human, or mammalian.

A wide variety of host cell systems can be used, such as for example Cos-7, CHO, BHK, 3T3, HEK293 cells. Microorganisms such as bacteria (eg E. coli or B. subtilis) can also be used. ; yeast (eg Saccharomyces, Pichia); insect cells, such as Sf9 or Sf21. The nucleic acid can be stably or transiently transfected by any method known to those skilled in the art, for example by calcium phosphate, DEAE-dextran, liposome, virus, electroporation, or microinjection.

In this method, the level of expression of the TACE gene can be determined by evaluating the level of transcription of said gene, or its level of translation.

By level of transcription of a gene is meant the amount of the corresponding mRNA produced.

By translation level of a gene is meant the amount of protein produced.

Those skilled in the art are familiar with techniques for the quantitative or semi-quantitative detection of the mRNA of a gene of interest. Techniques based on hybridization mRNA with specific nucleotide probes are the most common (Northern Blot, RT-PCR, Rnase protection assay). It may be advantageous to also use detection markers, such as fluorescent, radioactive, enzymatic or other ligands (eg, avidin / biotin).

The level of translation of the gene is evaluated for example by methods of the immunoassay type, mass spectrometry analysis (MALDI-TOF and thin layer analysis), etc. For an immunological assay of the product of said gene, the antibodies used may be of polyclonal or monoclonal type. Their production is based on conventional techniques. The immunoassay can be carried out in solid phase or in homogeneous phase; in a time or in two stages; sandwich method or competitive method, by way of non-limiting examples. According to a preferred embodiment, the capture antibody is immobilized on a solid phase. As non-limiting examples of solid phase, it is possible to use microplates, in particular polystyrene microplates, or particles or solid beads, paramagnetic beads.

ELISA assays, radioimmunoassays, or any other detection technique can be used to reveal the presence of the antigen-antibody complexes formed. Characterization of antigen / antibody complexes, and more generally isolated or purified but also recombinant proteins (obtained in vitro and in vivo) can be performed by mass spectrometric analysis (proteomics).

According to a third embodiment, the biological samples or reaction mixtures of step a) are reaction mixtures comprising a TACE enzyme or, preferably, an N-terminal fragment of 610 acids of the recombinant human TACE enzyme, and a substrate of the enzyme. The screening method then comprises contacting a compound (step b) with these mixtures and then determining the enzymatic activity (step c)). The selection of step d) is done by the observation of a decrease in activity, compared to a control carried out in the absence of the compound. Such a decrease indicates the utility of the compound for the preventive or curative treatment of acne.

An enzymatic activity test is described in Jin et al, Anal Biochem. 2002 Mar 15; 302 (2): 269-75. This test can also be modified using a different substrate. Thus, it is possible to use as fluorogenic substrate the MCA-PLAQAV peptide (Dpa) RSSSR-NH2 whose amino acid sequence surrounds the cleavage site of the TNFα precursor. The cleavage site is between Alanine and Valine residues. The fluorescent donor group is MCA ((7-methoxycoumarin-4-yl) acetyl) and the quenchant acceptor group is Dpa (3- (2,4-dinitrophenyl) -L-2,3-diaminopropionic amide). The cleavage activity of the substrate by the human recombinant enzyme TACE is followed by fluorescence by excitation at 320 nm and emission at 420 nm. Another way to measure the activity of TACE is to measure the production of TNFα, for example by an immunoassay using HTRF (homogeneous time resolved fluorescence) technology on keratinocytes or THP1 cell cultures. For this purpose, the cells are stimulated beforehand with LPS and / or TPA and the TNFα produced by the cells is assayed with the aid of two murine antibodies, each recognizing a different epitope of human TNFα. Each antibody is conjugated with either europium cryptate or XL665 allophycocyanin. This combination makes it possible to measure a FRET signal when the two antibodies each bind to one end of the TNFα produced.

Candidate TACE inhibitors can then be tested on acne models, or on patients with acne. The invention thus also provides a method for screening candidate compounds for the treatment and / or prevention of acne, said method comprising administering a TACE-inhibiting compound to a subject having acne-prone skin or acne-prone skin , and assessment of the condition of acne.

The administration is preferably carried out topically. Topically, we mean an application on the skin or the mucous membranes.

Enzyme inhibitors

The subject of the invention is also the use of an inhibitor of the human enzyme TACE, in particular an inhibitor obtained by the screening method described above, for the preparation of a medicinal product intended for preventive and / or curative treatment. acne. Preferably, the drug does not contain a cyclooxygenase-2 (COX-2) inhibitor. Preferably, the human enzyme inhibitor TACE is the only anti-acne active ingredient contained in the drug. Even more preferably, the inhibitor of the human enzyme TACE is the only active ingredient contained in the drug.

