5-HYDROXYTRYPTAMINE 1B RECEPTOR-STIMULATING AGENT FOR SKIN AND/OR HAIR REPAIR

Abstract

The present invention relates to the field of skin and/or hair repair, for therapeutic and cosmetic purposes. It more specifically relates to an agent stimulating the 5-hydroxytryptamine 1B receptor, for use for boosting skin and/or hair repair.

Claims

1. A 5-hydroxytryptamine 1B receptor (5-HT1 BR)-stimulating agent for a therapeutic use as a booster of skin and/or hair repair in patients suffering from pathological tissue, skin and/or hair loss, damage or impairment.

2. The agent for use according to claim 1, selected from the group consisting of antidepressant agents and antimigraine drugs, pharmaceutically acceptable derivatives, analogs, isomers, metabolites, salts, solvates, clathrates, polymorphs, and co-crystals thereof, and combinations thereof.

3. The agent for use according to claim 2, wherein said antidepressant agent is selected from the group consisting of: atypical antidepressants, preferably bisarylsulfanyl amines such as vortioxetine, and tianeptine, agomelatine, nefazodone, trazodone, buspirone, tandospirone, and ketamine; selective serotonin reuptake inhibitors (SSRIs), preferably fluoxetine, citalopram, escitalopram, sertraline, norsertraline, fluvoxamine, femoxetine, indalpine, alaproclate, cericlamine, ifoxetine, zimelidine, dapoxetine, etoperidone, and metabolites thereof such as desmethylcitalopram, didesmethylcitalopram, and seproxetine; serotonine and norepinephrine reuptake inhibitors (SNRIs), preferably duloxetine, venlafaxine, desvenlafaxine, milnacipran, levominalcipran, and sibutramine; serotonin-norepinephrine-dopamine reuptake inhibitor (SNDRIs), preferably bicifadine, brasofensine, tesofensine, and nomifensine; tricyclic antidepressants (TCAs), preferably clomipramine, amoxapine, nortriptyline, maprotiline, trimipramine, imipramine, desipramine and protriptyline; monoamine oxidase inhibitors (MAOs), preferably iproniazide, phenelzine, tranylcipromine, moclobemide, selegiline and rasagiline; and noradrenergic and specific serotoninergic antidepressants (NaSSAs), preferably mirtazapine, mianserin, aptazapine, esmirtazapine, setiptiline and S32212 (also known as N-[4-methoxy-3-(4-methylpiperazin-1-yl)phenyl]-1,2-dihydro-3H-benzo[e]indole-3-carbo-xamide).

4. The agent for use according to claim 2, wherein said antimigraine drug is ergotamine or a triptan, said triptan being preferably selected from the group consisting of sumatriptan, rizatriptan, zolmitriptan, eletriptan, almotriptan, frovatriptan, naratriptan avitriptan, and donitriptan.

5. The agent for use according to any one of claims 1 to 4, wherein said 5-hydroxytryptamine 1B receptor (5-HT1 BR)-stimulating agent is an atypical antidepressant, preferably a bisarylsulfanyl amine such as vortioxetine, tianeptine, agomelatine, nefazodone, trazodone, buspirone, tandospirone, and ketamine.

6. The agent for use according to any one of claims 1 to 5, wherein said agent is modified to comprise at least one charged chemical moiety, preferably positively charged.

7. The agent for use according to claim 6, wherein said positively charged chemical moiety is a quaternary ammonium group or a tertiary sulfonium group.

8. The agent for use according to claim 7, wherein said quaternary ammonium group has the formula (I)
(NR.sub.1R.sub.2R.sub.3).sup.+Z.sup.(I) wherein Z is an organic or inorganic anion; and R.sub.1, R.sub.2 and R.sub.3 are each independently selected from the group consisting of alkyl, aryl and cycloalkyl; or said tertiary sulfonium group has the formula (II)
(SR.sub.4R.sub.5).sup.+Z.sup.(II) wherein Z is an organic or inorganic anion; and R.sub.4 and R.sub.5 are each independently selected from the group consisting of alkyl, aryl and cycloalkyl.

9. The agent for use according to any one of claims 6 to 8, wherein said agent is a positively charged vortioxetine selected from the group consisting of salts of vortioxetine, vortioxetine coupled to a positively charged amino acid such as histidine, arginine or lysine, pyrrolidinium-vortioxetine, pyperazinium-vortioxetine, dimethylammonium-vortioxetine, sulfonium-vortioxetine, N-oxide-vortioxetine, sulfoxide-vortioxetine, and phosphonium-vortioxetine.

