5-HYDROXYTRYPTAMINE 1B RECEPTOR-STIMULATING AGENT FOR ENHANCING IN VIVO ENGRAFTMENT POTENTIAL

Abstract

The present invention relates to the field of regenerative medicine, and more particularly to the improvement of the in vivo engraftment potential of biological material to be administered to a subject in need thereof.

Claims

1. A method for enhancing the in vivo engraftment potential of a biological material, comprising at least the step of in vitro and/or ex vivo contacting an isolated biological material with at least one 5-hydroxytryptamine 1B receptor (5-HT1 BR)-stimulating agent.

2. The method according to claim 1, wherein said agent is 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 method 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, paroxetine, fluvoxamine, femoxetine, indalpine, alaproclate, cericlamine, ifoxetine, zimelidine, dapoxetine, etoperidone, and metabolites thereof 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 method 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 method according to any one of claims 1 to 4, wherein said agent is modified to comprise at least one charged chemical moiety, preferably positively charged.

6. The method according to claim 5, wherein said positively charged chemical moiety is a quaternary ammonium group or a tertiary sulfonium group.

7. The method according to claim 6, 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.

8. The method according to any one of claims 5 to 7, wherein said agent is a positively charged vortioxetine selected from the group consisting of salts of vortioxetine, vortoxietine coupled to at least one 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.

9. The method according to any one of claims 1 to 8, wherein said biological material is selected from the group consisting of cells, tissues, organs, derivatives thereof, and combinations thereof.

10. An isolated biological material with enhanced in vivo engraftment potential, obtainable according to the method as defined in any one of claims 1 to 9.

11. A medical device comprising at least the biological material according to claim 10 and optionally at least one pharmaceutically acceptable excipient.

12. The biological material according to claim 10, or the medical device according to claim 11, further comprising at least one active agent.

13. An isolated biological material as defined in claim 10 or 12, or a medical device as defined in claim 11 or 12, for use as a medicament.

14. An isolated biological material as defined in claim 10 or 12, or a medical device as defined claim 11 or 12, for use in a subject in need of cell, tissue or organ regeneration.

15. A 5-hydroxytryptamine 1B receptor (5-HT1 BR)-stimulating agent as defined in any one of claims 1 to 8, for a pre-therapeutic use in a subject in need of cell, tissue or organ regeneration.

16. A 5-hydroxytryptamine 1B receptor (5-HT1 BR)-stimulating agent as defined in any one of claims 1 to 8, for a pre-therapeutic use in a subject in need of a treatment intended for cell, tissue or organ regeneration, and co- and/or post-therapeutic use in said subject undergoing said treatment.

17. A 5-hydroxytryptamine 1B receptor (5-HT1 BR)-stimulating agent as defined in any one of claims 1 to 8, for a co-therapeutic and/or post-therapeutic use in a subject undergoing cell, tissue or organ regeneration.

18. An isolated biological material and a 5-hydroxytryptamine 1B receptor (5-HT1 BR)-stimulating agent as defined in any one of claims 1 to 8, as a combined preparation for simultaneous, separate or sequential administration in a treatment intended to regenerate cells, tissues or organs in a subject in need thereof.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0332] FIG. 1: Fluoxetine and vortioxetine increase the engraftment potential of the muscle stem cells. (a) Scheme of injection of fluoxetine. Rag.sup.−/−γC.sup.−/− mice were treated with fluoxetine for 6 weeks, injured with cardiotoxin and grafted 24 hours post-injury with FACS cell sorted satellite cells. 28 days post injury mice were sacrificed and tibialis anterior were fixed. (b-c) Transversal cut of the tibialis anterior immunostained with Laminin and FLAP on (b) placebo and (c) fluoxetine treated mice. (d) Histogram representing the percentage of PLAP+ fibres among all the fibres in placebo and fluoxetine treated animals. (e) Scheme of injection of vortioxetine. Rag.sup.−/−γC.sup.−/− mice were treated with vortioxetine for 12 days, injured with cardiotoxin and grafted 24 hours post-injury with FACS cell sorted satellite cells. 28 days post injury mice were sacrificed and tibialis anterior were fixed. (f-g) Transversal cut of the tibialis anterior immunostained with Laminin and PLAP on (f) placebo and (g) vortioxetine treated mice. (h) Histogram representing the percentage of PLAP+ fibres among all the fibres in placebo and fluoxetine treated animals. Means±SD are displayed * p<0.05.

