Agents, compositions and methods for improving viability and function of cells, tissues and organs

Abstract

Described herein are compounds, compositions, and methods for improving cell viability and / or function or for protecting cells, tissues, grafts, or organs from various damage in vitro, ex vivo, or in vivo. The reagents and compositions are based on the activation of heat shock reactions and / or anti-oxidation reactions, and include HSP90 cofactor inhibitors, such as celastrol or celastrol analogs, for example, used alone or in combination with an adjuvant (e.g., an NRF-2 activator, an antioxidant, etc.). The therapeutic enhancement may also include an increase in paracrine effector production and signaling. Also described herein are methods for improving the resistance of a cell, tissue, graft, or organ to damage or stress, such as hypoxia or oxidative stress-induced cell death, and / or for improving the viability and retention of transplanted or infused cells. Also provided are therapeutic treatments or prophylaxis of ischemic injury (e.g., myocardial infarction, ischemia / reperfusion injury) and related stressors (hypoxia, oxidative stress, inflammation, sepsis / shock, etc.

Description

[0001] Related applications

[0002] This application is a divisional application of the invention patent application filed on June 15, 2017, with application number "201780049693.5" and invention title "Reagents, compositions and methods for improving the vitality and function of cells, tissues and organs".

[0003] This application claims the benefit of U.S. Provisional Serial No. 62 / 350,258, filed June 15, 2016, the entire contents of which are incorporated herein by reference. Technical Field

[0004] This invention provides reagents, compositions, and methods for regulating the state of cells, cell preparations, tissues, grafts, or organs in vitro, in vitro, or in vivo. The invention also relates to reagents, compositions, and methods for improving cell viability and / or function or for protecting cells, tissues, grafts, or organs from various forms of damage in vitro, in vitro, or in vivo. These reagents and compositions may include HSP90 cofactor inhibitors, such as celastrol or celastrol analogues, alone or in combination with adjuvants (e.g., NRF-2 activators, antioxidants, etc.).

[0005] This invention specifically relates to the treatment or prevention of ischemic injury and related stressors (hypoxia, oxidative stress, inflammation, shock, etc.). This invention also specifically relates to cell-based and tissue-based therapies (e.g., regenerative medicine, transplantation, transplantation procedures, etc.). Background Technology

[0006] Despite numerous technological advancements in cardiology over the past few decades, ischemic heart disease remains a leading cause of morbidity and mortality worldwide. Following myocardial infarction (MI), reperfusion of the ischemic heart induces additional stress due to a significant increase in free radical production, inflammatory cell infiltration, and local pH changes. With appropriate cardioprotective measures, the final infarct size can be significantly reduced. Cardiac pre- and post-modulation have been investigated, including mechanical techniques (repetitive short-cycle ischemia / reperfusion (I / R) via coronary artery clamping), but the simplest and more clinically transferable technique is pharmacological modulation. In this case, cardioprotective effects will involve a reduction in I / R-induced cell death, accompanied by inhibition of mitochondrial permeability transition pore (mPTP) opening and a decrease in oxidative stress, leading to a reduction in infarct size and preservation of ventricular function.

[0007] The applicant investigated the effects of triptolide (a plant triterpenoid) on hypoxic cultures of H9c2 rat cardiomyocytes and a rat model of myoblastic leukemia (MI), and evaluated the therapeutic efficacy using echocardiography and histological analysis. The applicant found that in H9c2 cells, triptolide triggered reactive oxygen species (ROS) formation within minutes and induced nuclear translocation of the transcription factor heat shock factor 1 (HSF1), leading to a heat shock response (HSR) and consequently increased expression of heat shock proteins (HSPs), including HSP70 and HSP32 (heme oxygenase-1, HO-1). Triptolide improved H9c2 survival under hypoxic stress, and functional analysis revealed that HSF1 and HO-1 are key effectors induced by triptolide to promote cell and tissue protection. In rat ischemic myocardium, daily triptolide treatment improved cardiac function and reduced adverse left ventricular remodeling on day 14. Tripterygium wilfordii triggers the expression of cardioprotective HO-1 and inhibits fibrosis and infarct size expansion. In the peri-infarct region, triptolide reduces myofibroblast and macrophage infiltration while attenuating the upregulation of TGF-β and collagen.

[0008] The applicant was the first to report that triptolide treatment promotes cardiomyocyte survival, reduces damage and adverse remodeling while maintaining cardiac function, and concluded that triptolide represents a novel, potent pharmacological cardioprotective agent that mimics ischemic regulation and could have a valuable impact on the treatment of myocardial infarction (S. Der Sarkissian et al., British J. Pharmacol., 2014, 171:5265-5279).

[0009] The applicant also investigated the role of triptolide in the in vitro and ex vivo protection of stem cells for the reconstruction of damaged myocardium. In fact, stem cell transplantation has been proposed as a novel therapeutic approach for tissue engineering and regenerative medicine targeting various disease states. Stem cell-based therapies have been explored in preclinical animal models of ischemic disorders or diseases and are already in early clinical trials for ischemic disorders such as stroke, myocardial infarction (MI), and peripheral artery disease (PAD).

[0010] Inflammation occurs very rapidly following myocardial infarction (MI), and is indicated by high levels of ROS and necrotic cellular debris in resident and circulating leukocytes. These cells abrade damaged tissue and further release ROS, proteolytic enzymes, pro-inflammatory and cytotoxic diffusive factors, and participate in the phagocytosis of necrotic cells and the destruction of extracellular matrix (ECM) components. While clearing affected cells and debris is important, excessive or chronic inflammation can lead to infarct enlargement, poor remodeling, and poor patient outcomes (Steffens, S. et al. Thrombosis and Haemostasis, (2009), 102(2), 240-247; Jiang, B. et al. Journal of Cardiovascular Translational Research, (2010), 3(4), 410-416; Sun, Y. et al. Cardiovascular Research, (2009), 81(3), 482-490; Dobaczewski, M. et al. Journal of Molecular and Cellular Cardiology, (2010), 48(3), 504-511).

[0011] Because the myocardium typically possesses only a very limited regenerative capacity, the space vacated by cardiomyocyte death is replaced by fibrotic scarring, which adversely affects cardiac function. Therefore, research into promoting myocardial regeneration using cell-based therapeutic strategies, such as stem cell transplantation, is of great interest. To date, most of the positive effects of stem cells have been demonstrated through paracrine signaling in existing tissues rather than stem cell differentiation, incorporation, or cell fusion within the lesion site. Paracrine signaling in implanted cells can function by reducing apoptosis, inflammation, and fibrosis, and stimulating angiogenesis and other repair processes. 1,2

[0012] Stem cell therapy has the potential to improve healing of ischemic heart disease, rebuild damaged myocardium, and restore cardiac function. It offers a treatment solution that transcends the limitations of conventional therapies and holds the promise of a cure. The immense hope and potential of stem cell therapy are well understood, and autologous CD133 has already been demonstrated for the treatment of heart failure in animal models and clinical trials (e.g., IMPACT-CABG and COMPARE-AMI). +Feasibility and safety of stem cell transplantation. (Forcillo, J. et al., The Canadian journal of cardiology. 2013;29:441-7; Mansour S et al., Bone Marrow Res. 2011;385-124) However, recent trials of cell therapies involving cardiovascular disease have yielded mixed results, and inconsistent data have once again sparked interest in unresolved questions regarding the mechanisms that contribute to the efficacy of stem cell therapies. In fact, the biggest obstacle to the clinical effectiveness of cell therapies is the poor viability and retention of transplanted cells, especially those in ischemic tissue. Regardless of cell type, less than 1% of transplanted cells survive in ischemic myocardium several days after transplantation. (Pagani FD. et al., J Am Coll Cardiol. 2003;41:879-88; Toma C. et al., Circulation. 2002;105:93-8)

[0013] The applicant found that triptolide rapidly and strongly activates endogenous cytoprotective properties and increases stem cell survival under hypoxic and oxidative conditions mimicking the ischemic transplant microenvironment. The applicant observed significant and rapid activation of the PI3K / Akt and p44 / 42MAPK (ERK1 / 2) pathways, with upregulation of effectors and key genes involved in cell protection and survival, including Hif1a, HO-1 (HSP32), HSP27, HSP70, and VEGF, and increased translocation of Hsf1 from the cytoplasm to the nucleus. The applicant concluded that pre-conditioning stem cells with triptolide could be a safe and effective therapy for clinical applications such as ischemic cardiovascular disease (Der Sarkissian et al., Pre-Conditioning of Stem Cells With Celastrol to Enhance their Therapeutic Potential, Circulation 2011; 124:A14198).

[0014] However, there remains an urgent need for effective compounds, compositions, and methods to improve cell survival and function in in vitro, ex vivo, or in vivo environments by administering the compounds or compositions to ischemic sites or through pre-regulated cells.

[0015] This invention specification refers to multiple documents, the contents of which are incorporated herein by reference in their entirety. Summary of the Invention

[0016] In its first aspect, the present invention provides a composition or pharmaceutical composition comprising one or more compounds that activate heat shock response and antioxidant response; one or more compounds that activate heat shock protein 90 (HSP90) inhibitor and Kelch-like ECH-associated protein 1 (KEAP-1) inhibitor; one or more compounds that activate the heat shock factor protein 1 (HSF1) pathway and the nuclear factor (erythrocyte-derived protein 2)-like 2 (NRF2) pathway; and one or more compounds that activate heat shock proteins (HSPs) and antioxidant effectors.

[0017] In a more specific aspect, the present invention provides a composition or pharmaceutical composition that may comprise one or more compounds selected from the group consisting of: natural triterpenes, synthetic triterpenoid analogs and / or adjuvants.

[0018] Compounds that can be used to support this invention may include, for example, triptolide, triptolide analogs, solanine family compounds or analogs thereof, limonene family compounds (e.g., gadunin) or analogs thereof, bardosolone / CDDO compounds or analogs thereof (e.g., CDDO-methyl), curcuminoids (e.g., curcumin) or analogs thereof, sarsinol or analogs thereof, tert-butylhydroquinone (tBHQ) or analogs thereof, bis(2-hydroxybenzyl)acetone (2HBA) or analogs thereof, acetylenic tricyclic bis(cyanone) (TBE-31) or analogs thereof, epigallocatechin gallate (EGCG) or analogs thereof, gambogeylic acid or analogs thereof, neomycin or analogs thereof, chlordamine or analogs thereof, andrographolide or analogs thereof, edaravone or analogs thereof, ascorbic acid or analogs thereof, or any combination thereof.

[0019] More specifically, the present invention can be implemented by using one or more compounds of formula I.

[0020]

[0021] R1 can be selected from, for example, the group consisting of: -H, -F, -Cl, -Br, -I, -CN, -aryl, alkyl, imidazole, -ORa, -NRbRc, -(CH2). n OH and -(CH2) n NH2;

[0022] Ra can be selected from the group consisting of: H, substituted or unsubstituted straight-chain alkyl groups having 1 to 6 carbon atoms, substituted or unsubstituted branched alkyl groups having 3 to 6 carbon atoms, and protecting groups;

[0023] Rb and Rc can be independently selected from the following groups: -H, -OH, -OCH3, substituted (e.g., -CH2CH2OH, etc.) or unsubstituted straight-chain alkyl groups having 1 to 6 carbon atoms, substituted or unsubstituted branched alkyl groups having 3 to 6 carbon atoms, and protecting groups.

[0024] Where n can be 0, 1, 2, 3 or 4;

[0025] R2 and R3 can be independently selected from the following groups: -H, -ORd, =O, -C(=O)OH, -C(=O)ORx, and -C(=O)Rx;

[0026] Rd can be H or a lower alkyl group having 1 to 3 carbon atoms;

[0027] Rx can be H or a lower alkyl group having 1 to 3 carbon atoms;

[0028] R4 can be selected from the following groups: -H, -OH and lower alkyl groups having 1 to 3 carbon atoms.

[0029] According to the present invention, the compound may include, for example, a compound of formula Ia, a pharmaceutically acceptable salt, stereoisomer, tautomer, or prodrug.

[0030]

[0031] R1 can be selected from, for example, the group consisting of: -H, -F, -Cl, -Br, -I, -CN, -aryl, alkyl, imidazole, -ORa, -NRbRc, -(CH2). n OH and -(CH2) n NH2;

[0032] Ra can be selected from the group consisting of: H, substituted or unsubstituted straight-chain alkyl groups having 1 to 6 carbon atoms, substituted or unsubstituted branched alkyl groups having 3 to 6 carbon atoms, and protecting groups;

[0033] Rb and Rc can be independently selected from the group consisting of: -H, -OH, -OCH3, substituted or unsubstituted straight-chain alkyl groups having 1 to 6 carbon atoms, substituted or unsubstituted branched alkyl groups having 3 to 6 carbon atoms, and protecting groups.

[0034] Where n can be 0, 1, 2, 3 or 4;

[0035] R2 and R3 can be independently selected from the following groups: -H, -ORd, =O, -C(=O)OH, -C(=O)ORx, and -C(=O)Rx;

[0036] Rd can be H or a lower alkyl group having 1 to 3 carbon atoms;

[0037] Rx can be H or a lower alkyl group having 1 to 3 carbon atoms;

[0038] R4 can be selected from the following groups: -H, -OH and lower alkyl groups having 1 to 3 carbon atoms.

[0039] According to the present invention, the compound of formula I or Ia covers the following compounds, wherein R1 may be more specifically selected from the group consisting of -ORa and -NRbRc; Ra may be selected from the group consisting of: H, substituted or unsubstituted straight-chain alkyl groups having 1 to 6 carbon atoms, substituted or unsubstituted branched alkyl groups having 3 to 6 carbon atoms, and protecting groups; Rb and Rc may be independently selected from the group consisting of: -H, -OH, -OCH3, substituted or unsubstituted straight-chain alkyl groups having 1 to 6 carbon atoms, substituted or unsubstituted branched alkyl groups having 3 to 6 carbon atoms, and protecting groups; R2 and R3 may be independently selected from the group consisting of: -H, -ORd, and =O, where Rd may be H or a lower alkyl group having 1 to 3 carbon atoms; and / or R4 may be -H or CH3.

[0040] Further according to the invention, the compound of formula I or Ia covers the following compounds, wherein R1 may be more specifically selected from the group consisting of -ORa and -NRbRc; Ra may be selected from the group consisting of H and a lower alkyl group having 1 to 3 carbon atoms; Rb and Rc may be independently selected from the group consisting of -H, -OH, and -CH2CH2OH; R2 and R3 may be independently selected from the group consisting of -H, -OH, -OCH3 and =O; and / or R4 may be -H or CH3.

[0041] Furthermore, according to the present invention, the compound of formula I or Ia covers the following compounds, wherein R1 may be more specifically selected from the group consisting of -ORa and -NRbRc; Ra may be selected from the group consisting of H and -CH3; Rb and Rc may be independently selected from the group consisting of -H, -OH, and -CH2CH2OH; R2 and R3 may be independently selected from the group consisting of -OH and =O; and / or R4 may be -H.

[0042] Exemplary embodiments of the compounds covered in this invention include those of formula I or Ia, wherein R1 is NRbRc.

[0043] Exemplary embodiments of the compound are provided in Figure 23The term "triptodextrin" may include, for example, triptodextrin analogues identified as analogue 1, analogue 2, analogue 3, analogue 4, analogue 5, analogue 6, or dihydrocelastrol.

[0044] The triptolide analogues particularly considered in this invention include analogue 1, analogue 2, analogue 3, analogue 4, and dihydrotriptolide. Analogue 1 is considered even more particularly.

[0045] In some cases, triptolide can be specifically excluded from certain aspects of the invention. For example, compositions of triptolide alone or its use as the sole compound in methods for treating myocardial ischemia or protecting stem cells have been disclosed in the literature. (Der Sarkissian et al., Pre-Conditioning of Stem CellsWith Celastrol to Enhance their Therapeutic Potential, Circulation 2011; 124:A14198; S. Der Sarkissian et al., British J. Pharmacol., 2014, 171:5265-5279).

[0046] Therefore, when the composition, method, and use contain triptolide, it is preferably a compound of formula I or Ia other than triptolide, or used in combination with another compound (i.e., a compound of formula I or Ia and / or an adjuvant).

[0047] Alternatively, the present invention may be carried out using compounds of formula I or Ia having R1, R2, R3 or R4 groups as defined herein (these groups are different from the corresponding R1, R2, R3 or R4 groups of triptolide), as pharmaceutically acceptable salts, stereoisomers, tautomers or prodrugs.

[0048] According to one embodiment of the present invention, the adjuvant may be, for example, 2HBA, andrographolide, ascorbic acid, caffeol, salsaponin-badosol-imidazolium (CDDO-im), chalcone, N6-[2-[[4-(2,4-dichlorophenyl)-5-(1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]-3-nitro-2,6-pyridinediamine (CHIR98014), conglobatin, curcumin, cycloastragalool, 1,2-dithiacyclopenten-3-thione (D3T), damamod, edaravone, EGCG, gambogeylic acid, ganesespib, ganetespib, IQ-1, limonene, chlordamine, melatonin, benzamide tetrahydroindole, N886, alkylaminobiphenylamide, neomycin, pyridoxal 5′-phosphate (P 5'-P), hydroxypyridinethione, quercetin, rhizocarpine, resveratrol, N-(2-cyano-3,12-dioxo-28-noroleanane-1,9(11)-dien-17-yl)-2,2-difluoro-propionamide or omaveloxolone (RTA-408), 4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)-1H-imidazolium (SB202190), 3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione (SB216763), SNX-5422 (PF-04929113), sodium butyrate, sulforane, tetrabromobenzotriazole (TBB), tert-butylhydroquinone (tBHQ), valproic acid (valporic acid) (acid), solanine A, solanine, ergosterol, lupene ketone, and any analogues of such adjuvants.

[0049] More specifically, adjuvants may include, for example, tBHQ, caryophyllin, curcumin, 2HBA, or EGCG or their analogues.

[0050] In another aspect, the present invention provides a composition or pharmaceutical composition which may comprise, for example, a) one or more compounds of formula I, Ia, alone or in combination with one or more adjuvants, a pharmaceutically acceptable salt, stereoisomer, tautomer or prodrug, and b) a carrier or a pharmaceutically acceptable carrier or excipient.

[0051] This invention provides a method for regulating the state of cells (isolated cells), cell preparations, tissues (isolated tissues), grafts (isolated grafts), or organs (isolated organs). Such regulation can be performed to increase or maintain viability, or resistance to death, damage, and / or stress, as well as functionality. This regulation can be carried out in a manner that increases resilience to cell death, enhances resistance to oxidative and / or hypoxic stressors, and / or strengthens protein expression characteristics (which may consist of increased secretion of various proteins, including but not limited to heat shock proteins, antioxidant proteins, and growth factors, and / or reduced harmful expression of such peptide mediators associated with cellular senescence).

[0052] The method may include contacting the cell, cell preparation, tissue, graft, or organ with: a) a composition comprising one or more compounds of formula I or Ia, pharmaceutically acceptable salts, stereoisomers, tautomers, or prodrugs thereof; b) a combination comprising one or more compounds of formula I or Ia, pharmaceutically acceptable salts, stereoisomers, tautomers, or prodrugs thereof, and adjuvants; c) a different cell preparation that has been or has been contacted with one or more compounds of formula I or Ia, pharmaceutically acceptable salts, stereoisomers, tautomers, or prodrugs thereof, or combinations thereof; or d) a secretory proteome or cell culture medium of a different cell preparation that has been contacted with one or more compounds of formula I or Ia, pharmaceutically acceptable salts, stereoisomers, tautomers, or prodrugs thereof, or combinations thereof.

[0053] In another aspect, the present invention provides a method for protecting cells, tissues, grafts, or organs from damage or stressors (e.g., oxidative, hypoxic, inflammatory, thermal, osmotic, mechanical) that may occur during various pathological conditions (e.g., ischemia, ischemia / reperfusion, shock, sepsis) or procedures (e.g., freeze / thaw cycles, cell encapsulation, amplification, enrichment and purification steps, graft preparation / manufacturing, etc.).

[0054] The method may include contacting cells, tissues, grafts, or organs with a composition comprising one or more compounds that activate heat shock and antioxidant responses, one or more compounds that activate HSP90 inhibition and KEAP-1 inhibition pathways, one or more compounds that activate HSF1 and NRF2 pathways, one or more compounds that activate HSPs, and an antioxidant.

