TNF inhibitor combination therapy

Abstract

The present invention provides a combination therapy comprising a TNF inhibitor compound and a mitochondrial complex I modulator (MCIM) compound that is useful in the medical field of inflammatory and progressive disorders such as RA, IBD, idiopathic pulmonary fibrosis, interstitial lung disease, and MS.

Description

[Technical Field]

[0001] The present invention relates to combination therapies that can have restorative effects against a range of progressive and / or degenerative diseases. In certain cases, the combination therapy can be used to treat autoimmune diseases such as arthritis, specifically rheumatoid arthritis. In certain cases, the combination therapy comprises a tumor necrosis factor inhibitor and a therapeutic agent that can act by binding to mitochondrial complex I. The therapeutic agent that can act by binding to mitochondrial complex I is referred to herein as a mitochondrial complex I modulator compound (or MCIM compound). [Background technology]

[0002] Mitochondrial complex I (also known as NADH:ubiquinone oxidoreductase, type I NADH dehydrogenase, respiratory complex I, or simply “complex I”) is the first enzymatic complex of the respiratory chain (Yoga et al, 2021, the whole of which is incorporated herein by reference). Complex I is a very large protein complex containing 45 highly conserved subunits in eukaryotes and prokaryotes (Gutierrez-Fernandez, 2020). In eukaryotes, it links electron transfer from NADH to coenzyme ubiquinone (Q) with proton transposition across the inner mitochondrial membrane, although the mechanism linking spatially distinct proton transposition and electron transfer remains unknown (Gutierrez-Fernandez, 2020).

[0003] In humans, the most common form of ubiquinone is known as "Q10" because it has 10 isoprenyl subunits in its "tail" region. Q10 is hydrophobic and enters the complex I enzyme from the inner mitochondrial membrane via a long binding channel often called the "Q tunnel" (Bridges et al, 2020). The Q tunnel is long and inherently heterogeneous. While various compounds are known to bind within the Q tunnel, there is no single consensus site for compound binding. For example, piericidine A has been reported to bind within the Q tunnel as a "short form" of ubiquinone with interactions at multiple residues from the top of the Q tunnel to its midpoint (Gutierrez-Fernandez, 2020, Bridges et al, 2020, Chung et al, 2021, each of which is incorporated herein by reference in whole). Quinone-like compounds such as oleotin and pyridabene have also been observed to bind to similar sites in T. thermophilus (Gutierrez-Fernandez, 2020; Chung et al, 2021). IACS-2858 and BAY-87-2243 function like a "cork in a bottle," binding to clusters of residues of subunits ND1 and NDUFS7 in the central charged region of the ubiquinone-binding pocket of mouse complex I (Chung et al, 2021; Kurelac et al, 2022). Biguanides such as metformin are similarly thought to interact with Phe244 of ND1 and Arg77 of NDUFS7 in the Q tunnel in a manner dependent on the activity or inactivity state of the enzyme. Furthermore, evidence suggests that biguanides may be non-selective in binding, having putative additional binding sites on complex IV, another subunit of the electron transport chain (LaMoia et al, 2021). The binding sites of several complex I inhibitors have been outlined by Schiller and Zickermann (2022).

[0004] Classical C1 inhibitors, as described above, often induce cytotoxicity and cell death. For example, piericidine A is insecticidal and antibacterial, pyridaben is a miticide (kills ticks and mites), and aureotin exhibits antitumor, antifungal, and insecticidal activity. IACS-010579 and IM156 (also known as HL156A) have been reported to have antitumor effects, as they significantly affect cancer cell survival (Tsogbadrakh et al, 2018 and Izreig et al, 2020). However, despite these properties, none of the known classical C1 inhibitors are used as approved therapeutic agents. Indeed, these drugs exhibit mechanism-based toxicity (Yap, T et al, 2023) and have been used to induce disease in mouse models of neurodegenerative conditions such as Parkinson's disease (PD; Xiong et al, 2012, the whole of which is incorporated herein by reference) and Alzheimer's disease (AD; Joh et al, 2017, the whole of which is incorporated herein by reference). Thus, identifying a suitable approach for the therapeutic use of classical complex I inhibitors that provides benefits without toxicity has proven challenging. Alternative approaches that can produce benefits without adverse effects may have potential benefits in the treatment of various progressive diseases.

[0005] *** The goal of therapeutic tissue repair is to restore tissue to its original structural and functional state (Krafts, 2010 and Paul and Sharma, 2021). Achieving this goal has proven difficult, with the only effective examples being organ transplantation or surgical implants using natural or biomimetic structures, such as aortic valves or joint replacements. Furthermore, while research into strategies for achieving repair in different organ systems has increased significantly, these approaches aim to eliminate the primary drivers of tissue damage (e.g., calcified heart valves) or replace a dysfunctional matrix environment with one that favors homing of repair cells or local enhancement of soluble repair-promoting factors (e.g., fibrin). Examples include biomimetic scaffolds in orthopedics that simulate normal structural matrices for cell homing, and cell products that simulate soluble matrix microenvironments for skin and eye wound repair. Thus, identifying therapeutic agents that attenuate the drivers of tissue damage while simultaneously promoting a repair-promoting microenvironment has proven challenging.

[0006] Tissue repair (healing) is a highly coordinated and complex process involving a series of overlapping events that occur at precisely the right times. This process has three broad stages, first described in the 19th century (Virchow, 1859), and has since been enhanced by additional data on their cellular genotypes, phenotypes, and molecular mediators (Liehn, 2011, Takeo, 2015, and Somer et al, 2021). However, available data on the mechanisms supporting repair in most chronic disease settings are limited, if any (Peyrin-Biroulet, 2020), and the current understanding of the tissue repair process has not led to substantial improvements in the clinical care of tissue injury (Eming et al, 2014).

[0007] As shown in Figure 1, the three stages of tissue repair are as follows: 1. Inflammation 2. Proliferation (fibrosis and angiogenesis) 3.Organizational restructuring (Lokmic et al, 2012) In a healthy functional healing / repair process, these stages proceed in an overlapping sequence, with inflammation dissipation occurring in parallel with proliferation and matrix reconstruction, restoring tissue structure. In chronic disease states, this process is interrupted or dysregulated, leading to persistent, non-healing tissue damage.

[0008] Functional repair involves the dissipation of the primary inflammatory response to injury and the simultaneous activation of the inflammatory response resulting from the repair. These stages exhibit different qualitative and dynamic characteristics; the first stage induces a proliferation response in both infiltrating and resident cells, while the second stage involves the repolarization of infiltrating cells. Furthermore, cells involved in the repair process exhibit different responses to stress depending on the stage of repair progression. For example, myeloid immune cells (short-lived cells with high bioenergy demands) respond to stress by reducing proliferation and activating apoptotic pathways, so that the overall myeloid phenotype is anti-inflammatory, and in the context of repair, the reduced population exhibits immunomodulatory effects. In contrast, mesenchymal and epithelial cells respond to similar microenvironmental stressors by activating effector pathways. For example, mesenchymal cells typically respond to hypoxic partial pressure (hypoxia) by expanding and activating the repair phenotype.

[0009] There is an urgent need for therapies that exhibit a dual pharmacological approach: supporting tissue repair while reducing disease progression in chronic, progressive diseases that worsen over time without medical intervention. Notable examples include autoimmune diseases such as rheumatoid arthritis (RA), psoriasis, and inflammatory bowel disease (IBD), as well as progressive fibroses such as idiopathic pulmonary fibrosis (IPF), non-alcoholic fatty liver disease (NAFLD) / non-alcoholic steatohepatitis (NASH), and chronic kidney disease. Without treatment, these conditions typically worsen over time and can be fatal, as in the case of pulmonary fibrosis.

[0010] Current treatments for these conditions inhibit disease progression. However, generally, these drugs typically fail to promote tissue repair by directly influencing the repair process through effector responses. In fact, their suppression of pro-inflammatory effector cellular and molecular responses may progress cyclically over months or years. For example, in rheumatoid arthritis, persistent inflammation within bone erosions hinders healing (Berardi et al, 2021), while similar unresolved inflammation hindering repair is seen in chronic skin wounds (Li et al, 2021), ulcerative colitis, and Crohn's disease. An example of a condition in which effective anti-inflammatory effects from therapeutic drugs do not lead to effective repair is: • Suppression of synovitis and pannus formation does not result in complete suppression of erosion and bone loss in rheumatoid arthritis. • Suppression of mucosal inflammation does not lead to complete suppression of ulcers in inflammatory bowel disease. Examples include neurodegenerative diseases such as multiple sclerosis (MS), where suppression of the inflammatory drive to demyelination does not result in a simultaneous increase in oligodendrocyte or Schwann cell-induced remyelination. Furthermore, in other chronic diseases, the dynamic interaction between the drive to damage and the continuous activation of repair often leads to the generation of "futile" repair, i.e., inadequate repair of tissue reconstruction to a normal state. Such futile repair is seen in settings such as osteoarthritis and fibrosis.

[0011] The result of either a failed or futile repair is that most patients with chronic diseases experience persistent symptoms and disease progression despite treatment. Therefore, there is an urgent need for a treatment with dual pharmacology that can act on both the inflammatory and repairing arms of the lesion. Such a treatment would be... • It exhibits pharmacologically dependent differential transduction of microenvironmental stress signals. • Reduce the drive to injury, while simultaneously enhancing and utilizing the stressor events that constitute the inflammatory phase of tissue repair. • To restore key aspects of tissue function, repair should be coordinated in an anatomically appropriate manner (Eming et al, 2014).

[0012] In addition, pharmacological interventions can induce cellular changes that are consistent with the cellular changes necessary to modulate a controlled repair response. For example, this approach can induce controlled production of major basement membrane collagen IV, along with the production of growth factors crucial for angiogenesis, epithelialization, and matrix remodeling, such as VEGF, FGF21, and GDF15. If successful, this approach would induce repair in a pathology-independent manner; that is, the repair response would be observed in multiple settings, regardless of the nature of the original injury. Moreover, it can alter the activation response of commensal cells, desensitizing the microenvironment to the effects of pro-inflammatory cell infiltration and thus "allowing" repair.

[0013] One proposed approach to triggering a tissue repair response was to repair the cells first (Fu, 2021). Induction of the integrated stress response (ISR) may be an effective means of achieving this goal. The ISR is a cytoprotective mechanism that maintains cellular proteostasis (i.e., protein homeostasis) in response to stress conditions. The ISR is highly conserved across cell types and is induced in response to changes in mitochondrial function (Savu and Moisoi, 2022). Controlled ISR activation has been shown to have beneficial effects in several disease settings. For example, in a mouse model of multiple sclerosis, the ISR can be used to protect oligodendrocytes and myelin during inflammation (Way and Popko, 2016). In addition, ISR has been shown to regulate the health of cardiac progenitor cells by removing unhealthy cells, thereby preventing their differentiation and self-regeneration (Searfoss et al, 2019), a property that may be shared among progenitor cells in other settings, such as oligodendrocyte progenitor cells in the brain and spinal cord, type II alveolar epithelial cells (AT2) in lung and mesenchymal stem cells, and myeloid progenitor cells. The role of ISR in obesity, neurodegeneration, and heart failure has also been proposed. Importantly, to achieve these outcomes, ISR should be moderately and strictly regulated, such as riostat, to avoid directing cells toward apoptosis (Kaspar et al, 2021).

[0014] Drugs that can both control symptoms and induce tissue repair / healing may offer greater benefits to patients than existing therapies and may result in improved treatment outcomes.

[0015] *** Tumor necrosis factor alpha (formerly known as TNF-α, and referred to herein as TNF) is a cytokine with multifaceted effects on various cell types. TNF is a major regulator of inflammation, and dysregulation and / or hyperstimulation of TNF can lead to the development of pathological conditions such as chronic inflammation and various autoimmune diseases. For example, TNF overproduction is associated with rheumatoid arthritis (RA), psoriatic arthritis (PsA), psoriasis, inflammatory bowel disease (IBD), Crohn's disease (CD), and non-infectious uveitis (NIU) (Jang, DI., et al 2021).

[0016] Due to the involvement of TNF in the aforementioned diseases, several TNF inhibitors have been studied and are clinically applied, for example, for the treatment of CD and RA. These drugs include antibody-based therapies such as Remicade (infliximab), Humira (adalimumab), Cimzia (certolizumab pegol), and Simponi (golimumab), as well as the fusion protein-based therapy Enbrel (etanercept). However, despite their widespread availability, these therapies suffer from several limitations. For example, they are associated with high failure rates, diminished response, and the inability to achieve the ultimate goal of therapy, which is to induce deep and sustained remission that would allow the therapy to eventually be removed. Patients receiving these drugs further experience persistent symptoms and disease progression, as well as the potential for serious adverse effects such as increased infection rates and malignancies.

[0017] *** The inventors have previously discovered that compounds capable of binding to and regulating the activity of mitochondrial complex I (see European Patent Application No. 23162131.9, incorporated herein by reference) are beneficial for the treatment of inflammatory and / or progressive diseases such as inflammatory bowel disease (IBD), interstitial lung disease, or pulmonary fibrosis, multiple sclerosis (MS), or rheumatoid arthritis (RA). The inventors have demonstrated that such compounds can slow and even prevent disease progression in mice with the condition. However, even more surprisingly, there has been observation that these compounds further promote the repair of affected tissue, thereby reversing disease progression.

[0018] While this newly discovered class of compounds offers a promising new avenue for treating inflammatory and / or progressive diseases, there remains a need to improve the effectiveness of these new drug therapy regimens to further enhance patients' quality of life.

[0019] This invention has been devised in light of the above considerations. [Overview of the Initiative]

[0020] Current treatments for chronic and progressive conditions inhibit disease progression but fail to promote tissue repair. Therefore, patients with chronic diseases experience persistent symptoms and progression, as well as a reduced quality of life. Despite the allure of restoring normal tissue structure as a means of treating chronic diseases, pharmacological interventions to achieve this have not been studied, and consequently, practical applications of this approach do not yet exist. One potential approach to achieving pharmacological tissue repair lies in altering mitochondrial function. However, current literature has shown that inducing alterations in mitochondrial function inhibits repair and promotes inflammation (Cai et al, 2022), and in practice, alterations in mitochondrial function that alter disease progression through the control of inflammation, i.e., tissue remodeling, have not been extensively studied. The mitochondrial complex I modulator (MCIM) compounds disclosed in this invention and EP23162131.9 modulate the activity of mitochondrial complex I in a manner distinct from conventional complex I inhibitors. This leads to a selection of cellular fate and triggers adaptive stages that mimic the microenvironment, such as wound repair. Phenotypically, this can control inflammation, alter the activation response of commensal cells, desensitize the microenvironment to the effects of pro-inflammatory cell infiltration, and simultaneously initiate repair signals in affected tissues such as the lungs and joints. In addition, MCIM compounds can stimulate the production of major growth factors such as VEGF, as well as collagen I and basal collagen IV. Collectively, these mechanisms support the reduction of inflammation and the restoration of tissue structure in multiple organ / tissue settings.

[0021] In addition to the effects of MCIM compounds described in EP23162131.9, the inventors have further found that MCIM compounds can be used in combination with TNF inhibitors to further improve the treatment of diseases in which TNF dysregulation / hyperstimulation plays a role. Such combinations result in remarkable synergistic improvements in the level of protection against disease pathology progression and observed tissue repair compared to when either the MCIM compound or the TNF inhibitor is administered alone. For example, when mice were treated with the MCIM compound and the TNF inhibitor etanercept of the present invention, a synergistic reduction in the level of bone damage was observed in a mouse model of arthritis.

[0022] In its broadest sense, the present invention provides combinations of MCIM compounds and TNF inhibitor compounds. These combinations may be used for therapeutic purposes. The MCIM compounds and TNF inhibitor compounds may be administered separately, sequentially, or simultaneously, or in any order. When used to treat inflammatory and / or progressive diseases in which TNF plays a role in disease pathology, these combinations may achieve restorative effects. Preferably, the MCIM compounds can bind to complex I and modulate the function of complex I.

[0023] Accordingly, in a first aspect, the present invention provides a combination of pharmaceuticals comprising a mitochondrial complex I modulator (MCIM) compound and a TNF inhibitor compound. The combination of pharmaceuticals may be formulated as a single composition comprising the MCIM compound and the TNF inhibitor compound. The combination of pharmaceuticals may also be formulated as two separate compositions, each of which comprises either the MCIM compound or the TNF inhibitor compound. In some embodiments, the combination may comprise two or more TNF inhibitor compounds. If the combination comprises two or more TNF inhibitor compounds, the combination of pharmaceuticals may comprise a single composition comprising the two or more TNF inhibitor compounds, or the two or more TNF inhibitor compounds may comprise separate compositions.

[0024] In a second aspect, the present invention provides a pharmaceutical composition comprising a mitochondrial complex I modulator (MCIM) compound and a TNF inhibitor compound.

[0025] In a third embodiment, the combination according to the first embodiment or the composition according to the second embodiment may be used as a pharmaceutical agent.

[0026] In a fourth aspect, the present invention provides a pharmaceutical composition comprising a mitochondrial complex I modulator (MCIM) compound for use in the treatment of a target inflammatory and / or progressive disease. The treatment comprises separate, sequential, or concurrent administration of the pharmaceutical composition and the TNF inhibitor compound to the target. The treatment may achieve disease control, elimination, or tissue repair, or any combination thereof.

[0027] In a fifth aspect, the present invention further provides a pharmaceutical composition comprising a TNF inhibitor compound for use in the treatment of a target inflammatory and / or progressive disease. The treatment comprises the individual, sequential, or concurrent administration of the pharmaceutical composition and the mitochondrial complex I modulator (MCIM) compound to the target. The treatment may achieve disease control, elimination, or tissue repair, or any combination thereof.

[0028] In a related sixth aspect, the present invention further provides a pharmaceutical composition comprising a mitochondrial complex I modulator (MCIM) compound and a TNF inhibitor compound for use in the treatment of a target inflammatory and / or progressive disease. The treatment comprises administering the composition to a target. The treatment may achieve disease control, elimination, or tissue repair, or any combination thereof.

[0029] In a seventh aspect, the present invention further provides a method for treating an inflammatory and / or progressive disease of a subject, the method comprising administering a pharmaceutical composition comprising an MCIM compound to the subject. The method further comprises separate, sequential, or concurrent administration of a TNF inhibitor compound to the subject. The treatment may achieve disease control, elimination, or tissue repair, or any combination thereof.

[0030] In an eighth aspect, the present invention further provides a method for treating an inflammatory and / or progressive disease of a subject, the method comprising administering a pharmaceutical composition comprising a TNF inhibitor compound to the subject. The treatment further comprises separate, sequential, or concurrent administration of MCIM compounds to the subject. The treatment may achieve disease control, cessation, or tissue repair, or any combination thereof.

[0031] In a ninth aspect, the present invention further provides a method for treating an inflammatory and / or progressive disease of interest, the method comprising administering a pharmaceutical composition comprising an MCIM compound and a TNF inhibitor compound to the subject. The treatment may achieve disease control, elimination, or tissue repair, or any combination thereof.

[0032] In some embodiments of the present invention, the treatment induces disease control, remission, or tissue repair, or any combination thereof. In some embodiments, the treatment achieves disease control, remission, or tissue repair, or any combination thereof. In some embodiments, the treatment initiates an adaptive response in a particular cell type that leads to pharmacodynamic evidence of disease control, remission, or tissue repair, or any combination thereof. In some embodiments, the adaptive response results in tissue repair and / or disease remission. In some embodiments, tissue repair and / or disease remission induces the restoration of tissue structure toward its healthy state, characterized by anatomically normal structure. In some embodiments of the present invention, disease control includes inhibition of disease progression. In some embodiments, inhibition of disease progression includes prevention of disease progression. In some embodiments, inhibition of disease progression includes a reduced rate of disease progression. In some embodiments, disease control includes prevention of loss of anatomically normal tissue structure or a reduced rate of loss of anatomically normal tissue structure. In some embodiments, tissue repair and / or disease remission is characterized by an increased clinical repair score and / or increased wound healing.

