Non-immunosuppressive FK506 analogs and uses thereof

Abstract

The present invention provides a new class of non-immunosuppressive FK506 analogs, which are FKBP-selective, small molecule BMP agonists with modest therapeutic potential for tissue repair and regeneration. Methods for making and using these compounds are also disclosed herein.

Description

REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 795,700, filed January 23, 2019, and incorporated by reference for all purposes as if fully set forth herein. [Background technology]

[0002] Injuries due to accidents, diseases, and armed conflicts are among the most common medical problems. The cost of treatment for chronic non-healing wounds associated with severe burns and diseases such as diabetes mellitus was estimated to exceed $50 billion per year (Fife et al., 2012). Chronic wounds predispose the body to infection and increase the risk of introducing opportunistic pathogens that may lead to sepsis. Accelerating wound healing (WH) can therefore reduce the risk of infection and improve the mortality and morbidity of wound patients. However, options to shorten wound healing are limited, and the development of new treatments is required.

[0003] We have previously reported the discovery of synergistic drug combinations to accelerate cutaneous WH (Lin et al., 2014) and induce long-term allograft survival with 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 complete healing time in mice with four circular full-thickness excision wounds by 25%, which is unprecedented in existing therapeutic modalities. Accelerated WH is accompanied by the mobilization of bone marrow (BM)-derived stem cells (CD133, CD34, and cKit) and recruitment of CD133 stem cells to the wound site, as well as enhanced release of stromal-derived factor 1 (SDF-1), fibroblast growth factor (FGF), and vascular endothelial growth factor (VEGF) in granulation tissue ( Lin et al., 2014 ).

[0004] The underlying molecular mechanisms by which the combination of FK506 and AMD3100 (AF) accelerates WH are less studied. AMD3100 is a selective antagonist of the chemokine receptor CXCR4 (Hatse et al., 2002) and is used clinically to drive hematopoietic stem cells (HSCs) from bone marrow (BM) into peripheral blood (Liles et al., 2003), where they can be restored and stored until ablative irradiation and / or chemotherapy is completed. In addition to HSCs, infusion of AMD3100 enhanced the mobilization of BM-derived endothelial progenitor cells (EPCs), which was associated with more rapid angiogenesis and functional recovery after myocardial infarction in mice (Jujo et al., 2010; Balaji et al., 2013). However, increasing the number of circulating stem cells by AMD3100 treatment alone resulted in only slightly faster healing due to reduced recruitment at the wound site ( Lin et al., 2014 ).

[0005] In contrast to AMD3100, the exact role played by low-dose FK506 in the combination treatment (AF) has remained enigmatic. FK506 is a macrolide produced by the bacterium Streptomyces tsukubaensis and is an immunosuppressant widely used to prevent transplant rejection as well as treat certain autoimmune diseases (Tanaka et al., 1987; Fung et al., 2004). The underlying mechanisms for the immunosuppressive activity of FK506 are well established. At the cellular level, FK506 inhibits the activation of T helper cells. At the pathway level, it blocks intracellular signaling emanating from the T cell receptor, resulting in the transcriptional activation of IL-2 and other cytokine genes. At the molecular level, the binary FKBP-FK506 complex associates with and inhibits the protein phosphatase activity of calcineurin before it binds to FKBP12 and other members of the FKBP family, preventing the calcium-dependent dephosphorylation of nuclear factor of activated T-cells (NFAT) (Liu et al., 1991; Griffith et al., 1995; Kissinger et al., 1995).

[0006] A possible underlying mechanism for FK506 in WH is through the inhibition of calcineurin. However, topical FK506 has been shown to have deleterious effects on WH (Schaffer et al., 1998). Furthermore, we found that animals treated with low dose FK506 (0.1 mg / kg) alone showed slightly faster healing compared to the saline control group, whereas standard dose FK506 (1 mg / kg) for immunosuppression delayed healing time, leaving the question of whether calcineurin inhibition is responsible for the effect of FK506 on WH unanswered.

[0007] FKBP12 plays a supporting role in the immunosuppressive activity of FK506, but has also been shown to inhibit the activation of BMP type 1 receptors (Wang et al., 1996). Importantly, this interaction could be lessened by FK506 (Spiekerkoetter et al., 2013). Although it has been reported that epithelial cells downregulate many BMP receptors in response to injury, BMP signaling has not yet been directly linked to any stage of wound healing (Lewis et al., 2014). Conversely, it has recently been reported that enhanced BMP signaling in myofibroblasts could promote scarless wound healing (Plikus et al., 2017). BMPs have been demonstrated to generate a pro-inflammatory phenotype in endothelial cells, which increases 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 major target genes of these SMADs is inhibitor of differentiation 1 (ID-1), which represses the transcription of several genes related to embryonic development and stem cell self-renewal. Previous studies have investigated some of the downstream effects of BMP receptor activation following FK506 treatment, which involves an increase in SMAD1 and SMAD5 (denoted SMAD1 / 5) and / or SMAD8 (denoted SMAD1 / 5 / 8) phosphorylation in skeletal muscle cells (Spiekerkoetter et al., 2013) and human synovial stromal (hSSC) cells (Tateishi et al., 2007). Additionally, an increase was observed in MAPKK phosphorylation and ID-1 expression, and the activity of FK506 was sufficient to rescue endothelial dysfunction in mice induced by conditional BMP receptor type 2 (BMPR2) knockout (Spiekerkoetter et al., 2013).It has been reported that FK506 upregulates phosphorylation of SMADs downstream of the TGF-β signaling pathway (SMAD 2 and 3) in smooth muscle cells (Giordano et al. Cardiovasc Res. 2008; Bennett 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). Another study showed that FK506 increased the expression of the TGF-β type 3 coreceptor endoglin and stimulated both endothelial cell migration and angiogenic activity (Albinana et al., 2011). Overall, these observations raised the possibility that FK506 might exert its WH effects through FKBP12 independent of calcineurin inhibition. Summary of the Invention [Problem to be solved by the invention]

[0008] Thus, there remains a need for compounds and compositions that can improve wound healing. [Means for solving the problem]

[0009] We have synthesized novel non-immunosuppressive FK506 analogs that retain FKBP binding and lack calcineurin inhibitory activity (Figure 1). The newly synthesized FK506 analogs were found to activate BMP signaling in lymphocytes and endothelial cells through disruption of FKBP12-BMPR1 interaction. Furthermore, we 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.

[0010] Newly Synthesized FK506 AnalogsWhile the FK506 analogs display a range of immunosuppressive properties, they all activate BMP pathway reporters in Jurkat cells with potency similar to that of FK506.Furthermore, the derivatives display structure-dependent effects in NFAT reporter suppression in Jurkat cells.

[0011] According to one embodiment, the present invention provides a compound of formula I: [ka] where R is a cycloalkyl, aryl, or heteroaryl group and is selected from the group consisting of H, halo, N, O, P, C1-C6 alkyl, imidazoyl, cycloalkyl, R , and heterocycloalkyl One or more selected from the group consisting of and said heteroaryl group and said heterocycloalkyl group contain at least one heteroatom, preferably 1 to 4 heteroatoms selected from nitrogen, oxygen, and sulfur), or a salt, solvate, or isomer, or derivative thereof.

[0012] According to one embodiment, the present invention provides a compound of formula I, wherein R is selected from the group consisting of: [ka] [ka] [ka]

[0013] According to one embodiment, the present invention provides a compound of formula I: [ka] where R is a cycloalkyl, aryl, or heteroaryl group and is selected from the group consisting of H, halo, N, O, P, C1-C6 alkyl, imidazoyl, cycloalkyl, R , and heterocycloalkyl One or more selected from the group consisting ofand said heteroaryl and heterocycloalkyl groups contain at least one heteroatom, preferably 1 to 4 heteroatoms selected from nitrogen, oxygen, and sulfur, or a salt, solvate, or isomer, or derivative thereof, and a pharma- ceutically acceptable carrier.

[0014] According to one embodiment, the present invention provides a composition comprising a compound of formula I, wherein R is selected from the group consisting of: [ka] [ka]

[0015] According to further embodiments, the present invention provides compositions comprising a compound of formula 1 or a compound of formula 1 and at least one or more biologically active agents.

[0016] According to one embodiment, the present invention provides a compound of formula I: [ka] where R is a cycloalkyl, aryl, or heteroaryl group and is selected from the group consisting of H, halo, N, O, P, C1-C6 alkyl, imidazoyl, cycloalkyl, R , and heterocycloalkyl One or more selected from the group consisting of and wherein said heteroaryl group and said heterocycloalkyl group contain at least one heteroatom, preferably 1 to 4 heteroatoms selected from nitrogen, oxygen, and sulfur, or a salt, solvate, or isomer, or derivative thereof, at least one stem cell mobilizer, and a pharma- ceutically acceptable carrier.

[0017] According to one embodiment, the present invention provides a composition comprising a compound of formula I, wherein R is selected from the group consisting of: [ka] [ka] [ka]

[0018] In certain embodiments, the stem cell mobilizer comprises a CXCR4 antagonist. For example, the CXCR4 antagonist can comprise AMD3100, TG-0054, or AMD3465.

[0019] According to one embodiment, the present invention provides an effective amount of a compound of formula I: [ka] where R is a cycloalkyl, aryl, or heteroaryl group and is selected from the group consisting of H, halo, N, O, P, C1-C6 alkyl, imidazoyl, cycloalkyl, R , and heterocycloalkyl One or more selected from the group consisting of and wherein said heteroaryl group and said heterocycloalkyl group contain at least one heteroatom, preferably 1 to 4 heteroatoms selected from nitrogen, oxygen, and sulfur, or a salt, solvate, or isomer, or derivative thereof; and an effective amount of at least one stem cell mobilizer.

[0020] According to another embodiment, the present invention provides a method for treating tissue injury 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 mobilizer, and a pharma- ceutically acceptable carrier.

[0021] In some embodiments, the present invention provides a method for treating tissue injury in a subject, 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: [ka] [ka]

[0022] According to further embodiments, the present invention provides a method for treating tissue injury 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 mobilizer, at least one additional biologically active agent, and a pharma- ceutically acceptable carrier.

[0023] According to one embodiment, the present invention provides a method for making 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 and a base in a polar aprotic solvent in the presence of a palladium catalyst; b) heating the mixture for a sufficient time such that the aryl halide reacts with the FK506 to produce an aryl substitution on the FK506 molecule; and c) isolating the aryl-substituted FK506 product. [Brief description of the drawings]