[0333] FIG. 2: Fluoxetine and vortioxetine increase the engraftment potential of hematopoietic stem cells. (a) Scheme of injection of fluoxetine. Rag.sup.−/−γC.sup.−/− mice were treated with fluoxetine for 3 weeks and irradiated at 95cGy. 2 hours post irradiation mice were injected intra venously with fresh bone marrow coming from Tg:ActinGFP. 5 weeks post grafting the Rag.sup.−/−γvC.sup.−/− bone marrow was flushed and analysed by cytometry. (b) Percentage of GFP+ cells among the Rag.sup.−/−γC.sup.−/− bone marrow 5 weeks after grafting. (c) Cytometry dot plot analysis of Rag.sup.−/−γC.sup.−/− bone marrow 5 weeks after grafting. (d) Scheme of injection of vortioxetine. Rag.sup.−/−γC.sup.−/− mice were treated with vortioxetine for 12 days and irradiated at 95cGy. 2 hours post irradiation mice were injected intra venously with fresh bone marrow coming from Tg:ActinGFP. 5 weeks post grafting the Rag.sup.−/−γC.sup.−/− bone marrow was flushed and analysed by cytometry. (e) Percentage of GFP+ cells among the Rag.sup.−/−γC.sup.−/− bone marrow 5 weeks after grafting. (f) Scheme of injection of vortioxetine. In this condition the treatment with vortioxetine started the day of the irradiation. 2 hours post irradiation mice were injected intra venously with fresh bone marrow coming from Tg:ActinGFP. 5 weeks post grafting the Rag.sup.−/−γC.sup.−/− bone marrow was flushed and analysed by cytometry. (g) Representative histogram of the count and quantification of GFP+ cells by cytometry after vortioxetine treatment compared with placebo. Means±SD are displayed *p<0.05; **p<0.005; ns: statistically not significant.

[0334] FIG. 3. Fluoxetine and vortioxetine treatment stimulate the 5TH1B receptor Scatter plot representing the percentage of PLAP+ fibers (coming from the graft) after grafting muscle stem cells in placebo, fluoxetine treated, and fluoxetine+GR127935 5HT1B inhibitor treated Rag2−/−γC−/− immunodeficient host mice.

EXAMPLES

Fluoxetine and Vortioxetine Improve the Engraftment Potential of Muscle Stem Cells and Hematopoietic Stem Cells

1. Material and Methods

1.1. Mice Injection and Injury

[0335] All protocol used were approved by local ethic comity. Mice were anesthetised and injured with cardiotoxin 18 hours before grafting in the tibialis anterior. Before grafting the mice were anesthetised again and grafted with 10,000 cells in 40 nl in PBS. Mice were kept for 28 days until muscle regeneration was complete. For bone marrow grafting, mice were irradiated at 95cGy and grafted intra venous with non-irradiated bone marrow. Mice were sacrificed 5 weeks after the injection.

1.2. Histological Analysis

[0336] Tibialis anterior was carefully dissected and snap frozen in liquid-nitrogen-cooled isopentane for a few minutes and stored at −80° C. prior to cryosectioning (10 μm 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 PLAP antibody manually.

[0337] The slides were assessed by double blinding and automated when possible.

1.3. FACS Analysis

[0338] For bone marrow analysis, animals were sacrificed and bone marrow was flushed and filtered through 40 μm cell strainer. The bulk was then directly assessed using Gallios A94303 (Beckman Coulter) and analysed with Kaluza flow cytometry software.

1.4. Image Analysis

[0339] For image analysis, ImageJ 1.46r software was using between 10 different photos randomly taken per section and 3 sections minimum per experimental group. The pictures were converted in a binary image and then the pixel values were collected. For fibre size, the sections were immunostained with rabbit anti Laminin (Sigma-Aldrich #L9393) diluted at 1/200, overnight at 4° C. Secondary Donkey ant Rabbit 488 (DL488 JacksonImmuno #711486152) were used at 1/200 45 minutes at room temperature. The fibre perimeter was done automatically by using Pixcavator® software.

1.5. Statistical Analysis

[0340] 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 tested±SD or ±SEM for RT-qPCR, or as indicated.