[0033] Inflammatory and / or progressive diseases can be any condition in which there is an imbalance between cellular pathology and cellular repair. In some embodiments, inflammatory and / or progressive diseases are associated with or caused by the overexpression, secretion, or stimulation of tumor necrosis factor (TNF). In some embodiments, inflammatory and / or progressive diseases are autoimmune diseases. In some embodiments, the disease or disorder may be an autoimmune disorder such as rheumatoid arthritis (RA), inflammatory bowel disease (IBD), ulcerative colitis, or Crohn's disease; a fibrous condition such as interstitial lung disease; a neurological disorder such as multiple sclerosis (MS) or amyotrophic lateral sclerosis (ALS); or a bone disorder such as osteoarthritis or osteoporosis. In some embodiments, the disease or disorder may be RA, psoriatic arthritis, inflammatory arthritis, ankylosing spondylitis, juvenile idiopathic arthritis, reactive arthritis, gout, septic arthritis, colitis, or osteoarthritis. In some embodiments, tissue repair and / or disease resolution are characterized by an increased clinical repair score and / or increased wound healing.

[0034] Clinical repair scores can be used to objectively assess clinical repair in inflammatory and / or progressive diseases. An increased clinical repair score can be indicated by a decreased disease score. In some embodiments, disease control is characterized by a decrease in the rate of change of the disease score. In some embodiments, disease control is characterized by no change in the disease score after treatment. For example, disease control may be characterized by inhibition of disease progression associated with an increase in the disease score. The disease score can be calculated using any preferred method known in the art. For example, in some embodiments where the disease is arthritis, tissue repair and / or disease clearance may be characterized by a decrease in the mean arthritis index score, ACR / EULAR score, DAS38 score, HAQ-DI score, CDAI score, SDAI score, ACR20 / 50 / 70 score, EULAR score, mTSS score, or RAPID3 score, or any combination thereof, as described herein. In some embodiments, tissue repair and / or disease resolution may be characterized by i) a decrease in serum concentration of C-reactive protein (CRP), ii) a decrease in serum concentration of tartrate-resistant acid phosphatase 5 (TRAP5), iii) an increase in serum concentration of procollagen 1 intact N-terminus (P1NP), iv) an increase in serum concentration of osteocalcin, or v) any combination of i) to iv).

[0035] In some embodiments, disease control or remission, and / or tissue repair, includes an increased cell count of repair cells and / or a decreased cell count of pathologically driven cells. In some embodiments, disease control or remission, and / or tissue repair remission, includes an increased count of mesenchymal and / or epithelial cells. In some embodiments, the adaptive response is characterized by an increased count of mesenchymal and / or epithelial cells. In some embodiments, the adaptive response is characterized by increased differentiation of mesenchymal and / or epithelial cells. For example, if the inflammatory and / or progressive disease is IBD, repair cells may include epithelial and / or myxoid cells. For example, in the gastrointestinal tract, cells may include fibroblasts or epithelial cells, or alternatively, repair may be indicated by an increased overall number of myxoid cells, indicating normal progression of epithelial differentiation. In bone, an increase in the number or activity of osteoblasts and / or myeloid cells, such as M2 macrophages, may be observed. In addition, in the bone, a decrease in the number or activity of osteoclasts, transformed fibroblasts, fibroblast-like synovial cells, pro-inflammatory macrophages, effector memory T cells, plasmacytoid dendritic cells, or transformed fibroblasts, or any combination thereof, may be observed.

[0036] In some embodiments, the adaptive response involves changes in the function of mesenchymal or epithelial cells. For example, in IBD, cells may include PAS-positive cells or surfactants that produce epithelial cells. In some embodiments, tissue repair and / or disease clearance involves a reduced cell count or reduced function of activated immune cell subtypes. For example, if the inflammatory and / or progressive disease is RA, disease control, tissue repair, and / or disease clearance may involve a decrease in the number of myeloid cells such as macrophages or osteoclasts, lymphocytes such as T-, B-, or Th17 cells, or fibroblasts such as FLS cells. Preferably, a decrease in immune cell activity or the number of activated immune cells does not involve a decrease in the activity, function, or number of corresponding mesenchymal or epithelial cells. If the inflammatory and / or progressive disease is MS, repair cells may include oligodendrocyte progenitor cells (OPCs).

[0037] In some embodiments, tissue repair and / or disease remission induces the restoration of tissue structure toward a healthy state characterized by anatomically normal structure. For example, in some embodiments, the inflammatory and / or progressive disease is RA, and tissue repair and / or disease remission optionally includes increased osteogenesis and / or decreased bone resorption, along with reduced edema and / or erythema. In some embodiments, disease control includes reducing bone resorption and / or preventing further loss of anatomically normal structure. In some embodiments, disease control, tissue repair, and / or disease remission includes reducing inflammatory cytokine production from pro-inflammatory myeloid cells. In some embodiments, disease control, tissue repair, and / or disease remission includes increasing growth factors important for angiogenesis, epitheliogenesis, and matrix remodeling, such as VEGF, FGF21, and / or GDF15. Cytokine and growth factor levels can be measured by any preferred method, e.g., by ELISA or ELISpot as described herein. In some embodiments, disease control, tissue repair, and / or disease remission includes increasing basal collagen IV. Collagen IV levels can be measured by any preferred method, for example, by ELISA and / or immunohistochemical analysis as described herein.

[0038] In connection with this, the present invention also provides a pharmaceutical composition comprising an MCIM compound for use in combination with a TNF inhibitor to increase repair cells and / or decrease destructive cells in subjects having inflammatory and / or progressive diseases, thereby achieving disease control and / or tissue repair, and consequently, disease, disappearance, or cessation, or symptom control and improvement of quality of life. In connection with this, the present invention also provides a pharmaceutical composition comprising an MCIM compound and a TNF inhibitor for use in combination with an MCIM compound to increase repair cells and / or decrease destructive cells in subjects having inflammatory and / or progressive diseases, thereby achieving disease control and / or tissue repair, and consequently, disease, disappearance, or cessation, or symptom control and improvement of quality of life. In connection with this, the present invention also provides a pharmaceutical composition comprising an MCIM compound and a TNF inhibitor to increase repair cells and / or decrease destructive cells in subjects having inflammatory and / or progressive diseases, thereby achieving disease control and / or tissue repair, and consequently, disease, disappearance, or cessation, or symptom control and improvement of quality of life. In some embodiments, the pharmaceutical composition induces an adaptive response in subjects having inflammatory and / or progressive diseases. The present invention also provides methods for increasing repair cells and / or decreasing destructive cells in subjects with inflammatory and / or progressive diseases, which include administering a pharmaceutical composition to achieve disease control, tissue repair, and / or disease remission or elimination, or improved symptom control and quality of life. In some embodiments, the methods increase the adaptive response of mesenchymal or epithelial cells and / or decrease the activation or number of inflammatory / fibrous / erosive cells in subjects with inflammatory and / or progressive diseases. In connection with this, the present invention also provides pharmaceutical compositions for use in reducing cytokine production from pro-inflammatory myeloid cells in subjects with inflammatory and / or progressive diseases to achieve disease control, tissue repair, and / or disease remission. The present invention also provides methods for reducing cytokine production from pro-inflammatory myeloid cells in subjects with inflammatory and / or progressive diseases, which include administering a pharmaceutical composition to achieve disease control, tissue repair, and / or disease remission.Compounds and inflammatory and / or progressive diseases are defined herein.

[0039] Preferably, the compound binds to complex I and modulates its activity. Modulation of complex I activity can be determined by detecting a reduction in cellular O2 consumption. In some embodiments, the reduction in cellular O2 consumption is not associated with a decrease in cell viability. O2 consumption can be measured by any standard technique known in the art, for example, using a real-time cell metabolism analyzer (e.g., Seahorse Analyzer). Modulation of complex I activity can also result in a reversible reduction of cell proliferation. Reversibility of the reduction in cell proliferation means that the reduction in proliferation is reversed when the compound is removed. This is in contrast to the reduction in proliferation observed after treatment of the same cell type with classical complex I binders, where the reduction in cell proliferation is not reversed by the removal of classical complex I binders. To assess the reversibility of the reduction in cell proliferation, MCIM is applied to a cell culture at a concentration that substantially reduces cell proliferation for 24 hours at 37°C / 5% CO2. After 24 hours, the cell culture is washed and cultured under conditions that promote cell growth and proliferation. The recovery of cell proliferation is measured after 24 hours of incubation under these “growth” conditions (37°C / 5% CO2, MCIM absent). A recovery of cell proliferation is observed. This is in contrast to observations made with the same cells that underwent the same culture / wash / growth cycle as the classical complex I binder in the culture process. The reversible reduction of cell proliferation can be evaluated using human primary lung fibroblasts, for example, as described in Example 4 (as shown in Figure 6).

[0040] Preferably, the compound interacts with complex I at a binding site on the upper or outer side of the Q tunnel. In some embodiments, the binding site includes one or more amino acid residues from NDUFS2 (SEQ ID NO: 1) and / or NDUFS7 (SEQ ID NO: 2). In some embodiments, the compound interacts with at least one amino acid residue in NDUFS2 (SEQ ID NO: 1), for example, His92, Gly85, Tyr141, His88, Leu95, Asp193, or Phe458. In some embodiments, the compound interacts with one or more amino acid residues in NDUFS2 (SEQ ID NO: 1) selected from Tyr141, His92, and Asp193.

[0041] In some embodiments, the response may include the enhancement of a repair phenotype over the same time course as the control of inflammation. The control of inflammation induced by the compounds of the present invention may differ from the control of inflammation induced by other anti-inflammatory agents that rely on the suppression of inflammation before the activation of tissue repair as a secondary effect.

[0042] In some embodiments, the compound includes four or more pharmacophore features from the pharmacophore model shown in Figure 24. The three-dimensional arrangement of the pharmacophore features may be as shown in Tables 7-A, 7-B, and 7-C.

[0043] In some embodiments, the compound is a compound of the following formula, or a pharmaceutically acceptable salt, hydrate, or solvate thereof: [ka] As defined in claim 1 of WO2010 / 032009, the following: During the ceremony, -A is independently the following: [ka] -Ar is independently phenyl, pyridinyl, or pyrimidinyl. p is an independent integer between 0 and 3. q is independently an integer from 0 to 3, -R SN is independently -H or saturated aliphatic C 1-4 alkyl, -DQ is independently -D 1- Q 1 or -D 2 =O, -D 1- is independently cyclopentane-di-yl, cyclohexane-di-yl, cycloheptane-di-yl, bicyclo[3.1.1]heptane-di-yl, or bicyclo[3.2.1]octane-di-yl, optionally substituted with one or more groups -R D and -D 2 = is independently cyclopentane-yl-idene, cyclohexane-yl-idene, cycloheptane-yl-idene, bicyclo[3.1.1]heptane-yl-idene, or bicyclo[3.2.1]octane-yl-idene, optionally substituted with one or more groups -R D and each -R D is independently -F, -Cl, -Br, -I, -R DD , -CF3, -OH, -OR DD , -NH2, -NHR DD , and -NR DD 2, each -R DD is independently saturated aliphatic C 1-4 alkyl, -Q 1 is independently selected from

Chemical formula

[0044] Claim 1 of WO2010 / 032009 is incorporated herein by reference. Furthermore, WO2010 / 032009 is incorporated herein by reference in its entirety.

[0045] In one embodiment, the compound is a compound as defined in WO2014 / 207445A1 (which is incorporated herein by reference in its entirety), or a pharmaceutically acceptable salt, hydrate, or solvate thereof. For example, in some embodiments, the compound is a compound selected from the following formulas, or a pharmaceutically acceptable salt, hydrate, or solvate thereof: [ka] [ka]

[0046] In some embodiments, the compound is a compound of the following formula, or a pharmaceutically acceptable salt, hydrate, or solvate thereof: [ka]

[0047] In one embodiment, the compound is a compound as defined in WO2016 / 097001A1 (which is incorporated herein by reference in its entirety), or a pharmaceutically acceptable salt, hydrate, or solvate thereof. For example, in some embodiments, the compound is a compound selected from the following formulas, or a pharmaceutically acceptable salt, hydrate, or solvate thereof: [ka]

[0048] It should be noted that, as defined in WO2014 / 207445A1 and WO2016 / 097001A1, and as stated above, for "HMC" compounds, substituents on one side of the cyclohexyl ring (i.e., the right-hand OH and CH3) may be in a "trans" / "cis" or "cis" / "trans" configuration relative to the rest of the molecule (i.e., the rest of the compound bonded at the para position of the cyclohexyl ring on the cyclohexyl ring to which they are bonded). [ka]

[0049] Unless otherwise indicated, all such conformations are intended to be encompassed by references to compounds that do not specify a particular conformation.

[0050] In some embodiments, the compound is a compound of the following formula, or a pharmaceutically acceptable salt, hydrate, or solvate thereof: [ka] (As defined in claim 1 of WO2010 / 032010), in the formula, -A is independently the following: [ka] -Ar is independently phenyl, pyridinyl, or pyrimidinyl. p is an independent integer between 0 and 3. q is an independent integer between 0 and 3. -R SN These are independently -H or saturated aliphatic C 1-4 It is alkyl, -R S1 These are independently -H or saturated aliphatic C 1-4 It is alkyl, -R S2 These are independently -H or saturated aliphatic C 1-4 It is alkyl, -R S3 These are independently -H or saturated aliphatic C 1-4 It is alkyl, -R S4 These are independently -H or saturated aliphatic C 1-4 It is alkyl, -Q is independently selected from the following: [ka] During the ceremony, Each-R 1N These are independently -H or -R CN And, Each-R 2N These are independently -H or -R CN And, Each-R CN These are, independently, saturated aliphatic C 1-4 Is it alkyl? or -NR 1N R 2N These are independently azetidino, pyrrolidino, imidazolidino, pyrazolidino, piperidino, piperarino, morpholino, thiomorpholino, azepino, or diazepino, each of which is optionally saturated aliphatic C 1-4 Substituted with one or more groups independently selected from alkyl groups, -R 1A These are independently -H and -R C , or -R F And, -R 2A These are independently -H and -R C , or -R F Is it, or -R 1A and -R 2A Together, saturated aliphatic C 2-4 Forms an alkylene group, -R 3A -R is independent of the above. C , -R F , or -R J And, -R 4A These are independently -H and -R C , or -R F Is it, or -R 3A and -R 4A Together, saturated aliphatic C 2-4 Forms an alkylene group, -R 5A -R is independent of the above. C or -R F And, -R 6A These are independently -H and -R C , or -R F Is it, or -R 5A and -R 6A Together, saturated aliphatic C 2-4 Forms an alkylene group, -R 1B These are independently -H and -R C , or -R F And, -R 2B These are independently -H and -R C , or -R F Is it, Or -R 1B and -R 2B Together, saturated aliphatic C 2-4 Forms an alkylene group, -R 3B These are independently -H and -R C , -R F -OH, or -OR O And, -R 4B These are independently -H and -R C , or -R F Is it, Or -R 3B and -R 4B Together, saturated aliphatic C 2-4 Forms an alkylene group, -R 5B These are independently -H and -R C , or -R F And, -R 6B These are independently -H and -R C , or -RF Is it, Or -R 5B and -R 6B Together, saturated aliphatic C 2-4 Forms an alkylene group, Each-R C These are, independently, saturated aliphatic C 1-4 It is alkyl, Each-R F These are, independently, saturated aliphatic C 1-4 It is a fluoroalkyl, -R O These are, independently, saturated aliphatic C 1-4 It is alkyl, -R J These are independently -NH2 and -NHR JN1 ,- NR JN1 2, or -NR JN2 R JN3 And, Each-R JN1 -R is independent of the above. J1 , -R J2- OH, -R J2- Ure J1 And, Each-R J1 These are, independently, saturated aliphatic C 1-4 It is alkyl, Each-R J2- These are, independently, saturated aliphatic C 2-4 It is alkylene, -NR JN2 R JN3 These are independently azetidino, pyrrolidino, imidazolidino, pyrazolidino, piperidino, piperarino, morpholino, thiomorpholino, azepino, or diazepino, each of which is optionally saturated aliphatic C 1-4 Substituted with one or more groups independently selected from alkyl groups, Each-R X Independently, -F, -Cl, -Br, -I, -R XX , -OH, -OR XX , -SH, -SR XX , -CF3、-OCF3、-SCF3、 -NH2、-NHR XX 、-NR XX 2、-NR YY R ZZ 、 -C(=O)R XX 、-OC(=O)R XX 、 -C(=O)OH、-C(=O)OR XX 、 -C(=O)NH2、-C(=O)NHR XX 、-C(=O)NR XX 2、-C(=O)NR YY R ZZ 、 -OC(=O)NH2、-OC(=O)NHR XX 、-OC(=O)NR XX 2、-OC(=O)NR YY R ZZ 、 -NHC(=O)R XX 、-NR XX C(=O)R XX 、 -NHC(=O)OR XX 、-NR XX C(=O)OR XX 、 -NHC(=O)NH2、-NHC(=O)NHR XX 、-NHC(=O)NR XX 2、-NHC(=O)NR YY R ZZ 、 -NR XX C(=O)NH2、-NR XX C(=O)NHR XX 、-NR XX C(=O)NR XX 2、-NR XX C(=O)NR YY R ZZ 、 -CN、 -NO2、 -S(=O)2NH2、-S(=O)2NHR XX 、-S(=O)2NR XX 2、-S(=O)2NR YY R ZZ 、 -S(=O)R XX -S(=O)2R XX -OS(=O)2R XX -S(=O)2OH, or -S(=O)2OR XX And, During the ceremony, Each-R XX These are, independently, saturated aliphatic C 1-6 Alkyl, phenyl, or benzyl, wherein the phenyl and benzyl are optionally -F, -Cl, -Br, -I, -CF3, -OCF3, -R XXX -OH, -OR XXX , or -SR XXX Replaced with one or more groups selected from, each -R XXX These are, independently, saturated aliphatic C 1-4 It is alkyl, Each-NR YY R ZZ These are independently azetidino, pyrrolidino, imidazolidino, pyrazolidino, piperidino, piperarino, morpholino, thiomorpholino, azepino, or diazepino, each of which is optionally saturated aliphatic C 1-4 It is substituted with one or more groups independently selected from the alkyl group.

[0051] Claim 1 of WO2010 / 032010 is incorporated herein by reference. Furthermore, WO2010 / 032010 is incorporated herein by reference in its entirety.