[0024] [Figure 1] 1 depicts the chemical structures of FK506 and FKVP. [Diagram 2] Figures 2a-2c show that FK506 analogs exhibit various immunosuppressive properties. (2a) Cell viability after 72 h 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 suppression in Jurkat cells. Error bars represent standard deviation from the mean for all measurements (n=3), and absorbance / luminescence values ​​were normalized to DMSO-treated cells. [Diagram 3] Dose-response curves for BMP reporter activation by three non-immunosuppressive analogs (lb, 2b, and 3b) and FK506 are plotted. [Figure 4]Figure 4a-4b show details of binding to calcineurin. (4a) Close-up of the terminal olefin of FK-506 by calcineurin. (4b) Steric effect of FKVP by calcineurin. [Diagram 5] Figures 5a-5c (la-lc) illustrate the de novo synthesis of non-immunosuppressive analogs (FKVP) by modifying FK506 at the C40 position. (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 is 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 by normalization to DMSO control values ​​(n=3). [Figure 6] Schematic diagram of the synthesis of FVKP via ruthenium-catalyzed cross-metathesis. [Figure 7] Figures 7a-7b depict FVKP treatment of Jurkat cells. [Figure 8] Figures 8a-8b show that FKVP competes with both FK506 and rapamycin for binding to FKBP12. Both FK506 and rapamycin require FKBP12 for activity. (8a) FKVP blocks FK506 inhibition of calcineurin-mediated NFAT dephosphorylation (lower band), (8b) as well as inhibition of mTOR-mediated S6 phosphorylation by rapamycin. [Figure 9]Figures 9a-9c show the accelerated wound healing of diabetic GK rats treated with a combination of AMD3100 and FK506 or FKVP. (9a) Wound model: Four circular excision wounds (8 mm in diameter) were created on the dorsum of GK rats. (9b) Representative photos of wounds in GK rats for each treatment group (AF=AMD3100+FK506, AV=AMD3100+FKVP), days 0, 6, 12, 18 and 24. (9c) Quantitative analysis of complete healing time in GK rats. All data are expressed by mean ± SEM. [Figure 10] Figures 10a-10c show that non-immunosuppressive analogs, such as compounds of formula I, activate the ID-1 reporter and pSMAD1 / 5 phosphorylation through 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 is strongly inhibited by the addition of 100 nM LDN-193182. TGF-β1 serves as a negative control, whereas BMP4 shows a strong induction of luciferase expression after 18 hours. Relative luciferase activity was determined by normalization to DMSO control values. Error bars represent standard deviation from the mean. (10b) Dose-dependent induction of SMAD1 / 5 phosphorylation by FKVP in Jurkat cells. (10c) The BMPR1 selective inhibitor LDN-193189 inhibits SMAD1 / 5 phosphorylation induced by either BMP-4 or FKVP in Jurkat cells. [Figure 11] Figure 1 shows that unlike SMAD / 16, FKVP does not activate SMAD2 / 3 phosphorylation. Jurkat cells show no appreciable increase in SMAD2 / 3 phosphorylation after 2 hours. FKVP treatment compared to positive control (TGF-β1). [Figure 12]Figure 2 shows that AMD-3100 does not affect BMP signaling alone or in combination with FKVP. FKVP and AMD3100 alone and in combination treatment in Jurkat BMP FKVP luciferase assay (n=3). Relative luciferase activity was determined by normalization to DMSO control values. Error bars represent standard deviation from the mean. [Figure 13] Figure 1 shows that FKVP activates BMP signaling in the presence of the BMP inhibitor, Noggin. Combined treatment of BMP-4, FVKP, FK506 and Noggin in the Jurkat BMP activation assay (n=3). Relative luciferase activity was determined by normalization to DMSO control values. Error bars represent standard deviation from the mean. [Figure 14] Illustrates that FKVP enhances the sensitivity of cells to BMP-4 stimulation. BMP-4, FVKP, and combined treatments in the Jurkat BMP activation assay (n=3). Relative luciferase activity was determined by normalization to DMSO control values. Error bars represent standard deviation from the mean. [Figure 15] Figures 15a-15c depict that FKVP activates SMAD1 / 5 phosphorylation and ID-1 expression in HUVEC. (15a) Western blot of SMAD1 / 5 phosphorylation 2 hours after treatment with combined treatment of BMP-4, FKVP, and LDN in HUVEC cells. (15b) Time course western blot of HUVEC cells treated with 200 nM FKVP and blotted for SMAD1 / 5 phosphorylation and (15c) ID-1 expression. [Figure 16-1]Figures 16a-16d show 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 and different FKBP isoform knockout cells. (16b) Activation of BMP pathway reporter by FKVP and FK506 and suppression of reporter gene activation by LDN in different FKBP isoform knockout cells (n=3). Relative luciferase activity was determined upon normalization to DMSO control values. Error bars represent standard deviation from the mean. [Figure 16-2] (16c) Expression of FKBP12-SNAP fusion protein restores BMP pathway activation by FKVP and FK506 in FKBP12KO Jurkat cells (n=3). (16d) Pull-down of ALK receptor using FKBP12-SNAP in combination with SNAP-functionalized beads in the absence and presence of FKVP. [Figure 17] Illustrates that FKBP12-SNAP interacts with both mTOR and calcineurin. SNAP-tag pull-down and Western blots show that FKBP12-SNAP efficiently binds to both calcineurin and mTOR in the presence of FK506 and rapamycin, respectively. As a negative control, FKVP does not induce calcineurin association. BMP protein treatment was not examined for this experiment. [Figure 18]Figures 18a-18c show that blockade of BMP signaling prevents the beneficial effects of AF combination therapy on wound healing. (18a) Representative photographs of wounds from GK rats treated with saline, LDN, AF or AF plus LDN starting from day 12 show the differences between AF and AF plus LDN. (18b) Quantitative analysis of complete healing time in GK rats. All data are expressed as mean ± SEM. (18b) Representative immunohistochemical staining for 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, whereas LDN treatment dramatically reduced the number of CD133 stem cells at the wound site. [Figure 19] Without being bound to any particular theory, this example provides a mechanistic overview of enhanced healing by an exemplary compound of formula I of the present invention, FKVP, and a stem cell stimulator, AMD3100. AMD3100 releases CD133+ stem cells into the circulation, where FKVP-mediated BMP activation influences their recruitment to wound tissue. Systemic inhibition of FKBP12 by FKVP allows BMP-related gene expression in both recruited stem / progenitor cells and endothelial cells without affecting the necessary immune response to the wound. [Figure 20] The structure of FKVP and the mass spectrum of the compound. [C49H72N2O12+H] 881.5 (HRMS-ESI (m / z): calculated for C49H72N2O12 [FKVP+H]+ 881.5164, found 881.5135). [Figure 21] NMR spectra for the characterization of the FKVP structure are depicted. (21a) H-NMR spectra of FKVP and (21b) FK506 (CDCL3), with details from 5.5 ppm-6.0 ppm, showing the loss of the terminal alkene in FKVP (present in FK506, highlighted in yellow). DETAILED DESCRIPTION OF THE PRESENT EMBODIMENT DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] The following examples are included to provide guidance to those of ordinary skill in the art to practice representative embodiments of the presently disclosed subject matter. In light of this disclosure and the general level of skill in the art, the skilled artisan can recognize that the following examples are intended to be exemplary only, and that numerous changes, modifications, and variations can be adopted without departing from the scope of the presently disclosed subject matter. The following general description and specific examples are intended for illustrative purposes only, and should not be interpreted in any manner as limiting the preparation of the compounds of the present disclosure by other methods.

[0026] According to one embodiment, the present invention provides a compound of formula I: [ka] where R is a cycloalkyl, aryl, or heteroaryl group and is selected from the group consisting of H, halo, N, O, P, C1-C6 alkyl, imidazoyl, cycloalkyl, R , and heterocycloalkyl One or more selected from the group consisting of and said heteroaryl group and said heterocycloalkyl group contain at least one heteroatom, preferably 1 to 4 heteroatoms selected from nitrogen, oxygen, and sulfur), or a salt, solvate, or isomer, or derivative thereof.

[0027] The non-immunosuppressant compounds of formula I are derived from the immunosuppressant, tacrolimus. Tacrolimus (also FK-506 or Fujimycin) is an immunosuppressant drug used primarily after allogeneic organ transplantation to reduce the activity of a patient's immune system and thus the risk of organ rejection. It reduces the production of interleukin 2 (IL-2) by T cells. It is also used in topical preparations in 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 lactone discovered in 1984 from the fermentation broth of a Japanese soil sample that contained the bacterium Streptomyces tsukubaensis. The drug is marketed under the trade name Prograf. (R) ( (R) Prograf is given twice daily (intravenously) and Advagraf is given twice daily (intravenously). (R) (Advagraf) sustained release formulation allows for once-daily administration (oral), and Protopic (R) It is sold under the topical formulation Protopic.

[0028] According to one embodiment, the present invention provides a compound of formula I, wherein R is selected from the group consisting of: [ka] [ka]

[0029] The term "aliphatic" is an art-recognized term and includes linear, branched, and cyclic alkanes, alkenes, or alkynes. In certain embodiments, aliphatic groups herein are linear or branched and have from 1 to about 20 carbon atoms.

[0030] The term "alkyl" is art-recognized and inclusive of 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 has about 30 or fewer carbon atoms in its backbone (e.g., C1-C4 for a straight chain). 30 , for branched chains C3-C 30 ), and alternatively have up to about 20 carbon atoms. Likewise, cycloalkyls have from about 3 to about 10 carbon atoms in their ring structure, and alternatively about 5, 6 or 7 carbons in the ring structure.

[0031] Furthermore, the term "alkyl" (or "lower alkyl") includes both "unsubstituted alkyl" and "substituted alkyl," the latter of which refers to alkyl moieties having substituents replacing a hydrogen on one or more (also referred to simply as one or more) carbons of the hydrocarbon backbone. Such substituents can include, for example, halogen, hydroxyl, carbonyl (such as, for example, carboxyl, alkoxycarbonyl, formyl, or acyl), thiocarbonyl (such as, for example, thioester, thioacetate, or thioformate), alkoxyl, phosphoryl, phosphonate, phosphinate, amino, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate (also referred to as sulfate salt), sulfonate (also referred to as sulfonate salt), sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, or aromatic or heteroaromatic moieties (also referred to as (bisecting) moieties). It will be understood by those skilled in the art that the moieties substituted on the hydrocarbon chain can themselves be substituted, if appropriate. By way of example, substituents of substituted alkyls can include substituted and unsubstituted forms of amino, azido, imino, amido, phosphoryl (including phosphonates and phosphinates), sulfonyl (including sulfates, sulfonamides, sulfamoyl and sulfonates), and silyl groups, as well as ethers, alkylthio, carbonyl (including ketones, aldehydes, carboxylates, and esters), --CF3, --CN, and the like. Exemplary substituted alkyls are described below. Cycloalkyls can be further substituted with alkyls, alkenyls, alkoxys, alkylthios, aminoalkyls, carbonyl-substituted alkyls, --CF3, --CN, and the like.

[0032] The term "aralkyl" is art-recognized and includes aryl groups, e.g., aromatic or heteroaromatic groups.

[0033] The terms "alkenyl" and "alkynyl" are art-recognized and include, in organic molecules, atoms of any element other than mostly carbon or hydrogen. Exemplary heteroatoms include boron, nitrogen, oxygen, phosphorus, sulfur, and selenium.

[0034] The term "aryl" is art-recognized and includes 5-, 6-, and 7-membered monocyclic aromatic groups which may contain from 0 to 4 heteroatoms, such as benzene, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine, and pyrimidine, and the like. Those aryl groups having heteroatoms in the ring structure may be referred to as "aryl heterocycles" or "heteroaromatics." The aromatic ring may be substituted at one or more ring positions with such substituents as those described above, for example, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfidyl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamide, ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromatic moieties, --CF3, --CN or others of the like. 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 (the rings are "fused rings"), at least one of the rings is aromatic, and examples include rings to which the other cyclic rings are linked by cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, and / or heterocyclyl, or acyclic moieties.

[0035] The terms "ortho," "meta," and "para" are art-recognized and apply to 1,2-, 1,3-, and 1,4-disubstituted cyclohexanes, respectively. For example, the names 1,2-dimethylbenzene and orthodimethylbenzene are synonymous.

[0036] The terms "heterocyclyl" and "heterocyclic group" are art-recognized and include 3- to about 10-membered ring structures, such as 3- to about 7-membered rings, whose ring structures contain one to four heteroatoms. Heterocycles can also be polycyclic. Heterocyclyl groups include, for example, thiophene, thianthrene, furan, pyran, isobenzofuran, chromene, xanthene, phenoxanthine, 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, lactones, lactams such as azetidinones and pyrrolidinones, sultams, sultones, and the like. The heterocycle may be substituted at one or more positions with substituents as described above, such as, for example, halogen, alkyl, aralkyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphine, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromatic moieties, -CD, -CN, or the like.

[0037] The terms "polycyclyl" and "polycyclic group" are art-recognized and include structures having two or more rings (e.g., cycloalkyl, cycloalkenyl, cycloalkynyl, aryl and / or heterocyclyl) in which two or more carbons are common to two adjacent rings, e.g., the rings are "fused rings." Rings that are joined through non-adjacent atoms, e.g., a ring in which three or more atoms are common to both rings, are referred to as "bridged" rings. Each ring of a polycycle can be substituted with substituents as described above, such as, for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromatic moieties, -CD3, -CN, or the like.

[0038] The term "carbocycle" is art-recognized and includes aromatic or non-aromatic rings in which each atom of the ring is carbon. The following art-recognized terms have the following meanings: "nitro" means -NO2; the term "halogen" refers to -F, -Cl, -Br, or -I; the term "sulfhydryl" means -SH; the terms "hydroxyl" or "hydroxy" mean -OH; and the term "sulfonyl" means -SO2-.

[0039] The terms "amine" and "amino" are art-recognized and include both unsubstituted and substituted amines. A primary amine has two hydrogens, a secondary amine, one hydrogen and another substituent, and a tertiary amine, two hydrogens are replaced. The substituents on one or both of the hydrogens can be, for example, alkyl, alkenyl, and aryl, cycloalkyl, cycloalkenyl, heterocycle, polycycle, etc. When both hydrogens are replaced with a carbonyl, the nitrogen surrounded by the carbonyl forms an imide.

[0040] The term "alkylamine" includes an amine group, as defined above, having a substituted or unsubstituted alkyl attached thereto.

[0041] The term "amide" is art recognized as an amino substituted carbonyl.

[0042] The term "alkylthio" is art-recognized and includes an alkyl group, as defined above, having a sulfur radical attached thereto. In certain embodiments, the "alkylthio" moiety is represented by one of -S-alkyl, -S-alkenyl, -S-alkynyl, and the like. Representative alkylthio groups include methylthio, ethylthio, and the like.

[0043] The term "carbonyl" is art-recognized and includes the C=0 structure. Carbonyls are involved in esters, carboxyl groups, formates, thiocarbonyls, thioesters, thiocarboxylic acids, thioformates, ketones, and aldehydes.

[0044] The terms "alkoxyl" and "alkoxy" are art-recognized and include an alkyl group, as defined above, having an oxygen radical attached thereto. Representative alkoxyl groups include methoxy, ethoxy, propyloxy, tert-butoxy and the like.

[0045] An "ether" is two hydrocarbons covalently linked by an oxygen. Thus, the substituent of an alkyl that makes it an ether is, for example, an alkoxyl or the like, as may be represented by one of -O-alkyl, -O-alkenyl, -O-alkynyl, etc.

[0046] The term "sulfonate (also called sulfonate)" is art-recognized and includes a moiety in which a sulfur atom has two doubly bonded oxygens and one single bonded oxygen.

[0047] The term "sulfate" (also called sulfate salt) is art recognized and includes a moiety that is similar to a sulfonate, but contains two single-bonded oxygens.

[0048] The terms "sulfonamido," "sulfamoyl," "sulfonyl," and "sulfoxide" are art-recognized and each can include the various R group substituents described herein.

[0049] The terms "phosphoramidite" and "phosphonamidite" are art-recognized.

[0050] The term "selenoalkyl" is art-recognized and includes an alkyl group having a substituted seleno group attached thereto. Exemplary "selenoethers" that can be substituted on the alkyl are selected from one of -Se-alkyl, -Se-alkenyl, -Se-alkynyl, and the like.

