Non-immunosuppressive FK506 analogs and uses thereof

Abstract

The present application relates to non-immunosuppressive FK506 analogs and uses thereof. The present invention provides a new class of non-immunosuppressive FK506 analogs, which are FKBP selective small molecule BMP agonists, with moderate therapeutic efficacy for tissue repair and regeneration. Also disclosed herein are methods for making and using these compounds.

Description

[0001] This application is a divisional application of an application with an application date of January 23, 2020, application number 202080022171.8, and invention name “Non-immunosuppressive FK506 analogs and uses thereof”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 795,700, filed on January 23, 2019, and is hereby incorporated by reference for all purposes as if fully set forth herein. Background of the Invention

[0005] Trauma caused by accidents, diseases and armed conflicts is one of the most common medical problems. The cost of care for chronic non-healing wounds associated with severe burns and diseases such as diabetes has been estimated to be more than $50 billion per year (Fife et al., 2012). Chronic wounds make the human body more susceptible to infection and increase the risk of acquiring opportunistic pathogens that may lead to sepsis. Therefore, accelerating wound healing (WH) can reduce the risk of infection and improve mortality and morbidity in trauma patients. However, the options for shortening wound healing are limited and new therapies need to be developed.

[0006] The present inventors have previously reported the following findings: a synergistic drug combination for accelerating cutaneous WH (Lin et al., 2014) and inducing long-term allograft survival through host repopulation (Okabayashi et al., 2011, Hu et al., 2016, Cameron et al., 2016). The combination of two FDA-approved drugs, Tacrolimus (FK506) and Plerixafor (AMD3100), reduced the time to complete healing by 25% in mice with four circular full-thickness excisional wounds, which is unprecedented with existing treatment modalities. Accelerated WH was accompanied by the mobilization of bone marrow (BM)-derived stem cells (CD133, CD34, and cKit) and the recruitment of CD133 stem cells into the wound site, as well as the enhanced release of stromal cell-derived factor 1 (SDF-1), fibroblast growth factor (FGF), and vascular endothelial growth factor (VEGF) in granulation tissue (Lin et al., 2014).

[0007] The potential molecular mechanism that the combination of FK506 and AMD3100 (AF) accelerates WH has not been widely studied.AMD3100 is a selective antagonist of chemokine receptor CXCR4 (Hatse et al., 2002), and has been used clinically to drive hematopoietic stem cells (HSC) outside bone marrow (BM) to enter peripheral blood (Liles et al., 2003), in which they can be recovered and preserved until ablative irradiation and / or chemotherapy are completed.In addition to HSC, the injection of AMD3100 also increases the migration of endothelial progenitor cells (EPC) in BM source, which is related to faster neovascularization and functional recovery after myocardial infarction in mice (Jujo et al., 2010, Balaji et al., 2013).However, due to the recruitment reduced at the wound site, the increased circulating stem cell number treated by single AMD3100 only presents slightly faster healing (Lin et al., 2014).

[0008] Compared to AMD3100, the exact role of low-dose FK506 in combination therapy (AF) remains a mystery. FK506, a macrolide produced by the bacterium Streptomyces tsukubaensis, is an immunosuppressant widely used to prevent transplant rejection and treat certain autoimmune disorders (Tanaka et al., 1987; Fung et al., 2004). The potential mechanisms of the immunosuppressive activity of FK506 have been well established. At the cellular level, FK506 inhibits the activation of T helper cells. At the pathway level, it blocks intracellular signal transduction from the T cell receptor, which leads to transcriptional activation of IL-2 and other cytokine genes. At the molecular level, FK506 binds to FKBP12 and other members of the FKBP family, preventing calcium-dependent dephosphorylation of nuclear factor of activated T cells (NFAT), before the binary FKBP-FK506 complex binds to and inhibits the protein phosphatase activity of calcineurin (Liu et al., 1991; Griffith et al., 1995; Kissinger et al., 1995).

[0009] The possible underlying mechanism of FK506 in WH is through the inhibition of calcineurin. However, it has been shown that topical FK506 has adverse effects on WH ( et al., 1998). Furthermore, we have shown that animals treated with low doses of FK506 alone (0.1 mg / kg) presented slightly faster healing compared to saline controls, but the standard dose of FK506 used for immunosuppression (1 mg / kg) delayed healing time, leaving the unanswered question of whether calcineurin inhibition is responsible for the effects of FK506 on WH.

[0010] Although FKBP12 plays an auxiliary role in the immunosuppressive activity of FK506, it has also been shown to inhibit the activation of BMP type 1 receptors (Wang et al., 1996). Importantly, FK506 can mitigate this interaction (Spiekerkoetter et al., 2013). BMP signaling has not been directly linked to any stage of wound healing, although epithelial cells have been reported to downregulate many BMP receptors in response to injury (Lewis et al., 2014). In contrast, it has recently been reported that enhanced BMP signaling in myofibroblasts can promote scarless wound healing (Plikus et al., 2017). BMPs have been shown to produce a pro-inflammatory phenotype in endothelial cells, thereby increasing leukocyte adhesion and SDF-1 secretion (Csiszar et al., 2006; Young et al., 2012). Upon activation, BMP receptors phosphorylate and activate SMAD transcription factors 1, 5, and 8. One of the main target genes of these SMADs is inhibitor of differentiation 1 (ID-1), which inhibits the transcription of several genes related to embryogenesis and stem cell self-renewal. Previous studies have examined some of the downstream effects of BMP receptor activation after FK506 treatment, which is accompanied by increased phosphorylation of SMAD1 and SMAD5 (denoted as SMAD1 / 5) and / or SMAD8 (denoted as SMAD1 / 5 / 8) in skeletal muscle cells (Spiekerkoetter et al., 2013) and human synovial stromal (hSSC) cells (Tateishi et al., 2007). In addition, increases in MAPKK phosphorylation and ID-1 expression were observed, and the activity of FK506 was sufficient to rescue endothelial dysfunction induced by conditional knockout of type 2 BMP receptor (BMPR2) in mice (Spiekerkoetter et al., 2013). FK506 has been reported to upregulate phosphorylation of SMADs (SMAD 2 and SMAD 3) downstream of the TGF-β signaling pathway in smooth muscle cells (Giordano et al. Cardiovasc Res. 2008; Bennet et al. J Clin Med. 2016). However, downstream transcriptional activity was only seen in the presence of supplemented exogenous TGF-β (Spiekerkoetter et al., 2013; Wang et al., 1996). In another study, it was shown that FK506 increased the expression of the TGF-β type 3 co-receptor endoglin and stimulated both the migratory and angiogenic activities of endothelial cells ( et al., 2011). Together, these observations raise the possibility that FK506 may exert its WH effects via FKBP12 independently of calcineurin inhibition.

[0011] Therefore, there remains a need for compounds and compositions that can improve wound healing. SUMMARY OF THE INVENTION

[0013] The present inventors have now synthesized novel non-immunosuppressive FK506 analogs that retain FKBP binding and lack calcineurin inhibitory activity ( Figure 1 ). Newly synthesized FK506 analogs were found to activate BMP signaling in lymphocytes and endothelial cells by disrupting the FKBP12-BMPR1 interaction. Furthermore, the inventors can now show that the combination of these FK506 analogs and AMD3100 was found to accelerate wound healing in diabetic rats in a BMP receptor-dependent manner.

[0014] The newly synthesized FK506 analogs exhibited different immunosuppressive properties, while all of them activated the BMP pathway reporter in Jurkat cells with similar potency as FK506. In addition, the derivatives showed a structure-dependent effect in the inhibition of the NFAT reporter in Jurkat cells.

[0015] According to an embodiment, the present invention provides a compound of formula (I):

[0016]

[0017] or a salt, solvate, isomer or derivative thereof, wherein R is a cycloalkyl, aryl or heteroaryl group substituted by H, halogen, N, O, P, C1-C6 alkyl, imidazolyl, cycloalkyl and heterocycloalkyl, wherein the heteroaryl and heterocycloalkyl groups contain at least one heteroatom, preferably 1 to 4 heteroatoms selected from nitrogen, oxygen and sulfur.

[0018] According to an embodiment, the present invention provides a compound of formula (I), wherein R is selected from the group consisting of:

[0019]

[0020]

[0021]

[0022] According to an embodiment, the present invention provides a composition comprising a compound of formula (I):

[0023]

[0024] or a salt, solvate, isomer or derivative thereof, wherein R is a cycloalkyl, aryl or heteroaryl group substituted by H, halogen, N, O, P, C1-C6 alkyl, imidazolyl, cycloalkyl and heterocycloalkyl, wherein the heteroaryl and heterocycloalkyl groups contain at least one heteroatom, preferably 1 to 4 heteroatoms selected from nitrogen, oxygen and sulfur; and a pharmaceutically acceptable carrier.

[0025] According to an embodiment, the present invention provides a composition comprising a compound of formula (I), wherein R is selected from the group consisting of:

[0026]

[0027]

[0028] According to further embodiments, the present invention provides a compound of formula (I), or a composition comprising a compound of formula (I) and at least one or more biologically active agents.

[0029] According to an embodiment, the present invention provides a composition comprising a compound of formula (I):

[0030]

[0031] Or a salt, solvate, isomer or derivative thereof, wherein R is a cycloalkyl, aryl or heteroaryl group substituted by H, halogen, N, O, P, C1-C6 alkyl, imidazolyl, cycloalkyl and heterocycloalkyl, wherein the heteroaryl and heterocycloalkyl groups contain at least one heteroatom, preferably 1 to 4 heteroatoms selected from nitrogen, oxygen and sulfur; at least one stem cell migration agent; and a pharmaceutically acceptable carrier.

[0032] According to an embodiment, the present invention provides a composition comprising a compound of formula (I), wherein R is selected from the group consisting of:

[0033]

[0034]

[0035]

[0036] In a specific embodiment, the stem cell migration agent comprises a CXCR4 antagonist. For example, the CXCR4 antagonist can be selected from the group consisting of: AMD3100, TG-0054 or AMD3465.

[0037] According to an embodiment, the present invention provides a method for treating tissue damage in a subject, the method comprising administering to the subject an effective amount of a compound of formula (I):

[0038]

[0039] Or a salt, solvate, isomer or derivative thereof, wherein R is a cycloalkyl, aryl or heteroaryl group substituted by H, halogen, N, O, P, C1-C6 alkyl, imidazolyl, cycloalkyl and heterocycloalkyl, wherein the heteroaryl and heterocycloalkyl groups contain at least one heteroatom, preferably 1 to 4 heteroatoms selected from nitrogen, oxygen and sulfur; and an effective amount of at least one stem cell migration agent.

[0040] According to another embodiment, the present invention provides a method for treating tissue damage in a subject, comprising administering to the subject a composition comprising an effective amount of a compound of formula (I), an effective amount of at least one stem cell migration agent, and a pharmaceutically acceptable carrier.

[0041] In some embodiments, the present invention provides a method for treating tissue damage in a subject, the method comprising administering to the subject a composition comprising an effective amount of a compound of formula (I), wherein R is selected from the group consisting of:

[0042]

[0043]

[0044] According to another embodiment, the present invention provides a method for treating tissue damage in a subject, the method comprising administering to the subject a composition comprising an effective amount of a compound of formula (I), an effective amount of at least one stem cell migration agent, at least one additional bioactive agent, and a pharmaceutically acceptable carrier.

[0045] According to an embodiment, wherein the tissue injury is selected from the group consisting of chronic wounds, diabetic ulcers, burns, scalds, frostbite and puncture wounds.

[0046] According to an embodiment, the present invention provides a method for preparing an aryl-substituted FK506 molecule, comprising the steps of: a) adding a sufficient amount of FK506 to a mixture containing a sufficient amount of an aryl halide in the presence of a palladium catalyst and a base in a polar aprotic solvent; b) heating the mixture for a sufficient time to react the aryl halide with FK506 to produce an aryl substitution on the FK506 molecule; and c) isolating the aryl-substituted FK506 product. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 Depict the chemical structures of FK 506 and FKVP.

[0049] Figure 2A-2CFK506 analogs showing different immunosuppressive properties are shown. (2A) Cell viability after 72 hours of analog treatment in HUVEC cells. (2B) All analogs activate BMP pathway reporters in Jurkat cells with similar potency as FK506. (2C) Derivatives show structure-dependent effects in NFAT reporter inhibition in Jurkat cells. Error bars represent standard deviations of all measurements from the mean (n=3), and absorbance / luminescence values ​​are normalized to DMSO-treated cells.

[0050] Figure 3 Dose-response curves for activation of the BMP reporter by three non-immunosuppressive analogs (lb, 2b, and 3b) and FK506 are depicted.

[0051] Figure 4A-4B Details of binding to calcineurin are shown. (4A) Close-up of the terminal olefin of FK-506 and calcineurin. (4B) Steric effect of FKVP on calcineurin.

[0052] Figure 5A-Figure 5C (1a-1c) Schematic representation of the novel synthesis of non-immunosuppressive analogs (FKVP) of FK506 by modification at position C40. (5A) Chemical structures of FK506, FKVP, SLF and CsA. (5B) Resazurin-based cell viability assay of Jurkat cells after 3 days of FKVP or FK506 treatment (n=3). Absorbance values ​​were normalized to DMSO control. Error bars represent standard deviation. (5C) NFAT-luciferase reporter activity of PMA / ionomycin-activated Jurkat cells was inhibited by FK506 and CsA, but not by FKVP and SLF. Dose response curves were obtained by treating Jurkat cells expressing the NFAT luciferase reporter gene with serial dilutions of the indicated compounds, and relative luciferase activity was determined after normalization to DMSO control values. (n=3).

[0053] Figure 6 Schematic diagram showing the synthesis of FVKP via ruthenium-catalyzed cross-metathesis. Synthesis scheme of FVKP.

[0054] Figure 7A-7B Depicted are FVKP treatment of Jurkat cells.

[0055] Figure 8A-BFKVP is shown to compete with both FK506 and rapamycin for FKBP12 binding. Both FK506 and rapamycin require FKBP12 for activity. (8A) FKVP blocks FK506 inhibition of calcineurin-mediated NFAT dephosphorylation (lower band), (8B) and inhibition of mTOR-mediated S6 phosphorylation by rapamycin.

[0056] Figure 9A-9C Figures show accelerated wound healing in diabetic GK rats treated with a combination of AMD3100 and FK506 or FKVP. (9A) Wound model: Four circular excisional wounds (8 mm in diameter) were produced on the dorsal side of GK rats. (9B) Representative photographs of wounds in GK rats of each treatment group (AF = AMD3100 + FK506, AV = AMD3100 + FKVP) at day 0, day 6, day 12, day 18, and day 24. (9C) Quantitative analysis of complete healing time in GK rats. All data are represented by mean ± SEM.

[0057] Figure 10A-10C Non-immunosuppressive analogs, such as compounds of formula (I), are shown to activate ID-1 reporters and pSMAD1 / 5 phosphorylation by BMP type 1 receptor activation. (10A) BMP response element (BRE) reporter activity in Jurkat cells after treatment with increasing amounts of FK506 and FKVP. This activity was strongly inhibited by adding 100nM LDN-193182. TGF-β1 was used as a negative control, while BMP4 showed a strong induction of luciferase expression after 18 hours. Relative luciferase activity was determined after normalization to DMSO control values. Error bars represent standard deviations from the mean. (10B) Dose-dependent induction of SMAD1 / 5 phosphorylation by FKVP in Jurkat cells. (10C) BMPR1-selective inhibitor LDN-193189 inhibits SMAD1 / 5 phosphorylation induced by BMP-4 or FKVP in Jurkat cells.

[0058] Fig.11 It is shown that, unlike SMAD / 16, FKVP does not activate SMAD2 / 3 phosphorylation. Jurkat cells show no significant increase in SMAD2 / 3 phosphorylation after 2 hours. FKVP treatment compared to positive control (TGF-β1).

[0059] Fig.12AMD-3100 alone or in combination with FKVP was shown to not affect BMP signaling. FKVP and AMD3100 single and combined treatments in the Jurkat BMPFKVP luciferase assay (n=3). Relative luciferase activity was determined after normalization to DMSO control values. Error bars represent standard deviation from the mean.

[0060] Fig.13 It is shown that FKVP activates BMP signaling in the presence of the BMP inhibitor Noggin. Combination treatment of BMP-4, FVKP, FK506 and Noggin in Jurkat BMP activation assay (n=3). Relative luciferase activity was determined after normalization to DMSO control values. Error bars represent standard deviation from the mean.

[0061] Fig.14 The graph shows that FKVP increases the sensitivity of cells to BMP-4 stimulation. BMP-4, FVKP and combination treatments in Jurkat BMP activation assay (n=3). Relative luciferase activity was determined after normalization to DMSO control values. Error bars represent standard deviation from the mean.

[0062] Figure 15A-Figure 15C Depicts FKVP activation of SMAD1 / 5 phosphorylation and ID-1 expression in HUVECs. (15A) Western blot of SMAD1 / 5 phosphorylation in HUVEC cells after 2-hour treatment with a combination of BMP-4, FKVP, and LDN. (15B) Time course Western blot of HUVEC cells treated with 200 nM FKVP and blotted for SMAD1 / 5 phosphorylation, and (15C) ID-1 expression.