[0052] In some embodiments, the compound is a compound of the following formula, or a pharmaceutically acceptable salt, hydrate, or solvate thereof: [ka] (As defined in claim 1 of WO2020 / 035560 A1), in the formula, =X- is independently -CH= or -N=, -R 1 These are independently -H or -R 1X And, -R 1X These are independently -F, -Cl, and -R1C , -R 1F , or -CN, -R 1C These are independently saturated linear or branched C 1-3 It is alkyl, -R 1F These are independently saturated linear or branched C 1-3 It is a fluoroalkyl, -R 2 These are independently -H or -R 2X And, -R 2X These are independently -F, -Cl, and -R 2C , -R 2F , or -CN, -R 2C These are independently saturated linear or branched C 1-3 It is alkyl, -R 2F These are independently saturated linear or branched C 1-3 It is a fluoroalkyl, -R 3 These are independently -H or -R 3X And, -R 3X These are independently -F, -Cl, and -R 3C , -R 3F , or -CN, -R 3C These are independently saturated linear or branched C 1-3 It is alkyl, -R 3F These are independently saturated linear or branched C 1-3 It is a fluoroalkyl, -R 4 These are independently -H or -R 4X And, -R 4X These are independently -F, -Cl, and -R 4C , -R 4F , or -CN, -R 4C These are independently saturated linear or branched C 1-3 It is alkyl, -R 4F These are independently saturated linear or branched C 1-3It is a fluoroalkyl, -R 5 These are independently -H or -R 5X And, -R 5X These are independently -F and -R 5C , or -R 5F And, -R 5C These are independently saturated linear or branched C 1-3 It is alkyl, -R 5F These are independently saturated linear or branched C 1-3 It is a fluoroalkyl, -R 6 These are independently -H or -R 6X And, -R 6X These are independently -F and -R 6C , or -R 6F And, -R 6C These are independently saturated linear or branched C 1-3 It is alkyl, -R 6F These are independently saturated linear or branched C 1-3 Is it a fluoroalkyl group? Or -R 5 and -R 6 Together with the carbon atoms to which they bond, they form saturated C 3-6 It forms a cycloalkyl group.

[0053] Claim 1 of WO2020 / 035560A1 is incorporated herein by reference. Furthermore, WO2020 / 035560A1 is incorporated herein by reference in its entirety.

[0054] In some embodiments, the compound is a compound of the following formula, or a pharmaceutically acceptable salt, hydrate, or solvate thereof: [ka] (As defined in claim 1 of WO2020 / 212581A1), in the formula, -X= is independently -CH= or -N=, "m" is independently 0, 1, 2, or 3. Each -RA is independently -F, -Cl, -RAC, -RAF, or -CN. -RAC is independently a saturated linear or branched C1-3 alkyl group. -RAF is independently a saturated linear or branched C1-3 fluoroalkyl group. "n" is independently 0, 1, or 2. Each -RB is independently -F, -Cl, -RBC, -RBF, or -CN. -RBC is independently a saturated linear or branched C1-3 alkyl group. -RBF is independently a saturated linear or branched C1-3 fluoroalkyl group. -R1 is independently either -H or -R1X. -R1X is independently -F, -R1C, or -R1F. -R1C is independently a saturated linear or branched C1-3 alkyl group. -R1F is independently a saturated linear or branched C1-3 fluoroalkyl group. -R2 is independently either -H or -R2X. -R2X is independently -F, -R2C, or -R2F. -R2C is independently a saturated linear or branched C1-3 alkyl group. -R2F is independently a saturated linear or branched C1-3 fluoroalkyl group. Alternatively, -R1 and -R2, together with the carbon atoms to which they are bonded, form a saturated C3-6 cycloalkyl group. -R3 is independently either -H or -R3X. -R3X is independently -R3C or -R3F. -R3C is independently a saturated linear or branched C1-3 alkyl group. -R3F is independently a saturated linear or branched C1-3 fluoroalkyl group. -R4 is independently -R4C, -R4CC, or -N(R4N1)(R4N2), -R4C is independently a saturated linear or branched C1-6 alkyl group. -R4CC is independently a saturated C3-6 cycloalkyl, -R4N1 is independently -H or -R4N1C. -R4N1C is independently a saturated linear or branched C1-4 alkyl group. -R4N2 is independently -H or -R4N2C. -R4N2C is independently a saturated linear or branched C1-4 alkyl group. Alternatively, -N(R4N1)(R4N2) is independently azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl, or morpholinyl, optionally substituted with one or more saturated linear or branched C1-4 alkyl groups.

[0055] Claim 1 of WO2020 / 212581A1 is incorporated herein by reference. Furthermore, WO2020 / 212581A1 is incorporated herein by reference in its entirety.

[0056] In some embodiments, the compound is a compound of the following formula, or a pharmaceutically acceptable salt, hydrate, or solvate thereof: [ka]

[0057] In some embodiments, the compound is a compound of the following formula, or a pharmaceutically acceptable salt, hydrate, or solvate thereof: [ka]

[0058] In some embodiments, the compound is a compound of the following formula, or a pharmaceutically acceptable salt, hydrate, or solvate thereof: [ka]

[0059] In some embodiments, the compound is a compound of the following formula, or a pharmaceutically acceptable salt, hydrate, or solvate thereof: [ka] In some embodiments, the compound is a compound of the following formula, or a pharmaceutically acceptable salt, hydrate, or solvate thereof: [ka]

[0060] In some embodiments, the compound is a compound of the following formula, or a pharmaceutically acceptable salt, hydrate, or solvate thereof: [ka]

[0061] In some embodiments, the compound is a compound of the following formula, or a pharmaceutically acceptable salt, hydrate, or solvate thereof: [ka]

[0062] In some embodiments, the compound is a compound of the following formula, or a pharmaceutically acceptable salt, hydrate, or solvate thereof: [ka]

[0063] In some embodiments, the compound is a compound of the following formula, or a pharmaceutically acceptable salt, hydrate, or solvate thereof: [ka]

[0064] In some embodiments of aspects of the invention, the TNF inhibitor compound is an antibody or a fusion protein. In some embodiments, the TNF inhibitor compound is etanercept, infliximab, adalimumab, golimumab, or certolizumab.

[0065] The present invention includes combinations of the described aspects and preferred features, except where such combinations are clearly not permitted or are explicitly avoided.

[0066] Here, embodiments and experiments exemplifying the principles of the present invention will be considered with reference to the accompanying drawings.

Brief Description of the Drawings

[0067] [Figure 1] Graph of the time-dependent response of each of three stages of repair (inflammation, proliferation, and tissue remodeling) that are proposed to be widely similar among tissues. The cell types and soluble factors involved in each stage are shown below the graph along with changes in the extracellular matrix. [Figure 2] The high-throughput integrated biological platform (BioMAP®) profiles of the MCIM compounds of the present invention against multiple disease-related regulatory pathways identified that these compounds have the potential to regulate the inflammatory response and type IV collagen, which is a tissue remodeling collagen, for ABD599 (A), and HMC-C-01-A (B) and (C). [Figure 3] The high-throughput integrated biological platform (BioMAP®) profiles of the approved TNF inhibitors against multiple disease-related regulatory pathways identified that these compounds exhibit specific and restricted inflammatory responses against adalimumab and etanercept (A) and infliximab (B). [Figure 4]Electron micrographs of human primary bone marrow cells (osteoclasts) treated with the Complex I binder of the present invention show an adaptive response through changes in mitochondrial morphology, accompanied by an increase in mitochondrial area, without a clear increase in mitochondrial mass. [Figure 5] A) Three graphs showing changes in intracellular ATP levels (top left), nuclear counts (top center), and ATP readout / cell (top right) in response to increasing doses of MCIM compounds in standard medium, glucose supplement medium (square), medium supplemented with glucose and L-glutamine (white circles), or medium supplemented with glucose, L-glutamine, and pirubate (black circles). B) Graph showing changes in VEGF secretion in response to increasing concentrations of MCIM compounds in standard medium, glucose and L-glutamine supplement medium containing pirubate (black circles), or medium containing glucose and L-glutamine but without pirubate (white circles). The MCIM compounds of the present invention promote adaptive responses by increasing VEGF secretion in metabolically restricted human primary lung fibroblasts without changing cell number. [Figure 6] Cell proliferation inhibition, as measured by BrdU incorporation (A) and nuclear count (B), as a function of increasing dose of any of the following: rotenone (square), IACS-010759 (black circle), or MCIM compound (white circle). Comparison of unwashed cells and washed cells with the test compound in cell proliferation measurements by BrdU incorporation (C) and nuclear count (D). Unwashed and washed rotenone are shown in gray and white-out gray, respectively. Unwashed and washed IACS-010759 are shown in black and white-out black, respectively. Unwashed and washed MCIM compounds are shown in checkerboard fill and white-out fill patterns, respectively. The MCIM compounds of the present invention reversibly inhibit cell proliferation, but cells treated with typical complex I inhibitors IACS-010759 and rotenone do not regain cell proliferation capacity after washing off the compounds. [Figure 7]Seven graphs showing the mean arthritis index as a function of time (day of administration) for each of the following: (A) HMS-C-02-A, (B) HMS-C-01-A, (C) HMC-N-02-A, (D) HMC-N-01-A, (E) NASMP-01-A, (F) CHMSA-01-A, and (G) CHMSA-03-A, for each test compound (white circles) and control (black circles) administered by oral force-feeding at a dose of 10 mg / kg / day. [Figure 8] Graph showing the mean bone resorption count in mice with collagen-induced arthritis, treated with (A) vehicle control, 3 mg / kg / day etanercept, or 10 mg / kg / day of any of HMC-C-01-A, HMC-C-01-B, or HMC-N-01-B, and (B) vehicle control, or 10 mg / kg / day of any of ABD900, NASMP-01, CHMSA-03-A, or NASMP-06. The MCIM compounds of the present invention protect against bone resorption in mice with collagen-induced arthritis at a level equivalent to or exceeding that of approved treatment etanercept. Data are mean ± sem. ** p<0.01 *** p<0.005 vs vehicle §§§ p<0.005 vs etanercept. [Figure 9] Graph showing (A) mean osteoid count in mice with collagen-induced arthritis treated with vehicle control, 3 mg / kg / day etanercept, or 10 mg / kg / day of HMC-C-01-A, HMC-C-01-B, or HMC-N-01-B, and (B) mean osteoid band in mice with collagen-induced arthritis treated with vehicle control or 10 mg / kg / day of ABD900, NASMP-01, CHMSA-03-A, or NASMP-06. The MCIM compound of the present invention promotes osteoid formation, an indicator of bone formation, in mice with established collagen-induced arthritis. The new bone formation in animals with existing bone erosion shows a reparative effect that occurs to a much greater extent than seen with the approved therapeutic agent etanercept. Data are mean ± sem. ** p<0.01 *** p<0.005 vs. vehicle §§§ p<0.005 vs. etanercept. [Figure 10] Histological sections of the limbs from mice with collagen-induced arthritis treated with either a vehicle control (top panel), a 10 mg / kg / day MCIM compound (middle panel), or 3 mg / kg / day etanercept (bottom panel) (160x magnification, stained with toluidine blue). The MCIM compound of the present invention promotes osteogenesis in established arthritis and exhibits an adaptive repair response. The top panel shows bone from collagen-induced arthritis mice treated with the vehicle, which shows no clear signs of osteogenesis. The middle panel shows bone from collagen-induced arthritis mice with clear signs of structured osteoid (new bone) formation, as indicated by the black arrows. In contrast, mice treated with etanercept show only low levels of responsiveness and sporadic new bone formation. [Figure 11] Graph showing relative inflammation (two bars on the left) and osteoid (two bars on the right) scores in mice with collagen-induced arthritis treated for 14 days with either a vehicle control or a very low dose of the MCIM compound at 0.03 mg / kg per day. The MCIM compound of the present invention promotes an adaptive response leading to repair (osteoid formation) in mice with established collagen-induced arthritis at doses that do not control inflammation, demonstrating that the response is an independent adaptive response rather than a consequence of inflammation control. Data are mean ± sem. *** p < 0.005 vs. vehicle. [Figure 12] Graphs showing the mean changes in (A) synovitis score, (B) cartilage damage, (C) bone resorption, and (D) osteopathology in mice with collagen-induced arthritis treated with either 1 mg / kg / day etanercept, 0.03 mg / kg / day MCIM compound, or a combination of 1 mg / kg / day etanercept and 0.03 mg / kg / day MCIM compound. The mean changes were calculated compared to mice with collagen-induced arthritis treated with a vehicle control. Data are mean ± sem. *p<0.05 **p<0.01 vs vehicle. [Figure 13]This graph shows the mean change in osteoid score in mice with collagen-induced arthritis treated with either 3 mg / kg / day of etanercept (black bars), 0.03 mg / kg / day of an MCIM compound (white bars), or a combination of 3 mg / kg / day of etanercept and 0.03 mg / kg / day (gray bars). The mean change was calculated compared to mice with collagen-induced arthritis treated with a vehicle control. The data represent an average doubling compared to the vehicle control. [Figure 14] Graph showing a comparison of disease activity indices in mice with DSS-induced colitis treated with vehicle control, sulfasalazine, etanercept, and MCIM compounds. The MCIM compounds of the present invention reduce the severity of symptoms in mice with DSS-induced colitis compared to mice treated with vehicle control, sulfasalazine, or etanercept. Data are mean ± sem. *p<0.05, ***p<0.005 vs vehicle, §§§p<0.005 vs sulfasalazine, aaa p<0.005 vs etanercept. [Figure 15] Two graphs showing a comparison of mucosal erosion in mice with DSS-induced colitis treated with (A) vehicle control, sulfasalazine, or ABD900, and (B) vehicle control, sulfasalazine, or HMC-C-01-A. The MCIM compounds of the present invention inhibit mucosal erosion to a greater extent in mice with established DSS-induced colitis compared to mice treated with vehicle control, sulfasalazine (A), or etanercept (B). Data are mean ± sem. *p<0.05 vs vehicle §p<0.05 vs sulfasalazine. [Figure 16]Two graphs showing a comparison of glandular loss in mice with DSS-induced colitis treated with (A) vehicle control, sulfasalazine, or ABD900, and (B) vehicle control, sulfasalazine, or HMC-C-01-A. The MCIM compounds of the present invention reduce glandular loss to a greater extent in mice with established DSS-induced colitis compared to mice treated with vehicle control, sulfasalazine (A), or etanercept (B). Data are mean ± sem. **p<0.01 vs vehicle, §p<0.05 vs sulfasalazine. [Figure 17] Two graphs showing a comparison of epithelial hyperplasia in mice with DSS-induced colitis treated with (A) vehicle control, sulfasalazine, or ABD900, and (B) vehicle control, sulfasalazine, or HMC-C-01-A. The MCIM compounds of the present invention can promote epithelial hyperplasia to a comparable level to mice treated with sulfasalazine (A), but to a greater extent than mice treated with etanercept (B), in established DSS-induced colitis mice. Data are mean ± sem. *p<0.05 vs vehicle. [Figure 18] Two graphs showing a comparison of fibrous proliferation indicating tissue or wound repair in mice with DSS-induced colitis treated with (A) vehicle control, sulfasalazine, or ABD900, and (B) vehicle control, sulfasalazine, or HMC-C-01-A. The MCIM compounds of the present invention promote increased "healthy" fibrous proliferation in mice with established DSS-induced colitis compared to mice treated with vehicle control, sulfasalazine (A), or etanercept (B). Data are mean ± sem. ***p<0.005 vs vehicle, §§§p<0.005 vs sulfasalazine, aaa p<0.005 vs etanercept. [Figure 19]Histological sections from mice with DSS-induced colitis treated with either a vehicle control (upper panel), etanercept (middle panel), or HMC-C-01-A (lower panel). Ulceration, structural loss, edema / inflammation, and erosion are observed in the vehicle control (arrows). Etanercept, the positive control, shows general preservation of tissue structure, but with underlying inflammation and edema (arrows). The MCIM of the present invention shows general preservation of structure, without inflammation or edema, and with a radial distribution of repair (arrows). [Figure 20] A homology model of complete Complex I constructed from publicly available structures of five different organisms. The proposed target dissipated across all five structures. [Figure 21] An in silico homology model of NDUFS2. Drug potential evaluation was performed using SiteMap, and two binding pockets were identified within the complex I subunit NDUFS2 (sphere). [Figure 22] In silico SiteFinder model of NDUFS2 in complex I. Narrow channels for drug-like compound binding in Q10 and Q tunnels were identified. Spheres are used to illustrate the space / channels around NDUFS2 in the complex I structure. [Figure 23] An in silico model of the MCIM compound of the present invention docked to the Q site of complex I. This model reveals the interaction between the complex I inhibitor of the present invention and NDUFS2, as well as additional interactions with the adjacent complex I subunit NDUFS7. [Figure 24] 3D display of a pharmacophore model constructed from an in silico model of drug docking in complex I. [Figure 25] An overlay of the MCIM compound of the present invention on a ligand-protein pharmacophore model, demonstrating successful docking of the MCIM compound of the present invention in the Q tunnel of complex I. [Figure 26]Overlay of the MCIM compound CHMSA-02-A of the present invention on a ligand-protein pharmacophore model (top panel) and chemical structure of CHMSA-02-A (bottom panel). This shows the successful docking of CHMSA-02-A in the Q tunnel of complex I. [Figure 27] A graph showing a quantitative structure-activity relationship (QSAR) model used to identify further complex I binders of the present invention and predict their activity in vivo. The predicted pActs of the complex I binders of the present invention correlate well with their experimentally validated pActs, with a coefficient of determination (R²) value of 0.8322, demonstrating that this QSAR model can be used to accurately identify the complex I binders of the present invention. [Modes for carrying out the invention]

[0068] Hereinafter, aspects and embodiments of the present invention will be discussed with reference to the accompanying drawings. Further aspects and embodiments will be apparent to those skilled in the art. All documents referenced herein are incorporated herein by reference.

[0069] This disclosure provides combinations of pharmaceuticals and pharmaceutical compositions comprising an MCIM compound and a TNF inhibitor compound. These combinations or compositions may be used as pharmaceuticals. The disclosure also provides compositions comprising an MCIM compound for use in a method for treating or preventing inflammatory and / or progressive diseases, the method further comprising administering a TNF inhibitor compound. Furthermore, pharmaceutical compositions comprising a TNF inhibitor compound for use in a method for treating or preventing inflammatory and / or progressive diseases are also provided, the method further comprising administering an MCIM compound. Furthermore, pharmaceutical compositions comprising an MCIM compound and a TNF inhibitor compound for use in a method for treating or preventing inflammatory and / or progressive diseases are also provided. Additionally, the use of an MCIM compound in the manufacture of a pharmaceutical for use in a therapeutic method, for example, in a method for treating or preventing inflammatory and / or progressive diseases, the method further comprising administering a TNF inhibitor compound. Also provided is the use of TNF inhibitor compounds in the manufacture of agents for use in therapeutic methods, for example, in methods for treating or preventing inflammatory and / or progressive diseases, the method further comprising administering MCIM compounds.

[0070] A method for treating or preventing inflammatory and / or progressive diseases is further provided, comprising administering to a subject in need of treatment a therapeutic or prophylactic effective amount of (i) an MCIM compound and (ii) a TNF inhibitor compound.

[0071] The present disclosure also provides MCIM compounds and TNF inhibitor compounds for use in a method of treating or preventing an inflammatory and / or progressive disease (e.g., in the form of a combination of medicaments or a pharmaceutical composition comprising an MCIM compound and a TNF inhibitor compound). Also provided is the use of MCIM compounds and TNF inhibitor compounds in the manufacture of a medicament for use in a method of treating or preventing an inflammatory and / or progressive disease (e.g., in the form of a combination of medicaments or a pharmaceutical composition comprising an MCIM compound and a TNF inhibitor compound). Also provided is a method of treating or preventing an inflammatory and / or progressive disease, the method comprising administering to a subject in need of treatment a therapeutically or prophylactically effective amount of an MCIM compound and a TNF inhibitor compound (e.g., in the form of a combination of medicaments or a pharmaceutical composition comprising an MCIM compound and a TNF inhibitor compound).

[0072] In some aspects and embodiments, the MCIM compound and the TNF inhibitor compound may be provided as a combination therapy. In some embodiments, the MCIM compound and the TNF inhibitor compound may be administered simultaneously or sequentially. Simultaneous administration refers to, for example, as a pharmaceutical composition containing both drugs (i.e., as a combined preparation), or immediately after each other (e.g., within 1, 4, 6, 8, or 12 hours), and optionally via the same route of administration, e.g., into the same artery, vein, or other blood vessel, administering two or more drugs together. Sequential administration refers to administering one of the drugs followed by a separate administration of another drug after a given time interval. It is not necessary for the drugs to be administered by the same route, although this may apply in some embodiments. The time interval may be any time interval.