[0051] Substitutions can be made to alkenyl and alkynyl groups to produce, for example, aminoalkenyls, aminoalkynyls, amidoalkenyls, iminoalkenyls, iminoalkynyls, thioalkenyls, thioalkynyls, carbonyl-substituted alkenyls or alkynyls.

[0052] Hydrocarbon is an art-recognized term and includes all permissible compounds having at least one hydrogen and 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.

[0053] The phrase "protecting group" is art-recognized and includes temporary substituents that protect potentially reactive functional groups from undesired chemical transformations. Examples of such protecting groups include esters of carboxylic acids, silyl ethers of alcohols, and acetals and ketals of aldehydes and ketones, respectively. The field of protecting group chemistry has been reviewed, for example, in Greene et al., Protective Groups in Organic Synthesis 2nd ed., Wiley, New York, (1991).

[0054] In each formula, example, and the like, a definition of 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 otherwise indicated explicitly or by context.

[0055] The terms triflyl, tosyl, mesyl, and nonaflyl are art-recognized and refer to trifluoromethanesulfonyl, p-toluenesulfonyl, methanesulfonyl, and nonafluorobutanesulfonyl groups, respectively. The terms triflate, tosylate, mesylate, and nonaflate are art-recognized and refer to trifluoromethanesulfonate ester, p-toluenesulfonate ester, methanesulfonate ester, and nonafluorobutanesulfonate ester functional groups and molecules containing said groups, respectively.

[0056] The abbreviations Me, Et, Ph, Tf, Nf, Ts, and Ms are art-recognized and represent methyl, ethyl, phenyl, trifluoromethanesulfonyl, nonafluorobutanesulfonyl, p-toluenesulfonyl, and methanesulfonyl, respectively. A more comprehensive list of abbreviations utilized by organic chemists of ordinary skill in the art appears in the first issue of each volume of the Journal of Organic Chemistry; this list is typically presented in a table entitled Standard List of Abbreviations.

[0057] It will be understood that "substituted" or "substituted by" includes the implicit proviso that such substitution is subject to the allowed valences of the substituted atom and the substituted moieties, 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.

[0058] The term "substituted" is also intended to include all permissible substitution moieties of organic compounds, such as, for example, imide reagents of interest. In a broad aspect, permissible substitution moieties include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. Exemplary substitution moieties include, for example, those described herein. Permissible substitution moieties may be one or more for appropriate organic compounds and may be the same or different. For purposes of this invention, heteroatoms, such as, for example, nitrogen, may have hydrogen substituents to fill the valence of the heteroatom and / or any permissible substitution moiety of organic compounds described herein. This invention is not intended to be limited in any manner by the permissible substitution moieties of organic compounds.

[0059] According to one embodiment, the present invention provides a method for making 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 for the aryl halide to react with FK506 to create an aryl substitution on the FK506 molecule; and c) isolating the aryl-substituted FK506 product.

[0060] As used herein, the present invention provides a novel method for generating aryl substitutions on the FK506 molecule at the terminal alkenyl carbon (carbon 40) of the molecule. The present invention uses the Heck reaction. The Heck reaction (also called the Mizoroki-Heck reaction) is a chemical reaction of an unsaturated halide (or triflate) with an alkene in the presence of a base and a palladium catalyst (or a palladium nanomaterial-based catalyst) to form a substituted alkene. 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, which he shared with Ei-ichi Negishi and Akira Suzuki. This reaction is the first example of a carbon-carbon bond-forming reaction following a Pd(0) / Pd(II) catalytic cycle, the same catalytic cycle seen in other Pd(0)-catalyzed cross-coupling reactions.

[0061] In some embodiments, the methods of the invention are used to prepare aryl-substituted compounds of FK506 (compounds of formula I).

[0062] In some embodiments, the catalysts used in the methods of the present invention can be typical catalysts and pre-catalysts including tetrakis(triphenylphosphine)palladium(0), palladium chloride, and palladium(II) acetate.

[0063] In some embodiments, the supporting ligands used in the methods of the invention are triphenylphosphine, PHOX, and BINAP.

[0064] In some embodiments, the base used in the method of the present invention is triethylamine, potassium carbonate, tris(o-tolyl)phosphine, and sodium acetate.

[0065] In some embodiments, the aryl electrophiles used in the methods of the invention are halides, as well as triflates, as well as benzyl or vinyl halides.

[0066] In some embodiments, the methods of the present invention occur in the absence of oxygen.

[0067] In some embodiments, the methods of the present invention occur in polar aprotic solvents. Examples of such solvents include N-methylpyrrolidone, THF, ethyl acetate, acetone, DMF, acetonitrile, DMSO, and propylene carbonate.

[0068] In some embodiments, the methods of the present invention heat the reactants in the range of 80-130° C. In a preferred embodiment, the reactants are heated to about 100° C.

[0069] In some embodiments, a sufficient amount of aryl halide relative to FK506 ranges from 1 to 3 molar equivalents. In some embodiments, a sufficient amount of palladium catalyst is between about 2 to about 20 mol %. In some embodiments, a sufficient amount of base is between about 5 to about 30 mol % of tris(o-tolyl)phosphine or trimethylamine.

[0070] In some embodiments, the aryl halide used in the methods of the present invention is selected from those shown in Table 1 below.

[0071] Table 1: Schematic representation of the method of the invention and aryl halides that can be used in the method of the invention. [Table 1]

[0072] According to one embodiment, the present invention provides a compound of formula I: [ka] where R is a cycloalkyl, aryl, or heteroaryl group and is selected from the group consisting of H, halo, N, O, P, C1-C6 alkyl, imidazoyl, cycloalkyl, R , and heterocycloalkyl One or more selected from the group consisting of and said heteroaryl and heterocycloalkyl groups contain at least one heteroatom, preferably 1 to 4 heteroatoms selected from nitrogen, oxygen, and sulfur, or a salt, solvate, or isomer, or derivative thereof, and a pharma- ceutically acceptable carrier.

[0073] According to one embodiment, the present invention provides a composition comprising a compound of formula I, wherein R is selected from the group consisting of: [ka] [ka] [ka]

[0074] As used herein, the term "effective" means adequate to achieve a desired, expected, or intended result. More specifically, "effective amount" or "therapeutically effective amount" are used interchangeably and refer to the amount of stem cell mobilizer and / or immunosuppressant, possibly in combination with yet another therapeutic agent, required to provide an amount effective to provide the desired "treatment" (as defined herein) or therapeutic effect, e.g., preventing, alleviating, treating, or ameliorating the symptoms of tissue injury or wounding. 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 subject's age, general condition, the severity of the condition being treated, the particular compound and / or composition administered, and the like. An appropriate "therapeutically effective amount" in any individual case can be determined by those of ordinary skill in the art by reference to the relevant documents and literature and / or by using routine experimentation.

[0075] The pharmaceutical composition of the present invention is in a biologically compatible form suitable for topical or in vivo administration to a subject. The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the US Pharmacopeia or other generally recognized pharmacopoeias for use in animals, more particularly in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the stem cell mobilizer and / or compound of formula I is administered. Such pharmaceutical carriers may be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, 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 the carrier. When the pharmaceutical composition is administered intravenously, saline and aqueous dextrose may be the carrier. Saline and aqueous dextrose and glycerol solutions can be employed as liquid carriers for injectable solutions.Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried slim milk, glycerol, propylene, glycol, water, ethanol, and the like.The pharmaceutical composition can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.

[0076] The pharmaceutical composition of the present invention can take the form of a solution, suspension, emulsion, tablet, pill, capsule, powder, sustained release formulation, and the like. The composition can be formulated as a suppository, using traditional binders and carriers, such as triglycerides. Oral formulations can include standard carriers, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. In certain embodiments, the pharmaceutical composition includes an effective amount of stem cell mobilizer and / or compound of formula I together with an appropriate amount of a pharma- ceutical acceptable carrier to provide a form for proper administration to a patient. The formulation must be compatible with the mode of administration. A preferred formulation is a subcutaneous formulation.

[0077] According to one embodiment, the present invention provides an effective amount of a compound of formula I: [ka] where R is a cycloalkyl, aryl, or heteroaryl group and is selected from the group consisting of H, halo, N, O, P, C1-C6 alkyl, imidazoyl, cycloalkyl, R , and heterocycloalkyl One or more selected from the group consisting of and wherein said heteroaryl and said heterocycloalkyl groups contain at least one heteroatom, preferably 1 to 4 heteroatoms selected from nitrogen, oxygen, and sulfur, or a salt, solvate, or isomer, or derivative thereof.

[0078] According to another embodiment, the present invention provides a method for treating tissue injury 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 mobilizer.

[0079] According to a further embodiment, the present invention provides a method for treating tissue injury 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 mobilizer, and a pharma- ceutically acceptable carrier.

[0080] In some embodiments, the present invention provides a method for treating tissue injury in a subject, 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: [ka] [ka]

[0081] According to further embodiments, the present invention provides a method for treating tissue injury 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 mobilizer, at least one additional biologically active agent, and a pharma- ceutically acceptable carrier.

[0082] The pharmaceutical compositions of the present invention may be administered orally, parenterally, subcutaneously, intramuscularly, intravenously, intraarticularly, intrabronchially, intraabdominally, intracapsularly, intrachondrally, intracavitaryly, intracelially, intracelerally, intracerebellarly, intraventricularly, intracolicly, intrahepatically ... Administration can be by any specific route of administration, including enteral, intracolonic, intracervical, intragastric, intrahepatic, intramyocardial, intraosteal, intraosseous, intrapelvic, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, bolus, vaginal, rectal, buccal, sublingual, intranasal, iontophoretic, or transdermal. The most suitable route is oral administration or injection. In certain embodiments, subcutaneous injection is preferred.

[0083] In general, pharmaceutical compositions comprising stem cell mobilizers and compounds of formula I disclosed herein may be used alone (e.g., formulations comprising stem cell mobilizers and compounds of formula I) or in concert with other therapeutic agents at appropriate dosages as determined by routine testing to obtain optimal efficacy while minimizing potential toxicity. Dosage regimens utilizing the pharmaceutical compositions of the present invention may be selected according to a variety of factors, including the type, species, age, weight, sex, and medical condition of the patient; the severity of the condition being treated; the route of administration; the renal and hepatic function of the patient; and the particular pharmaceutical composition employed. A physician of ordinary skill can readily determine and prescribe the effective amount of the pharmaceutical composition (and potentially other agents, including therapeutic agents) required to prevent, counter, or arrest the progression of the condition.

[0084] Optimal precision in achieving a concentration of a treatment regimen (e.g., a pharmaceutical composition comprising a stem cell mobilizer and / or a compound of formula I in combination with another therapeutic agent) within a range that results in 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, equilibrium, and elimination of the pharmaceutical composition may be considered. The dosage of the pharmaceutical compositions disclosed herein may be adjusted when combined to achieve a desired effect. Alternatively, the dosage of the pharmaceutical composition and various therapeutic agents may be independently optimized and combined to achieve a synergistic result in which pathology is reduced more than if either was used alone.

[0085] 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, e.g., LD 50 (a dose lethal to 50% of the population) and ED 50 (the dose that is therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, and it is the LD 50 / ED 50 It may be expressed as a ratio. Except when cytotoxicity of the pharmaceutical composition is the desired activity or therapeutic outcome, pharmaceutical compositions that exhibit large therapeutic indices are preferred. Although pharmaceutical compositions that exhibit toxic side effects may be used, the delivery system can target such compositions to the site of affected tissues to minimize potential damage to non-infected cells and thereby reduce side effects. In most cases, the pharmaceutical compositions of the present invention can be administered in a manner that maximizes efficacy and minimizes toxicity.

[0086] Data obtained from cell culture assays and animal studies can be used in formulating a range of dosages for use in humans. Dosing of such compositions is preferably ED with little or no toxicity. 50The therapeutically effective dose for any composition used in the methods of the invention can be estimated initially from cell culture assays. The IC as determined in cell culture is within the range of circulating concentrations including: 50 Doses can be formulated in animal models to achieve a circulating plasma concentration range that includes (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.

[0087] Furthermore, the dosage regimen of the compositions 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 define the pharmacokinetic / pharmacodynamic profile of one or more dosage regimens. Then, one of the dosage regimens can be selected for administration, which achieves a desired pharmacokinetic / pharmacodynamic response based on the particular pharmacokinetic / pharmacodynamic profile. See International Publication No. WO 00 / 67776, which is expressly incorporated herein by reference in its entirety.

[0088] "Agent" refers to any substance that may be used as or in a pharmaceutical composition, or may be such as a compound, e.g., small synthetic or naturally occurring organic compounds, nucleic acids, polypeptides, antibodies, fragments, isoforms, variants, or other materials that may be used independently for such purposes, all in accordance with the present invention.

[0089] "Antagonist" refers to an agent that downregulates (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 an enzyme substrate. An antagonist can also be a compound that downregulates expression of a gene or that reduces the amount of expressed protein present.

[0090] "Hematopoiesis" refers to the highly organized process of blood cell development and homeostasis. Before birth, hematopoiesis occurs in the yolk sack, then the liver, and finally the bone marrow. In normal adults, it occurs in the bone marrow and lymphoid tissues. All blood cells arise from pluripotent stem cells, which differentiate into stem cells that are committed to three, two, or one hematopoietic differentiation pathways. However, none of these stem cells are morphologically distinct.

[0091] The term "immunosuppressant" refers to an agent that suppresses, slows, or reverses the activity of the immune system. Immunosuppressants act by suppressing the function of immune cells (including, for example, T cells) to respond either directly (e.g., by acting on immune cells) or indirectly (e.g., by acting on other intermediary cells).

