Meclozine derivatives and diclazuril derivatives for use in the prevention and/or the treatment of disorders associated to the inflammation induced by <i>P. acnes</i>

Abstract

The present invention relates to compounds of the following general formula (I) or (II) or a pharmaceutically acceptable salt and/or solvate thereof, for use in the prevention and/or the treatment of disorders associated to the inflammation induced by P. acnes, in particular in the prevention and/or the treatment of acne, psoriasis, chronic urticaria, urticaria pigmentosa, cutaneous autoinflammatory diseases, hidradenitis or atopic dermatis. ##STR00001##

Claims

1. A method for treating disorders associated to the inflammation induced by P. acnes comprising administering to a subject in need thereof an effective amount of a compound of following general formula (Ib): ##STR00012## or a pharmaceutically acceptable salt and/or solvate thereof, wherein R.sub.1 to R.sub.4 are, independently of one another, hydrogen atom or a group selected from halo, —NO.sub.2, —CN, —OR.sub.25, —NR.sub.26R.sub.27, —C(O)OR.sub.28, —S(O).sub.2R.sub.29, or a (C.sub.1-C.sub.6)alkyl group optionally substituted with one or several groups selected from halo or —OR.sub.30, R.sub.5 to R.sub.9 are, independently of one another, hydrogen atom, halo, —CN, —NO.sub.2, —OR.sub.12, —NR.sub.13R.sub.14, —C(O)OR.sub.15, —C(O)NR.sub.16R.sub.17, —S(O).sub.2NR.sub.18R.sub.19, —S(O).sub.2R.sub.20, —NHS(O).sub.2R.sub.21, —NHC(O)R.sub.22, or a group selected from (C.sub.1-C.sub.6)alkyl, aryl, (C.sub.1-C.sub.6)alkyl-aryl, heterocycle, (C.sub.1-C.sub.6)alkyl-heterocycle, said group being optionally substituted with one or several groups selected from halo, (C.sub.1-C.sub.6)alkyl, —OR.sub.23 or —OC(O)R.sub.24; or the couple R.sub.5-R.sub.6, R.sub.6-R.sub.7, R.sub.7-R.sub.8 or R.sub.8-R.sub.9 form together with the carbon atoms to which they are chemically linked, an heterocycle, while the others are hydrogen atoms, R.sub.12 to R.sub.30 are, independently of one another, hydrogen atom, halo, or a group selected from (C.sub.1-C.sub.6)alkyl, aryl, (C.sub.1-C.sub.6)alkyl-aryl, heterocycle, (C.sub.1-C.sub.6)alkyl-heterocycle, said group being optionally substituted with one or several groups selected from halo, (C.sub.1-C.sub.6)alkyl, CF.sub.3 or —OR.sub.31, and R.sub.31 is hydrogen atom, halo or a (C.sub.1-C.sub.6)alkyl group.

2. The method of claim 1, wherein R.sub.1 to R.sub.4 are, independently of one another, hydrogen atom or a group selected from halo, —NO.sub.2, —CN, —OR.sub.25, —NR.sub.26R.sub.27 or a (C.sub.1-C.sub.6)alkyl group; R.sub.25 to R.sub.27 being as defined in claim 1.

3. The method of claim 1, wherein R.sub.5 to R.sub.9 are, independently of one another, hydrogen atom, halo, —CN, —NO.sub.2, —OR.sub.12, —NR.sub.13R.sub.14, —C(O)NR.sub.16R.sub.17, —S(O).sub.2NR.sub.18R.sub.19, or a group selected from (C.sub.1-C.sub.6)alkyl, aryl, (C.sub.1-C.sub.6)alkyl-aryl, heterocycle or (C.sub.1-C.sub.6)alkyl-heterocycle; and R.sub.12 to R.sub.14 and R.sub.16 to R.sub.19 are defined as in claim 1.

4. The method of claim 1, wherein R.sub.12 to R.sub.30 are, independently of one another, hydrogen atom, halo, or a group selected from (C.sub.1-C.sub.6)alkyl.

5. The method of claim 1, wherein said compound of formula (I) is a compound of the following formula (Ic): ##STR00013## or a pharmaceutically acceptable salt and/or solvate thereof.

6. A method for treating disorders associated to the inflammation induced by P. acnes comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising at least one compound of claim 1 at least one pharmaceutically acceptable excipient.

7. The method of claim 6, wherein said composition further comprises another active principle.

8. The method of claim 1, wherein said disorders associated to the inflammation induced by P. acnes are acne, psoriasis, chronic urticaria, urticaria pigmentosa, cutaneous autoinflammatory diseases, hidradenitis, or atopic dermatitis.

9. The method of claim 1, wherein said compound is the dihydrochloride salt of compound (Ic): ##STR00014##

10. The method of claim 6, wherein said disorders associated to the inflammation induced by P. acnes are acne, psoriasis, chronic urticaria, urticaria pigmentosa, cutaneous autoinflammatory diseases, hidradenitis, or atopic dermatitis.

11. The method of claim 7, wherein the another active principle is selected from topical antibiotic, topical anti-inflammatory, topical anti-seborrheic, zinc derivatives, cyclins, and isotretinoin.

12. The method of claim 7, wherein the another active principle is selected from erythromycine, dalacine, benzoyl peroxydes derivatives, tretinoin, adapalene, zinc gluconate, cyclins, and isotretinoin.

Description

DESCRIPTION OF THE FIGURES

(1) FIGS. 1A-1D: Dose-dependent inhibition of IL-8 production and cell viability evaluation by keratinocytes pre-treated with Diclazuril and Meclozine. HaCaT cell were incubated for 24 h with (A, C) diclazuril and (B, D) meclozine alone at concentrations ranging from 0.39 to 50 μM (gray bar) and stimulated with P. acnes (dark bar). Controls experiments were done with HaCaT cell untreated and unstimulated (hatched bar) and with HaCaT cells stimulated with P. acnes only (horizontal line bar). Measurement of IL-8 production was realized by ELISA and cytotoxicity was determined by the MTT assay as described in Materials and Methods. Data are means±S.D. of three separate experiments. Statistical significance is indicated by * (P<0.05), ** (P<0.01), *** (P<0.001), and **** (P<0.0001), respectively.