[0073] Also provided is the use of an MCIM compound in the manufacture of a medicament for use in the treatment or prevention of an inflammatory and / or progressive disease, the treatment or prevention of the inflammatory and / or progressive disease further comprising administering a TNF inhibitor compound.

[0074] This disclosure further provides pharmaceutical compositions comprising MCIM compounds for use in therapeutic methods, the methods comprising administering TNF inhibitor compounds. This disclosure further provides pharmaceutical compositions comprising MCIM compounds disclosed herein for use in therapeutic methods, the methods further comprising administering TNF inhibitor compounds, the TNF inhibitor compounds comprising antibodies or fusion proteins. This disclosure further provides pharmaceutical compositions comprising MCIM compounds for use in therapeutic methods, the methods further comprising administering etanercept, infliximab, adalimumab, golimumab, or certolizumab. This disclosure further provides pharmaceutical compositions comprising TNF inhibitor compounds for use in medicine, the methods further comprising administering MCIM compounds disclosed herein. This disclosure further provides pharmaceutical compositions comprising TNF inhibitor compounds, the TNF inhibitor compounds comprising antibodies or fusion proteins for use in medicine, the use further comprises administering MCIM compounds disclosed herein. This disclosure further provides pharmaceutical compositions comprising etanercept, infliximab, adalimumab, golimumab, or certolizumab for medical use, the method further comprising administering the MCIM compounds disclosed herein. This disclosure further provides pharmaceutical compositions comprising the MCIM compounds disclosed herein and TNF inhibitor compounds for medical use. This disclosure further provides pharmaceutical compositions comprising the MCIM compounds disclosed herein and etanercept, infliximab, adalimumab, golimumab, or certolizumab for medical use.

[0075] Pharmacophore The term "pharmacophore" is used herein as defined in Wermuth, CG, Ganellin, CR, Lindberg, P., Mitscher, LA; Glossary of Terms Used in Medicinal Chemistry (IUPAC Recommendations 1998); Pure & Appl. Chem. 70:5 (1998) 1129-1143: A pharmacophore is an ensemble of aromatic stereochemical and electronic features necessary to ensure optimal supramolecular interactions with a particular biological target and to induce (or block) its biological response. This ensemble of aromatic stereochemical and electronic features represents the so-called "pharmacophore features." Typical pharmacophore features include, for example, hydrogen bond donors, hydrogen bond acceptors, hydrophobicity, aromaticity, and positive and negative ionization regions.

[0076] As used herein, the term “pharmacophore model” refers to a pharmacophore hypothesis relating to binding interactions at a particular active site. A pharmacophore model consists of a set of interconnected annotation points in 3D space. The annotation points indicate the location and type of biologically significant atoms and groups; that is, each annotation point is associated with a pharmacophore feature of the model. Each annotation point is associated with a radius that describes the allowable variation in 3D space for the location of a given pharmacophore feature.

[0077] As used herein, the phrase “fits a pharmacophore model” means that the compounds described herein bind to a target binding site (i.e., NDUSF2 and / or NDUSF7 binding site) in a 3D conformation (i.e., “pause”), thereby occupying four or more annotation points of the pharmacophore model described herein by corresponding features of the MCIM compounds described herein, as determined by the unified annotation scheme of Molecular Operating Environment (MOE), 2022.02 Chemical Computing Group ULC, 1010 Sherbooke St. West, Suite #910, Montreal, QC, Canada, H3A 2R7, 2022. In some embodiments, four or more annotation points of the pharmacophore model described herein are occupying the corresponding features of the MCIM compounds described herein. In some embodiments, five or more, six or more, seven or more, eight or more, or all nine of the annotation points of the pharmacophore model described herein are occupied by the corresponding features of the MCIM compounds described herein. The fit of the compounds described herein with the pharmacophore features is determined using the unified annotation scheme in Molecular Operating Environment (MOE), 2022.02 Chemical Computing Group ULC, 1010 Sherbooke St. West, Suite #910, Montreal, QC, Canada, H3A 2R7, 2022. Annotation points can be broadly divided into three categories: atoms, projections, and centroids. Annotation points are determined for a given compound according to the unified annotation scheme of Molecular Operating Environment (MOE), 2022.02 Chemical Computing Group ULC, 1010 Sherbooke St. West, Suite #910, Montreal, QC, Canada, H3A 2R7, 2022.

[0078] The annotation points used in this specification are described below.

[0079] atom Don: H-bonded donor Acc: H bond acceptor Atomic annotations are located directly on the atoms of a molecule and typically indicate functions related to protein-ligand binding.

[0080] The Don annotation is applied to heavy atoms that are H-bond donors. The Don annotation is added to all oxygen and nitrogen atoms that have at least one (possibly implicit) hydrogen added.

[0081] Acc annotates H bond acceptor heavy atoms. O, S, and N elements can be hydrogen bond acceptors, provided they conform to the following rules. 1. Sulfur atoms are not acceptors except for sulfur in S=C groups and anionic sulfur, which are given the Acc annotation. 2. Nitrogen atoms are acceptors and are given the Acc annotation, provided they are not buried. Buried nitrogen is one of {=N=, -N#, >N=, >N<} or a non-tricyclic conjugated nitrogen in the form of {>N-π, >N-[C+], >NB, >NS=O, >NP=O}. 3. Oxygen atoms are acceptors and are given the Acc annotation, provided they are not buried and do not belong to any of the following exceptions. Buried oxygen is one of {=O=, -O#;>O=, >O<} or non-tricyclic conjugated oxygen in the form of {>O-π, >OB, >O-[C+]}. Unburied oxygen atoms are acceptors unless they are part of any of the following exceptions. [ka]

[0082] projection Don2: Projected Donor Acc2: Projected Acceptor The projected annotations are (typically) located along the implicit lone pair or implicit hydrogen direction and are used to annotate the locations of possible hydrogen bonds or metal ligation partners, or possible R-group atom locations.

[0083] The projected Don2 annotations are added according to the hybridization and donor heavy atom coordination. In the following table, 'd' represents a Don2 feature. [Table 1]

[0084] The projected annotation for hydrogen bond Acc2 is added to eligible heavy atoms as H bond acceptors (see above), and the Acc annotation is given. The projected annotation for Acc2 is added to the same positions as that for the projected annotation for Don2, following the same rules. (i.e., donors and acceptors are projected using the same angles and distances.) Atoms that are both Don and Acc are annotated with the projected annotation "Don2&Acc2".

[0085] Projected annotations such as Don2 and Acc2 are located at potential heavy atom positions. For example, Don2 indicates a potential hydrogen bonding partner heavy atom. In reality, this partner atom cannot have too much overlap with any atom in the molecule that generates the projected annotation. This condition depends on the specific conformation of the molecule and cannot be reliably predicted by topological means. Therefore, solvent exposure tests must be applied to verify any hits resulting from pharmacophore searches, i.e., tests to verify that the relevant projected feature is not covered by other parts of the conformation (this prevents the estimated projected atom from occupying an intended position).

[0086] Centroid Aroaromatic Hydrophobic Hydro Centroid annotations (Aro, Hyd) are located at the geometric center of a subset of atoms in a molecule.

[0087] Aro annotation centroids are used for aromatic and pseudo-aromatic rings. Aro annotation centroids are placed in the centroid of each aromatic ring (for example, two centroids in naphthalene).

[0088] The definition of aromaticity is generous (daylight style definition), with each ring treated individually and the Huckel 4n+2 rule applied. C=O carbons are counted with 0 electrons, otherwise C=R is counted with 1, N=X nitrogens are counted with 1, and >N-nitrogens and -O-oxygens are counted with 2 electrons. Thus, the following are treated as (pseudo) aromatic and given an Aro annotation centroid at the center of the ring. [ka]

[0089] Hydrophobic atoms are annotated with HydA, and hydrophobic centroids are annotated with Hyd. Hydrophobic groups are determined by graph theory algorithms.

[0090] The basic rules for determining whether an atom is hydrophobic are summarized below. 1. The nitro nitrogen atom (not the nitrate anion) is hydrophobic. 2. A divalent sulfur atom having two heavy neighboring groups bonded only to carbon or sulfur is hydrophobic. 3. Halogens are hydrophobic. 4. Carbon atoms are hydrophobic, a) an aliphatic carbon π bonded to a noncarbon, or b) A π carbon atom adjacent to a monovalent oxygen atom, or c) Aromatic carbon adjacent to the aromatic oxygen of the 5th ring, or d) A carbon atom adjacent to two or more {N,O} atoms, e) Remove the anionic carbon in c1cccc1. The assignment of hydrophobic annotations proceeds by first applying the preceding hydrophobic atom type rules, but excluding fluorine atoms because they are small and do not affect the arrangement of the annotations.

[0091] The unified scheme provides atomic center hydrophobic annotation, HydA, and centroid hydrophobic feature, Hyd. HydA annotation is used for hydrophobic atoms that are considered to have a sufficiently high (potential) exposure to potential acceptors. This means, for example, that sp3 carbons with four heavy neighbors are not marked (because they are buried), and aromatic carbons with two heavy neighbors and two ortho substituents are not annotated.

[0092] Hyd annotation is assigned by a procedure that groups connected hydrophobic atoms and assigns a centroid weighted by an estimate of the potentially exposed surface area of ​​each hydrophobic atom, meaning that the Hyd centroid is positioned closer to the exposed hydrophobic atom in the hydrophobic group.

[0093] The following grouping algorithm is used. 1. Remove fluorine. Remove all fluorine that is not bonded to {H,F} from further consideration. Note that the estimated exposed surface area should be calculated using fluorine-inhibiting molecules. 2. Rings. Find all 5, 6, 7, and 8-membered rings that are not composed of smaller rings. For each such ring, extract each continuous stretch of hydrophobic atoms with at least three atoms having a sufficiently high total exposed surface area to generate surface area-weighted centroid annotations. Remove all annotated ring atoms from further examination. 3. Components. From the remaining hydrophobic material, find all connected components (single-linked clusters), and exclude clusters whose total exposed surface area is considered too small (less than -CH2- groups). 4. Small components. For the remaining hydrophobic material having three or fewer atoms, generate exposed surface area weighted centroid annotations. 5. Larger constituents. Identify the center of the constituent graph and generate centroids weighted by the center containing its neighboring groups. Divide the constituents, excluding the annotated atoms, and group the hydrophobicity (recursively) by applying the smaller constituent process and / or the larger constituent process.

[0094] The MCIM compounds described herein conform to the pharmacophore models described herein.

[0095] MCIM compounds intended for use in combination with the TNF inhibitor compounds described herein (e.g., adalimumab, etanercept, infliximab, and / or certolizumab) conform to the pharmacophore models described herein.

[0096] Inflammatory and / or progressive diseases As described herein, combinations of mitochondrial complex I modulator (MCIM) compounds and TNF inhibitor compounds are suitable for use in the treatment of inflammatory and / or progressive diseases.

[0097] The disease may be a chronic, progressive disease associated with fibrosis of the affected tissue, including: interstitial pulmonary fibrosis (ILD), idiopathic pulmonary fibrosis (IPF); pulmonary fibrosis; hepatic fibrosis; non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD); renal fibrosis; chronic kidney disease (CKD); cardiac fibrosis; ischemia-reperfusion injury; heart failure with reduced ejection fraction, heart failure with maintained ejection fraction; myelofibrosis; retroperitoneal fibrosis; atherosclerosis; myocardial infarction; stroke; neurodegenerative diseases; multiple sclerosis; frontotemporal dementia (FTD); and muscular atrophy. Lateral sclerosis (ALS), Alzheimer's disease (AD); osteoporosis, osteopenia; osteoarthritis; endometriosis; bone loss associated with endometriosis; bone neoplasms (including, for example, those as primary or metastatic tumors, including, for example, bone cancer; osteosarcoma; or osteoma); cancer-related bone diseases (including, for example, metastatic bone diseases associated with breast cancer, lung cancer, prostate cancer, or multiple myeloma; bone calcification and density associated with cancer, including, for example, cancer-related hypercalcemia); and bone metastases (including, for example, osteolytic bone metastases).

[0098] The diseases may include autoimmune diseases such as: rheumatoid arthritis (RA); psoriatic arthritis; ankylosing spondylitis; spondyloarthritis; reactive arthritis; infectious arthritis; systemic lupus erythematosus; scleroderma; juvenile idiopathic arthritis; psoriasis; systemic lupus erythematosus; lupus nephritis; uveitis; systemic sclerosis; scleroderma; hepatitis; Sjögren's syndrome; inflammatory bowel disease; ulcerative colitis; Crohn's disease; multiple sclerosis; arteriosclerosis; chronic obstructive pulmonary disease (COPD); uveitis; allergic diseases (e.g., atopy, allergic rhinitis, atopic dermatitis, anaphylaxis, allergic bronchopulmonary aspergillosis, allergic gastroenteritis, hypersensitivity pneumonitis); type 1 diabetes mellitus; celiac disease; oophoritis; primary biliary tract disease Juicy cirrhosis; insulin-resistant diabetes; Behçet's disease; myasthenia gravis; autoimmune polyneuritis; pemphigus; rheumatic carditis; Goodpasture syndrome; post-cardiac surgery syndrome; polymyositis; dermatomyositis; irritable bowel syndrome; pancreatitis; gastritis; chronic pneumonia; alveolar septitis; polycystic kidney disease; cryopyrin-associated periodic syndromes (CAPS); Mackle-Wells syndrome; Guillain-Barré syndrome; chronic inflammatory demyelinating polyneuritis; dermatitis; atopic dermatomyositis; Graves' disease; autoimmune (Hashimoto) thyroiditis; bronchitis; cystic fibrosis; pulmonary embolism; sarcoidosis; emphysema; respiratory failure; acute respiratory distress syndrome; BENTA disease; or polymyositis; SSC-ILD; hidradenitis suppurative, alopecia areata, atopic dermatitis. The disease may be an autoinflammatory disorder such as Crohn's disease, gout, Behçet's disease, or Mackle-Wells syndrome.

[0099] Disease control can be achieved by treatment with the compositions of the present invention. Disease control may include inhibiting disease progression and / or controlling disease symptoms. Inhibition of disease progression includes preventing disease progression and slowing disease progression. Standard methods in the art may be used to determine disease progression.

[0100] Evaluation of disease repair in humans Chronic autoimmune diseases are suitable for treatment with the combination of complex I modulator (MCIM) compounds and TNF inhibitor compounds described herein. Such diseases include rheumatoid arthritis (RA), inflamed dysplasia (IBD), ulcerative colitis (UC), psoriatic arthritis (PsA), and psoriasis. As described herein, MCIM compounds can induce tissue repair and disease remission.

[0101] In some embodiments, histopathological evaluation can be used to determine the effect of MCIM compounds on arthritis in combination with TNF inhibitor compounds: For this evaluation of arthritis, the following signs are monitored three times a week in the fingers or limbs of each subject and totaled to generate an arthritis index (AI). (The maximum AI for one animal is 16): 0: No visible effects of arthritis. 1: Edema and / or erythema of one toe. 2: Edema and / or erythema of two toes. 3: Edema and / or erythema of more than two toes. 4: Severe arthritis of the entire foot and toes. In some embodiments, MCIM compounds combined with TNF inhibitor compounds reduce the mean score compared to negative controls, subjects treated with MCIM compounds alone, and / or TNF inhibitor compounds alone.

[0102] In subjects suffering from arthritis, inhibition of disease progression may be indicated by a stable arthritis index (AI) score or any other suitable method known in the art. Other suitable clinical scoring methods known in the art include the ACR / EULAR2010 scoring criteria (ACR / EULAR score), the "28 Joints Disease Activity Score" criteria (DAS28 score), the "Health Assessment Questionnaire Disability Index" (HAQ-DI score), the "Clinical Disease Activity Index" (CDAI score), the "Simplified Disease Activity Index" (SDAI score), the "American College of Rheumatology Response Criteria" (also known as the "ACR20 / 50 / 70 Response"; ACR20 / 50 / 70 score), the European Union Response Criteria for Rheumatism (EULAR score), the "Modified total Sharp / van der Heijde score" (mTSS score), and / or the "routine assessment of patient index data 3 score" (RAPID3 score). For example, inhibition of disease progression may be determined by comparing the subject's AI score, ACR / EULAR score, DAS38 score, HAQ-DI score, CDAI score, SDAI score, ACR20 / 50 / 70 score, EULAR score, mTSS score, and / or RAPID3 score before, during, and / or after treatment with MCIM compounds and TNF inhibitor compounds. Inhibition of disease progression may be indicated by the AI ​​score, ACR / EULAR score, DAS38 score, HAQ-DI score, CDAI score, SDAI score, ACR20 / 50 / 70 score, EULAR score, mTSS score, and / or RAPID3 score remaining stable (i.e., unchanged) over time after treatment. Inhibition of disease progression may be indicated by the AI ​​score, ACR / EULAR score, DAS38 score, HAQ-DI score, CDAI score, SDAI score, ACR20 / 50 / 70 score, EULAR score, mTSS score, and / or RAPID3 score progressing or increasing at a slower rate after treatment. Disease control in arthritis may be determined by any preferred method known in the art.In some embodiments, MCIM compounds combined with TNF inhibitor compounds are used to treat arthritis to reduce mean arthritis scores (e.g., AI score, ACR / EULAR score, DAS38 score, HAQ-DI score, CDAI score, SDAI score, ACR20 / 50 / 70 score, EULAR score, mTSS score, and / or RAPID3 score) compared to administration of MCIM compounds or TNF inhibitor compounds alone. In some embodiments, MCIM compounds combined with TNF inhibitor compounds reduce the target AI score, ACR / EULAR score, DAS38 score, HAQ-DI score, CDAI score, SDAI score, ACR20 / 50 / 70 score, EULAR score, mTSS score, and / or RAPID3 score by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

[0103] In IBD, repair / healing can be clinically assessed using intestinal transit time, occult blood, endoscopy, histopathology, electrolytes, and / or by measuring biomarkers such as pANCA, ASCA, GP2, CUZD1, CHI3L1, GM-CSF, ACA, PS / PT, ALCA, ACCA, AMCA, OmpC, I2, CBir1, laminarin, chitin, IFI16, IL-1β, IL-6, IL-8, IL-9, IFN-γ, TNF, CCL2, IL-22, IL-2, and / or IL-6, as disclosed in Chen et al (2020) (which is incorporated herein by reference in its entirety).

[0104] In some embodiments, histopathological evaluation can be used to determine the effect of MCIM compounds on IBD in combination with MCIM compounds and TNF inhibitor compounds: Ileocecal tissue sections can be stained with hematoxylin and eosin (H&E), and parameters for inflammation, mucosal erosion, epithelial hyperplasia, epithelial dysplasia, myxocyte dysplasia, and fibrous proliferation are evaluated on a scale of 0 to 5 as follows: 0: Normal. 1: Minimal, localized. 2: Moderate, localized. 3: Moderate, multifocal or diffuse. 4: Marked, localized. 5: Marked, multifocal or diffuse. In some embodiments, MCIM compounds in combination with TNF inhibitor compounds reduce the mean score compared to negative controls, subjects treated with MCIM compounds alone, and / or TNF inhibitor compounds alone. In some embodiments, MCIM compounds in combination with TNF inhibitor compounds are intended for use in the treatment of IBD to reduce the mean IBD score compared to MCIM or TNF inhibitor compounds alone. In some embodiments, the mean score decreases by at least 1, at least 2, at least 3, at least 4, or 5.

[0105] Disease control can be achieved through treatment with MCIM compounds and TNF inhibitor compounds. Disease control may include inhibiting disease progression, controlling disease symptoms, and / or supporting tissue repair. Inhibition of disease progression includes preventing disease progression and slowing disease progression. Standard methods in the art may be used to determine disease progression.