[0092] 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 have the ability to renew themselves through cell division, sometimes after long periods of inactivity. Second, under certain physiological or experimental conditions, stem cells can be induced to become tissue- or organ-specific cells with special functions. In some organs, such as the digestive tract and bone marrow, stem cells divide periodically to repair and replace worn out or damaged tissues. However, in other organs, such as the pancreas and heart, stem cells only divide under special conditions.

[0093] The term "stem cell" can refer to a pluripotent stem cell that has the ability to differentiate into all blood cells, including red blood cells, white blood cells, and platelets. By way of example, "hematopoietic stem cells" or "stem cells" as used in the present invention include not only in bone marrow, but also in umbilical cord blood-derived cells.

[0094] A "patient," "subject," or "host" treated by the method of the present invention refers to either a human or non-human animal, such as, for example, primates, mammals, and vertebrates.

[0095] A "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, peptide, polypeptide, peptide mimetic, carbohydrate, lipid, or other organic (carbon-containing) or 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.

[0096] As used herein, terms such as "treatment", "treating", "treat" and the like refer to obtaining a desired pharmacological and / or physiological effect. The term is also used in the context of application of a "therapeutically effective amount" of an agent, e.g., a stem cell mobilizer and / or an immunosuppressant. The effect may be prophylactic in that it fully or partially prevents a particular outcome, disease or its symptoms, and / or may be therapeutic in that it partially or fully cures the disease and / or adverse effects caused by the disease. As used herein, "treatment" covers any treatment of disease in a subject, particularly a human, and includes: (a) preventing the disease from occurring in a subject, who may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., preventing its onset; and (c) relieving the disease, e.g., causing regression of the disease, e.g., fully or partially eliminating symptoms of the disease. In certain embodiments, 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, wounds, burn victims, and autoimmune diseases, including IBD.

[0097] As used herein, the term "tissue injury" refers to acute or chronic wounds or ulcers of the skin or soft tissue. Examples of such wounds include diabetic sores and ulcers, burns, scalds, frostbite, puncture wounds, abrasions, and the like.

[0098] In some embodiments, the compounds and pharmaceutical compositions described herein can be used in preparing a medicament for use in modulating wound healing response in mammalian skin, including topical or subcutaneous application of the composition in an effective amount for a sufficient period of time.As used herein, the term "modulating wound healing response" refers to the prevention or downregulation of pathological responses to tissue injury, such as those characterized by the formation of keloids and hypertrophic scars in the skin, tendon adhesions, conduction block following nerve injury, scleroderma, Crohn's disease, esophageal strictures, urethral strictures, capsules around breast implants, liver cirrhosis, atherosclerosis, and fibrous adhesion failure in bone.Chronic non-healing skin ulcers are also examples of pathological responses to tissue injury, and are alleviated by the compounds of formula I.

[0099] Stem Cell Mobilizer

[0100] As used herein, "stem cell mobilizer", "hematopoietic stem or progenitor cell mobilizer" or "mobilize" (used interchangeably) refers to any compound, whether it is a small organic molecule, synthetic or natural, or 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 that acts to enhance stem cell migration from bone marrow to peripheral blood. Stem cell mobilizers can increase the number of hematopoietic stem cells or hematopoietic progenitor / precursor cells in peripheral blood, thus allowing a more accessible source of stem cells for use in organ transplant recipients, burn victims, treatment of IBD and / or promotion of wound healing. In certain embodiments, stem cell mobilizers refer to any agent that mobilizes CD34+ and / or CD133+ stem cells. In other embodiments, stem cell mobilizers disrupt CXCL12 (SDF-1)-mediated chemoattraction of CXCR4-expressing cells.

[0101] The present invention provides pharmaceutical compositions comprising at least one stem cell mobilizer and at least one immunosuppressant. Generally, stem cell mobilizers include, but are not limited to, small organic molecules, polypeptides, nucleic acids, and carbohydrates.

[0102] In the case of polypeptides, stem cell mobilizers can include cytokines, colony stimulating factors, proteases, or chemokines. More specifically, cytokines 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).

[0103] In the case of colony-stimulating factors, stem cell mobilizers may include, but are 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 combinations thereof.

[0104] In another embodiment, protease stem cell mobilizers may include, but are 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 (or) CD26).

[0105] In yet another embodiment, chemokine stem cell mobilizers may include, but are not limited to, CXCL12, IL-8, Mip-1α, and GROβ.

[0106] In yet another embodiment, the nucleic acid stem cell mobilizer is a DNA or RNA molecule. In even more specific embodiments, the nucleic acid can be a small interfering RNA (siRNA) molecule or an antisense molecule specific for CXCL12.

[0107] In the case of carbohydrates, the stem cell mobilizer can be a sulfated carbohydrate, including, but 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, for example, 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).

[0108] Stem cell mobilizers 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 Inhibitor Factor-1 (MPIF-1) (see U.S. Patent Application Publication No. 20080274109); and Very Late Antigen (VLA-4) antagonists (e.g., alpha-4 integrin antagonists, such as natalizumab or anti-phosphointegrin α4 (Ser988) containing antibodies, such as clone 6.33 (Upstate Cell Signaling)). The antibodies may further include a peptide (e.g., phenylacetyl-leu-asp-phe-D-prolinamide (Cytel Corp., San Diego Calif.))).

[0109] In certain embodiments, the stem cell mobilizer comprises a CXCR4 antagonist. In certain embodiments, the CXCR4 antagonist is TG-504 (Burixafor; phosphonic acid, p-(2-(4-(6-amino-2-(((trans-4-(((3-(cyclohexylamino)propyl)amino)methyl)cyclohexyl)methyl)amino)-4-pyrimidinyl)-1-piperazinyl)ethyl)-) (TaiGenBiotechnology Co., Ltd., Taipei, Taiwan). In other specific embodiments, the CXCR4 antagonist is AMD3465 (N-(pyridin-2-ylmethyl)-1-[4-(1,4,8,11-tetrazacyclotetradec-1-ylmethyl)phenyl]methanamine). In yet other embodiments, the CXCR4 antagonist is AMD3100. AMD3100 (1,1'-[1,4-phenylenebis(methylene)]bis-1,4,8,11-tetraazacyclotetradecane) is a symmetric bicyclam, prototypic non-peptide antagonist of the CXCR4 chemokine receptor. See U.S. Patent Nos. 6,835,731 and 6,825,351. The terms "AMD" or "AMD3100" include Plerixafor, rINN, USAN, JM3100, and its trade name Mozobil. TM ( TM is used interchangeably with its trademark designation in the United States etc. (Mozobil). For convenience, the term "Plerixafor" is used throughout to refer to CXCR4 antagonists.

[0110] The present invention also contemplates the use of mimetics of AMD3100. Mutational substitutions at 16 positions located in TM-III, -IV, -V, -VI, and -VII lining the main ligand binding pocket of the CXCR4 receptor identified three acid residues: Asp, Asp2, Asp3, Asp4, Asp5, Asp6, Asp7, Asp8, Asp9, Asp10, Asp11, Asp12, Asp13, Asp14, Asp15, Asp16, Asp17, Asp18, Asp19, Asp11, Asp11, Asp15, Asp16, Asp19, Asp19, Asp11, Asp12, Asp13, Asp14, Asp15, Asp16, Asp17, Asp18, Asp19, Asp19, Asp19, Asp16, Asp19, Asp1 171 (AspIV:20), Asp 262 (AspVI:23), and Glu 288(GluVII:06). Molecular modeling showed that one of the cyclam rings of AMD3100 is Asp 171 The other ring interacts with Asp in TM-VI and -VII, respectively. 262 and Glu 288 This suggests that the CXCR4 receptor is sandwiched between the carboxylic acid groups of the CXCR4 and CXCR5. One study found that introduction of only Glu at position VII:06 and removal of the neutralizing Lys residue at position VII:02 resulted in a 1000-fold increase in the affinity of AMD3100 to within 10-fold of its affinity at CXCR4. Therefore, mimetics, such as peptide or non-peptide antagonists with improved oral bioavailability, can be designed to efficiently and selectively block the CXCR4 receptor.

[0111] In another embodiment, the stem cell mobilizer is BKT140 (Biokin Therapeutics, Ltd., Rehovot, Israel). BKT140 (4F-benzoyl-TN14003) binds and inhibits the CXCR4 chomokin receptor with high affinity, with an IC of ~1 nmol / L compared to that obtained with AMD3100. 50 In addition, the BKT140 is a Plerixafor IC 50 value was 51 ± 17 nmol / L, whereas IC 50 At concentrations in the range of 0.5-2.5 nmol / L, it inhibits CXCL12-stimulated cell migration, suggesting ahigh mobilization capacity. See Peled et al., 20 CLIN. CANCER RES. 469-79 (2013).

[0112] FK binding protein ligand

[0113] In conjunction with at least one stem cell mobilizer, the pharmaceutical composition includes non-immunosuppressive FK binding protein ligand(s). In addition to the compounds of Formula I disclosed herein, other examples of non-immunosuppressive ligands include meridamycin, antascomycin, and synthetic ligands of FKBP (SLF).

[0114] The standard dose 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 standard dose, 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).

[0115] In certain embodiments, the stem cell mobilizer is AMD3100. In such embodiments, the pharmaceutical composition can include a typical dose of AMD3100, which is typically administered to human patients at about 0.12-0.24 mg / kg. For a patient weighing 60 kg, the dosage of AMD3100 is about 0.24 mg / kg / day by subcutaneous injection.

[0116] The pharmaceutical composition can be described in terms of the ratio of (a) a compound of formula I or a non-immunosuppressive FKBP ligand to (b) a stem cell mobilizer (e.g., a CXCR antagonist). In certain embodiments, the ratio is 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 / 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 / 76, 1 / 77, 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 / 76, 1 / 77, 1 / It can be 78, 1 / 79, 1 / 80, 1 / 81, 1 / 82, 1 / 83, 1 / 84, 1 / 85, 1 / 86, 1 / 87, 1 / 88, 1 / 89, 1 / 90, 1 / 91, 1 / 92, 1 / 93, 1 / 94, 1 / 95, 1 / 96, 1 / 97, 1 / 98, 1 / 99, 1 / 100, or more.

[0117] The pharmaceutical composition comprises (a) a compound of formula I or a non-immunosuppressive FKBP ligand and (b) a stem cell mobilizer, the compound comprising at least one of: 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 / 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 / 10-1 / 34, 1 / 10-1 / 33, 1 / 10-1 / 32, 1 / 10-1 / 31, 1 / 10-1 / 30, 1 / 10-1 / 29, 1 / 10-1 / 28, 1 / 10-1 / 27, 1 / 10-1 / 26, 1 / 10-1 / 25, 1 / 10-1 / 24, 1 / 10-1 / 23, 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.

[0118] In an alternative embodiment, the pharmaceutical composition comprises (a) a compound of formula I or a non-immunosuppressive FKBP ligand and (b) a stem cell mobilizer at 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 / 15-1 / 84, 1 / 1 5-1 / 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 / 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 / 15-1 / 38, 1 / 15-1 / 1 / 15-1 / 37, 1 / 15-1 / 36, 1 / 15-1 / 35, 1 / 15-1 / 34, 1 / 15-1 / 33, 1 / 15-1 / 32, 1 / 15-1 / 31, 1 / 15-1 / 30, 1 / 15-1 / 29, 1 / 15-1 / 28, 1 / 15-1 / 27, 1 / 15-1 / 26, 1 / 15-1 / 25, 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 ratio ranges.

[0119] The ratio range of (a) the compound of formula I or the non-immunosuppressive FKBP ligand to (b) the stem cell mobilizer in the pharmaceutical composition is 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 / 87, 1 / 20-1 / 88, 1 / 20-1 / 89, 1 / 20-1 / 89, 1 / 20-1 / 88, 1 / 20-1 / 87, 1 / 20-1 / 86, 1 / 20-1 / 85, 1 / 20-1 / 89 ... / 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 / 6 3, 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- The period may include 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.

[0120] In other embodiments, the ratio range of (a) the compound of formula I or the non-immunosuppressive FKBP ligand to (b) the stem cell mobilizer in the pharmaceutical composition is 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 / 4 9, 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.

[0121] In a further embodiment, the pharmaceutical composition comprises (a) a compound of formula I or a non-immunosuppressive FKBP ligand and (b) a stem cell mobilizer at about 1 / 40-1 / 100, 1 / 40-1 / 99, 1 / 40-1 / 98, 1 / 40-1 / 97, 1 / 40-1 / 96, 1 / 40-1 / 95, 1 / 40-1 / 94, 1 / 40-1 / 93, 1 / 40-1 / 92, 1 / 40-1 / 91, 1 / 40-1 / 9 0, 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 / 40-1 / 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 ratio ranges.

[0122] In an alternative embodiment, the pharmaceutical composition comprises (a) a compound of formula I or a non-immunosuppressive FKBP ligand and a stem cell mobilizer in an amount of about 1 / 50-1 / 100, 1 / 50-1 / 99, 1 / 50-1 / 98, 1 / 50-1 / 97, 1 / 50-1 / 96, 1 / 50-1 / 95, 1 / 50-1 / 94, 1 / 50-1 / 93, 1 / 50-1 / 92, 1 / 50-1 / 91, 1 / 50-1 / 90, 1 / 50-1 / 89, 1 / 50-1 / 88, 1 / 50-1 / 87, 1 / 50-1 / 86, 1 / 50-1 / 85, 1 / 50-1 / 84, 1 / 50-1 / 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 / 85, 1 / 60-1 / 84, 1 / 6 The ranges of ratios may include: 0-1 / 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.