10. A therapeutic composition for treating or repairing pathological skin and/or hair loss, damage or impairment, wherein said composition comprises at least one 5-hydroxytryptamine 1B receptor (5-HT1 BR)-stimulating agent as defined in any one of claims 1 to 9 and at least one acceptable excipient.

11. A package product comprising a first product for local administration of the agent defined in any one of claims 3 to 9 and a second product for systemic administration of the agent defined in any one of claims 3 to 9, wherein both first and second products are packaged for simultaneous, separate or concomitant use in patients suffering from pathological tissue, skin and/or hair loss, damage or impairment, wherein the first product is formulated for local administration at the areas receiving the engraftment of a skin, tissue or scalp prior, after, or both to the engraftment and the second product is for a systemic administration such as oral route or subcutaneous injection in the areas to be treated.

12. A package product according to claim 11 wherein the graft material, such as skin or scalp, is in vivo pretreated with the agent defined in any one of claims 3 to 9 before surgery.

13. A package product according to claim 11, comprising a third product consisting of a product for ex vivo pretreating a graft material, such as skin or scalp, with the agent defined in any one of claims 3 to 9 before surgery.

14. A package product comprising the first and third product or the second and third product as defined in claim 11, 12 or 13.

15. A package product according to anyone of claims 11 to 14 wherein the agent is fluoxetine or vortioxetine.

16. A pre-containing seringe comprising the agent defined in any one of claims 3 to 9 in an effective amount to accelerate tissue, hair or skin repairs, to improve the tissue, hair, or skin function or to improve tissue, hair, or skin structure to an extent leading to restoring significantly the original and natural appearance, structure, function of said tissue, hair, or skin, or the three of them.

17. A pre-containing seringe according to claim 16, wherein said agent is fluoxetine or vortioxetine.

18. A package kit comprising at least 6, 12 or 24 pre-contained seringes as defined in claim 16 or 17.

19. The agent for use according to any one of claims 1-9, or the therapeutic composition for use according to claim 10, wherein said pathological skin and/or hair loss, damage or impairment is selected the group consisting of skin redness or soreness, irritated skin, blisters, wounds, burns, abscess, bedsores and skin ulcer.

20. The agent for use according to any one of claims 1-9, or the composition for use according to claim 10, wherein said pathological skin and/or hair loss, damage or impairment is alopecia, preferably alopecia involving inflammation of the scalp, such as scarring alopecia.

21. Cosmetic use of a 5-hydroxytryptamine 1B receptor (5-HT1 BR)-stimulating agent as defined in claim 2-9 for repairing an induced skin and/or hair loss, damage or impairment that has been made for example by a means selected from the group consisting of: abrasion such as micro-abrasion or microdermabrasion, incision such as micro-incision, injection such as microinjection, perforation such as microperforation, peeling, hair depilation or shaving, electrical stimulation, laser, hair or skin graft, surgery, and combinations thereof.

22. Cosmetic use of a 5-hydroxytryptamine 1B receptor (5-HT1 BR)-stimulating agent as defined in claim 2-9 for boosting and/or promoting and/or accelerating skin and/or hair repair or for slowing, attenuating and/or countering natural skin and/or hair loss, damage or impairment in a subject.

23. Cosmetic use of a 5-hydroxytryptamine 1B receptor (5-HT1 BR)-stimulating agent as defined in claim 2-9 for rejuvenating the skin and/or for limiting natural loss of hair in a subject.

24. A 5-hydroxytryptamine 1B receptor (5-HT1 BR)-stimulating agent as defined in any one of claims 1 to 9 and an active agent, as a combined preparation for simultaneous, separate or sequential administration for treating a subject suffering from pathological skin and/or hair loss, damage or impairment.

25. A 5-hydroxytryptamine 1B receptor (5-HT1 BR)-stimulating agent as defined in any one of claims 1 to 9 combined with a means for inducing skin and/or hair loss, damage or impairment as defined in claim 13, for simultaneous, separate or sequential administration for treating a subject suffering from a pathological skin and/or hair loss, damage or impairment.