[0106] For example, inhibition of disease progression in subjects suffering from IBD may be determined by the histopathological methods described above, or by any other suitable method known in the art. For example, inhibition of disease progression may be determined by comparing the histopathological outcomes from subjects before and after treatment with the compositions disclosed herein. Inhibition of disease progression may be indicated by histopathological scores that remain stable (i.e., unchanged) over time after treatment. Inhibition of disease progression may be indicated by histopathological scores that progress at a slower rate after treatment compared to disease progression before treatment.

[0107] In some embodiments, the TNF inhibitor compound comprises an antibody or a fusion protein, and optionally, the TNF inhibitor compound is selected from etanercept, infliximab, adalimumab, golimumab, or certolizumab, or any combination thereof, and the inflammatory and / or progressive disease is rheumatoid arthritis (RA). In some embodiments, the TNF inhibitor compound comprises an antibody or a fusion protein, and optionally, the TNF inhibitor compound is selected from etanercept, infliximab, adalimumab, golimumab, or certolizumab, or any combination thereof, and the inflammatory and / or progressive disease is inflammatory bowel disease such as Crohn's disease and ulcerative colitis. In some embodiments, the TNF inhibitor compound comprises an antibody or a fusion protein, and optionally, the TNF inhibitor compound is selected from etanercept, infliximab, adalimumab, golimumab, or certolizumab, or any combination thereof, and the inflammatory and / or progressive disease is lupus erythematosus. In some embodiments, the TNF inhibitor compound comprises an antibody or a fusion protein, and optionally, the TNF inhibitor compound is selected from etanercept, infliximab, adalimumab, golimumab, or certolizumab, or any combination thereof, and the inflammatory and / or progressive disease is psoriatic arthritis. In some embodiments, the TNF inhibitor compound comprises an antibody or a fusion protein, and optionally, the TNF inhibitor compound is selected from etanercept, infliximab, adalimumab, golimumab, or certolizumab, or a combination thereof, and the inflammatory and / or progressive disease is reactive arthritis.

[0108] In some embodiments, tissue repair, disease remission, disease control (including, e.g., prevention or delay of disease progression), increased repair cells, decreased destructive cells, and / or reduction of cytokine production from peripheral mononuclear cells or lymphocytes (such as T, B, or NK cells) can be determined as defined herein. For example, in some embodiments, MCIM compounds reduce the levels of inflammatory mediators such as TNFα, eSEL, CD38, CD40, CD69, sIgG, sIL-17A, sIL-17F, sIL-2, and / or sIL-6 produced by target cells. In some embodiments, MCIM compounds reduce the levels of type I collagen and / or MMP1 and / or increase the production of type IV collagen. In some embodiments, MCIM compounds increase ETC efficiency without increasing biomass. In some embodiments, MCIM compounds decrease ETC efficiency. In some embodiments, MCIM compounds reduce cell proliferation without reducing ATP concentration / cell and viability, depending on the environmental composition, such as the absence of piruvate. In some embodiments, MCIM compounds induce adaptive / repair responses under metabolic stress conditions. For example, MCIM compounds simultaneously induce adaptive / repair responses by modulating the activity of complex I, attenuating high-energy processes such as proliferation and / or differentiation, and increasing the production of pro-angiogenic / repair factors such as VEGF to restore tissue metabolic homeostasis, particularly under metabolic stress conditions. In some embodiments, MCIM compounds reduced cell viability, particularly in cell types that are highly dependent on complex I metabolism and lack metabolic flexibility to adapt. Each of these effects on tissue repair, disease control, disease remission, increased repair cells, decreased destructive cells, and / or reduced cytokine production from pro-inflammatory myeloid cells can be determined by comparing the effects in the presence and absence of MCIM compounds.

[0109] In some embodiments, lung fibroblasts are used as target cells to determine the effects of MCIM compounds. For example, cell adaptation may be determined in human primary lung fibroblasts by measuring, for instance, vascular endothelial growth factor (VEGF) secretion. (VEGF may be induced in cells that do not receive sufficient oxygen or nutrients to support ATP production. This can illustrate how metabolic signaling pathways interact and integrate with angiogenic signaling events and repair.) In some embodiments, VEGF secretion is measured in primary human lung fibroblasts at 2 × 10⁶ 3 Cells / well can be measured by plating them in 100 μL of DMEM complete medium in a 96-well plate containing 1 g / L glucose and 110 mg / L pirubate, or 1 g / L glucose without pirubate (supplemented with 1% penicillin-streptomycin and 10% thermoinactivated fetal bovine serum, respectively), and incubating for 24 hours in a humidified 5% CO2 37°C incubator. The test compound is prepared as a 10-fold final concentration solution in the culture medium, added to the final concentration, and then the cells are further incubated at 37°C / 5% CO2 for 24 hours. VEGF secretion is measured in the cell supernatant using the Quantikine® ELISA Human VEGF Kit according to the manufacturer's instructions. Absorbance at 450 nm is measured using a BMG plate reader (CLARIOstar plus) with path length correction. Background absorbance is measured at 540 nm. Preferably, treatment with the test compound results in increased VEGF secretion in the absence of pirubate, but not in the presence of pirubate as a cellular metabolic substrate. This indicates the induction of an adaptive / repair response under metabolic stress conditions by attenuating high-energy-consuming processes such as proliferation and the simultaneous production of pro-angiogenic / repair factors such as VEGF, in order to restore metabolic homeostasis of the tissue.

[0110] In some embodiments, histopathological evaluation can be used to determine the effect of MCIM compounds on IBD in combination with MCIM compounds and TNF inhibitor compounds: Ileocecal tissue sections can be stained with hematoxylin and eosin (H&E), and parameters for inflammation, mucosal erosion, epithelial hyperplasia, epithelial dysplasia, myxocyte dysplasia, and fibrous proliferation are evaluated on a scale of 0 to 5 as follows: 0: Normal. 1: Minimal, localized. 2: Moderate, localized. 3: Moderate, multifocal or diffuse. 4: Marked, localized. 5: Marked, multifocal or diffuse. In some embodiments, MCIM compounds combined with TNF inhibitor compounds reduce the mean score compared to negative controls, subjects treated with MCIM compounds alone, and / or TNF inhibitor compounds alone.

[0111] Disease control can be achieved by treatment with the compositions of the present invention. Disease control may include inhibiting disease progression, controlling disease symptoms, and / or supporting tissue repair. Inhibition of disease progression includes preventing disease progression and slowing disease progression. Standard methods in the art may be used to determine disease progression.

[0112] For example, inhibition of disease progression in subjects suffering from IBD may be determined by the histopathological methods described above, or by any other suitable method known in the art. For example, inhibition of disease progression may be determined by comparing the histopathological outcomes from subjects before and after treatment with the composition of the present invention. Inhibition of disease progression may be indicated by a histopathological score that remains stable (i.e., unchanged) over time after treatment. Inhibition of disease progression may be indicated by a histopathological score that progresses at a slower rate after treatment compared to disease progression before treatment.

[0113] In some embodiments, histopathological evaluation can be used to determine the effect of MCIM compounds on arthritis in combination with TNF inhibitor compounds: For this evaluation of arthritis, the following signs are monitored three times a week in the fingers or limbs of each subject and totaled to generate an arthritis index (AI). (The maximum AI for one animal is 16): 0: No visible effects of arthritis. 1: Edema and / or erythema of one toe. 2: Edema and / or erythema of two toes. 3: Edema and / or erythema of more than two toes. 4: Severe arthritis of the entire foot and toes. In some embodiments, MCIM compounds combined with TNF inhibitor compounds reduce the mean score compared to negative controls, subjects treated with MCIM compounds alone, and / or TNF inhibitor compounds alone.

[0114] In subjects suffering from arthritis, inhibition of disease progression may be indicated by a stable arthritis index (AI) score or by any other preferred method known in the art. For example, inhibition of disease progression may be determined by comparing AI scores from subjects before and after treatment with the composition of the present invention. Inhibition of disease progression may be indicated by an AI score that remains stable (i.e., unchanged) over time after treatment. Inhibition of disease progression may be indicated by an AI score that progresses or increases at a slow rate after treatment. Disease control in arthritis may be determined by any preferred method known in the art.

[0115] *** In some embodiments, tissue repair, disease remission, disease control, increased repair cells, decreased destructive cells, and / or reduced cytokine production from pro-inflammatory myeloid cells can be determined as defined herein. For example, in some embodiments, the combination therapy reduces the levels of inflammatory mediators such as TNFα, eSEL, CD38, CD40, CD69, sIgG, sIL-17A, sIL-17F, sIL-2, and / or sIL-6 produced by target cells. In some embodiments, the combination therapy increases the expression of Coll IV. In some embodiments, the combination therapy increases ETC efficiency without increasing biomass. In some embodiments, the combination therapy reduces cell proliferation in the absence of piruvate without reducing ATP concentration / cells and viability. In some embodiments, the combination therapy induces an adaptive / repair response under metabolic stress conditions and reduces cell death. For example, MBS compounds simultaneously induce adaptive / repair responses by modulating the activity of complex I, attenuating high-energy processes such as proliferation, and increasing the production of pro-angiogenic / repairing factors such as VEGF to restore tissue metabolic homeostasis, particularly under metabolic stress conditions. Each of these effects on tissue repair, disease remission, increased repair cells, decreased destructive cells, and / or reduced cytokine production from pro-inflammatory myeloid cells can be determined by comparing the effects in and out of the combination therapy.

[0116] *** Combinations of medicines The pharmaceutical combinations disclosed herein refer to combinations comprising two or more active compounds. A pharmaceutical combination may comprise two or more compositions, each comprising at least one active compound. In such embodiments, the two or more compositions may be administered separately, sequentially, or simultaneously. For example, a pharmaceutical combination may comprise a first composition comprising an MCIM compound such as HMC-C-01-A and a second composition comprising a TNF inhibitor compound such as an antibody or fusion protein, the TNF inhibitor compound being optionally selected from etanercept, infliximab, adalimumab, golimumab, or certolizumab, or any combination thereof. The first and second compositions may be administered separately, sequentially, or simultaneously.

[0117] Alternatively, the combination of pharmaceuticals may comprise a single composition comprising two or more active compounds. For example, the combination may comprise a single composition comprising an MCIM compound and a TNF inhibitor compound. In some embodiments, the MCIM compound comprises HMC-C-01-A. In some embodiments, the TNF inhibitor compound comprises one or more compounds selected from antibodies or fusion proteins, and optionally, the TNF inhibitor compound is selected from etanercept, infliximab, adalimumab, golimumab, or certolizumab, or any combination thereof. In some embodiments, the composition comprises the MCIM compound HMC-C-01-A and a TNF inhibitor compound selected from antibodies or fusion proteins, and optionally, the TNF inhibitor compound is selected from etanercept, infliximab, adalimumab, golimumab, or certolizumab, or any combination thereof.

[0118] Pharmaceutical composition Pharmaceuticals and pharmaceutical compositions in the manner disclosed herein may be formulated for administration by several routes, including, but not limited to, parenteral, i.e., parenteral routes (e.g., by injection, subcutaneous, intravenous, intraarterial, intramuscular, or intratumoral; topical or intradermal; by inhalation or intranasal; or rectal), and oral, i.e., oral routes. The pharmaceuticals and compositions may be formulated in solid, semi-solid, or liquid dosage forms. Pharmaceutical compositions according to the present invention may be delivered by routes that facilitate exposure in the systemic circulation, or by routes or methods that result in localized, targeted delivery of the active compound to a selected area in the body. Pharmaceutical compositions according to the present invention may also be administered to humans or animals.

[0119] The dose is preferably a "therapeutic dose," which is sufficient to demonstrate benefit to the individual. The actual amount administered, as well as the rate and time course of administration, depends on the nature and severity of the disease being treated. For example, the rate of release of the active compound from the pharmaceutical composition may be immediate, sustained, sustained-release, controlled, pulsating, or follow a pattern that is optimal for the intended therapeutic use. The dosing frequency may be fixed or variable to provide the desired therapeutic effect, depending on the rate of drug release, systemic circulation, or required level at the target site. Determining treatment instructions, such as dosage, is the responsibility of the physician and other physicians, typically taking into account the disorder being treated, the individual patient's condition, the site of delivery, the method of administration, and other factors known to the practitioner. Examples of the techniques and protocols described above can be found in Remington's Pharmaceutical Sciences, 23rd Edition, 2020, pub. Lippincott, Williams & Wilkins. This disclosure provides a pharmaceutical combination comprising an MCIM compound and a TNF inhibitor compound. In some embodiments, the combination may comprise a single composition comprising an MCIM compound and a TNF inhibitor compound. In other embodiments, the pharmaceutical combination may comprise a first composition comprising an MCIM compound and a second composition comprising a TNF inhibitor compound. In other embodiments, the combination comprises a first composition comprising an MCIM compound and a second composition comprising two or more TNF inhibitor compounds. In embodiments in which the combination comprises two or more TNF inhibitor compounds, the two or more TNF inhibitor compounds may be formulated as individual compositions comprising a single TNF inhibitor compound.

[0120] Pharmaceutical compositions may be prepared using pharmaceutically acceptable “carriers” consisting of materials considered safe and effective. “Pharmaceutically acceptable” means “generally considered safe,” for example, physiologically tolerable and typically not causing allergic reactions or similar adverse reactions, such as stomach upset, when administered to humans. In some embodiments, this term refers to molecular entities and compositions approved by a U.S. federal or state regulatory agency as GRAS (Generally Recognized As Safe) under Sections 204(s) and 409 of the Federal Food, Drug and Cosmetic Act, subject to premarket review and approval by the FDA or similar lists, U.S. Pharmacopeia, or another generally recognized pharmacopoeia for use in animals, more specifically in humans.

[0121] The term “carrier” refers to diluents, binders, lubricants, and disintegrants. Those skilled in the art are familiar with such pharmaceutical carriers and methods of formulating pharmaceutical compositions using such carriers. When a pharmaceutical composition is formulated as a solid dosage form, it may be further coated or left uncoated to impart aesthetic features, physical protection, enhance the physical and / or chemical stability of the active compound or other components of the pharmaceutical composition, and / or alter the rate of dissolution and release of the active compound from the pharmaceutical composition. When a pharmaceutical composition is formulated as a parenteral injection dosage form, it may also contain aqueous or non-aqueous solvents, co-solvent mixtures, buffers, surfactants, tonic modifiers, chelating agents, pH modifiers, viscosity modifiers, and / or suspensions. When a pharmaceutical composition is formulated as a parenteral inhalation dosage form, it may also contain, depending on the inhalation delivery device used, a particle carrier for pulmonary delivery of the active compound, absorption and permeation enhancers, and / or propellants. Those skilled in the art are familiar with such pharmaceutical carriers and excipients therein, as well as methods for incorporating these excipients into pharmaceutical compositions. Suitable excipients for use in pharmaceutical compositions can be found in standard pharmaceutical texts, such as Handbook of Pharmaceutical Excipients, 9th edition, Pharmaceutical Press, American Pharmaceutical Association, 2020.

[0122] The pharmaceutical compositions provided herein may contain one or more excipients, such as solvents, solubility enhancers, suspending agents, buffers, isotonic agents, antioxidants, or antimicrobial preservatives. When used, the excipients of the composition do not adversely affect the stability, bioavailability, safety, and / or efficacy of the active ingredient, i.e., the MCIM compound and / or TNF inhibitor compound used in the composition. Accordingly, those skilled in the art will understand that compositions without incompatibility among any of the components of the dosage form are provided. The excipients may be selected from the group consisting of buffers, solubilizers, isotonic agents, chelating agents, antioxidants, antimicrobial agents, and preservatives.

[0123] It may be convenient or desirable to prepare, purify, and / or handle the corresponding solvates of active compounds. The term "solvate" is used herein in its conventional sense to refer to a complex of solute (e.g., active compound, salt of active compound) and solvent. When the solvent is water, the solvate may conveniently be called a hydrate, e.g., monohydrate, dihydrate, trihydrate, etc.

[0124] Unless otherwise specified, references to specific compounds include their solvates.

[0125] It may be convenient or desirable to prepare, purify, and / or handle pharmaceutically acceptable salts of corresponding salts of active compounds, e.g., MCIM compounds, e.g., HMC-C-01-A, or TNF inhibitor compounds. Examples of pharmaceutically acceptable salts are discussed in Berge et al., 1977, “Pharmaceutically Acceptable Salts,” J. Pharm. Sci., Vol. 66, pp. 1-19. For example, if a compound is anionic or has a functional group that can be anionic (e.g., COOH can be COO), the salt may be formed with a suitable cation. If a compound is cationic or has a functional group that can be cationic (e.g., NH2 can be NH3+), the salt may be formed with a suitable anion. Unless otherwise specified, references to specific compounds include their salt forms.

[0126] Specific co-products The pharmaceutical compositions according to the present invention include, but are not limited to, dosage forms in which the active compound is uniformly co-formulated in a common excipient base such as a conventional monolayer tablet or conventional powder-in-capsule for oral administration, a solution, suspension, or dispersion for parenteral administration, a lotion, cream, or ointment for topical administration, a transdermal patch, microneedle, or autoinjector system for transdermal administration, a dry powder, solution, or suspension for inhalation administration, and a solution or suspension for intranasal administration.

[0127] The present invention provides alternative pharmaceutical compositions particularly useful for solid dosage forms for oral administration, in which active compounds are physically separated within a single pharmaceutical composition to avoid potential physical and chemical interactions and incompatibility. Such pharmaceutical compositions include, but are not limited to, two-layer / multilayer tablets, where the formulation of each layer is optimized for each active compound, which can be combined into a single pharmaceutical composition by conventional tablet compression. Such pharmaceutical compositions may also include single-layer or two-layer / multilayer tablets, where one of the active compounds is contained within a layer coated on the outside of a single-layer or two-layer / multilayer compressed tablet core, thus providing physical separation of the active compounds and their associated excipient systems. A further alternative pharmaceutical composition according to the present invention is a multi-particle capsule, in which the formulation of each active compound in particle / granular form can be optimized and then combined and encapsulated in a conventional capsule as a single pharmaceutical composition.

[0128] The method for preparing the pharmaceutical composition according to the present invention is well known to those skilled in the art and is adequately described in general pharmaceutical textbooks such as Remington, The Science and Practice of Pharmacy, Editor: Adeboye Adejare, 23rd edition, 2020, publisher: Elsevier; Aulton's Pharmaceutics, The Design and Manufacture of Medicines, editors: Kevin Taylor and Michael Aulton, 6th edition, 2021, publisher: Elsevier; and Lachman / Liebermans: The Theory and Practice of Industrial Pharmacy, Editors: Roop Khar, SP Vyas, Farnham Ahmad, Gaurav Jain, 4th edition, 2014, publisher: CBS.