[0055] The method may also include contacting cells, tissues, grafts or organs with a composition comprising one or more compounds selected from the group consisting of: natural triterpenes, synthetic triterpenoid analogs and / or adjuvants.

[0056] The method may more specifically include contacting cells, tissues, grafts, or organs with a composition comprising one or more compounds of formula I, Ia, pharmaceutically acceptable salts thereof, stereoisomers, tautomers, or prodrugs.

[0057] According to the present invention, the method can be performed in vitro, in vitro, or in vivo.

[0058] Therefore, according to the present invention, the method can be an ex vivo method performed on a cell preparation, tissue, or organ. The modified cell preparation, tissue, or organ can then be administered / transplanted into a mammal in need (e.g., a human mammal). The method can also be an in vivo method performed by administering a composition, combination, different cell preparations, or secreted proteome to a mammal in need (e.g., a mammal suffering from or susceptible to ischemic diseases, undergoing surgery or medical intervention, or suffering from degenerative diseases with cell and tissue loss).

[0059] Degenerative diseases can include cardiomyopathy, liver diseases such as non-alcoholic fatty liver disease, non-alcoholic steatohepatitis (NAFLD / NASH), cirrhosis, lung diseases such as chronic obstructive pulmonary disease (COPD), osteoarthritis, pancreatic diseases such as diabetes, and neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, dementia, and amyotrophic lateral sclerosis (ALS).

[0060] Compositions, combinations, different cell preparations, or secreted proteomes can be administered immediately before, during, or after surgery or medical intervention, and can be administered, for example, systemically or locally.

[0061] This method can also be performed on cultured cells or in vitro before freezing.

[0062] Cells, cell preparations, tissues, grafts, or organs may be exposed to the compound, combination, different cell preparations, or secreted proteome for, for example, at least 5 to 180 minutes prior to use.

[0063] According to one embodiment of the present invention, the compound of formula I or formula Ia can be in the form of 10 -6 M to 10 -10 Use at the concentration of M.

[0064] This method may include the local or systemic administration of compounds, combinations, cellular agents, or secreted proteomes. For example, when administering compounds, combinations, or secreted proteomes, they may be administered systemically (e.g., intravenously) or locally (e.g., topically at the site of injury). When administering cellular agents, they may be preferentially administered locally (e.g., at sites of suspected ischemia, at sites requiring cell protection, etc.).

[0065] The cells can be immortalized or primary cells. Cells can be, for example, stem cells, cardiomyocytes, myoblasts, myocytes, kidney cells, pancreatic cells, hepatocytes, neurons, endothelial cells, and epithelial cells.

[0066] Stem cells can be embryonic pluripotent stem cells. Exemplary embodiments of stem cells may include mesenchymal stem cells, hematopoietic stem cells, and induced pluripotent stem cells. According to the present invention, these cells are non-cancerous cells.

[0067] Cells are preferably derived from mammals, such as humans. Cells are suitable for allogeneic or autologous stem cell transplantation. Cells can be derived from commercial sources or can be isolated from a donor or host. Cells can be selected based on specific and desired markers.

[0068] According to the invention, the method may include additionally contacting cells, tissues, grafts, or organs with one or more adjuvants. Thus, cells, tissues, grafts, or organs may be contacted with a composition comprising a) a compound of formula I or Ia (or a pharmaceutically acceptable salt, stereoisomer, tautomer, or prodrug thereof) and b) an adjuvant mixed together, or the compound and adjuvant may be added one after the other.

[0069] Cells, tissues, grafts, or organs may also come into contact sequentially with compounds of formula I or Ia (or their pharmaceutically acceptable salts, stereoisomers, tautomers, or prodrugs) and adjuvants.

[0070] In one aspect, the present invention further provides a method for preventing or treating ischemic diseases, including, for example, ischemic cardiovascular diseases, stroke, myocardial infarction (MI), peripheral artery disease (PAD), or diseases involving cell and tissue loss or degeneration (e.g., diabetes, liver disease, lung disease, etc.).

[0071] The method may include administering to a mammal in need a composition comprising one or more compounds that activate heat shock response activation and antioxidant response activation, one or more compounds that activate HSP90 inhibition and KEAP-1 inhibition pathways, one or more compounds that activate HSF1 activation and NRF2 pathways, one or more compounds that activate HSP, and an antioxidant, or a secretory proteome of a cell preparation regulated by such composition.

[0072] The method may also include administering to mammals in need a stem cell preparation pre-conditioned with a composition comprising one or more compounds that activate heat shock response activation and antioxidant response activation, one or more compounds that activate HSP90 inhibition and KEAP-1 inhibition pathways, one or more compounds that activate HSF1 and NRF2 pathways, one or more compounds that activate HSP, and an antioxidant.

[0073] In addition, the method may include administering to a mammal in need a composition comprising one or more compounds selected from the group consisting of: natural triterpenes, synthetic triterpenoid analogs and / or auxiliaries.

[0074] The method may also include administering to a mammal in need a stem cell preparation pre-conditioned with a composition comprising one or more compounds selected from the group consisting of: natural triterpenes, synthetic triterpenoid analogs and / or adjuvants.

[0075] The method may also include administering to a mammal in need a secretory proteome or culture medium of a stem cell preparation treated with a composition comprising one or more of the compounds and / or adjuvants described herein.

[0076] The method may more specifically include administering to a mammal in need the following: a) a composition comprising one or more compounds of formula I or Ia, pharmaceutically acceptable salts, stereoisomers, tautomers, or prodrugs thereof; b) a stem cell preparation pre-regulated with one or more compounds of formula I or Ia, pharmaceutically acceptable salts, stereoisomers, tautomers, or prodrugs thereof; or c) a cell preparation regulated with such a composition containing the secretory proteome.

[0077] According to the present invention, the method covers administering a composition comprising a) a pharmaceutically acceptable salt, stereoisomer, tautomer or prodrug of a compound of formula I or Ia, and b) an adjuvant, administering stem cells pre-regulated with a composition comprising such a combination, or administering a secretory proteome of a cell preparation regulated with such a combination.

[0078] According to one embodiment of the invention, the stem cell preparation may be an autologous stem cell preparation isolated from a mammal in need. According to another embodiment of the invention, the stem cell preparation may be an allogeneic stem cell preparation isolated from a mammalian donor. For suitability for administration to mammals, the allogeneic stem cell preparation is preferably HLA-matched. The stem cell preparation may also be immune-exempt, low-immunogenic, or immune-evading.

[0079] In another aspect, the present invention also provides cell preparations or isolated cell, tissue, graft, or organ preparations pre-regulated with the secretory proteome of different cell preparations modified with such compositions, the compositions comprising one or more compounds that activate heat shock response activation and antioxidant response activation, one or more compounds that activate HSP90 inhibition and KEAP-1 inhibition pathways, one or more compounds that activate HSF1 activation and NRF2 pathways, and one or more compounds that activate HSP activation and antioxidant response activation.

[0080] In another aspect, the present invention provides preparations of isolated cells, tissues, grafts or organs pre-conditioned with compositions comprising one or more compounds selected from the group consisting of: natural triterpenes, synthetic triterpenoid analogs and / or adjuvants.

[0081] More specifically, in another aspect thereto, the present invention provides cell preparations or isolated cell, tissue, graft or organ preparations pre-regulated with the secretory proteome of different cell preparations modified with such compositions, the compositions comprising one or more compounds of formula I, formula Ia, pharmaceutically acceptable salts, stereoisomers, tautomers or prodrugs thereof.

[0082] According to the present invention, preparations of isolated cells, tissues, grafts or organs can be pre-modified with a composition comprising a) a pharmaceutically acceptable salt, stereoisomer, tautomer or prodrug of a compound of formula I or Ia and b) an adjuvant.

[0083] According to the present invention, preparations of isolated cells, tissues, grafts or organs can be modulated with the secretory proteome of cells, said cells being pre-modified with a composition comprising a) a pharmaceutically acceptable salt, stereoisomer, tautomer or prodrug of a compound of formula I or Ia and b) a combination of an adjuvant.

[0084] According to one embodiment of the invention, the composition may be washed out of the formulation before use, or may be retained as part of the formulation.

[0085] According to an exemplary embodiment, the cell preparation may be a stem cell preparation, such as in vitro processed stem cells or stem cells harvested from a donor who has previously received systemic treatment.

[0086] According to another embodiment of the invention, the cell preparation can be a cell suspension. According to another embodiment of the invention, the cell preparation can be in the form of a three-dimensional scaffold. To obtain the three-dimensional scaffold, cells can be cultured as cell aggregates in the presence of microcarriers, on alginate microcapsules, in hydrogels (e.g., thermally reversible hydrogels, chitosan-based hydrogels, etc.), or in nanostructure scaffolds composed of self-assembled peptides (Meng, X. et al., SpringerPlus, 2014, 3:80).

[0087] The present invention further relates to a method for reducing cell damage during the transplantation of stem cells, tissues, grafts or organs, the method comprising contacting stem cells, tissues, grafts or organs with a composition or a cell preparation modified with such a composition containing one or more compounds that activate heat shock response activation and antioxidant response activation, one or more compounds that activate HSP90 inhibition and KEAP-1 inhibition pathways, one or more compounds that activate HSF1 activation and NRF2 pathways, and one or more compounds that activate HSP activation and antioxidant response activation.

[0088] The present invention further relates to a method for reducing cell damage in stem cell, tissue, graft, or organ transplantation, the method comprising contacting the stem cell, tissue, graft, or organ with a composition comprising one or more compounds selected from the group consisting of: natural triterpenes, synthetic triterpenoid analogs, and / or adjuvants.

[0089] More specifically, the present invention further provides a method for reducing cell damage during the transplantation of stem cells, tissues, grafts or organs, the method comprising contacting the stem cells, tissues, grafts or organs with a composition or a secretory proteome of a cell preparation modified with such a composition before and / or during and / or after transplantation, the composition comprising at least one compound of formula I or Ia, a pharmaceutically acceptable salt, stereoisomer, tautomer or prodrug thereof.

[0090] According to one embodiment of the present invention, the method may include administering a) a pharmaceutically acceptable salt, stereoisomer, tautomer, or prodrug of a compound of formula I or Ia, and b) an adjuvant.

[0091] In another aspect, the present invention relates to the use of one or more compounds that activate heat shock response activation and antioxidant response activation, one or more compounds that activate HSP90 inhibition and KEAP-1 inhibition pathways, one or more compounds that activate HSF1 activation and NRF2 pathways, and one or more compounds that activate HSP activation and antioxidant response activation for the protection of cells, tissues, grafts or organs from stress or damage.

[0092] In another aspect, the present invention relates to the use of one or more compounds selected from the group consisting of natural triterpenes, synthetic triterpenoid analogs and / or adjuvants for the protection of cells, tissues, grafts or organs from stress or damage.

[0093] In a more specific aspect, the present invention relates to the use of one or more compounds of formula I or Ia, pharmaceutically acceptable salts, stereoisomers, tautomers or prodrugs thereof for the protection of cells, tissues, grafts or organs from stress or damage.

[0094] According to another aspect of the invention, the invention relates to the use of a) a pharmaceutically acceptable salt, stereoisomer, tautomer or prodrug of a compound of formula I or Ia and b) an adjuvant.

[0095] In another aspect, the present invention provides a method for treating patients who require surgery or medical intervention or who are susceptible to cellular stress or damage.

[0096] According to one embodiment of the invention, the method may include administering a composition comprising one or more compounds as defined herein.

[0097] According to a more specific embodiment of the invention, the method may include administering a composition comprising a compound of formula I, formula Ia, a pharmaceutically acceptable salt, stereoisomer, tautomer, or prodrug before and / or during surgery or medical intervention.

[0098] In another embodiment of the invention, the method may include administering a) a pharmaceutically acceptable salt, stereoisomer, tautomer, or prodrug of a compound of formula I or Ia, and b) an adjuvant.

[0099] According to the invention, the composition can be administered topically, for example, at the site of surgery or medical intervention. According to another embodiment of the invention, the composition can be administered systemically.

[0100] According to the present invention, the compound or composition can be administered directly to the mammal in need at sites susceptible to cellular damage. Therefore, the compound or composition can be administered to prevent or treat cellular damage.

[0101] The present invention also relates to a kit that may include a first vial and a second vial, the first vial containing a compound of formula I, formula Ia, a pharmaceutically acceptable salt thereof, a stereoisomer, a tautomer, or a prodrug, and the second vial containing cells.

[0102] According to another embodiment of the invention, the kit may include a third vial containing adjuvants. According to one embodiment of the invention, the cells may be stem cells.

[0103] The present invention also relates to kits for pre-mixing different components.

[0104] The present invention also provides a device suitable for administering compositions, combinations, different cellular agents, or secreted proteomes into mammals in need. This device may be, for example, a pre-filled syringe, or a syringe having a compartment for receiving the composition, combination, different cellular agent, or secreted proteome.

[0105] The present invention also provides an apparatus suitable for cell and tissue preparations. This apparatus may be, for example, a cell sorting or cell culture or amplification device, such as a bioreactor, which can contain compositions, combinations, different cell preparations, or secreted proteomes.

[0106] Other aspects of the invention are also provided in the following entries 1 to 31:

[0107] 1. A method for improving cell resistance to cell death, the method comprising contacting cells with an effective amount of one or more compounds that activate the HSF1 and NRF2 pathways.

[0108] 2. A method for generating a regulated cell population, said cells having increased resilience to cell death, greater resistance to oxidative and / or hypoxic stressors, and / or enhanced protein expression characteristics (including enhanced secretion of at least one of heat shock proteins, antioxidant proteins, and / or growth factors, anti-inflammatory cytokines, chemokines, and / or reduced expression of harmful peptide mediators (e.g., aging-related proteins)), said method comprising contacting the cells with an effective amount of one or more compounds that activate the HSF1 and NRF2 pathways.

[0109] 3. A method for improving the viability and retention of transplanted or infused cells, the method comprising contacting the cells with an effective amount of one or more compounds that activate the HSF1 and NRF2 pathways before and / or after transplantation or infusion.

[0110] 4. The method described in the entry described herein, wherein the method includes ex vivo or in vitro contact with cells prior to transplantation or infusion.

[0111] The methods described in the entries described herein include in vivo contact with cells.

[0112] 5. The method described in the entry described herein, wherein the method includes in vivo contact with cells before or after transplantation or infusion.

[0113] 6. A method of treating a subject requiring cell transplantation or infusion, the method comprising (a) contacting cells to be transplanted with an effective amount of one or more compounds that activate the HSF1 and NRF2 pathways; and (b) transplanting or infusing the cells of (a) into the subject.

[0114] 7. The method described in the entry herein, further comprising contacting the transplanted or infused cells with an effective amount of the one or more compounds after transplantation.

[0115] 8. The method described in the entry herein, wherein the subject suffers from organ or tissue ischemia or degeneration and / or wherein the organ or tissue ischemia is myocardial ischemia. Degeneration includes loss of pancreatic cells, cardiomyocytes, muscle cells, neurons, kidney cells, hepatocytes, etc.

[0116] 10. The method described in the entry described herein, wherein the subject has a myocardial infarction (MI).

[0117] 11. The method described in the entries herein, wherein at least one of one or more compounds is an HSP90 inhibitor.

[0118] 12. The method described in the entry described herein, wherein the HSP90 inhibitor is an HSP90N or C-terminal inhibitor, and more specifically an HSP90 cofactor inhibitor.

[0119] 13. The method described in the entries herein, wherein at least one of one or more compounds has NRF2-inducing activity or antioxidant activity.

[0120] 14. The method described in the entries herein, wherein said one or more compounds comprise triptolide or an analogue thereof, solanine family compounds or an analogue thereof, limonene family compounds (e.g., gadurin) or an analogue thereof, bardosolone / CDDO compounds or an analogue thereof (e.g., CDDO-methyl), curcuminoids (e.g., curcumin) or an analogue thereof, sarsaparilla phenol or an analogue thereof, tert-butylhydroquinone (tBHQ) or an analogue thereof, bis(2-hydroxybenzyl)acetone (2HBA) or an analogue thereof, acetylenic tricyclic bis(cyanone) (TBE-31) or an analogue thereof, epigallocatechin gallate (EGCG) or an analogue thereof, gambogeylic acid or an analogue thereof, neomycin or an analogue thereof, chlordamine or an analogue thereof, andrographolide or an analogue thereof, edaravone or an analogue thereof, ascorbic acid or an analogue thereof, gamendazole or an analogue thereof, sulforaphane or an analogue thereof, sulphoxythiocarbamate alkyne (STCA) or its analogues or any combination thereof.

[0121] 15. The method described in the entries herein, wherein said one or more compounds comprise (i) (a) triptolide or an analogue thereof; and (b) EGCG or an analogue thereof; (ii) (a) triptolide or an analogue thereof; and (b) tBHQ or an analogue thereof; (iii) (a) triptolide or an analogue thereof; and (b) 2HBA or an analogue thereof; (iv) (a) triptolide or an analogue thereof; and (b) curcumin or an analogue thereof; (v) (a) triptolide or an analogue thereof; and (b) sarstigmolol or an analogue thereof; (vi) (a) gadunin or an analogue thereof; and (b) EGCG or an analogue thereof; or (vii) (a) gadunin or an analogue thereof; and (b) tBHQ or an analogue thereof; (viii) (a) gadunin or an analogue thereof; and (b) 2HBA or an analogue thereof; (ix) (a) gadunin or an analogue thereof; and (b) curcumin or an analogue thereof; (x) (a) gadurin or its analogues; and (b) sarstilin or its analogues.

[0122] 16. The method described in the entry described herein, wherein the cell is a stem / pluripotent / progenitor cell or a differentiated cell.

[0123] 17. The method described in the entry described herein, wherein the stem / pluripotent / progenitor cell is a mesenchymal stem cell, CD34 + Cells or CD133 + cell.

[0124] 18. The method described in the entry described herein, wherein the cell is a differentiated cell.

[0125] 19. The method described in the entry described herein, wherein the cells are present in a tissue or organ.

[0126] 20. The method described in the entry herein, wherein the contact comprises adding a single dose or multiple doses of one or more compounds to the culture medium.

[0127] 21. The method described in the entry described herein, wherein the contact comprises administering a single or multiple doses of one or more compounds to a subject.

[0128] 22. A method for identifying one or more compounds, said compounds being used to improve cell resistance to cell death and / or improve the viability and retention of transplanted or infused cells, said method comprising (i) contacting cells with said one or more compounds; (ii) determining whether the HSF1 and NRF2 pathways are activated in said cells, said activation of the pathways indicating that said one or more compounds are used to improve cell resistance to cell death and / or improve the viability and retention of contacted cells, and / or said one or more compounds are used to improve cell function and / or improve the paracrine proteome of contacted cells.

[0129] 23. The method described in the entry described herein, wherein the cell is a stem / progenitor cell or a differentiated cell.

[0130] 24. The method described in the entry described herein, wherein the stem / progenitor cells are mesenchymal stem cells, CD34 + Cells or CD133 + cell.

[0131] 25. The method described in the entry described herein, wherein the cell is a differentiated cell.

[0132] 26. The method described in the entry described herein, wherein the cells are present in a tissue or organ.

[0133] 27. The method described in the entry herein, wherein the step of determining whether the HSF1 and NRF2 pathways are activated in the cell comprises measuring the expression of one or more genes under the transcriptional control of HSF1 and / or NRF2, wherein increased expression of the one or more genes indicates that the HSF1 and / or NRF2 pathways are activated.

[0134] 28. The method described in the entry described herein, wherein one or more of the genes expressed upon activation of HSF1 or NRF2 are heat shock proteins (HSPs) (e.g., HSP90, HSP70), glutathione S-transferase, NADPH-quinone oxidoreductase 1 (NQO1), growth factors (e.g., VEGF, FGF2, HGF, IGF), heme oxygenase 1 (HO1), superoxide dismutase 1-3 (SOD1-3), thioredoxin (TRX), catalase (CAT), and / or glutathione peroxidase (GPx).