26. Use of a cosmetic method comprising: a) administering to a subject at least one 5-hydroxytryptamine 1B receptor (5-HT1 BR)-stimulating agent; and b) further administering to said subject at least one means inducing skin and/or hair loss, damage or impairment, simultaneously, separately or sequentially to (i.e. before or after) step a), for boosting and/or promoting and/or accelerating skin and/or hair repair.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0377] FIG. 1: Fluoxetine and vortioxetine speed up the wound healing process. Macroscopic observations. (A) Scheme of fluoxetine and vortioxetine delivery and sampling times. (B) Size of the wound through time in the treated and placebo mice. The treatment was given prior to the injury and continued during the healing process. (C-K) Illustrations of wounds at different time points under different treatments. (L) Size of the wound after injury and topical application of vortioxetine. No pre-treatment was performed. Scale bar is 4 mm, *p0.05; **p0.01; nothing: not significant. All statistics are done with Mann-Whitney test and compared with placebo.

[0378] FIG. 2: Fluoxetine and vortioxetine speed up the wound healing process. Histological observations. (A-C) H&E staining of the wounds at T0, right after the injury, the biopsies were fixed in formol and H&E stained. Arrows are pointing the vessels. (D) Quantification of the number of vessels per mm.sup.2 in the placebo, fluoxetine and vortioxetine treated mice. (E-G) H&E of skin around the wound 5 days post injury. Arrows represent the size of the wound. (H-J) H&E of skin around the wound 16 days post injury. Arrows represent the size of the wound. Scale bar is 2 mm, *p0.05; **p0.01; nothing: not significant. All statistics are done with Mann-Whitney test and compared with placebo.

[0379] FIG. 3: Cellular behaviour at the site of the wound healing after fluoxetine and vortioxetine treatment. (A) Histological measurement of the wound area. (B) Distance between epithelial tips 5 days post injury. (C) Length of the epithelial tongue 5 days post injury. (D) Cell infiltration in the dermis 5 days post injury. (E) Percentage of Ki67+ representing the cycling cells 5 and 14 days post injury in the fluoxetine, vortioxetine and placebo treated mice. *p0.05; **p0.01; nothing: not significant. All statistics are done with Mann-Whitney test and compared with placebo.

[0380] FIG. 4: No changes in hair length is detected under treatment but hair growth after shaving is faster. (A) Length of hairs measured one by one (n=50 hairs per mice and n=4 mice) in the placebo, fluoxetine and vortioxetine treated mice. (B) Size of the hair follicle after treatment. (C-E) H&E representing a transversal cut of the skin after treatment. Arrow pointing the hair follicle. (F) Thickness of the skin after placebo, fluoxetine and vortioxetine treatment. (G) Representative picture of the 2 protocol used on the same mouse. Right part is shaved with electric clipper; left part is shaved with wax. (H-M) pictures of the skin of mice in placebo, fluoxetine and vortioxetine treated animals, 5 and 10 days after depilation. (N) Relative intensity of darkness of the skin in the placebo, fluoxetine and vortioxetine treated mice measured after photography and image J relative intensity of luminescence. (O) Skin thickness in placebo, fluoxetine and vortioxetine treated mice 5 and 10 days after shaving.

[0381] FIG. 5: Size of the wound 6 days after injury in control mice, mice topically treated with CP 94253 (0.5 mol) and mice topically treated with the 5HT1 BR agonist CP 94253 and the 5HT1 BR antagonist GR127935 as disclosed in example 3.

EXAMPLES

Fluoxetine and Vortioxetine Accelerate Wound Healing and Hair Growth

1. Material and Methods

1.1. Mice and Injections

[0382] All procedures in this study were approved by the Animal Care and Use committee at the Institut Pasteur (CETEA 2016-0007). Unless specified, 6 weeks old male mice C57Bl/6Rj were used in this study and housed on a 12:12 light/dark cycle in a pathogen free facility with controlled temperature and humidity. Food and drink were given ad libitum. Fluoxetine was dissolved in 0.9% NaCl and delivered either by intra peritoneal injections or per os at 18 mg/Kg. Control group received daily injections of 0.9% NaCl. For vortioxetine mice also received the product either dissolved in 96% Ethanol and then diluted 1/200 in water ad libitum at 20 mg/kg or dissolved in 96% Ethanol and then diluted 1/200 in 0.9% NaCl, final concentration 20 mg/kg body weight corrected. For topical applications vortioxetine was dissolved in 0.9% Nacl and directly applied in the wound the 3 first days and then on top on the wound the following days, every day.