[0129] Sequence List Human NDUFS2 amino acid sequence SEQ ID NO: 1: MAALRALCGFRGVAAQVLRPGAGVRLPIQPSRGVRQWQPDVEWAQQFGGAVMYPSKETAHWKPPPWNDVDPPKDTIVKNITLNFGPQHPAAHGVLRLVMELSGEMVRKCDPHIGL LHRGTEKLIEYKTYLQALPYFDRLDYVSMMCNEQAYSLAVEKLLNIRPPPRAQWIRVLFGEITRLLNHIMAVTTHALDLGAMTPFFWLFEEREKMFEFYERVSGARMHAAYIRPGG VHQDLPLGLMDDIYQFSKNFSLRLDELEELLTNNRIWRNRTIDIGVVTAEEALNYGFSGVMLRGSGIQWDLRKTQPYDVYDQVEFDVPVGSRGDCYDRYLCRVEEMRQSLRIIAQC LNKMPPGEIKVDDAKVSPPKRAEMKTSMESLIHHFKLYTEGYQVPPGATYTAIEAPKGEFGVYLVSDGSSRPYRCKIKAPGFAHLAGLDKMSKGHMLADVVAIIGTQDIVFGEVDR Human NDUFS7 amino acid sequence, SEQ ID NO: 2: MAVLSAPGLRGFRILGLRSSVGPAVQARGVHQSVATDGPSSTQPALPKARAVAPKPSSRGEYVVAKLDDLVNWARRSSLWPMTFGLACCAVEMMHMAAPRYDMDRF GVVFRASPRQSDVMIVAGTLTNKMAPALRKVYDQMPEPRYVVSMGSCANGGGYYHYSYSVVRGCDRIVPVDIYIPGCPPTAEALLYGILQLQRKIKRERRLQIWYRR The features disclosed in the foregoing specification, or the following claims, or the accompanying drawings, either in their specific forms or in terms of means for carrying out the disclosed functions, or the methods or processes for obtaining the disclosed results, may be used, individually or in any combination of such features, as necessary, to realize the present invention in its various forms.

[0130] While the present invention has been described in conjunction with the exemplary embodiments described above, many equivalent modifications and variations will become apparent to those skilled in the art when this disclosure is given. Therefore, the exemplary embodiments of the present invention described above are illustrative and not limiting. Various modifications to the embodiments described can be made without departing from the spirit and scope of the invention.

[0131] To avoid any doubt, the theoretical explanations provided herein are provided for the purpose of improving the reader's understanding. The inventors do not wish to be bound by any of these theoretical explanations.

[0132] Any section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described herein.

[0133] Unless the context requires otherwise, throughout this Spec., including the following claims, the terms “comprise” and “include,” as well as variations such as “comprises,” “comprising,” and “including,” are understood to mean including the integer or process, or group of integers or processes, described herein, but not to mean excluding any other integer or process, or group of integers or processes.

[0134] Where used herein and in the appended claims, the singular forms “a,” “an,” and “the” should be noted to include multiple references unless explicitly indicated otherwise by context. Ranges may be expressed herein as “about” one particular value to and / or “about” another particular value. Where such ranges are expressed, another embodiment includes one particular value to and / or another particular value. Similarly, where values ​​are expressed as approximations by the use of the antecedent “about,” understand that a particular value forms another embodiment. The term “about” in relation to numbers is optional and means, for example, + / - 10%. [Examples]

[0135] Example 1 - BioMAP profiling To profile the effects of MCIM compounds on multiple disease-related regulatory pathways, a high-throughput integrated biological platform (BioMAP®) was used. BioMAP®, as described in US6656695 (the entirety of which is incorporated herein), is a method developed to evaluate the efficacy, safety, and mechanism of action of drugs in multiple human cell types stimulated by inflammatory loads. The BioMAP system reflects human disease pathology and has the ability to detect and differentiate the effects of approved drugs and human therapeutic compounds in clinical trials. BioMAP technology enables rapid determination of the efficacy, side effects, and mechanism of action of drug candidates.

[0136] BioMAP® provides an unbiased, target-independent, and data-driven approach to understanding the effects of compounds or combination therapies on human disease models and translational biomarkers. The system is validated with clinically approved drugs and known test agents. The assay principle involves testing compounds in a human primary cell-based disease system and comparing the data to a reference database of over 4,500 compounds. By comparing the compound profile to the reference compound, it is possible to determine whether the biological activity of the test item is distinct from the reference.

[0137] The activity of MCIM compounds was determined using three BioMAP systems: a fibrosis panel, an autoimmune panel (HDFSAg), and Diversity Plus.

[0138] Using 13 primary human cell and co-culture assays, the effects of MCIM compounds on clinically relevant protein biomarkers of inflammation, cell growth, and fibrosis were evaluated as part of the quality-controlled BioMAP® Diversity PLUS, a commercially available service (Eurofins DiscoverX Corporation, Freemont, CA, USA; for more information, see https: / / www.discoverx.com). In short, the BioMAP® panel consists of human primary cell-based systems designed to model different aspects of the human body in an in vitro format. The 12 cell assays utilized in the Diversity PLUS panel enable unbiased characterization of agent responses across a broad set of systems modeling various human disease states compared to historical controls. The BioMAP® panel is constructed from primary cell types from healthy human donors and stimulated (e.g., cytokines or growth factors) to capture relevant signaling networks that naturally occur in human tissue or pathological states.

[0139] MCIM compounds were tested in these assays at four concentrations: 4000 nM, 1300 nM, 400 nM, and 150 nM. Human primary cells used in the BioMAP® system were passaged to level 4 or earlier, derived from multiple donors (n=2-6), and were commercially available and handled according to the manufacturer's recommendations. Human blood-derived CD14+ monocytes were differentiated into macrophages in vitro and then added to an LPS system (Eurofins DiscoverX Corporation).

[0140] The human cell types and stimuli used in each assay system were as follows: 3C system [human umbilical vein endothelial cells (HUVEC) + (IL-1β, TNFα, and IFNγ)], 4H system [HUVEC + (IL-4 and histamine)], lipopolysaccharide (LPS) system [peripheral blood monocytes (PBMC) and HUVEC + LPS (TLR4 ligand)], Sag system [peripheral blood mononuclear cells, PBMC, and HUVEC + TCR ligand], HDFSAg system [peripheral blood mononuclear cells, PBMC, and human neonatal dermal fibroblasts + TCR ligand], BT system [CD19 + B cells and PBMC + (α- IgM and TCR ligands), BF4T lineage [tracheal epithelial cells and human neonatal dermal fibroblasts, HDFn, + (TNFα and IL-4)], BE3C lineage [tracheal epithelial cells + (IL-1β, TNFα, and IFNγ)], CASM3C lineage [coronary artery smooth muscle cells (IL-1β, TNFα, and IFRγ)], HDF3CGF lineage [HDFn + (IL-1β, TNFα, IFNγ, EGF, bFGF, and PDGF-BB)], KF3CT lineage [keratinocytes and HDFn + (IL-1β, TNFα, IFNγ, and TGFβ)], MyoF lineage [differentiated lung myofibroblasts + (TNFα (and TGFβ), SAEMyoF system [small airway epithelial cells and differentiated pulmonary myofibroblasts + (TNFα and TGFβ)], ReMyoF system [renal epithelial cells and differentiated pulmonary myofibroblasts + (TNFα and TGFβ)], and lMphg system [HUVEC and M1 macrophages + zymosan (TLR2 ligand)].

[0141] The assays were derived from either single-cell types or co-culture systems. Adherent cell types were cultured in 96 or 384-well plates until confluence, followed by the addition of PBMCs (Sag and LPS systems). The BT system consisted of CD19+ B cells co-cultured with PBMCs and stimulated with a BCR activator and low levels of TCR stimulation. Test agents prepared with either DMSO (small molecule; final concentration ≤0.1%) or PBS (biological agent) were added at the indicated concentrations one hour prior to stimulation, and cells were maintained during 24 hours of culture, or otherwise as instructed [48 hours for the MyoF system; 72 hours for the BT system (soluble readout); 168 hours for the BT system (secreted IgG)]. Each assay plate contained an appropriate negative control (e.g., unstimulated conditions) and vehicle control (e.g., 0.1% DMSO) for each system. Biomarker levels of cell-associated targets and cell membrane targets were measured using direct ELISA. Soluble factors from the supernatant were quantified using either HTRF® detection, a bead-based multiplex immunoassay, or capture ELISA. The apparent adverse effects (cytotoxicity) of the compounds on cell proliferation and viability were detected by sulforhodamine B (SRB) staining for adherent cells and alamarBlue® reduction for cells in suspension. For proliferation assays, individual cell types were cultured in subconfluence and measured at time points optimized for each system (48 hours: 3C and CASM3C systems; 72 hours: BT and HDF3CGF systems; 96 hours: HDFSAg and Sag systems). Cytotoxicity against adherent cells was measured at the indicated time points by SRB (24 hours: 3C, 4H, LPS, Sag, HDFSag, BF4T, BE3C, CASM3C, HDF3CGF, KF3CT, and lMphg system; 48 hours: MyoF system) and by alamarBlue® staining for cells in suspension (24 hours: HDFSAg and Sag system; 42 hours: BT system).

[0142] Biomarker measurements in treated samples were divided by the mean of the control samples (at least six vehicle controls from the same plate), and then log10-converted ratios were generated. The significance predictor envelope was calculated using proprietary historical vehicle control data with 95% confidence intervals. Biomarker activity was annotated when two or more consecutive concentrations changed in the same direction compared to the vehicle control, were outside the significance envelope, and had at least one concentration with an effect magnitude >20% (log10 ratio >0.1). Major biomarker activities were described as regulated if these activities increased in some systems but decreased in others. Cytotoxic conditions were described when total protein levels decreased by more than 50% (log10 ratio of SRB or alamarBlue® levels <0.3) and were indicated by thin black arrows on the X axis. If cytotoxicity was detected in three or more systems, the compound was considered to have broad-spectrum cytotoxicity. Concentrations of test agents with broadly detectable cytotoxicity were excluded from biomarker activity annotation and downstream benchmarking, similarity searches, and cluster analysis. Antiproliferative activity was defined by an SRB or alamar Blue® log10 ratio value <-0.1 from cells plated at lower densities and indicated by a gray arrow on the X-axis. Cytotoxicity and antiproliferative arrows require only one concentration to meet the thresholds indicated for profile annotation.

[0143] Figure 2 shows the BioMAP profiles of several MCIM compounds. (A) ABD599, tested with a complete bioMAP profile, showed tissue remodeling with reduced inflammation and immunomodulation, as well as increased IV collagen levels. (B) HMC-C-01-A showed anti-inflammatory and immunomodulatory activity in the BT and Sag systems, and (C) HMC-C-01-A increased type IV collagen in a fibrosis panel.

[0144] Figure 3 shows the BioMAP profiles of three anti-TNF compounds in the HDFSAg inflammatory system. (A) Adalimumab, a humanized monoclonal antibody against TNF approved for the treatment of Crohn's disease, psoriatic arthritis, plaque psoriasis, rheumatoid arthritis, and ulcerative colitis, and etanercept, a biofusion protein that blocks TNF approved for the treatment of rheumatoid arthritis, plaque psoriasis, psoriatic arthritis, juvenile idiopathic arthritis, and ankylosing spondylitis, show anti-inflammatory activity and reduced tissue remodeling. (B) Infliximab, a chimeric monoclonal antibody against TNF approved for the treatment of psoriasis, Crohn's disease, ankylosing spondylitis, psoriatic arthritis, and ulcerative colitis, shows anti-inflammatory activity and reduced tissue remodeling.

[0145] Table I shows the bioMAP activity of compounds and known TNF inhibitors.

[0146] The data demonstrated that MCIM compounds possess distinct phenotypic profiles with multimodal activity and exhibit specific effects in different cell types after stimulation by different inflammatory mediators. In particular, MCIM compounds modulated immunoactivity by reducing levels of inflammatory mediators such as TNFα, eSEL, CD38, CD40, CD69, sIgG, sIL-17A, sIL-17F, sIL-2, and sIL-6, similar to TNF inhibitors. Importantly, however, the compounds increased Coll IV, a crucial basement membrane collagen involved in tissue repair and remodeling activity. This activity was distinct compared to TNF inhibitors. [Table 2-1] [Table 2-2]

[0147] The data demonstrate that MCIM compounds possess distinct phenotypic profiles with multimodal activity and exhibit specific effects in different cell types after stimulation by different inflammatory mediators. In particular, MCIM compounds modulate immunoactivity by reducing levels of inflammatory mediators such as TNFα, e-selectin, CD38, CD40, CD69, sIgG, sIL-17A, sIL-17F, sIL-2, and sIL-6. In addition, MCIM compounds exhibit potential for tissue remodeling, as indicated by the reduction of Coll I and MMP1. Notably, MCIM compounds increase the production of Coll IV, a key basement membrane collagen involved in tissue repair and remodeling activity. This activity was distinguished compared to TNF inhibitors.

[0148] Example 2 - Evaluation of mitochondrial phenotype in osteoclasts The ability of mitochondria to undergo fusion and fission processes is essential for mitochondrial function and cellular health. Qualitative and / or quantitative changes in the mitochondrial network have also been observed under pathological conditions caused by genetic mutations in mitochondrial DNA or the nuclear OXPHOS gene, suggesting a close relationship between mitochondrial structure and function. In addition, some evidence suggests that the injury response of damaged cells may be improved by the presence of healthy mitochondria (Jin, et al, 2019, the whole of which is incorporated herein by reference). Therefore, studying mitochondrial morphology and function can provide important insights into the potential of cells and tissues to recover from injury. Osteoclasts are a high-energy cell type sensitive to changes in mitochondrial metabolism, making them suitable for evaluating such relationships.

[0149] Peripheral blood mononuclear cells were isolated from human whole blood by differential centrifugation using Ficoll-Paque PLUS (GE Healthcare Biosciences). CD14+ monocytes were purified from freshly isolated PBMCs using a CD14+ selection kit (StemCells.UK) by positive magnetic selection according to the manufacturer's instructions. 1 × 10⁶ cells were added to Complete Minimal Essential Medium-Alpha supplemented with 10% thermo-inactivated fetal bovine serum (FBS, Invitrogen, UK), 2 mM glutamine (Invitrogen, UK), 20U mL-1 penicillin, and 100 ug / mL streptomycin (Sigma ALdrich, UK), along with 25 ng mL-1 recombinant human M-CSF (Peprotech, UK) and 25 ng mL-1 RANKL. 6 The cells were differentiated into osteoclasts by adding mL-1 cells for 6 days. On day 6, the cells were treated with the test compound (final concentration 0.03-1 μM, 0.05% DMSO) or the control rotenone (100 nM).

[0150] Adhering cells were fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) for 1 hour, post-fixed with 1% osmium tetroxide (Electron Microscopy Science), dehydrated in a stepwise series of ethanol, and embedded in Epon (Electron Microscopy Science). The embedded samples were sectioned using an ultramicrotome (Ultracut E, Richert-Jung, Leica Microsystem). Thin sections (90 nm thick) were collected on a 300-mesh nickel grid and stained with uranyl acetate (Electron Microscopy Science) and lead citrate. Samples were observed using a Zeiss EM 109 instrument (Zeiss). Images were captured using a Nikon digital camera Dmx 170 1200F and ACT-1 software.

[0151] Representative images are shown in Figure 4. (A) Control cells show a heterogeneous and dynamic population of mitochondria (mitochondria (M) and endoplasmic reticulum (ER)) with a good balance of fusion and division. (B) Cells treated with the prototype complex I inhibitor rotenone show an increased number of abnormal mitochondria that are rounder, have condensed cristae with evidence of fragmentation, and show evidence of nearby lysosomes (L). (C) 0.03 mM ABD900 showed increased tubular mitochondria with evidence of protruding seedlings, consistent with adaptive changes in mitochondrial structure to increase mitochondrial area without an apparent increase in organelle biomass. Dumbbell morphology consistent with the formation of electron transport chain (ETC) supercomplexes was also observed. (D) 0.1 mM ABD900 showed a similar profile with evidence of cristae refraction. (E) 0.3 mM ABD900 showed filamentous elongation in many mitochondria, consistent with an attempt to expand cristae volume. (F) 1 mM ABD900 exhibits a heterogeneous population of mitochondria with a rounded morphology and condensed cristae.

[0152] The results demonstrate that, in contrast to the classical complex I blocker rotenone, the MCIM compounds of the present invention induce a mitochondrial phenotype consistent with maturity-based differentiation. For example, older mitochondria, normally removed by mitophagy, are retained as part of the integrative stress response. Structural adaptations occur to maintain cellular free energy without an apparent increase in biomass, thereby increasing ETC efficiency.

[0153] Example 3 - Cell metabolism and viability The in vitro effects of the test compound on cell metabolism and viability were determined by incubation with human primary lung fibroblasts, followed by measurement of cellular ATP concentration and cell count.

[0154] ATP is an organic compound that can be produced by several cellular processes, including glycolysis and oxidative phosphorylation. However, if oxygen levels or substrates that promote oxidative phosphorylation are insufficient, cells can reprogram their metabolism toward glycolysis or other pathways to maintain ATP availability. Depending on the cellular environment, such metabolic changes may be accompanied by adaptive changes in gene expression. For example, under certain culture conditions, cells may upregulate an "adaptive response" gene known as vascular endothelial growth factor (VEGF), which encodes a pro-angiogenic protein that functions to induce new angiogenesis in search of new sources of oxygen and nutrients. This is crucial for triggering a functional repair response. By modulating the activity of complex I, the MCIM compounds of the present invention modulate ATP production and thereby induce a cellular adaptive response.

[0155] The effects of MCIM compounds on cellular adaptation were evaluated in vitro in human primary lung fibroblasts (HLFs). Cells were cultured under different metabolic substrates (glucose, L-glutamine, and pirubate) to explore potential changes in response to the cellular microenvironment. Cells cultured under these conditions were treated with various concentrations of the compounds, and intracellular ATP concentration, nuclear count, and VEGF secretion were measured to quantify cell viability, metabolism, and indicators of adaptive response.

[0156] Human primary lung fibroblasts were placed in 96-well plates in 100 μL of DMEM complete medium (5.5 mM glucose, 2 mM L-glutamine, and 1 mM pirubate) containing 1% penicillin-streptomycin and 10% thermoinactivated fetal bovine serum, with 2 × 10⁶ cells per well. 3Cells were plated at a concentration of cells / well. Cells were incubated overnight at 37°C / 5%CO2 to allow cell adhesion. On the day of treatment, the cell culture medium was removed and the cells were washed three times with Gibco® DMEM, glucose-free, L-glutamine-free, pirubate-free, phenol red-free + 1% penicillin-streptomycin and 10% thermo-inactivated fetal bovine serum (referred to as “basic” medium). Fresh cell cultures supplemented with 5.5 mM glucose alone, 5.5 mM glucose and 2 mM L-glutamine, or 5.5 mM glucose, 2 mM L-glutamine and 1 mM pirubate were added to the wells. The test compounds were prepared as 10-fold final concentration solutions in each appropriate culture medium. The compounds were added to the cultures at 1-fold final concentration and incubated at 37°C / 5%CO2 for 1 or 3 days. On day 1, VEGF secretion was measured from the cell culture supernatant, and 72 hours after treatment, the cells were assayed for nuclear count and ATP production.

[0157] To evaluate nuclear counts, formaldehyde was added to a final concentration of 4%. After a further 20-minute incubation at room temperature, the medium was aspirated, and the cells were washed twice with 200 μL of TBS-T (1×TBS + 0.1% Tween20). The nuclei were stained with 50 μL of PBS containing 10% thermoinactivated fetal bovine serum, 0.1% Triton-X-100, and 2 μM Hoechst dye. After a 30-minute incubation at room temperature protected from light, the cells were washed twice with TBS-T, and the cells were counted in 100 μL of PBS solution using an ImageXpress Pico system with a DAPI channel (50 ms exposure, -3 digital confocal setting) for stitched plate acquisition (4x magnification).

[0158] To evaluate ATP concentration, 50 μL of reconstituted ATPlite substrate solution (ATPlite first-step luminescence assay system, Perkin Elmer) was added to the cells. After incubation at room temperature for 5 minutes on a plate shaker, luminescence was measured on a BMG plate reader (PHERAstar) using a LUM plus module, gain 3000, and CR 96 / 384 opening spoon (type A3).

[0159] Next, the average values ​​across the tested concentrations were plotted, and the data was analyzed using GraphPad Prism software (v9) for 4-parameter IC analysis. 50 By fitting the equation, the half-maximal inhibitory concentration (IC) of the effect on ATP or nuclear count can be determined. 50 The following calculations were performed. The ATP readout per nuclear count was calculated by dividing the ATPlite emission readout value by the Hoechst-stained nuclear count per test concentration.