(2) FIGS. 2A-2D: Dose-dependent inhibition of IL-1β production and cell viability evaluation by monocytes pre-treated with Diclazuril and Meclozine. ThP-1 cell were incubated for 24 h with (A, C) diclazuril and (B, D) meclozine alone at concentrations ranging from 0.39 to 50 μM (gray bar) and stimulated with P. acnes (dark bar). Controls experiments were done with ThP-1 cell untreated and unstimulated (hatched bar) and with ThP-1 cells stimulated with P. acnes only (horizontal line bar). Measurement of IL-1β production was realized by ELISA and cytotoxicity was determined by the MTT assay as described in Materials and Methods. Data are means±S.D. of three separate experiments. Statistical significance is indicated by * (P<0.05), ** (P<0.01), *** (P<0.001), and **** (P<0.0001), respectively.

(3) FIGS. 3A and 3B: Diclazuril and meclozine inhibit P. acnes-induced IL-8 and IL-1β mRNA productions. (A) HaCaT cells and (B) ThP-1 cells were pre-treated for 24 h by diclazuril (light gray bar) and meclozine (dark gray bar) at 10 μM and then stimulated for 5 h with P. acnes suspension (OD.sub.600 nm=0.3). Control experiments were done with unpre-treated and unstimulated cells (cell alone) and with cell stimulated by P. acnes only (white bar). Total RNA was extracted and IL-8/IL-1β mRNA levels were determined by real-time RT-PCR. IL-8 and IL-1β mRNA levels were compared with GAPDH mRNA level (used as control) and are expressed as fold-change. Data are means±S.D. of three separate experiments. Statistical significance is indicated by * (P<0.05), ** (P<0.01), *** (P<0.001), and **** (P<0.0001), respectively.

(4) FIGS. 4A-4C: Diclazuril and meclozine inhibit inflammatory signaling pathways. HaCaT cells were pre-treated for 24 h with diclazuril and meclozine at 10 μM and then stimulated with P. acnes for 15, 30, 60, 120 and 180 min. At each time measurement of IL-8 production was realized by ELISA supernatant (A) and whole-cell lysates were prepared and used for IκB (B), (C) p-ERK, western blot analysis, using the appropriate antibodies.

(5) FIGS. 5A and 5B: Dose-dependent inhibition of IL-8 production by keratinocytes pre-treated with Diclazuril and Meclozine and stimulated with PGN. HaCaT cells were incubated for 24 h with (A) diclazuril and (B) meclozine at concentrations ranging from 0.39 to 25 μM and then stimulated with PGN at 5 (light gray curve), 10 (middle gray curve), and 20 μg/ml (dark gray curve). Controls experiments were done with untreated HaCaT cells. Measurement of IL-8 production was realized by ELISA as described in Materials and Methods. Data are means±S.D. of three separate experiments.

(6) FIGS. 6A-6D: Dose-dependent inhibition of IL-1β production by monocytes pre-treated with Diclazuril and Meclozine and stimulated with PGN and LTA. ThP-1 cells were incubated for 24 h with (A, C) diclazuril and (B, D) meclozine at concentrations ranging from 0.39 to 25 μM and then stimulated with PGN (A, B) and LTA (C, D) at 5 (light gray curve), 10 (middle gray curve), and 20 μg/ml (dark gray curve). Controls experiments were done with untreated ThP-1 cells. Measurement of IL-1β production was realized by ELISA as described in Materials and Methods. Data are means±S.D. of three separate experiments.

(7) FIGS. 7A-7D: Time-dependent effect of diclazuril and meclozine pre-treatment. HaCaT cells were pre-treated with diclazuril (A, C) and meclozine (B, D) at concentrations ranging from 0.39 to 12.5 μM for 1 h (light gray curve), 6 h (middle gray curve), 24 h (dark gray curve), and 48 h (black curve), and then stimulated by P. acnes for 18 h. Measurement of IL-8 production was realized by ELISA and cytotoxicity was determined by the MTT assay as described in Materials and Methods. Data are means±S.D. of three separate experiments.

(8) FIGS. 8A and 8B: Comparison effect on IL-8 production between diclazuril and meclozine with common commercial molecules used in acne treatment. HaCaT cells were pre-treated with diclazuril, meclozine, erythromycine, clindamycine, Luperox (benzoyl peroxide), isotretinoine, adapalene and retinoic acid at 6 μM (A) and 12.5 μM (B) for 24 h, and then stimulated by P. acnes for 18 h. Controls experiments corresponded to unpre-treated but P. acnes stimulated HaCaT cell (PA alone). Measurement of IL-8 production (gray bar) was realized by ELISA and cytotoxicity (black bar) was determined by the MTT assay as described in Materials and Methods. Data are means±S.D. of three separate experiments. Statistical significance is indicated by * (P<0.05), ** (P<0.01), *** (P<0.001), and **** (P<0.0001), respectively.

(9) FIGS. 9A and 9B: Effect of VNPA-A2 gel topical application on P. acnes-induced inflammation in vivo. Ears of mice were intradermally injected with P. acnes (OD.sub.620 nm=1.0 corresponding to 2.107 CFU/20 μl in PBS) to induce inflammation. Subsequently, 1.3% diclazuril gel was applied on the ear skin surface of mice each day for 3 days. (A) The score corresponding to the ear thickness, the peeling and the redness, was measured every day for a period of 96 h. Data are means±S.D. of 10 individual experiments. PBS corresponds to the non-treated group injected with PBS. PA+Vehicle corresponds to P. acnes injected in ears treated with vaseline alone. PA+diclazuril corresponds to P. acnes injected in ears treated with 1.3% diclazuril mixed with vaseline. Statistical significance is indicated by * (P<0.05), ** (P<0.01), *** (P<0.001), and **** (P<0.0001), respectively.

(10) FIGS. 10A and 10 B: Dose-dependent inhibition of IL-8 production by keratinocytes stimulated by P. acnes pre-treated with the RCL PH000645-PH analogue. HaCaT cell were incubated for 24 h with RCL PH000645-PH (A) and diclazuril (B) at concentrations ranging from 2.5 to 6.25 μM (gray bar) and stimulated with P. acnes (dark bar). Controls experiments were done with unstimulated HaCaT cell (hatched bar) and HaCaT stimulated with P. acnes (horizontal bar). Measurement of IL-8 production was realized by ELISA as described in Materials and Methods. Data are means±S.D. of three separate experiments. Statistical significance is indicated by * (P<0.05), ** (P<0.01), *** (P<0.001), and **** (P<0.0001), respectively.

