Alkyl-TPP compounds for mitochondria targeting and Anti-cancer treatments

Abstract

To provide novel therapeutic compounds that target and inhibit a mitochondrial function, and may serve as anti-cancer therapeutics.SOLUTION: Alkyl-triphenylphosphonium compounds having a saturated, linear alkyl chain of about 9 to about 18 carbons may be used as therapeutic agents to treat cancer. Demonstrative compound dodecyl-TPP (d-TPP) dose-dependently inhibits the propagation of breast cancer stem cells and targets the bulk of the adherent of cancer cells, by decreasing MCF-7 cell viability. Further, d-TPP potently inhibits a mitochondrial oxygen consumption rate, while simultaneously shifting cell metabolism toward a glycolytic pathway. This shift to a strict metabolic dependency on glycolysis may be used to eradicate the residual glycolytic CSC population, by using additional metabolic stressors. For example, d-TPP may be combined with a glycolytic inhibitor or an OXPHOS inhibitor, through co-administration or sequential administration, to treat cancer and eradicate CSCs.SELECTED DRAWING: Figure 2

Description

[Technical field]

[0001] Related Applications This application is a continuation of U.S. Provisional Patent Application No. 62 / 834,932, filed April 16, 2019, and and claims the benefit of U.S. Provisional Patent Application No. 62 / 842,893, filed May 3, 2019. No. 6,399,433, both of which are incorporated by reference in their entireties into this application.

[0002] The present disclosure relates to the use of alkyltriphenylphosphonium compounds such as dodecyl TPP to The present invention relates to inhibiting mitochondrial function and eradicating cancer, particularly cancer stem cells. [Background technology]

[0003] Researchers are working to develop new anti-cancer treatments. Conventional cancer therapies (e.g., radiation, Alkylating agents such as cyclophosphamide, and metabolic antagonists such as 5-fluorouracil) By interfering with cellular mechanisms involved in growth and DNA replication, rapidly growing cancer cells Other cancer therapies attempt to selectively detect and eradicate the rapidly growing Immunotherapy that selectively binds mutant tumor antigens to cancer cells (e.g., monoclonal antibodies Unfortunately, these therapies are followed by recurrences at the same site or in one or more different areas. Tumors often recur in the same area, indicating that not all cancer cells have been eradicated. Relapse may be due to inadequate chemotherapy dose and / or resistance to therapy. This may be due to the emergence of cancer clones with the same functions. Therefore, new cancer treatment strategies are needed. do.

[0004] Advances in mutation analysis allow detailed study of genetic mutations that occur during cancer progression Despite knowledge of the genomic landscape, modern oncology has However, it has been difficult to identify primary driver mutations across all subtypes of HIV-1. The harsh reality is that each patient's tumor is unique and a single tumor may contain multiple different clonal cells. This seems to be due to the possibility that the cells may contain different This new approach focuses on the commonalities between different types of cancer. Targeting metabolic differences between the two is a promising new cancer treatment strategy. Analysis of transcriptional profiling data from samples revealed that mitochondrial biogenesis and / or The study revealed increased expression of over 95 mRNA transcripts related to mitochondrial translation. Finally, more than 35 of the 95 upregulated mRNAs were found to be mitochondrial ribosomal proteins. Mitochondrial ribosomal protein (MRP) Similarly, proteomic analysis of human breast cancer stem cells identified multiple mitochondria that encode Significant overexpression of mitochondrial proteins and other proteins involved in mitochondrial biogenesis overexpression was evident.

[0005] Mitochondria function constantly to meet cellular requirements and adapt to the cellular microenvironment. The cells split, elongate, and connect with each other to form a tubular network or fragmented granules. Mitochondrial fusion and fission are highly dynamic organelles in the cell. Determine mitochondrial morphology, abundance, function, and spatial distribution, and thus ATP production, mitochondrial function, and Mitochondrial-dependent mechanisms of mitophagy, apoptosis, and calcium homeostasis Mitochondrial dynamics affect many biological processes. It can be regulated by mitochondrial metabolism, respiration, and oxidative stress. Not surprisingly, an imbalance in fusion activity has deleterious effects on multiple pathological conditions, including cancer. Cancer cells often exhibit fragmented mitochondria and show increased or decreased division. Mitochondrial dysfunction is often associated with cancer, but how mitochondrial dynamics contributes to tumorigenesis remains unclear. There remains a need for a comprehensive mechanistic understanding of how this effect occurs.