[0160] To evaluate VEGF secretion, the cell supernatants from three replication wells were combined, and VEGF secretion was measured according to the instructions provided by the Quantikine® ELISA Human VEGF Kit manufacturer. Absorbance at 450 nm was measured using a BMG plate reader (CLARIOstar plus) with path length correction. Background absorbance was measured at 540 nm.

[0161] The data were normalized to the VEGF standard curve and expressed as the mean (pg / mL) of the control well. The data were plotted, and informed consent (IC) was issued regarding the effect on VEGF secretion. 50 The data was then processed using GraphPad Prism software (v9) to obtain data for a 4-parameter IC. 50 The calculation was performed by fitting the data to the equation.

[0162] The results are shown in Figure 5.

[0163] Figure 5A consists of three panels showing intracellular ATP (left panel), nuclear count (center panel), and ATP readout per cell (right panel) after 72 hours of incubation with MCIM compounds. Cells cultured in glucose-supplemented medium (square) showed no effect of MCIM compounds on intracellular ATP levels, nuclear count, or ATP readout per cell. Cells cultured in glucose and glutamine-supplemented medium (white circles) showed increased vehicle-supplemented intracellular ATP levels and nuclear counts compared to cells cultured with glucose alone. Treatment of cells cultured in glucose and glutamine-supplemented medium with MCIM compounds reduced intracellular ATP levels and nuclear counts in a concentration-dependent manner without affecting ATP levels per cell. When the basal culture medium was supplemented with glucose and L-glutamine was concentrated with pirubate (black circles), the effect of MCIM compounds on intracellular ATP and nuclear counts observed in glucose and L-glutamine-supplemented medium alone was eliminated.

[0164] The results show that MCIM compounds did not exhibit cytotoxicity and did not affect cell viability in cells cultured in a glucose-containing basal medium. When cells were cultured in a medium supplemented with glucose and L-glutamine, intracellular ATP levels and nuclear counts increased, reflecting L-glutamine's role as an essential precursor amino acid for cell proliferation. The reduction in intracellular ATP and nuclear counts observed in cells cultured with glucose and L-glutamine, as well as the lack of effect on ATP levels per cell, indicates that cells adapted to the metabolic effects of MCIM compounds' regulation of Complex I by limiting highly energy-intensive processes such as cell proliferation in order to maintain intracellular ATP levels. In addition, the reversal of this effect when the medium was further supplemented with pirubate (glucose + L-glutamine + pirubate conditions) suggests that the observed adaptive response (reduced cell proliferation) is dependent on nutrient availability to the cells.

[0165] Figure 5B shows that in complete medium, MCIM compounds had no effect on VEGF secretion (black circles (●)), but in the absence of pirubate in the medium (white circles (○)), MCIM compounds caused a concentration-dependent increase in VEGF secretion (at the semi-maximal inhibitory concentration (IC)). 50 This shows that ) = 112nM).

[0166] The results indicate that by regulating complex I, in the absence of excess substrate, cells induce an adaptive response that leads to growth and repair by upregulating VEGF. The lack of effect when pirubate is present in the culture medium suggests that the adaptive repair response is also dependent on nutrient availability.

[0167] In summary, the results show that treatment with the MCIM compound of the present invention induces an adaptive response that enables cells to maintain their ATP supply. When cells are treated with the MCIM compound, they attempt to restore tissue homeostasis by reducing energy-intensive activities such as proliferation and increasing the production of growth factors such as VEGF. This occurs without any effect on cell viability.

[0168] Example 4 Classical complex I inhibitors often cause cytotoxicity and cell death, and despite reports of antitumor effects for compounds such as IACS-010579, known inhibitors have not found use as approved therapeutic agents, primarily due to mechanism-based toxicity (Yap, T et al, 2023). Thus, identifying a suitable approach for complex I inhibition that provides benefits without toxicity has proven challenging. Alternative approaches that do not cause adverse effects may have potential benefits in the treatment of various progressive diseases. In this study, we evaluated whether the MCIM compound of the present invention showed differences in cellular behavior compared to the known complex I inhibitors IACS-010579 and rotenone.

[0169] Human primary lung fibroblasts were placed in 96-well plates in 100 μL of DMEM complete medium (5.5 mM glucose, 2 mM L-glutamine, and 1 mM pirubate) containing 1% penicillin-streptomycin and 10% thermoinactivated fetal bovine serum, with 5 × 10⁶ cells per well. 3 Cells were plated at the cell / well concentration. Cells were incubated overnight at 37°C / 5% CO2 to allow cell adhesion. On the day of treatment, the cell culture medium was removed and the cells were washed three times with Gibco® DMEM, glucose-free, L-glutamine-free, pirubate-free, phenol red-free + 1% penicillin-streptomycin and 10% thermoinactivated fetal bovine serum (referred to as "basic" medium). Fresh cell culture medium (pirubate-free) was added to the wells. The test compound was prepared as a 10-fold final concentration solution in pirubate-free medium. After 24 hours of compound treatment (in a cell incubator at 5% CO2, 37°C), the used medium was removed from all wells and thoroughly washed three times with basal cell medium. For the wash assay conditions, 100 μL of pirubate-free medium was added per well. Without washout, 90 μL of pirubate-free medium and 10 μL of the test agent (or assay medium) prepared at 10 times the final assay concentration were added per well. Recovery of cell proliferation was measured after incubation at 37°C / 5% CO2 for 24 hours using either pirubate-free medium (without re-addition of the compound) or pirubate-free medium with re-addition of the compound.

[0170] To evaluate nuclear counts, formaldehyde was added to a final concentration of 4%. After a further 20-minute incubation at room temperature, the medium was aspirated, and the cells were washed twice with 200 μL of TBS-T (1×TBS + 0.1% Tween20). The nuclei were stained with 50 μL of PBS containing 10% thermoinactivated fetal bovine serum, 0.1% Triton-X-100, and 2 μM Hoechst dye. After a 30-minute incubation at room temperature protected from light, the cells were washed twice with TBS-T, and the cells were counted in 100 μL of PBS solution using an ImageXpress Pico system with a DAPI channel (50 ms exposure, -3 digital confocal setting) for stitched plate acquisition (4x magnification).

[0171] As cells grow and divide (called proliferation), they progress through the cell cycle, a tightly controlled process consisting of two main activities: DNA replication and mitosis / cell division. As a method for accurately quantifying cell proliferation rate, DNA replication can be measured using the pyrimidine analog BrdU. BrdU can be incorporated into newly synthesized DNA in place of thymidine. After incubating proliferating cells with BrdU for 20 hours, the labeling solution was completely removed, and the cells were fixed with 70 μL of fixative solution at room temperature for 30 minutes. The fixative solution was aspirated, and anti-BrdU-Eu antibody was added and incubated in the dark at room temperature for 2 hours. The wells were washed three times with washing solution, 70 μL of DELFIA inducer was added per well, and incubated in the dark for 30 minutes. Eu fluorescence was measured using a time-resolved method with PHERAstar FSX (Ex337 / Em620nm). Data were plotted and curves fitted using a four-parameter equation with GraphPad Prism software.

[0172] The results are shown in Figure 6. The figure shows four panels: the effect of compound washing on cell proliferation, measured by BrdU uptake (A) and nuclear count (B), and by BrdU uptake (C) and nuclear count (D), at the highest test concentration. In panels A and B, ABD900 is shown as a black circle (●), rotenone as a gray square (■), and IACS-010759 as a white circle (○). In panels C-D, cells without washing are shown in black (IACS-010759) and gray (rotenone), and washed cells are shown as white-out black (IACS-010759) and gray (rotenone) bars. For ABD900, washed cells are shown as a white-out fill pattern, and cells without washing are shown as a checkerboard fill. Data are mean ± sem.

[0173] The results show that typical complex I inhibitors, such as IACS-010759 and rotenone, reduce cell proliferation (BRdU incorporation and nuclear count) when cells are cultured under piruvate-restricted conditions. For known complex I inhibitors such as IACS-010759 and rotenone, there is no recovery from these effects if the compound is washed away. However, for the MCIM compound, there is a recovery of cell proliferation (BrdU incorporation and nuclear count) after the compound is washed away. Overall, the data together show that the MCIM compound of the present invention exhibits different cellular effects compared to known archetypal inhibitors of complex I.

[0174] Example 5 - Monotherapy and combination therapy in mice with collagen-induced arthritis Male DBA / 1j mice aged 7-8 weeks were used in all procedures. The animals were housed in groups of 10, with free access to food and water, and maintained at 21°C ± 2°C in a 12-hour light / dark cycle. Complete Freund's adjuvant (CFA) was prepared by emulsifying a 4 mg / mL suspension of Mycobacterium tuberculosis, H37Ra in incomplete Freund's adjuvant (IFA) (0.85 mL of paraffin oil and 0.15 mL of monooleate mannide) with 4 mg / mL of bovine type II collagen in a 1:1 (v / v) ratio. All mice were subcutaneously immunized with 200 μg of bovine type II collagen in CFA. After 21 days, all mice were subcutaneously immunized with 100 μg of bovine type II collagen in IFA. The mice began to develop signs and symptoms of arthritis after "booster" immunization.

[0175] For macroscopic assessment of arthritis, the following signs were monitored three times a week in each paw of each mouse, and the sum was used to generate an arthritis index (AI) (the maximum AI for one animal is 16). 0 = No visible effects on arthritis. 1 = Edema and / or erythema of one toe. 2 = Edema and / or erythema of the two toes. 3. Edema and / or erythema exceeding 2 toes. 4 = Severe arthritis affecting the entire foot and toes.

[0176] Figure 7 shows seven graphs of the mean arthritis index as a function of time (days of administration) for each of the following: (A) HMC-C-02-A, (B) HMC-C-01-A, (C) HMC-N-02-A, (D) HMC-N-01-A, (E) NASMP-01-A, (F) CHMSA-01-A, (G) CHMSA-03-A. These data demonstrate that the MCIM compounds described herein exhibit excellent oral in vivo activity in preventing the progression of established severe arthritis.

[0177] The animals were classified into treatment groups with a mean arthritis index of 2.5, and then administered the compound once daily for 14 days by oral force-feeding. On day 14, the animals were slaughtered, and their limbs were fixed in 10% neutral buffered formalin.

[0178] The fixed limbs were prepared as paraffin blocks, sectioned, and then stained with toluidine blue.

[0179] For the histopathological evaluation of the biological effects of MCIM compounds in arthritis, bone resorption was assessed by direct counting per bone in areas showing clear Haussip's fossa or active osteolytic lesions. To assess the frequency of new bone formation, total counts of the cancellous bone girdles were performed.

[0180] Data were analyzed by generating total scores for each parameter in each treatment group. The treatment groups were compared to the vehicle group by ANOVA using the Kruskal-Wallis test statistic with accurate p-value comparisons. Data are summarized in Table 3. Data are shown as sums from all limbs per animal per group. *p<0.05, **p<0.01, ***p<0.005 vs. vehicle. §§§ p<0.005 vs. etanercept. [Table 3]

[0181] Data for some of the compounds are also shown in Figures 7 and 8.

[0182] Figures 8 and 9 show the reduction in bone resorption by the approved drugs etanercept (Figure 8A) and tofacitinib (Figure 8C), as well as various MCIM compounds (Figures 8A and 8B). Figure 9A shows a moderate increase in bone formation in mice treated with etanercept compared to the vehicle control. In contrast, mice treated with HMC-C-01-A, HMC-C-01-B, and HMC-N-01-B showed a significant increase in bone formation compared to the vehicle control and etanercept (Figure 9A). Figure 10 shows the appearance of osteoid / new bone formed in response to treatment with the vehicle (top panel), the approved disease-modifying antirheumatic drug (etanercept, bottom panel), and the compound HMC-C-01-A (center panel). The central panel of Figure 10 shows that the new bone formed in response to treatment with HMC-C-01-A has a regular appearance with preservation of the highest point (arrow). This indicates that the formed bone is responsive to pressure and possesses structural integrity, in contrast to the reactive and sporadic deposits produced with etanercept (lower panel) or tofacitinib (not shown).

[0183] Figure 11 shows that osteoid improvement is achieved in mice treated with HMC-C-01 without controlling inflammation. This demonstrates a direct reconstructive effect that is independent of inflammation control.

[0184] Figures 7–11 show the effects of MCIM compounds in models of joint inflammation and bone loss. The results indicate that MCIM compounds alone reduce total bone resorption and localized areas of bone resorption as well as, or better than, antibiotics such as etanercept or Janus kinase (JAK) inhibitors such as tofacitinib. In addition, MCIM compounds induce an adaptive repair response, resulting in increased new bone deposition (osteoid) in both areas of osteoid counting and osteoid formation.

[0185] The new bone formed in response to treatment with MCIM compounds has a regular appearance with preservation of the highest point. This indicates that the formed bone is responsive to pressure and possesses structural integrity, in contrast to the reactive and sporadic deposits produced with etanercept.

[0186] In summary, the above data show that MCIM compounds exhibit excellent oral in vivo activity in preventing the progression of bone loss in established severe arthritis, but importantly, they also increase bone formation, which is a form of repair in established arthritis.

[0187] Considering the improved response of mice to treatment with MCIM compounds, we then investigated whether combining MCIM compounds with approved drugs used to treat arthritis would further improve arthritis pathology in the mouse model. The data are summarized in Table 4 and illustrated in Figure 12. [Table 4]

[0188] Figure 12 shows four graphs demonstrating the anti-arthritis effects of the TNF inhibitor etanercept, the MCIM compound HMC-C-01, and the combination of etanercept and HMC-C-01 on the overall mean change in (A) synovitis, (B) cartilage damage, (C) bone resorption, and (D) osteopathology.

[0189] Figure 13 shows a graph demonstrating the effects of the TNF inhibitor compound etanercept, the MCIM compound HMC-C-01, and the combination of etanercept and HMC-C-01 on osteoid or new bone formation.

[0190] These data indicate that etanercept and HMC-C-01 have additive effects on synovitis, bone resorption, and cartilage damage. However, importantly, the combination of HMC-C-01 and etanercept significantly reduces overall osteopathology compared to either drug alone, and a synergistic effect is observed when used in combination compared to either compound alone (Figure 11D). Furthermore, HMC-C-01, when administered alone, shows a strong effect on osteoid formation, which is an indicator of its repair-promoting phenotype. The TNF inhibitor compound etanercept shows a limited effect on osteoid formation. However, importantly, the combination of HMC-C-01 and etanercept significantly improves overall bone osteoid formation compared to either drug alone. The effect of the combination of MCIM and TNF inhibitors on reducing bone lesions is thought to be due to the reconstructive and repair activity of the MCIM compound, which enhances the control of pathology compared to what can be achieved simply through the control of synovitis.

[0191] Example 6 - Reinforcement therapy in an IBD model DSS-induced colitis is a widely used model of IBD (Chassaing et al, 2015, the entire work is incorporated herein by reference). Female C57Bl / 6 mice aged 8-9 weeks were used for all treatments. The animals were housed in groups of 10, with free access to food and water, and maintained at 21°C ± 2°C with a 12-hour light / dark cycle. Dextran sulfate (DSS) was prepared by dissolving DSS in water to a final concentration of 1.5%. All mice were given optional access to water containing DSS for 6 hours before being administered by oral force-feeding once daily for 8 days either a vehicle control, 300 mg / kg of sulfadiazine, 3 mg / kg of etanercept, or 10 mg / kg of the MCIM test compound. Mice began to develop signs and symptoms of colitis within 1 day.

[0192] To assess colitis, mouse body weight, fecal consistency, and the presence or absence of blood in the feces were monitored. Based on the severity of changes in each of these observed parameters, mice were assigned a score according to the criteria in Table 5. The disease scores were summed to generate a Disease Activity Index (DAI) (maximum DAI for a single animal is 9). Data are presented as mean sem for the entire group, and statistical analysis was performed using two-way ANOVA with multiple comparisons (GraphPad Prism v 9.2.0). *p<0.05, ***p<0.005 vs. vehicle. §§§ p<0.005 vs. sulfasalazine, aaa p<0.005 vs. etanercept. [Table 5]

[0193] Figure 14 shows the mean disease activity index of mice with DSS-induced colitis after treatment with vehicle, 300 mg / kg / day of sulfasalazine, 3 mg / kg / day of etanercept, or 10 mg / kg / day of the MCIM compound HMC-C-01-A. These data demonstrate that the MCI compounds described herein exhibit superior in vivo activity in preventing the progression of established DSS-induced colitis. Furthermore, the data show that the MCIM compounds have superior efficacy compared to both conventional treatments, sulfasalazine, and etanercept.

[0194] To evaluate the histopathological effects of MCIM compounds in colitis, the colon from the rectum to the ileocecal junction was removed on day 9, and its length was recorded. Fecal matter was then removed, and the weight of the colon was recorded. The colon was preserved in 10% neutral buffered formalin and treated as a paraffin block.

[0195] Next, tissue sections were stained with hematoxylin and eosin (H&E), and parameters for inflammation, mucosal erosion, epithelial hyperplasia, epithelial dysplasia, myxocyte dysplasia, and fibrous proliferation were evaluated on a scale of 0 to 5 as follows. 0: Normal 1: Minimal, localized 2: Moderate, localized 3: Moderate, multifocal, or diffuse 4: Prominent, localized 5: Prominent, multifocal, or diffuse Data were analyzed by generating mean histopathology scores across each treatment group. The treatment groups were compared to the vehicle and positive control group using a two-way ANOVA (Prism 9.2.0) with adjustments for multiple comparisons. Data are presented as mean ± sem. * p<0.05, ** p<0.01, *** p<0.005 vs. vehicle. aaa p<0.005 vs. sulfasalazine. §p<0.05, §§p<0.01, §§p<0.005 vs. etanercept. Data are summarized in Figures 12-16.

[0196] Figure 14 shows one graph showing the mean disease activity index for vehicle control, 300 mg / kg / day of sulfasalazine, 3 mg / kg / day of etanercept, and 10 mg / kg / day of HMC-C-01-A. Figure 15 shows two graphs for each of the mean mucosal erosion scores: (A) vehicle control, 300 mg / kg / day of sulfasalazine and 10 mg / kg / day of ABD900, and (B) vehicle control, 3 mg / kg / day of etanercept and 10 mg / kg / day of HMC-C-01-A. Figure 16 shows two graphs for each of the mean gland loss scores for each of the following: (A) Vehicle control, 300 mg / kg / day sulfasalazine, and 10 mg / kg / day ABD900, and (B) Vehicle control, 3 mg / kg / day etanercept, and 10 mg / kg / day HMC-C-01-A. Figure 17 shows two graphs for each of the following: (A) Vehicle control, 300 mg / kg / day sulfasalazine, and 10 mg / kg / day ABD900, and (B) Vehicle control, 3 mg / kg / day etanercept, and 10 mg / kg / day HMC-C-01-A. Figure 18 shows two graphs for each of the following: (A) Vehicle control, 300 mg / kg / day of sulfasalazine, and 10 mg / kg / day of ABD900; (B) Vehicle control, 3 mg / kg / day of etanercept, and 10 mg / kg / day of HMC-C-01-A.