(11) FIGS. 11A and 11B: Evaluation of cell viability after treatment with the RCL PH000645-PH analogue on keratinocytes. HaCaT cell were incubated for 24 h with RCL PH000645-PH (A) and diclazuril (B) at concentrations ranging from 2.5 to 6.25 μM (gray bar) and stimulated with P. acnes (dark bar). Controls experiments were done with unstimulated HaCaT cell (hatched bar) and HaCaT stimulated with P. acnes (horizontal bar). Measurement of cell viability was realized by the MTT assay as described in Materials and Methods. Data are means±S.D. of three separate experiments.

(12) FIG. 12: Effect of Meclozine on P. acnes-induced inflammation in vivo. Ears of mice were intradermally injected with P. acnes (OD.sub.620 nm=1.0 corresponding to 2.10.sup.7 CFU/20 μl in PBS) to induce inflammation. Subsequently, 1% meclozine gel was applied on the ear skin surface of mice each day for 3 days. The score corresponding to the ear thickness, the peeling and the redness, was measured every day for a period of 96 h. Data are means±S.D. of 8 individual experiments. PBS corresponds to the non-treated group injected with PBS. PA+Vehicle corresponds to P. acnes injected in ears treated with Vaseline containing DMSO (150 μl), Solutol HS153070/water (30:70, w/w) (300 μl). PA+meclozine corresponds to P. acnes injected in ears treated with 1% meclozine mixed with Vaseline containing DMSO (150 μl), Solutol HS153070/water (30:70, w/w) (300 μl). Statistical significance is indicated by * (P<0.05), ** (P<0.01).

(13) FIG. 13: Effect of Meclozine topical application on P. acnes-induced inflammation in vivo. Ears of mice were intradermally injected with P. acnes (OD.sub.620 nm=1.0 corresponding to 2.10.sup.7 CFU/20 μl in PBS) to induce inflammation. Subsequently, 1% meclozine gel was applied on the ear skin surface of mice each day for 3 days. PBS corresponds to the non-treated group injected with PBS. PA+Vehicle corresponds to P. acnes injected in ears treated with Vaseline containing DMSO (150 μl), Solutol HS153070/water (30:70, w/w) (300 μl). PA+meclozine corresponds to P. acnes injected in ears treated with 1% meclozine mixed with Vaseline containing DMSO (150 μl), Solutol HS153070/water (30:70, w/w) (300 μl).

(14) FIG. 14: Histopathological analysis of mouse ears. Ears of mice were intradermally injected with P. acnes (OD.sub.620 nm=1.0 corresponding to 2.10.sup.7 CFU/20 μl in PBS) to induce inflammation. Subsequently, 1% meclozine gel was applied on the ear skin surface of mice each day for 3 days. Ears were formalin-fixed and embedded in paraffin. Detection of cells was made by hematoxylin and eosin staining. PBS corresponds to the non-treated group injected with PBS. PA+Vehicle corresponds to P. acnes injected in ears treated with vaseline containing DMSO (150 μl), Solutol HS153070/water (30:70, w/w) (300 μl). PA+meclozine corresponds to P. acnes injected in ears treated with 1% meclozine mixed with vaseline containing DMSO (150 μl), Solutol HS153070/water (30:70, w/w) (300 μl).

(15) FIG. 15: Diclazuril and meclozine inhibit inflammatory signaling pathways. HaCaT cells were pre-treated for 24 h with diclazuril and meclozine at 25 μM and then stimulated with P. acnes for 30, 60, 120, 180 min and 18, 24 h. At each time whole-cell lysates were prepared and used for p-IκB/IκB, p-ERK/ERK, p-p38/p38, p-JNK/JNK, p-PKC/PKC, p-Akt/Akt, p-mTOR/mTOR western blot analysis, using the appropriate antibodies. Protein load (25 μg protein per lane) was assessed by using β-actin.

(16) FIG. 16: Diclazuril and meclozine inhibit inflammatory signaling pathways. HaCaT cells were pre-treated for 24 h with diclazuril and meclozine at 25 μM and then stimulated with P. acnes for 30, 60, 120, 180 min and 18, 24 h. At each time whole-cell lysates were prepared and used for p-PI3K/PI3K, ICAM-1, Cox2, PPAR alpha, PPAR beta, PPAR gamma western blot analysis, using the appropriate antibodies. Protein load (25 μg protein per lane) was assessed by using β-actin.

(17) FIG. 17: Evaluation of keratinocyte viability after treatment with meclozine and diclazuril in the in vitro psoriasis-like model. Human cutaneous primary keratinocytes (NHDK) were stimulated by the solution M5 consisting by a combination of IL-17A, OSM, TNF-α, IL-22, IL-1 IL-1α (10 ng/ml) and treated for 48 h with JAK inhibitor at 6.25 μM (positive control) or meclozine and diclazuril at concentrations ranging from 0.39 to 12.5 μM. Control experiments were done with untreated unstimulated NHDK cells (NHDK alone) and untreated NHDK stimulated by M5 (NHDK+M5). Measurement of cytotoxicity was determined by the MTT assay as described in the Materials and Methods. Data are means±S.D. of three individual experiments. Statistical significance (versus NHDK alone) is indicated by * (P<0.05), ** (P<0.01), and *** (P<0.001), respectively.

(18) FIG. 18: IL-8 production by meclozine-treated keratinocytes in the in vitro psoriasis-like model. Human cutaneous primary keratinocytes (NHDK) were stimulated by the solution M5 consisting by a combination of IL-17A, OSM, TNF-α, IL-22, IL-1α (10 ng/ml) and treated for 48 h with JAK inhibitor at 6.25 μM (positive control) or meclozine and diclazuril at concentrations ranging from 0.39 to 12.5 μM. Control experiments were done with untreated unstimulated NHDK cells (NHDK alone) and untreated NHDK stimulated by M5 (NHDK+M5). Measurement of IL-8 was realized by ELISA as described in the Materials and Methods. Data are means±S.D. of three individual experiments. Statistical significance (versus NHDK+M5) is indicated by **** (P<0.0001).