[0063] Figures 16A-16D It is shown that FKBP12 alone is required for FK506- and FKVP-induced SMAD1 / 5 phosphorylation. (16A) Induction of SMAD1 / 5 phosphorylation by FKVP and BMP-4 in wild-type cells and different FKBP isoform knockout cells. (16B) Activation of BMP pathway reporters by FKVP and FK506 in different FKBP isoform knockout cells, and inhibition of reporter gene activation by LDN (n=3). Relative luciferase activity was determined after normalization to DMSO control values. Error bars represent standard deviations from the mean. (16C) Expression of FKBP12-SNAP fusion protein restored activation of the BMP pathway by FKVP and FK506 in FKBP12KO Jurkat cells (n=3). (16D) Pulldown of ALK receptor using FKBP12-SNAP in combination with SNAP-functionalized beads in the absence and presence of FKVP.

[0064] Fig.17 The diagram shows that FKBP12-SNAP interacts with both mTOR and calcineurin. SNAP tag pull-down and Western blot showed that FKBP12-SANP effectively associates with both calcineurin and mTOR in the presence of FK506 and rapamycin, respectively. As a negative control, FKVP did not induce calcineurin association. BMP protein treatment was not explored in this experiment.

[0065] Figures 18A-18C Blockade of BMP signaling abolishes the beneficial effects of AF combination therapy in wound healing. (18A) Representative photographs of wounds of GK rats treated with saline, LDN, AF, or AF plus LDN, showing the differences between AF and AF plus LDN starting from day 12. (18B) Quantitative analysis of complete healing time in GK rats. All data are represented by mean ± SEM. (18C) Representative immunohistochemical staining of the stem cell marker CD133 in granulation tissue of GK rats on day 7. Rats receiving AF treatment had significantly higher numbers of CD133 cells (brown) in granulation tissue compared to the saline control group, while LDN treatment significantly reduced the number of CD133 stem cells in the wound site.

[0066] Fig.19 , without being bound by any particular theory, this figure shows an overview of the mechanism of enhanced healing by an exemplary compound of formula (I) of the present invention, FKVP and stem cell stimulator AMD3100. AMD3100 releases CD133+ stem cells into the circulation, where FKVP-mediated BMP activation affects the recruitment of wound tissue. Systemic inhibition of FKBP12 by FKVP allows BMP-related genes to be expressed in both migrating stem / progenitor cells and endothelium without affecting the necessary immune response to the wound.

[0067] Fig. 20 The structure of FKVP and the mass spectrum of the compound ([C 49 H 72 N2O 12 +H] 881.5 (HRMS-ESI (m / z): for C 49 H 72 N2O 12 [FKVP+H]+ calculated value 881.5164, measured value 881.5135).

[0068] Figure 21A-21B NMR spectra depicting the structural characterization of FKVP. (21A) FKVP and (21B) FK506(CDCl3) 1H-NMR spectrum, including details from 5.5 ppm to 6.0 ppm showing the disappearance of the terminal olefin in FKVP (present in FK506, highlighted in yellow). DETAILED DESCRIPTION OF THE INVENTION

[0070] The following examples have been included to provide guidance to those of ordinary skill in the art for practicing representative embodiments of the subject matter disclosed herein. Based on the present disclosure and the general level of those skilled in the art, it will be appreciated by those skilled in the art that the following examples are intended to be exemplary only, and that many variations, modifications, and changes may be used without departing from the scope of the subject matter disclosed herein. The subsequent comprehensive description and specific examples are intended only for illustrative purposes and should not be construed as limiting in any way the compounds of the present disclosure prepared by other methods.

[0071] According to an embodiment, the present invention provides a compound of formula (I):

[0072]

[0073] or a salt, solvate, isomer or derivative thereof, wherein R is a cycloalkyl, aryl or heteroaryl group substituted by H, halogen, N, O, P, C1-C6 alkyl, imidazolyl, cycloalkyl and heterocycloalkyl, wherein the heteroaryl and heterocycloalkyl groups contain at least one heteroatom, preferably 1 to 4 heteroatoms selected from nitrogen, oxygen and sulfur.

[0074] The non-immunosuppressive compound of formula (I) is derived from the immunosuppressant tacrolimus. Tacrolimus (also FK-506 or Fujimycin) is an immunosuppressive drug that is primarily used after allogeneic organ transplantation to reduce the activity of the patient's immune system and thus reduce the risk of organ rejection. It reduces interleukin-2 (IL-2) production by T cells. It is also used in topical preparations for the treatment of severe atopic dermatitis (eczema), severe refractory uveitis after bone marrow transplantation, and the skin condition vitiligo. It is a 23-membered macrolide that was discovered in 1984 from a fermentation broth of a Japanese soil sample containing the bacterium Streptomyces tsukuba. The drug is sold under the following trade names: Administer twice daily (intravenously); Sustained-release formulations that allow once-daily dosing (oral); and Topical preparations.

[0075] According to an embodiment, the present invention provides a compound of formula (I), wherein R is selected from the group consisting of:

[0076]

[0077]

[0078] The term "aliphatic" is a well-known term in the art and includes straight chain, branched chain and cyclic alkanes, alkenes or alkynes. In certain embodiments, the aliphatic group in the present invention is straight chain or branched chain and has from 1 to about 20 carbon atoms.

[0079] The term "alkyl" is well known in the art and includes saturated aliphatic groups, including straight chain alkyl groups, branched chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl groups. In certain embodiments, a straight chain or branched chain alkyl group has about 30 or fewer carbon atoms in its backbone (e.g., C1-C2 for a straight chain). 30 , for the branched chain C3-C 30 ), and optionally, about 20 or less carbon atoms. Likewise, cycloalkyl has from about 3 to about 10 carbon atoms in its ring structure, and optionally has about 5, 6 or 7 carbons in the ring structure.

[0080] In addition, the term "alkyl" (or "lower alkyl") includes both "unsubstituted alkyl" and "substituted alkyl", where substituted alkyl refers to an alkyl moiety having a substituent replacing hydrogen on one or more carbons of the hydrocarbon backbone. Such substituents may include, for example, halogen, hydroxyl, carbonyl (such as carboxyl, alkoxycarbonyl, formyl or acyl), thiocarbonyl (such as thioester, thioacetate or thioformate), alkoxy, phosphoryl, phosphonate, phosphite, amino, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclic, aralkyl or aromatic or heteroaromatic moieties. It will be appreciated by those skilled in the art that the moiety substituted on the hydrocarbon chain may itself be substituted if appropriate. For example, the substituent of substituted alkyl can include substituted and unsubstituted forms of amino, azido, imino, amido, phosphoryl (including phosphonate and phosphite), sulfonyl (including sulfate, sulfonamido, sulfamoyl and sulfonate) and silyl groups, as well as ether, alkylthio, carbonyl (including ketone, aldehyde, carboxylate and ester),-CF ,-CN and similar groups. Exemplary substituted alkyl is described below. Cycloalkyl can also be substituted by alkyl, alkenyl, alkoxy, alkylthio, aminoalkyl, alkyl substituted by carbonyl,-CF ,-CN and similar groups.

[0081] The term "aralkyl" is art-recognized and includes aryl groups (eg, aromatic or heteroaromatic groups).

[0082] The terms "alkenyl" and "alkynyl" are art-recognized and, in organic molecules, typically include atoms of any element other than carbon or hydrogen. Exemplary heteroatoms include boron, nitrogen, oxygen, phosphorus, sulfur and selenium.

[0083] The term "aryl" is well known in the art and includes 5-, 6- and 7-membered monocyclic aromatic groups that may contain from 0 to 4 heteroatoms, such as benzene, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine and similar groups. Those aryl groups having heteroatoms in the ring structure may also be referred to as "aryl heterocycles" or "heteroaromatics". The aromatic ring may be substituted at one or more ring positions by substituents such as those described above, such as halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxy, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, ester, heterocyclic radical, aromatic or heteroaromatic moieties, -CF3, -CN or similar groups. The term "aryl" also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjacent rings (these rings are "fused rings"), wherein at least one of the rings is aromatic, e.g., the other cyclic rings can be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl and / or heterocyclyl, or rings connected by non-cyclic moieties.

[0084] The terms "ortho", "meta" and "para" are well known in the art and apply to 1,2-disubstituted cyclohexanes, 1,3-disubstituted cyclohexanes and 1,4-disubstituted cyclohexanes, respectively. For example, the names 1,2-dimethylbenzene and ortho-dimethylbenzene are synonyms.

[0085] The terms "heterocyclyl" and "heterocyclic group" are well known in the art and include 3 to about 10 membered ring structures, such as 3 to about 7 membered rings, whose ring structures contain 1 to 4 heteroatoms. The heterocycle can also be polycyclic. The heterocyclyl group includes, for example, thiophene, thianthrene, furan, pyran, isobenzofuran, chromene, xanthene, phenoxanthin, pyrrole, imidazole, pyrazole, isothiazole, isoxazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine , pyrimidine, phenanthroline, phenazine, phenarsazine, phenothiazine, furazan, phenoxazine, pyrrolidine, oxolane, thiolane, oxazole, piperidine, piperazine, morpholine, lactone, lactam such as azetidinone and pyrrolidone, sultam, sultone and similar groups. The heterocycle can be substituted at one or more positions by a substituent such as described above, for example, halogen, alkyl, aralkyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, heterocyclic radical, aromatic or heteroaromatic moiety, -CD3, -CN or similar groups.

[0086] The terms "polycyclic radical" and "polycyclic group" are well known in the art, and include structures with two or more rings (e.g., cycloalkyl, cycloalkenyl, cycloalkynyl, aryl and / or heterocyclic radical), wherein two or more carbons are shared for two adjacent rings, e.g., the ring is a "condensed ring". The ring connected by non-adjacent atoms, e.g., two rings share three or more atoms, is referred to as a "bridge" ring. Each ring in the polycyclic ring can be substituted by a substituent such as described above, e.g., halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, heterocyclic radical, aromatic moiety or heteroaromatic moiety, -CD3, -CN or similar groups.

[0087] The term "carbocycle" is well known in the art and includes aromatic or non-aromatic rings in which each atom in the ring is carbon. The following terms well known in the art have the following meanings: "nitro" means -NO2; the term "halogen" means -F, -Cl, -Br or -I; the term "thiol" means -SH; the term "hydroxyl" or "hydroxyl" means -OH; and the term "sulfonyl" means -SO2-.

[0088] The terms "amine" and "amino" are well known in the art and include both unsubstituted and substituted amines. Primary amines carry two hydrogens, secondary amines carry one hydrogen and another substituent, and two hydrogens of tertiary amines are substituted. Substituents for one or both hydrogens may be, for example, alkyl, alkenyl and aryl, cycloalkyl, cycloalkenyl, heterocycle, polycycle, etc. If both hydrogens are replaced by carbonyls, the nitrogen of the carbonyl framework forms an imide.

[0089] The term "alkylamine" embraces an amine group as defined above having a substituted or unsubstituted alkyl group attached thereto.

[0090] The term "acylamino" is art-recognized as an amino-substituted carbonyl group.

[0091] The term "alkylthio" is well known in the art and includes an alkyl group as defined above having a thio group attached thereto. In certain embodiments, the "alkylthio" moiety is represented by one of -S-alkyl, -S-alkenyl, -S-alkynyl, etc. Representative alkylthio groups include methylthio, ethylthio, and the like.

[0092] The term "carbonyl" is art-recognized and includes the structure C=0. Esters, carboxyl groups, formates, thiocarbonyls, thioesters, thiocarboxylic acids, thioformates, ketones, and aldehydes contain carbonyl groups.

[0093] The terms "alkoxyl" and "alkoxy" are art-recognized and include an alkyl group as defined above having an oxygen group attached thereto. Representative alkoxy groups include methoxy, ethoxy, propoxy, tert-butoxy, and the like.

[0094] An "ether" is two hydrocarbons covalently linked by oxygen. Thus, a substituent of the alkyl group that makes an alkyl group an ether is or resembles an alkoxy group, such as may be represented by one of -O-alkyl, -O-alkenyl, -O-alkynyl, and the like.

[0095] The term "sulfonate" is art-recognized and includes moieties in which the sulfur atom has two doubly-bonded oxygens as well as a single-bonded oxygen.

[0096] The term "sulfate" is art-recognized and includes moieties similar to sulfonates but containing two single-bonded oxygens.

[0097] The terms "sulfonamide," "sulfamoyl," "sulfonyl," and "sulfoxide" are art-recognized, and each may contain a variety of R group substituents as described herein.

[0098] The terms "phosphoramidite" and "phosphonamidite" are well known in the art.

[0099] The term "selenoalkyl" is well known in the art and includes an alkyl group having a substituted selenium group attached thereto. Exemplary "selenoethers" that may be substituted on the alkyl group are selected from one of -Se-alkyl, -Se-alkenyl, -Se-alkynyl, and the like.

[0100] Alkenyl and alkynyl groups can be substituted to produce, for example, aminoalkenyl, aminoalkynyl, amidoalkenyl, iminoalkenyl, iminoalkynyl, thioalkenyl, thioalkynyl, carbonyl substituted alkenyl or alkynyl groups.

[0101] Hydrocarbon is a term well known in the art and includes all permissible compounds having at least one hydrogen atom and at least one carbon atom. For example, permissible hydrocarbons include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic organic compounds, which may be substituted or unsubstituted.

[0102] The term "protecting group" is well known in the art and includes temporary substituents that protect potential reactive functional groups from undesirable chemical transformations. Examples of such protecting groups include esters of carboxylic acids, silyl ethers of alcohols, and acetals and ketals respectively of aldehydes and ketones. The field of protecting group chemistry has been reviewed, for example Greene et al., Protective Groups in Organic Synthesis, 2nd edition, Wiley, New York, (1991).

[0103] The definition of each expression such as alkyl, aryl, etc., when it occurs more than one time in any structure, is intended to be independent of its definition elsewhere in the same structure, unless expressly stated otherwise or the context indicates otherwise.

[0104] The terms triflyl, tosyl, mesyl and nonaflyl are well known in the art and refer to trifluoromethanesulfonyl, p-toluenesulfonyl, methanesulfonyl and nonafluorobutanesulfonyl groups, respectively. The terms triflate, tosylate, mesylate and nonaflate are well known in the art and refer to trifluoromethanesulfonate ester, p-toluenesulfonate ester, methanesulfonate ester and nonafluorobutanesulfonate ester functional groups, respectively, and molecules comprising said groups.

[0105] The abbreviations Me, Et, Ph, Tf, Nf, Ts, and Ms are well known in the art and represent methyl, ethyl, phenyl, trifluoromethanesulfonyl, nonafluorobutanesulfonyl, p-toluenesulfonyl, and methylsulfonyl, respectively. A more comprehensive list of abbreviations used by organic chemists of ordinary skill in the art appears in the first issue of each volume of the Journal of Organic Chemistry; the list is usually presented in a table entitled "Standard List of Abbreviations".

[0106] It will be understood that "substituted" or "substituted by" includes the implicit proviso that such substitution is in accordance with the allowed valencies of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., one that does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination or other reaction.

[0107] The term "substituted" also contemplates all permissible substituents including organic compounds (such as imide reagents of interest). In a broad aspect, permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of organic compounds. Illustrative substituents include, for example, those described herein. For suitable organic compounds, permissible substituents may be one or more and may be the same or different. For purposes of the present invention, heteroatoms such as nitrogen may have hydrogen substituents and / or any permissible substituents of organic compounds described herein that satisfy the valence of heteroatoms. The present invention is not intended to be limited in any way by the permissible substituents of organic compounds.

[0108] According to an embodiment, the present invention provides a method for preparing an aryl-substituted FK506 molecule, the method comprising the steps of: a) adding a sufficient amount of FK506 to a mixture containing a sufficient amount of an aryl halide in the presence of a palladium catalyst and a base in a polar aprotic solvent; b) heating the mixture for a sufficient time to react the aryl halide with FK506 to produce an aryl substitution on the FK506 molecule; and c) isolating the aryl-substituted FK506 product.

[0109] As used herein, the present invention provides a novel method for producing aromatic substitution at the molecular terminal alkenyl carbon (carbon 40) of FK506 molecules. The present invention uses the Heck reaction. The Heck reaction (also known as the Mizoroki-Heck reaction) is a chemical reaction of an unsaturated halide (or triflate) with an olefin in the presence of a base and a palladium catalyst (or a catalyst based on palladium nanomaterials) to form a substituted olefin. It is named after Tsutomu Mizoroki and Richard F.Heck. Heck was awarded the 2010 Nobel Prize in Chemistry for the discovery and development of this reaction, and he shared this award with Ei-ichi Negishi and Akira Suzuki. The reaction is the first carbon-carbon bond formation reaction that follows the Pd(0) / Pd(II) catalytic cycle, which is the same catalytic cycle seen in other Pd(0)-catalyzed cross-coupling reactions.

[0110] In some embodiments, the methods of the present invention are used to prepare aryl-substituted compounds of FK506 (compounds of formula (I)).

[0111] In some embodiments, the catalyst used in the method of the present invention may be a typical catalyst and pre-catalyst including tetrakis(triphenylphosphine)palladium(0), palladium chloride, and palladium(II) acetate.

[0112] In some embodiments, the supporting ligands used in the methods of the invention are triphenylphosphine, PHOX, and BINAP.

[0113] In some embodiments, the base used in the methods of the present invention is triethylamine, potassium carbonate, tri(o-tolyl)phosphine, and sodium acetate.

[0114] In some embodiments, the aryl electrophiles used in the methods of the invention are halides, as well as triflates and benzyl halides or vinyl halides.

[0115] In some embodiments, the methods of the invention occur in the absence of oxygen.