[0197] Figure 19 shows representative histological sections of the colon taken from mice with DSS-induced colitis treated with a vehicle, 3 mg / kg / day of etanercept, or 10 mg / kg / day of HMC-C-01-A, respectively. In vehicle-treated mice, there is clear ulceration (upper right panel, arrows) and general loss of colonic tissue structure, as indicated by visible edema / inflammation and erosion (upper left panel, arrows). Mice treated with etanercept show general preservation of tissue structure but still exhibit moderate inflammation and edema (center panel, arrows). In contrast, the colons of mice treated with HMC-C-01-A have preserved tissue structure and no visible signs of inflammation or edema. Surprisingly, treatment with HMC-C-01-A stimulates adaptive repair of the colon that is not seen with etanercept treatment (lower panel, arrows). Histological sections from mice treated with HMC-C-01-A demonstrate that this reaction is organized and localized within the lamina propria, aligned along the basal layer along with the expansion / maintenance of the basement membrane and the maintenance of foveal structures.

[0198] In summary, these data indicate that the HMC-C-01-A and ABD900 compounds possess excellent activity in preventing the progression of established colitis and can stimulate the repair of damaged tissue. Administration of MCIM compounds inhibited the major histological outcomes of mucosal erosion / ulceration, as shown in Figure 15. Importantly, administration also increased epithelial hyperplasia, suggesting induction of a repair response. This is supported by the unique findings of fibrous proliferation in the MCIM compound-treated group (Figures 17A and 17B).

[0199] Overall, Figures 14–19 show the effects of MCIM compounds in a model of gastrointestinal disease. The results indicate that MCIM compounds significantly reduce disease signs and symptoms and protect underlying tissue damage compared to approved drugs, sulfasalazine, or the anti-TNF bioagent etanercept, and that MCIM compounds significantly promote the repair response of epithelial hyperplasia, myxocyte dysplasia, and fibrous proliferation compared to sulfasalazine and etanercept.

[0200] Example 7 - Cell thermal shift assay (CETSA) and multiplex quantitative mass spectrometry Following observations that MCIM compounds can influence mitochondrial morphology and regulate cellular metabolism, we investigated whether the repair properties of MCIM compounds may be due to their binding to / regulation of mitochondrial proteins and / or complexes. To this end, MCIM compounds were evaluated using a cell thermal shift assay (CETSA) combined with quantitative mass spectrometry (MS) to determine which pathways are regulated by MCIM compounds.

[0201] Thp-1 cells were incubated for 4 hours in the presence of 2 μM MCIM compound (ABD900) or DMSO (vehicle control). After the incubation period, the samples were heated to one of the following temperatures: 40.0, 42.9, 46.0, 49.6, 53.2, 56.8, 60.8, 64.0, 67.1, and 70.0°C. Each test condition was performed in pairs.

[0202] After heating, the cells were lysed, and the samples were digested with trypsin. The digested soluble peptide fractions corresponding to each temperature were then labeled with different isotopic tags using the TMT10plex system described in Bantscheff, M., et al (2007), Bantscheff, M., et al (2012), and Franken, H., et al (2015) (each of which is incorporated in its entirety by reference). Labeling the fractions from each temperature with individual TMT10Plex ​​tags allows for sample pooling and analysis by mass spectrometry in a single run.

[0203] After labeling with TMT10Plex ​​and pooling, samples were fractionated using liquid chromatography with hydrophilic strong anion exchange (hSAX) (24 fractions per sample) and tandem mass spectrometry (LC-MS / MS). LC-MS / MS data were analyzed to identify proteins whose thermal stability shifted in the presence of the MCIM compound, using the TPP R Package (Franken, H., et al (2015)) according to the procedure described in Savitski, MM et al (2014). Generally, when a compound binds, the protein becomes more stable and therefore more resistant to thermal denaturation. Thus, the shift in thermal stability of the protein in the described CETSA assay may indicate direct binding of the compound to a protein with shifted thermal stability. Alternatively, the shift in thermal stability may indicate that the protein is involved in downstream events from the bound protein, such as post-translational modifications resulting from altered metabolic or signaling pathways.

[0204] In short, the criteria for selecting target candidates were as follows: • Minimum p-value < 0.4 (Benjamini-Hochberg correction) • The DTm for execution 1 (R1) and execution 2 (R2) have the same symbol. • DTm (drug vs. DMSO) > DTm (DMSO R1 vs. DMSO R2) Minimum slope less than -0.06 Using the above criteria, we identified 105 proteins that exhibited a thermal shift, indicating either stabilization or destabilization when incubated with MCIM compounds. Of these 105 proteins, 73 were classified as high-confidence hits and 32 as moderate-confidence hits.

[0205] Functional protein interactions and / or associations of 105 identified candidate proteins were searched using the freely available STRING software (https: / / string-db.org). STRING analysis identified candidates involved in oxidative phosphorylation (e.g., NDUFA6 and SDHB), mitochondrial function, and the ER-Golgi interface.

[0206] In addition, thermal shifts were observed in proteins involved in adaptive stress responses (e.g., YME1L1, OXSR1, MKNK1) and other proteins that play a role in NF-κB signaling (e.g., OXSR1, RASA1, and BIRC2).

[0207] Therefore, in line with the data from Example 3, we concluded that incubation with MCIM compounds alters cellular metabolic activity through the specific regulation of oxidative phosphorylation, as indicated by the thermal shift of NDUFA6 (a component of respiratory complex 1). We hypothesized that such regulation of complex 1 and the oxidative phosphorylation pathway alters intracellular NFκB and adaptive response pathways, as indicated by the thermal shifts observed for proteins such as YMEL1L1, OXSR1, MKNK1, RASA1, and BIRC2.

[0208] Example 8 - Photoaffinity labeling (PAL) and quantitative stable isotope labeling with amino acids (SILAC) in cell culture To investigate the binding partners of MCIM compounds and the cellular activities they regulate, photoaffinity labeling (PAL) and quantitative stable isotope labeling with amino acids in cell culture (SILAC) were performed. To do this, clickable linker probes of MCIM compounds were generated.

[0209] This identified NDUFS2, a subunit of mitochondrial complex I, as a binder for the clickable linker probe MCIM compound. Collectively, the CETSA and PAL / SILAC-MS results strongly suggest that the MCIM compound can modulate oxidative phosphorylation and stress response pathways by binding to and regulating the activity of mitochondrial complex I.

[0210] Example 9 - Computational modeling for identifying binding sites in NDUFS2 Considering that both CETSA and PAL / SILAC-MS methods identified the complex I subunit as a target for the MCIM compound, an in silico computer modeling approach was used to determine the binding site of the MCIM compound to the complex I subunit.

[0211] Initially, the homology model was constructed from publicly available structures of mitochondrial complex I from five organisms, including humans (Table 6). The proposed target was completely lost in the five structures. Mitochondrial complex I used in the following modeling approach is shown in Figure 20. [Table 6]

[0212] Homology modeling revealed a lid pocket within the NDUFS2 subunit of complex I in contact with the Q tunnel. In four of the models, the lid pocket was found to be in an "open" conformation, while in the remaining models, it was in a "closed" conformation.

[0213] We constructed an open confirmation homology model and then used SiteFinder to map NDUFS2 in both open and closed conformations. In particular, we constructed a model of the "drugability" of NDUFS2 using SiteFinder (Halgren TA, 2009), as measured by the volume of buried nonpolar available surface area (ASA; Figure 21). This model identified two binding sites on NDUFS2. The first binding site ("Pocket A") is located on the lid pocket in the open conformation and is represented by a cluster of spheres in Figure 19. Pocket A has a buried nonpolar available surface area percentage of 72%. The second binding site ("Pocket B") is located on the "backside" of mitochondrial complex I relative to the positions of mitochondrial complex I subunits NDUFS7 and ND1 and is represented by a second cluster of spheres. Pocket B has a buried nonpolar available surface area percentage of 71%.

[0214] Further modeling of Q10, the intrinsic ligand for the Q tunnel within the Q site of complex 1, using SiteFinder, revealed that the space for binding within this pocket is limited (Figure 22). The sphere in Figure 22 shows the space and channels around NDUFS2 when NDUFS2 is associated with complex 1. This modeling approach also revealed additional ligand binding sites at the junction between NDUFS2 and NDUFS7 (Figure 23). In particular, MCIM compounds are predicted to interact with His38 and Tyr141 of NDUFS2.

[0215] Using these protein models, the optimal binding sites for ABD900, HMC-C-01-A, and HMC-N-01-A were determined to be the NDUFS2 lid pocket (as shown in Figure 19, "Pocket A"), specifically the lid pocket with an open conformation (Glide score approximately 6) near the Q site. More specifically, the inventors were able to predict which amino acid residues in NDUFS2 contributed to the binding of the compounds: The H bond between the carbonyl skeleton of Gly85, the amine skeleton of Leu95, and the carboxylic acid of Asp193. • H bonding to Tyr141 and His38 • π-π stacking using Phe458 and His88 In conclusion, analysis of the Q site in NDUFS 2 indicates that this site must be Q10 or an open confirmation site for accommodating small drug-like compounds.

[0216] Example 10 - Virtual screening to evaluate the structure-activity relationship for NDUFS 2 pockets As discussed in Example 9, the optimal binding site for the MCIM compound has been determined to be within the lid pocket of NDUFS2 adjacent to the Q tunnel. From the data of Example 9, a ligand-protein pharmacophore model was constructed identifying nine pharmacophore features (Figure 24 and Tables 7A-7C). Table 7-A describes the relationship between the type of pharmacophore feature and the acceptable variation in 3D space for the position of a given pharmacophore feature. Table 7-B shows a distance matrix describing the 3D relationships between the centers of each pharmacophore feature. Table 7-C describes the angles between each triplet combination of pharmacophore features, with the column "Y" describing the vertices of each angle. Using the parameters listed in Tables 7-A-7-C, a visual representation of the pharmacophore model was created using a unified annotation scheme in the (MOE) software tool (Figure 24). (MOE) can also be used with parameters to determine whether a molecule fits the pharmacophore model, i.e., whether four or more annotation points of the pharmacophore model are occupied by corresponding annotation points located on the test molecule. [Table 7A] [Table 7B] [Table 7C-1] [Table 7C-2] [Table 7C-3]

[0217] Using the Molecular Operating Environment (MOE), 2022.02 Chemical Computing Group ULC,1010 Sherbooke St.West, Suite #910, Montreal, QC, Canada, H3A 2R7,2022 MOE docking method, 117 compounds were evaluated for their ability to dock to the binding sites predicted using the pharmacophore model described above. For docking to be considered successful, the molecule must hit at least four features of the pharmacophore model shown in Figure 22 and reach a semi-maximal inhibitory concentration (IC). 50 It is necessary that the concentration be ≤ 1 μM.

[0218] Figure 25 shows MCIM compounds that fit the pharmacophore model and satisfy seven of the nine annotation points determined to be important for binding to complex I. Surprisingly, of the 117 compounds evaluated for binding ability in the pharmacophore model, only 13 compounds showed IC. 50 It was also found that they had >1 μM, indicating that these compounds do not dock at the expected binding site. The remaining 104 compounds hit at least four of the pharmacophore features and IC 50 It is predicted to have ≤1 μM. Figure 26 shows a representative compound CHMSA-02-A, which satisfies the pharmacophore model and has approximately 7 pAct(-Log(IC)). 50 It is predicted that it will have ).

[0219] Example 11 - Ultra-high throughput virtual screening (uHTVS) Following the construction of the pharmacophore model, the inventors then wanted to use this model to identify additional NDUFS2 conjugates.

[0220] The 3D model of the entire complex 1 described in Example 9 (Figure 20) was validated by docking Q10 and the active MCIM compound. This made it possible to establish the bioactive conformation of the MCIM compound when docked to the Q tunnel of complex 1. This further enabled the generation of structure-based hypotheses, the rationalization of the structure-activity relationship (SAR) of the compound, and the construction of the pharmacophore model and the QSAR model for activity prediction (Figure 27) described in Example 10. The QSAR model is a linear regression model that includes docking scores and parameters related to ligand energy and electrostatics.

[0221] For uHTVS, the compound library was screened against the pharmacophore model described in Example 10. This model identified 37.6 million compounds from the compound library, generally hitting 3 to 6 pharmacophore features. Of these 37.6 million compounds, those with molecular weights up to approximately 350 Da were effectively docked to the 3D complex I model without imposing pharmacophore restrictions. 67,000 compounds were predicted to dock to complex I and were retained for further screening.

[0222] Next, the retained compounds were selected using a more theoretically rigorous docking method and evaluated using a QSAR model to predict the biological activity of each compound (Figure 27). A GBVI / WSA ΔG force field-based scoring function (Naim et al. 2007) was used on the MOE dock (Molecular Operating Environment (MOE), 2022.02 Chemical Computing Group ULC, 1010 Sherbooke St. West, Suite #910, Montreal, QC, Canada, H3A 2R7). Using the described screening process, 756 compounds were identified as potential drug candidates targeting NDUFS2 / NDUFS7 of complex 1.

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Claims

1. A combination of pharmaceuticals containing a mitochondrial complex I modulator (MCIM) compound and a TNF inhibitor compound.

2. A pharmaceutical composition comprising a mitochondrial complex I modulator (MCIM) compound and a TNF inhibitor compound.

3. A combination of pharmaceuticals or pharmaceutical composition according to claim 1 or 2, for use as a pharmaceutical agent.

4. A pharmaceutical composition comprising a mitochondrial complex I modulator (MCIM) compound for use in the treatment of a subject requiring treatment for an inflammatory and / or progressive disease, wherein the treatment comprises separate, sequential, or simultaneous administration of the pharmaceutical composition and a TNF inhibitor compound to the subject.

5. A pharmaceutical composition comprising a TNF inhibitor compound for use in the treatment of a subject requiring treatment for an inflammatory and / or progressive disease, wherein the treatment comprises separate, sequential, or simultaneous administration of the pharmaceutical composition and a mitochondrial complex I modulator (MCIM) compound to the subject.

6. A pharmaceutical composition comprising a mitochondrial complex I modulator (MCIM) compound and a TNF inhibitor compound for use in the treatment of a subject requiring treatment for an inflammatory and / or progressive disease, wherein the treatment comprises administering the composition to the subject.

7. A composition for use according to any one of claims 4 to 6, wherein the treatment achieves control, elimination, or tissue repair of a disease, or any combination thereof.

8. The composition for use according to claim 7, wherein the control of the disease includes inhibiting the progression of the disease.

9. A composition for use according to claim 7 or 8, wherein disease control includes supporting tissue repair.

10. A composition for use according to any one of claims 7 to 9, wherein disease control includes preventing the loss of anatomically normal tissue structure or reducing the rate of anatomically normal tissue structure loss.

11. A composition for use according to any one of claims 7 to 10, comprising disease control, disappearance, or tissue repair, or any combination thereof, including an increased repair score and / or increased wound healing.

12. The composition for use according to any one of claims 7 to 11, wherein the control or resolution of the disease and / or tissue repair comprises an increased cell count of repairing cells and / or a decreased cell count of pathologically driven cells.

13. A composition for use according to any one of claims 7 to 12, wherein the control or elimination of the disease and / or tissue repair induces the restoration of anatomically normal tissue structure.

14. The composition for use according to any one of claims 4 to 13, wherein the inflammatory and / or progressive disease is related to or caused by the overexpression, secretion, or stimulation of TNF.

15. The composition for use according to any one of claims 4 to 14, wherein the inflammatory and / or progressive disease is an autoimmune disease or an autoinflammatory disease.

16. The composition for use according to any one of claims 4 to 15, wherein the inflammatory and / or progressive disease is arthritis, inflammatory bowel disease (IBD), atopic dermatitis, psoriasis, plaque psoriasis, idiopathic pulmonary fibrosis (IPF), non-alcoholic fatty liver disease (NAFLD) / non-alcoholic steatohepatitis (NASH), chronic kidney disease, or non-infectious uveitis (NIU).

17. The composition for use according to any one of claim 16, wherein the arthritis is rheumatoid arthritis (RA), psoriatic arthritis, inflammatory arthritis, ankylosing spondylitis, juvenile idiopathic arthritis, reactive arthritis, gout, septic arthritis, enteroarthritis, or osteoarthritis.

18. A composition for use according to any one of claims 7 to 16, wherein the tissue repair, disease control, and / or disease resolution include increased bone formation and / or decreased bone resorption.

19. The composition for use according to any one of claims 12 to 18, wherein the inflammatory and / or progressive disease is arthritis, and the pathological driving cells include osteoclasts (OCs) and / or transformed fibroblasts.

20. The composition for use according to any one of claims 12 to 19, wherein the inflammatory and / or progressive disease is arthritis, and the repair cells comprise osteoblasts (OBs) and / or non-transformed fibroblasts.

21. The composition for use according to claim 161, wherein the inflammatory and / or progressive disease is inflammatory bowel disease (IBD), and the repair cells include epithelial cells and / or mucinous cells.

22. The composition for use according to claim 21, wherein the IBD is Crohn's disease or ulcerative colitis.

23. A composition for use according to any one of claims 7 to 22, wherein the control, resolution, or tissue repair of the disease, or any combination thereof, includes a reduction in cytokine production from pro-inflammatory myeloid cells.

24. The binding of the MCIM compound to complex I modulates the activity of complex I, and this regulation of complex I does not reduce cell viability in cells O ２ A combination according to claim 1, a composition according to claim 2, a composition for use according to any one of claims 3 to 23, or a combination for use, determined by detecting a reduction in consumption.

25. The combination, composition, combination for use, or composition for use according to any one of the prior claims, wherein the binding of the MCIM compound to complex I modulates the activity of complex I and results in a reversible reduction of cell proliferation.

26. The combination, composition, combination for use, or composition for use according to any one of the prior claims, wherein the MCIM compound interacts with complex I at a binding site outside the Q tunnel.

27. The combination, composition, combination for use, or composition for use according to any one of the prior claims, wherein the MCIM binding site comprises one or more amino acid residues from NDUSF2 and / or NDUSF7.

28. The combination, composition, combination for use, or composition for use according to claim 27, wherein the MCIM compound interacts with one or more amino acid residues in NDUFS2 selected from His92, Gly85, Tyr141, His88, Leu95, Asp193, and Phe458.

29. The combination, composition, combination for use, or composition for use according to claim 27, wherein the MCIM compound interacts with at least one amino acid residue in NDUFS2 selected from Tyr141, His92, and Asp139.

30. A combination, composition, combination for use, or composition for use according to any one of the prior claims, wherein the 3D conformation of the MCIM compound annotated by the Molecular Operating Environment (MOE) 2022 Unified Annotation Scheme includes four or more pharmacophore features that conform to the pharmacophore models shown in Figure 22 and Tables 7-A, 7-B, and 7-C.

31. The MCIM compound is defined in claim 1 of WO2010 / 032009 as having the following formula: 【Chemistry 1】 A combination, composition, combination for use, or composition for use according to any one of the prior claims, which is an MCIM compound, or a pharmaceutically acceptable salt, hydrate, or solvate thereof.

32. The MCIM compound is given by the following formula: 【Chemistry 2-1】 【Chemistry 2-2】 [Chemistry 2-3] 【Chemistry 2-4】 The combination, composition, combination for use, or composition for use according to claim 31, which is an MCIM compound selected from the compounds, or a pharmaceutically acceptable salt, hydrate, or solvate thereof.

33. The MCIM compound is defined in claim 1 of WO2020 / 035560A1 as having the following formula: 【Transformation 3】 A combination, composition, combination for use, or composition for use according to any one of the prior claims, which is an MCIM compound, or a pharmaceutically acceptable salt, hydrate, or solvate thereof.

34. The MCIM compound is defined in claim 1 of WO2020 / 212581A1 as having the following formula: 【Chemistry 4】 A combination, composition, combination for use, or composition for use according to any one of the prior claims, which is an MCIM compound, or a pharmaceutically acceptable salt, hydrate, or solvate thereof.

35. The combination, composition, combination for use, or composition for use according to any one of the prior claims, wherein the TNF inhibitor compound is an antibody or a fusion protein, and optionally the TNF inhibitor compound is selected from etanercept, infliximab, adalimumab, golimumab, or certolizumab.

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