1.2. Wound Biopsy

[0383] Mice were anesthetized using Isoflurane, initially in an induction chamber. The anaesthetised mice were transferred to the anaesthetic nose-cone and maintained on a heated block throughout the procedure (typically for about 5 minutes). Analgesic was administered by subcutaneous injection e.g. buprenorphine 1.5 l of 5% w/v solution, diluted in 30 l sterile water. The area of the biopsy was shaved. Then an area of dorsal skin was rendered sterile by swabbing with Videne surgical scrub followed by sterile water. A 8 mm sterile punch biopsy was used to create an incision through the epidermis/dermis to the muscle fascia. Any bleeding at this step was carefully avoided. The punch biopsy was removed and the skin separated from muscle connective tissues using forceps or blunted scissors. Mice were then removed from the anesthetic nose-cone and housed in sterilised cages containing paper liner and paper bedding. They were kept warm (using a cage placed on a heated mat) until fully recovered from the effects of the anaesthetic.

1.3. Mice Shaving

[0384] 2 different protocols were used to remove the hair from the mice. Mice were anesthetized using Isoflurane, initially in an induction chamber. The anaesthetised mice were transferred to the anaesthetic nose-cone and maintained on a heated block throughout the procedure (typically for about 5 minutes). In protocol 1, shaving was performed by cutting the hair: the mice were gently restrained manually and half of their back was shaved with portable electric hair clippers. In protocol 2, some hair was firstly removed with forceps and wax was used to unstack the hair. The wax was applied twice for maximum efficiency.

1.4. Histological Analysis

[0385] Tibialis anterior muscle was carefully dissected and snap frozen in liquid-nitrogen-cooled isopentane for a few minutes and stored at 80 C. prior to cryosectioning (10 m sections). Sections were kept at room temperature overnight before staining. Sections were then rehydrated in PBS for 10 minutes and fixed in 10% formalin for 3 minutes. The sections were then routinely stained with haematoxylin and eosin (H&E) using an automated stain machine. The slides were assessed by double blinding and automated when possible (fibre diameter, cell count).

1.5. Re-Epithelization and Measure of Epithelial Tongue

[0386] Width of the wound and distance of the traversed epithelium were measured on H&E.

[0387] Percent of re-epithelization was calculated according to the following formula: [distance covered by epithelium]/[distance between wound bed]100.

[0388] The extent of epithelialization and granulation tissue formation was determined on H&E-stained paraffin tissue sections. The length of the epithelial tongue was determined as the distance that the neo-epithelium had migrated from the margin of the wound as defined by the presence of hair follicles in non-wounded skin.

1.6. Skin Color

[0389] The skin color of the mouse skin was measured three times in 5 different locations of the shaved skin randomly and averaged within a 1616 mm2 field, using ImageJ. The luminance measured reflects the relative darkness ranging from white (0) to black (1 0 0).

1.7. Immunostainings

[0390] Immunostaining was performed on formol fixed sections. Permeabilized with 0.5% Triton X-100 20 min at room temperature, washed, and blocked with 10% BSA for 30 min. Sections were incubated with primary antibodies overnight at 4 C. and with Alexa-conjugated secondary antibodies 1/250 and Hoechst for 45 minutes. Sections were then analysed using an automated axioscan (Zeiss) or inverted Observer.Z1 Apotome (Zeiss).

1.8. Statistical Analysis

[0391] Statistical analysis was performed using GraphPad Prism software using appropriate tests (non-parametric Mann-Whitney unless specified) and a minimum of 95% confidence interval for significance; p values indicated on figures are <0.05 (*), <0.01(**), and <0.001 (***). Figures display average values of all animals testedSD or SEM for RT-qPCR, or as indicated.

2. Results

2.1. Macroscopic Observations of the Wound Healing

[0392] In order to assess the regenerative potential of the skin, a 8 mm biopsy of the skin was performed in C57Bl/6 mice, which were either treated per os with fluoxetine at 18 mg/kg or with vortioxetine at 20 mg/kg for 6 weeks and 12 days respectively (FIG. 1A) and during the healing process.