[0116] In some embodiments, the methods of the invention occur in a polar aprotic solvent. Examples of such solvents include N-methylpyrrolidone, THF, ethyl acetate, acetone, DMF, acetonitrile, DMSO, and propylene carbonate.

[0117] In some embodiments, the methods of the present invention heat the reactants in the range of 80° C. to 130° C. In a preferred embodiment, the reaction is heated to about 100° C.

[0118] In some embodiments, a sufficient amount of aryl halide is in the range of 1 to 3 molar equivalents for FK506. In some embodiments, a sufficient amount of palladium catalyst is about 2 mol% to about 20 mol%. In some embodiments, a sufficient amount of base is tri(o-tolyl)phosphine or trimethylamine between about 5 mol% to about 30 mol%.

[0119] In some embodiments, the aryl halide used in the methods of the present invention is selected from those shown in Table 1 below.

[0120] Table 1: Schematic diagram of the process of the invention and aryl halides that can be used in the process of the invention.

[0121]

[0122] According to an embodiment, the present invention provides a composition comprising a compound of formula (I):

[0123]

[0124] or a salt, solvate, isomer or derivative thereof, wherein R is a cycloalkyl, aryl or heteroaryl group substituted by H, halogen, N, O, P, C1-C6 alkyl, imidazolyl, cycloalkyl and heterocycloalkyl, wherein the heteroaryl and heterocycloalkyl groups contain at least one heteroatom, preferably 1 to 4 heteroatoms selected from nitrogen, oxygen and sulfur; and a pharmaceutically acceptable carrier.

[0125] According to an embodiment, the present invention provides a composition comprising a compound of formula (I), wherein R is selected from the group consisting of:

[0126]

[0127]

[0128]

[0129] As used herein, the term "effective" means sufficient to achieve a desired, predicted or expected result. More particularly, "effective amount" or "therapeutically effective amount" are used interchangeably and refer to the amount of stem cell migration agent and / or immunosuppressant (possibly further combined with another therapeutic agent) necessary to provide the desired "treatment" (defined herein) or therapeutic effect, for example, an amount effective for preventing, alleviating, treating or alleviating the symptoms of tissue damage or trauma. As will be appreciated by those of ordinary skill in the art, the exact amount required will vary from subject to subject, depending on the age, general condition of the subject, the severity of the condition being treated, the specific compound and / or composition being administered, and the like. Those of ordinary skill in the art can determine the appropriate "therapeutically effective amount" in any individual case by reference to relevant texts and literature and / or by using routine experiments.

[0130] The pharmaceutical composition of the present invention is in a biocompatible form, suitable for topical or in vivo administration to a subject. The pharmaceutical composition may also include a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable" means approved by a regulatory agency of the federal government or state government or listed in the U.S. Pharmacopoeia or other generally recognized pharmacopoeias for use in animals and more particularly in humans. The term "carrier" refers to a diluent, adjuvant, excipient or vehicle, with which the stem cell migration agent and / or the compound of formula (I) are administered. Such a pharmaceutical carrier may be a sterile liquid, such as water and oil, including oils of petroleum origin, animal origin, plant origin or synthetic origin, including but not limited to peanut oil, soybean oil, mineral oil, sesame oil and the like. When the pharmaceutical composition is administered orally, water may be a carrier. When the pharmaceutical composition is administered intravenously, saline and aqueous dextrose may be carriers. Saline solutions and aqueous dextrose and glycerol solutions may be used as liquid carriers for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol and the like. The pharmaceutical composition may also contain a small amount of a wetting agent or emulsifier, or a pH buffer.

[0131] The pharmaceutical composition of the present invention can take the form of solution, suspension, emulsion, tablet, pill, capsule, powder, sustained release formulation and the like. The composition can be formulated as a suppository, containing traditional binders and carriers such as triglycerides. Oral formulations can include standard carriers, such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. In a specific embodiment, the pharmaceutical composition includes an effective amount of a stem cell migration agent and / or a compound of formula (I) and an appropriate amount of a pharmaceutically acceptable carrier to provide a suitable form of administration for the patient. The preparation should be suitable for the mode of administration. The preferred preparation is a subcutaneous preparation.

[0132] According to an embodiment, the present invention provides a method for treating tissue damage in a subject, the method comprising administering to the subject an effective amount of a compound of formula (I):

[0133]

[0134] or a salt, solvate, isomer or derivative thereof, wherein R is a cycloalkyl, aryl or heteroaryl group substituted by H, halogen, N, O, P, C1-C6 alkyl, imidazolyl, cycloalkyl and heterocycloalkyl, wherein the heteroaryl and heterocycloalkyl groups contain at least one heteroatom, preferably 1 to 4 heteroatoms selected from nitrogen, oxygen and sulfur.

[0135] According to another embodiment, the present invention provides a method for treating tissue damage in a subject, the method comprising administering to the subject a composition comprising an effective amount of a compound of formula (I) and an effective amount of at least one stem cell migration agent.

[0136] According to another embodiment, the present invention provides a method for treating tissue damage in a subject, comprising administering to the subject a composition comprising an effective amount of a compound of formula (I), and an effective amount of at least one stem cell migration agent and a pharmaceutically acceptable carrier.

[0137] In some embodiments, the present invention provides a method for treating tissue damage in a subject, the method comprising administering to the subject a composition comprising an effective amount of a compound of formula (I), wherein R is selected from the group consisting of:

[0138]

[0139]

[0140] According to another embodiment, the present invention provides a method for treating tissue damage in a subject, the method comprising administering to the subject a composition comprising an effective amount of a compound of formula (I), an effective amount of at least one stem cell migration agent, at least one additional bioactive agent and a pharmaceutically acceptable carrier.

[0141] The pharmaceutical composition of the present invention can be administered by any specific route of administration, including but not limited to oral, parenteral, subcutaneous, intramuscular, intravenous, intraarticular, intrabronchial, intraperitoneal, intracapsular, intracartilaginous, intracavitary, intracelial, intracerebellar, intraventricular, intracervical, intragastric, intrahepatic, intramyocardial, intraosteal, intramedullary, intraosseous, intrapelvic, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, rectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, bolus, vaginal, rectal, buccal, sublingual, intranasal, iontophoresis or transdermal administration. The most suitable route is oral administration or injection. In certain embodiments, subcutaneous injection is preferred.

[0142] In general, the pharmaceutical compositions disclosed herein comprising a stem cell migration agent and a compound of formula (I) can be used alone (e.g., a formulation comprising a stem cell migration agent and a compound of formula (I)) or together with other therapeutic agents at appropriate doses determined by routine testing to obtain optimal efficacy while minimizing any potential toxicity. The dosage regimen using the pharmaceutical compositions of the present invention can be selected based on a variety of factors, including the type, race, age, weight, sex, medical condition of the patient; the severity of the condition to be treated; the route of administration; the patient's renal and liver function; and the specific pharmaceutical composition used. A physician of ordinary skill can easily determine and prescribe an effective amount of the pharmaceutical composition (and potential other agents, including therapeutic agents) required to prevent, counteract, or stop the progression of a condition.

[0143] The optimal precision of achieving the concentration of a treatment regimen (e.g., a pharmaceutical composition comprising a stem cell migration agent and / or a compound of formula (I) and another therapeutic agent in combination) within the range of producing maximum efficacy with minimal toxicity may require a regimen based on the kinetics of the availability of the pharmaceutical composition to one or more target sites. When determining the optimal concentration of a treatment regimen, the distribution, balance, and elimination of the pharmaceutical composition may be considered. The dosage of the pharmaceutical composition disclosed herein can be adjusted when combined to achieve the desired effect. On the other hand, the dosage of the pharmaceutical composition and various therapeutic agents can be independently optimized and combined to achieve a synergistic result, in which the condition will be reduced more than if any one is used alone.

[0144] In particular, the toxicity and therapeutic efficacy of the pharmaceutical compositions disclosed herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, to determine the LD 50 (a dose lethal to 50% of the population) and ED50 The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD 50 / ED 50 Pharmaceutical compositions that exhibit large therapeutic indices are preferred unless cytotoxicity of the composition is the desired active or therapeutic outcome. Although pharmaceutical compositions that exhibit toxic side effects may be used, delivery systems may target such compositions to the site of the affected tissue in order to minimize potential damage to uninfected cells and thereby reduce side effects. In general, the pharmaceutical compositions of the present invention may be administered in a manner that maximizes efficacy and minimizes toxicity.

[0145] The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. Preferably, the dosage of such compositions lies within the range of ED with little or no toxicity. 50 The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any composition used in the methods of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve an IC value including as determined in cell culture. 50 (the concentration of the test composition that achieves half-maximal inhibition of symptoms). Such information can be used to accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography.

[0146] In addition, the dosage administration of the composition of the present invention can be optimized using a pharmacokinetic / pharmacodynamic modeling system. For example, one or more dosage regimens can be selected, and a pharmacokinetic / pharmacodynamic model can be used to determine the pharmacokinetic / pharmacodynamic profile of one or more dosage regimens. Next, based on a specific pharmacokinetic / pharmacodynamic profile, one of the dosage regimens for administration that achieves a desired pharmacokinetic / pharmacodynamic response can be selected. See WO 00 / 67776, which is expressly incorporated herein by reference in its entirety.

[0147] "Agent" refers to all materials that can be used as or in a pharmaceutical composition, or can be a compound such as a small synthetic or naturally derived organic compound, nucleic acid, polypeptide, antibody, fragment, isoform, variant or other material that can be used independently for these purposes, all according to the present invention.

[0148] "Antagonist" refers to an agent that down-regulates (e.g., suppresses or inhibits) at least one biological activity of a protein. An antagonist can be a compound that inhibits or reduces the interaction between a protein and another molecule (e.g., a target peptide or enzyme substrate). An antagonist can also be a compound that down-regulates the expression of a gene or reduces the amount of expressed protein present.

[0149] "Hematopoiesis" refers to the highly coordinated process of blood cell development and homeostasis. Before birth, hematopoiesis occurs in the yolk sac, then the liver, and finally the bone marrow. In normal adults, it occurs in the bone marrow and lymphoid tissues. All blood cells develop from pluripotent stem cells. Pluripotent cells differentiate into stem cells for three, two, or one hematopoietic differentiation pathway. However, none of these stem cells are morphologically distinguishable.

[0150] The term "immunosuppressant" refers to an agent that inhibits, slows or reverses the activity of the immune system. Immunosuppressants work by inhibiting the function of responsive immune cells (including, for example, T cells) directly (e.g., by acting on immune cells) or indirectly (by acting on other regulatory cells).

[0151] The terms "stem cell" and "hematopoietic stem cell" are used interchangeably herein. Stem cells are distinguished from other cell types by two important characteristics. First, stem cells are unspecialized cells that are able to self-renew through cell division, sometimes after long periods of inactivation. Second, under certain physiological or experimental conditions, stem cells can be induced to become tissue-specific cells or organ-specific cells with specific functions. In some organs, such as the intestine and bone marrow, stem cells divide regularly to repair and replace worn or damaged tissues. However, in other organs, such as the pancreas and heart, stem cells only divide under specific conditions.

[0152] The term "stem cell" may refer to a pluripotent stem cell that can differentiate into all blood cells including red blood cells, white blood cells and platelets. For example, the "hematopoietic stem cell" or "stem cell" used in the present invention is contained not only in the bone marrow but also in cells derived from umbilical cord blood.

[0153] A "patient," "subject," or "host" to be treated by the methods of the present invention refers to a human or non-human animal, such as primates, mammals, and vertebrates.

[0154] "Small molecule" refers to a composition having a molecular weight of less than about 3 kilodaltons (kDa), less than about 1.5 kilodaltons, or less than about 1 kilodalton. A small molecule can be a nucleic acid, a peptide, a polypeptide, a peptide mimetic, a carbohydrate, a lipid, or other organic (carbon-containing) molecule or an inorganic molecule. A "small organic molecule" is an organic compound (or an organic compound complexed with an inorganic compound (e.g., a metal)) having a molecular weight of less than about 3 kilodaltons, less than about 1.5 kilodaltons, or less than about 1 kDa.

[0155] As used herein, the terms "treatment", "treating", "treat" and similar terms refer to obtaining a desired pharmacological effect and / or physiological effect. These terms are also used in the context of administering a "therapeutically effective amount" of an agent, such as a stem cell migration agent and / or an immunosuppressant. The effect can be preventive in terms of preventing a specific outcome, disease or its symptoms in whole or in part, and / or can be therapeutic in terms of partially or completely curing a disease and / or side effects attributable to the disease. "Treatment" as used herein encompasses any treatment of a disease in a subject, particularly a human being, and includes: (a) preventing the disease from occurring in a subject who may be susceptible to the disease but has not yet been diagnosed with the disease; (b) inhibiting the disease, i.e., preventing its development; and (c) alleviating the disease, such as causing the disease to regress, such as to completely or partially remove the symptoms of the disease. In a specific embodiment, the term is used in the context of promoting or improving wound healing in a patient. In other embodiments, the term is used in the context of treating organ transplant recipients, trauma, burn victims, and autoimmune diseases including IBD.

[0156] As used herein, the term "tissue injury" means an acute or chronic wound or ulcer of the skin or soft tissue. Examples of such wounds include diabetic sores and ulcers, burns, scalds, frostbite, puncture wounds, abrasions, and the like.

[0157] According to some embodiments, the compounds and pharmaceutical compositions described herein can be used to prepare a medicament for regulating the wound healing response of mammalian skin, including topical or subcutaneous application of an effective amount of a composition for a sufficient period of time. As used herein, the term "regulating the wound healing response" means preventing or downregulating a pathological response to tissue damage, which is characterized by fibrosis, including, for example, the production of keloids and hypertrophic scars in the skin, tendon adhesions, transmission blockade after nerve damage, scleroderma, Crohn's disease, esophageal strictures, urethral strictures, capsules around breast implants, cirrhosis of the liver, atherosclerosis, and fibrotic non-union in bones. Chronic non-healing skin ulcers are also examples of pathological responses to tissue damage and are alleviated by compounds of formula (I).

[0158] Stem cell migration agent

[0159] As described herein, "stem cell migratory agent", "migratory agent for hematopoietic stem or progenitor cells" or "migration agent" (used interchangeably) refers to any compound that acts to enhance the migration of stem cells from the bone marrow to the peripheral blood, whether it is a synthetic or naturally derived small organic molecule, or a polypeptide, a polypeptide such as a growth factor or colony stimulating factor or an active fragment or mimetic thereof, a nucleic acid, a carbohydrate, an antibody or any other agent. Stem cell migratory agents can increase the number of hematopoietic stem cells or hematopoietic progenitor / precursor cells in the peripheral blood, thereby allowing a more readily available source of stem cells for treating organ transplant recipients, burn victims, IBD and / or promoting wound healing. In specific embodiments, a stem cell migratory agent refers to any agent that migrates CD34+ and / or CD133+ stem cells. In other embodiments, the stem cell migratory agent interferes with the chemoattraction of CXCR4-expressing cells mediated by CXCL12 (SDF-1).

[0160] The present invention provides a pharmaceutical composition comprising at least one stem cell mobilizing agent and at least one immunosuppressive drug. Generally, stem cell mobilizing agents include, but are not limited to, small organic molecules, polypeptides, nucleic acids, and carbohydrates.

[0161] In the case of polypeptides, stem cell migration agents may include cytokines, colony stimulating factors, proteases or chemokines. More particularly, cytokines may include, but are not limited to, interleukin-1 (IL-1), interleukin-3 (IL-3), interleukin-6 (IL-6), interleukin-11 (IL-11), interleukin-7 (IL-7) and interleukin-12 (IL12).

[0162] In the case of colony stimulating factors, the stem cell migration agent may include, but is not limited to, granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), stem cell factor, FLT-3 ligand, or a combination thereof.

[0163] In another embodiment, the protease stem cell migration agent may include, but is not limited to, metalloproteinases (such as MMP2 or MMP9), serine proteases (such as cathepsin G or elastase), cysteine ​​proteases (such as cathepsin K), and dipeptidyl peptidase-1 (DDP-1 or CD26).

[0164] In yet another embodiment, chemokine stem cell migration agents may include, but are not limited to, CXCL12, IL-8, Mip-1α, and GROβ.

[0165] In yet another embodiment, the nucleic acid stem cell mobilization agent is a DNA molecule or an RNA molecule. In a more specific embodiment, the nucleic acid can be a small interfering RNA (siRNA) molecule or an antisense molecule specific for CXCL12.

[0166] In the case of carbohydrates, the stem cell migration agent can be a sulfated carbohydrate, which can include but is not limited to fucoidan and sulfated dextran. Fucoidan is a carbohydrate composed of L-fucose, sulfate and acetate in a molar ratio of 1:1.23:0.36, and can be isolated from the Pacific brown seaweed Fucus evanescens. See Bilan et al., 337 (8) CARBOHYDRATE RESEARCH 719-30 (2002). Sulfated dextran refers to a series of polysaccharides with different sulfation patterns. See, e.g., Pomin et al., 15(12) GLYCOBIOLOGY 1376-1385 (2005); Melo et al., 279(2) J. BIOL. CHEM. 20824-20835 (2004); and Farias et al., 275(38) J. BIOL. CHEM. 29299-29307 (2000).