[0135] 29. The method described in the entry described herein, wherein the step of determining whether the NRF2 pathway is activated in the cell includes measuring the expression of one or more pro-inflammatory genes, wherein a decrease in the expression of said one or more genes indicates that the HSF1 and / or NRF2 pathway is activated.

[0136] 30. The method described in the entry described herein, wherein one or more inflammatory genes are IL-1β and / or TNFα.

[0137] 31. The method described in the entry described herein, wherein the method includes measuring cell death under hypoxic and / or oxidative, hypoxic / reoxidative stress conditions.

[0138] According to the present invention, representative embodiments of HSP90 inhibitors may include, for example, triptolide or an analogue of formula I.

[0139] On the other hand, the present invention relates to compositions comprising triptolide or an analogue of formula I in combination with one or more protective compounds.

[0140] According to the present invention, one or more protective compounds may be adjuvants, such as NRF2 activators or antioxidants, and may include, for example,

[0141] Tripterygium wilfordii, 2HBA, andrographolide, ascorbic acid, caffeol, badoxaclofen-imazole (CDDO-im), chalcone, N6-[2-[[4-(2,4-dichlorophenyl)-5-(1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]-3-nitro-2,6-pyridinediamine (CHIR98014), cytosolic acid, curcumin, cycloastragalool, 1,2-dithiacyclopenten-3-thione (D3T), damaride, edaravone, EGCG, gambogeylic acid, ganesespib, ganetespib, IQ-1, limonene, chlorpheniramine, melatonin, benzamide tetrahydroindole, N886, alkylaminobiphenylamide, neomycin, pyridoxal 5′-phosphate (P5′-P), hydroxypyridinethione Quercetin, rhizocarpine, resveratrol, N-(2-cyano-3,12-dioxo-28-noroleanane-1,9(11)-dien-17-yl)-2,2-difluoro-propionamide or omaveloxolone (RTA-408), 4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)-1H-imidazolium (SB202190), 3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione (SB216763), SNX-5422 (PF-04929113), sodium butyrate, sulforane, tetrabromobenzotriazole (TBB), tert-butylhydroquinone (tBHQ), valproic acid (valporic acid) (acid), solanine A, solanine, ergosterol, lupene ketone, and any analogues of such adjuvants.

[0142] In another aspect, the present invention relates to cell preparations containing live cells that, upon contact with the compositions described herein, may possess improved characteristics. These improved characteristics may include improved function, increased gene expression related to cell protection, increased viability, increased survival rate, and increased resistance to cell damage from stressors (e.g., oxidative, hypoxic, inflammatory, thermal, osmotic, mechanical) that may arise during various pathological conditions (e.g., ischemia, ischemia / reperfusion, sepsis, shock) or procedural processes (e.g., freeze / thaw cycles, cell encapsulation, expansion, enrichment and purification steps, graft preparation / manufacturing, etc.).

[0143] Cellular preparations may contain stem cells, such as stem cells suitable for preventing or repairing damage caused by ischemia. Stem cells may be derived from commercial sources, isolated from individuals in need of treatment (i.e., autologous), or isolated from compatible donors (i.e., allogeneic).

[0144] According to one embodiment of the invention, cell or tissue preparations may be pre-conditioned using the compositions or reagents described herein. According to another embodiment of the invention, cell preparations may comprise a culture medium containing the compositions or reagents described herein.

[0145] Other objects, advantages, and features of the invention will become clearer from the following non-limiting description, which is given only by way of specific embodiments with reference to the accompanying drawings. Attached Figure Description

[0146] In the attached diagram:

[0147] Figure 1 Examples of the activities of various HSP90 inhibitors and triptolide-like compounds (A, B) and reporter-based screening (C) are shown. The Perkin Elmer Operetta® high-content screening system can detect various fluorophores and cell morphology changes. The Perkin Elmer Victor Multilabel Counter or EnVision® Multilabel Reader photometer can detect luciferin metabolism. It can also quantitatively characterize EC using various reporter cell lines. 50 And maximum multiple induction.

[0148] Figure 2A and 2BTripterygium wilfordii preconditioning showed increased in vivo viability / retention of MSCs. MSCs were treated with the conditioning compound or mordant for 60 min. Cells were then washed, labeled with fluorescent cell trackers, and injected into the left ischemic hindlimb of rats at t = 0. Transplanted cells were imaged in vivo using Optix® up to day 9. 3 million MSCs injected into the left quadriceps femoris muscle at t = 0 were tracked using the Optix in vivo imaging system up to day 9 (logarithmic scale).

[0149] Figure 3 The study demonstrated the enhanced therapeutic effect of triptolide-preregulated stem cell therapy. As observed by laser Doppler scanning, triptolide-preregulated MSCs more effectively reconstructed blood flow in a hindlimb ischemia model.

[0150] Figure 4 It showed that triptolide (10) -6 Cell-protective proteins, growth / survival factors, and antioxidant proteins released by human mesenchymal stem cells (hMSCs) one hour after M) treatment. The regulated cell culture medium was collected and concentrated, proteins were separated by gel electrophoresis, analyzed by mass spectrometry, and identified by database search.

[0151] Figure 5 Some of the compounds used in the experiments described herein are shown. Figure 6 -twenty two).

[0152] Figure 6 Treatment of rat mesenchymal stem cells (rMSCs) with HSP90 inhibitors (triptolide, gadurin, and rhizocarpine) (instead of NRF2 activators alone (EGCG, tBHQ)) for 1 hour protected rMSCs from hypoxia-induced cell death for 48 hours. EGCG enhanced the protective effect mediated by triptolide.

[0153] Figure 7 The study demonstrated that pre-regulation of H9c2 cells with medium derived from rMSCs (treated with a high dose of HSP90 cofactor inhibitors (triptoside; gadurin)) for 48 hours protected H9c2 cells from 48 hours of hypoxia-induced cell death.

[0154] Figure 8A Treatment of rMSCs with NRF2-activated compounds (triptolide, EGCG) (but essentially not HSF1 activators (gerdromycin, rhizocarpine)) for 1 hour showed that treatment protected rMSCs from oxidative stress-induced death. EGCG enhanced the protective effect induced by triptolide.

[0155] Figure 8BTreatment of H9c2 cardiomyocytes with triptolide in combination with 2HBA, tBHQ, or EGCG for 1 hour resulted in a synergistic increase in cell viability after oxidative activation (incubation in 1 mM H2O2 for 1 hour).

[0156] Figure 9 Pre-conditioning with medium derived from rMSCs (treated with NRF2 activators (Gaudenine, EGCG, tBHQ)) for 24 hours demonstrated protection of H9c2 cells from oxidative stress-induced cell death. Individual potent HSF1 activators (rhizocarpine, triptolide) showed no protective effect.

[0157] Figure 10 The expression of HSP70 mRNA in rMSCs was shown after 1 hour of treatment and 3 hours of clearance. EGCG and tBHQ produced a synergistic increase in triptolide-induced HSP70 expression.

[0158] Figure 11 The expression of HSP32(HO-1) mRNA in rMSCs was shown after 1 hour of treatment and 3 hours of clearance. EGCG and tBHQ produced a synergistic increase in triptolide-induced HSP32 expression.

[0159] Figure 12 The expression of HSP70 mRNA in MSCs (hMSCs) was shown after 1 hour of treatment and 3 hours of clearance. EGCG produced a synergistic increase in HSP70 expression induced by triptolide.

[0160] Figure 13 The expression of HSP32 (HO-1) mRNA in MSCs (hMSCs) was shown after 1 hour of treatment and 3 hours of clearance. EGCG produced a synergistic increase in HSP32 expression induced by triptolide.

[0161] Figure 14 The study showed FGF2 mRNA expression in rMSCs after 1 hour of treatment and 3 hours of clearance. EGCG and tBHQ produced a synergistic increase in triptolide-induced FGF2 expression. The combination of TBHQ and gadurin also increased FGF2 mRNA expression.

[0162] Figure 15 The expression of VEGFα mRNA in rMSCs was shown after 1 hour of treatment and 3 hours of clearance. EGCG produced a synergistic increase in triptolide-induced VEGF expression.

[0163] Figure 16 The expression of catalase (CAT) mRNA in rMSCs was shown after 1 hour of treatment and 3 hours of clearance. EGCG and tBHQ produced a synergistic increase in triptolide-induced CAT expression.

[0164] Figure 17 The expression of glutathione peroxidase (GPx) mRNA in rMSCs was shown after 1 hour of treatment and 3 hours of clearance. EGCG and tBHQ produced a synergistic increase in triptolide-induced GPx expression.

[0165] Figure 18 The expression of glutathione reductase (GR) mRNA in rMSCs was shown after 1 hour of treatment and 3 hours of clearance. EGCG produced a synergistic increase in GR expression induced by triptolide. tBHQ showed a synergistic effect only at low doses.

[0166] Figure 19 Superoxide dismutase 1 (SOD1) mRNA expression in rMSCs was shown after 1-hour treatment and 3-hour clearance. EGCG produced a synergistic increase in triptolide-induced SOD1 expression.

[0167] Figure 20 The expression of IL-1β mRNA in rMSCs was shown after 1 hour of treatment and 3 hours of clearance. Tripterygium wilfordii and gadurin downregulated IL-1β expression induced by EGCG and tBHQ.

[0168] Figure 21 The expression of TNFα mRNA in rMSCs was shown after 1 hour of treatment and 3 hours of clearance. EGCG and tBHQ synergistically enhanced the downregulation of TNFα induced by triptolide and gadunin.

[0169] Figure 22A The graph shows the results of rMSC activity and expression (checkmarks indicate activation level; x indicates level of inhibition; ≠ indicates no effect; blank indicates no experiment was performed).

[0170] Figure 22B A compilation diagram showing the paracrine activity of H9c2 cells (checkmarks indicate activation level; x indicates inhibition level; ≠ indicates no effect; blank indicates no experiment was performed).

[0171] Figure 23 The tested triptolide analogues are shown.

[0172] Figure 24 A list of potential adjuvants for testing is displayed.

[0173] Figure 25A This is a table showing the mRNA expression of selected genes in human mesenchymal stem cells (hMSCs) regulated by triptolide alone or in combination with selected adjuvants, as measured by real-time PCR. Results are expressed as fold changes relative to the adjuvant-treated cells.

[0174] Figure 25B This is a table showing the mRNA expression of selected genes in human mesenchymal stem cells (hMSCs) regulated alone or in combination with selected triptolide analogs, as measured by real-time PCR. Results are expressed as fold changes relative to the agent-treated cells.

[0175] Figures 26A to 26C The results show the mRNA expression of cellular protective genes in human mesenchymal stem cells (hMSCs) co-treated with triptolide or triptolide analogs and selected helper NRF2 activators, as measured by real-time PCR. Only treatment combinations that showed a ≥ 1.5-fold increase in VEGF, FGF2, HO1, and SDF1 gene expression compared to the causative-treated cells were retained. Results are expressed as fold changes relative to the causative-treated cells. (Any angiogenesis index: (VEGFa + FGF2 + SDF1)*2) (A = additive effect; S = synergistic effect).

[0176] Figure 26D The TaqMan group (Thermo Fisher Scientific) shows the effects of treating human mesenchymal stem cells (hMSCs) one hour with triptolide (1 uM) alone or in combination with 2 HBA (1 uM), followed by incubation for 24 hours under normoxic or hypoxic (< 1% O2) and low serum (0.2% FBS) conditions, as can be observed in infarcted microenvironments, on the remodeling of mRNA gene expression by antioxidants, growth factors, and matrix. Results are expressed as fold changes in hMSCs relative to the mediator treatment.

[0177] Figure 26E Real-time PCR expression of HO-1 (top panel) and VEGFa (bottom panel) gene mRNAs was shown after H9c2 cardiomyocytes were treated with triptolide (1 uM) alone or in combination with adjuvant treatments (1 uM HBA; 10 uM EGCG or 5 uM curcumin) for one hour, followed by incubation for 3 hours under normoxic or hypoxic (< 1% O2) and low serum (0.2% FBS) and low glucose conditions, as can be observed in infarct microenvironments. Results are expressed as fold changes relative to the agent-treated h9c2.

[0178] Figure 26FThe results show the VEGFa protein content measured by human mesenchymal stem cells (hMSCs) in the culture medium (Thermo Fisher Scientific; BioPlex 200, Bio Rad) after one hour of co-treatment with triptolide (1 uM) and a selected auxiliary compound (1 uM) followed by 48 hours of incubation under normoxic or hypoxic (< 1% O2) and low serum (1% FBS) conditions as can be observed in the infarcted microenvironment.

[0179] Figure 27A The study showed that pre-conditioning with triptolide for 1 hour before cryopreservation increased rMSC activity after thawing.

[0180] Figure 27B This study demonstrated that triptolide treatment in vivo modulates rMSCs in a dose-dependent manner to resist oxidative stress-induced death.

[0181] Figure 28 Images obtained from confocal microscopy (Olympus, FV1000MPE / BK61WF) show porcine arterioles conditioned overnight in HBSS medium without triptolide (A) or with triptolide (1 μM) (B). Vascular sections were stained using the LIIVE / DEAD kit. Arrows point to dead cells and degraded endothelial areas in the medium containing the mordant, while representative vascular images show that endothelial cell viability and integrity are maintained in the medium supplemented with triptolide.

[0182] Figure 29A and 29B It is a protein blot that shows protein expression in total cell extracts or nuclear and cytoplasmic fractions in different cell types after stimulation with triptolide (1 uM) for 5 to 120 minutes, followed by recovery for 0 to 24 hours.

[0183] Figure 29C This is a histogram representing the viability index of insulin-secreting INS-1 cells pre-regulated for 30 minutes with 0.25 μM or 0.50 μM triptolide or rhizocarpine after 6 hours of hypoxia stimulation. The results were compared with those of mediator-treated cells.

[0184] Figure 30 This is a graph showing that triptolide 1 mg / kg prevented a lethal drop in blood pressure in rats that received a dose of LPS of 10 mg / kg.

[0185] Figure 31These are images of Western blots showing that a single injection of triptolide (1 mg / kg) induced the expression of Hsp32 (HO-1) in rat kidneys within 60 minutes (see a). Triptolide increased the expression of Hsp70 in the unligated control kidneys of rats experiencing renal ischemia, indicating increased systemic sensitivity and cellular protective response (see b).

[0186] Figure 32 This is a table summarizing the effects of triptolide (triptolide 1c: purchased from a commercial source; triptolide 2 and 3 were used to produce synthetic analogs) and triptolide analogs on H9c2 cell viability during hypoxic stress, oxidative stress, and hypoxia / reoxidative stress.

[0187] Figure 33 According to A to 33H indicators, compared with DMSO treatment following warm systemic myocardial ischemia-reperfusion, triptolide (10) -7 mol / L, optimal dose) and analogue 1 (10 -8 The treatment (at a mol / L dose) protected the heart from I / R-induced systolic dysfunction, as shown by changes in A, B) + / - dP / dt, C) generated pressure (maximum-minimum pressure), D) end-diastolic pressure (EDP), and E) systolic index. F) coronary reserve flow (CRF) was preserved with treatment, G) high-sensitivity troponin T (TNT-hs) release, and H) the infarct area was significantly reduced by TTC staining in the triptolide and analogue treatment groups compared to hearts treated with I / R injury mediators (DMSO).

[0188] Figure 34 According to A to 34D, compared with animals treated with DMSO, triptolide (1 mg / Kg) and its analogue 1 (1 mg / Kg) protected cardiac function after I / R injury by preserving A) ejection fraction (EF), B) cardiac output (CO), C) shortened fraction (FS) and D) stroke volume (SV).

[0189] Figure 35 This is a table summarizing the effects of triptolide (triptolide 1c: purchased from a commercial source; triptolide 2 and 3 were used to produce synthetic analogs) and triptolide analogs on heat shock response (HSR) and antioxidant response (AR) as measured by luciferase reporter assay. EC is reported. 50 Maximum fold induction and activation at a fixed dose of 1 μM were assessed. Compounds were classified according to their efficacy and potency in stimulation of the HSR and AR pathways.

[0190] Figure 36This is a schematic diagram showing the proposed mechanism by which triptolide induces HSR and AR, leading to the upregulation of cell-protective genes. Summary of the Invention

[0192] In this application, the applicant demonstrates that triptolide can be used to protect cells and tissues from different types of damage and to increase the survival rate of cells as part of complex tissues (e.g., grafts and organs). For example, stem cells pre-regulated with triptolide alone or in combination with adjuvants protect cells from stress and / or damage and stimulate the secretion of paracrine mediators and growth factors that enhance the therapeutic properties of cells.

[0193] The applicant also identified triptolide analogs with similar or enhanced cytoprotective effects and identified several therapeutic combinations with triptolide and / or triptolide analogs that have synergistic or additive cytoprotective effects.

[0194] The mechanism by which triptolide induces HSR and antioxidant responses, leading to upregulation of protective HSPs, is as follows: Figure 36 As shown.

[0195] More specifically, the mechanism of action of triptolide includes 1) activating rapid and transient cellular defense mechanisms. More specifically, triptolide regulates the activity of KEAP1, a repressor of the transcription factor nuclear factor erythrocyte-associated factor 2 (NRF2), thereby allowing NRF2 to translocate to the nucleus and bind to AREs (antioxidant response elements), which activate the transcription of protective antioxidant mediators and enzymes, including HO1 (HSP32)

[0196] The mechanism of action of triptolide also includes 2) activation of cellular pathways to ensure prolonged protection and survival: triptolide antagonizes the essential co-chairman of HSP90, namely Cdc37, which leads to the dissociation of HSF1 from its molecular chairman repressor HSP90. This results in phosphorylation, trimerization, nuclear translocation, and binding to HSE (heat shock element) of HSF1, thereby inducing the re-transcription of cellular protective heat shock proteins (HSPs), including HSP27, HSP32, and HSP70.

[0197] The mechanism of action of triptolide can be further included in 3) induction / amplification of protective signal transduction: triptolide produced by stimulating ROS can activate the above mechanism, thereby leading to the activation of protective signal transduction pathways.

[0198] Unless otherwise indicated herein or clearly contradicted by the context, in the context of describing the invention (especially in the context of the claims), the terms “a” and “the”, and similar designations, shall be interpreted to include both the singular and the plural.

[0199] Unless otherwise stated, the terms “comprising,” “having,” “including,” and “containing” should be interpreted as open-ended terms (i.e., meaning “including but not limited to”).

[0200] The range of values ​​cited herein is intended only as a shorthand for individually referring to each individual value falling within that range, unless otherwise stated herein, and each individual value is incorporated into this specification as if it were individually cited herein. Subsets of all values ​​within the range are also incorporated into this specification as if individually cited herein.

[0201] Similarly, the general chemical structures of various substituents and groups listed for those substituents are intended as a concise method of referring individually to each molecule obtained by any combination of groups with any substituent. Each individual molecule is incorporated into the specification as if cited separately herein. Furthermore, all subsets of molecules within the general chemical structures and all structures / molecules belonging to the same compound family are also incorporated into the specification as if cited separately herein.

[0202] Any and all combinations and sub-combinations of the embodiments and features disclosed herein are included in this invention.

[0203] Here, the term "about" has its general meaning. The term "about" is used to indicate that the value includes inherent error variations in the means or methods used to determine the value, or contains a value close to said value, for example, within 10% or 5% of said value (or the range of values).

[0204] All methods described herein can be performed in any suitable order, unless otherwise stated herein or otherwise clearly contradicted by the context.

[0205] Any and all instances or exemplary language (e.g., "for example") provided herein are intended only to better illustrate the invention and not to limit the scope of the invention as otherwise claimed.

[0206] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0207] This invention provides a method for improving cellular resistance to cell death (e.g., resistance to oxidative and / or hypoxic stress-induced death; resistance to other physical, chemical, or mechanical stressors, including heat, oxidation, H2O2, hypoxia, encapsulation, etc.), said method comprising contacting cells with an effective amount of one or more compounds that activate the HSF1 and NRF2 pathways. This invention provides the use of one or more compounds that activate the HSF1 and NRF2 pathways for improving cellular resistance to cell death and improving cellular function.

[0208] The present invention also provides a method for improving the viability and retention of transplanted or infused cells, the method comprising contacting the cells with an effective amount of one or more compounds that activate the HSF1 and NRF2 pathways before and / or after transplantation or infusion.