(19) FIG. 19: Inhibition of IL-8 production by diclazuril in the in vitro psoriasis-like model. Human cutaneous primary keratinocytes (NHDK) were stimulated by the solution M5 consisting by a combination of IL-17A, OSM, TNF-α, IL-22, IL-1α (10 ng/ml) and treated for 48 h with JAK inhibitor at 6.25 μM (positive control) or meclozine and diclazuril at concentrations ranging from 0.39 to 12.5 μM. Control experiments were done with untreated unstimulated NHDK cells (NHDK alone) and untreated NHDK stimulated by M5 (NHDK+M5). Data are means±S.D. of three individual experiments. Statistical significance (versus NHDK+M5) is indicated by **** (P<0.0001).

(20) FIG. 20: hBD-2 production by meclozine-treated keratinocytes in the in vitro psoriasis-like model. Human cutaneous primary keratinocytes (NHDK) were stimulated by the solution M5 consisting by a combination of IL-17A, OSM, TNF-α, IL-22, IL-1α (10 ng/ml) and treated for 48 h with JAK inhibitor at 6.25 μM (positive control) and meclozine at concentrations ranging from 0.39 to 12.5 μM. Control experiments were done with untreated unstimulated NHDK cells (NHDK alone) and untreated NHDK stimulated by M5 (NHDK+M5). Data are means±SD. of three individual experiments. Statistical significance is indicated by ** (P<0.01), *** (P<0.001) and **** (P<0.0001), respectively.

(21) FIG. 21: Inhibition of hBD-2 production by diclazuril in the in vitro psoriasis-like model. Human cutaneous primary keratinocytes (NHDK) were stimulated by the solution M5 consisting by a combination of IL-17A, OSM, TNF-α, IL-22, IL-1α (10 ng/ml) and treated for 48 h with JAK inhibitor at 6.25 μM (positive control) and diclazuril at concentrations ranging from 0.39 to 12.5 μM. Control experiments were done with untreated unstimulated NHDK cells (NHDK alone) and untreated NHDK stimulated by M5 (NHDK+M5). Data are means±S.D. of three individual experiments. Statistical significance is indicated by ** (P<0.01), and **** (P<0.0001), respectively.

EXAMPLES

Example 1: Biological Activities of the Compounds According to the Invention

(22) Materials and Methods

(23) Bacterial strain and conditions of growth. P. acnes strain 6919 was obtained from the American Type Culture Collection (Manassas, Va.) and P. acnes strains RON and PIE were isolated from patient with joint infection. All strains were grown under anaerobic conditions in reinforced clostridial liquid and solid medium (RCM) (Difco Laboratories, Detroit, Mich.). P. acnes was transferred from the bacterial stock onto RCM agar plate and incubated for 5 days under anaerobic condition by using a GasPak™ EZ Anaerobic Container System (Becton Dickinson & Co, Sparks Md., USA). A single colony was transferred into 100 ml RCM and grown as described above. Bacterial suspension was then store frozen at −80° C. in presence of 10% glycerol final. This stock was called «start stock» and used for all the experiments. For routine culture, 100 ml of RCM was used and bacteria were harvested after 5 days at 37° C. by centrifugation at 7,000×g for 10 min at 4° C. Pellets were pooled and washed in about 30 ml of cold sterile PBS [1.5 mM KH.sub.2PO.sub.4, 2.7 mM Na.sub.2HPO.sub.4.7H.sub.2O, 0.15 M NaCl (pH 7.4)] and centrifuged again as described above. Finally, the bacterial pellet was suspended in sterile PBS (1:10 from volume culture).

(24) Cell culture, pretreatment and stimulation. The immortalized human keratinocyte cell line HaCaT, fibroblast MRC5 were grown in Dulbecco's modified Eagle's medium-Glutamax-I (DMEM) with 1 mM sodium pyruvate. The immortalized human monocytic cell line ThP1 was grown in Roswell Park Memorial Institute 1640 Medium-Glutamax-I (RPMI). DMEM and RPMI were supplemented with 0.1% and 10% heat-inactivated fetal calf serum (Invitrogen), and an antibiotic/antimycotic solution (10 U/ml Pencillin, 10 μg/ml Streptomycin, 0.25 μg/ml Amphoterin) at 37° C. in humidified atmosphere containing 5% CO2 as described (Life Technologie). Primary human keratinocytes (NHDK) and fibroblast (HDF) were grown in the KGM-Gold and in FGM-2 Bullet Kit, respectively, as described by the manufacturer (Lonza). The immortalized cell lines were routinely tested to assess the absence of Mycoplasma infection. Cells, cultivated in 6- or 96-well polystyrene plates, were pretreated with appropriate molecule solution for 1 to 48 h at 37° C. in the dark at the appropriate concentration. Then, for stimulation, cells were incubated for 15 min to 24 h with the P. acnes suspension adjusted at the appropriate concentration at 37° C. in 5% CO2. For experiences using an in vitro model of psoriasis, the primary human keratinocytes (NHDK) were grown in culture medium for 24 hours. The medium was removed and replaced with culture medium containing meclozine, diclazuril and JAK inhibitor (used as positive control) at the concentrations of 0.39, 0.78, 1.56, 3.12, 6.25 and 12.5 μM and the pro-inflammatory mixture M5 (combination of IL-17A, OSM, TNF-α, IL-22, IL-1α at 10 ng/ml) was added to the cells followed by an incubation for 48 or 72 hours.

(25) Cell viability assays. Viability of cells was estimated by using the MTT assay where cells were incubated with a 0.2% MTT solution in cell culture medium for 4 h at 37° C. The MTT solution was then discarded and DMSO added to solubilize the MTT-formazan cristals produced in living cells. After thorough mixing, the absorbance was measured at 540 nm.

(26) ELISA. Human IL-1β, IL-8, hBD-2 and TNF-α protein concentration were measured in the supernatants of stimulated cells using various ELISA Sets (all from Ready-Set-Go from eBioscience, except hBD-2 measurements: Human DEFB4A/BD-2 ELISA Kit from LSBio) according to the manufacturer's instructions. We used serial dilutions of recombinant human IL-1β, IL-8, hBD-2 and TNF-α for standard curve. The optical density was determined at 450 nm at a wavelength correction of 540 nm.