[0393] No difference was noted in the size of the wound for the first 24 hours. Two days post injury, a smaller size in the wound of fluoxetine was observed in treated animals compared with placebo (FIG. 1B). Four days post injury, the wound was smaller in both fluoxetine and vortioxetine treated animals compared with placebo group (FIG. 1B). In the course of wound closure, the size was constantly smaller in fluoxetine and vortioxetine treated animals until 16 days post injury (FIG. 1B-K). Six days post injury, the area of the wound was 15.67 mm.sup.24.2 in the placebo, 11.48 mm.sup.23.1 in the fluoxetine treated and 10.10 mm.sup.24.4 in the vortioxetine treated animals (FIG. 1B, D, G). Sixteen days post injury, the skin of the fluoxetine and vortioxetine treated animals was virtually completely regenerated at the macroscopic level. Indeed the area of the wound was still 0.5 mm.sup.20.03 in the placebo against 0.05 mm.sup.20.004 in the fluoxetine treated animals and vortioxetine (FIG. 1B, H, K).

[0394] To further confirm the data, the same protocol was performed on C57Bl/6 mice wounded with 8 mm biopsy, but this time the mice were not pre-treated and received topical (300 l) applications of vortioxetine at a concentration of 20 mg/ml diluted in 96% ethanol. The placebo received the same amount of 96% ethanol. No differences between the sizes of the wounds were observed from T0 (when the wound is created) to 4 days post injury. Six days post injury, the size of the wound was smaller in the animals topically treated with vortioxetine (FIG. 1L). Eight days post injury, the size of the wound was 10 times lower than the placebo (FIG. 1L). Sixteen days post injury, the size of the wound was 0.20 mm.sup.20.07 whereas it was virtually completely closed in the topical vortioxetine treated (FIG. 1L).

2.2. Histological Observations 5 and 14 Days Post Injury

[0395] To further characterize the wound closure, histological analysis was performed at the time of the biopsy (T0), 5 days post injury and 14 days post injury.

[0396] At the time of the biopsy,the skin was collected from the wound and the number of vessels was counted by H&E staining. Animals treated with fluoxetine or vortioxetine had more vessels than mice that received the placebo (FIG. 2A-D). Indeed, the placebo displayed 4.81.3 vessels/mm.sup.2 whereas the fluoxetine-treated mice displayed 8.22.7 vessels/mm.sup.2 and the vortioxetine-treated mice displayed 8.73.5 vessels/mm.sup.2 (FIG. 2D).

[0397] To further confirm these macroscopic observations, an H&E staining was also performed on the injured skin 5 and 14 days post biopsy. A smaller open wounded area was observed in the fluoxetine and vortioxetine-treated mice both at 5 days post injury (FIG. 2E-J). Measurement of the wound area on digital images showed that the differences between fluoxetine and vortioxetine treated mice and placebo mice were statistically significant on day 5 and day 14 post injury (FIG. 3A).

[0398] Another readout of skin regeneration is the extent of re-epithelization. On H&E-stained paraffin sections, representing the longitudinal diameter of the wound, significantly shorter distances between the tips of the epithelial tongues were measured for the wounds of fluoxetine and vortioxetine mice compared with placebo animals on days 5 wounding (FIG. 3B). The length of the epithelial tongue itself was determined as the distance that the neo-epithelium had migrated from the margin of the wound as defined by the presence of hair follicles in non-wounded skin. Likewise, the length of the epithelial tongues was significantly increased in fluoxetine and vortioxetine treated mice in comparison with placebo mice on day 5 after injury (FIG. 3C). Interestingly more cells were detected in the fluoxetine and vortioxetine treated mice when compared with placebo treated mice 5 days post injury (FIG. 3D). This infiltration was due to higher cell division and immune cell infiltration (inflammation). A first evidence came from the number of K167+ cells (marker of proliferating cells) that is higher in the wound of fluoxetine and vortioxetine treated mice compared with placebo 5 days post injury (FIG. 3E). No differences were detected 14 days post injury in the number of cycling cells (FIG. 3E).

2.3. Hair Growth is Correlated to Wound Healing

[0399] The hair follicle (HF) is one of the most functionally diverse mini-organs in the mammalian body, which mediates functions as varied as thermal homeostasis, structural protein and hormone biosynthesis, immune defense, UV protection, sensory perception (Schneider et al., 2009). Over several decades, intensive research has characterized the phenotypical flexibility and immense proliferative capacity of HF cells under conditions of homeostasis. In contrast, although correlation between the HF and skin wound healing/regeneration has long been noted, only recently has the molecular and cellular basis for these observations begun to be addressed. Early evidence revealed that shallow wounds, where portions of HFs remained intact, healed more quickly. Recent studies have shown that a substantial portion of the neoepidermis in wounded skin is provided by epithelial stem cells residing in the HF (Ito et al., 2005; Levy et al., 2007; Snippert et al., 2010). This response seems specific to wounding, as the HF does not appear to contribute to epithelial homeostasis under physiological conditions.