[0006] Increased bioenergetics and metabolism of cancer cells, especially as they direct tumor growth and metastatic dissemination In order to support the demands of biosynthesis, intact and enhanced metabolic functions are required. However, mitochondrial-dependent metabolic pathways extract energy from multiple fuel sources. By doing so, it provides a biochemical platform for cancer cells.

[0007] Recently, energy metabolism and mitochondrial function have been identified as specific pathways involved in the maintenance and proliferation of CSCs. These CSCs are involved in tumor initiation, metastatic spread, and anti-cancer CSCs are a distinct subpopulation of cells within a tumor mass that are involved in resistance to therapy. A specific and unique increase in mitochondrial mass and enhanced mitochondrial biogenesis; and These behaviors indicate a stronger activation of mitochondrial protein translation. Consistent with these observations, elevated Enhanced mitochondrial metabolic function and OXPHOS were detected in CSCs from multiple tumor types. Similarly, the fluorescent mitochondrial dye MitoTracker has been shown to enrich and differentiate CSCs. This dye accumulates in CSCs and is used to purify them, demonstrating their anchorage-independence in vitro. Allows characterization of cancer cell subpopulations based on their extent of growth and high tumor-initiating ability in vivo In addition, during asymmetric cell division, young mitochondria retain a stem-like phenotype. The daughter cells that are involved in differentiation lose their stemness and become more differentiated. This is the most important step to support stemness. We further explored the idea of ​​selecting for functionally intact, viable, and undamaged mitochondria. It is something to support.

[0008] Based on these observations, we aimed to target mitochondria in CSCs. Novel pharmacological approaches have been proposed, some of which are in preclinical and clinical studies. For example, mitochondrial proteins have been successfully used as off-target effects. Doxycycline, an antibiotic that reduces protein translation, selectively targets CSCs Its potential has been suggested for clinical management of early stage breast cancer patients. The key to success is identifying candidate cancer therapeutic compounds that target and inhibit mitochondrial function. do.

[0009] However, adaptive mechanisms may be adopted by tumor masses to overcome the lack of mitochondrial function. Therefore, the use of anti-mitochondrial agents alone in cancer therapy has certain limitations. These adaptive mechanisms include, for example, intrinsic and extrinsic mechanisms within the tumor cell and the surrounding niche. In a multidirectional process of metabolic plasticity driven by both factors, One of the key features of CSCs is their ability to shift to alternative energy pathways. Manipulation of such metabolic flexibility could be advantageous from a therapeutic perspective. Preventing or otherwise exploiting the metabolic shift in inhibiting cancer cell proliferation , a therapeutic approach. Summary of the Invention [Problem to be solved by the invention]

[0010] The objective of the present disclosure is to develop a method for targeting and inhibiting mitochondrial function to serve as an anti-cancer therapy. Another object of the present disclosure is to identify and describe novel therapeutic compounds that can inhibit the proliferation and proliferation of cancer cells. and / or exploiting said metabolic shift to inhibit cancer cell proliferation; To identify and describe novel therapeutic approaches using the above therapeutic compounds to eradicate CSCs. And so. [Means for solving the problem]

[0011] Elevated mitochondrial metabolism is involved in tumor initiation, dissemination at secondary sites, and therapeutic resistance A distinctive feature of cancer stem cells (CSCs) is Using mitochondrial disrupting agents, we can effectively disrupt the maintenance and propagation of CSCs and prevent neoplastic diseases. This can lead to better control of the triphenylphosphonium (TPP)-based Mitochondrial targeting compounds are small, generally inorganic, molecules that accumulate within the mitochondria of living cells. and anti-cancer therapeutics for blocking the growth and proliferation of CSCs. A candidate for the law.

[0012] Described herein are novel alkyl triphenyl ethers, such as dodecyl TPP (d-TPP). Cancer using the selective mitochondrial inhibitory properties of a phenylphosphonium compound (A-TPP) A-TPP compounds are cationic and are usually taken as salts (e.g., d-TPP bromide salt). It should be understood that the present invention can be used as a pharmaceutical Pharmaceutical compositions containing an effective amount of an A-TPP compound, such as d-TPP, and methods for treating cancer and inhibiting cancer growth inhibiting the expression of IL-1, eradicating CSCs, preventing or reducing the likelihood of tumor recurrence, and preventing metastasis. In some embodiments, the method may take the form of a method for preventing or reducing the likelihood of A-TPP compounds such as .-TPP are used as the first metabolic inhibitor to inhibit glycolysis and / or OXPHOS. Such combinations may be used in combination with a second metabolic inhibitor that interferes with the The use of these compounds as therapeutics can treat cancer, inhibit cancer growth, eradicate CSCs, and inhibit tumor growth. Prevent or reduce the likelihood of tumor recurrence and prevent or reduce the likelihood of metastasis. Cut.