[0123] In other embodiments, the ratio range of (a) the compound of formula I or the non-immunosuppressive FKBP ligand to (b) the stem cell mobilizer in the pharmaceutical composition is 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 / 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 / 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 / 8 These can include 0-1 / 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.

[0124] In certain embodiments, the pharmaceutical composition of the present invention may be administered at least once a week for several weeks. In one embodiment, the pharmaceutical composition is administered at least once a week for several weeks to several months. In another embodiment, the pharmaceutical composition is administered once a week for 4-8 weeks. In yet another embodiment, the pharmaceutical composition is administered once a week for 4 weeks.

[0125] More specifically, the pharmaceutical composition is administered once a day for at least about 2 days, at least once a day for at least about 3 days, at least once a day for at least about 4 days, at least once a day for at least about 5 days, at least once a day for at least about 6 days, at least once a day for at least about 7 days, at least once a day for at least about 8 days, at least once a day for at least about 9 days, at least once a day for at least about 10 days, at least once a day for at least about 11 days, at least once a day for at least about 12 days, at least once a day for at least about 13 days, at least once a day for at least about 14 days, at least once a day for at least about 15 days, at least once a day for at least about 16 days, It may be administered 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.

[0126] In other embodiments, the pharmaceutical composition may 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.

[0127] Alternatively, the pharmaceutical composition may be administered about once every 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.

[0128] Alternatively, the pharmaceutical compositions of the present invention may 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.

[0129] Alternatively, the pharmaceutical compositions of the present invention may be administered about once per month, about once per two months, about once per three months, about once per four months, about once per five months, about once per six months, about once per seven months, about once per eight months, about once per nine months, about once per ten months, about once per eleven months, or about once per twelve months.

[0130] Alternatively, the pharmaceutical composition may 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.

[0131] Alternatively, the pharmaceutical composition may 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.

[0132] In a particular embodiment, the present invention relates to the use of FDA approved drugs, such as stem cell mobilizing agent AMD3100 and the compound of formula I, either sequentially or in combination for the treatment of various tissue injuries (wounds), organ transplants, and inflammatory or autoimmune diseases, such as inflammatory bowel disease (IBD). Thus, pharmaceutical compositions comprising a combination of stem cell mobilizing agent AMD3100 and immunosuppressant FK506 are useful for the treatment of various disorders.

[0133] AMD3100. AMD3100 (Plerixafor or Mozobil) is a CXCR4 antagonist that was originally developed as an anti-HIV drug, but was found to potently mobilize CD34 and other stem cells from their bone marrow niches. AMD3100 was first approved by the FDA in 2008 for use in multiple myeloma cancer patients for stem cell banking prior to myeloablative chemotherapy. Today, Plerixafor is often used with neupogen (G-CSF) to mobilize hematopoietic stem cells in multiple myeloma cancer patients for banking prior to myeloablative chemotherapy. The mobilized stem cells are then transplanted into the patient after cancer treatment. The drug is therefore well established to be safe and effective.

[0134] FK506.FK506 (Tacrolimus or Prograph) was discovered in 1987 from the soil bacterium Streptomyces tsukubaensis. FK506 reduces peptidyl-prolyl isomerase activity by binding to the immunophilin FKBP12 (FK506-binding protein) to create a new complex. FK506 was first approved by the FDA for use in liver transplantation in 1994; this has been expanded to include kidney, heart, small intestine, pancreas, lung, trachea, skin, and cornea, bone marrow, and limb transplants. The compounds of formula I also reduce peptidyl-prolyl isomerase activity by binding to the immunophilin FKBP12, but do not possess the immunosuppressive effects of FK506.

[0135] The pharmaceutical compositions described herein provide potent, synergistic activity of AMD3100 and the compound of formula I in mobilizing, recruiting and retaining stem cells at the site of injury. The combined treatment induces organ transplant acceptance, promotes wound healing and promotes regeneration of the intestinal mucosa in inflammatory bowel disease. In certain embodiments, the ratio of the compound of formula I to AMD3100 is about 1 / 10 to 1 / 100 in the composition. In a particular embodiment, the pharmaceutical composition comprises only two active ingredients, the first active ingredient is AMD3100 and the second active ingredient is a compound of formula I, the composition comprises 10-40 mg of AMD3100 and 0.1-4 mg of the compound of formula I, the compound enhances the efficacy of the AMD3100, and the pharmaceutical composition further comprises an excipient. The composition is formulated for subcutaneous or intravenous injection, or possibly for oral or topical administration.

[0136] The dosage of AMD3100 and the compound of formula I can be determined individually. In the prior art treatment regimen, AMD3100 is typically administered to human patients at about 0.12-0.24 mg / kg / day. The compound of formula I is typically administered intravenously at about 0.01-0.05 mg / Kg / day or orally at 0.1-0.2 mg / Kg / day to prevent immune mediated rejection after organ transplantation. The dosage of the compound of formula I is adjusted according to the clinical response and observed FK506 whole blood trough concentrations (6-20 ng / ml). In animals (rodents), AMD3100 is typically administered at about 1 mg / kg / day to effectively mobilize bone marrow stem cells. Compounds of formula I are typically administered to rodents at about 0.5-3 mg / Kg / day to prevent immune-mediated rejection after organ transplantation. In the methods and compositions of the present invention, compounds of formula I will typically be used at lower dosages than those given above (e.g., blood trough concentrations of about 0.5-4 ng / ml). Thus, AMD3100 can be used in humans at dosages of 0.12-0.24 mg / kg / day (1 mg / Kg / day in animals), and compounds of formula I can be used at dosages of 0.001-0.005 mg / Kg (subcutaneously).

[0137] 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 over a period of time after administration. Controlled or sustained release compositions include formulations in lipophilic depots (e.g., fatty acids, waxes, oils). In another embodiment, the compositions are immediate release compositions, i.e., compositions in which all or substantially all of the compounds are released immediately after administration.

[0138] In yet another embodiment, the compound of the present invention can be delivered in a controlled release system.For example, the drug can be administered using intravenous infusion, an implantable osmotic pump, a transdermal patch, or other modes of administration.In one embodiment, a pump can be used.In one embodiment, a polymeric material can be used.

[0139] 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 illustrative only, and are not limiting of the remainder of the disclosure in any way whatsoever. EXAMPLES

[0140] The following examples are presented to provide one of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, articles, devices, and / or methods described and claimed herein are made and evaluated, and are intended to be purely illustrative, and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers (examples, amounts, temperatures, etc.), but some errors and deviations should be accounted for herein. Unless otherwise indicated, parts are parts by weight, temperatures are degrees Celsius or ambient, and pressures are at or near atmospheric pressure. There are many variations and combinations of reaction conditions, examples being component concentrations, desired solvents, solvent mixtures, temperatures, pressures, and other reaction ranges and conditions that can be used to optimize purity and yield of products obtained from the described processes. Only reasonable and routine experimentation will be required to optimize such process conditions.

[0141] Chemical synthesis of compounds of formula I.

[0142] We synthesized the first novel non-immunosuppressive FK506 analog, named FKVP, which retains FKBP binding and lacks calcineurin inhibitory activity (Figure 1). FKVP was found to activate bone morphogenic protein (BMP) signaling in lymphocytes and endothelial cells through disruption of FKBP12-BMPR1 interaction. Furthermore, the combination of FKVP and AMD3100 was found to promote wound healing in diabetic rats in a BMP receptor-dependent manner.

[0143] FKVP synthesis and formulation for animal studies

[0144] FK506 (100 mg, 0.120 mmol) and 40 mol% of Zhanlb (also called Zhang 1b, Zhan 1b, etc.) catalyst RuCl2[C 21 H 26 N2][C 12 H 17 To a solution of [NO3S] in 3 mL of anhydrous DCE was added 4-vinylpyridine (14.2 μL, 0.132 mmol). The mixture was stirred for 30 s and then irradiated in a microwave 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 NMR were performed to confirm that the new compound was >99% free of the parent compound (FK506). 1 H-NMR experiments were performed. The product was analyzed by Hi-Resolution MS (Figure 20) and 1 H-NMR (FIGS. 21A-B) and then dissolved in DMSO or used in formulations for animal studies.

[0145] For animal studies, FKVP powder was dissolved in 80% EtOH / 20% Cremophor RH60 solution at 5 mg / mL. This stock was diluted 1:50 in saline prior to subcutaneous injection.

[0146] Table 2: Antibody information [Table 2]

[0147] Chemicals: Research grade FK506 (>98%) was purchased from APExBIO. LDN-193189 hydrochloride, 4-vinylpyridine, and solvents were purchased from MilliporeSigma. Zhan-lb ruthenium catalyst (CAS: 918870-76-5) was purchased from STREM Chemicals.

[0148] Cell culture and transfection

[0149] Jurkat (E6.1, ATCC) cells were cultured in RPMI containing 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 / antibiotic-free RPMI with 0.5% DMSO. 30 min after transfection, cells were transferred to complete RPMI and rested overnight. Before plating, cells were resuspended in fresh medium and 0.5 × 10 6Cells were diluted to 1000 cells / mL. HEK293T cells were cultured in DMEM containing 10% FBS, 1% PennStrep, and 500 μg / mL G418 (Coming). Cells were transfected using SuperFect reagent and the transfection protocol is provided. 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 with 5% CO2.

[0150] Cell viability assay

[0151] Jurkat or HUVEC cells were plated at 1000 cells / well in 180 μL of growth medium before adding 20 μL of 10× drug / protein stock. After 72 h of incubation, 22 μL of resazurin sodium salt solution (0.1 mg / mL stock in water) was added to each well and incubated at 37° C. Metabolic conversion of resazurin dye was monitored by absorbance at 570 nm after 6 h. After background subtraction (medium only + dye), absorbance values ​​were left as arbitrary absorbance units or normalized to values ​​obtained from cells treated with DMSO.

[0152] Western blot

[0153] Jurkat T cells were harvested by centrifugation (300g, 5 min), washed with PBS, and lysed by sonication in RIPA buffer containing protease and phosphatase inhibitors (Cell Signaling). Lysates were normalized using the DC assay (BioRad) and run on SDS-PAGE gels. Proteins were transferred to nitrocellulose membranes overnight at 100 mA. After blocking with 5% milk in TBS-T for 20 min, membranes were incubated with primary antibodies overnight at 4°C (Table 2). After washing three times with TBS-T, membranes were incubated with secondary antibodies (Table 2) for 1 h. After three further washes, blots were visualized using either ECL substrate (Thermo) or SynGene with laser excitation and filters (647 nm).

[0154] FKBP12-SNAP pulldown

[0155] FKBP12-SNAP was cloned using pSNAPf vector (New England Bio) and PCR amplified FKBP12 with added EcoRI and BsrGI restriction sites (a gift from Tobias Meyer, Addgene plasmid #20175). Gel purified plasmid was ligated using T4 DNA ligase (Thermo), transformed into DH5α, and plated on LB agar plus ampicillin for selection of single colonies, sequenced, and plasmid purified.

[0156] Alkl-V5 plasmid was generated by Gateway cloning of pDONR223-ACVRL1(Alkl) (a gift from William Hahn & David Root, Addgene plasmid #23873) and pEF-DEST51 ​​(Thermo) vectors using LR Clonase enzyme mix (Thermo). Plasmids were transformed into DH5α and plated on LB agar plus ampicillin for single colony selection, sequencing, and plasmid purification. HA-tagged Alk2, Alk3, and Alk6 plasmids were gifts from Aristidis Moustakas (Addgene plasmid #80870, 80873, 80882).

[0157] FKBP 12-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 minutes. After 1 hour, cells were lysed in a vessel with 1 mL of lysis buffer (150 mM NaCl, 50 mM Tris-HCl, 0.1% Triton-100, 5% glycerol, protease and phosphatase inhibitors) and a plate scraper. Lysates were transferred to 2 mL eppendorfs and rotated for 20 minutes at 4°C. Lysates were centrifuged at 14000 g for 10 minutes and ~1 mL of supernatant was transferred to a new tube with 200 nM drug or 0.2% DMSO. 20 μL of input was taken and mixed with 20 μL of 2× loading buffer before boiling. 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 for 1 hour at 4° C. The beads were washed three times with 1 mL of lysis buffer and then boiled in 100 μL of 2× loading buffer. The boiled lysates were vortexed and centrifuged, and the lysates (~90 μL) were carefully removed from the beads and used for Western blotting.

[0158] The same method was used for calcineurin-FK506 and mTOR-rapamiximide down-regulation following transfection of 10 μg of FKBP12-SNAP cDNA alone.

[0159] FKBP Knockout Line

[0160] Jurkat T and Hek293T cells were transfected with all-in-one CRISPR / Cas9 (mCherry tagged) plasmids (Genecopeia) containing guide RNAs for FKBP12 (HCP267023-CG01-3-B), FKBP51 (HCP257374-CG01-3-B), or FKBP52 (HCP205551-CG01-3-B) as previously described. After 48 h, cells were sorted into 96-well plates (1 cell / well) for mCherry fluorescence (650 nm laser). After 2 weeks of culture, single clones were verified by Western blotting.