(27) RT-qPCR assay. Cells were grown in 6 wells polystyrene plate and pretreated for 24 h with diclazuril and meclozine at 10 μM and stimulated 5 h by P. acnes as described previously. Total RNA was isolated using the NucleoSpin RNA and treated with DNAse I, according to the manufacturer's instructions (Macherey-Nagel, Hoerdt, France). RNA concentration was determined at 260 nm on a nanodrop (Labtech, France) and the ratios for all samples were ranging between 1.6 and 1.9. Complementary DNA were generated from 100 ng of total RNA at 50° C. for 10 min followed by the quantitative PCR analysis, carried out in the LightCycler Nano (Roche), and performed with the iTaq Universal SYBR Green One-Step kit (Bio-Rad Laboratories, Hercules, Calif., USA) with a 2-step cycles conditions set at 95° C. for 60 s followed by 40 cycles of 95° C. for 15 s, 68° C. for 60 s, and ended by a melting curve at 65-95° C., 60 s with 0.1° C./s. From the amplification curves, the threshold cycles (Ct) are determined for the studied genes. The amount of relative RNA in stimulated cells relative to control cells is calculated according to the method of 2Act and expressed as a relative fold change expression normalized to gene expression of internal control (GAPDH). IL-8 primers were used: sens 5′-TCTTGGCAGCCTTCCTGATT-3′, anti-sens 5′-TTTCGTGTTGGCGCAGTGT-3′ and GAPDH primers: sens 5′-GCCACATCGCTCAGACAC-3′, GADPH anti-sens 5′-GCCCAATACGACCAAATCC-3′. Sample quantification was made in triplicate.

(28) Western Blot analysis. Whole cell protein extracts (25 μg) were separated by electrophoresis (LDS-PAGE) under denaturing conditions with NuPAGE Novex 4-12% Bis-Tris gel (1 mm, 12 wells, Invitrogen, UK) and proteins were transferred onto nitrocellulose membranes and saturated in 20 ml of saturation buffer consisting of TBS 1× (Tris Buffered Saline) containing 200 mM Tris, 1.4 M NaCl (pH 7.6), 5% no fat milk, 0.1% Tween 20 for 1 h. After washing three times for 15 min with 15 ml of TBS/T buffer [1×TBS, 0.1% Tween-20], membranes were incubated overnight with gentle mixing at 4° C. with 10 ml of rabbit polyclonal primary antibodies against human ICAM-1 (SC-7891, 1:500), PPARα (SC-398394, 1:250), PPARβ (SC-74517, 1:200), PPARγ (SC-7196, 1:500), IκB (SC-371, 1:500), p-IκB (SC-7977, 1:500), Cox-2 (SC-7951, 1:250), p-mTOR (CS, Ref 2974, 1:1000), mTOR (CS, Ref 2972, 1:1000), p-p38 (SC-17852, 1:500), p38 (SC-535, 1:250), ERK (SC-94, 1:500), JNK (SC-571, 1:500), and mouse monoclonal primary antibodies against human p-PI3 kinase (CS, Ref 4228, 1:250), PI3 kinase (CS, Ref 4257, 1:250), p-Akt1/2/3 (SC-81433, 1:500), p-ERK (SC-7383, 1:500), p-JNK (SC-6254, 1:500), -actin used to control loading, (SC-47778, 1:1000) diluted in TBS/T supplemented with 5% BSA (antibodies were purchased from Santa Cruz Biotechnology, Inc., Santa Cruz, Calif., USA abbreviated SC; and from Cell Signaling Technology, Inc. Leiden, The Netherlands abbreviated CS above). After washing to remove unbound antibodies, bound primary antibodies were detected by incubation for 1 h using secondary antibody against rabbit- and mouse IgG (Santa Cruz Biotechnology, SC-2357, 1:5000 and SC-2005, 1:5000, respectively). Unbound material was removed by washing and peroxidase activity was detected in a chemiluminescence assay (WesternBright ECL, Advansta, Menlo Park, USA).

(29) Statistical analysis. The statistical significance of differences between data from experimental groups was analyzed by paired Student's-test. A level of P<0.05 was accepted as significant. Statistical significance is indicated by * (P≤0.05), ** (P≤0.01), and *** (P≤0.001), respectively.

(30) Results

(31) 1. Diclazuril and Meclozine Dose-Dependently Inhibits P. acnes-Induced IL-8 Production in Keratinocytes.

(32) Both molecules, diclazuril and meclozine, were purchased separately from Sigma and tested independently on immortalized keratinocytes HaCaT cell for their capacity to inhibit the IL-8 production in a dose-dependent manner. HaCaT cells were pre-treated with diclazuril and meclozine, at the concentrations ranging from 0.39 to 50 μM, for 24 h and then stimulated with P. acnes suspension as described in Materials and Methods. The production of IL-8 was measured on culture supernatant by ELISA and the viability of cells was estimated by MTT assay (FIG. 1). For both molecules, diclazuril and meclozine, their capacity to inhibit the production of IL-8 in a dose-dependent manner with an IC50 at about 6 μM (P=0.011) and 3 μM (P=0.0024), respectively (FIG. 1A, B) is confirmed, while no change was observed in pretreated cells without being stimulated. Moreover, no cytotoxicity was observed at the IC50 concentrations (FIG. 1 C, D).

(33) Same results were obtained with two other P. acnes strains (RON, PIE) on primary keratinocytes NHDK and fibroblast (HDF) cell lines.

(34) 2. Diclazuril and Meclozine Dose-Dependently Inhibits P. acnes-Induced IL-1p Production in Monocytes.

(35) Both molecules, were tested to inhibit the production of IL-1β by the monocytic cell line. ThP-1 cells were pretreated with diclazuril and meclozine, at the concentrations ranging from 0.39 to 50 μM, for 24 h and then stimulated with P. acnes suspension as described in Materials and Methods. The production of IL-1β was measured on culture supernatant by ELISA and the viability of cells was estimated by MTT assay (FIG. 2). It has been shown that diclazuril and meclozine were able to inhibit the production of IL-1β in a dose-dependent manner with an IC50 at about 3 μM for diclazuril (P=5.8.Math.10.sup.−6) and 6 μM for meclozine (P=6.9.Math.10.sup.−6) (FIG. 2A, B), with no cytotoxicity for diclazuril (FIG. 2C) and moderate cytotoxicity at 64% for meclozine (FIG. 2D). In parallel we tested the ability of both molecules, diclazuril and meclozine, to inhibit the production of TNF-α by the monocytic cell line and shown no effect (Data not shown).