2. Results

2.1. Fluoxetine and Vortioxetine Treatment Triggers an Increase in Engraftment Potential of Muscle Stem Cells in an Injured Paradigm

[0341] In order to investigate the potential effect of fluoxetine and vortioxetine on the muscle engraftment potential, Rag.sup.−/−γC.sup.−/− immunodeficient mice (Sanchez et al., 2013) were used in this study. Mice were injured with cardiotoxin in the tibialis anterior (TA) and injected with 10,000 satellite cells (SC) 24 hours after the injury. The satellite cells (SC) were obtained from Tg:Pax7nGFP::PLAP mice (Deprimo et al., 1996; Rocheteau et al., 2012), allowing the prospective isolation of a pure population of SC by FACS (Pax7 being a specific marker of SC) and the follow up (PLAP placental alkaline phosphatase being an ubiquitous marker). The TA had been sampled 28 days post engraftment and the number of PLAP fibres was counted.

[0342] The Rag.sup.−/−γC.sup.−/− were treated for 6 weeks with fluoxetine per os at 18 mg/Kg at the time of the graft and the treatment continued during the regeneration process (FIG. 1a). When regeneration was completed 28 days post injury, the percentage of PLAP positive fibres was 20.5%±6.5 against 36%±6.2 in the treated animals (p=0.016; n=5, FIG. 1b-d). In other words, the engraftment potential of the injected SC almost doubled when injected in fluoxetine treated animals.

[0343] To investigate the potential effect of vortioxetine on the engraftment potential, the same approach was used. The Rag.sup.−/−γC.sup.−/− were treated for 12 days by intra peritoneal injections at 20 mg/Kg. Twelve days after the beginning of the treatment, the mice were injured and grafted with Pax7nGFP:PLAP SC. The treatment was continuous during the entire regenerative process (FIG. 1e). 28 days post injury the TA were sampled and stained for Laminin and PLAP. 19%±7.7 fibres were PLAP+ after placebo treatment against 36.1%±5.7 in the vortioxetine treated animals (p=0.028, n=4, FIG. 1f-h). In other words, the engraftment potential of the injected SC more than doubled when injected in vortioxetine treated animals.

2.2. Fluoxetine and Vortioxetine Treatment Triggers an Increase in Engraftment Potential of the Bone Marrow after Irradiation

[0344] In order to investigate the potential effect of fluoxetine and vortioxetine on the bone marrow engraftment potential, Rag.sup.−/−γc.sup.−/− immunodeficient mice were used. These mice were irradiated at 95cGy and then injected intravenously with 2 million cells taken from Tg:ActinGFP mouse (Okabe et al., 1997), to track the fate of the grafted cells.

[0345] Rag.sup.−/−γc.sup.−/− mice were treated with fluoxetine per os at 18 mg/Kg for 6 weeks. After this period of time the mice were irradiated at 95cGy and grafted with Tg:ActinGFP freshly isolated bone marrow cells (2 millions) (FIG. 2a). The number of GFP+ cells were investigated after 5 weeks in the bone marrow (the treatment was never discontinued). 63.7%±6.5 of GFP+ cells were detected in the placebo mice and 80.7%±8.4 GFP+ cells in the fluoxetine treated mice (p=0.0023, n=7, FIG. 2b,c). There was thus a 25% increase in engraftment efficiency in the fluoxetine treated animals.

[0346] To investigate the potential effect of vortioxetine on the engraftment potential, the same approach was used. The Rag.sup.−/−γc.sup.−/− were treated for 12 days by intra peritoneal injections at 20 mg/Kg. Twelve days after the beginning of the treatment, the mice were irradiated at 95cGy and intravenously grafted with 2 million freshly isolated bone marrow cells from Tg:ActinGFP. 5 weeks after grafting the mice were sacrificed and bone marrow was flushed and analysed (FIG. 2d). The number of GFP+ cells was investigated after 5 weeks in the bone marrow (the treatment was never discontinued). 65.8%±5.5 cells were found in the placebo mice while 80.2%±5.8 cells were found in the vortioxetine treated animals (p=0.0079, n=5, FIG. 2e). There was thus a 21% increase in engraftment potential of bone marrow cells after vortioxetine treatment.

[0347] To further characterise the timing of vortioxetine beneficial effects, the previous experiment was repeated without 12 days of pre-treatment (FIG. 2e,f). Rag.sup.−/−γc.sup.−/− were irradiated and grafted 2 hours post irradiation fresh bone marrow from Tg:ActinGFP mice. The same placebo was used displaying 65.8%±5.5 of GFP+ cells and 77.2%7.6 GFP+ cells in the vortioxetine treated animals at the time of the irradiation (p=0.04, n=5, FIG. 2e,g). A similar increase in engraftment potential (17%) was observed when animals were not pre-treated.