[0209] The present invention also provides a method for improving the therapeutic characteristics and functionality of stem cells (activating beneficial proteins, including growth factors, antioxidant enzymes, heat shock proteins, chemokines and anti-inflammatory cytokines, for paracrine improvement of the transplantation microenvironment), said method comprising contacting stem cells with an effective amount of one or more compounds that activate the HSF1 and NRF2 pathways.

[0210] The present invention also provides a method for identifying one or more compounds that can be used to improve cell resistance to cell death (e.g., hypoxia and / or oxidative and / or hypoxia / reoxidative stress-induced cell death) and / or improve the viability and retention of transplanted or infused cells, the method comprising (i) contacting cells with the one or more compounds; and (ii) determining whether the HSF1 and NRF2 pathways are activated in the cells, wherein activation of the pathways indicates that the one or more compounds can be used to improve cell resistance to cell death and / or improve the viability and retention of transplanted or infused cells.

[0211] The present invention also provides a method for treating a subject requiring cell transplantation or infusion, the method comprising (a) contacting cells to be transplanted with an effective amount of one or more compounds that activate the HSF1 and NRF2 pathways; and (b) transplanting or infusing the cells of (a) into the subject.

[0212] The term "compound that activates the heat shock factor protein 1 (HSF1) pathway" refers to any agent (small molecule, peptide, protein, antibody, oligomer, etc.) that can directly or indirectly increase the release of HSF1 from the molecular chaperone repressor HSP90 complex and / or activate its translocation to the nucleus or increase its cellular content, thereby increasing HSF1-mediated transcription. It includes agents that antagonize one or more co-chaperones of HSP90 (e.g., Cdc37 and p23), leading to the dissociation / activation of HSF1 or the HSF1 protein itself.

[0213] The term "compound that activates the nuclear factor (erythrocyte-derived protein 2)-like 2 (NRF2) pathway" refers to any agent (small molecule, peptide, antibody, oligomer, etc.) that can directly or indirectly increase the release of NRF2 from the repressor Kelch-like ECH-associated protein 1 (KEAP1) and / or activate its translocation to the nucleus or increase its cellular content, thereby increasing NRF2-mediated transcription. KEAP1 contains six Kelch repeat sequences (residues 327-372, 373-423, 424-470, 471-517, 518-564, and 565-611), which mediate interaction with NRF2. Residues 69-84 of NRF2, and more specifically residues 76-84 (which contain a conserved ETGE motif), are involved in the interaction with KEAP1. Examples of compounds that activate the NRF2 pathway include triptolide, tBHQ, CDDO, caryophyllene, andrographolide, caffeol, sulforaphane, curcumin, EGCG, hydroxypyridinethione, resveratrol, guarjunin, quercetin, di(2-hydroxybenzylidene)acetone (2-HBA) or HBB2, 1,2-dithiacyclopenten-3-thione (D3T), acetylenic tricyclic bis(cyanone) (TBE31), anthothecol, solanine and its analogues, or alkaloids, quinones and quinone methylates, gambogeylic acid, limonenes, rotenones, terpenoids, furans, cathechins, alkenyl groups, carbohydrates, flavonoids, or aromatic members.

[0214] In one embodiment, the method includes using a compound that activates both the HSF1 and NRF2 pathways. In another embodiment, the method includes using two or more molecules that activate both the HSF1 and NRF2 pathways, such as a first compound that activates the HSF1 pathway and a second compound that activates the NRF2 pathway, or a first compound that activates both the HSF1 and NRF2 pathways and a second compound that activates only the NRF2 pathway, etc.

[0215] In one embodiment, the one or more compounds comprise triptolide or its analogues, solanine family compounds (e.g., solanine A, lycopene A) or their analogues, limonene family compounds (e.g., gadunin) or their analogues, curcuminoids (e.g., curcumin) or their analogues, sulforaphane or its analogues, sulphoxythiocarbamate alkyne (STCA) or its analogues, neomycin or its analogues, chlordamine or its analogues, gamendazole or its analogues, bardosorlon / CDDO compounds (e.g., CDDO-im) or their analogues, tert-butylhydroquinone (tBHQ) or its analogues, (1E,4E)-1,5-bis(2-hydroxyphenyl)-1,4-pentadien-3-one (HBB2) or its analogues, acetylene tricyclic bis(cyanone) (TBE-31) or its analogues, epigallocatechin gallate (EGCG) or its analogues, or any combination thereof. As used herein, the term "analyte" refers to a compound having the basic or skeletal structure of a reference compound but containing one or more modifications (e.g., bond sequence, absence or presence of one or more atoms and / or groups, and combinations thereof) that do not eliminate biological activity on the HSF1 and / or NRF2 pathways. For example, triptolide analogs (pentacyclic triterpenoids) are described in Klaic et al., ACS Chem Biol. [ACS Chemical Biology] 2012 May 18; 7(5):928-937 and PCT Publication No. WO2015 / 148802.

[0216] In one embodiment, at least one of the one or more compounds is an HSP90 inhibitor, an HSP90 N- or C-terminal inhibitor, preferably an HSP90 cofactor inhibitor.

[0217] In one embodiment, at least one of the one or more compounds has NRF2-inducing activity.

[0218] In one embodiment, a combination of compounds that have an enhancing (e.g., synergistic) activity relative to the activity of compounds used alone is used. Examples of such combinations include (i) (a) triptolide or an analogue thereof; and (b) EGCG or an analogue thereof; (ii) (a) triptolide or an analogue thereof; and (b) tBHQ or an analogue thereof; (iii) (a) gadunin or an analogue thereof; and (b) EGCG or an analogue thereof; (iv) (a) gadunin or an analogue thereof; and (b) tBHQ or an analogue thereof; (v) (a) triptolide or an analogue thereof; and (b) 2HBA or an analogue thereof; (vi) (a) triptolide or an analogue thereof; and (b) curcumin or an analogue thereof; or (vii) (a) triptolide or an analogue thereof; and (b) sarsaparilla or an analogue thereof.

[0219] As used herein, the term "adjuvant" refers to an agent that can increase cell survival, viability, or resistance to stress or damage. "Adjuvants" can modulate, for example, cell phenotype and may include antioxidants and NRF2 activators. Adjuvants include, for example, 2HBA, andrographolide, ascorbic acid, caffeol, caryophylloxol-badosol-imidazolium (CDDO-im), chalcone, N6-[2-[[4-(2,4-dichlorophenyl)-5-(1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]-3-nitro-2,6-pyridinediamine (CHIR98014), cytosolic acid, curcumin, cycloastragalool, 1,2-dithiacyclopenten-3-thione (D3T), damamod, edaravone, EGCG, gambogeylic acid, ganetespib, ganetene, IQ-1, limonene, lonidamide, melatonin, benzamide tetrahydroindole, N886, alkylaminobiphenylamide, neomycin, pyridoxal 5′-phosphate (P5′-P), hydroxypyridinethione, quercetin. , rhizocarpine, resveratrol, N-(2-cyano-3,12-dioxo-28-noroleanane-1,9(11)-dien-17-yl)-2,2-difluoro-propionamide or omaveloxolone (RTA-408), 4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)-1H-imidazolium (SB202190), 3-(2, (4-Dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione (SB216763), SNX-5422 (PF-04929113), sodium butyrate, sulforane, tetrabromobenzotriazole (TBB), tert-butylhydroquinone (tBHQ), valproic acid, solanine A, ergosterol, lupene ketone, and any analogues of such adjuvants.

[0220] As used herein, the term "secretory proteome" refers to the organic molecules and / or inorganic elements secreted by biological cells, tissues, organs, and organisms.

[0221] As used in this article, the term "regulation of cellular state" refers to changes in cell phenotype, expression patterns of certain genes, or secretion patterns of certain proteins.

[0222] As used herein, a straight-chain alkyl group having 1 to 6 carbon atoms (i.e., C1-C6 alkyl) refers to a saturated monovalent hydrocarbon group having a straight-chain or branched moiety and containing 1 to 6 carbon atoms. The term "branched alkyl group" refers to an alkyl group containing one or more tertiary or quaternary carbon atoms. Alkyl groups can be substituted (OH, NH2, I, F, Cl, Br, CN) or unsubstituted. Examples of such groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, and tert-butyl.

[0223] As used herein, the term “substituted” refers to a group in which one or more hydrogen atoms are independently replaced by a substituent selected from the following: methyl, ethyl, n-propyl, isopropyl, hydroxy, methoxy, ethoxy, fluorine, chlorine, bromine, iodine, cyano, nitro, amino, alkylamino, dialkylamino, carboxyl, chloromethyl, trichloromethyl, trifluoromethyl, methoxyethyl, etc.

[0224] As used herein, the term "lower alkyl group having 1 to 3 carbon atoms" refers to methyl, ethyl, and propyl.

[0225] In one embodiment, the one or more compounds are present in a pharmaceutical composition, which further comprises one or more pharmaceutically acceptable carriers, excipients, and / or diluents. As used herein, “pharmaceuticalally acceptable” (or “biologically acceptable”) means a material characterized by the absence (or limited presence) of toxicity or adverse biological effects in vivo. It refers to compounds, compositions, and / or dosage forms that, to the extent of proper medical judgment, are suitable for contact with the biological fluids and / or tissues and / or organs of a subject (e.g., human, animal) without excessive toxicity, irritation, allergic reactions, or other problems or complications, in proportion to a reasonable benefit / risk ratio.

[0226] The term “pharmaceutically acceptable carriers, excipients and / or diluents” refers to additives commonly used in the preparation of pharmaceutical compositions, and includes, for example, solvents, dispersion media, saline solutions, surfactants, solubilizers, lubricants, emulsifiers, coatings, antibacterial and antifungal agents, chelating agents, pH adjusters, soothing agents, buffers, reducing agents, antioxidants, isotonic agents, absorption delaying agents, etc. (see, for example, Rowe et al., Handbook of Pharmaceutical Excipients, Pharmaceutical Press; 6th edition, 2009).

[0227] In one embodiment, the one or more compounds may be combined / mixed with scaffold materials used for cell transplantation / tissue engineering (e.g., biomaterials, polymers and / or matrices commonly used as stem cell scaffolds).

[0228] One or more compounds can be formulated for administration via any conventional route, such as intravenous, oral, transdermal, intraperitoneal, subcutaneous, mucosal, intramuscular, intranasal, intrapulmonary, parenteral, or local administration. The preparation of such formulations is well known in the art (see, for example, Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins; 21st ed., 2005).

[0229] Furthermore, as illustrated in the following examples, culture media derived from cells treated with one or more compounds (e.g., secretory proteomes) can confer protection against cell death, and therefore, in one embodiment, the method described herein includes culturing cells (e.g., mesenchymal stem cells, CD133) in the presence of one or more compounds. + A population of cells (including pancreatic cells, kidney cells, epithelial cells, and endothelial cells) is obtained from the culture, and the culture medium or supernatant is collected from the culture; and the cells are contacted in the presence of the culture medium or supernatant.

[0230] In one embodiment, the cell is a stem / pluripotent / progenitor cell or a differentiated cell, such as hematopoietic stem cells (HSCs), hematopoietic progenitor cells (HPCs), pluripotent progenitor cells (MPPs), lymphoid progenitor cells, myeloid progenitor cells, mesenchymal stem cells (MSCs), adipose-derived stem cells (ADSCs), etc. In another embodiment, the cell is a differentiated cell.

[0231] The starting cell population can be obtained from an organism or an organ containing suitable cells. The collected cells can be obtained in a manner known to those skilled in the art (e.g., based on certain markers such as CD34).+ CD133 + The expression of MSCs can be enriched into cells with certain characteristics. Furthermore, the starting cell population can be used directly or frozen and stored for use at a later time point. Therefore, the cell population can be first enriched or purified, including negative and / or positive selection of cells based on adhesion to plastic containers based on specific cell markers, to provide a starting cell population, such as providing a starting cell population rich in MSCs. Methods for separating the starting cell population based on specific cell markers can use fluorescence activated cell sorting (FACS) technology or solid or insoluble substrates that bind antibodies or ligands that interact with specific cell surface markers. For example, cells can be contacted with a solid substrate containing antibodies (e.g., bead columns, flasks, magnetic particles) and any unbound cells can be removed. When using solid substrates containing magnetic or paramagnetic beads, cells bound to the beads can be easily separated using a magnetic separator (e.g., magnetic cell sorting devices manufactured by Miltenyi Biotec®, MACS®, CliniMacs® product line).

[0232] Cells can be cultured in media suitable for cell maintenance, growth, or proliferation, or under normal culture conditions in a bioreactor, for example, for large-scale production. The culture conditions for cell populations will vary depending on various factors, particularly the initial cell population. Suitable media and conditions are well known in the art. The method of the present invention can be carried out in natural, semi-synthetic, or synthetic media (in terms of composition), and can be a solid, semi-solid, or liquid media (in terms of form), as well as any nutrient medium or defined medium for cell culture, which may be supplemented with one or more suitable factors. Such media typically contain sodium, potassium, calcium, magnesium, phosphorus, chlorine, amino acids, vitamins, cytokines, hormones, antibiotics, serum, fatty acids, sugars, etc. Other chemical or biological components may be incorporated into the culture, alone or in combination, as needed. Such components incorporated into the culture medium can include fetal bovine serum, human serum, horse serum, insulin, transferrin, lactoferrin, cholesterol, ethanolamine, sodium selenite, thioglycerol, 2-mercaptoethanol, bovine serum albumin, sodium pyruvate, polyethylene glycol, various vitamins, various amino acids, agar, agarose, collagen, methylcellulose, various cytokines, and various growth factors. The culture medium can be a chemically defined, serum-free, and / or heterologous system.

[0233] Cells can be cultured under conditions suitable for maintenance, growth, and / or proliferation during or after treatment with one or more compounds.

[0234] The amount of one or more compounds used to mediate the above-described effects can be determined by a person skilled in the art. In one embodiment, the concentration is about 1 nM to about 1 mM, about 10 nM to 100 μM, or about 100 nM to about 10 μM. The invention also includes 10 -5 M to 10 -10 M (individually including concentrations from 1 μM to 10 μM and from 5 μM to 10 mM).

[0235] In one embodiment, the above contact includes adding one or more doses of one compound to the culture medium.

[0236] The compound can be administered in vivo to patients and cells, tissues, and organs, and can then be collected as described above.

[0237] The cell population can then be washed to remove one or more compounds and / or any other components from the cell culture and resuspended in a suitable cell suspension medium, washed off or kept in contact with a medium for short-term use or in a medium suitable for long-term storage (e.g., a medium suitable for cryopreservation).

[0238] Subjects who may benefit from transplanted / infused cells, and especially stem cells, include those with the following conditions: heart failure, angina, myocardial infarction (e.g., myocardial / myocardial ischemia), arrhythmia, valvular heart disease, myocardial / pericardial disease, congenital heart disease (e.g., atrial septal defect, ventricular septal defect, ductus arteriosus, tetralogy of Fallot), arterial disease (e.g., arteriosclerosis, aneurysm, etc.), venous disease (e.g., varicose veins), severe limb or organ ischemia (e.g., hepatic ischemia), degenerative joint disease, osteoarthritis, rheumatoid arthritis, and bone diseases (e.g., osteoporosis). Inflammation, osteoporosis, osteoarthritis, osteosarcoma), skin diseases (e.g., psoriasis, eczema, skin cancer), corneal diseases (e.g., keratoconus, keratitis), liver diseases (e.g., acute and chronic liver failure, hepatitis, genetic defects including urea cycle disorders), lung diseases (e.g., COPD, ARDS, pneumonia), kidney diseases (e.g., CKD), musculoskeletal injuries, tendinitis, systemic diseases (e.g., sepsis), cancer, disorders, degenerative diseases (including CNS diseases such as Alzheimer's disease, Parkinson's disease, dementia, ALS), and spinal cord injuries.

[0239] The above-described one or more compounds may be used in combination with other therapies / medications for treating the above-described diseases / disorders / conditions. The above-described one or more compounds may be administered in any conventional dosage form or co-administered (e.g., continuously, simultaneously, or at different times). In the context of this invention, co-administration refers to administering more than one therapeutic agent in a coordinated treatment process to achieve improved clinical outcomes. Such co-administration may also be co-extended, i.e., occurring within overlapping time periods. For example, the above-described one or more compounds may be administered to the subject before, simultaneously, before and after, or after the administration of another active agent or therapy. In one embodiment, the active agent (e.g., one or more compounds and another active agent) may be combined / formulated in a single composition and thus administered simultaneously.

[0240] Any suitable amount of one or more compounds or pharmaceutical compositions comprising them may be administered to a subject. The dosage and frequency of administration depend on many factors, including the manner of administration. For the prevention, treatment, or reduction of the severity of a given disease or condition, the appropriate dosage of one or more compounds / compositions will depend on the type of disease or condition to be treated, the severity and duration of the disease or condition, whether the compound / composition is administered for preventative or therapeutic purposes, prior treatment, the patient's clinical history and response to the compound / composition, and the discretion of the attending physician. The compound / composition is suitable for administration to the patient once or in a series of treatments. Preferably, it is desirable to determine the dose-response profile in an in vitro setting and in a useful animal model prior to testing in humans. The present invention provides dosages of compounds and compositions comprising them. For example, depending on the type and severity of the disease, from about 1 μg / kg to 1000 mg / kg (mg / kg body weight) per day. In addition, the effective dose can be approximately 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 1 mg / kg, 5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, 35 mg / kg, 40 mg / kg, 45 mg / kg, 50 mg / kg, 55 mg / kg, 60 mg / kg, 70 mg / kg, 75 mg / kg, 80 mg / kg, 90 mg / kg, 100 mg / kg, 125 mg / kg, 150 mg / kg, 175 mg / kg, or 200 mg / kg, and can be increased in 25 mg / kg increments up to 1000 mg / kg, or the range can be between any two of the foregoing values. Depending on the above factors, a typical daily dose range can be from approximately 1 μg / kg to 100 mg / kg or higher. For repeated administration over several days or longer, depending on the condition, treatment should continue until the desired symptom suppression or clinical endpoint is achieved. One or more of the above compounds can be administered at an appropriate frequency, such as once, once daily, twice weekly, once weekly, once every two weeks, or once monthly. However, other dosing regimens may also be useful. Progression of the therapy can be easily monitored using routine techniques and assays. These are merely guidelines, as the actual dose must be carefully selected and titrated by the attending physician based on each patient's unique clinical factors. The optimal dose will be determined using methods known in the art and will be influenced by factors such as patient age and other clinically relevant factors. Furthermore, the patient may be taking medications to treat other diseases or conditions.

[0241] Similarly, when administering pre-conditioned cells to a patient, the number of cells infused will take into account factors such as sex, age, weight, type of disease or condition, stage of condition, percentage of the cell population required, and the number of cells needed to produce a therapeutic benefit. In one specific embodiment, the composition is administered via intravenous infusion and contains at least about 1 × 10⁻⁶ cells. 4 Cells / kg or at least about 1 × 10⁻⁶ 5 cells / kg, for example from about 1 × 10 4 cells / kg to approximately 1 x 10 8 cells / kg or from approximately 1 × 10 4 One cell / kg to approximately 1 × 10⁻⁶ 7 Cells / kg

[0242] Subjects who may be treated using the methods described herein are mammals, including but not limited to cattle, sheep, goats, horses, dogs, cats, guinea pigs, rats, monkeys, or other bovine, sheep, equine, canine, feline, rodent, or rat species, primates (and preferably humans), male or female.

[0243] Other applications of the methods described in this article include, for example, the preservation of tissues, grafts (e.g., vascular grafts), and ex vivo organ perfusion (e.g., lung perfusion (EVLP)). Detailed Implementation

[0244] The invention will be further illustrated by the following non-limiting examples.

[0245] Example 1: HSP90 cochaperone inhibitors as cell regulators

[0246] To identify regulatory compounds that can ensure cell viability in vivo, based on preliminary evidence using triptolide, more than a dozen structurally similar compounds identified through literature and the Sigma-Aldrich® online structure search tool were screened. The ability of the compounds to improve human MSC viability was measured, including MSC stimulation under hypoxic stress. The screened molecules (most of which were natural compounds) were categorized into triterpenoids, limonenes, solanines, sterols, isoprene / diterpenes, and flavonoids. Classic HSP90 inhibitors, such as those directly targeting HSP90, were also included in the screening.