Here is described a method of preventive and / or curative treatment of acne, said method comprising administering a therapeutically effective amount of a modulator of the human enzyme TACE to a patient in need of such treatment.

The term "inhibitor" refers to a compound or chemical substance that substantially eliminates or reduces the enzymatic activity of TACE. The term "substantially" means a reduction of at least 20%, preferably at least 30%, more preferably at least 50%, and more preferably at least 70% or 90%. More particularly, it may be a compound that interacts with, and blocks, the catalytic site of the enzyme. A preferred inhibitor interacts with the enzyme in solution at inhibitor concentrations of less than 1 μM, preferably less than 0.1 μM, more preferably less than 0.01 μM.

The modulator compound may be an anti-TACE inhibitory antibody, preferably a monoclonal antibody. Advantageously, such an inhibitory antibody is administered in an amount sufficient to obtain a plasma concentration of about 0.01 μg per ml to about 100 μg / ml, preferably from about 1 μg per ml to about 5 μg / ml.

Other preferred inhibitors are shown in Table 1 below:

Table 1: TACE Inhibitors



The names, CAS number, and references of even more preferred inhibitors are shown in Table 2 below:

Table 2: TACE inhibitor names:

Preferred TACE inhibitors include W-3646, Ro-32-7315, GW-3333, apratastat or (3S) -N-hydroxy-4 - [[4 - [(4-hydroxybut-2-ynyl) oxy] phenyl] sulfonyl] -2,2-dimethylthiomorpholine-3-carboxamide, GW-4459, CGS-33090A, DPC-333, TNF-484, WTACE2, SP-057, SL-422, FYK-1388, and KB-R7785.

Also advantageous are the following TACE inhibitors: 3- [3- [N-isopropyl-N- (4-methoxyphenylsulfonyl) amino] -phenyl] -3- (3-pyridyl) -2 (E) -propenohydroxamic acid, ( 2R, 3S) -3- (formylhydroxyamino) -4-methyl-2- (2-methylpropyl) -N - [(1S, 2S) -2-methyl-1 - [(2-pyridinylamino) carbonyl] butyl] pentanamide, (2R, 3S) -3- (formylhydroxyamino) -N- [(IS) -4-

[[imino (nitroamino) -methyl] amino] -1 - [(2-thiazolylamino) carbonyl] butyl] -2- (2-methylpropyl) -hexanamide, (αR, 1α, 4β) -α; - [[( 4-ethoxyphenyl) -sulfonyl] (4-pyridylmethyl) amino] -N-hydroxy-4-propoxy-cyclohexaneacetamide, and (αR) -N-hydroxy-α, 3-dimethyl-2-oxo-3- [4- 2-methyl-4-quinolinyl-methoxy) phenyl] -1-pyrrolidineacetamide.

The following table 3 gives the references of the patent applications describing various TACE inhibitors: Table 3: TACE Inhibitor Reference

The inhibiting compounds are formulated within a pharmaceutical composition, in association with a pharmaceutically acceptable vehicle. These compositions may be administered, for example, orally, enterally, parenterally, or topically. Preferably, the pharmaceutical composition is applied topically. Orally, the pharmaceutical composition may be in the form of tablets, capsules, dragees, syrups, suspensions, solutions, powders, granules, emulsions, suspensions of microspheres or nanospheres or lipid vesicles or polymers for controlled release. Parenterally, the pharmaceutical composition may be in the form of solutions or suspensions for infusion or for injection.

Topically, the pharmaceutical composition is more particularly intended for the treatment of skin and mucous membranes and may be in the form of ointments, creams, milks, ointments, powders, soaked swabs, solutions, gels , sprays, lotions or suspensions, patches. It may also be in the form of suspensions of microspheres or nanospheres or lipid or polymeric vesicles or polymeric patches or hydrogels allowing controlled release. This composition for topical application may be in anhydrous form, in aqueous form or in the form of an emulsion. In a preferred variant, the pharmaceutical composition is in the form of a gel, a cream or a lotion.

The pharmaceutical composition may further contain inert additives or combinations of these additives, such as

- wetting agents;

- flavor enhancers;

preserving agents such as esters of parahydroxybenzoic acid; stabilizing agents;

humidity regulating agents;

pH regulating agents;

osmotic pressure modifying agents; emulsifying agents;

UV-A and UV-B filters;

and antioxidants, such as alpha-tocopherol, butylhydroxyanisole or butylhydroxytoluene, superoxide dismutase, ubiquinol or certain metal chelators.

Claims

1. A m in vitro method for screening candidate compounds for the preventive and / or curative treatment of acne, comprising determining the ability of a compound to inhibit the expression or activity of the TACE enzyme, or the expression of its gene or the activity of at least one of its promoters.