[0013] The present disclosure demonstrates that TPP derivative compounds inhibit CSC proliferation more than twice as effectively as other TPP derivative compounds described below. The present invention relates to compositions and treatment strategies that include d-TPP, which has the potential to Targets the majority of cancer cells, reducing their viability but not against normal fibroblasts In cancer cells, especially CSCs, the mitochondrial membrane potential is higher than that of normal or Therefore, using a TPP-based strategy, Mitochondria and "bad" mitochondria are primarily classified into two subtypes: healthy and diseased. These organelles can be distinguished based on the unique chemical and physical characteristics of the organelles. TPP treatment determined a shift in energy metabolism towards activation of the glycolytic pathway, which was associated with increased d‐T This may be a compensatory response to the anti-mitochondrial effects induced by PP. This metabolic shift may lead to a higher severity of cancer cells' glycolysis after d-TPP treatment. In CSCs, manipulation of such metabolic flexibility has therapeutic implications. For example, synchronizing CSCs to specific metabolic dependencies can be advantageous. Therefore, blocking the ability of CSCs to shift between multiple energy pathways could lead to the eradication of CSCs. As a result, the approach of the present invention may represent a useful strategy for the treatment of residual CSCs. This involves the use of a second metabolic inhibitor (glycolysis or OXPHOS) to further starve the population. This includes "two-hit" treatment strategies.

[0014] In a "two-hit" strategy embodiment of the approach of the present invention, A-TPP such as d-TPP is The compound was used as a first metabolic inhibitor to impair CSC mitochondria and induce glycolysis in cancer cells. This will then deprive cancer cells of their bioenergy sources, facilitating their spread and recurrence. and a second metabolic inhibitor (glycolysis or OXPHOS) that inhibits metastasis. It should be understood that the metabolic inhibitors may be administered simultaneously or sequentially. TPP may be administered first to induce a metabolic shift of CSCs towards glycolysis, followed by a second A metabolic inhibitor of the present invention may be administered.

[0015] The metabolic and biological effects of d-TPP on cancer cells, especially breast cancer cells, were investigated. Using a 3D tumor mass assay in MCF-7 cells, d-TPP was We demonstrated that the treatment dose-dependently inhibited the propagation of breast CSCs in suspension. These results suggest that d-TPP may play a role in the survival of cancer cells by reducing the viability of MCF-7 cells. This indicates that the target is the "bulk" of the attached seahorse. Metabolic flux analysis using Xfe96 demonstrated that d-TPP inhibits mitochondrial oxygen consumption. These results suggest that α-amycin is a potent inhibitor of β-amycin, and at the same time shifts cellular metabolism towards glycolysis.

[0016] Following this metabolic shift, the dependence of CSCs on glycolysis can be used to counteract additional metabolic stressors. By combining d-TPP with a second metabolic inhibitor, the remaining glycolytic CSC population can be eradicated. Evidence-based combinations were used to validate the "two-hit" therapeutic strategy of the present approach. The secondary metabolic inhibitors identified included natural and synthetic compounds, some of which are FDA approved. can be used, and glycolytic inhibitors (e.g., vitamin C, 2-deoxy-glucose or 2DG) or with OXPHOS inhibitors (e.g. doxycycline, niclosamide, berberine chloride) These inhibitors are known to behave as inhibitors of β-actin. The concentration of d-TPP that is toxic to cancer cells but not to normal cells At high concentrations, the toxicity was reduced by 100% CSC proliferation in normal human fibroblasts (hTER T-BJ1) and chicken chorioallantoic membrane assays were used to evaluate.

[0017] The results disclosed herein demonstrate that d-TPP inhibits the associated desirable functions in normal cells. It halts the proliferation of CSCs and inhibits the majority of cancer cells without inducing unwanted off-target effects. These observations support the idea of ​​targeting TPP-inducing molecules in cancer therapy. This opens the way to further explore the potential of the body. The potential to distinguish between 'bad' and 'bad' mitochondria needs to be explored. This is because distinct biochemical and metabolic changes in these organelles are responsible for specific These findings suggest that the phenotype may precede a normal or pathological phenotype. The data suggest that manipulation of the energy machinery may be a useful tool for controlling the stemness of cancer cells. Verify that this is the case.