2.3. Fluoxetine and Vortioxetine Treatment Stimulate the 5TH1B Receptor

[0348] Fluoxetine treatment was provided at 18 mg/Kg for 6 weeks to Rag.sup.−/−γc.sup.−/− mice. The tibialis anterior (TA) was injured and 18 h post injury 10,000 satellite cells (SC) coming from Tg:Pax7nGFP:PLAP:MyHC2E3FLacZ triple transgenic mice were grafted to the Rag.sup.−/−γc.sup.−/− mice pre-treated with fluoxetine. After 21 days, 25.7%±8.7 of the fibers observed were PLAP+ in the placebo group meaning that said fibers come from the graft, against 39%±6.6 PLAP+ in animals treated with fluoxetine (n=8 animals per condition) (FIG. 3).

[0349] When immunodeficient Rag.sup.−/−γc.sup.−/− host animals were treated with fluoxetine at 18 mg/Kg but also with a 5HT1BR inhibitor (GR127935), 29.9%±7.8 of fibers were PLAP+, i.e. similar to the placebo situation (n=7 animals) (FIG. 3).

[0350] These data indicate that the inhibition of 5HT1BR in vivo is sufficient to lose the beneficial effect of fluoxetine, and that stimulation of 5HT1BR is the mode of action of fluoxetine for enhancement of the engraftment potential.

3. Discussion

[0351] The number of applications of cell or stem cell based therapies is limited to the clinics but the number of cell based clinical trials is currently growing (776 referenced world-wide, including 590 in the USA and 101 in Europe). Thus, having a drug capable of improving the engraftment potential of cell therapies (e.g. embryonic cells, adult cells, or stem cells) in order to better control diseases and the reconstitution of the tissue is key for a successful use of this emerging new type of medicine.

[0352] Here, the present results show that the use of two different drugs (fluoxetine and vortioxetine) that are known to stimulate the 5HT1B receptor, in two different paradigms (namely, the engraftment of muscle stem cells and the engraftment of bone marrow cells), is beneficial and that after grafting cells, the percentage or area of the tissue that was reconstituted from the donor cells was higher. It was further observed that the potential of vortioxetine in this respect was greater than the one of fluoxetine, due to the earlier onset of its beneficial effects on in vivo cell engraftment. After delivery of a 5HT1B inhibitor, it was observed that the effect of these drugs was obtained via the stimulation of the 5HT1B receptor.

[0353] These results are promising and can find direct applications in graft of bone marrow in leukaemia or graft of muscle stem cells in dystrophic patients, in order to improve said therapies.

[0354] Another important field of application comes from the mesenchymal stem cells (MSC) field. Indeed, safety and regulatory concerns surrounding allogeneic cell preparations make autologous and minimally manipulated cell therapies an attractive option for many regenerative, anti-inflammatory and autoimmune applications. MSCs have been shown to have interesting trophic properties (anti-scarring, anti-apoptotic, angiogenic, mitogenic, immuno-modulatory effects, anti-Microbial effects. So as a result, the MSCs are nowadays used in many studies and many clinical trials such as orthopaedics and spine therapies: fracture repair, osteonecrosis, spine fusion, cartilage repair, arthrisis; cardiovascular therapies: cardiac, vascular diseases; wounds and tissue repair: wounds and ulcer, burns, neural disorders: multiple sclerosis and amyotrophic lateral sclerosis, Parkinson's disease, stroke, spinal cord injury; autoimmune disorders: rheumatoid arthritis, Crohn's disease, lupus erythematosus, asthma; oncology leukaemia. One of the big advantages of those MSCs is the fact that they are not necessary immunogenic, they can be used in autograft but allograft has already proven to be also efficient. All those studies could benefit from the drugs that are under development by improving the engraftment and preparing the ground for better engraftability of the cells.

[0355] Other applications that are of main interest are cardiac tissue regenerative medicine that involves cardiomyocyte regeneration, neovascularization, and paracrine cytokines, which have anti-inflammatory, anti-apoptotic, and anti-remodelling effects. During the last decade, stem cells have become promising candidates for regenerative medicine not only because of their capacity of differentiation toward cardiomyocyte and vascular cell lineages but also their capacity for releasing such paracrine factors and their anti-arrhythmic effects. Paracrine cytokines and chemokines play pivotal roles in stem cell-related cardiac repair mechanisms. All studies demonstrating a beneficial effect of stem cell therapy on myocardial infarction (MI) agreed to stress the importance of the cardioprotective effects of those cells.

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