(36) 3. Diclazuril and Meclozine Inhibit P. acnes-Induced IL-8 and IL-1β mRNA Production.

(37) As it has been shown that P. anes-induced IL-8 and IL-1β protein production were inhibited by diclazuril and meclizine, it has been investigated whether IL-8 and IL-1β traductions were also regulated at the mRNA level. RT-qPCR analysis are used to assess the effect of diclazuril and meclozine on the level of IL-8 and IL-1β mRNA production in HaCaT keratinocyte cells and ThP-1 monocyte cells lines stimulated by P. acnes (FIG. 3A). Both cell lines were pretreated for 24 h with diclazuril and meclozine at 10 μM and stimulated for 5 h with P. acnes. Controls experiments were done with unpretreated/unstimulated cell and with P. acnes stimulated cells only. We showed that P. acnes strongly induced IL-8 and IL-1β mRNA productions. However, when cells were pretreated with diclazuril and meclozine, mRNA-IL-8 and -IL-1β productions were inhibited by up to 97% in HaCaT cells (P=0.002, P=0.002) (FIG. 3A), and by up to 91% and 38% in ThP-1 cells (P=0.002, P=0.047) (FIG. 3B).

(38) 4. Diclazuril and Meclozine Inhibition of MAPK Pathways.

(39) The molecular basis for the inhibition of IL-8 production by diclazuril and meclozine has been investigated, in particular it has been evaluated if they interfered with signaling pathways known to be activated when keratinocytes are stimulated with P. acnes. It has first been confirmed the activation of IL-8 production when cells were stimulated by P. acnes while pre-treatment with both, diclazuril and meclozine, inhibit such production (FIG. 4A). It has also been confirmed that the activation of TLR-2 by P. acnes led to IκB degradation and the stimulation of MAPK pathways in HaCaT cells as a time lag in the response of the HaCaT cell was observed for both the IkB and ERK pathways (FIG. 4B, C, panels P. acnes alone). It has been shown that pre-treating HaCaT cells keratinocytes with diclazuril before P. acnes stimulation did not prevented the degradation of IκB (FIG. 4B). However, both molecules prevented the phosphorylation of ERKs induced by P. acnes (FIG. 4C, panels 10 μM diclazuril, meclozine). Stripping and subsequent reprobing of the blot with antibodies against total ERKs demonstrated no change in total protein levels following P. acnes stimulation, suggesting that P. acnes activated pre-existing ERKs. These data suggest that the inhibition by diclazuril and meclozine of P. acnes-induced IL-8 production in keratinocytes involves downregulation of the MAPK pathways.

(40) 5. Diclazuril and Meclozine Inhibit PGN- and LTA-Induced IL-8 and IL-1β Production.

(41) Previously it has been shown that diclazuril and meclozine were able to inhibit IL-8 and IL-1β productions at the transcriptional and traditional levels in pretreated cells stimulated with P. acnes. Here, it is investigated if the nature of the cell stimuli would have an impact on the diclazuril and meclozine effects on the IL-8 and IL-1β productions. Both, HaCaT and ThP-1 cell lines were pretreated with several concentrations of diclazuril and meclozine ranging from 0.39 to 25 μM for 24 h, and then stimulated with 3 different concentrations of peptidoglycanne (PGN) and lipoteicoic acid (LTA) at 5, 10 and 20 μg/ml for 18 h at 37° C. Levels of IL-8 and IL-1β productions were measured by ELISA on the culture supernatants and shown in FIGS. 5 and 6. It is showed that IL-8 production dose-dependently increase when cell are only stimulated with various concentrations of PGN (5, 10, 20 μg/ml) starting at about 70 pg/ml, raising up to 130 and 180 pg/ml. Pretreatment of keratinocytes with diclazuril decreased the production of IL-8 by an average of 65% at 6.25 μM regardless of the initial PGN dose used (FIG. 5A). Same results were obtained with meclozine and at 6.25 μM the IL-8 production was reduced by 60% (FIG. 5B).

(42) To assess the effect of diclazuril and meclozine on the IL-1β production, monocytic ThP-1 cell line stimulated either by PGN and LTA is used. It has been shown that the production of IL-1β was induced from 170 to 210 pg/ml with PGN (FIG. 6A, B) and from 9 to 12 pg/ml with LTA (FIG. 6C, D). Pretreatment of cells with diclazuril and meclozine, dose-dependently inhibited the IL-1β production. Pretreatment at 6.25 μM with diclazuril decreased the IL-1l production by an average of 64% (FIG. 6A, C), and with meclozine by an average of 36% (FIG. 6B, D).

(43) 6. Time-Dependent Effect of Keratinocyte Pre-Treatment on IL-8 Production.

(44) In this experiment the effect of various time of keratinocytes pretreatment by diclazuril and meclozine on the IL-8 production is evaluated. Keratinocyte HaCaT cell line was pretreated for 1, 6, 24 and 48 h with diclazuril and meclozine at concentrations ranging from 0.39 to 12.5 μM and then stimulated with P. acnes (FIG. 7). For 1 and 6 h pre-treatment, both molecules did not reduce significantly the IL-8 production (FIG. 7A, B). After 24 h pretreatment, we confirmed the effect of diclazuril and meclozine by reducing the IL-8 production by 36% (P=0.03) and 63% (P=0.00004) at 6.25 μM, respectively. Interestingly, when cell pre-treatment was raised to 48 h the IL-8 production decrease by 65% (P=0.0004) with diclazuril and with 78% (P=0.0002) with meclozine (FIG. 7A, B) without any major impact on cell viability (FIG. 7C, D). Here we shown that increasing pre-treatment time of cell contribute to enhance the IL-8 production.

(45) 7. Comparison Between Diclazuril and Meclozine with Molecules Used in Acne Treatment.