[0167] Stem cell migration agents may also include, but are not limited to, AMD3100; stromal cell derived factor (SDF-1); SDF-1 analogs (e.g., CTCE-0214 (Chemokine Therapeutics Corp.)); anti-SDF-1 antibodies; cyclophosphamide; stem cell factor (SCF); filgrastim; ancestim; Myeloid Progenitor Inhibitory Factor-1 (MPIF-1) (see U.S. Patent Publication No. 20080274109); and Very Late Antigen (VLA-4) antagonists (e.g., α-4 integrin antagonists, such as antibodies including natalizumab or anti-phospho-Integrin α4 (Ser988), clone 6.33 (Upstate Cell Signaling Solutions) or peptides (e.g., phenylacetyl-leu-asp-phe-D-prolinamide (Cytel Corp., San Diego Calif.))).

[0168] In a specific embodiment, the stem cell migration agent includes a CXCR4 antagonist. In a specific embodiment, the CXCR4 antagonist is TG-0054 (Burixafor; phosphonic acid, p-(2-(4-(6-amino-2-(((trans-4-(((3-(cyclohexylamino)propyl)amino)methyl)cyclohexyl)methyl)amino)-4-pyrimidinyl)-1-piperazinyl)ethyl)-)(TaiGen Biotechnology Co., Ltd. (Taipei, Taiwan, China)). In other specific embodiments, the CXCR4 antagonist is AMD3465 (N-(pyridin-2-ylmethyl)-1-[4-(1,4,8,11-tetraazacyclotetradecane-1-ylmethyl)phenyl]methylamine). In yet other embodiments, the CXCR4 antagonist is AMD3100. AMD3100 (1,1'-[1,4-phenylenebis(methylene)]bis-1,4,8,11-tetraazacyclo-tetradecane) is a symmetrical bicyclam, a prototype non-peptide antagonist of the CXCR4 chemokine receptor. See U.S. Patent Nos. 6,835,731 and 6,825,351. The term "AMD" or "AMD3100" is synonymous with plerixafor, rINN, USAN, JM3100 and its trade name Mozobil TM For convenience, the term "plerixafor" is used throughout to refer to CXCR4 antagonists.

[0169] The present invention also contemplates the use of mimetics of AMD3100. Mutational substitutions at 16 positions in TM-III, TM-IV, TM-V, TM-VI and TM-VII lining the primary ligand binding pocket of the CXCR4 receptor have identified three acid residues: Asp 171 (AspIV:20), Asp 262 (AspVI:23) and Glu 288 (GluVII:06) as the main interaction point of AMD3100. Molecular modeling showed that a cycloamine ring of AMD3100 interacts with Asp in TM-IV. 171 interaction, while the other loop is clamped between Asp from TM-VI and TM-VII, respectively. 262 and Glu 288 In one study, it was found that the introduction of only Glu at position VII:06, and the removal of the neutralizing Lys residue at position VII:02, resulted in a 1000-fold increase in the affinity of AMD3100, to within 10 times of its affinity in CXCR4. Thus, mimetics, such as peptide antagonists or non-peptide antagonists with improved oral bioavailability, can be designed to effectively and selectively block the CXCR4 receptor.

[0170] In other embodiments, the stem cell migration agent is BKT140 (Biokin Therapeutics, Ltd. (Rehovot, Israel)). BKT140 (4F-benzoyl-TN14003) binds to and inhibits the CXCR4 chemokine receptor with high affinity, showing an IC of ∼1 nmol / L compared to the value obtained with AMD3100. 50 In addition, compared with the IC of 51nmol / L+17nmol / L of plerixafor 50 Compared with the IC values ​​of BKT140, which ranged from 0.5 nmol / L to 2.5 nmol / L 50 The cell migration stimulated by CXCL12 was blocked within 10% of the values, indicating a high migratory capacity. See Peled et al., 20 CLIN. CANCER RES. 469-79 (2013).

[0171] FK binding protein ligand

[0172] In conjunction with at least one stem cell mobilization agent, the pharmaceutical composition comprises a non-immunosuppressive FK binding protein ligand. In addition to the compounds of formula (I) disclosed herein, other examples of non-immunosuppressive ligands include meridamycin, antascomicins, and synthetic ligands of FKBP (SLF).

[0173] The normal dosage of the compound of formula (I) is about 0.1 mg / kg / day-0.3 mg / kg / day (oral) and about 0.01 mg / kg / day-0.05 mg / kg / day (IV). In certain embodiments, the low dose of tacrolimus is about one tenth of the normal dosage, for example, about 0.01 mg / kg / day-0.03 mg / kg / day (oral) and about 0.001 mg / kg / day-0.005 mg / kg / day (IV).

[0174] In certain embodiments, the stem cell migration agent is AMD3100. In such embodiments, the pharmaceutical composition may include a typical dose of AMD3100. This drug is typically administered to human patients at about 0.12 mg / kg-0.24 mg / kg. In patients with a body weight of 60 kg, the dosage of AMD3100 injected subcutaneously is about 0.24 mg / kg / day.

[0175] The pharmaceutical composition can be described by the ratio of (a) a compound of formula (I) or a non-immunosuppressive FKBP ligand to (b) a stem cell migration agent (e.g., a CXCR antagonist). In certain embodiments, the ratio can be 1 / 1, 1 / 2, 1 / 3, 1 / 4, 1 / 5, 1 / 6, 1 / 7, 1 / 8, 1 / 9, 1 / 10, 1 / 11, 1 / 12, 1 / 13, 1 / 14, 1 / 15, 1 / 16, 1 / 17, 1 / 18, 1 / 19, 1 / 20, 1 / 21, 1 / 22, 1 / 23, 1 / 24, 1 / 25, 1 / 26, 1 / 27, 1 / 28, 1 / 29, 20, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 2219, 222 1 / 25, 1 / 26, 1 / 27, 1 / 28, 1 / 29, 1 / 30, 1 / 31, 1 / 32, 1 / 33, 1 / 34, 1 / 35, 1 / 36, 1 / 37, 1 / 38, 1 / 39, 1 / 40, 1 / 41, 1 / 42, 1 / 43, 1 / 44, 1 / 45, 1 / 46, 1 / 47, 1 / 48, 1 / 49, 1 / 50 , 1 / 51, 1 / 52, 1 / 53, 1 / 54, 1 / 55, 1 / 56, 1 / 57, 1 / 58, 1 / 59, 1 / 60, 1 / 61, 1 / 62, 1 / 63, 1 / 64, 1 / 65, 1 / 66, 1 / 67, 1 / 68, 1 / 69, 1 / 70, 1 / 71, 1 / 72, 1 / 73, 1 / 74, 1 / 75, 1 1 / 94, 1 / 95, 1 / 96, 1 / 97, 1 / 98, 1 / 99, 1 / 100 or more.

[0176] The pharmaceutical composition may comprise (a) a compound of formula (I) or a non-immunosuppressive FKBP ligand and (b) a stem cell migratory agent in a ratio ranging from about 1 / 10-1 / 100, 1 / 10-1 / 99, 1 / 10-1 / 98, 1 / 10-1 / 97, 1 / 10-1 / 96, 1 / 10-1 / 95, 1 / 10-1 / 94, 1 / 10-1 / 93, 1 / 10-1 / 92, 1 / 10-1 / 91, 1 / 10-1 / 90, 1 / 10-1 / 89, 1 / 10-1 / 88, 1 / 10-1 / 87, 1 / 10-1 / 86, 1 / 10-1 / 85, 1 / 10-1 / 84, 1 / 10-1 / 83, 1 / 10-1 / 84. 0-1 / 82, 1 / 10-1 / 81, 1 / 10-1 / 80, 1 / 10-1 / 79, 1 / 10-1 / 78, 1 / 10-1 / 77, 1 / 10-1 / 76, 1 / 10-1 / 75, 1 / 10-1 / 74, 1 / 10-1 / 73, 1 / 10-1 / 72, 1 / 10-1 / 71, 1 / 10-1 / 70, 1 / 10-1 / 69, 1 / 10-1 / 68, 1 / 10-1 / 67, 1 / 10-1 / 66, 1 / 10-1 / 65, 1 / 10-1 / 64, 1 / 10-1 / 63, 1 / 10-1 / 62, 1 / 10-1 / 61, 1 / 10-1 / 60, 1 / 10-1 / 59, 1 / 1 / 10-1 / 58, 1 / 10-1 / 57, 1 / 10-1 / 56, 1 / 10-1 / 55, 1 / 10-1 / 54, 1 / 10-1 / 53, 1 / 10-1 / 52, 1 / 10-1 / 51, 1 / 10-1 / 50, 1 / 10-1 / 49, 1 / 10-1 / 48, 1 / 10-1 / 47, 1 / 10-1 / 46, 1 / 10-1 / 45, 1 / 10-1 / 44, 1 / 10-1 / 43, 1 / 10-1 / 42, 1 / 10-1 / 41, 1 / 10-1 / 40, 1 / 10-1 / 39, 1 / 10-1 / 38, 1 / 10-1 / 37, 1 / 10-1 / 36, 1 / 10-1 / 35, 1 1 / 10-1 / 22, 1 / 10-1 / 21, 1 / 10-1 / 20, 1 / 10-1 / 19, 1 / 10-1 / 18, 1 / 10-1 / 17, 1 / 10-1 / 16, 1 / 10-1 / 15, 1 / 10-1 / 14, 1 / 10-1 / 13, 1 / 10-1 / 12, or 1 / 10-1 / 11.

[0177] In alternative embodiments, the pharmaceutical composition may comprise (a) a compound of formula (I) or a non-immunosuppressive FKBP ligand and (b) a stem cell migration agent in a ratio ranging from about 1 / 15-1 / 100, 1 / 15-1 / 99, 1 / 15-1 / 98, 1 / 15-1 / 97, 1 / 15-1 / 96, 1 / 15-1 / 95, 1 / 15-1 / 94, 1 / 15-1 / 93, 1 / 15-1 / 92, 1 / 15-1 / 91, 1 / 15-1 / 90, 1 / 15-1 / 89, 1 / 15-1 / 88, 1 / 15-1 / 87, 1 / 15-1 / 86, 1 / 15-1 / 85, 1 / 1 5-1 / 84, 1 / 15-1 / 83, 1 / 15-1 / 82, 1 / 15-1 / 81, 1 / 15-1 / 80, 1 / 15-1 / 79, 1 / 15-1 / 78, 1 / 15-1 / 77, 1 / 15-1 / 76, 1 / 15-1 / 75, 1 / 15-1 / 74, 1 / 15-1 / 73, 1 / 15-1 / 72, 1 / 15-1 / 71, 1 / 15-1 / 70, 1 / 15-1 / 69, 1 / 15-1 / 68, 1 / 15-1 / 67, 1 / 15-1 / 66, 1 / 15-1 / 65, 1 / 15-1 / 64, 1 / 15-1 / 63, 1 / 15-1 / 62, 1 / 1 / 15-1 / 61, 1 / 15-1 / 60, 1 / 15-1 / 59, 1 / 15-1 / 58, 1 / 15-1 / 57, 1 / 15-1 / 56, 1 / 15-1 / 55, 1 / 15-1 / 54, 1 / 15-1 / 53, 1 / 15-1 / 52, 1 / 15-1 / 51, 1 / 15-1 / 50, 1 / 15-1 / 49, 1 / 15-1 / 48, 1 / 15-1 / 47, 1 / 15-1 / 46, 1 / 15-1 / 45, 1 / 15-1 / 44, 1 / 15-1 / 43, 1 / 15-1 / 42, 1 / 15-1 / 41, 1 / 15-1 / 40, 1 / 15-1 / 39, 1 1 / 15-1 / 24, 1 / 15-1 / 23, 1 / 15-1 / 22, 1 / 15-1 / 21, 1 / 15-1 / 20, 1 / 15-1 / 19, 1 / 15-1 / 18, 1 / 15-1 / 17, or 1 / 15-1 / 16.

[0178] The ratio range of (a) the compound of formula (I) or the non-immunosuppressive FKBP ligand to (b) the stem cell migration agent in the pharmaceutical composition may include about 1 / 20-1 / 100, 1 / 20-1 / 99, 1 / 20-1 / 98, 1 / 20-1 / 97, 1 / 20-1 / 96, 1 / 20-1 / 95, 1 / 20-1 / 94, 1 / 20-1 / 93, 1 / 20-1 / 92, 1 / 20-1 / 91, 1 / 20-1 / 90, 1 / 20-1 / 89, 1 / 20-1 / 88, 1 / 20-1 / 87, 1 / 20-1 / 86, 1 / 20-1 / 85, 1 / 20-1 / 86 1 / 20-1 / 84, 1 / 20-1 / 83, 1 / 20-1 / 82, 1 / 20-1 / 81, 1 / 20-1 / 80, 1 / 20-1 / 79, 1 / 20-1 / 78, 1 / 20-1 / 77, 1 / 20-1 / 76, 1 / 20-1 / 75, 1 / 20-1 / 74, 1 / 20-1 / 73, 1 / 20-1 / 72, 1 / 20-1 / 71, 1 / 20-1 / 70, 1 / 20-1 / 69, 1 / 20-1 / 68, 1 / 20-1 / 67, 1 / 20-1 / 66, 1 / 20-1 / 65, 1 / 20-1 / 64, 1 / 20 -1 / 63, 1 / 20-1 / 62, 1 / 20-1 / 61, 1 / 20-1 / 60, 1 / 20-1 / 59, 1 / 20-1 / 58, 1 / 20-1 / 57, 1 / 20-1 / 56, 1 / 20-1 / 55, 1 / 20-1 / 54, 1 / 20-1 / 53, 1 / 20-1 / 52, 1 / 20-1 / 51, 1 / 20-1 / 50, 1 / 20-1 / 49, 1 / 20-1 / 48, 1 / 20-1 / 47, 1 / 20-1 / 46, 1 / 20-1 / 45, 1 / 20-1 / 44, 1 / 20-1 / 43, 1 / 20-1 / 42, 1 / 20-1 / 41, 1 / 20-1 / 40, 1 / 20-1 / 39, 1 / 20-1 / 38, 1 / 20-1 / 37, 1 / 20-1 / 36, 1 / 20-1 / 35, 1 / 20-1 / 34, 1 / 20-1 / 33, 1 / 20-1 / 32, 1 / 20-1 / 31, 1 / 20-1 / 30, 1 / 20-1 / 29, 1 / 20-1 / 28, 1 / 20-1 / 27, 1 / 20-1 / 26, 1 / 20-1 / 25, 1 / 20-1 / 24, 1 / 20-1 / 23, 1 / 20-1 / 22, or 1 / 20-1 / 21.

[0179] In other embodiments, the ratio of (a) the compound of formula (I) or the non-immunosuppressive FKBP ligand to (b) the stem cell migration agent in the pharmaceutical composition may include about 1 / 30-1 / 100, 1 / 30-1 / 99, 1 / 30-1 / 98, 1 / 30-1 / 97, 1 / 30-1 / 96, 1 / 30-1 / 95, 1 / 30-1 / 94, 1 / 30-1 / 93, 1 / 30-1 / 92, 1 / 30-1 / 91, 1 / 30-1 / 90, 1 / 30-1 / 89, 1 / 30-1 / 88, 1 / 30-1 / 87、1 / 30-1 / 86、1 / 30-1 / 85、1 / 30-1 / 84、1 / 30-1 / 83、1 / 30-1 / 82、1 / 30-1 / 81、1 / 30-1 / 80、1 / 30-1 / 79、1 / 30-1 / 78、1 / 30-1 / 77、1 / 30-1 / 76、1 / 30-1 / 75、1 / 30-1 / 74、1 / 30-1 / 73、1 / 30-1 / 72、1 / 30-1 / 71、1 / 30-1 / 70、1 / 30-1 / 69、 1 / 30-1 / 68、1 / 30-1 / 67、1 / 30-1 / 66、1 / 30-1 / 65、1 / 30-1 / 64、1 / 30-1 / 63、1 / 30-1 / 62、1 / 30-1 / 61、1 / 30-1 / 60、1 / 30-1 / 59、1 / 30-1 / 58、1 / 30-1 / 57、1 / 30-1 / 56、1 / 30-1 / 55、1 / 30-1 / 54、1 / 30-1 / 53、1 / 30-1 / 52、1 / 30-1 / 51、1 / 30-1 / 50、 1 / 30-1 / 49, 1 / 30-1 / 48, 1 / 30-1 / 47, 1 / 30-1 / 46, 1 / 30-1 / 45, 1 / 30-1 / 44, 1 / 30-1 / 43, 1 / 30-1 / 42, 1 / 30-1 / 41, 1 / 30-1 / 40, 1 / 30-1 / 39, 1 / 30-1 / 38, 1 / 30-1 / 37, 1 / 30-1 / 36, 1 / 30-1 / 35, 1 / 30-1 / 34, 1 / 30-1 / 33, 1 / 30-1 / 32 or 1 / 30-1 / 31.