[0247] Roe SM. et al., J Med Chem [Journal of Medicinal Chemistry] 1999 Jan 28;42(2):260-6. (NT inhibitor) Root erythritol in the ATP-binding pocket at the NT region. Screening showed that the compound enhanced cell viability during hypoxic stress. Figure 1(A, B) and, as seen by reporter-based assays, their variable power in inducing HSP expression ( Figure 1 C). For some of the highest-scoring compounds with structural similarity to triptolide (including solanine and gadurin), emerging evidence suggests that many of these potent regulatory compounds belong to the HSP90 regulator family that targets the interaction of HSP90 co-chair molecules.

[0248] Patwardhan CA et al., J Biol Chem. [Journal of Medicinal Chemistry] 8 March 2013; 288(10):7313-25.

[0249] Sreeramulu S. et al., Angew Chem Int Ed Engl. [Applied Chemistry International Edition] 2009;48(32):5853-5.

[0250] Gu M. et al. Invest New Drugs. Feb 2014;32(1):68-74.

[0251] Example 2: Tripterygium wilfordii enhances the retention of transplanted cells in vivo and strengthens paracrine function.

[0252] The results described below show that triptolide increased the in vivo viability and retention of transplanted cells. In short, rat MSCs were treated with the regulatory compound in suspension for 60 minutes. The cells were then washed, labeled with fluorescent cell trackers, and injected into the ischemic left hindlimb of the rat at t = 0. The transplanted cells were imaged in vivo up to day 9 using an Optix™ MX3 molecular imaging system from ART (Figure 2: A, B). The results showed improved in vivo viability and retention of the pretreated stem cells, which translated into a trend toward improved revascularization, as observed by laser Doppler scanning. Figure 3 Proteomics analysis showed that triptolide treatment of human MSCs maintained and enhanced stem cell function, as evidenced by increased levels of heat shock proteins (HSP90a increased 7.0-fold; HSP90b increased 2.2-fold; HO1 increased 2.2-fold; HSP70 increased 1.8-fold), growth factors and cytokines (MCSF increased 24.4-fold, HGF increased 2.1-fold), and antioxidant response-related genes (GSH increased 2.8-fold, TRX increased 2.5-fold, CAT increased 1.7-fold) in the culture medium. Figure 4 ).

[0253] In summary, rat MSCs were incubated for one hour in low-serum medium (αMEM 1X, 1% fetal bovine serum (FBS), 1% penicillin-streptomycin (PS)) containing triptolide (10E-6M-10E-8M) or a mediator (dimethyl sulfoxide (DMSO)). The medium was then aspirated, and the cells were washed with three different media (αMEM 1X, 1% FBS, 1% PS). Cells were trypsinized and stained with Vybrant CFDA (Thermo Fisher Scientific) according to the manufacturer's protocol, which allows for detection in fluorescence imaging (Optix MX2). Cells were then counted using a Countess II FL automated cell counter (Thermo Fisher Scientific), and 3 million viable MSCs were injected into an ischemic rat hindlimb model at 5 different sites in a total volume of 200 μL using a 26G needle and syringe.

[0254] Sprague-Dawley rats (CD CRL: Charles River Laboratories) were anesthetized (isoflurane 2.5%–3.0% (Abbott Laboratories, Abbott Science Park, Illinois), 1 L / min oxygen) and bupivacaine was injected into the thigh at the incision site (2 mg / kg sc qd). The left common femoral artery was removed, and the distal portion of the saphenous artery and all its collateral branches and veins were excised. The proximal and distal portions of the artery between the inguinal ligament and the genu were excised. Thus, no collateral branches of the femoral artery could form. The right hind limb of each animal was left intact and used as a control. The wound was sealed with Vicryl 5-0. Animals were given buprenorphine hydrochloride (0.05 mg / kg sc bid 3 days) and placed in cages on diamond pads. The day after surgery, PBS or MSCs pretreated with the mediator or experimental compound were injected. The animals were anesthetized (isoflurane 2.5%-3.0% (Abbott Laboratories, Abbott Science Park, Illinois), 1 L / min oxygen) and the cells were injected directly into the quadriceps femoris muscle at 5 different points using a 26G needle and syringe.

[0255] Transplanted cells were imaged in vivo using the Optix™ MX3 molecular imaging system from ART until day 9. Simultaneously, hindlimb Doppler scans of rats (Moor instrument) were performed, which allowed for the study of blood flow restoration (below the knee) in a rat lower limb (n = 3) ischemia model following transplantation of regulated rMSCs.

[0256] Compared with catalyst-treated cells, triptolide-regulated cells increased the viability and retention of stem cells implanted in the ischemic hindlimbs of rats and improved the gradual recovery of blood flow in the affected limb (as shown in logarithmic pattern).

[0257] Example 3: Identification of stem cell drug optimization agents

[0258] Standards for identifying stem cell drug optimizers

[0259] The criteria used to identify stem cell drug optimizers rely on the ability of treatments to satisfy two main conditions:

[0260] 1. Cell treatment preferably confers increased in vivo viability and retention properties, particularly in the context of stem cell transplantation in hypoxic and / or oxidative microenvironments such as those observed in ischemic tissues; and

[0261] 2. Cell treatment preferably allows for the maintenance of normal cell phenotype and / or function as much as possible. However, desirable drug optimizers can also enhance cell function to contribute to beneficial or therapeutic phenotypes. In the case of stem cells involving paracrine activity and tissue repair, drug optimizers can preferably enhance the secretion of beneficial proteins and / or reduce the production of harmful proteins, thereby having a favorable balance and influence on the transplant environment.

[0262] Methods for testing candidate stem cell drug optimizers

[0263] To test the primary conditions, MSCs (derived from rats or humans) were treated with candidate drug optimizers, washed, and subjected to either hypoxia / serum starvation (48–72 hours in a hypoxic chamber < 1% O2 in a low-serum medium) or oxidative stress (1 hour incubation in a medium incorporating 0–2 mM H2O2), which mimic the primary lethal stressors present in the ischemic transplant microenvironment. Cell viability was assessed using the LIVE / DEAD Viability / Cytotoxicity Kit (Life Technologies™), and results were quantified using the Operetta High Content Screening (HCS) device equipped with Harmony automated analysis software (Perkin Elmer™).

[0264] To test the second primary condition, MSCs were treated with the candidate drug optimizer for 1 hour, followed by elimination for 3 hours. Cell mRNA was extracted and the expression of the target gene was quantified by real-time PCR. In a separate MSC culture, cells were treated for 1 hour, washed, and cultured in low-serum medium for 24 hours. The H9c2 cardiomyogenic cell line was then contacted with medium containing paracrine factors secreted by MSCs. The H9c2 cells were then subjected to hypoxia / serum starvation (48 hours in a hypoxic chamber <1% O2 in low-serum medium) or oxidative stress (1 hour incubation in medium incorporating 0-1 mM H2O2). The viability of H9c2 cells was assessed using the LIVE / DEAD assay as detailed above.

[0265] To identify the MSCs responsible for protective regulation and the mechanisms by which H9c2 is protected via paracrine mechanisms when incubated with regulated MSC culture media, various molecules were selected as regulators targeting two major cellular pathways: the heat shock pathway via HSP90 targeting (HSF1 activation) and / or the nuclear factor (erythrocyte-derived protein 2)-like 2 (NRF2) pathway.

[0266] The tested compounds ( Figure 5 ):

[0267] ● Tripterygium wilfordii: a potent HSF1 activator (HSP90-cofactor inhibitor); an NRF2 activator

[0268] ●Gaudurin: HSF1 activator (HSP90-cofactor inhibitor); NRF2 activator

[0269] ● Rhizocarpine: HSF1 activator (HSP90 classic ATP inhibitor); no NRF2 activity reported.

[0270] ●EGCG: No reports of HSF1 activation (HSP90-CT inhibition); NRF2 activator

[0271] ●tBHQ: No HSF1 activation reported; NRF2 activator

[0272] The results of these experiments, such as Figures 6 to 9 The findings reported can be summarized as follows:

[0273] ● Compounds that can induce HSF1 (triptolide, gadurin, and rhizocarpine) protect cells from hypoxia-induced death. Figure 6 );

[0274] ●Mediators of paracrine effects of HSP90 cofactor inhibitors (triptolide, gadurin) Figure 6 , 7 );

[0275] ●HSP90 NT inhibitors (rhizobacterin) are mediators that do not produce paracrine effects. Figure 7 );

[0276] ● The HSP90 CT inhibitor (EGCG) does not protect treated cells from hypoxia, but it has an additive effect on the protection induced by triptolide. Figure 6 );

[0277] ● NRF2 activators (EGCG, ganoderic acid, triptolide) protect treated cells from oxidative stress-induced cell death. EGCG has a cumulative effect on the protection induced by triptolide (Figure 8); and

[0278] ●In addition to triptolide, NRF2 activators (EGCG, tBHQ, gadurin) produce paracrine mediators that prevent oxidative stress-induced cell death. Figure 9 ).

[0279] In summary, these results demonstrate that the optimal pharmacological regulatory treatment for enhancing cell viability combines the activity of an HSP90 cofactor inhibitor capable of inducing HSF1 (resilience against hypoxia-induced death through direct and paracrine effects) with the activity of an antioxidant NRF2 pathway inducer (resilience against oxidative stress-induced death through direct and paracrine effects). EGCG was found to enhance the cytoprotective effect of triptolide stimulation.

[0280] In addition to enhanced vitality (the primary criterion for stem cell drug optimization), enhanced expression profiles were assessed by quantifying the mRNA expression of inflammation-related HSPs, growth factors (GFs), antioxidant proteins / enzymes, and cytokines (the secondary criterion for stem cell drug optimization). Single and combined treatments were tested.

[0281] Combined processing of tests - First experiment:

[0282] Tripterygium wilfordii (1 µM) or gadurin (1 µM) + EGCG (1 µM, 10 µM) or TBHQ (1 µM, 5 µM)

[0283] The results of these experiments, such as Figures 10 to 21 The findings reported can be summarized as follows:

[0284] HSP Expression

[0285] ●EGCG and tBHQ synergistically increase the expression of HSP70 and HSP32 induced by triptolide ( Figure 10 , 11 In two other rMSC cell lines, EGCG also produced a synergistic increase in triptolide-induced HSP70 expression, while tBHQ had no effect on triptolide-induced changes.

[0286] ●EGCG induces a synergistic dose-dependent increase in the expression of HSP70 and HSP32 induced by triptolide in human MSCs. Figure 12 , 13 );

[0287] Expression of growth factors (GF)

[0288] ●EGCG synergistically enhances the expression of triptolide-stimulated FGF2 and VEGF, while TBHQ increases and decreases the expression of triptolide and gadurin in FGF2 and VEGF, respectively. Figure 14 , 15 ).

[0289] Expression of antioxidants

[0290] ●EGCG synergistically enhances the expression of triptolide-stimulated CAT, GPx, GR, and SOD1. Figure 16 , 17 18, 19).

[0291] ●tBHQ synergistically enhances the expression of triptolide-stimulated CAT, GPx, and GR (with low dose tBHQ) and has no effect on SOD1 expression.

[0292] Expression of inflammatory cytokines

[0293] ● Tripterygium wilfordii and gadurin downregulate IL1β-induced expression via EGCG and tBHQ ( Figure 20 );

[0294] ●EGCG and tBHQ enhanced the downregulation of TNFα induced by triptolide and gadunin ( Figure 21 ).

[0295] In summary, these results indicate that EGCG synergistically enhances the expression of beneficial factors for stem cell function stimulated by triptolide. Without being bound by theory, this could be due to EGCG inhibiting unknown functional repressors of triptolide or stabilizing the HSP90 conformation to enhance the triptolide effect. A synergistic effect could also be at least partially secondary to the activation of NRF2 mediators and / or the normalization of cellular redox balance. Indeed, tBHQ, which lacks HSP90 inhibitory activity, showed some synergistic or additive effects on triptolide-stimulated expression in at least one MSC rat line. Finally, co-treatment with EGCG and tBHQ, along with triptolide or gadurin, further downregulated inflammatory cytokines.

[0296] The compilation of vitality and expression results in Figure 22A and 22B These results provide evidence that optimal pharmacological modulation of treatments, which provide a suitable transplant microenvironment, combines:

[0297] 1. HSP90 inhibitory effects (capable of inducing HSP90 cofactor inhibition of HSF1), which have been shown to enhance cell viability against hypoxia-induced death and promote increased expression of various beneficial paracrine factors (HSP, GF, antioxidant molecules) and decreased expression of inflammatory mediators; and

[0298] 2. NRF2 activity (with potential HSP90 CT inhibition) shows enhanced resistance to oxidative stress-induced death and enhances (additively or synergistically) the expression of certain beneficial paracrine factors and / or further downregulates inflammatory cytokine mediators.

[0299] Interestingly, the applicant found that when combined with triptolide to increase the viability of H9c2 cardiomyocytes stimulated by oxidative stress (incubation in 1 mM H2O2 for 1 hour), 2HBA, tBHQ, and EGCG also showed a synergistic increase. Figure 8B ).

[0300] Example 4: Identification of triptolide analogues as stem cell drug optimizers

[0301] Using a similar approach to that described in Example 3, the applicant tested several triptolide analogues, including Figure 23 The examples cited in the text illustrate this point, and several analogues that can act as stem cell drug optimizers were identified. Figure 24 , 25A 25B, 26A-26D).

[0302] The applicant also tested triptolide or triptolide analogues with potential adjuvants (e.g., NRF-2 activators and / or antioxidants). Figure 24 Several combinations of treatments were administered, and several effects on HSP, growth factors (GF), antioxidant proteins / enzymes, cytokines, and matrix remodeling proteins were identified. Figure 25A , 25B and Figures 26A to 26D The expression of HSP90 exhibits a combination of synergistic (S) and additive (A) effects. These data indicate that analogs 3, 1, dihydrotripterin, and tripterin are the best HSP90 inhibitors, and 2HBA, EGCG, curcumin, tBHQ, and sauropodophyllin are the best adjuvants for identification.

[0303] Furthermore, after normal culture for 48 hours, treatment of human mesenchymal stem cells (hMSCs) with triptolide combined with 2HBA, EGCG, tBHQ, or curcumin for 1 hour resulted in a synergistic increase in VEGF protein expression. In contrast, the same cells cultured for 48 hours under hypoxic conditions encountered in an infarct microenvironment produced even higher VEGF protein expression. Figure 26F Essentially the same observations were observed, namely, increased mRNA expression of HO-1 antioxidant and VEGFa angiogenic factor in H9c2 cardiomyocytes treated with triptolide combined with 2HBA and EGCG adjuvants for 1 hour, followed by clearance under normoxic or hypoxic conditions for 3 hours. Figure 26E ).

[0304] Example 5: Tripterygium wilfordii increases the activity of cryopreserved rMSCs

[0305] Proper cryopreservation is an important aspect of cell processing laboratories, requiring them to demonstrate that their cryopreservation protocols produce acceptable post-thaw viability (≥ 70%) prior to transplantation. For example, studies have shown that cryopreservation induces significant alterations in thawed hepatocytes and impairs their viability, attachment, and function. Figure 27A The results presented showed that pre-conditioning rMSCs with triptolide for one hour before cryopreservation increased the activity of thawed rMSCs.

[0306] Mesenchymal stem cells (MSCs) were isolated from the hind limb bone marrow of male rats (175–200 g) and expanded as described. Briefly, bone marrow mononuclear cells (BMNCs) were isolated by Ficoll-Paque gradient centrifugation (Amersham) and cultured in Minimum Essential Medium α 1X (αMEM 1X: Gibco 12571) containing 10% FBS (Gibco) and 1% penicillin-streptomycin (PS: Invitrogen 15140). After 48 hours, non-adherent cells were discarded, and the cells were washed with fresh medium. MSCs were separated from hematopoietic cells based on their preferential attachment to polystyrene surfaces. The multi-lineage potential of MSCs was confirmed by in vitro adipogenesis and osteogenic / chondrogenic differentiation assays under specific culture conditions and staining. Immunophenotyping was performed using multiparameter flow cytometry (FACScan). ® (Becton Dickinson, Mountain View, California, USA) used monoclonal antibodies against surface antigens such as CD29, CD34, CD45, CD90, and CD105 (Coulter Immunology, Hyeria, Florida, USA). For in vitro experiments, MSCs will be used between passage 4 and passage 10.

[0307] Cell viability protocols (freeze / thaw cycles)

[0308] MSCs were resuspended in 10% serum medium (αMEM 1X, 10% FBS, 1% PS) and seeded at a density of 4000 cells per well in 96-well plates using multichannel pipettes, and incubated at 37ºC. Triples were prepared for each experimental and control condition. The next day, the medium was gently aspirated and replaced with low-serum medium (αMEM 1X, 1% FBS, 1% PS) containing triptolide (10E-6M) or DMSO for one hour. The medium was then aspirated, and the cells were washed with three different media (αMEM 1X, 1% FBS, 1% PS). Cells were digested with trypsin and counted using a Countess II FL automated cell counter (Thermo Fisher Scientific).

[0309] Cell freezing: After trypsin digestion and counting steps as described above, aliquot the cells at the desired density, add DMSO (1:10 final dilution) to the cell concentrate and transfer to pre-labeled cryovials. Place the cryovials in a freezing container and incubate at -80ºC overnight, and then incubate at -150ºC.

[0310] Thawing cells: Frozen cells were thawed by pouring pre-warmed culture medium onto the frozen aliquots. The vials were centrifuged (200 × g; 3 min), the supernatant was aspirated, and the cells were resuspended in pre-warmed 10% serum medium (αMEM 1X, 10% FBS, 1% PS). Trypan blue dilution was added to the cell aliquots, and cell viability was measured using a Countess II FL automated cell counter (Thermo Fisher Scientific).

[0311] Example 6: Tripterygium wilfordii increases the body's resistance to oxidative stress-induced death.

[0312] Figure 27B The results presented showed that intraperitoneal injection of triptolide (1 mg / kg; 12 hours apart) in one or two intraperitoneal injections regulated bone marrow cells in Sprague-Dürer rats in a dose-dependent manner to resist oxidative stress-induced death (details below).

[0313] As described in Example 5, mesenchymal stem cells (MSCs) are isolated.

[0314] Internal regulation

[0315] Sprague-Dürer rats (CD CRL; Charles River Laboratory) were administered triptolide (1 mg / kg) or a carboxin via intraperitoneal injection once or twice at 12-hour intervals. The rats were then sacrificed, and MSCs were isolated as described above. MSCs were cultured by resuspending them in 10% serum medium (αMEM 1X, 10% FBS, 1% PS) and seeded at a density of 4000 cells per well in 96-well plates using multichannel pipettes, and incubated at 37ºC. Triples were prepared for each experimental and control condition.

[0316] Cells were then challenged by incubation for 60 minutes with 1% serum containing 0 mM, 0.5 mM, 0.75 mM, or 1 mM hydrogen peroxide (ACP Chemicals, H7000). Cells were then gently washed twice with warm αMEM 1X medium stained with the LIVE / DEAD kit (Thermo Fisher Scientific) according to the manufacturer's protocol. Images were captured and analyzed using the Operetta High Content Screening (HCS) system, running Harmony High Content Imaging and Analysis Software version 4.1 (Perkin Elmer, Waltham, MA). The results presented in this paper demonstrate that in vivo treatment with triptolide in Sprague-Dürer rats dose-dependently modulates bone marrow cells to resist oxidative stress-induced death.

[0317] Example 7: Tripterygium wilfordii preserves endothelial cell viability

[0318] Porcine carotid arteries were harvested, cut into closed loops and semi-circular open loops, and incubated overnight in Hank's Balanced Salt Solution (HBSS) or HBSS containing triptolide at a final concentration of 10E-6M. The carotid artery loops were stained using the LIVE / DEAD kit (Thermo Fisher Scientific) according to the manufacturer's protocol. The tissues were imaged using a confocal microscope (Olympus, FV1000MPE / BK61WF) with a 20X immersion objective. Z-stacking of the carotid artery endothelial cell surface was also obtained. Figure 28 The results show that, compared to the negative control without triptolide (where arrows point to dead cells and loss of endothelial integrity), the group with triptolide maintained endothelial cell viability and integrity. Figure 28 ).