[0018] In the present approach, the pharmaceutical composition comprises a pharma- ceutical effective amount of an A-TPP derivative such as d-TPP. and pharma- ceutically acceptable compounds for the above compounds (including pharma- ceutically acceptable salts thereof). Some embodiments of the pharmaceutical composition may include a carrier, diluent, or excipient. Also includes a pharma- tically effective amount of a second metabolic inhibitor compound, such as a glycolysis inhibitor or an OXPHOS inhibitor. The second metabolic inhibitor compound may, in some embodiments, be a separate pharma- ceutically acceptable The compounds according to the present approach can be used as anti-cancer therapeutic agents. A pharma- ceutical effective amount of the compound according to the present approach can be used in combination with any of the techniques known in the art. d-TPP can be administered to a subject in stages. In some embodiments, d-TPP can be administered to a subject in stages. Alternatively, d-TPP may be administered prior to and / or after a second metabolic inhibitor. Optionally, it can be administered prior to and together with the second metabolic inhibitor. By administering these compounds, it is possible to treat cancer, eradicate CSCs, and prevent tumor recurrence. and can prevent or reduce the likelihood of metastasis. In some embodiments, cancer is ameliorated by administering a pharmacologic effective amount of d-TPP. In some embodiments, a pharmacologic effective amount of d-T By administering PP, the efficacy of chemotherapy can be increased. In this study, administration of a pharmacologic effective amount of d-TPP reduced tumor recurrence, metastasis, and drug resistance. and / or radiotherapy resistance. The possibility of this being the case can be reduced. [Brief description of the drawings]

[0019] [Figure 1] FIG. 1 shows the structure of d-TPP (structure A) and the structure of b-TPP (structure B). [Diagram 2] FIG. 2 shows the results of a tumor mass assay for MCF-7 cells exposed to various concentrations of d-TPP and b-TPP. [Diagram 3] FIG. 3 shows the results of a tumor mass assay for MDA-MB-231 cells exposed to various concentrations of d-TPP. [Figure 4A-4C] Figures 4A-4C compare the results of cell viability studies using various concentrations of d-TPP on MCF-7 breast cancer cells and h-TERT normal cells, with Figure 4A showing the results after 24 hours, Figure 4B showing the results after 48 hours, and Figure 4C showing the results after 72 hours. [Figure 5A-5B] 5A-5B show the oxygen consumption rate for MCF-7 cells treated with various concentrations of d-TPP. [Figure 6A-6B] 6A-6B show ECAR results for multiple concentrations of d-TPP ranging from 50 nM to 500 nM, and a vehicle-only control. [Figure 7A-7E] 7A-7E show the results of a tumor mass assay for 100 nM d-TPP in combination with a second inhibitor compound according to some embodiments of the inventive approach. [Figure 8A-8D] 8A-8D show the results of the xCELLigence analysis. Results for 48 hours of d-TPP treatment are shown in Figures 8A and 8B, and Figures 8C and 8D show the results for 72 hours of d-TPP treatment. [Figure 9] FIG. 9 shows the tumor weight results from the CAM assay for the various treatment groups. [Figure 10]FIG. 10 shows the metastatic invasion results from the CAM assay for the various treatment groups. [Figure 11] FIG. 11 shows embryo survival from the CAM assay for the various treatment groups. [Figure 12] FIG. 12 is a Kaplan-Meier curve showing survival rates obtained from the CAM assay for the various treatment groups. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] The following description provides an embodiment of the inventive approach, and is provided to illustrate a method and system for practicing the inventive approach. The inventive approach is described in sufficient detail to provide a Although described with reference to the present invention, it will be understood that the inventive approaches can be embodied in different forms. This description is intended to encompass any appended claims as specific embodiments described herein. Rather, these embodiments should not be construed as limiting the scope of the present disclosure. and complete, and will fully convey the scope of the present invention to those skilled in the art. As such, it is provided.

[0021] This description uses various terms that will be understood by those of ordinary skill in the art. The following explanation is provided to avoid any misunderstanding. "treat," "treated," "treating" g)" and "treatment" means the condition, disorder or disease being treated; Reduction or amelioration of at least one symptom, particularly associated with or caused by cancer In certain embodiments, the treatment includes treating a cancer associated with or caused by the cancer being treated. At least one symptom of the disease is reduced and / or ameliorated by the compounds of the present invention. In some embodiments, the treatment includes treating a particular cancer in the host's body by administering a therapeutic agent to the host. This includes inducing the death of target cells, e.g., CSCs, which can be used to Preventing further spread of cancer cells by depriving them of their energy generating mechanisms; and / or This can be achieved by inhibiting CSC function. For example, treatment can be one or more of the following: This may include a reduction in symptoms of the cancer, or the complete eradication of the cancer.