[0161] BMP and NFAT pathway reporters

[0162] Jurkat T cells transfected with BRE-Luc were plated in 96-well plates (80 μL / well of 0.5 × 10 6 Cells were divided into 100 μL of lysis buffer (per well) and treated with the aforementioned compounds / proteins (20 μL of 5× stock in RPMI, 0.5% DMSO) for 18 h. rBMP-4 and rTGF-β1 (R&D) were used as positive and negative controls, respectively. Plates were centrifuged at 3000 rpm for 10 min and then carefully aspirated. Cells were resuspended in 100 μL of lysis buffer (per well) and placed on a plate shaker for 30 min. An 80 μL aliquot of lysate was transferred to a white-walled 96-well plate and luminescence was recorded 2 seconds after automated injection of luciferase substrate. Luminescence values ​​were background subtracted (lysis buffer + substrate) and normalized to DMSO control values.

[0163] FKBP12KO Jurkat T cells transfected with FKBP12-SNAP plasmid were selected with 1200 μg / mL G418 for 7 days before use in the BRE-Luc assay.

[0164] NFAT-Luc-transfected Jurkat T cells were plated in a 96-well plate (80 μL / well of 0.5 × 10 6 Cells were split into 1000 μl / mL (cells / mL) and treated with the indicated compounds (20 μl of 5× stock in RPMI, 0.5% DMSO) 30 min prior to activation with PMA / Ionomycin (40 nM / 1 μM). After 6 h, wells were aspirated, lysed, and luminescence measured as described above. FK506 and CsA served as positive controls, while DMSO and non-activated wells provided negative control values. With the exception of knockout cell experiments, Jurkat cells used for each experiment were co-transfected and cultured together overnight until plating and treatment the next day.

[0165] Ethical animal care and use statement

[0166] Goto-Kakizaki (GK) type 2 diabetic rats obtained from Charles River (Boston, MA) were housed in a pathogen-free facility and cared for according to NIH guidelines and protocols approved by the Johns Hopkins University Animal Care and Use Committee (ACUC). Both male and female GK rats at 4-5 months of age were used in this study.

[0167] In vivo excision wound model

[0168] Full-thickness wounds were created in the dorsal skin of rats using a sterile disposable biopsy punch (8 mm 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 wounding and every other day until complete healing, defined as complete re-epithelialization of the wound area. To evaluate the role of BMP signaling, animals were injected intraperitoneally (ip) with LDN-193189 (2 mg / kg / day) in addition to standard saline or AF treatment. Wounds were evaluated daily as previously described (Lin et al., 2014).

[0169] immunohistochemistry

[0170] For immunohistochemical staining, 5-μm cut sections were prepared from frozen tissues. 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, specimens were incubated with anti-CD133 (1:300, abl9898; Abcam) overnight at 4°C. Tissue sections were then incubated with biotin-conjugated goat anti-rabbit IgG (1:200, #14708S Cell Signaling, Danvers, MA) for 30 min at room temperature. VectaStain Elite ABC kit (HRP) (Vector Laboratories, Burlingame, CA) was used to increase the sensitivity of staining. Diaminobenzidine tetrahydrochloride (5 min, D4293, Sigma-Aldrich, St. Louis, MO) was used as the chromogen and Mayer's Hematoxylin (30 sec, Dako, S3309) was used for counterstaining.

[0171] statistics

[0172] When comparing days of wound healing, one-way analysis of variance (ANOVA) was used to determine statistical differences in wound healing between AF, AF+LDN, saline, and S+LDN groups, or between AF, AV, and saline groups. Bonferroni-Holm post-hoc procedure was used to adjust p values. p<0.05 was considered significantly different. Example 1

[0173] To conduct structure-activity relationship studies, we synthesized new analogs of FKVP containing a pyridine core structure. Previously, we and others have used ruthenium-catalyzed cross-metathesis (CM) to modify the terminal alkene of FK506 (C40) to generate non-immunosuppressive FK506 analogs. Unfortunately, FKVP and other nitrogen-containing analogs could only be obtained in very low yields. This is most likely due to the nitrogen lone pair competitively coordinated to the ruthenium metal center. The use of soluble tosylated salts of amines could improve the yields, but it was not helpful in the case of FKBP.

[0174] To address the issues associated with nitrogenous base-containing 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 nitrogen-containing 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%) in DMF at 100 °C gave the best yield (66%).

[0175] As an example, FK-506 (0.0500 mmol, 40 mg, 1.0 equiv), aryl halide (2.0 equiv) and mixture of Pd(OAc)2 (0.00500 mmol, 1.1 mg, 0.10 equiv) and P(o-tol)3 (0.0100 mmol, 3.0 mg, 0.20 equiv) in a flame-dried 10 mL-Schlenk tube were added with dry DMF (1.0 mL) and Et3N (0.10 mL) dried over K2CO3 under argon balloon protection, and the mixture was stirred at 100 °C. The reaction was monitored by mass spectrometry. When the reaction was completed, the reaction mixture was cooled to room temperature and purified by flash column with gradient solvent (dichloromethane and methanol) to give the corresponding product. PLC was used as further purification to separate epimers if necessary.

[0176] Using FKVP as a lead, seven nitrogen-containing heterocyclic halides bearing a pyridine core and four halides bearing substituted anilines were selected as substrates for the Heck reaction. Encouragingly, all halide substrates were successfully coupled to FK506 in moderate to good yields (see Table 1).

[0177] Halide substrates showed distinct reactivity in the Heck reaction. First, bromides and iodides gave similar yields. Second, electron-withdrawing groups of pyridine and quinoline appeared to increase yields (Table 1, entries 4, 6, 7). Third, unprotected anilines showed the lowest yields (Table 1, entries 9-11). Importantly, the unreacted FK506 starting material in the reaction mixture was easily separated from the more polar nitrogen-containing products by flash chromatography. Example 2

[0178] With the FK506 analogs in hand, we assessed their effects on cell viability, BMP activation, and NFAT activation at two concentrations (Figure 2). In the HUVEC cell viability assay, we found that the quinoline analogs (5b-7b) inhibited cell proliferation at 10 μM (Figure 2a), while the other compounds were comparable to FK506 in their cytotoxicity.

[0179] 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 the new analogs could activate the BMP signaling pathway. Initial screening of the compounds showed that most analogs had similar activity to FK506 or FKVP. This was somewhat expected, since we previously showed that FKBP12 binding is necessary and sufficient for activation of BMP signaling (Figure 2b).

[0180] To determine the effect of analogs on calcineurin, we used 8d We employed a PMA / ionomycin-activated Nuclear Factor of Activated T-cell (NFAT) reporter in IL-2 (luciferase under the control of the IL-2 promoter) (Fig. 2c). Two analogs (2b, 3b), as well as FKVP(lb), did not cause significant inhibition of the NFAT-luciferase reporter at concentrations up to 10 μM. Surprisingly, most other analogs showed either partial or near-complete inhibition of the NFAT reporter at 1 μM (Fig. 2c). It is noteworthy that some immunosuppressive analogs, including 5b, 6b, and 7b, have bulkier substituents than 2b and 3b due to the presence of fused aromatic rings. It remains to be determined how those bulkier groups are accommodated in the binding site of calcineurin. Example 3

[0181] We found that the EC of three non-immunosuppressive analogs (lb-3b) was significantly higher in the BMP luciferase assay than in the control. 50 All three analogs were found to be slightly more potent than FK506 (Figure 3), which may be due to increased solubility due to the more polar pyridine substituent. Among the three analogs, FKVP had an IC of 12.4 nM. 50 remained the most active. Example 4

[0182] The structure of the FKBP12-FK506-calcineurin complex was previously determined by X-ray crystallography. In this complex, the terminal alkene 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(lb) sterically clashes with residue M118 of calcineurin (Figure 4b), which explains the exclusion of calcineurin binding by analog lb-3b. However, the structure cannot explain how several other analogs containing bulkier substituents than FKVP at the same position maintain their immunosuppressive properties.

[0183] After investigating the pKa values ​​of all nitrogen-containing analogs, we noticed that compounds with higher pKa values ​​(lb, 2b, 3b, 5b) showed less calcineurin inhibition. Conversely, those with lower pKa caused by electron-withdrawing groups were all immunosuppressive at 1 μM (4b, 6b, 7b). The pKa value of aniline (9b-1lb) is lower than that of pyridine (lb), which may also explain its retention of inhibitory activity against calcineurin. These observations suggest that the formation of a positively charged pyridinium appendage at the terminal alkene of FK506 plays a more important role in disrupting the interaction between the terminal alkene of FK506 and the hydrophobic pocket in calcineurin. These observations provide an alternative and complementary mechanism for the loss of calcineurin inhibition in non-immunosuppressive FK506 analogs, which was previously rationalized by a large molecular "bump" that sterically hinders calcineurin binding. The same binding pocket in calcineurin may have significant conformational flexibility to accommodate uncharged bulky aromatic rings such as those present in 4b, 6b and 7b.

[0184] In summary, we developed the method of the present invention, which involves one-step synthesis of FK506 analogs containing nitrogenous bases using the Heck reaction. We identified three non-immunosuppressive analogs with even higher potency in activating the BMP signaling pathway. We found that the key factor that interferes with the FK506-calcineurin interaction is electrostatic rather than steric interactions. Given the role of BMP signaling in wound healing and tissue regeneration, the newly developed synthetic route to FKVP and analogs will facilitate the development of non-immunosuppressive analogs of FK506 for regenerative medicine. Example 5

[0185] Modification of FK506 with C40 led to a non-immunosuppressive analogue - FKVP.

[0186] It has been established that the in vivo immunosuppressive activity of FK506 is mediated through the inhibition of calcineurin (Bueno et al., 2002). Previous studies have shown that modification of FK506 with a terminal alkene could block calcineurin inhibition while retaining FKBP binding (Clemons et al., 2002). To determine whether calcineurin inhibition is required for the promotion of healing by AF combinations, we designed and synthesized a non-immunosuppressive analog of FK506 by fusing a vinylpyridine moiety to the terminal alkene as a "bump" in the effector domain of FK506 using cross-metathesis. The resulting analog was named FKVP (Figure 5a). The newly added pyridine moiety was intended to increase water solubility while providing steric bulk to disrupt the interaction with calcineurin (Figure 6). The cytotoxicity of FKVP was evaluated and compared to that of FK506 in both Jurkat T (Figure 5b, Figure 7a) and primary HUVEC cells (Figure 7b). Similar to FK506, FKVP did not affect cell viability at concentrations up to 10 μM (Figure 5b). We then determined the effect of FKVP on a PMA / ionomycin-activated NFAT-luciferase reporter gene in Jurkat T cells (Clemons et al., 2002). While both FK506 and CsA showed strong repression of the reporter, FKVP did not cause significant repression at concentrations up to 10 μM (Figure 5c), suggesting that FKVP is no longer capable of repressing calcineurin. To confirm that FKVP retained the ability to bind FKBP, we applied it to a competition assay combining FK506 and rapamycin, since sequestration of free FKBP prevents the formation of active FKBP12-FK506 or FKBP-rapamycin complexes and therefore antagonizes the activity of both drugs (Rao et al., 1997; Abraham et al., 1996). The effect of FK506 on calcineurin was determined using it as a readout of the phosphorylation status of NFATc2. Hence, FK506 blocked the dephosphorylation of NFATc2 in response to stimulation with PMA and ionomycin (Figure 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 status of its substrate p70s6k. Again, high concentrations of FKVP reversed the inhibition of rapamycin on p70s6k phosphorylation (Figure 8b). Taken together, these results clearly demonstrated that FKVP is capable of antagonizing the activities of both FK506 and rapamycin through competitive binding to endogenous FKBP protein. Example 6

[0187] FKVP combined with AMD3100 promoted wound healing

[0188] We previously reported the synergistic effect of AMD3100 and low-dose FK506 (AF) in promoting wound healing after full-thickness skin excision (Lin et al., 2014). To determine whether FKVP has a comparable effect, we performed a wound healing experiment in a type 2 diabetic rat model. Four full-thickness wounds were created by circular excision with a diameter of 8 mm on the shaved back of diabetic GK rats, and each wound site was digitally photographed at specified time intervals (Figure 9a). Re-epithelialization of the entire wound area was used as the defining parameter of complete healing, and the complete healing time of the four wounds in each animal was calculated in days (Figure 9b).

[0189] Wounded rats were randomly divided 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 a mean sufficient healing time of 26 days, animals treated with AF showed significantly faster healing as wounds reached sufficient re-epithelialization on day 21, which is consistent with our report in a nondiabetic rodent model of surgical excision wounds (Lin et al., 2014). Importantly, 10 rats receiving AV therapy displayed comparable effects to AF with a significantly reduced time to sufficient healing from 26 days to 20 days (Figure 9c). These results strongly suggest that inhibition of calcineurin is not involved in the synergistic activity of AMD3100 and FK506 in promoting wound healing. Example 7

[0190] FKVP activates ID-1 reporter and SMAD1 / 5 phosphorylation through the BMP type 1 receptor

[0191] Having ruled out calcineurin as a relevant mediator of the WH-accelerating activity of FK506, we turned to FKBP12 and the BMP signaling pathway that it has been reported to regulate. We first began by determining whether both FK506 and FKVP were capable of activating the BMP signaling pathway by employing a BMP response element (BRE) pathway reporter (luciferase under the control of the ID1 gene promoter) (Spiekerkoetter et al., 2013) in Jurkat [E6.1] T cells. The Jurkat line was found to express high levels of FKBP12 and CXCR4 as well as functional components of BMP signaling (BMPR, SMAD1 / 5), making it an excellent model system. To confirm reporter selectivity, rBMP-4 and rTGF-β1 were used as positive and negative controls, respectively. Treatment with both FKVP and FK506 activated the reporter in a dose-dependent manner, and the increase in luciferase activity was fully blocked by the selective BMP1R inhibitor LDN-193189 (LDN) (Figure 10a). In contrast to FK506 and FKVP, cyclosporine A (CsA) did not activate the reporter (Figure 10a), consistent with the idea that calcineurin is not involved in the activation of BMPR1 kinase by FK506.