The method of claim 1, comprising the steps of: a. Preparation of at least two biological samples or reaction mixtures; b. Contacting one of the samples or reaction mixtures with one or more of the test compounds; vs. Measuring the expression or the activity of the TACE enzyme, the expression of its gene or the activity of at least one of its promoters, in biological samples or reaction mixtures; d. Selection of compounds for which an inhibition of the expression or activity of the TACE enzyme, or an inhibition of the expression of its gene or an inhibition of the activity of at least one of its promoters, is measured in the sample or mixture treated in (b) in relation to the untreated sample or mixture.

Use of an inhibitor of the enzyme TACE, for the preparation of a medicament for the preventive and / or curative treatment of acne, said medicament not containing a cyclooxygenase-2 inhibitor.

4. Use according to claim 3, characterized in that the inhibitor of the enzyme TACE is chosen from E-3- [3- [N- (4-methoxyphenylsulfonyl) -N-isopropylamino] phenyl] -3 acid. - (3-pyridyl) -2 (E) -propenohydroxamic acid, 2- (2-methylpropyl) -2- (methylsulfonyl) hydrazide (2R, 3S, 5E) -3-

[(hydroxyamino) carbonyl] -2- (2-methylpropyl) -6-phenyl-5-hexenoic acid, (2R, 3S) -3- (formyl-hydroxyamino) -4-methyl-2- (2-methylpropyl) - N - [(1S, 2S) -2-methyl-1 - [(2-pyridinylamino) carbonyl] butyl] pentanamide, apratastate, (2R, 3S) -3- (formyl-hydroxyamino) -N - [( 1S) -4 - [[imino (nitroamino) methyl] amino] -1 - [(2-thiazolylamino) carbonyl] butyl] -2- (2-methylpropyl) hexaneamide, (αR, 1α, 4β) -α - [[(4-ethoxyphenyl) -sulfonyl] (4-pyridinylmethyl) amino] -N-hydroxy-4- propoxycyclohexaneacetamide, (αR) -N-hydroxy-α, 3-dimethyl-2-oxo-3- [4- (2-methyl-4-quinolinylmethoxy) phenyl] -1-pyrrolidineacetamide, TNF-484, WTACE2,

(8S, 11R, 12S) -N, N-hydroxy-1- (1- (2-methylpropyl) -N8- [2- (4-morpholinyl) -2-oxoethyl] -2,10-dioxo-1-oxa-3 9-diazacyclopentadecane-8,12-dicarboxamide, (6S, 7R, 10S) -N6-hydroxy-N10- [2- (methylamino) -2-oxoethyl] -7- (2-methylpropyl) -8-oxo-2 -oxa-9-azabicyclo [10.2.2] hexadeca-12,14,15-triene-6,10-dicarboxamide, [2R- [1 (S ^* ), 2R ^* , 3S ^* ]] monoacetate - N1- [ 1 - [[4- (aminoiminomethyl) amino] phenyl] methyl] -2- (methylamino) -2-oxoethyl] -N4-hydroxy-2- (2-methylpropyl) -3- (3-phenylpropyl) butanediamide, (2S , 3R) -N1-hydroxy-2-methyl-N4 - [(1S) -2- (methylamino) -2-oxo-1-phenylethyl] -3- (2-methylpropyl) butanediamide, the acid 3-

[3- [N-Isopropyl-N- (4-methoxyphenylsulfonyl) amino] -phenyl] -3- (3-pyridyl) -2 (E) -propenohydroxamic acid, (2R, 3S) -3- (formylhydroxyamino) - 4-methyl-2- (2-methylpropyl) -N - [(1S, 2S) -2-methyl-1 - [(2-pyridinylamino) carbonyl] butyl] pentanamide, (2R, 3S) -3- (formylhydroxyamino) N - [(1 S) -4 - [[imino (nitroamino) methyl] amino] -1 - [(2-thiazolylamino) carbonyl] butyl] -2- (2-methylpropyl) hexanamide, (αR, 1α, 4β) -α- [[(4-ethoxyphenyl) -sulfonyl] (4-pyridinylmethyl) amino] -N-hydroxy-4-propoxy-cyclohexaneacetamide, and (αR) -N-hydroxy-α, 3-dimethyl-2 -oxo-3- [4- (2-methyl-4-quinolinylmethoxy) phenyl] -1-pyrrolidineacetamide.

Use according to claim 3 or 4, wherein the TACE enzyme inhibiting compound is the sole active ingredient.

6. Use according to one of claims 3 to 5, wherein the inhibiting compound is a compound identified by the method defined in one of claims 1 or 2.

The use according to one of claims 3 to 6, wherein the medicament is for topical administration.