[0319] Example 8: Tripterygium wilfordii-induced cell protection mediators

[0320] Tripterygium wilfordii similarly induces cell-protective mediators (survival kinases: pAkt / Akt, pERK / ERK; antioxidant HO1; heat shock response proteins: HSF1, HSP70) and protein expression kinetics (for 10) in various cell types. -6 The testing time for M doses was 5 to 120 minutes, and the treatment was performed with 10 -6 Treatment with M triptolide for 1 hour followed by recovery (0 to 24 hours). Figure 29A and 29B This indicates Western blotting and activity assays performed in various rat and human cell lines (H9c2 rat cardiomyocytes; human and rat MSCs; rat neonatal cardiomyocytes; and INS-1 insulin-producing rat β-cell lines).

[0321] More specifically, INS-1 cells were cultured in complete medium containing 10% FBS (supplemented with RPMI containing the following filtered solutions: 1 mM sodium pyruvate + 50 μM β-mercaptoethanol, 10 mM Hepes Ultra, 2 mM L-glutamine). Cells were digested with trypsin (0.25% trypsin-EDTA), centrifuged at 1500 RPM for 5 min, and resuspended in complete INS medium supplemented with 1% FBS. Cells were counted using a hemocytometer, and 1–2 million cells were resuspended in Sarstedt tubes containing 9 ml of 1% FBS complete INS medium. 2.5 μL or 5.0 μL of a 1 mM Hsp90 inhibitor (triptolide: Cayman Chemicals 70950, gerdemycin: Cayman Chemicals 13355, or rhizobacterin: Cayman Chemicals 13089) was resuspended in DMSO. Control samples were prepared similarly, but instead of the Hsp90 inhibitor, 2.5 μL or 5.0 μL of DMSO medium was used. The cell suspension was filled to a final volume of 10 mL with 1% FBS complete INS medium. The suspension was gently mixed by inverting the tube several times, and the tubes were incubated at 37ºC for 30 minutes. The inversion was repeated after 15 minutes. The tubes were then centrifuged at 1500 RPM for 5 minutes, the medium was gently aspirated, and the cells were washed with 12 mL of warm RPMI solution. The rotation and washing cycle was repeated twice more, and the cells were resuspended in 2.5 mL or 5.0 mL of 1% FBS complete INS medium to obtain pellets containing 1 or 2 million cells, respectively. Next, using multichannel pipettes, 100 μL of cell suspension aliquots (containing 40,000 cells / ampoule) were plated into 96-well plates. Each experimental and control condition was plated four times and incubated for 6 hours under normoxic or hypoxic conditions. Hypoxia (< 1% oxygen) was achieved by placing the culture plates in an airtight hypoxic chamber (Billups-Rothenberg) and flushing them with a 5% CO2 gas mixture balanced with 95% N2 at a flow rate of 15–20 L / min for 10 minutes. After 6 hours of incubation, the medium from the hypoxia-induced and normoxic control cultures was replaced with 90 μL of 10% FBS complete INS medium, and 10 μL of PrestoBlue reagent was added to each well. After 60 minutes of incubation, the activity was quantified by fluorescence acquisition using a microplate reader.

[0322] These data indicate that transient pre-regulation of INS-1 cells with triptolide when subjected to lethal hypoxic stress can protect cell viability. Figure 29C ).

[0323] Example 9: Tripterygium wilfordii rescues rats from LPS-induced lethal hypotension

[0324] In addition to the effects of triptolide on reducing infarct size and maintaining cardiac function in rats, the applicant also observed blood pressure regulation in treated rats. Given the potential of the compounds and combinations described herein to preserve organs and tissues from damage (i.e., ischemic, oxidative, inflammatory, and necrotic damage) and to preserve systemic pressure / perfusion, their application in shock models (i.e., purulent, cardiogenic models) would be desirable, considering the high levels of organ failure and mortality associated with these conditions. Indeed, in the United States, more than 750,000 cases of severe sepsis are diagnosed annually with a mortality rate of 25%–30% (CritCare Med 29(7): 1303Y1310, 2001), caused by multi-organ failure (including heart failure) as a key manifestation (Circulation 116(7): 793Y802, 2007). We propose adding a septic shock model (an endotoxin model induced by bacterial lipopolysaccharide (LPS)) to rats (Life Sci., 1997; 60 (15): 1223-30).

[0325] In short, SD rats were anesthetized with 2.5%–3.0% isoflurane (Abbott Laboratories, Abbott Science Park, Illinois), 1 L / min oxygen, and placed on a heating blanket to prevent hypothermia. The left external carotid artery was cannulated with a gelco #20 and connected to a pressure sensor. Alternatively, the left femoral artery was cannulated, depending on size. The rats were kept in 2% isoflurane, 1 L / min oxygen, and baseline pressure measurements were collected. The barokinetic response to intravenous injection of LPS via the jugular vein (20–50 mg / kg) and intraperitoneal injection of triptolide (1 mg / kg) was determined. It is noteworthy that the dose of LPS in the animals can vary and also depends on the age of the animals (Infection and Immunity, March 1996, Vol. 64, No. 3, p. 769). In our experiments, both rats received a first high-dose bolus of LPS at 10 mg / kg. After a brief drop in blood pressure (BP) in both rats, BP stabilized and returned to baseline. Then, different rats were administered a large-dose bolus injection of either 300 μL triptolide (1 mg / kg) or a carrier (10% DMSO, 70% hydrogenated castor oil EL / ethanol (3:1), 20% PBS) intraperitoneally (see arrow). Figure 30 In two rats, prior to re-injection of a high-dose bolus of 10 mg / kg LPS, a lethal drop in blood pressure was observed in carcass-treated rats, while blood pressure was maintained in rats receiving a single injection of triptolide. Figure 30 ).

[0326] Example 10: Tripterygium wilfordii-induced expression of Hsp32(HO-1) in rat kidneys

[0327] The applicant previously demonstrated that triptolide promotes cardiomyocyte survival, reduces damage and adverse remodeling, while preserving cardiac function in ischemic myocardium of rats. The applicant tested whether this protective effect could be observed in other types of ischemic diseases.

[0328] Rats were anesthetized with 2.0%–3.0% isoflurane in 1 L / min oxygen (Abbott Laboratories) and placed in a supine position on a heating pad. Rats received a single large-dose injection via the external jugular vein at a dose of 1 mg / kg of either a mediator solution or triptolide (Cayman Chemicals 70950) 50 mM stock solution resuspended in a 0.2 μM filter sterile mediator: DMSO (Sigma 154938) (4% total volume), PBS 1X (96% total volume)). Rats were shaved, ophthalmic ointment was applied to the cornea, and bupivacaine (2 mg / kg s.c.) was injected through a 3–4 cm incision site beginning at the base of the sternum to the umbilicus. The incision was kept open with a retractor, and the intestine was wrapped with sterile saline-dampened gauze. The left kidney (K) was dissected. L The renal artery was closed with a vascular clamp. The intestine was replaced in the abdominal cavity, and the skin was sutured with temporary 3-0 sutures (Ethicon). Rats were given buprenorphine hydrochloride (0.05 mg / kg sc) and maintained in ischemia for 30 minutes with 1.0%–2.0% isoflurane (Abbott Laboratories) at 1 L / min oxygen, followed by reperfusion for another 45 minutes after clamp removal. Blood was drawn from rats and organs were collected by perfusion with saline supplemented with 40 mM KCl, rinsed in cold phosphate-buffered saline (PBS 1X), and stored overnight in 10% formalin buffered with PBS for paraffin embedding for histological / immunohistoscopic sections or rapidly frozen in liquid nitrogen for Western blot expression analysis.

[0329] The results of Western blot analysis presented in this article demonstrate that a single injection of triptolide (1 mg / kg) induces the expression of Hsp32 (HO-1) in the kidneys of rats within 60 minutes. Figure 31 Furthermore, triptolide increased the number of unligated control kidneys (K+) in rats that had experienced renal ischemia. R The expression of Hsp70 in the cells indicates increased sensitivity and cellular protective responses. This phenomenon is particularly interesting because it suggests the possibility of conferring systemic cellular protection during clinical interventions, similar to remote modulation, potentially generating systemic neuro-humoral protective mediators that could reduce potential associated complications (such as stroke induced by surgery).

[0330] Example 11: Effects of triptolide and triptolide analogues on cell viability and protection against stress and damage

[0331] The applicant tested the effects of triptolide or triptolide analogues on myoblasts of H9c2 rat cardiomyocytes and hypoxic cultures in a rat model of myocardial infarction. Combinations of triptolide or triptolide analogues with adjuvants can be tested in a similar manner.

[0332] In vitro studies

[0333] Cell culture and stimulation:

[0334] For survival against hypoxia and oxidative stress, H9c2 cardiomyocytes were subjected to hypoxia / serum starvation (48 hours in a hypoxic chamber < 1% O2 in a low-serum medium) or oxidative stress (1 hour incubation in a medium incorporating 0–1 mM H2O2). The viability of H9c2 cells was assessed using the LIVE / DEAD assay.

[0335] Next, hypoxia / reoxidation stimulation was performed. Briefly, as previously reported, viability analysis was performed in rat H9c2 cardiomyocytes. For hypoxia / reoxidation stress, cells were cultured in glucose-free DMEM (Life Technologies) and serum-starved and placed under hypoxic conditions (< 1% O2) for 18 hours. Under reoxidation (noroxic conditions), cells were subjected to triptolide (10... -10 Up to 10 -6 mol / L, Cayman Chemicals, Ann Arbor, Michigan), triptolide analogue or catalyst (dimethyl sulfoxide (DMSO), Sigma-Aldrich, Oakville, Ontario; final concentration <1% v / v) was treated in DMEM high glucose 1% FBS for 1 hour, followed by further oxidation in DMEM high glucose for 5 hours.

[0336] Figure 32 The results presented show that triptolide (1 uM) (triptolide 1c: purchased from a commercial source; triptolide 2 and 3 were used to produce synthetic analogs) and triptolide analogs 3 (1 uM), 1 (1 uM), 2 (1 uM) and 4 (1 uM) effectively protect H9c2 cardiomyocytes from hypoxia and hypoxia / reoxidative stress.

[0337] Example 12: Tripterygium wilfordii and its analogues are used to treat ischemic diseases.

[0338] Lewis rats (250–300 g, Charles River Laboratories, St. Constant, Quebec, QC) were used for all in vitro and in vivo experiments. All animals were handled in accordance with laboratory animal care and usage guidelines.

[0339] In vitro studies

[0340] Preparation of isolated perfused heart:

[0341] Rats were randomly assigned to the following groups: propagator (n = 6), triptolide or triptolide analogue 10 -8 10 -7 Or 10 -6 mol / L (n = 5 per group). Under isoflurane anesthesia, rats were injected with heparin (IP, 1000 IU, Novartis, Dorval, Quebec (QC)) and their hearts were harvested and immediately immersed in ice-cold Krebs buffer (in mmol / L: NaCl 113, KCl 4.5, NaH2PO4 1.6, CaCl2 1.25, MgCl2+6H2O 1, D-glucose 5.5, NaHCO3 25). The hearts were retrogradely perfused using a Langendorff system (Radnoti, Monrovia, CA) at a constant aortic pressure of 60–70 mmHg, using Krebs buffer (37ºC) and bubbling with 5% CO2 equilibrated O2. A latex balloon connected to a pressure sensor was inserted into the left ventricle (LV) and adjusted to 15 mmHg (LV preload). Set your heart rate to 300 bpm and allow it to stabilize for 20 minutes.

[0342] Indoor pressure was continuously measured using a Power Lab 8 / 30 multi-wave recorder (AD Instruments, Colorado Springs, Colorado), and recorded and analyzed using LabChart pro v.7.3.7 (AD Instruments).

[0343] To ensure contact between triptolide, triptolide analogues, or mediators (DMSO) and the heart at the start of reperfusion, the system is initiated during the induction of warm systemic ischemia, achieved by stopping cardiac pacing and perfusion for 30 minutes. Krebs buffer and triptolide (10... -8 10 -7 Or 10 -6 Reperfusion was initiated with a solution of mol / L or a medium for 10 minutes, followed by continued reperfusion (Krebs buffer) for a total duration of 120 minutes.

[0344] At the end of stabilization, cardiac effluent was collected for 5 minutes after reperfusion, and then every 15 minutes thereafter for a total of 60 minutes. The volume was measured and the sample was held at -80ºC until analysis.

[0345] At the end of reperfusion, transverse sections of the heart (1–2 mm) were prepared and stained for 20 minutes at 37°C with 5% 2,3,5-triphenyltetrazol chloride in phosphate-buffered saline (TTC, Sigma-Aldrich, Canada) at pH 7.4. 15 Sections were weighed and then imaged using a Stemi 508 stereomicroscope coupled to an AxioCam ERc 5s camera, and processed using Zen 2.3 imaging software (Carl Zeiss, Toronto, Ontario, Canada). Analysis was performed using ImageJ 1.51h free software (NIH, Bethesda, Maryland). Infarct area was normalized to the weight of the cardiac tissue section. One section / heart was rapidly frozen for gene and protein expression.

[0346] Figure 33 The results shown in A to 33H indicate that, compared with DMSO treatment following warm systemic myocardial ischemia-reperfusion, triptolide (10... -7 mol / L, optimal dose) and analogue 1 (10 -8 The treatment (at a mol / L dose) protected the heart from I / R-induced systolic dysfunction, as shown by changes in A, B) + / - dP / dt, C) generated pressure (maximum-minimum pressure), D) end-diastolic pressure (EDP), and E) systolic index. F) coronary reserve flow (CRF) was preserved with treatment, G) high-sensitivity troponin T (TNT-hs) release, and H) the infarct area was significantly reduced by TTC staining in the triptolide and analogue treatment groups compared to hearts treated with I / R injury mediators (DMSO).

[0347] In vivo studies

[0348] Rats were randomly assigned to the following groups: sham-operated group (n = 6), catalyst group (n = 8), triptolide 1 mg / kg (n = 6), or triptolide analogue. Baseline echocardiography was performed as described using a Sonos 5500 imaging system (Philips, Philips Healthcare, Andover, Massachusetts, USA) with a 12 MHz transducer under 2% isoflurane anesthesia. 12All measurements were taken by the same experienced observer who was unaware of the treatment. For each measurement, three to five cardiac cycles were analyzed and their average was calculated.

[0349] Following echocardiography, the animal was intubated and mechanically ventilated, then bupivacaine 2 mg / kg was injected, and a left thoracotomy was performed to expose the heart. The left anterior descending coronary artery was closed using a 5-0 filamentous slipknot. Visual whitening and ECG changes confirmed myocardial ischemia. In sham-operated animals, the sutures were not ligated. After 30 minutes, the sutures were loosened and closed. Tripterygium wilfordii, a triptolide analogue, or a thoracolytic was administered intraventricularly for acute systemic delivery, followed by thoracotomy. Buprenorphine (0.05 mg / kg sc) and carbofenone (5 mg / kg sc) were administered at the end of the procedure. The animal was allowed to recover for 24 hours, followed by a second echocardiogram. The animal was euthanized, the heart tissue was rapidly frozen, and blood was collected in heparinized tubes and centrifuged at 4ºC. Plasma was collected, rapidly frozen, and maintained at -80ºC until analysis.

[0350] Figure 34 The results indicated that, compared with animals treated with DMSO, triptolide (1 mg / Kg) and its analogue 1 (1 mg / Kg) protected cardiac function after I / R injury by preserving A) ejection fraction (EF), B) cardiac output (CO), C) fractional shortening (FS), and D) stroke volume (SV). These in vivo protective effects of triptolide were associated with a significant increase in tissue expression of cardioprotective HSP70 and HO-1.

[0351] Statistical analysis

[0352] Data are presented as mean ± standard error or median, with a confidence interval of 95%. ANOVA was used for group comparisons of non-repeated measures. For repeated measures, a linear mixed-effects model was used to compare groups (MIXED program in SAS software, version 9.3; SAS Institute, Cary, NC, USA). Differences between groups were assessed. For non-normally distributed measurements, such as indices and ratios, a logarithmic transformation of the measurements was used. For hemodynamic measurements, up to 200 measurements / rat / time points were used in the model, with each individual measurement weighted accordingly (i.e., 1 / 200). For all analyses, P < 0.05 was considered statistically significant.

[0353] Example 13: Tripterygium wilfordii and its analogues regulate gene expression under the control of HSR and ARE elements.

[0354] Following the manufacturer's protocol, H9c2 rat cardiomyocytes were seeded at a density of 5,000 cells per well in 96-well plates containing DMEM 10% FBS complete medium and transfected using a Liposome-based Cignal Reporter Assay Kit for Heat Shock and Antioxidant Responses (SABiosciences, Qiagen). The following day, tripterygin, its analogues, and various other compounds were added triplicate to wells containing DMEM 1% FBS medium for 4 hours at doses ranging from 10E-5 to 10E-10M. The complete medium was then removed for 3 hours, after which signal transduction activity was measured using a dual-luciferase assay (Promega). Figure 35 The results summarized in the study showed that triptolide (triptolide 1c: purchased from a commercial source; triptolide 2 and 3 were used to produce synthetic analogs) and triptolide analogs 1, 3 and 4 belong to the most potent and effective test compounds for stimulating the expression of reporter genes partially controlled by health shock response elements (HSR) or antioxidant response elements (ARE).

[0355] Although the invention has been described above by way of specific embodiments, modifications may be made thereto without departing from the spirit and essence of the invention as defined in the appended claims. In the claims, the word “comprising” is used as an open-ended term, essentially equivalent to the phrase “including but not limited to.” Unless otherwise expressly indicated by the context, the singular forms “a / an” and “the” include the corresponding plural indicators.

[0356] project

[0357] Item 1. A composition or pharmaceutical composition comprising one or more compounds that activate heat shock response and / or antioxidant response.

[0358] Item 2. A composition or pharmaceutical composition comprising a) one or more compounds of formula I, a pharmaceutically acceptable salt thereof, a stereoisomer, a tautomer, or a prodrug; b) one or more adjuvants; and c) a carrier or a pharmaceutically acceptable carrier.

[0359]

[0360] R1 is selected from the following groups: -H, -F, -Cl, -Br, -I, -CN, aryl, alkyl, imidazole, -ORa, -NRbRc, -(CH2). n OH and -(CH2) n NH2;

[0361] Ra is selected from the group consisting of: H, substituted or unsubstituted straight-chain alkyl groups having 1 to 6 carbon atoms, substituted or unsubstituted branched alkyl groups having 3 to 6 carbon atoms, and protecting groups;

[0362] Rb and Rc are independently selected from the group consisting of: -H, -OH, -OCH3, substituted or unsubstituted straight-chain alkyl groups having 1 to 6 carbon atoms, substituted or unsubstituted branched alkyl groups having 3 to 6 carbon atoms, and protecting groups.

[0363] Where n is 0, 1, 2, 3 or 4;

[0364] R2 and R3 are independently selected from the following groups: -H, -ORd, =O, -C(=O)OH, -C(=O)ORx, and -C(=O)Rx;

[0365] Where Rd is H or a lower alkyl group having 1 to 3 carbon atoms;

[0366] Wherein Rx is H or a lower alkyl group having 1 to 3 carbon atoms;

[0367] R4 is selected from the following groups: -H, -OH and lower alkyl groups having 1 to 3 carbon atoms.

[0368] Item 3. A composition or pharmaceutical composition as described in Item 2, wherein the compound comprises formula Ia

[0369]

[0370] R1 is selected from the following groups: -H, -F, -Cl, -Br, -I, -CN, aryl, alkyl, imidazole, -ORa, -NRbRc, -(CH2). n OH and -(CH2) n NH2;

[0371] Ra is selected from the group consisting of: H, substituted or unsubstituted straight-chain alkyl groups having 1 to 6 carbon atoms, substituted or unsubstituted branched alkyl groups having 3 to 6 carbon atoms, and protecting groups;

[0372] Rb and Rc are independently selected from the group consisting of: -H, -OH, -OCH3, substituted or unsubstituted straight-chain alkyl groups having 1 to 6 carbon atoms, substituted or unsubstituted branched alkyl groups having 3 to 6 carbon atoms, and protecting groups.