[0192] To study downstream BMPR1 signaling events, we examined the effects of FK506 and FKVP on the phosphorylation of SMAD1 / 5. Both compounds induced SMAD1 / 5 phosphorylation in a dose-dependent manner (Figure 10b), an effect abolished by LDN treatment (Figure 10c). In contrast, there was no increase in phosphorylated SMAD2 / 3 under the same conditions (Figure 11), suggesting that while FKBP12 can bind to the TGF-β receptor (Chen et al., 1997), its dissociation is insufficient to activate receptor kinase activity in the absence of TGF-β. This result is consistent with previous studies showing that FK506 and nonfunctional analogs are not able to activate TGF-β pathway reporters without the addition of exogenous ligand (Spiekerkoetter et al., 2013; Wang et al., 1996). In the BRE-luciferase reporter, AMD3100 was found to have no effect on ID-1 promoter stimulation alone or in combination with FKVP (Figure 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 the BMP reporter (Figure 13). These observations suggest that endogenous BMPs are not required for signaling activation by FK506 or FKVP, and that these compounds were able to activate BMPs in tissues with underexpression of BMP proteins or overexpression of extracellular inhibitors such as Noggin. In addition, FKVP additively increased the sensitivity of cells to BMP-4 stimulation, suggesting that inhibition of FKBP12 binding promotes receptor activation by the endogenous ligand (Figure 14). Both SMAD1 / 5 phosphorylation and ID-1 expression were significantly increased in HUVEC cells after FKVP treatment (Figure 15), providing evidence that more than one cell type is sensitive to BMP activation by FKVP. Taken together, these results indicate that both FK506 and FKVP are capable of activating BMPR1 kinase signaling, raising the possibility that this activation plays an important role in the pro-healing activity of FK506 in AF combination therapy. Example 8

[0193] A separate FKBP12 is required for FKVP-induced SMAD1 / 5 phosphorylation.

[0194] FKBP12 is a member of the FKBP superfamily of proteins. Previous work showed that FKBP12 associates with BMPR1 activin-like kinase 2 (ALK2). However, attempts to knock down several FKBPs failed to reveal any specific effect on BMPR1 signaling (Spiekerkoetter et al., 2013), likely 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 FK506 (Kozany et al., 2009), and in BRE-luciferase reporter assays, it was found that only FKBP12 knockout cells lost sensitivity to FK506 and FKVP (Figures 16a, 16b). It can also be seen that FKBP12KO cells showed constitutively elevated levels of SMAD1 / 5 phosphorylation (Figure 16a), explaining the lower values ​​of BMP-4 treatment in FKBP12KO cells compared to the already elevated DMSO-treated samples. Furthermore, reconstitution of FKBP12 with a novel SNAP-tagged fusion construct restored FK506 / FKVP sensitivity in KO cells to that of the original parental lineage (Figure 16c). We were able to use this fusion protein to pull down calcineurin and mTOR in the presence of FK506 and rapamycin, respectively, suggesting that the fused SNAP tag did not interfere with the interaction of FKBP12-FK506 and FKBP12-rapamycin complexes with calcineurin or mTOR (Figure 17). We then applied the same construct to pull down V5- or HA-tagged ALK receptor 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 by FKVP (Fig. S4d). These observations strongly suggest that the FKBP12-BMPR1 interaction is solely responsible for mediating the effects of FKVP-induced BMP activation. Example 9

[0195] The combined effect of AF in accelerating wound healing requires BMP signaling

[0196] To determine whether BMP activation by FK506 is involved in the acceleration of wound healing, a selective BMPR1 kinase inhibitor, LDN-193189 (2 mg / kg / day, ip) was administered to wounded GK rats treated with saline or AF combination. LDN has been shown to be effective in vivo (Cuny et al., 2008; Sun et al., 2013) and has been shown to have no effect on wound healing alone. Interestingly, LDN abolished the beneficial effect of AF combination therapy and extended the time to complete healing from 21 to 25 days (Figure 18a, 18b). We have reported that FK506 plays an important role in the recruitment of AMD3100-mobilized CD133 stem cells to wound sites (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 blockade of BMP signaling suppresses stem cell recruitment, we performed immunohistochemical staining for CD133 in wound tissue sections collected from animals on day 7 after surgery. We identified a small number (two, three) of CD133+ cells in wound tissue sections from animals treated with saline (Figure 18c). The number of CD133+ cells was significantly increased in the newly formed granulation tissue of wounds in animals receiving AF combination therapy. Strikingly, administration of the BMP inhibitor LDN dramatically reduced the number of CD133+ cells in wounds in animals receiving AF combination treatment. Taken together, these results suggest that recruitment of more CD133+ stem cells to the wound site by AF combination therapy depends on BMP activation by FK506, and that blockade of BMP signaling by LDN abolishes the beneficial effects of AF combination therapy.

[0197] We investigated the mechanism by which FK506 accelerates WH when used in combination with AMD3100. Using FKVP, a novel, non-immunosuppressive analogue of FK506, we ruled out calcineurin as a mediator of both WH accelerations, raising the possibility that FKBPs are the primary targets of both effects. Furthermore, we demonstrated that macrocyclic FKBP ligands activate BMP signaling by relieving the inhibition of BMPR1 by endogenous FKBP12. We showed that FKBP12 plays an essential role in the BMP signaling pathway, an effect that can be mediated without calcineurin inhibition through the use of a non-immunosuppressive FK506 analogue. We found that BMP receptor signaling is required for the enhancement of wound healing by FK506, and that blocking this activation resulted in fewer stem cells being recruited to the wound area. BMP signaling may manipulate several cell types in wound healing mechanisms, such as stem cell chemotaxis or endothelial adhesion of mobilized cells in wound tissue (Figure 19).

[0198] FKBP12 is the founding member of the FKBP superfamily and has been shown to possess multiple cellular and physiological functions in addition to its role in mediating the inhibition of calcineurin and T cell activation by FK506. Association with and inhibition by FKBP12 adds another layer of BMPR1 kinase regulation. That relief of FKBP12 inhibition by FK506 or FKVP is sufficient to activate the ID-1 luciferase reporter gene suggests that there is a basal level of activity of BMPR, which is normally repressed by FKBP12, and that relief of this inhibition leads to sufficient, albeit moderate, activation of the signaling pathway relative to BMP-4 binding. Therefore, BMPR may exist in three distinct activation states upon release of FKBP12 inhibition, upon BMP binding, and both. The present invention reveals that pharmacological partial activation of BMP by FK506 or FKVP is sufficient to accelerate wound healing in combination with AMD3100.

[0199] Type 2 BMP receptors have been reported to constitutively phosphorylate the GS domain of type 1 receptors (ALKs). FKBP12 is thought to suppress random activation of ALKs by binding to residues in the GS domain (Chaikuad et al., 2012). Our results showed that this association was competed by FKVP for all BMP-specific ALKs. Loss of FKBP12 was shown to result in elevated basal phosphorylation of SMAD1 / 5, suggesting a partially activated state of ALKs in the absence of its endogenous intracellular inhibitor FKBP12. The concomitant additive increase in ID-1 reporter stimulation from FKVP and rBMP treatment is also consistent with the three activation states of BMPR1. Furthermore, addition of the BMP inhibitor Noggin did not prevent ID-1 reporter stimulation by FKVP, suggesting that regulation of BMPR by endogenous FKBP12 is independent of BMP protein receptor binding.

[0200] Inhibition of calcineurin by FK506 was shown to be responsible for both its potent immunosuppressive activity and multiple of its side effects, including nephrotoxicity and neurotoxicity (Bechstein et al., 2000; Naesens et al., 2009). By de novo placement of a molecular “bump” at the calcineurin-interacting effector domain of FK506, the resulting FKVP lost its immunosuppressive activity as judged by NFAT reporter gene assays. Compared to calcineurin, loss of function of FKBP12 and other members appears to have a much smaller, and dramatically less impact on both yeast and mammalian cells. Apart from BMP receptors, FKBP12 was reported to regulate calcium flux at inositol 1,4,5-triphosphate (IP3) and ryanodine receptors (Cameron et al., 1995; Jayaraman et al., 1992), suggesting that FK506 may affect vascular or cardiac smooth muscle contractility. However, calcineurin inhibition alone was recognized as a significant enhancer of hypertension (Hoorn et al., 2012). As such, the non-immunosuppressive FKBP ligands of the present invention should have fewer side effects, resulting in safer and more selective pharmacological BMP agonists. Furthermore, FKBP52 inhibition with FK506 has been shown to enhance nerve regeneration (Gold, BG, 1999; Gold, BG et al., 1999), and it has been suggested that this effect may synergize with FKBP12-mediated tissue regeneration through BMP signaling.

[0201] One of the compounds of formula I, FKVP, is effective in enhancing WH in the same way that FK506 is, which also has important clinical implications due to its lack of immunosuppressive effects. In patients at higher risk of infection, such as those with diabetes, treatment with FKVP would provide effective treatment without the risks associated with immunosuppressants. This is highlighted in our use of Goto-Sakazaki rats for this study; a rat model that spontaneously develops type 2 diabetes after 3-4 months of age and suffers from many of the same physiological symptoms that affect humans with the disease, including significant impairment of 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 its application to clinical use. Our demonstration that FKVP recapitulates the WH efficacy of FK506 in a challenging rat model makes FKVP an attractive lead compound, and we can show that other non-immunosuppressive compounds of formula I lack the same inhibitory activity against calcineurin as FKVP, and have similar beneficial effects. Furthermore, the synergistic action of AMD3100 and FKVP demonstrates a regenerative therapy that can be applied to several other types of tissue injury. Beyond WH, our laboratory has shown improved liver regeneration after partial hepatectomy and AF combination treatment (Zhai et al., 2018). Therefore, the compound of formula I, FKVP and other non-immunosuppressive FKBP12 ligands, may find use in both wound healing and regenerative therapies.

[0202] All references cited herein, including publications, patent applications, and patents, are hereby incorporated by reference to the same extent as if each reference was individually and specifically indicated to be incorporated by reference and was set forth in its entirety herein.