[0180] In other embodiments, the pharmaceutical composition may comprise (a) a compound of formula (I) or a non-immunosuppressive FKBP ligand and (b) a stem cell migration agent in a ratio ranging from about 1 / 40 to 1 / 100, 1 / 40 to 1 / 99, 1 / 40 to 1 / 98, 1 / 40 to 1 / 97, 1 / 40 to 1 / 96, 1 / 40 to 1 / 95, 1 / 40 to 1 / 94, 1 / 40 to 1 / 93, 1 / 40 to 1 / 92, 1 / 40 to 1 / 91, 1 / 40 to 1 / 92, 1 / 40 to 1 / 93, 1 / 40 to 1 / 94, 1 / 40 to 1 / 95, 1 / 40 to 1 / 96, 1 / 40 to 1 / 97 ... / 40-1 / 90, 1 / 40-1 / 89, 1 / 40-1 / 88, 1 / 40-1 / 87, 1 / 40-1 / 86, 1 / 40-1 / 85, 1 / 40-1 / 84, 1 / 40-1 / 83, 1 / 40-1 / 82, 1 / 40-1 / 81, 1 / 40-1 / 80, 1 / 40-1 / 79, 1 / 40-1 / 78, 1 / 40-1 / 77, 1 / 40-1 / 76, 1 / 40-1 / 75, 1 / 40-1 / 74, 1 / 40-1 / 73, 1 / 40-1 / 72, 1 / 40-1 / 71, 1 / 40-1 / 70, 1 / 40-1 / 69, 1 / 40-1 / 68, 1 / 40-1 / 67, 1 / 40-1 / 66, 1 / 40-1 / 65, 1 / 40-1 / 64, 1 / 40-1 / 63, 1 / 40-1 / 62, 1 / 40-1 / 61, 1 / 40-1 / 60, 1 / 40-1 / 59, 1 / 40-1 / 58, 1 / 4 0-1 / 57, 1 / 40-1 / 56, 1 / 40-1 / 55, 1 / 40-1 / 54, 1 / 40-1 / 53, 1 / 40-1 / 52, 1 / 40-1 / 51, 1 / 40-1 / 50, 1 / 40-1 / 49, 1 / 40-1 / 48, 1 / 40-1 / 47, 1 / 40-1 / 46, 1 / 40-1 / 45, 1 / 40-1 / 44, 1 / 40-1 / 43, 1 / 40-1 / 42, or 1 / 40-1 / 41.

[0181] In alternative embodiments, the pharmaceutical composition may comprise (a) a compound of formula (I) or a non-immunosuppressive FKBP ligand and (b) a stem cell migratory agent in a ratio ranging from about 1 / 50 to 1 / 100, 1 / 50 to 1 / 99, 1 / 50 to 1 / 98, 1 / 50 to 1 / 97, 1 / 50 to 1 / 96, 1 / 50 to 1 / 95, 1 / 50 to 1 / 94, 1 / 50 to 1 / 93, 1 / 50 to 1 / 92, 1 / 50 to 1 / 91, 1 / 50 to 1 / 90, 1 / 50 to 1 / 89, 1 / 50 to 1 / 88, 1 / 50 to 1 / 87, 1 / 50 to 1 / 86, 1 / 50 to 1 / 85, 1 / 50 to 1 / 84, 1 / 50 to 1 / 86 1 / 83, 1 / 50-1 / 82, 1 / 50-1 / 81, 1 / 50-1 / 80, 1 / 50-1 / 79, 1 / 50-1 / 78, 1 / 50-1 / 77, 1 / 50-1 / 76, 1 / 50-1 / 75, 1 / 50-1 / 74, 1 / 50-1 / 73, 1 / 50-1 / 72, 1 / 50-1 / 71, 1 / 50-1 / 70, 1 / 50-1 / 69, 1 / 50-1 / 68, 1 / 50-1 / 67, 1 / 50-1 / 66, 1 / 50-1 / 65, 1 / 50-1 / 64, 1 / 50-1 / 63, 1 / 50-1 / 62, 1 / 50-1 / 61, 1 / 50-1 / 60, 1 / 50-1 / 59, 1 / 50-1 / 58, 1 / 50-1 / 57, 1 / 50-1 / 56, 1 / 50-1 / 55, 1 / 50-1 / 54, 1 / 50-1 / 53, 1 / 50-1 / 52, 1 / 50-1 / 51, 1 / 60-1 / 100, 1 / 60-1 / 99, 1 / 60-1 / 98, 1 / 60-1 / 97, 1 / 60-1 / 96, 1 / 60-1 / 95, 1 / 60-1 / 94, 1 / 60-1 / 93, 1 / 60-1 / 92, 1 / 60-1 / 91, 1 / 60-1 / 90, 1 / 60-1 / 89, 1 / 60-1 / 88, 1 / 60-1 / 87, 1 / 60-1 / 86, 1 / 60-1 / 8 5. 1 / 60-1 / 84, 1 / 60-1 / 83, 1 / 60-1 / 82, 1 / 60-1 / 81, 1 / 60-1 / 80, 1 / 60-1 / 79, 1 / 60-1 / 78, 1 / 60-1 / 77, 1 / 60-1 / 76, 1 / 60-1 / 75, 1 / 60-1 / 74, 1 / 60-1 / 73, 1 / 60-1 / 72, 1 / 60-1 / 71, 1 / 60-1 / 70, 1 / 60-1 / 69, 1 / 60-1 / 68, 1 / 60-1 / 67, 1 / 60-1 / 66, 1 / 60-1 / 65, 1 / 60-1 / 64, 1 / 60-1 / 63, 1 / 60-1 / 62, 1 / 60-1 / 61.

[0182] In other embodiments, the ratio of (a) the compound of formula (I) or the non-immunosuppressive FKBP ligand to (b) the stem cell migration agent in the pharmaceutical composition may include about 1 / 70-1 / 100, 1 / 70-1 / 99, 1 / 70-1 / 98, 1 / 70-1 / 97, 1 / 70-1 / 96, 1 / 70-1 / 95, 1 / 70-1 / 94, 1 / 70-1 / 93, 1 / 70-1 / 92, 1 / 70-1 / 91, 1 / 70-1 / 92, 1 / 70-1 / 93, 1 / 70-1 / 94, 1 / 70-1 / 95, 1 / 70-1 / 96, 1 / 70-1 / 97 ... 1 / 90, 1 / 70-1 / 89, 1 / 70-1 / 88, 1 / 70-1 / 87, 1 / 70-1 / 86, 1 / 70-1 / 85, 1 / 70-1 / 84, 1 / 70-1 / 83, 1 / 70-1 / 82, 1 / 70-1 / 81, 1 / 70-1 / 80, 1 / 70-1 / 79, 1 / 70-1 / 78, 1 / 70-1 / 77, 1 / 70-1 / 76, 1 / 70-1 / 75, 1 / 70-1 / 74, 1 1 / 70-1 / 73, 1 / 70-1 / 72, 1 / 70-1 / 71, 1 / 80-1 / 100, 1 / 80-1 / 99, 1 / 80-1 / 98, 1 / 80-1 / 97, 1 / 80-1 / 96, 1 / 80-1 / 95, 1 / 80-1 / 94, 1 / 80-1 / 93, 1 / 80-1 / 92, 1 / 80-1 / 91, 1 / 80-1 / 90, 1 / 80-1 / 89, 1 / 80-1 / 88, 1 / 80- 1 / 87, 1 / 80-1 / 86, 1 / 80-1 / 85, 1 / 80-1 / 84, 1 / 80-1 / 83, 1 / 80-1 / 82, 1 / 80-1 / 81, 1 / 90-1 / 100, 1 / 90-1 / 99, 1 / 90-1 / 98, 1 / 90-1 / 97, 1 / 90-1 / 96, 1 / 90-1 / 95, 1 / 90-1 / 94, 1 / 90-1 / 93, 1 / 90-1 / 92 or 1 / 90-1 / 91.

[0183] In a specific embodiment, the pharmaceutical composition of the present invention can be administered at least once per week over the course of several weeks. In one embodiment, the pharmaceutical composition is administered at least once per week over a period of several weeks to several months. In another embodiment, the pharmaceutical composition is administered once per week over a period of 4 weeks to 8 weeks. In yet another embodiment, the pharmaceutical composition is administered once per week over a period of 4 weeks.

[0184] More specifically, the pharmaceutical composition can be administered at least once a day for about 2 days, at least once a day for about 3 days, at least once a day for about 4 days, at least once a day for about 5 days, at least once a day for about 6 days, at least once a day for about 7 days, at least once a day for about 8 days, at least once a day for about 9 days, at least once a day for about 10 days, at least once a day for about 11 days, at least once a day for about 12 days, at least once a day for about 13 days, at least once a day for about 14 days, at least once a day for about 15 days, at least once a day for about 16 days, times, at least once a day for about 17 days, at least once a day for about 18 days, at least once a day for about 19 days, at least once a day for about 20 days, at least once a day for about 21 days, at least once a day for about 22 days, at least once a day for about 23 days, at least once a day for about 24 days, at least once a day for about 25 days, at least once a day for about 26 days, at least once a day for about 27 days, at least once a day for about 28 days, at least once a day for about 29 days, at least once a day for about 30 days, or at least once a day for about 31 days.

[0185] In other embodiments, the pharmaceutical composition can be administered every other day for about 2 days, every other day for about 3 days, every other day for about 4 days, every other day for about 5 days, every other day for about 6 days, every other day for about 7 days, every other day for about 8 days, every other day for about 9 days, every other day for about 10 days, every other day for about 11 days, every other day for about 12 days, every other day for about 13 days, every other day for about 14 days, every other day for about 15 days, every other day for about 16 days. every other day for about 17 days, every other day for about 18 days, every other day for about 19 days, every other day for about 20 days, every other day for about 21 days, every other day for about 22 days, every other day for about 23 days, every other day for about 24 days, every other day for about 25 days, every other day for about 26 days, every other day for about 27 days, every other day for about 28 days, every other day for about 29 days, every other day for about 30 days, or every other day for about 31 days or more.

[0186] Alternatively, the pharmaceutical composition can be administered about once a day, about once every 2 days, about once every 3 days, about once every 4 days, about once every 5 days, about once every 6 days, about once every 7 days, about once every 8 days, about once every 9 days, about once every 10 days, about once every 11 days, about once every 12 days, about once every 13 days, about once every 14 days, about once every 15 days, about once every 16 days, about once every 17 days, about once every 18 days, about once every 19 days, about once every 20 days, about once every 21 days, about once every 22 days, about once every 23 days, about once every 24 days, about once every 25 days, about once every 26 days, about once every 27 days, about once every 28 days, about once every 29 days, about once every 30 days, or about once every 31 days. In certain embodiments, the pharmaceutical composition is administered every other day.

[0187] Alternatively, the pharmaceutical composition of the present invention can be administered about once a week, about once every 2 weeks, about once every 3 weeks, about once every 4 weeks, about once every 5 weeks, about once every 6 weeks, about once every 7 weeks, about once every 8 weeks, about once every 9 weeks, about once every 10 weeks, about once every 11 weeks, about once every 12 weeks, about once every 13 weeks, about once every 14 weeks, about once every 15 weeks, about once every 16 weeks, about once every 17 weeks, about once every 18 weeks, about once every 19 weeks, or about once every 20 weeks.

[0188] Alternatively, the pharmaceutical composition of the present invention can be administered about once a month, about once every 2 months, about once every 3 months, about once every 4 months, about once every 5 months, about once every 6 months, about once every 7 months, about once every 8 months, about once every 9 months, about once every 10 months, about once every 11 months, or about once every 12 months.

[0189] Alternatively, the pharmaceutical composition can be administered at least once a week for about 2 weeks, at least once a week for about 3 weeks, at least once a week for about 4 weeks, at least once a week for about 5 weeks, at least once a week for about 6 weeks, at least once a week for about 7 weeks, at least once a week for about 8 weeks, at least once a week for about 9 weeks, at least once a week for about 10 weeks, at least once a week for about 11 weeks, at least once a week for about 12 weeks, at least once a week for about 13 weeks, at least once a week for about 14 weeks, at least once a week for about 15 weeks, at least once a week for about 16 weeks, at least once a week for about 17 weeks, at least once a week for about 18 weeks, at least once a week for about 19 weeks, or at least once a week for about 20 weeks.

[0190] Alternatively, the pharmaceutical composition can be administered at least once a week for about 1 month, at least once a week for about 2 months, at least once a week for about 3 months, at least once a week for about 4 months, at least once a week for about 5 months, at least once a week for about 6 months, at least once a week for about 7 months, at least once a week for about 8 months, at least once a week for about 9 months, at least once a week for about 10 months, at least once a week for about 11 months, or at least once a week for about 12 months.

[0191] In a specific embodiment, the present invention relates to FDA-approved drugs such as stem cell mobilization agent AMD3100 and compounds of formula (I) for use in treating a variety of tissue injuries (trauma), organ transplantation and inflammatory disorders or autoimmune disorders such as inflammatory bowel disease (IBD) in sequence or in combination. Therefore, a pharmaceutical composition comprising a combination of stem cell mobilization agent AMD3100 and immunosuppressant FK506 can be used to treat a variety of disorders.

[0192] AMD3100.AMD3100 (Plerixafor or Mozobil) is a CXCR4 antagonist that was originally developed as an anti-HIV drug but was found to be effective in mobilizing CD34 and other stem cells from their bone marrow niche. AMD3100 was first approved by the FDA in 2008 for use in patients with multiple myeloma for stem cell reserving prior to myeloablative chemotherapy. Today, plerixafor is often used with neupogen (G-CSF) to mobilize hematopoietic stem cells in patients with multiple myeloma for reserving prior to myeloablative chemotherapy. Following cancer treatment, the mobilized stem cells are then transplanted back into the patient. Thus, the drug is well established to be safe and effective.

[0193] FK506. FK506 (Tacrolimus or Prograph) was discovered in 1987 from a soil bacterium, Streptomyces tsukuba. FK506 reduces peptidyl prolyl isomerase activity by binding to the immunophilin FKBP12 (FK506 binding protein) to produce a new complex. FK506 was first approved by the FDA in 1994 for liver transplantation; this has been expanded to include kidney transplantation, heart transplantation, small intestine transplantation, pancreas transplantation, lung transplantation, tracheal transplantation, skin transplantation and corneal transplantation, bone marrow transplantation and limb transplantation. The compound of formula (I) also reduces peptidyl prolyl isomerase activity by binding to the immunophilin FKBP12, but does not have the immunosuppressive effect of FK506.

[0194] Pharmaceutical compositions described herein provide effective synergistic activity of AMD3100 and compounds of formula (I) in terms of migration, recruitment and retention of stem cells at the site of injury. In inflammatory bowel disease, combined treatment induces organ transplantation acceptance, accelerates wound healing and promotes intestinal mucosal regeneration. In certain embodiments, the ratio of the compound of formula (I) to AMD3100 in the composition is about 1 / 10 to 1 / 100. In a specific embodiment, the pharmaceutical composition only comprises two active ingredients, wherein the first active ingredient is AMD3100, and the second active ingredient is a compound of formula (I), wherein the composition comprises 10mg-40mg of AMD3100 and 0.1mg to 4mg of the compound of formula (I), wherein the compound enhances the effectiveness of the AMD3100, and wherein the pharmaceutical composition also comprises an excipient. The composition is formulated for subcutaneous injection or intravenous injection, or may be formulated for oral administration or topical application.

[0195] The dosage of AMD3100 and the compound of formula (I) can be determined separately. In the treatment scheme of the prior art, AMD3100 is usually applied to human patients at about 0.12mg / kg / day-0.24mg / kg / day. The compound of formula (I) is usually applied intravenously at about 0.01mg / kg / day-0.05mg / kg / day or orally at 0.1mg / kg / day-0.2mg / kg / day to prevent immune-mediated rejection after organ transplantation. The dosage of the compound of formula (I) is adjusted according to clinical response and observed FK506 whole blood trough concentration (6ng / ml-20ng / ml). In animals (rodents), AMD3100 is usually applied at about 1mg / kg / day to effectively migrate bone marrow stem cells. The compound of formula (I) is usually applied to rodents at about 0.5mg / kg / day-3mg / kg / day to prevent immune-mediated rejection after organ transplantation. In the methods and compositions of the present invention, the compound of formula (I) will generally be used at a lower dosage than that given above (e.g., a blood trough concentration of about 0.5 ng / ml-4 ng / ml). Thus, AMD3100 can be used in humans at a dosage of 0.12 mg / kg / day-0.24 mg / kg / day (1 mg / kg / day for animals), and the compound of formula (I) can be used in a dosage of 0.001 mg / kg-0.005 mg / kg (subcutaneously).

[0196] In one embodiment, the compositions of the invention provided herein can be controlled release compositions, i.e., compositions in which one or more compounds are released within a period of time after administration. Controlled release compositions or sustained release compositions include formulations in lipophilic depots (lipophilic depots) (e.g., fatty acids, waxes, oils). In another embodiment, the composition is an immediate release composition, i.e., a composition that releases all or substantially all compounds immediately after administration.

[0197] In yet another embodiment, the compounds of the invention can be delivered in a controlled release system. For example, the agent can be administered using intravenous infusion, an implantable osmotic pump, a transdermal patch, or other modes of administration. In an embodiment, a pump can be used. In one embodiment, a polymeric material can be used.

[0198] Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent.The following examples are merely illustrative, and are not intended to limit the remainder of the disclosure in any way whatsoever. Example

[0199] The following examples are presented to provide a person of ordinary skill in the art with a complete disclosure and description of how to prepare and evaluate the compounds, compositions, articles, devices and / or methods described and claimed herein, and the following examples are intended to be purely illustrative and are not intended to limit the scope of the invention that the inventors regard as their invention. Efforts have been made to ensure the accuracy of numbers (e.g., amounts, temperatures, etc.), but some errors and deviations should be considered herein. Unless otherwise indicated, parts are parts by weight, temperatures are in degrees Celsius or are at ambient temperature, and pressures are at or near atmospheric pressure. There are many variations and combinations of reaction conditions, such as component concentrations, desired solvents, solvent mixtures, temperature, pressure, and other reaction ranges and conditions that can be used to optimize the product purity and yield obtained by the described process. Only reasonable and conventional experiments will be required to optimize such process conditions.