(46) In this experiment we compared the effect of diclazuril and meclozine with the most common molecules (antibiotics, benzoyl peroxide, retinoids) used in the treatment of acne on the IL-8 production. Keratinocyte HaCaT cell was pretreated for 24 h with all molecules at 6.25 and 12.5 μM and then stimulated with P. acnes (FIG. 8). As we shown before, diclazuril and meclozine were able to decease the IL-8 production by >60% with no toxicity. In parallel, antibiotics erythromycine and clindamycine as well as benzoyl peroxide have very low none significative or no effect on the IL-8 production with no toxicity. However, we show that retinoids are able to decrease moderately the IL-8 production. Isotretinoin and retinoic acid are able to decrease IL-8 production by 5% and adapalene significantly by 20%. All retinoids exhibits cytotoxicity at about 20% for isotretinoin and retinoic acid and at about 60% for adapalene (FIG. 8A). Moreover, when the concentration of all molecules tested was raised up to 12.5 μM (FIG. 8B), we confirmed the strong effect of diclazuril and meclozine on IL-8 production with no cytotoxicity. However, the other molecules have no effect beside the adapalene witch shown a IL-8 reduction by 90% but associated with 75% cytotoxicity.

(47) 8. Capacity to Inhibit the Inflammatory Reaction Induced by P. acnes in an In Vivo Model of Inflammation.

(48) According to previous results showing in vitro efficacy of both molecules on the anti-inflammatory response, we tested their capacity to inhibit the inflammatory reaction induced by P. acnes in an in vivo model of inflammation.

(49) This model is based on the capacity of mouse ears to react while P. acnes is intradermally injected. The inflammatory reaction is evaluated each day over a period of 4 days after P. acnes injection by measuring the thickness of the ears, the redness as well as the presence of a desquamation and/or small pustules. At the end of the experiment, final measurement of inflammation was realized and photographic pictures of ears were taken. Then, mice were euthanized and ears were immediately fixed in a formalin-containing buffer for a future histological analysis.

(50) The experimental design consisted of 3 groups containing 10 mice each. 1) PBS corresponds to the non-treated group injected with PBS. 2) PA+Vehicle TOPIC corresponds to P. acnes injected in ears treated with Vaseline alone. 3) PA+diclazuril corresponds to P. acnes injected in ears treated with 1.3% diclazuril mixed with Vaseline.

(51) The preparation of the 1.3% diclazuril gel consisted of extemporaneously gently mixing 6.5 mg of diclazuril with 0.5 mg of Vaseline for 1 min at room temperature (21° C.) and then directly applied to the mouse ears.

(52) The results are exposed in FIG. 9. It has been shown that diclazuril VNPA-A2 is able to decrease the ear inflammation in topical application by 37% (FIG. 9A).

(53) 9—Complementary Data for Chemical Diclazuril Analogue Testing

(54) Diclazuril-Analogue RCL PH000645-PH Dose-Dependently Inhibits P. acnes-Induced IL-8 Production in Keratinocytes.

(55) Both molecules, diclazuril and RCL PH000645-PH, were purchased from Sigma and tested independently on immortalized keratinocytes HaCaT cell for their capacity to inhibit the IL-8 production. HaCaT cells were pre-treated with RCL PH000645-PH and diclazuril (used as reference), at the concentrations ranging from 0.39 to 6.25 μM, for 24 h and then stimulated with P. acnes suspension as described in Materials and Methods. The production of IL-8 was measured on culture supernatant by ELISA (FIG. 10) and the viability of cells was estimated by the MTT assay (FIG. 11). We confirmed the inhibition of IL-8 production by diclazuril and shown that the analogue RCL PH000645-PH was able to inhibit the production of IL-8 in a dose-dependent manner with an IC50 at about 3 μM (P=0.000069) (FIG. 10A, B), while no change was observed in pretreated cells without being stimulated. In parallel we tested cell viability and shown no cytotoxicity at the IC50 concentration (FIG. 11).

(56) 10—Complementary Data for Chemical Meclozine Analogue Testing

(57) Lidoflazine, GBR 12909 dihydrochloride, Chlorcyclizine hydrochloride and Lomerizine were purchased from Prestwick and tested independently on immortalized keratinocytes HaCaT cell for their capacity to inhibit the IL-8 production. HaCaT cells were pre-treated with Lidoflazine, GBR 12909 dihydrochloride, Chlorcyclizine hydrochloride and Lomerizine at the concentration of 10 μM, for 24 h and then stimulated with P. acnes suspension as described in Materials and Methods. The production of IL-8 was measured on culture supernatant by ELISA and the viability of cells was estimated by the MTT assay. The results are shown on Table 1 below.

(58) TABLE-US-00001 TABLE 1 Dose-dependent inhibition of IL-8 production by Evaluation of cell keratinocytes stimulated viability after treatment Meclozine by P. acnes pre-treated with meclozine analogues analogues with meclozine analogues on keratinocytes. Lidoflazine 65% 107% GBR 12909 78%  78% dihydrochloride, Chlorcyclizine 54%  81% hydrochloride Lomerizine 58%  95%

(59) We shown that the meclozine analogues Lidoflazine, GBR 12909 dihydrochloride, Chlorcyclizine hydrochloride and Lomerizine were able to inhibit the production of IL-8 ranging from 54 to 78% (Table 1). In parallel we tested cell viability and shown no (95, 107%) or very weak (78, 81%) cytotoxicity at 10 μM concentration (Table 1).

(60) 11—Complementary Data for In Vivo Inflammation Model Testing

(61) According to previous results showing in vitro efficacy of both molecules on the anti-inflammatory response, we tested their capacity to inhibit the inflammatory reaction induced by P. acnes in an in vivo model of inflammation.

(62) This model is based on the capacity of mouse ears to react while P. acnes is intradermally injected. The inflammatory reaction is evaluated each day over a period of 4 days after P. acnes injection by measuring the thickness of the ears, the redness as well as the presence of a desquamation and/or small pustules. At the end of the experiment, final measurement of inflammation was realized and photographic pictures of ears were taken. Then, mice were euthanized and ears were immediately fixed in a formalin-containing buffer for a future histological analysis.

(63) The experimental design consisted of 3 groups containing 8 mice each. 1) PBS corresponds to the non-treated group injected with PBS. 2) PA+Vehicle corresponds to P. acnes injected in ears treated with the vehicle alone. 3) PA+meclozine corresponds to P. acnes injected in ears topically treated with 1% meclozine mixed with vehicle. The preparation of the 1% meclozine gel consisted of extemporaneously solubilizing 5 mg of meclozine in 150 μl of DMSO and in 300 μl of Solutol HS153070/water (30:70, w/w) to finally gently incorporated in 1.5 g of Vaseline for 1 min at room temperature (21° C.) and then directly applied to the mouse ears.