[0203] Use of the terms "a" and "an" and "the" and similar referents in the context of describing the present invention (particularly in the context of the claims below) should be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" should be construed as open-ended terms (i.e., meaning "including, but not limited to"), unless otherwise stated. Recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of individually referring to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated in the specification as if it were individually set forth herein. All methods described herein can be performed in any suitable order, unless otherwise indicated herein or clearly contradicted by context. Any and all examples provided herein, or the use of exemplary language (eg, "such as"), are intended merely to better clarify the invention and do not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0204] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of these preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect that skilled artisans will employ such variations as they deem appropriate, and the inventors intend that the invention be practiced other than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0205] References H. Tanaka, A. et al. J. Am. Chem. Soc. (Journal of the American Chemical Society), 1987, 109, 5031; (b) J. J. Fung, Transplantation, 2004, 77, S41. S. Barik Cell Mol. Life Sci., 2006, 63, 2889. J. Liu, JD Farmer, WS Lane, J. Friedman, I. Weissman and SL Schreiber, Cell 1991, 66, 807; (b) JP Griffith et al. Cell 1995, 82, 506; (c) CR Kissinger et al. Nature 1995, 378, 641. Y. Chen, F. Liu, and J. Massague, EMBO J. 1997, 16, 3866; (b) M. Huse, Y. Chen, J. Massague, and J. Kuriyan, Cell 1999, 96, 425; (c) T. Wang et al. Cell 1996, 86, 435; (d) MS Rahman, N. Akhtar, HM Jamil, RS Banik and S. M Asaduzzaman, Bone Res. 2015, 3, 15005. E. Spiekerkoetter et al. J. Clin. Invest. (Journal of Clinical Investigation) 2013, 123, 3600. T. Kawahara et al. Oncotarget 2015, 6, 1582. BJ Peiffer and others unpublished. PS Marinec, CG Evans, GS Gibbons, MA Tarnowski, DL Overbeek and JE Gestwicki. Bioorg. Med. Chem. 2009, 17, 5763; (b) M. Nambu. et al. Bioorg. Med. Chem. Lett. 2017, 27, 2465; (c) Z. Guo, R. Zhang and F. Liang, RSC Adv. 2014, 4, 11400; (d) PA Clemons, BG Gladstone, A. Seth, ED Chao, MA Foley, and S. L. Schreiber Chemistry & Biology 2002, 9, 49. P. Compain, Adv. Synth. Catal. 2007, 349, 1829. CP Woodward, ND Spiccia, WR Jackson and AJ Robinson, Chem. Commun. 2011, 47, 779 RF Heck and JP Nolley, J. Org. Chem. 1972, 37, 2320; (b) T. Mizoroki, K. Mori and A. Ozaki, Bull. Chem. Soc. Jap. 1971, 44, 581; (c) RF Heck, Org. React. 1982, 27, 345; (d) A. de Meijere and FE Meyer, Angew. Chem. Int. Ed. Engl. 1994, 33, 2379; (e) IP Beletskaya and AV Cheprakov, Chem. Rev. 2000, 100, 3009. Abraham, RT, and Wiederrecht, GJ (1996). Immunopharmacology of rapamycin. Annu. Rev. Immunol. 14, 483-510. Albinana, V., Sanz-Rodriguez, F., Recio-Poveda, L., Bernabeu, C., and Botella, LM (2011). Immunosuppressor FK506 increases endoglin and activin receptor-like kinase 1 expression and modulates transforming growth factor-β1 signaling in endothelial cells. Mol. Pharmacol. 79, 833-843. Balaji, S., King, A., Crombleholme, TM, and Keswani, SG (2013). The Role of Endothelial Progenitor Cells in Postnatal Vasculogenesis: Implications for Therapeutic Neovascularization and Wound Healing. Adv Wound Care (New Rochelle) 2, 283-295. Bechstein, WO (2000). Neurotoxicity of calcineurin inhibitors: impact and clinical management. Transpl. Int. 13, 313-326. Bennett, J., Cassidy, H., Slattery, C., Ryan, MP, and McMorrow, T. (2016). Tacrolimus Modulates TGF-β Signaling to Induce Epithelial-Mesenchymal Transition in Human Renal Proximal Tubule Epithelial Cells. Journal of Clinical Medicine 5, 50. Bueno, OF, Brandt, EB, Rothenberg, ME, and Molkentin, JD (2002). Defective T cell development and function in calcineurin A beta -deficient mice. Proc. Natl. Acad. Sci. USA 99, 9398-9403. Cameron, AM, Steiner, JP, Sabatini, DM, Kaplin, AI, Walensky, LD, and Snyder, SH (1995). Immunophilin FK506 binding protein associated with inositol 1,4,5-trisphosphate receptor modulates calcium flux. PNAS 92, 1784-1788. Cameron, AM, Wesson, RN, Ahmadi, AR, Singer, AL, Hu, X., Okabayashi, T., Wang, Y., Shigoka, M., Fu, Y., Gao, W., et al. (2016). Chimeric Allografts Induced by Short-Term Treatment With Stem Cell Mobilizing Agents Result in Long-Term Kidney Transplant Survival Without Immunosuppression: II, Study in Miniature Swine. Am. J. Transplant.(American Journal of Transplantation) 16, 2066-2076. Chaikuad, A., Alfano, I., Kerr, G., Sanvitale, CE, Boergermann, JH, Triffitt, JT, Delft, F. von, Knapp, S., Knaus, P., and Bullock, AN (2012). Structure of the Bone Morphogenetic Protein Receptor ALK2 and Implications for Fibrodysplasia Ossificans Progressiva. J. Biol. Chem. 287, 36990-36998. Chen, YG, Liu, F., and Massague, J. (1997). Mechanism of TGFbeta receptor inhibition by FKBP12. EMBO J 16, 3866-3876. Clemons, PA, Gladstone, BG, Seth, A., Chao, ED, Foley, MA, and Schreiber, SL (2002). Synthesis of calcineurin-resistant derivatives of FK506 and selection of compensatory receptors. Chem. Biol. 9, 49-61. Csiszar, A., Ahmad, M., Smith, KE, Labinskyy, N., Gao, Q., Kaley, G., Edwards, JG, Wolin, MS, and Ungvari, Z. (2006). Bone Morphogenetic Protein-2 Induces Proinflammatory Endothelial Phenotype. Am J Pathol 168, 629-638. Cuny, GD, Yu, PB, Laha, JK, Xing, X., Liu, J.-F., Lai, CS, Deng, DY, Sachidanandan, C., Bloch, KD, and Peterson, RT (2008). Structure-activity relationship study of bone morphogenetic protein (BMP) signaling inhibitors. Bioorg Med Chem Lett 18, 4388-4392. Fife, CE, and Carter(v), MJ (2012). Wound Care Outcomes and Associated Cost Among Patients Treated in US Outpatient Wound Centers: Data From the US Wound Registry. Wounds: A Compendium of Clinical Research and Practice 24, 10-17. Fung, JJ (2004). Tacrolimus and transplantation: a decade in review. Transplantation 77, S41-43. Giordano, A., Romano, S., Mallardo, M., D'Angelillo, A., Cali, G., Corcione, N., Ferraro, P., and Romano, MF (2008). FK506 can activate transforming growth factor-beta signalling in vascular smooth muscle cells and promote proliferation. Cardiovasc. Res. 79, 519-526. Gold, BG (1999). FK506 and the role of the immunophilin FKBP-52 in nerve regeneration. Drug Metab. Rev. 31, 649-663. Gold, BG, Densmore, V., Shou, W., Matzuk, MM, and Gordon, HS (1999). Immunophilin FK506-binding protein 52 (not FK506-binding protein 12) mediates the neurotrophic action of FK506. J. Pharmacol. Exp. Ther. 289, 1202-1210. Griffith, JP, Kim, JL, Kim, EE, Sintchak, MD, Thomson, JA, Fitzgibbon, MJ, Fleming, MA, Caron, PR, Hsiao, K., and Navia, MA (1995). X-ray structure of calcineurin inhibited by the immunophilin-immunosuppressant FKBP12-FK506 complex. Cell 82, 507-522. Hatse, S., Princen, K., Bridger, G., De Clercq, E., and Schols, D. (2002). Chemokine receptor inhibition by AMD3100 is strictly confined to CXCR4. FEBS Lett. 527, 255-262. Hoorn, EJ, Walsh, SB, McCormick, JA, Zietse, R., Unwin, RJ, and Ellison, DH (2012). Pathogenesis of calcineurin inhibitor-induced hypertension. J. Nephrol. 25, 269-275. Hu, X., Okabayashi, T., Cameron, AM, Wang, Y., Hisada, M., Li, J., Raccusen, LC, Zheng, Q., Montgomery, RA, Williams, GM, et al. (2016). Chimeric Allografts Induced by Short-Term Treatment With Stem Cell-Mobilizing Agents Result in Long-Term Kidney Transplant Survival Without Immunosuppression: A Study in Rats. Am. J. Transplant. 16, 2055-2065. Jayaraman, T., Brillantes, AM, Timerman, AP, Fleischer, S., Erdjument-Bromage, H., Tempst, P., and Marks, AR (1992). FK506 binding protein associated with the calcium release channel (ryanodine receptor). J. Biol. Chem. 267, 9474-9477. 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 bone marrow progenitor cell recruitment to the neovasculature and reduces mortality after myocardial infarction. Proc. Natl. Acad. Sci. USA 107, 11008-11013. Kissinger, CR, Parge, HE, Knighton, DR, Lewis, CT, Pelletier, LA, Tempczyk, A., Kalish, VJ, Tucker, KD, Showalter, RE, and Moomaw, EW (1995). Crystal structures of human calcineurin and the human FKBP12-FK506-calcineurin complex. Nature 378, 641-644. Kozany, C., Marz, A., Kress, C., and Hausch, F. (2009). Fluorescent probes to characterise FK506-binding proteins. Chembiochem: A European Journal of Chemical Biology 10, 1402-1410. Lewis, CJ, Mardaryev, AN, Poterlowicz, K., Sharova, TY, Aziz, A., Sharpe, DT, Botchkareva, NV, and Sharov, AA (2014). Bone morphogenetic protein signaling suppresses wound-induced skin repair by inhibiting keratinocyte proliferation and migration. J. Invest. Dermatol.(The Journal of Investigative Dermatology) 134, 827-837. Liles, WC, Broxmeyer, HE, Rodger, E., Wood, B., Hubel, K., Cooper, S., Hangoc, G., Bridger, GJ, Henson, GW, Calandra, G., et al. (2003). Mobilization of hematopoietic progenitor cells in healthy volunteers by AMD3100, a CXCR4 antagonist. Blood 102, 2728-2730. Lin, Q., Wesson, RN, Maeda, H., Wang, Y., Cui, Z., Liu, JO, Cameron, AM, Gao, B., Montgomery, RA, Williams, GM, et al. (2014). Pharmacological mobilization of endogenous 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, JD, Lane, WS, Friedman, J., Weissman, I., and Schreiber, SL (1991). Calcineurin is a common target of cyclophilin-cyclosporin A and FKBP-FK506 complexes. Cell 66, 807-815. Mueller, TD, and Nickel, J. (2012). Promiscuity and specificity in BMP receptor activation. FEBS Letters 586, 1846-1859. Naesens, M., Kuypers, DRJ, and Sarwal, M. (2009). Calcineurin Inhibitor Nephrotoxicity. CJASN (Clinical Journal of the American Society of Nephrology) 4, 481-508. Okabayashi, T., Cameron, AM, Hisada, M., Montgomery, RA, Williams, GM, and Sun, Z. (2011). Mobilization of host stem cells enables long-term liver transplant acceptance in a strongly rejecting rat strain combination. Am. J. Transplant. 11, 2046-2056. Plikus, MV, Guerrero-Juarez, CF, Ito, M., Li, YR, Dedhia, PH, Zheng, Y., Shao, M., Gay, DL, Ramos, R., Hsi, T.-C., et al. (2017). Regeneration of fat cells from myofibroblasts during wound healing. Science 355, 748-752. Rao, A., Luo, C., and Hogan, PG (1997). Transcription factors of the NFAT family: regulation and function. Annu. Rev. Immunol. 15, 707-747. Schafferr, MR, Fuchs, N., Proksch, B., Bongartz, M., Beiter, T., and Becker, HD (1998). Tacrolimus impairs wound healing: a possible role of decreased nitric oxide synthesis. Transplantation 65, 813-818. Shin, K., Lim, A., Zhao, C., Sahoo, D., Pan, Y., Spiekerkoetter, E., Liao, JC, and Beachy, PA (2014). Hedgehog Signaling Restrains Bladder Cancer Progression by Eliciting Stromal Production of Urothelial Differentiation Factors. Cancer Cell 26, 521-533. Spiekerkoetter, E., Tian, ​​X., Cai, J., Hopper, RK, Sudheendra, D., Li, CG, El-Bizri, N., Sawada, H., Haghighat, R., Chan, R., et al. (2013). FK506 activates BMPR2, rescues endothelial dysfunction, and reverses pulmonary hypertension. J. Clin. Invest. 123, 3600-3613. Sun, CC, Vaja, V., Chen, S., Theurl, I., Stepanek, A., Brown, DE, Cappellini, MD, Weiss, G., Hong, CC, Lin, HY, et al. (2013). A hepcidin lowering agent mobilizes iron for incorporation into red blood cells in an adenine-induced kidney disease model of anemia in rats. Nephrol Dial Transplant 28, 1733-1743. Tanaka, H., Kuroda, A., Marusawa, H., Hatanaka, H., Kino, T., Goto, T., Hashimoto, M., and Taga, T. (1987). Structure of FK506, a novel immunosuppressant isolated from Streptomyces. J. Am. Chem. Soc. 109, 5031-5033. Tateishi, K., Higuchi, C., Ando, ​​W., Nakata, K., Hashimoto, J., Hart, DA, Yoshikawa, H., and Nakamura, N. (2007). The immunosuppressant FK506 promotes development of the chondrogenic phenotype in human synovial stromal cells via modulation of the Smad signaling pathway. Osteoarthritis and Cartilage 15, 709-718. Wang, T., Li, BY, Danielson, PD, Shah, PC, Rockwell, S., Lechleider, RJ, Martin, J., Manganaro, T., and Donahoe, PK (1996). The immunophilin FKBP12 functions as a common inhibitor of the TGF beta family type I receptors. Cell 86, 435-444. Young, K., Conley, B., Romero, D., Tweedie, E., O'Neill, C., Pinz, I., Brogan, L., Lindner, V., Liaw, L., and Vary, CPH (2012). BMP9 regulates endoglin-dependent chemokine responses in endothelial cells. Blood 120, 4263-4273. Zhai, R., Wang, Y., Qi, L., Williams, GM, Gao, B., Song, G., Burdick, JF, and Sun, Z. (2018). Pharmacological Mobilization of Endogenous Bone Marrow Stem Cells Promotes Liver Regeneration after Extensive Liver Resection in Rats. Scientific Reports 8, 3587.

Claims

1. Formula I: 【Chemistry 1】 (Wherein, R is 【Chemistry 2】 or a salt or solvate thereof.

2. Formula I: 【Chemistry 3】 (Wherein, R is 【Chemistry 4】 or a salt or solvate thereof, and a pharma- ceutically acceptable carrier.

3. The composition of claim 2, further comprising at least one biologically active agent.

4. 13. A composition comprising a compound of claim 1, or a salt or solvate thereof, and at least one or more biologically active agents.

5. Formula I: 【Chemistry 5】 (Wherein, R is 【Chemistry 6】 or a salt or solvate thereof, at least one stem cell mobilizer, and a pharma- ceutically acceptable carrier.

6. The composition of claim 5 , wherein the stem cell mobilizer comprises a CXCR4 antagonist.

7. 7. The composition of claim 6, wherein the CXCR4 antagonist is selected from the group consisting of 1,1'-[1,4-phenylenebis(methylene)]bis-1,4,8,11-tetraazacyclotetradecane, p-(2-(4-(6-amino-2-(((trans-4-(((3-(cyclohexylamino)propyl)amino)methyl)cyclohexyl)methyl)amino)-4-pyrimidinyl)-1-piperazinyl)ethyl)-phosphonate, and N-(pyridin-2-ylmethyl)-1-[4-(1,4,8,11-tetrazacyclotetradec-1-ylmethyl)phenyl]methanamine.

8. Use of an effective amount of a compound according to claim 1, or a salt or solvate thereof, or a composition according to any one of claims 2 to 7, for the preparation of a medicament for treating tissue injury in a subject in need thereof.

9. 9. The use according to claim 8, wherein the compound according to claim 1, or a salt or solvate thereof, or the composition according to any one of claims 2 to 7, further comprises at least one additional biologically active agent.

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

Images