[0200] Chemical synthesis of compounds of formula (I)

[0201] The present inventors synthesized the first novel non-immunosuppressive FK506 analog, named FKVP, which retains FKBP binding and lacks calcineurin inhibitory activity ( Figure 1 ). It was found that FKVP activated bone morphogenetic protein (BMP) signaling in lymphocytes and endothelial cells by disrupting FKBP12-BMPR1 interaction. In addition, it was found that the combination of FKVP and AMD3100 accelerated wound healing in diabetic rats in a BMP receptor-dependent manner.

[0202] FKVP Synthesis and Formulation for Animal Studies

[0203] FK506 (100 mg, 0.120 mmol) and 40 mol% Zhan1b catalyst RuCl2[C 21 H 26 N2][C 12 H 17 NO3S] 4-vinylpyridine (14.2 μL, 0.132 mmol) was added to a solution in 3 mL of anhydrous DCE. The mixture was stirred for 30 seconds and then microwaved at 120°C for 20 min. The mixture was then purified using flash chromatography (0%-25% MeOH in DCM), preparative TLC (9:1 DCM:MeOH) and reverse phase HPLC (45%-85% ACN in H2O). Conversion = 25%, purification yield = 8%. LC-MS and 1 H-NMR experiments confirmed that the new compound was >99% free of the parent compound (FK506). The product was analyzed by Hi-Resolution MS ( Fig. 20 )and 1 H-NMR( Figure 21A-21B ) and then dissolved in DMSO or in formulations for animal experiments.

[0204] For animal experiments, FKVP powder was dissolved at 5 mg / mL in 80% EtOH / 20% Cremophor RH60 solution. This stock solution was diluted 1:50 into saline prior to subcutaneous injection.

[0205] Table 2: Antibody information

[0206]

[0207] Chemicals: Research grade FK506 (>98%) was purchased from APExBIO. LDN-193189 hydrochloride, 4-vinylpyridine and solvents were purchased from MilliporeSigma. Zhan-1b ruthenium catalyst (CAS: 918870-76-5) was purchased from STREM Chemicals.

[0208] Cell culture and transfection

[0209] Jurkat (E6.1, ATCC) cells were cultured in RPMI with 10% FBS and 1.5% PennStrep. Jurkat cells (1×10 6 ) were transfected with 10 μg of BRE luciferase (kindly provided by Martine Roussel & Peter ten Dijke) or NFAT luciferase cDNA (Promega) by electroporation (BioRad, square wave, 250 V, 950 μF) in 400 μL serum-free / antibiotic RPMI containing 0.5% DMSO. 30 minutes after transfection, cells were transferred to complete RPMI and left overnight. Prior to plating, cells were resuspended in fresh medium and diluted to 0.5×10 6 Cells / mL. HEK293T cells were cultured in DMEM with 10% FBS, 1% PennStrep and 500 μg / mL G418 (Corning). Cells were transfected using SuperFect reagent and the provided transfection protocol. HUVEC cells were cultured in Lonza Endothelial Cell Growth Medium (EGM-2) and used between passages 3 and 7. All cells were cultured at 37°C and 5% CO2.

[0210] Cell viability assay

[0211] Jurkat or HUVEC cells were plated at 1000 cells / well in 180 μL growth medium before adding 20 μL of 10× drug / protein stock solution. After incubation for 72 h, 22 μL of resazurin sodium salt solution (0.1 mg / mL stock solution in water) was added to each well and allowed to incubate at 37°C. Metabolic conversion of resazurin dye was monitored by absorbance at 570 nm after 6 h. After background subtraction (medium + dye only), absorbance values ​​were left as arbitrary absorbance units or normalized to those obtained from cells treated with DMSO.

[0212] Western blotting

[0213] Jurkat T cells are collected by centrifugation (300g, 5min), washed with PBS, and lysed with sonication in RIPA buffer containing protease and phosphatase inhibitors (Cell Signaling). Lysate is normalized using DC assay (BioRad) and run on SDS-PAGE gel. Protein is transferred to nitrocellulose membrane overnight at 100mA. After blocking with TBS-T containing 5% milk for 20min, membrane is incubated overnight at 4°C with primary antibody (Table 2). After washing three times with TBS-T, membrane is incubated with secondary antibody (Table 2) for 1 hour. After another 3 washings, use SynGene, use ECL substrate (Thermo) or laser excitation and filter (647nm) to make the trace visible.

[0214] FKBP12-SNAP pull-down

[0215] FKBP12-SNAP was cloned using the pSNAPf vector (New England Bio) and PCR amplified FKBP12 (a gift from Tobias Meyer, Addgene plasmid #20175) with the addition of EcoRI and BsrGI restriction sites. The gel-purified plasmid was ligated using T4 DNA ligase (Thermo), transformed into DH5α, and plated on LB-agar plus ampicillin for single colony selection, sequencing, and plasmid purification.

[0216] The Alk1-V5 plasmid was generated by gateway cloning of pDONR223-ACVRL1 (Alk1) (a gift from William Hahn & David Root, Addgene plasmid #23873) and pEF-DEST51 ​​(Thermo) vectors using LR Clonase enzyme mix (Thermo). The plasmid was transformed into DH5α and plated on LB-agar plus ampicillin for single colony selection, sequencing, and plasmid purification. The HA-tagged Alk2 plasmid, Alk3 plasmid, and Alk6 plasmid were gifts from Aristidis Moustakas (Addgene plasmids #80870, 80873, 80882).

[0217] FKBP12-SNAP (5 μg) and tagged ALK receptor plasmid (5 μg) were co-transfected into Hek293T cells using SuperFect and the provided protocol. After 48 hours, cells were treated with DMSO (0.1%) or 1 μM FKVP for 30 min. After 1 hour, cells were lysed in a container with 1 mL lysis buffer (150 mM NaCl, 50 mM Tris-HCl, 0.1% Trition-100, 5% glycerol, protease and phosphatase inhibitors) and a scraper. The lysate was transferred to a 2 mL eppendorf and rotated at 4 ° C for 20 min. The lysate was centrifuged at 14000 g for 10 min, and ~1 mL of the supernatant was transferred to a new tube with 200 nM drug or 0.2% DMSO. Take 20 μL of input and mix it with 20 μL of 2× loading buffer and then boil. Each lysate sample was then mixed with 250 μL of SNAP buffer (lysis buffer + 5 mM DTT) containing 40 μL of magnetic SNAP-capture beads (New England Bio) and rotated at 4°C for 1 hour. The beads were washed 3 times with 1 mL of lysis buffer and then boiled in 100 μL of 2× loading buffer. The boiled lysate was vortexed and centrifuged, and the lysate (~90 μL) was carefully removed from the beads and used for Western blotting.

[0218] The same approach was used for calcineurin-FK506 and mTOR-rapamycin pull-downs after transfection of 10 μg of FKBP12-SNAP cDNA only.

[0219] FKBP knockout lines

[0220] As previously described, Jurkat T cells and Hek293T cells were transfected with integrated CRISPR / Cas9 (mCherry tag) plasmids (Genecopeia), which contained guide RNAs for FKBP12 (HCP267023-CG01-3-B), FKBP51 (HCP257374-CG01-3-B) or FKBP52 (HCP205551-CG01-3-B). After 48 hours, cells were sorted into 96-well plates (1 cell / well) for mCherry fluorescence (650nm laser). After 2 weeks of cultivation, monoclonal clones were verified by Western blotting.

[0221] BMP and NFAT pathway reporters

[0222] Jurkat T cells transfected with BRE-Luc were distributed into 96-well plates (80 μL / well, 0.5×10 6 Cells / mL) were added and treated with the previously stated compounds / proteins (20 μL of 5× stock solutions in RPMI, 0.5% DMSO) for 18 h. rBMP-4 and rTGF-β1 (R&D) were used as positive and negative controls, respectively. The plate was centrifuged at 3000 rpm for 10 min and then carefully aspirated. The cells were resuspended in 100 μL lysis buffer (per well) and placed on a plate shaker for 30 min. An aliquot of 80 μL of the lysate was transferred to a white-walled 96-well plate and luminescence was recorded 2 seconds after the automatic injection of the luciferase substrate. The luminescence value was background subtracted (lysis buffer+substrate) and normalized to the DMSO control value.

[0223] FKBP12KO Jurkat T cells transfected with FKBP12-SNAP plasmid were selected with 1200 μg / mL G418 for 7 days before use in BRE-Luc assay.

[0224] Jurkat T cells transfected with NFAT-Luc were distributed into 96-well plates (80 μL / well, 0.5×10 6 Cells / mL) and treated with the specified compound (20 μL of 5× stock solution in RPMI, 0.5% DMSO) for 30 min, then activated with PMA / ionomycin (40 nM / 1 μM). After 6 h, the wells were aspirated, lysed and luminescence was measured as previously described. FK506 and CsA were used as positive controls, while DMSO and unactivated wells gave negative control values. Except for the knockout cell experiments, Jurkat cells used for each experiment were transfected simultaneously and cultured together overnight until plating and processing on the second day.

[0225] Ethical Statement on Animal Care and Use

[0226] Goto-Kakizaki (GK) type 2 diabetic rats obtained from Charles River (Boston, MA) were housed in a pathogen-free facility and were cared for according to NIH guidelines and a protocol approved by the Johns Hopkins University Animal Care and Use Committee (ACUC). In this study, both male and female GK rats aged 4-5 months were used.

[0227] In vivo excisional wound model

[0228] Full-thickness wounds were created in the dorsal skin of rats with a sterile disposable biopsy punch (8 mm in diameter). Animals were injected subcutaneously with saline, AMD3100 (1 mg / kg) plus FK506 (0.1 mg / kg) or AMD3100 (1 mg / kg) plus FKVP (0.1 mg / kg) immediately after the wound and every other day until complete healing, which was defined as complete epidermal cell regeneration in the wound area. To evaluate the role of BMP signaling, in addition to standard saline or AF treatment, animals were injected intraperitoneally (ip) with LDN-193189 (2 mg / kg / day). Wounds were evaluated daily according to previously described methods (Lin et al., 2014).

[0229] Immunohistochemistry

[0230] 5 μm sections were prepared from frozen tissue for immunohistochemical staining. The frozen sections were fixed with acetone at -20 ° C for 10 min and dried at room temperature for 1 h. After inactivating endogenous peroxidase and blocking nonspecific antibody binding, the samples were incubated with anti-CD133 (1:300, ab19898; Abcam) at 4 ° C overnight. The tissue sections were then incubated with biotin-conjugated goat anti-rabbit IgG (1:200, #14708S Cell Signaling, Danvers, MA) at room temperature for 30 minutes. VectaStain Elite ABC kit (HRP) (Vector Laboratories, Burlingame, CA) was used to improve the sensitivity of staining. Diaminobenzidine tetrahydrochloride (5min, D4293, Sigma-Aldrich, St.Louis, MO) was used as a chromogen, and Mayer's hematoxylin (30s, Dako, S3309) was used for counterstaining.

[0231] statistics

[0232] When comparing days to wound healing, one-way analysis of variance (ANOVA) was used to determine statistical differences in wound healing between the AF, AF+LDN, saline, and S+LDN groups or between the AF, AV, and saline groups. The Bonferroni-Holm post hoc procedure was used for p-value adjustment. p<0.05 was considered a significant difference.

[0233] Example 1

[0234] To conduct structure-activity relationship studies, the inventors synthesized new FKVP analogs containing a pyridine core structure. In the past, the inventors and others have relied on ruthenium-catalyzed cross-metathesis (CM) to modify the terminal olefin (C40) of FK506 to produce non-immunosuppressive FK506 analogs. Unfortunately, FKVP and other nitrogen-containing analogs can only be obtained in very low yields. This is most likely due to competition with the ruthenium metal center for coordinated nitrogen lone pairs. The use of soluble p-toluenesulfonates of amines can improve yields, but this did not help in the case of FKBP.

[0235] To address the issues associated with nitrogenous base substrates in the CM reaction, we turned to the Heck reaction as an alternative because the palladium catalyst used in the Heck reaction is compatible with nitrogenous heterocycles such as pyridine. After optimizing the reaction conditions, we found that reacting FK506 with 4-iodopyridine (2.0 equiv.) in the presence of Pd(OAc)2 (10 mol%) and P(o-tol)3 (20 mol%) at 100 °C in DMF gave the best yield (66%).

[0236] As an example, under the protection of an argon balloon, to a mixture of FK-506 (0.0500mmol, 40mg, 1.0 equivalent) in a flame-dried 100mL Schlenk tube, aryl halides (2.0 equivalents) and Pd(OAc) (0.00500mmol, 1.1mg, 0.10 equivalent) and P(o-tol) (0.0100mmol, 3.0mg, 0.20 equivalent) add dry DMF (1.0mL) and KCO dry EtN (0.10mL), and the mixture is stirred at 100°C. The reaction is monitored by mass spectrometry. When the reaction is complete, the reaction mixture is cooled to room temperature, and with a gradient solvent (dichloromethane and methanol) by rapid column purification, to give the corresponding product. If necessary, PLC is used as additional purification to separate diastereomers.

[0237] Using FKVP as a lead, 7 nitrogen-containing heterocyclic halides with a pyridine core and 4 halides with substituted anilines were selected as substrates for the Heck reaction. To our delight, all halide substrates were successfully coupled to FK506 in moderate to good yields (see Table 1).

[0238] Halide substrates show significant reactivity in the Heck reaction. First, bromides and iodides give similar yields. Second, the electron withdrawing groups on pyridine and quinoline appear to increase the yield (Table 1, entries 4, 6, 7). Third, unprotected aniline gives the lowest yield (Table 1, entries 9-11). Importantly, the unreacted FK506 starting material in the reaction mixture is easily separated from the more polar nitrogen-containing product by flash chromatography.

[0239] Example 2

[0240] In the case of FK506 analogs, their effects on cell viability, BMP activation and NFAT activation were evaluated at two concentrations ( Figure 2A-2C In a HUVEC cell viability assay, we found that quinoline analogs (5b-7b) inhibited cell proliferation at 10 μM ( Figure 2A ), while the cytotoxicity of the other compounds was comparable to that of FK506.

[0241] We used a BMP response element (BRE) pathway reporter (luciferase under the control of the ID1 gene promoter) in Jurkat T cells to determine whether new analogs were able to activate the BMP signaling pathway. Initial screening of compounds indicated that most analogs had similar activity to FK506 or FKVP. This was somewhat expected, as we have previously shown that FKBP12 binding is necessary and sufficient for activation of BMP signaling ( Figure 2B ).

[0242] To determine the effects of analogs on calcineurin, we used 8d A PMA / ionomycin-activated nuclear factor of activated T cells (NFAT) reporter (luciferase under the control of the IL-2 promoter) was used. Figure 2C ). Two analogs (2b, 3b) did not cause significant inhibition of the NFAT luciferase reporter at concentrations up to 10 μM, similar to FKVP (1b). Surprisingly, most of the other analogs showed partial or almost complete inhibition of the NFAT reporter at 1 μM ( Figure 2C ). Of note, some immunosuppressive analogs, including 5b, 6b, and 7b, have bulkier substituents than 2b and 3b due to the presence of fused aromatic rings. How those bulkier groups are accommodated in the calcineurin binding site remains to be determined.

[0243] Example 3

[0244] We determined the EC values ​​of three non-immunosuppressive analogs (1b-3b) in a BMP luciferase assay. 50 All three analogs were found to be slightly more potent than FK506 ( Figure 3 ), which may be attributed to the increased solubility due to the more polar pyridine substituent. Among the three analogs, FKVP remained the most active with an IC of 12.4 nM 50 .

[0245] Example 4

[0246] The structure of the FKBP12-FK506-calcineurin complex has previously been determined by X-ray crystallography. In this complex, the terminal olefin of FK-506 fits into a binding pocket in calcineurin formed by hydrophobic amino acids ( Figure 4A When modeled in place of FK506, the pyridine moiety in FKVP (1b) has a steric clash with residue M118 of calcineurin ( Figure 4B), which explains the abrogation of calcineurin binding by analogs 1b-3b. However, how some other analogs containing more bulky substituents than FKVP at the same positions remain immunosuppressive cannot be explained by structure.

[0247] After investigating the pKa values ​​of all nitrogen-containing analogs, we noticed that compounds with higher pKa values ​​(1b, 2b, 3b, 5b) showed lower calcineurin inhibition. Conversely, those compounds with lower pKa caused by electron withdrawing groups were all immunosuppressive at 1 μM (4b, 6b, 7b). The pKa values ​​of anilines (9b-11b) were lower than those of pyridines (1b), which may also explain their retention of calcineurin inhibitory activity. These observations suggest that the formation of the positively charged pyridinium appendage at the terminal olefin of FK506 plays a more important role in interfering with the interaction between the terminal olefin of FK506 and the hydrophobic pocket in calcineurin. These observations suggest an alternative and complementary mechanism for the loss of calcineurin inhibition in non-immunosuppressive FK506 analogs, which in the past was rationalized by the macromolecular "protrusions" sterically hindering calcineurin binding. It is possible that the same binding pocket in calcineurin has significant conformational flexibility to accommodate uncharged bulky aromatic rings such as those present in 4b, 6b, and 7b.