(64) We have shown that meclozine is able to decrease the ear inflammation in topical application by 29.5% (FIG. 12) as it is shown in the ears pictures (FIG. 13). Histological analysis revealed an increase in ear thickness and an important leukocyte infiltration in the P. acnes injected-ears (FIG. 14, panel PA+Vehicle) comparing to negative control (FIG. 14, Panel PBS). When ears were treated with meclozine, the ear thickness decrease as well as the leukocyte infiltration (FIG. 14, Panel PA+Meclozine).

(65) 12—Complementary Data for Signalling Pathways Analysis

(66) Diclazuril and meclozine modulation of inflammatory-related pathways. We investigated the molecular basis for the inhibition of IL-8 production by diclazuril and meclozine, in particular we evaluated if both molecules interfered with inflammatory-related signalling pathways as well as cell adhesion- and lipids-related pathways known to be activated when keratinocytes are stimulated with P. acnes.

(67) We shown that the activation of HaCaT keratinocytes by P. acnes led to the transient p-IκB degradation while IκB was steady. Pre-treating HaCaT keratinocytes with diclazuril and meclozine before P. acnes stimulation did not alter the degradation of p-IκB.

(68) We also shown that the activation of HaCaT keratinocytes by P. acnes led to the activation of p-ERK, p-p38, p-JNK, p-PKC, p-Akt, p-mTOR (FIG. 15, panel P. acnes alone) and p-PI3K (FIG. 16, panel P. acnes alone). However, both molecules prevented the phosphorylation of ERK-, p38-, PKC-, Akt-, and PI3K-induced by P. acnes, while they did not cause any changed in the phosphorylation of JNK-, and mTOR-induced P. acnes (FIGS. 15 and 16, panels diclazuril and meclozine). Interestingly, diclazuril was able to inhibits the phosphorylation of PKC and PI3K stronger than meclozine while meclozine hah a stronger effect on the inhibition of the phosphorylation of Akt. Stripping and subsequent reprobing of the blot with antibodies against total IκB, ERK, p38, JNK, PKC, Akt, mTOR, and PI3K demonstrated no change in total protein levels following P. acnes stimulation, suggesting that P. acnes activated pre-existing all of these proteins (FIGS. 15 and 16).

(69) These data suggested that the inhibition by diclazuril and meclozine of P. acnes-induced IL-8 production in keratinocytes involves downregulation of the MAPK, PKC, Akt and PI3 kinase pathways.

(70) Diclazuril and meclozine modulation of adhesion molecules- and lipids-related pathways. We investigated the up-regulation of adhesion molecules onto keratinocytes which play subsequently an important role in the infiltration of leukocytes into the skin during the inflammation reaction. Stimulating HaCaT keratinocytes by P. acnes increase the expression of the intercellular adhesion molecule-1 (ICAM-1). Pre-treating HaCaT cells with diclazuril had no effect on the P. acnes-induced ICAM-1 expression while meclozine decreased its expression (FIG. 16). Inflammatory acne is a multifactorial disease of the pilosebaceous unit where lipids and fatty acids present in sebum induced an inflammation reaction as well. Increase sebum production is essential to the development of acne and its serves as a nutrient source for P. acnes. It has been shown that the peroxisome-proliferator activated receptors (PPARs) influence lipid catabolism by increasing sebum production and are important mediators of inflammatory responses as well as prostaglandins (PG) induced by the activation of the cyclooxygenase (Cox-2) gene expression (Tsai 2013, Gupta 2015). We then analysed the expression of these markers (PPARs and Cox-2) and shown that the P. acnes-induced keratinocytes increase the expression of Cox-2, PPARα and PPARβ and no significant change in PPARγ expression. Pre-treating HaCaT cells with diclazuril decrease the expression of Cox-2 and no change for the PPARs expression. On the other hand, meclozine treatment strongly decrease the expression of Cox-2 as well as for the PPARα and PPARβ (FIG. 7)

(71) These data suggested that diclazuril inhibited weakly the ICAM-1 and the Cox-2/PPARs expressions, while meclozine had a stronger inhibitory capacity.

(72) 13—Complementary Data for Psoriasis Testing

(73) To assess the meclozine and diclazuril anti-inflammatory activities on psoriasis, we used an in vitro model of normal human epidermal keratinocytes (NHDK) stimulated by a pro-inflammatory mixture M5 mimicking a psoriasis-like phenotype. We then evaluated the meclozine and diclazuril abilities to inhibit the release of IL-8 and of β-defensin-2 protein (hBD-2) by the keratinocytes stimulated in this condition (Rabeony et al., 2014).

(74) The M5 stimulation on primary keratinocytes had no deleterious effect on cell viability. When cells are pre-treated with the JAK inhibitor we observed a decrease by 24.4% of the cell viability. Pre-treatment of cells with meclozine decrease the cell viability by 0.14 to 15%, while pre-treatment with diclazuril decrease the cell viability by 20 to 62% (FIG. 17).

(75) Meclozine and diclazuril activity on IL-8 production. In basal conditions, normal human epidermal keratinocytes (NHDK) produced a small amount of IL-8. The IL-8 production was greatly increased by the stimulation with the combination of 5 cytokines. The reference Jak Inhibitor I (positive control) moderately inhibited the stimulating effect of this association (49% inhibition) (FIGS. 18 and 19). Under the experimental conditions of this study, meclozine had no effect on the IL-8 production (FIG. 18) while diclazuril decreased the IL-8 production in a dose-dependent manner with an IC50 around 2-3 μM with a decrease by 66% at 3,125 μM (FIG. 19).

(76) Meclozine and diclazuril activities on hBD-2 production. In basal conditions, normal human epidermal keratinocytes released a very small amount of β-defensin-2 protein (hBD-2). The hBD-2 production was greatly increased by the treatment with the combination of 11-17, TNF-α and OSM. The reference Jak Inhibitor I (positive control) inhibited the stimulating effect of this association (38% inhibition) (FIGS. 20 and 21). Under the experimental conditions of this study, meclozine had no effect on the hBD-2 production (FIG. 20) while diclazuril decreased the hBD-2 production in a dose-dependent manner with a decrease by 68% at 12.5 μM (FIG. 21).

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