[0022] The terms "cancer stem cells" and "CSCs" refer to cancer cells that are capable of self-renewal, differentiation, and proliferation when transplanted into an animal host. "Bulk" refers to the subpopulation of cancer cells within a tumor that have the capacity to form tumors. Compared to cancer cells, CSCs have a greater mitochondrial mass and enhanced mitochondrial biogenesis. As used herein, mitochondrial protein translation is more activated. In this case, "circulating tumor cells" are cells that are These cancer cells flow from the tumor into the vascular system or lymphatic vessels and are carried throughout the body in the blood circulation. Using the ellSearch Circulating Tumor Cell Test circulating tumor cells can be detected.

[0023] As used herein, the phrase "a pharma- ceutical effective amount" refers to a The "effective amount" of α-glucose is a stimulant for the regulation, modulation, and mitigation of protein kinase activity. or inhibition, e.g., inhibition of the activity of a protein kinase, or a therapeutic result, such as the treatment of cancer. must be administered to a host or to a cell, tissue, or organ of a host to achieve A physician or veterinarian of ordinary skill in the art will be able to determine the amount of the pharmaceutical composition required. The effective amount can be readily determined and prescribed. For example, a physician or veterinarian can determine the amount of the compound contained in the pharmaceutical composition. Doses of the compounds of the invention may be initiated at levels below those required to obtain the desired therapeutic effect, and may be administered at any time up to 20 minutes. Dosage can be gradually increased until a therapeutic effect is achieved.

[0024] As used herein, the phrase "active compound" means refers to the A-TPP compounds described herein, including pharma- ceutically acceptable salts thereof, or The phrase "active compound" may also include isotopic analogs. In embodiments that include a second inhibitor, such as an S inhibitor, a second inhibitor compound may be included. The one or more active compounds may be, as known to those of ordinary skill in the art, It is understood that administration to a subject may be by any suitable approach. The amount of compound and the timing of its administration will depend on the individual subject being treated (among other factors). in particular age and weight), the method of administration, the pharmacokinetic properties of one or more particular active compounds, It should be understood that the results may be influenced by the individual's own judgment and the prescribing physician's judgment. Accordingly, any dosages set forth herein are intended as initial guidelines. and the physician may use the information to effectuate the treatment that the physician considers appropriate for the subject. The dosage of the compound can be titrated to achieve the desired degree of treatment. balance various factors such as age, weight, presence of pre-existing disease, and the presence of other diseases. As described in more detail below, pharmaceutical formulations include, but are not limited to, can be formulated for any desired route of administration, including oral, intravenous, or aerosol administration. do.

[0025] Inter- and intratumor heterogeneity is one of the main factors explaining tumor progression and therapy failure. CSCs play a hierarchical role in generating a full repertoire of multiple cell types within the tumor mass. The ability to produce differentially differentiated offspring generates and promotes the heterogeneity described above. Subsequently, pharmacological strategies to identify and selectively target CSCs will be of great value in the therapeutic landscape. Among the most promising, yet challenging, approaches to CSC have been implemented. To explain the origin, various theories have been proposed. According to the “tumor stemness” model, a specific metabolic phenotype determines the stemness of a tumor. This model shows that specific metabolic dynamics are essential for the differentiation of non-cancer or differentiated cancer cells. These results suggest that tumor metabolism may drive the acquisition of stemness in cancer. Based on these observations, we hypothesize that cancer metabolism can be mediated by a novel mechanism that selects CSCs. It is not surprising then that many see this as an opportunity to target companies more effectively.

[0026] CSCs across multiple cancer types display increased OXPHOS activity compared to non-stem cell cancer populations Specific metabolic pathways, including increased mitochondrial activity, elevated mitochondrial biogenesis, and higher mitochondrial mass Supporting these findings, we found that telomerase activity was high (hTERT h igh ), and thus cancer cells with the characteristic of high immortality are characterized by the expression of hTERT low The corresponding These observations specifically show increased mitochondrial mass compared to controls. These results suggest that CSC populations can be specifically inhibited using endorhizobial agents, which may be A path to identifying chemical strategies aimed at selectively targeting mitochondria in CSCs It opens the