[0248] In summary, the inventors have developed an inventive method involving the one-step synthesis of FK506 analogs containing nitrogen bases using the Heck reaction. Three non-immunosuppressive analogs with high potency in activating the BMP signaling pathway were identified. It was found that the key element to interfere with the FK506-calcineurin interaction is through electrostatic interactions, rather than steric interactions. In view of the role of BMP signaling in wound healing and tissue regeneration, the newly developed synthetic route of FKVP and analogs will promote the development of non-immunosuppressive analogs of FK506 for regenerative medicine.

[0249] Example 5

[0250] Modification of FK506 at C40 generates a non-immunosuppressive analogue - FKVP

[0251] The in vivo immunosuppressive activity of FK506 has been determined to be mediated through inhibition of calcineurin (Bueno et al., 2002). Previous studies have shown that modification of the terminal olefin of FK506 can block calcineurin inhibition while retaining FKBP binding (Clemons et al., 2002). To determine whether enhanced healing by AF combination requires calcineurin inhibition, we designed and synthesized a non-immunosuppressive analog of FK506 by fusing a vinyl pyridine moiety to the terminal olefin as a "protuberance" of the effector domain of FK506 using cross-metathesis. The resulting analog was named FKVP ( Figure 5A The newly added pyridine moiety is intended to increase water solubility while providing steric hindrance to interfere with its interaction with calcineurin ( Figure 6 In Jurkat T cells ( Figure 5B , Fig. 7A ) and primary HUVEC cells ( Figure 7B ) and compared with the cytotoxicity of FK506. Like FK506, FKVP did not affect cell viability at concentrations up to 10 μM ( Figure 5B We then determined the effect of FKVP on the PMA / ionomycin-activated NFAT luciferase reporter gene in Jurkat T cells (Clemons et al., 2002). While both FK506 and CsA exhibited potent inhibition of the reporter, FKVP did not induce significant inhibition at concentrations as high as 10 μM ( Figure 5C ), indicating that FKVP is no longer able to inhibit calcineurin. To confirm that FKVP retains the ability to bind FKBP, we applied it to a competition assay in combination with FK506 and rapamycin, since the sequestration of free FKBP will prevent the formation of active FKBP12-FK506 complexes or FKBP-rapamycin complexes and thereby antagonize the activity of both drugs (Rao et al., 1997, Abraham et al., 1996). The effect of FK506 on calcineurin was determined using the phosphorylation state of NFATc2 as a readout. Thus, FK506 blocked the dephosphorylation of NFATc2 in response to stimulation with PMA and ionomycin ( Fig. 8A ). The presence of 10 μM FKVP reversed the inhibitory effect of FK506 on NFATc2 dephosphorylation, suggesting a mutual antagonism between FKVP and FK506. Similarly, we examined the effect of rapamycin on mTOR activity as judged by the phosphorylation state of its substrate p70s6k. High concentrations of FKVP again reversed the inhibition of rapamycin on p70s6k phosphorylation ( Figure 8B). Taken together, these results clearly demonstrate that FKVP is able to antagonize the activity of both FK506 and rapamycin by competitive binding to endogenous FKBP protein.

[0252] Example 6

[0253] The combination of FKVP and AMD3100 accelerates wound healing.

[0254] We previously reported the synergistic activity of AMD3100 and low-dose FK506 (AF) in accelerating wound healing after full-thickness skin excision (Lin et al., 2014). To determine whether FKVP has an equivalent effect, we performed a wound healing experiment in a rat model of type 2 diabetes. Four full-thickness wounds were generated by circular excision of 8 mm diameter on the shaved back of diabetic GK rats, and each wound site was digitally photographed at specified time intervals ( Fig.9A Reepithelialization of the entire wound area was used as the defining parameter for complete healing, and the time to complete healing was calculated in days for four wounds in each animal ( Fig. 9B ).

[0255] Wounded rats were randomized into three experimental groups and received subcutaneous injections of saline, AF (AMD3100 (1.0 mg / kg) plus FK506 (0.1 mg / kg)) or AV (AMD3100 plus FKVP (0.1 mg / kg)) immediately after wounding and every other day until complete healing. While the saline control group showed an average complete healing time of 26 days, animals treated with AF presented significantly faster healing, as the wounds achieved complete epithelial regeneration on day 21, consistent with our report in a non-diabetic rodent model of surgical excisional wounds (Lin et al., 2014). Importantly, the 10 rats that received AV therapy showed a significant reduction in complete healing time from 26 days to 20 days, equivalent to the AF effect ( Fig. 9C ). These results strongly suggest that inhibition of calcineurin is not responsible for the synergistic activity of AMD3100 and FK506 in accelerating wound healing.

[0256] Example 7

[0257] FKVP activates ID-1 reporter and SMAD1 / 5 phosphorylation via BMP type 1 receptor

[0258] Having ruled out calcineurin as a relevant mediator of the WH-accelerating activity of FK506, we turned to FKBP12 and the BMP signaling pathway reportedly regulated by it. We began by determining whether both FK506 and FKVP were able to activate the BMP signaling pathway by using a BMP response element (BRE) pathway reporter (luciferase under the control of the ID1 gene promoter) in Jurkat [E6.1] T cells (Spiekerkoetter et al., 2013). It was found that in addition to high levels of FKBP12 and CXCR4, the Jurkat cell line also expressed working components of BMP signaling (BMPR, SMAD1 / 5), making it an excellent model system. To confirm the selectivity of the reporter, rBMP-4 and rTGF-β1 were used as positive and negative controls, respectively. Treatment with both FKVP and FK506 caused dose-dependent activation of the reporter, and the increase in luciferase activity was completely blocked by the selective BMP1R inhibitor LDN-193189 (LDN) ( Fig. 10A ). In contrast to FK506 and FKVP, cyclosporine A (CsA) did not activate the reporter ( Fig. 10A ), which is consistent with the finding that calcineurin is not involved in BMPR1 kinase activation by FK506.

[0259] To investigate downstream BMPR1 signaling events, we determined the effects of FK506 and FKVP on SMAD1 / 5 phosphorylation. Both compounds induced SMAD1 / 5 phosphorylation in a dose-dependent manner ( Fig. 10B ), an effect abolished by LDN treatment ( Fig. 10C In contrast, phosphorylated SMAD2 / 3 did not increase under the same conditions ( Fig.11 ), suggesting that while FKBP12 can bind to the TGF-β receptor (Chen et al., 1997), its dissociation is not sufficient to activate receptor kinase activity in the absence of TGF-β. This result is consistent with previous studies showing that FK506 and non-functional analogs are unable to activate TGF-β pathway reporters in the absence of added exogenous ligand (Spiekerkoetter et al., 2013, Wang et al., 1996). In the BRE-luciferase reporter, AMD3100 alone or in combination with FKVP was found to have no effect on ID-1 promoter stimulation ( Fig.12 ), suggesting that the synergistic activity of these drugs does not occur at the level of BMP signaling. Furthermore, we found that the extracellular BMP inhibitor protein Noggin was not effective in reducing FK506- or FKVP-mediated induction of BMP reporters ( Fig.13). These observations suggest that endogenous BMPs are not required for FK506 or FKVP to activate signaling and that these compounds can activate BMPs in tissues in the presence of underexpression of BMP proteins or overexpression of extracellular inhibitors such as Noggin. Furthermore, FKVP enhanced the sensitivity of cells to BMP-4 stimulation in an additive manner, suggesting that inhibition of FKBP12 binding promotes receptor activation via endogenous ligands ( Fig.14 ). After FKVP treatment, both SMAD1 / 5 phosphorylation and ID-1 expression in HUVEC cells were significantly increased ( Figure 15A-Figure 15C ), providing evidence that more than one cell type is sensitive to BMP activation via FKVP. Taken together, these results indicate that both FK506 and FKVP are able to activate BMPR1 kinase signaling, raising the possibility that this activation plays a key role in the healing-accelerating activity of FK506 in combination therapy for AF.

[0260] Example 8

[0261] FKBP12 alone is required for FKVP-induced SMAD1 / 5 phosphorylation.

[0262] FKBP12 is a member of the FKBP protein superfamily. In previous work, FKBP12 has been shown to be associated with BMPR1 activin-like kinase 2 (ALK2). However, attempts to knock out several FKBPs failed to reveal specific effects on BMPR1 signaling (Spiekerkoetter et al., 2013), which may be due to the relative stability and high abundance of FKBPs. To address this issue, we generated CRISPR-Cas9 knockouts of three cytosolic FKBPs, FKBP12, FKBP51, and FKBP52. All three proteins have been reported to bind to FK506 (Kozany et al., 2009), and in a BRE-luciferase reporter assay, only FKBP12 knockout cells were found to lose sensitivity to FK506 and FKVP ( Fig.16A , Fig. 16B ). It can also be seen that FKBP12KO cells showed substantially elevated SMAD1 / 5 phosphorylation levels ( Fig.16A ), which explains the low values ​​of BMP-4 treatment in FKBP12KO cells relative to the already elevated DMSO-treated samples. Furthermore, reconstitution of FKBP12 by a novel SNAP-tagged fusion construct restored FK506 / FKVP sensitivity in KO cells to that of the original parental cell line ( Fig. 16CWe were able to use this fusion protein to pull down calcineurin and mTOR in the presence of FK506 and rapamycin, respectively, indicating that the fused SNAP tag does not interfere with the interaction of the FKBP12-FK506 complex and the FKBP12-rapamycin complex with calcineurin or mTOR ( Fig.17 We then applied the same constructs to pull down V5-tagged or HA-tagged ALK receptors from transfected HEK293T cells. We observed that SNAP-FKBP12 and ALK receptors indeed interacted with each other and that this association was sensitive to competition with FKVP ( Fig.16D These observations strongly suggest that the FKBP12-BMPR1 interaction is solely responsible for mediating the effects of FKVP-induced BMP activation.

[0263] Example 9

[0264] BMP signaling is required for the effects of AF combination in accelerating wound healing.

[0265] To determine whether activation of BMPs by FK506 was responsible for the accelerated wound healing, the selective BMPR1 kinase inhibitor LDN-193189 (2 mg / kg / day, ip) was administered to GK rats with wounds treated with saline or AF combination. LDN has been shown to be effective in vivo (Cuny et al., 2008; Sun et al., 2013), and LDN alone has shown no effect on wound healing. Interestingly, LDN abolished the beneficial effects of AF combination therapy and increased the time to complete healing from 21 days to 25 days ( Fig.18A , Fig.18B ). We have reported that FK506 plays a key role in recruiting AMD3100-migrated CD133 stem cells to the site of trauma (Lin et al., 2014) or injured organs (Okabayashi et al., 2011; Hu et al., 2016; Cameron et al., 2016; Zhai et al., 2018). To further confirm whether blocking BMP signaling inhibits stem cell recruitment, we performed immunohistochemical staining for CD133 in wound tissue sections recovered from animals on day 7 after surgery. A small number of CD133+ cells were identified in wound tissue sections from animals treated with saline ( Fig. 18C). The number of CD133+ cells in the newly formed granulation tissue of the wound in animals receiving AF combination therapy was significantly increased. Strikingly, in animals treated with AF combination therapy, the administration of the BMP inhibitor LDN significantly reduced the number of CD133+ cells in the wound. Taken together, these results indicate that AF combination therapy recruits more CD133+ stem cells to the wound site depending on the activation of BMP by FK506, and that blocking BMP signaling with LDN eliminates the beneficial effects of AF combination therapy.

[0266] The inventors studied the mechanism by which FK506 accelerates WH when used in combination with AMD3100. Using FKVP, a novel non-immunosuppressive analog of FK506, we ruled out calcineurin as a mediator of both WH accelerations, raising the possibility that FKBP is the primary target of both effects. In addition, we demonstrated that macrocyclic FKBP ligands activate BMP signaling by alleviating the inhibition of BMPR1 by endogenous FKBP12. We show that FKBP12 plays an essential role in the BMP signaling pathway, an effect that can be mediated in the absence of calcineurin inhibition by using non-immunosuppressive FK506 analogs. We found that BMP receptor signaling is required for FK506 to enhance wound healing, and blocking this activation results in a smaller number of stem cells recruited to the wound area. BMP signaling may manipulate several cell types in the wound healing mechanism, such as chemotaxis of stem cells or endothelial adhesion of cells migrating in wound tissue ( Fig.19 ).

[0267] It has been shown that FKBP12, a basic member of the FKBP superfamily, has a variety of cellular and physiological functions in addition to its role in mediating calcineurin and T cell activation via the inhibition of FK506. The association with FKBP12 and the inhibition by FKBP12 add another layer of BMPR1 kinase regulation. Reducing the inhibition of FK506 or FKVP to FKBP12 is sufficient to activate the ID-1 luciferase reporter gene. This shows that there is a basal level of BMPR activity normally inhibited by FKBP12, and compared with BMP-4 combination, the reduction of this inhibition leads to a significant but moderate activation of the signal transduction pathway. Therefore, BMPR may exist in three different activation states, after FKBP12 inhibition is released, after BMP is combined, and both. The present invention discloses that in combination with AMD3100, the partial activation of BMP in pharmacology with FK506 or FKVP is sufficient to accelerate wound healing.

[0268] Type 2 BMP receptors are reported to substantially phosphorylate the GS domain of type 1 receptors (ALK). FKBP12 is thought to inhibit random activation of ALK by binding to residues in the GS domain (Chaikuad et al., 2012). The inventors' results indicate that this association is competed by FKVP for all BMP-specific ALKs. It was shown that the loss of FKBP12 resulted in elevated basal phosphorylation of SMAD1 / 5, indicating a partially activated state of ALK in the absence of FKBP12, an endogenous intracellular inhibitor of ALK. The additive increase in stimulation of the ID-1 reporter from concomitant FKVP and rBMP treatment is also consistent with the three activation states of BMPR1. In addition, the addition of the BMP inhibitor noggin did not prevent FKVP stimulation of the ID-1 reporter, indicating that endogenous FKBP12 regulation of BMPR is independent of BMP protein-receptor binding.

[0269] It has been shown that the inhibition of FK506 to calcineurin is responsible for its potent immunosuppressive activity and its many side effects including nephrotoxicity and neurotoxicity (Bechstein et al., 2000, Naesens et al., 2009). Through the novel positioning of the molecular "protrusion" on the calcineurin interaction effector domain of FK506, the resulting FKVP loses its immunosuppressive activity, as determined by the NFAT reporter gene assay. Compared with calcineurin, the loss of function of FKBP12 and other members appears to have a much smaller and more moderate impact on both yeast and mammals. In addition to BMP receptors, FKBP12 has been reported to also regulate calcium flux in 1,4,5-trisphosphate inositol (IP3) and ranolidine receptors (Cameron et al., 1995; Jayaraman et al., 1992), which suggests that FK506 may affect vascular or cardiac smooth muscle contractility. However, single calcineurin inhibition has been considered a key enhancing factor for hypertension (Hoorn et al., 2012). Therefore, the non-immunosuppressive FKBP ligands of the present invention should have fewer side effects, which leads to safer and more selective pharmacological BMP agonists. In addition, inhibition of FKBP52 by FK506 has been shown to enhance nerve regeneration (Gold, BG, 1999; Gold, BG et al., 1999), suggesting that this effect may be synergistic with FKBP12-mediated tissue regeneration through BMP signaling.

[0270] One of the compounds of formula (I), FKVP, is as effective as FK506 in enhancing WH, and also has important clinical significance due to its lack of immunosuppressive effects. In patients with a greater risk of infection, such as patients with diabetes, treatment with FKVP will provide effective treatment without the risks associated with immunosuppressants. This is emphasized in our use of Goto-Kakazaki rats for this study; this rat model spontaneously develops type 2 diabetes after 3-4 months of age and suffers from many of the same physiological manifestations that affect humans with the disease, including significantly impaired wound healing. By using such a model for this study, we illustrate the power of these compounds of formula (I) and their use in treatment and their application for clinical use. We demonstrate that FKVP reproduces the WH efficacy of FK506 in a challenging rat model, making FKVP an attractive lead compound, and we can show that other non-immunosuppressive compounds of formula (I) have no inhibitory activity on calcineurin similar to FKVP and have similar beneficial effects. Furthermore, the synergistic activity of AMD3100 and FKVP demonstrates that regenerative therapy can be applied to several other types of tissue injuries. In addition to WH, our laboratory has also shown improved liver regeneration after partial hepatectomy and AF combination therapy (Zhai et al., 2018). Therefore, FKVP and other non-immunosuppressive FKBP12 ligands of compounds of formula (I) may find use in both wound healing and regenerative therapy.

[0271] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0272] Unless otherwise indicated herein or clearly contradictory to the context, the terms "a" and "an" and "the" and similar indicators are used in the context of describing the present invention (especially in the context of the accompanying claims) to be interpreted as covering both the singular and the plural. Unless otherwise noted, the terms "comprising", "having", "including" and "containing" are interpreted as open terms (i.e., meaning "including but not limited to"). Unless otherwise indicated herein, the description of the range of values ​​herein is intended only to be used as a shorthand method for individually referring to each individual value falling within the range, and each individual value is incorporated into this specification as if it is individually described herein. Unless otherwise indicated herein or clearly contradictory to the context, all methods described herein can be performed in any suitable order. Unless otherwise stated, the use of any and all examples or exemplary language (e.g., "such as") provided herein is intended only to better illustrate the present invention and does not impose limitations on the scope of the present invention. The language in this specification should not be interpreted as indicating that any unclaimed element is essential to the practice of the present invention.