[0400] Considering that the present data demonstrate a better regeneration of the skin after a treatment with fluoxetine or vortioxetine, and with the existing strong links between HF and skin regeneration via its stem cells, it was further investigated whether the HF could benefit from such treatment.

[0401] To study the possible effect of fluoxetine and vortioxetine on hair growth, fluoxetine (at 18 mg/kg) or vortioxetine (at 20 mg/kg) was provided per os to mice for 6 weeks and 12 days, respectively. No differences in hair size was detected (FIG. 4A), neither in hair follicle length (FIG. 4B-E). Last, skin thickness, which has been shown to correspond to hair follicle length and defined as the distance from the epidermal granular layer to the top edge of the panniculus carnosus, did not change (FIG. 4F). However, when the hairs were challenged with shaving or depilation, a faster growth of hairs was observed

[0402] In order to study the influence of fluoxetine or vortioxetine on hair growth after a stress 2 different protocols were used on the same mouse. The half right part of the mouse back was shaved by cutting the hair with hairs clippers, and, in parallel, the hairs were removed using wax to unstack the hairs on the left part of the back (FIG. 4G). A faster growth of hairs was observed in both protocols (FIG. 4G-O). More advanced hair growth was observed 5 days after shaving in the animals treated with fluoxetine or vortioxetine compared with placebo (FIG. 4I,L,N). This was still true 10 days after shaving with longer hairs in the treated animals compared with placebo (FIG. 4J,M,N). However, 24 days after shaving, when hairs were supposed to be fully regenerated, no differences could be noted between the groups. The skin thickness measured by H&E staining confirmed the previous observation of a more advanced, faster regeneration of the hairs after shaving under fluoxetine and vortioxetine treatment (FIG. 4O). These observations are in favor of a faster growth and faster regeneration but not bigger or longer hairs.

2. Discussion

[0403] In this study, the inventors showed that the delivery of either fluoxetine or vortioxetine was speeding up the wound closure of injured mice. Indeed, the wound was in average 27% smaller after fluoxetine treatment and 35% smaller after vortioxetine treatment between the 4.sup.th and the 14.sup.th day post-injury. The same observation was also made when delivering the vortioxetine directly onto the skin (i.e. topically) without any pre-treatment, however longer delays were observed. Interestingly, when in quiescence (telogen phase) the hair did not seem to react to the treatment as the hair follicle or length of the hairs did not change. However, it seemed that the hair follicle growth was improved after shaving in treated mice compared with placebo.

[0404] Although the skin seemed to also be impacted after fluoxetine or vortioxetine treatment alone (without shaving or biopsy), this particular condition needed to be further investigated with more precise tools. For example, a clear increase in the number of vessels was observed, and the skin seems to be thicker. The inventors further showed that fluoxetine and vortioxetine were acting via the stimulation of 5-HT1b receptor.

3. Stimulating 5-HT1b Receptor Involves a Pro-Regenerative Process

[0405] Mice were treated with another 5HT1 BR agonist (CP 94253) directly on the wound (topic administration) at a concentration of 0.5 mol, 12 h after a wound injury.

[0406] No pretreatment was performed on this set of experiments and the drug was administered once a day for the totality of the duration of the wound closure. Controls group received PBS topically only, once a day for the total duration of the treatment.

[0407] This direct stimulation of 5HT1 BR triggers a faster regeneration and faster closure of the wound. Indeed 6 days post injury, the wound drops from 15.092.3 to 7.72.5 (p=0.0019) (FIG. 5, second column).

[0408] Interestingly, the concomitant administration of an antagonist against 5HT1 BR (GR127935 hydrochloride inhibitor), delivered by means of osmotic pumps at 4 mg/kg, blocked partially the positive effects observed with the agonist. The size of the wound decreased from 15.092.3 to 10.33.1 (p=0.01) (FIG. 5, last column).

[0409] These results show that the direct stimulation of 5HT1 BR receptor speeds up the closure of the wound.

[0410] The use of 5HT1 BR antagonist showed a partial decrease in this healing effect. This means that 5HT1 BR is sufficient to trigger the pro regenerative effect observed on the skin but that other receptors could be involved as well.

[0411] Taken together those results demonstrate that stimulation of 5HT1b is involved in the pro regenerative process but that other mechanisms may be at play.

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