[0373] Where n is 0, 1, 2, 3 or 4;

[0374] R2 and R3 are independently selected from the following groups: -H, -ORd, =O, -C(=O)OH, -C(=O)ORx, and -C(=O)Rx;

[0375] Where Rd is H or a lower alkyl group having 1 to 3 carbon atoms;

[0376] Wherein Rx is H or a lower alkyl group having 1 to 3 carbon atoms;

[0377] R4 is selected from the following groups: -H, -OH and lower alkyl groups having 1 to 3 carbon atoms.

[0378] Item 4. The composition or pharmaceutical composition as described in Item 2 or 3, wherein R1 is selected from the group consisting of -ORa and -NRbRc; wherein Ra is selected from the group consisting of H, a substituted or unsubstituted straight-chain alkyl group having 1 to 6 carbon atoms, a substituted or unsubstituted branched alkyl group having 3 to 6 carbon atoms, and a protecting group; wherein Rb and Rc are independently selected from the group consisting of -H, -OH, -OCH3, a substituted or unsubstituted straight-chain alkyl group having 1 to 6 carbon atoms, a substituted or unsubstituted branched alkyl group having 3 to 6 carbon atoms, and a protecting group; wherein R2 and R3 are independently selected from the group consisting of -H, -ORd, and =O, where Rd is H or a lower alkyl group having 1 to 3 carbon atoms; and / or wherein R4 is -H or CH3.

[0379] Item 5. The composition or pharmaceutical composition of any one of Items 1 to 4, wherein R1 is selected from the group consisting of -ORa and -NRbRc; wherein Ra is selected from the group consisting of H and a lower alkyl group having 1 to 3 carbon atoms; wherein Rb and Rc are independently selected from the group consisting of -H, -OH, and -CH2CH2OH; wherein R2 and R3 are independently selected from the group consisting of -H, -OH, -OCH3 and =O; and / or wherein R4 is -H or CH3.

[0380] Item 6. The composition or pharmaceutical composition of any one of Items 1 to 5, wherein R1 is selected from the group consisting of -ORa and -NRbRc; wherein Ra is selected from the group consisting of H and -CH3; wherein Rb and Rc are independently selected from the group consisting of -H, -OH, and -CH2CH2OH; wherein R2 and R3 are independently selected from the group consisting of -OH and =O; and / or wherein R4 is -H.

[0381] Item 7. The composition or pharmaceutical composition as described in any one of Items 2 or 6, wherein the adjuvant is 2HBA, andrographolide, ascorbic acid, caffeol, caryophyllotoxin, CDDO, chalcone, CHIR98014, cytosolicin, curcumin, cycloastragaloyl alcohol, 1,2-dithiacyclopentene-3-thione (D3T), damaride, edaravone, EGCG, gambogeylic acid, ganetespib, ganetene, IQ-1, limonene, lonidamide. Melatonin, benzamide tetrahydroindole, N886, alkylaminobiphenylamide, neomycin, pyridoxal 5′-phosphate (P5′-P), hydroxypyridinethione, quercetin, rhizobacterin, resveratrol, RTA-408, SB202190, SB216763, SNX-5422, sodium butyrate, sulforane, tetrabromobenzotriazole (TBB), tert-butylhydroquinone (tBHQ), valproic acid, solanine A or solanine, ergosterol, lupene ketone, and their analogues.

[0382] Item 8. The composition or pharmaceutical composition as described in any one of Items 2 or 6, wherein the adjuvant is tBHQ, caryophyllene, curcumin, 2HBA or EGCG.

[0383] Item 9. The composition or pharmaceutical composition of any one of Items 2 to 8, wherein the compound is triptolide.

[0384] Item 10. The composition or pharmaceutical composition as described in Items 2 to 8, wherein said compound is

[0385]

[0386] Item 11. The composition or pharmaceutical composition as described in Items 2 to 8, wherein said compound is

[0387]

[0388] Item 12. The composition or pharmaceutical composition as described in Items 2 to 8, wherein said compound is

[0389]

[0390] Item 13. The composition or pharmaceutical composition as described in Items 2 to 8, wherein said compound is

[0391]

[0392] Item 14. The composition or pharmaceutical composition as described in Items 2 to 8, wherein said compound is

[0393]

[0394] Item 15. A method for regulating the state of cells, cell preparations, tissues, grafts, or organs to increase or maintain viability, or resistance to death, damage, or stress, and functionality, said method comprising contacting said cells, cell preparations, tissues, grafts, or organs with: a) a composition comprising one or more compounds of Formula I, pharmaceutically acceptable salts, stereoisomers, tautomers, or prodrugs thereof; b) a combination comprising one or more compounds of Formula I, pharmaceutically acceptable salts, stereoisomers, tautomers, or prodrugs thereof, and adjuvants; c) different cell preparations that have been contacted with one or more compounds of Formula I, pharmaceutically acceptable salts, stereoisomers, tautomers, or prodrugs thereof, or combinations thereof; or d) secretory proteomes of different cell preparations that have been contacted with said one or more compounds of Formula I, pharmaceutically acceptable salts, stereoisomers, tautomers, or prodrugs thereof, or combinations thereof.

[0395]

[0396] R1 is selected from the following groups: -H, -F, -Cl, -Br, -I, -CN, -aryl, alkyl, imidazole, -ORa, -NRbRc, -(CH2). n OH and -(CH2) n NH2;

[0397] Ra is selected from the group consisting of: H, substituted or unsubstituted straight-chain alkyl groups having 1 to 6 carbon atoms, substituted or unsubstituted branched alkyl groups having 3 to 6 carbon atoms, and protecting groups;

[0398] Rb and Rc are independently selected from the group consisting of: -H, -OH, -OCH3, substituted or unsubstituted straight-chain alkyl groups having 1 to 6 carbon atoms, substituted or unsubstituted branched alkyl groups having 3 to 6 carbon atoms, and protecting groups.

[0399] Where n is 0, 1, 2, 3 or 4;

[0400] R2 and R3 are independently selected from the following groups: -H, -ORd, =O, -C(=O)OH, -C(=O)ORx, and -C(=O)Rx;

[0401] Where Rd is H or a lower alkyl group having 1 to 3 carbon atoms;

[0402] Wherein Rx is H or a lower alkyl group having 1 to 3 carbon atoms;

[0403] R4 is selected from the following groups: -H, -OH and lower alkyl groups having 1 to 3 carbon atoms.

[0404] Item 16. The method as described in Item 15, wherein the compound comprises formula Ia

[0405]

[0406] R1 is selected from the following groups: -H, -F, -Cl, -Br, -I, -CN, -aryl, alkyl, imidazole, -ORa, -NRbRc, -(CH2). n OH and -(CH2) n NH2;

[0407] Ra is selected from the group consisting of: H, substituted or unsubstituted straight-chain alkyl groups having 1 to 6 carbon atoms, substituted or unsubstituted branched alkyl groups having 3 to 6 carbon atoms, and protecting groups;

[0408] Rb and Rc are independently selected from the group consisting of: -H, -OH, -OCH3, substituted or unsubstituted straight-chain alkyl groups having 1 to 6 carbon atoms, substituted or unsubstituted branched alkyl groups having 3 to 6 carbon atoms, and protecting groups.

[0409] Where n is 0, 1, 2, 3 or 4;

[0410] R2 and R3 are independently selected from the following groups: -H, -ORd, =O, -C(=O)OH, -C(=O)ORx, and -C(=O)Rx;

[0411] Where Rd is H or a lower alkyl group having 1 to 3 carbon atoms;

[0412] Wherein Rx is H or a lower alkyl group having 1 to 3 carbon atoms;

[0413] R4 is selected from the following groups: -H, -OH and lower alkyl groups having 1 to 3 carbon atoms.

[0414] Item 17. The method as described in Item 15 or 16, wherein R1 is selected from the group consisting of -ORa and -NRbRc; wherein Ra is selected from the group consisting of H, substituted or unsubstituted straight-chain alkyl groups having 1 to 6 carbon atoms, substituted or unsubstituted branched alkyl groups having 3 to 6 carbon atoms, and protecting groups; wherein Rb and Rc are independently selected from the group consisting of -H, -OH, -OCH3, substituted or unsubstituted straight-chain alkyl groups having 1 to 6 carbon atoms, substituted or unsubstituted branched alkyl groups having 3 to 6 carbon atoms, and protecting groups; wherein R2 and R3 are independently selected from the group consisting of -H, -ORd, and =O, where Rd is H or a lower alkyl group having 1 to 3 carbon atoms; and / or wherein R4 is -H or CH3.

[0415] Item 18. The method of any one of items 15 to 17, wherein R1 is selected from the group consisting of -ORa and -NRbRc; wherein Ra is selected from the group consisting of H and lower alkyl groups having 1 to 3 carbon atoms; wherein Rb and Rc are independently selected from the group consisting of -H, -OH, and -CH2CH2OH; wherein R2 and R3 are independently selected from the group consisting of -H, -OH, -OCH3 and =O; and / or wherein R4 is -H or CH3.

[0416] Item 19. The method of any one of items 15 to 18, wherein R1 is selected from the group consisting of -ORa and -NRbRc; wherein Ra is selected from the group consisting of H and -CH3; wherein Rb and Rc are independently selected from the group consisting of -H, -OH, and -CH2CH2OH; wherein R2 and R3 are independently selected from the group consisting of -OH and =O; and / or wherein R4 is -H.

[0417] Item 20. The method of any one of Items 15 to 19, wherein the adjuvant is 2HBA, andrographolide, ascorbic acid, caffeol, caryophyllotoxin, CDDO, chalcone, CHIR98014, cytosolic acid, curcumin, cycloastragaloyl alcohol, 1,2-dithiacyclopenten-3-thione (D3T), damamode, edaravone, EGCG, gambogeylic acid, ganetespib, ganetene, IQ-1, limonene, chlordamide, melatonin, benzoyl peroxide Amide tetrahydroindole, N886, alkylaminobiphenylamide, neomycin, pyridoxal 5′-phosphate (P5′-P), hydroxypyridinethione, quercetin, rhizobacterin, resveratrol, RTA-408, SB202190, SB216763, SNX-5422, sodium butyrate, sulforane, tetrabromobenzotriazole (TBB), tert-butylhydroquinone (tBHQ), valproic acid, solanine A or solanine, ergosterol, lupene ketone, or analogues thereof.

[0418] Item 21. The method of any one of Items 15 to 19, wherein the adjuvant is tBHQ, caryophyllene, curcumin, 2HBA or EGCG.

[0419] Item 22. The method of any one of Items 15 to 19, wherein when the compound is triptolide, the compound is combined with an adjuvant.

[0420] Item 23. The method of any one of Items 15 to 21, wherein said compound is

[0421]

[0422] Item 24. The method of any one of Items 15 to 21, wherein said compound is

[0423]

[0424] Item 25. The method of any one of items 15 to 21, wherein said compound is

[0425]

[0426] Item 26. The method of any one of Items 15 to 21, wherein said compound is

[0427]

[0428] Item 27. The method of any one of Items 15 to 21, wherein said compound is

[0429]

[0430] Item 28. The method of any one of Items 15 to 21, wherein the compound of Formula I comprises an R1, R2, R3 or R4 group, said group being different from the corresponding R1, R2, R3 or R4 group of triptolide.

[0431] Item 29. The method of any one of Items 15 to 28, wherein the method is performed in vitro, ex vivo, or in vivo.

[0432] Item 30. The method of Item 29, wherein the method is performed in vitro on a cell preparation, tissue or organ.

[0433] Item 31. The method of Item 30, wherein the cell preparation comprises stem cells.

[0434] Item 32. The method as described in Item 31, wherein the stem cells are mesenchymal stem cells, CD34 +Cells, CD133 + Cells or stem cells, pluripotent cells, progenitor cells, or adult differentiated cells.

[0435] Item 33. The method of Item 32, wherein the stem cells are autologous stem cells isolated from a mammal in need.

[0436] Item 34. The method of Item 32, wherein the stem cells are allogeneic stem cells isolated from a mammalian donor.

[0437] Item 35. The method of Item 34, wherein the allogeneic stem cells from the mammalian donor are HLA-matched, immune-exempt, low-immunogenic, or immune-evading to the mammal in need.

[0438] Item 36. The method of Item 30, wherein the cell preparation, tissue or organ is suitable for transplantation in mammals.

[0439] Item 37. The method of Item 29, wherein the method is carried out in vivo by administering the composition, combination, different cell preparations, secretory proteome or cell culture medium to a mammal in need.

[0440] Item 38. The method as described in Item 37, wherein the mammal in need suffers from or is susceptible to ischemic or degenerative diseases.

[0441] Item 39. The method as described in Item 38, wherein the ischemic disease is stroke, myocardial infarction (MI), peripheral artery disease (PAD), transient ischemic attack, microangiopathy, ischemia of the brain, intestine, liver, lung, or kidney, or vascular dementia.

[0442] Item 40. The method described in Item 38, wherein the degenerative disease is cardiomyopathy, liver disease such as NAFLD / NASH, cirrhosis, lung disease such as COPD, osteoarthritis, pancreatic disorders such as diabetes, neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, dementia, and ALS.

[0443] Item 41. The method as described in Item 37, wherein the mammal in need is a patient undergoing surgery or medical intervention.

[0444] Item 42. The method of any one of items 37 to 41, wherein the administration is performed systemically or locally.

[0445] Item 43. The method as described in Item 29, wherein the method is performed in vitro on cells before freezing or during conditioning.

[0446] Item 44. The method of any one of items 15 to 43, wherein the cells, cell preparations, tissues, grafts or organs are contacted with the compound, combination, different cell preparations or secretory proteome for 5 to 180 minutes prior to use.

[0447] Item 45. The method as described in any one of items 15 to 44, wherein 10 -6 M to 10 -10 The concentration of M is achieved using the compound.

[0448] Item 46. A method for protecting cells, tissues, grafts, or organs from stress or damage, the method comprising contacting the cells, tissues, grafts, or organs with a composition comprising one or more compounds that activate heat shock response activation and / or antioxidant response, or with the secretory proteome of a cell preparation that has been contacted with the composition.

[0449] Item 47. A method for protecting cells, tissues, grafts, or organs from stress or damage, the method comprising contacting the cells, tissues, grafts, or organs with a composition comprising one or more compounds of Formula I, pharmaceutically acceptable salts, stereoisomers, tautomers, or prodrugs thereof, or with the secretory proteome of a cell preparation already contacted with the composition.

[0450]

[0451] R1 is selected from the following groups: -H, -F, -Cl, -Br, -I, -CN, -aryl, alkyl, imidazole, -ORa, -NRbRc, -(CH2). n OH and -(CH2) n NH2;

[0452] Ra is selected from the group consisting of: H, substituted or unsubstituted straight-chain alkyl groups having 1 to 6 carbon atoms, substituted or unsubstituted branched alkyl groups having 3 to 6 carbon atoms, and protecting groups;

[0453] Rb and Rc are independently selected from the group consisting of: -H, -OH, -OCH3, substituted or unsubstituted straight-chain alkyl groups having 1 to 6 carbon atoms, substituted or unsubstituted branched alkyl groups having 3 to 6 carbon atoms, and protecting groups.

[0454] Where n is 0, 1, 2, 3 or 4;

[0455] R2 and R3 are independently selected from the following groups: -H, -ORd, =O, -C(=O)OH, -C(=O)ORx, and -C(=O)Rx;

[0456] Where Rd is H or a lower alkyl group having 1 to 3 carbon atoms;

[0457] Wherein Rx is H or a lower alkyl group having 1 to 3 carbon atoms;

[0458] R4 is selected from the following groups: -H, -OH and lower alkyl groups having 1 to 3 carbon atoms.

[0459] Item 48. The method as defined in Item 47, wherein the compound comprises formula Ia

[0460]

[0461] R1 is selected from the following groups: -H, -F, -Cl, -Br, -I, -CN, -aryl, alkyl, imidazole, -ORa, -NRbRc, -(CH2). n OH and -(CH2) n NH2;

[0462] Ra is selected from the group consisting of: H, substituted or unsubstituted straight-chain alkyl groups having 1 to 6 carbon atoms, substituted or unsubstituted branched alkyl groups having 3 to 6 carbon atoms, and protecting groups;

[0463] Rb and Rc are independently selected from the group consisting of: -H, -OH, -OCH3, substituted or unsubstituted straight-chain alkyl groups having 1 to 6 carbon atoms, substituted or unsubstituted branched alkyl groups having 3 to 6 carbon atoms, and protecting groups.

[0464] Where n is 0, 1, 2, 3 or 4;

[0465] R2 and R3 are independently selected from the following groups: -H, -ORd, =O, -C(=O)OH, -C(=O)ORx, and -C(=O)Rx;

[0466] Where Rd is H or a lower alkyl group having 1 to 3 carbon atoms;

[0467] Wherein Rx is H or a lower alkyl group having 1 to 3 carbon atoms;

[0468] R4 is selected from the following groups: -H, -OH and lower alkyl groups having 1 to 3 carbon atoms.

[0469] Item 49. The method of any one of Items 47 or 48, wherein the method is performed in vitro or ex vivo.

[0470] Item 50. The method of any one of items 47 to 49, wherein the cell is a stem cell, a differentiated cell, a progenitor cell, or an adult cell.

[0471] Item 51. The method of any one of items 47 to 50, wherein the cells, tissues, grafts or organs are contacted with the compound for 5 to 180 minutes prior to use.

[0472] Item 52. The method as described in any one of items 47 to 51, wherein 10 -6 M to 10 -10 The concentration of M is achieved using the compound.

[0473] Item 53. The method of any one of items 47 to 52, wherein the compound is washed out from the cells, tissues, grafts or organs prior to administration.

[0474] Item 54. The method of any one of items 47 to 53, wherein the composition further comprises one or more adjuvants.

[0475] Item 55. A method for preventing or treating ischemic diseases or degenerative diseases, the method comprising administering to a mammal in need a composition comprising one or more compounds that activate heat shock response activation and / or antioxidant response, or administering a secretory proteome of a cellular preparation regulated by said composition.

[0476] Item 56. A method for preventing or treating ischemic diseases, the method comprising administering to a mammal in need: a) a composition comprising one or more compounds of Formula I, pharmaceutically acceptable salts, stereoisomers, tautomers, or prodrugs thereof; or b) a stem cell preparation pre-regulated with one or more compounds of Formula I, pharmaceutically acceptable salts, stereoisomers, tautomers, or prodrugs thereof; or c) a secretory proteome of a cell preparation regulated with said composition.

[0477]

[0478] R1 is selected from the group consisting of: -H, -F, -Cl, -Br, -I, -CN, -aryl, alkyl, imidazole, -CN, -aryl, alkyl, imidazole, -ORa, -NRbRc, -(CH2) n OH and -(CH2) n NH2;

[0479] Ra is selected from the group consisting of: H, substituted or unsubstituted straight-chain alkyl groups having 1 to 6 carbon atoms, substituted or unsubstituted branched alkyl groups having 3 to 6 carbon atoms, and protecting groups;

[0480] Rb and Rc are independently selected from the group consisting of: -H, -OH, -OCH3, substituted or unsubstituted straight-chain alkyl groups having 1 to 6 carbon atoms, substituted or unsubstituted branched alkyl groups having 3 to 6 carbon atoms, and protecting groups.

[0481] Where n is 0, 1, 2, 3 or 4;

[0482] R2 and R3 are independently selected from the following groups: -H, -ORd, =O, -C(=O)OH, -C(=O)ORx, and -C(=O)Rx;

[0483] Where Rd is H or a lower alkyl group having 1 to 3 carbon atoms;

[0484] Wherein Rx is H or a lower alkyl group having 1 to 3 carbon atoms;

[0485] R4 is selected from the following groups: -H, -OH and lower alkyl groups having 1 to 3 carbon atoms.

[0486] Item 57. The method as defined in Item 56, wherein the compound comprises formula Ia

[0487]

[0488] R1 is selected from the group consisting of: -H, -F, -Cl, -Br, -I, -CN, -aryl, alkyl, imidazole, -CN, -aryl, alkyl, imidazole, -ORa, -NRbRc, -(CH2) n OH and -(CH2) n NH2;

[0489] Ra is selected from the group consisting of: H, substituted or unsubstituted straight-chain alkyl groups having 1 to 6 carbon atoms, substituted or unsubstituted branched alkyl groups having 3 to 6 carbon atoms, and protecting groups;

[0490] Rb and Rc are independently selected from the group consisting of: -H, -OH, -OCH3, substituted or unsubstituted straight-chain alkyl groups having 1 to 6 carbon atoms, substituted or unsubstituted branched alkyl groups having 3 to 6 carbon atoms, and protecting groups.