[0027] Mitochondrial inhibition has been shown to be effective in inhibiting cancer recurrence and metastasis, as well as eradicating cancer cells, particularly CSCs. For example, the antibiotic doxycycline inhibits CSC activity. Long-term treatment with doxycycline , which turns on the glycolytic pathway in CSCs in response to mitochondrial dysfunction. Vitamin C, etc. The glycolysis inhibitor, doxycycline, was used to completely eradicate CSCs. The combination of doxycycline and vitamin C is an effective treatment for the clinical management of cancer patients. Doxycycline represents a safe approach. At biologically active concentrations, has a very low range of side effects, and vitamin C has potentially no side effects. In addition, a meta-analysis of 21 published studies found that Data from preclinical studies have shown that oral vitamin C reduces the risk of lung and breast cancer. It has been demonstrated that cellular cellular functions such as cellular metabolism, cellular metabolism, and cellular metabolism have been shown to reduce overall mortality and disease-specific mortality. Oral administration of ascorbate for 1 week prior to vaccination reduced lymphoma xenograft transfer. Tumor progression was reduced in explant models.

[0028] Clinical validation of these findings for doxycycline is planned by the inventors in a recent This pilot study included patients with early stage breast cancer receiving short-term (14-day) (between 2013 and 2016) were enrolled for preoperative doxycycline treatment. In particular, postoperative vs. preoperative breast tumor trials Analysis of the specimens revealed a significant increase in stem cell death in patients receiving doxycycline compared with controls. Selective reduction of the cellular markers CD44 and ALDH was demonstrated, which indicates that mitochondrial targeting We present clinical evidence that the use of a targeted strategy can effectively halt CSC dissemination. Nevertheless, the efficacy of doxorubicin in combination with vitamin C in breast cancer patients has not been shown to be significant. Further studies are needed to investigate the action of cyclins.

[0029] Triphenylphosphonium (TPP) ) is an example of a lipophilic cation that targets cellular mitochondria. It is possible to covalently bind a compound and deliver the bound therapeutic compound to mitochondria. The therapeutic compound was found to be more highly concentrated in mitochondria than in other parts of the cell. The present inventors have demonstrated that some compounds that possess a TPP moiety inhibit mitochondrial function. We demonstrated that the mitochondrial targeting signal reduces the spread of CSCs. The TPP moiety acts as a “cargo” therapeutic compound via covalent attachment. The inherent properties of the cargo molecule and its attachment to the TPP structure are Deeply affect the accumulation of therapeutic compounds in the mitochondria of living cells and subsequently their overall viability. It may affect the biological function.

[0030] Thus, covalent modification of compounds to the TPP cation allows probes and imaging agents to bind to mitochondria. This is a method of delivering the product to Doria. + Cations transport small molecules into mitochondria. It is a highly effective chemical "vehicle" for Chemical synthesis of thiones is easily achieved, and the extent of their accumulation in mitochondria is comparable to that of positively charged TP It is increased by the chemical attraction between P cations and the negative membrane potential of the inner mitochondrial membrane.

[0031] Certain chemical derivatives of TPP also target mitochondria and exert mitochondrial inhibitory activity. This makes it useful for targeting and eradicating cancer cells. No. PCT / US201, filed on Oct. 13, 2013, which is incorporated by reference in its entirety. No. 8 / 062174 describes examples of TPP derivatives having anticancer activity. The authors demonstrated that the TPP derivative compound 2-butene-1,4-bis-TPP (b-TPP) is demonstrated that it impairs mitochondrial metabolic function, which leads to inhibition of breast CSC activity. .

[0032] Described herein are saturated linear alkyl chains having from about 9 to about 18 carbons. A-TPP compounds that:

[0033] [ka]

[0034] (wherein "x" is 8 to 17, preferably 9 to 16, more preferably 11 to 14). A novel therapeutic approach using an integrated metabolic strategy to eradicate CSCs by exploiting their advantageous properties In an exemplary embodiment, x is 11 and is a d- TPP is obtained.

[0035] In a demonstrative embodiment of the approach of the present invention, the compound d-TPP inhibits the mitochondria of CSCs. It is used as a therapeutic agent that inhibits the cellular function of the inflammatory bowel disease. Other A-TPP compounds that are compatible with the present invention may be used without departing from the present approach. It should be understood that d-TPP, a compound that carries the mitochondrial targeting TPP moiety, The mitochondrial inhibitory effect results in a dose- and time-dependent reduction in cell viability.