[0273] Preferred embodiments of the present invention are described herein, including the best mode known to the inventor for carrying out the present invention. After reading the previous description, the variations of those preferred embodiments may become apparent to those of ordinary skill in the art. The inventor expects that the technician may utilize such variations as needed, and the inventor intends that the present invention be implemented in a manner different from that specifically described herein. Therefore, the present invention includes all modifications and equivalents of the subject matter narrated in the appended claims hereof permitted by applicable law. In addition, unless otherwise indicated herein or otherwise clearly contradictory with context, any combination of the above-described elements with all possible variations thereof is included in the present invention.

[0274] References

[0275] H. Tanaka, A. et al. J. Am. Chem. Soc., 1987, 109, 5031; (b) JJ Fung, Transplantation, 2004, 77, S41.

[0276] S. Barik Cell Mol. Life Sci., 2006, 63, 2889.

[0277] J.Liu,JDFarmer,WSLane,J.Friedman,I.Weissman and SLSchreiber,Cell 1991,66,807;(b)JPGriffith et al.Cell 1995,82,506;(c)CRKissinger etal.Nature 1995,378,641.

[0278] Y.Chen,F.Liu,and J.Massagué,EMBO J.1997,16,3866;(b)M.Huse,Y.Chen,J.

[0279] Massagué,and J.Kuriyan,Cell 1999,96,425;(c)T.Wang et al.Cell 1996,86,435;(d)M.

[0280] S.Rahman,N.Akhtar,HMJamil,RSBanik and SM Asaduzzaman,BoneRes.2015,3,15005.

[0281] E.Spiekerkoetter et al.J.Clin.Invest.2013,123,3600.

[0282] T.Kawahara et al.Oncotarget 2015,6,1582.

[0283] B.J. Peiffer et al.Unpublished.

[0284] PSMarinec,CGEvans,GSGibbons,MATamowski,DLOverbeek and JE

[0285] Gestwicki Bioorg.Med.Chem.2009,17,5763;(b)M.Nambu.etal.Bioorg.Med.Chem.Lett.2017,27,2465;(c)Z.Guo,R.Zhang and F.Liang,RSCAdv.2014,4,11400;(d)PAClemons,BGGladstone,A.Seth,EDChao,MAFoley,andS.L.Schreiber Chemistry&Biology 2002,9,49.

[0288] P.Compain,Adv.Synth.Catal.2007,349,1829.

[0289] CPWoodward,NDSpiccia,WRJackson and AJRobinson,Chem.Commun.2011,47,779

[0290] RFHeck and JPNolle,J.Org.Chem.1972,37,2320:(b)T.Mizoroki,K.MoriandA.

[0291] Ozaki,Bull.Chem.Soc.Jap.1971,44,581:(c)RFHeck,Org.React.1982,27,345:(d)A.

[0292] de Meijer and FEMeyer Angew.Chem.Int.Ed.Engl.1994,33,2379:(e)IPBeletskaya and AVCheprakov,Chem.Rev.2000,100,3009.

[0293] Abraham,RT,and Wiederrecht,GJ(1996).Immunopharmacology ofrapamycin.Annu.Rev.Immunol.14,483-510.

[0294] V.,Sanz-Rodriguez,F.,Recio-Poveda,L.,Bernabéu,C.,and Botella,LM(2011).

[0295] Immunosuppressor FK506 increases endoglin and activin receptor-likekinase 1 expression and modulates transforming growth factor-β1 signaling inendothelial cells.Mol.Pharmacol.79,833-843.

[0296] Balaji,S.,King,A.,Crombleholme,T.M.,and Keswani,S.G.(2013).The Roleof Endothelial Progenitor Cells in Postnatal Vasculogenesis:Implications forTherapeutic Neovascularization and Wound Healing.Adv Wound Care(New Rochelle)2,283-295.Bechstein,W.O.(2000).Neurotoxicity of calcineurin inhibitors:impactand clinical management.Transpl.Int.13,313-326.

[0297] Bennett,J.,Cassidy,H.,Slattery,C.,Ryan,M.P.,and McMorrow,T.(2016).Tacrolimus Modulates TGF-β Signaling to Induce Epithelial-MesenchymalTransition in Human Renal Proximal Tubule Epithelial Cells.Joumal of ClinicalMedicine 5,50.

[0298] Bueno,O.F.,Brandt,E.B.,Rothenberg,M.E.,and Molkentin,J.D.(2002).Defective T cell development and function in calcineurin A beta-deficient mice.Proc.Natl.Acad.Sci.U.S.A.99,9398-9403.

[0299] Cameron,A.M.,Steiner,J.P.,Sabatini,D.M.,Kaplin,A.I.,Walensky,L.D.,andSnyder,S.H.(1995).Immunophilin FK506 binding protein associated with inositol1,4,5-trisphosphate receptor modulates calcium flux.PNAS 92,1784-1788.

[0300] Cameron,A.M.,Wesson,R.N.,Ahmadi,A.R.,Singer,A.L.,Hu,X.,Okabayashi,T.,Wang,Y.,Shigoka,M,Fu,Y.,Gao,W.,et al.(2016).Chimeric Allografts Induced byShort-Term Treatment With Stem Cell Mobilizing Agents Result in Long-TermKidney Transplant Survival Without Immunosuppression:I1,Study in MiniatureSwine.Am.J.Transplant.16,2066-2076.

[0301] Chaikuad,A.,Alfano,I.,Kerr,G.,Sanvitale,C.E.,Boergermann,J.H.,Triffitt,J.T.,Delft,F.von,Knapp,S.,Knaus,P.,and Bullock,A.N.(2012).Structureof the Bone Morphogenetic Protein Receptor ALK2 and Implications forFibrodysplasia Ossificans Progressiva.J.Biol.Chem.287,36990-36998.

[0302] Chen,Y.G.,Liu,F.,and Massague,J.(1997).Mechanism of TGFbeta receptorinhibition by FKBP12.EMBO J 16,3866-3876.

[0303] Clemons,P.A.,Gladstone,B.G.,Seth,A.,Chao,E.D.,Foley,M.A.,andSchreiber,S.L.(2002).Synthesis of calcineurin-resistant derivatives of FK506and selection of compensatory receptors.Chem.Biol.9,49-61.

[0304] Csiszar,A.,Ahmad,M.,Smith,K.E.,Labinskyy,N.,Gao,Q.,Kaley,G.,Edwards,J.G.,Wolin,M.S.,and Ungvari,Z.(2006).Bone Morphogenetic Protein-2 InducesProinflammatory Endothelial Phenotype.Am J Pathol 168,629-638.

[0305] Cuny,GD.,Yu,P.B.,Laha,J.K.,Xing,X.,Liu,J.-F.,Lai,C.S.,Deng,D.Y.,Sachidanandan,C.,Bloch,K.D.,and Peterson,R.T.(2008).Structure-activityrelationship study of bone morphogenetic protein(BMP)signalinginhibitors.Bioorg Med Chem Lett 18,4388-4392.Fife,C.E.,and Carter,M.J.(2012).Wound Care Outcomes and Associated Cost Among Patients Treated in USOutpatient Wound Centers:Data From the US Wound Registry.Wounds 24,10-17.

[0306] Fung,J.J.(2004).Tacrolimus and transplantation:a decade inreview.Transplantation 77,S41-43.

[0307] Giordano,A.,Romano,S.,Mallardo,M.,D’Angelillo,A.,Cali,G.,Corcione,N.,Ferraro,P.,and Romano,M.F.(2008).FK506 can activate transforming growthfactor-beta signalling in vascular smooth muscle cells and promoteproliferation.Cardiovasc.Res.79,519-526.

[0308] Gold,B.G.(1999).FK506 and the role of the immunophilin FKBP-52 innerve regeneration.Drug Metab.Rev.31,649-663.

[0309] Gold,B.G.,Densmore,V.,Shou,W.,Matzuk,M.M.,and Gordon,H.S.(1999).

[0310] Immunophilin FK506-binding protein 52(not FK506-binding protein 12)mediates the neurotrophic action of FK506.J.Pharmacol.Exp.Ther.289,1202-1210.

[0311] Griffith,J.P.,Kim,J.L.,Kim,E.E.,Sintchak,M.D.,Thomson,J.A.,Fitzgibbon,M.J.,

[0312] Fleming,M.A.,Caron,P.R.,Hsiao,K.,and Navia,M.A.(1995).X-ray structureof calcineurin inhibited by the immunophilin-immunosuppressant FKBP 12-FK506complex.Cell 82,507-522.

[0313] Hatse,S.,Princen,K.,Bridger,G.,De Clercq,E.,and Schols,D.(2002).Chemokine receptor inhibition by AMD3100 is strictly confined to CXCR4.FEBSLett.527,255-262.

[0314] Hoom,E.J.,Walsh,S.B.,McCormick,J.A.,Zietse,R.,Unwin,R.J.,and Ellison,D.H.(2012).Pathogenesis of calcineurin inhibitor-inducedhypertension.J.Nephrol.25,269-275.

[0315] Hu,X.,Okabayashi.T.,Cameron,A.M.,Wang,Y.,Hisada,M.,Li,J.,Raccusen,L.C.,Zheng,Q.,Montgomery,R.A.,Williams,G.M.,et al.(2016).Chimeric AllograftsInduced by Short-Term Treatment With Stem Cell-Mobilizing Agents Result inLong-Term Kidney Transplant Survival Without Immunosuppression:A Study inRats.Am.J.Transplant.16,2055-2065.

[0316] Jayaraman,T.,Brillantes,A.M.,Timerman,A.P.,Fleischer,S.,Erdjument-Bromage,H.,Tempst,P.,and Marks,A.R.(1992).FK506 binding protein associatedwith the calcium release channel(ryanodine receptor).J.Biol.Chem.267,9474-9477.

[0317] Jujo,K.,Hamada,H.,Iwakura,A.,Thorne,T.,Sekiguchi,H.,Clarke,T.,Ito,A.,Misener,S.,Tanaka,T.,Klyachko,E.,et al.(2010).CXCR4 blockade augments bonemarrow progenitor cell recruitment to the neovasculature and reducesmortality after myocardial infarction.Proc.Natl.Acad.Sci.U.S.A.107,11008-11013.

[0318] Kissinger,C.R.,Parge,H.E.,Knighton,D.R.,Lewis,C.T.,Pelletier,L.A.,Tempczyk,A.,Kalish,V.J.,Tucker,K.D.,Showalter,R.E.,and Moomaw,E.W.(1995).Crystal structures of human calcineurin and the human FKBP 12-FK506-calcineufin complex.Nature 378,641-644.

[0319] Kozany,C., A.,Kress,C.,and Hausch,F.(2009).Fluorescent probes tocharacterise FK506-binding proteins.Chembiochem 10,1402-1410.

[0320] Lewis,C.J.,Mardaryev,A.N.,Poterlowicz,K.,Sharova,T.Y.,Aziz,A.,Sharpe,D.T.,Botchkareva,N.V.,and Sharov,A.A.(2014).Bone morphogenetic proteinsignaling suppresses wound-induced skin repair by inhibiting keratinocyteproliferation and migration.J.Invest.Dermatol.134,827-837.

[0321] Liles,W.C.,Broxmeyer,H.E.,Rodger,E.,Wood,B.,Hübel,K.,Cooper,S.,Hangoc,G.,

[0322] Bridget,G.J.,Henson,G.W.,Calandra,G.,et al.(2003).Mobilizationofhematopoietic progenitor cells in healthy voltunteers by AMD3100,a CXCR4antagonist.Blood 102,2728-2730.

[0323] Lin,Q.,Wesson,R.N.,Maeda,H.,Wang,Y.,Cui,Z.,Liu,J.O.,Cameron,A.M.,Gao,B.,Montgomery,R.A..Williams,G.M.,et al.(2014).Pharmacological mobilization ofendogenous stem cells significantly promotes skin regeneration after full-thickness excision:the synergistic activity of AMD3100 and tacrolimus.J.Invest.Dermatol.134,2458-2468.Liu,J.,Farmer,J.D.,Lane,W.S.,Friedman,J.,Weissman,I.,and Schreiber,S.L.(1991).

[0324] Calcineurin is a common target of cyclophilin-cyclosporin A and FKBP-FK506 complexes.Cell 66,807-815.

[0325] Mueller,T.D.,and Nickel,J.(2012).Promiscuity and specificity in BMPreceptor activation.FEBS Letters 586,1846-1859.

[0326] Naesens,M.,Kuypers,D.R.J.,and Sarwal,M.(2009).Calcineurin InhibitorNephrotoxicity.CJASN 4,481-508.

[0327] Okabayashi,T.,Cameron,A.M.,Hisada,M.,Montgomery,R.A.,Williams,G.M.,and Sun,Z.(2011).Mobilization of host stem cells enables long-term livertransplant acceptance in a strongly rejecting rat straincombination.Am.J.Transplant.11,2046-2056.

[0328] Plikus,M.V.,Guerrero-Juarez,C.F.,Ito,M.,Li,Y.R.,Dedhia,P.H.,Zheng,Y.,Shao,M.,Gay,D.L.,Ramos,R.,Hsi,T.-C.,et al.(2017).Regeneration of fat cellsfrom myofibroblasts during wound healing.Science 355,748-752.

[0329] Rao,A.,Luo,C.,and Hogan,P.G.(1997).Transcription factors of the NFATfamily:regulation and function.Annu.Rev.Immunol.15,707-747.

[0330] M.R.,Fuchs,N.,Proksch,B.,Bongartz,M.,Beiter,T.,and Becker,H.D.(1998).Tacrolimus impairs wound healing:a possible role of decreased nitricoxide synthesis.Transplantation 65,813-818.

[0331] Shin,K.Lim,A.,Zhao.C.,Sahoo,D.,Pan,Y.,Spiekerkoetter,E.,Liao,J.C.,andBeachy,P.A.(2014).Hedgehog Signaling Restrains Bladder Cancer Progression byEliciting Stromal Production of Urothelial Differentiation Factors.CancerCell 26,521-533.

[0332] Spiekerkoetter,E.,Tian,X.,Cai,J.,Hopper,R.K.,Sudheendra,D.,Li,C.G.,El-Bizri,N.,Sawada,H.,Haghighat,R.,Chan,R.,et al.(2013).FK506 activatesBMPR2,rescues endothelial dysfunction,and reverses pulmonaryhypertension.J.Clin.Invest.123,3600-3613.

[0333] Sun,C.C.,Vaja,V.,Chen,S.,Theurl,I.,Stepanek,A.,Brown,D.E.,Cappellini,M.D.,Weiss,G.,Hong,C.C.,Lin,H.Y.,et al.(2013).A hepcidin lowering agentmobilizes iron for incorporation into red blood cells in an adenine-inducedkidney disease model of anemia in rats.Nephrol Dial Transplant 28,1733-1743.

[0334] Tanaka,H.,Kuroda,A.,Marusawa,H.,Hatanaka,H.,Kino,T.,Goto,T.,Hashimoto,M.,and Taga,T.(1987).Structure of FK506,a novel immunosuppressantisolated from Streptomyces.J.Am.Chem.Soc.109,5031-5033.

[0335] Tateishi,K.,Higuchi,C.,Ando,W.,Nakata,K.,Hashimoto,J.,Hart,D.A.,Yoshikawa,H.,and Nakamura,N.(2007).The immunosuppressant FK506 promotesdevelopment of the chondrogenic phenotype in human synovial stromal cells viamodulation of the Smad signaling pathway.Osteoarthritis and Cartilage 15,709-718.

[0336] Wang,T.,Li,B.Y.,Danielson,P.D.,Shah,P.C.Rockwell,S.,Lechleider,R.J.,Martin,J.,Manganaro,T.,and Donahoe,P.K.(1996).The immunophilin FKBP12functions as a common inhibitor of the TGF beta family type I receptors.Cell86,435-444.

[0337] Young,K.,Conley,B.,Romero,D.,Tweedie,E.,O’Neill,C.,Pinz,I.,Brogan,L.,Lindner,V.,Liaw,L.,and Vary,C.P.H.(2012).BMP9 regulates endoglin-dependentchemokine responses in endothelial cells.Blood 120,4263-4273.

[0338] Zhai,R.,Wang,Y.,Qi,L.,Williams,G.M.,Gao,B.,Song,G.,Burdick,J.F.,andSun,Z.(2018).Pharmacological Mobilization of Endogenous Bone Marrow StemCells Promotes Liver Regeneration after Extensive Liver Resection inRats.Scientific Reports 8,3587.。

Claims

1. A compound of formula (I): or a salt thereof, wherein R is selected from the group consisting of:

2. A composition comprising a compound of formula (I): or a salt thereof, wherein R is selected from the group consisting of: and a pharmaceutically acceptable carrier.

3. The composition according to claim 2, further comprising at least one or more bioactive agents.

4. A composition comprising a compound of formula (I): or a salt thereof, wherein R is selected from the group consisting of: at least one stem cell mobilization agent; and a pharmaceutically acceptable carrier.

5. The composition of claim 4, wherein the stem cell migration agent comprises a CXCR4 antagonist.

6. The composition of claim 5, wherein the CXCR4 antagonist is selected from the group consisting of: AMD3100, TG-0054 or AMD3465.

7. Use of the compound of claim 1 or the composition of any one of claims 2 to 6 for the preparation of a medicament for treating tissue damage in a subject in need thereof.

8. Use according to claim 7, wherein the medicament further comprises at least one additional biologically active agent.

9. The use according to claim 7 or 8, wherein the tissue injury is selected from the group consisting of chronic wounds, diabetic ulcers, burns, scalds, frostbite and puncture wounds.

Images