[0491] Where n is 0, 1, 2, 3 or 4;

[0492] R2 and R3 are independently selected from the following groups: -H, -ORd, =O, -C(=O)OH, -C(=O)ORx, and -C(=O)Rx;

[0493] Where Rd is H or a lower alkyl group having 1 to 3 carbon atoms;

[0494] Wherein Rx is H or a lower alkyl group having 1 to 3 carbon atoms;

[0495] R4 is selected from the following groups: -H, -OH and lower alkyl groups having 1 to 3 carbon atoms.

[0496] Item 58. The method as described in Item 56 or 57, wherein the composition further comprises one or more adjuvants.

[0497] Item 59. The method as described in Item 58, wherein the adjuvant is 2HBA, andrographolide, ascorbic acid, caffeol, caryophyllotoxin, CDDO, chalcone, CHIR98014, cytosolic acid, curcumin, cycloastragaloyl alcohol, 1,2-dithiacyclopenten-3-thione (D3T), damamod, edaravone, EGCG, gambogeylic acid, ganetespib, ganetene, IQ-1, limonene, chlordamide, melatonin, and benzamide tetrahydrobenzamide. Indole, N886, alkylaminobiphenylamide, neomycin, pyridoxal 5′-phosphate (P5′-P), hydroxypyridinethione, quercetin, rhizocarpine, resveratrol, RTA-408, SB202190, SB216763, SNX-5422, sodium butyrate, sulforane, tetrabromobenzotriazole (TBB), tert-butylhydroquinone (tBHQ), valproic acid, solanine A or solanine, ergosterol, lupene ketone, and their analogues.

[0498] Item 60. The method as described in Item 59, wherein the adjuvant is tBHQ, caryophyllene, curcumin, 2HBA, or EGCG.

[0499] Item 61. The method of any one of Items 56 to 60, wherein the stem cell preparation is an autologous stem cell preparation isolated from a mammal in need.

[0500] Item 62. The method of any one of Items 56 to 60, wherein the stem cell preparation is an allogeneic stem cell preparation isolated from a mammalian donor.

[0501] Item 63. The method of Item 62, wherein the allogeneic stem cell preparation from the mammalian donor is HLA-matched, immune-exempt, low-immunogenic, or immune-evading for the mammal in need.

[0502] Item 64. The method of any one of Items 56 to 63, wherein the ischemic disease is stroke, myocardial infarction (MI) or peripheral artery disease (PAD), transient ischemic attack, microangiopathy, ischemia of the brain, intestine, liver, lung, or kidney, or vascular dementia.

[0503] Item 65. The method described in items 56 to 63, wherein the degenerative disease is cardiomyopathy, liver disease such as NAFLD / NASH, cirrhosis, lung disease such as COPD, osteoarthritis, pancreatic disorder such as diabetes, neurodegenerative disease such as Alzheimer's disease, Parkinson's disease, dementia, and ALS.

[0504] Item 66. An isolated cell, tissue, graft, or organ preparation, pre-conditioned with a composition comprising one or more compounds that activate heat shock response and / or have antioxidant effects.

[0505] Item 67. An isolated cell, tissue, graft, or organ preparation, pre-conditioned with a composition comprising one or more compounds of Formula I, their pharmaceutically acceptable salts, stereoisomers, tautomers, or prodrugs.

[0506]

[0507] R1 is selected from the following groups: -H, -F, -Cl, -Br, -I, -CN, -aryl, alkyl, imidazole, -ORa, -NRbRc, -(CH2). n OH and -(CH2) n NH2;

[0508] Ra is selected from the group consisting of: H, substituted or unsubstituted straight-chain alkyl groups having 1 to 6 carbon atoms, substituted or unsubstituted branched alkyl groups having 3 to 6 carbon atoms, and protecting groups;

[0509] Rb and Rc are independently selected from the group consisting of: -H, -OH, -OCH3, substituted or unsubstituted straight-chain alkyl groups having 1 to 6 carbon atoms, substituted or unsubstituted branched alkyl groups having 3 to 6 carbon atoms, and protecting groups.

[0510] Where n is 0, 1, 2, 3 or 4;

[0511] R2 and R3 are independently selected from the following groups: -H, -ORd, =O, -C(=O)OH, -C(=O)ORx, and -C(=O)Rx;

[0512] Where Rd is H or a lower alkyl group having 1 to 3 carbon atoms;

[0513] Wherein Rx is H or a lower alkyl group having 1 to 3 carbon atoms;

[0514] R4 is selected from the following groups: -H, -OH and lower alkyl groups having 1 to 3 carbon atoms.

[0515] Item 68. An isolated cell, tissue, graft, or organ preparation as described in Item 67, wherein said compound comprises formula Ia.

[0516] Item 69. An isolated cell, tissue, graft, or organ preparation as described in Item 67 or 68, wherein the cell is a stem cell.

[0517] Item 70. An isolated cell, tissue, graft, or organ preparation as described in Item 69, wherein the stem cells are mesenchymal stem cells or hematopoietic stem cells.

[0518] Item 71. An isolated cell, tissue, graft, or organ preparation as described in any one of Items 67 to 70, wherein the composition further comprises one or more adjuvants.

[0519] Item 72. An isolated cell, tissue, graft, or organ preparation as described in any one of Items 67 to 71, wherein said compound is a part of said preparation.

[0520] Item 73. An isolated cell, tissue, graft, or organ preparation as described in any one of Items 67 to 72, wherein the cell preparation is in a three-dimensional structural form or is integrated into a scaffold.

[0521] Item 74. An isolated cell, tissue, graft, or organ preparation as described in any one of Items 67 to 72, wherein the cell preparation is encapsulated in a medically acceptable biogel.

[0522] Item 75. An isolated cell, tissue, graft, or organ preparation as described in Item 74, wherein the medically acceptable biogel is a thermosensitive hydrogel.

[0523] Item 76. A method for reducing cell damage during transplantation of stem cells, tissues, grafts, or organs, the method comprising contacting the stem cells, tissues, grafts, or organs with a composition comprising one or more compounds that activate heat shock response activation and / or antioxidant response, or with a secretory proteome or cell culture medium of a cell preparation modified with said composition.

[0524] Item 77. A method for reducing cell damage during transplantation of stem cells, tissues, grafts, or organs, the method comprising contacting the stem cells, tissues, grafts, or organs with a composition comprising at least one compound of formula I, or with a secretory proteome of a cell preparation modified with said composition, before and / or during and / or after transplantation.

[0525]

[0526] R1 is selected from the following groups: -H, -F, -Cl, -Br, -I, -CN, -aryl, alkyl, imidazole, -ORa, -NRbRc, -(CH2). n OH and -(CH2) n NH2;

[0527] Ra is selected from the group consisting of: H, substituted or unsubstituted straight-chain alkyl groups having 1 to 6 carbon atoms, substituted or unsubstituted branched alkyl groups having 3 to 6 carbon atoms, and protecting groups;

[0528] Rb and Rc are independently selected from the group consisting of: -H, -OH, -OCH3, substituted or unsubstituted straight-chain alkyl groups having 1 to 6 carbon atoms, substituted or unsubstituted branched alkyl groups having 3 to 6 carbon atoms, and protecting groups.

[0529] Where n is 0, 1, 2, 3 or 4;

[0530] R2 and R3 are independently selected from the following groups: -H, -ORd, =O, -C(=O)OH, -C(=O)ORx, and -C(=O)Rx;

[0531] Where Rd is H or a lower alkyl group having 1 to 3 carbon atoms;

[0532] Wherein Rx is H or a lower alkyl group having 1 to 3 carbon atoms;

[0533] R4 is selected from the following groups: -H, -OH and lower alkyl groups having 1 to 3 carbon atoms.

[0534] Item 78. The method of Item 77, the method further comprising contacting the stem cells, tissue, graft or organ with the composition after the transplantation.

[0535] Item 79. The method as described in Item 77 or 78, wherein the composition further comprises one or more adjuvants.

[0536] Item 80. One or more compounds that activate heat shock response and / or antioxidant response for use in protecting cells, tissues, grafts or organs from stress or damage.

[0537] Item 81. Use of a compound of formula I, its pharmaceutically acceptable salt, stereoisomer, tautomer, or prodrug for the protection of cells, tissues, grafts, or organs from stress or damage.

[0538]

[0539] R1 is selected from the following groups: -H, -F, -Cl, -Br, -I, -CN, -aryl, alkyl, imidazole, -ORa, -NRbRc, -(CH2). n OH and -(CH2) n NH2;

[0540] Ra is selected from the group consisting of: H, substituted or unsubstituted straight-chain alkyl groups having 1 to 6 carbon atoms, substituted or unsubstituted branched alkyl groups having 3 to 6 carbon atoms, and protecting groups;

[0541] Rb and Rc are independently selected from the group consisting of: -H, -OH, -OCH3, substituted or unsubstituted straight-chain alkyl groups having 1 to 6 carbon atoms, substituted or unsubstituted branched alkyl groups having 3 to 6 carbon atoms, and protecting groups.

[0542] Where n is 0, 1, 2, 3 or 4;

[0543] R2 and R3 are independently selected from the following groups: -H, -ORd, =O, -C(=O)OH, -C(=O)ORx, and -C(=O)Rx;

[0544] Where Rd is H or a lower alkyl group having 1 to 3 carbon atoms;

[0545] Wherein Rx is H or a lower alkyl group having 1 to 3 carbon atoms;

[0546] R4 is selected from the following groups: -H, -OH and lower alkyl groups having 1 to 3 carbon atoms.

[0547] Item 82. As defined in Item 81, wherein the cell is an immortalized cell or a primary cell.

[0548] Item 83. As defined in Item 81 or 82, wherein the cells are derived from human.

[0549] Item 84. As defined in any of Items 81 to 83, wherein the cell is a stem cell, progenitor cell, cardiomyocyte or myoblast, or insulin-secreting cell.

[0550] Item 85. As defined in Item 84, wherein the stem cells are mesenchymal stem cells or hematopoietic stem cells.

[0551] Item 86. The use as defined in any of Items 81 to 85, wherein the cell, tissue, graft or organ is used for transplantation.

[0552] Item 87. As defined in any of Items 81 to 85, wherein the composition further comprises one or more adjuvants.

[0553] Item 88. A method for treating a patient requiring surgery or medical intervention, the method comprising administering a composition comprising one or more compounds that activate heat shock response activation and / or antioxidant response, or administering a secretory proteome or cell culture medium of a cell preparation modified with said composition.

[0554] Item 89. A method for treating a patient requiring surgery or medical intervention, the method comprising administering a composition comprising a compound of formula I or a cell preparation modified with said composition to a secretory proteome before and / or during and / or after said surgery or medical intervention.

[0555]

[0556] R1 is selected from the following groups: -H, -F, -Cl, -Br, -I, -CN, -aryl, alkyl, imidazole, -ORa, -NRbRc, -(CH2). n OH and -(CH2) n NH2;

[0557] Ra is selected from the group consisting of: H, substituted or unsubstituted straight-chain alkyl groups having 1 to 6 carbon atoms, substituted or unsubstituted branched alkyl groups having 3 to 6 carbon atoms, and protecting groups;

[0558] Rb and Rc are independently selected from the group consisting of: -H, -OH, -OCH3, substituted or unsubstituted straight-chain alkyl groups having 1 to 6 carbon atoms, substituted or unsubstituted branched alkyl groups having 3 to 6 carbon atoms, and protecting groups.

[0559] Where n is 0, 1, 2, 3 or 4;

[0560] R2 and R3 are independently selected from the following groups: -H, -ORd, =O, -C(=O)OH, -C(=O)ORx, and -C(=O)Rx;

[0561] Where Rd is H or a lower alkyl group having 1 to 3 carbon atoms;

[0562] Wherein Rx is H or a lower alkyl group having 1 to 3 carbon atoms;

[0563] R4 is selected from the following groups: -H, -OH and lower alkyl groups having 1 to 3 carbon atoms.

[0564] Item 90. The method of Item 89, wherein the composition is applied topically at the site of the surgical or medical intervention.

[0565] Item 91. The method of Item 89, wherein the composition is administered systemically.

[0566] Item 92. A reagent kit comprising a first vial and a second vial, the first vial containing a compound of formula I alone or in mixture with an adjuvant, and the second vial containing stem cells;

[0567]

[0568] R1 is selected from the following groups: -H, -F, -Cl, -Br, -I, -CN, -aryl, alkyl, imidazole, -ORa, -NRbRc, -(CH2). n OH and -(CH2) n NH2;

[0569] Ra is selected from the group consisting of: H, substituted or unsubstituted straight-chain alkyl groups having 1 to 6 carbon atoms, substituted or unsubstituted branched alkyl groups having 3 to 6 carbon atoms, and protecting groups;

[0570] Rb and Rc are independently selected from the group consisting of: -H, -OH, -OCH3, substituted or unsubstituted straight-chain alkyl groups having 1 to 6 carbon atoms, substituted or unsubstituted branched alkyl groups having 3 to 6 carbon atoms, and protecting groups.

[0571] Where n is 0, 1, 2, 3 or 4;

[0572] R2 and R3 are independently selected from the following groups: -H, -ORd, =O, -C(=O)OH, -C(=O)ORx, and -C(=O)Rx;

[0573] Where Rd is H or a lower alkyl group having 1 to 3 carbon atoms;

[0574] Wherein Rx is H or a lower alkyl group having 1 to 3 carbon atoms;

[0575] R4 is selected from the following groups: -H, -OH and lower alkyl groups having 1 to 3 carbon atoms.

[0576] Item 93. The kit as described in Item 92, the kit further comprising a container containing cells, tissues, grafts or organs.

[0577] Item 94. An apparatus suitable for in vivo administration of a composition as described in Items 1 to 14, a cell preparation modified with said composition, or a secretory proteome or cell culture medium of a cell preparation modified with said composition, said apparatus comprising a compartment filled with said composition, said cell preparation, or said secretory proteome.

[0578] Item 95. An apparatus suitable for cell regulation or fabrication using the composition or a cell preparation modified with the composition, such as a cell sorting or amplification bioreactor, the apparatus comprising a compartment filled with the composition, the cell preparation, or the secretory proteome.

Claims

1. A composition or pharmaceutical composition comprising one or more compounds that activate heat shock response and / or antioxidant response.

2. A composition or pharmaceutical composition comprising a) one or more compounds of formula I, a pharmaceutically acceptable salt thereof, a stereoisomer, a tautomer, or a prodrug; b) one or more adjuvants; and c) a carrier or a pharmaceutically acceptable carrier. R1 is selected from the following groups: -H, -F, -Cl, -Br, -I, -CN, aryl, alkyl, imidazole, -ORa, -NRbRc, -(CH2). n OH and -(CH2) n NH2; Ra is selected from the group consisting of: H, substituted or unsubstituted straight-chain alkyl groups having 1 to 6 carbon atoms, substituted or unsubstituted branched alkyl groups having 3 to 6 carbon atoms, and protecting groups; Rb and Rc are independently selected from the group consisting of: -H, -OH, -OCH3, substituted or unsubstituted straight-chain alkyl groups having 1 to 6 carbon atoms, substituted or unsubstituted branched alkyl groups having 3 to 6 carbon atoms, and protecting groups. Where n is 0, 1, 2, 3 or 4; R2 and R3 are independently selected from the following groups: -H, -ORd, =O, -C(=O)OH, -C(=O)ORx, and -C(=O)Rx; Where Rd is H or a lower alkyl group having 1 to 3 carbon atoms; Wherein Rx is H or a lower alkyl group having 1 to 3 carbon atoms; R4 is selected from the following groups: -H, -OH and lower alkyl groups having 1 to 3 carbon atoms.

3. The composition or pharmaceutical composition of claim 2, wherein the compound comprises formula Ia R1 is selected from the following groups: -H, -F, -Cl, -Br, -I, -CN, aryl, alkyl, imidazole, -ORa, -NRbRc, -(CH2). n OH and -(CH2) n NH2; Ra is selected from the group consisting of: H, substituted or unsubstituted straight-chain alkyl groups having 1 to 6 carbon atoms, substituted or unsubstituted branched alkyl groups having 3 to 6 carbon atoms, and protecting groups; Rb and Rc are independently selected from the group consisting of: -H, -OH, -OCH3, substituted or unsubstituted straight-chain alkyl groups having 1 to 6 carbon atoms, substituted or unsubstituted branched alkyl groups having 3 to 6 carbon atoms, and protecting groups. Where n is 0, 1, 2, 3 or 4; R2 and R3 are independently selected from the following groups: -H, -ORd, =O, -C(=O)OH, -C(=O)ORx, and -C(=O)Rx; Where Rd is H or a lower alkyl group having 1 to 3 carbon atoms; Wherein Rx is H or a lower alkyl group having 1 to 3 carbon atoms; R4 is selected from the following groups: -H, -OH and lower alkyl groups having 1 to 3 carbon atoms.

4. The composition or pharmaceutical composition of claim 2 or 3, wherein R1 is selected from the group consisting of -ORa and -NRbRc; wherein Ra is selected from the group consisting of H, substituted or unsubstituted straight-chain alkyl groups having 1 to 6 carbon atoms, substituted or unsubstituted branched alkyl groups having 3 to 6 carbon atoms, and protecting groups; wherein Rb and Rc are independently selected from the group consisting of -H, -OH, -OCH3, substituted or unsubstituted straight-chain alkyl groups having 1 to 6 carbon atoms, substituted or unsubstituted branched alkyl groups having 3 to 6 carbon atoms, and protecting groups; wherein R2 and R3 are independently selected from the group consisting of -H, -ORd, and =O, where Rd is H or a lower alkyl group having 1 to 3 carbon atoms; and / or wherein R4 is -H or CH3.

5. The composition or pharmaceutical composition according to any one of claims 1 to 4, wherein R1 is selected from the group consisting of -ORa and -NRbRc; wherein Ra is selected from the group consisting of H and a lower alkyl group having 1 to 3 carbon atoms; wherein Rb and Rc are independently selected from the group consisting of -H, -OH, and -CH2CH2OH; wherein R2 and R3 are independently selected from the group consisting of -H, -OH, -OCH3 and =O; and / or wherein R4 is -H or CH3.

6. The composition or pharmaceutical composition according to any one of claims 1 to 5, wherein R1 is selected from the group consisting of -ORa and -NRbRc; wherein Ra is selected from the group consisting of H and -CH3; wherein Rb and Rc are independently selected from the group consisting of -H, -OH, and -CH2CH2OH; wherein R2 and R3 are independently selected from the group consisting of -OH and =O; and / or wherein R4 is -H.

7. The composition or pharmaceutical composition of any one of claims 2 or 6, wherein the adjuvant is 2HBA, andrographolide, ascorbic acid, caffeol, caryophyllotoxin, CDDO, chalcone, CHIR98014, cytosolic acid, curcumin, cycloastragaloyl alcohol, 1,2-dithiacyclopenten-3-thione (D3T), damaride, edaravone, EGCG, gambogeylic acid, ganetespib, ganetene, IQ-1, limonene, or lonidamide. Melatonin, benzamide tetrahydroindole, N886, alkylaminobiphenylamide, neomycin, pyridoxal 5′-phosphate (P5′-P), hydroxypyridinium ketone, quercetin, rhizobacterin, resveratrol, RTA-408, SB202190, SB216763, SNX-5422, sodium butyrate, sulforane, tetrabromobenzotriazole (TBB), tert-butylhydroquinone (tBHQ), valproic acid, solanine A or solanine, ergosterol, lupene ketone, and their analogues.

8. The composition or pharmaceutical composition of any one of claims 2 or 6, wherein the adjuvant is tBHQ, caryophyllene, curcumin, 2HBA, or EGCG.

9. The composition or pharmaceutical composition according to any one of claims 2 to 8, wherein the compound is triptolide.

10. The composition or pharmaceutical composition according to claims 2 to 8, wherein said compound is

Images