[0036] These effects reduced the formation of 3D tumor-like masses, which was analyzed as a readout for CSC activity. Metabolic flux analysis showed that d-TPP increased basal mitochondrial respiration and ATP production. The combination of these effects results in the following: The reason for the decline in the functional capacity of CSCs, as observed in response to administration of d-TPP, is unclear. Rationale is provided. Breast cancer cells preferentially display a glycolytic phenotype in response to d-TPP. This metabolic switch allows a subset of CSCs to respond to the anti-mitochondrial effects of d-TPP. You will be able to deal with it.

[0037] In some embodiments, the approach of the present invention involves the use of a second metabolic inhibitor ( Targeting these dependencies through a "two-hit" combination with either glycolysis or OXPHOS The d-TPP compound further starves the remaining CSC population by As the first metabolic inhibitor to act as a hit (specifically as a mitochondrial disruptor) ) followed by a second metabolic inhibitor (e.g., glycolysis) that acts as a second hit. or OXPHOS inhibitors) are used.

[0038] Despite this acquisition of compensatory glycolytic behavior, d-TPP treatment did not suppress the glycolytic inhibitors and OXP Attenuating CSCs by increasing their sensitivity to the action of HOS inhibitors Therefore, these effects of d-TPP make it possible to perform a variety of combination therapies. In our study, administration of d-TPP together with one or more such inhibitors was shown to reduce the risk of CS Demonstration of a second metabolic inhibitor could provide a "two-hit" therapeutic approach to eradicate C. Notable examples are the glycolysis inhibitors vitamin C and 2-deoxy-D-glucose (2- DG), as well as the OXPHOS inhibitors doxycycline, azithromycin, and niclothionein. Examples include tetracycline, chlorotetracycline, tetracycline, tetracycline chloride ... The terramycin family, including erythrocycline, minocycline, and tigecycline; Erythromycin, including telithromycin, clarithromycin, and roxithromycin Other FDA-approved members of the cyclosporine family include eravacycline and sarcocycline. Recycline, omadacycline, etc. may also be used without departing from the approach of the present invention. A functional validation of the approach of the present invention is provided below, in which MCF-7 cells Simultaneous treatment with d-TPP and a second metabolic inhibitor resulted in almost complete inhibition of CSC activity. There was a significant loss.

[0039] As mentioned above, some derivatives of the TPP moiety have the potential to inhibit cancer growth and metastasis. Using the ATP depletion assay as a surrogate marker for mitochondrial dysfunction The present inventors have filed a patent application entitled "Compound for a cellular endothelial cell" which is hereby incorporated by reference in its entirety on November 21, 2018. In International Patent Application No. PCT / US2018 / 062174, several TPP derivatives were identified. For example, 2-butene-1,4-bis-TPP (b-TPP) It showed considerable potency in inhibiting the spread of CSCs (IC-50 ~ 500 nM). Accumulation of TPP derivatives in doria and the mitochondrial inhibition and anticancer efficacy of the compounds What is needed are additional TPs with anticancer efficacy. It is a P derivative compound.

[0040] The present inventors have identified another TPP derivative with considerable mitochondrial inhibition and anticancer potency. The metabolic and biological properties of d-TPP compounds were investigated. Furthermore, we aim to effectively target the flexible cellular energy machinery of CSCs to eradicate cancer. The combinatorial strategy that has been proposed is described below. is an organophosphorus compound containing a quaternary phosphate with three phenyl rings and a saturated 12-carbon alkyl chain. It is a cation.

[0041] [ka]

[0042] As a cation, d-TPP can be a salt, such as the bromide salt. The structure of d-TPP (structure A) is compared with 2-butene-1,4-bis-TPP (structure B). As can be seen, d-TPP has only one TPP moiety and is a hydrophobic long-chain alkyl ester. On the other hand, B-TPP has two TPP moieties linked by a butene.

[0043] Both d-TPP and b-TPP inhibited CSC proliferation, but d-TPP inhibited it more effectively. The tumor formation assay was performed using increasing concentrations of d-TPP or b-T Estrogen receptor (ER) positive treated with PP The results were used as a readout of CSC proliferation in MCF-7 breast cancer cells. The CSC proliferation inhibitory activity of d-TPP and b-TPP in the mouse model is compared in Figure 2. As shown, treatment with d-TPP increased the cellular cellular fate by up to the two highest concentrations tested. At low concentrations (500 nM and 1 μM), it caused an 80% reduction in CSC propagation, The small but significant reduction in tumor mass formation (20%) was extremely significant for d-TPP. This was already observed at low concentrations (50 nM and 100 nM). d-TPP was more than two-fold more potent in inhibiting tumor nodule formation.

[0044] The inhibitory effect of d-TPP was also examined in the triple-negative breast cancer cell line MDA-MB-231 A similar inhibitory tendency towards CSC proliferation was observed in the triple-negative breast cancer cell line MDA-M The tumor-like mass formation assay for MDA-MB-231 cells was performed. The results are shown in Figure 3. As can be seen, d-TPP was at the highest concentration tested, 50 nM. It showed inhibitory effects at very low concentrations, and achieved nearly 80% inhibition at 1 μM. Successful.

[0045] In our previous efforts, we demonstrated that a specific TPP derivative inhibits mitochondrial function in cancer cells. It can interfere with the metabolic activity of cancer cells, ultimately resulting in reduced survival rates of cancer cells. Interestingly, these effects of certain TPP compounds were observed in cancer cells. It is selectively induced in cancer cells and not in normal (i.e., healthy) cells. This study demonstrates the ability of d-TPP to reduce the rate but not cell viability of normal cells. To investigate the mechanism of action of IFN-γ in breast cancer cells, we investigated the viability of both MCF-7 breast cancer cells and normal human fibroblast cells (hTERT-BJ1). The survival rates were evaluated in various doses with increasing concentrations of d-TPP (from 50 nM to 1 μM). The results are summarized in Figures 4A-4C. Specifically, Figure 4A shows the results after 24 hours, Figure 4B shows the results after 48 hours, and Figure 4C shows the results after 24 hours. 4C shows the results after 72 hours. These data indicate that d-TPP inhibits the survival of MCF-7 cells. The results show that high concentrations of the compound (e.g. 250 nM, 500 nM, 1 μM) were evident only 24 hours after treatment. After 72 hours of treatment, cell viability was reduced by approximately 30%. The decrease in the rate was nearly 80% at the above high concentrations (250 nM, 500 nM, 1 μM). Even at low concentrations (50 nM, 100 nM), there was a slight (nearly 30%) but significant On the other hand, the cell viability of hTERT-BJ1 cells was relatively high. The decrease was observed only at low TPP concentrations (250 nM, 500 nM, 1 μM) and low concentrations (50 nM, 1 These results were not toxic when d-TPP was used at low concentrations. , demonstrating the ability to selectively target cancer but not normal cells.

[0046] Metabolic flux analysis showed that d-TPP blocked ATP production and promoted metabolic inflexibility. Some cancer cells have been shown to derive their energy source from OXPHOS. They have the ability to flexibly shift fuel sources depending on local availability. This seemingly innate ability to mediate abnormal cell proliferation, survival, and distant site proliferation is implicated. Impairing this flexibility of the cancer cell's energy machinery is a prerequisite for dissemination in the host. Pharmacological and / or metabolic approaches aimed at preventing and treating tumor progression have been shown to adversely affect It becomes.

[0047] To understand the effect of d-TPP treatment on the metabolism of cancer cells, we Metabolic flux analysis was performed using horse XFe96. The oxygen consumption rate for MCF-7 cells treated with various concentrations of d-TPP was The results of the optical cell count (OCR) are shown below. Basal respiration, proton leak, For ATP production-related respiration, maximum respiration, and spare respiratory capacity, Figure 5A shows the OCR (pmo 1 / min / SRB) over time, and FIG. 5B shows OCR as fold change relative to vehicle. After treatment with 50 nM d-TPP, a dramatic decrease in OCR was observed in MCF-7 cells. The decrease in OCR was observed as the d-TPP concentration increased from 50 nM to 500 nM. Basal mitochondrial respiration was therefore increased by approximately 250 nM IC50 (CSC The ATP levels were also decreased in a dose-dependent manner. was gradually depleted.

[0048] A clear opposite trend was observed for glycolysis, with d-TPP-treated MCF-7 cells showing a significantly increased glycolysis rate. ECAR (extracellular acidification rate) The EC values ​​were significantly and dose-dependently increased as shown by the analysis of the extracellular acidification rate (EC The AR results are shown in Figures 6A and 6B. In Figure 6A, the ECAR (mpH / min / SRB) was 5 Over time with d-TPP concentrations ranging from 0 nM to 500 nM and control (vehicle only) FIG. 6B shows the ECARs for glycolysis, glycolytic reserve, and glycolytic reserve capacity. As can be seen, glycolysis in MCF-7 cells was significantly increased by d-TP The d‐TPP treatment increased the β-amyloides concentration in the myocardium in the cerebrospinal fluid (C-TPP)-treated mice in a dose-dependent manner. ATP generation from oxidative phosphorylation in MCF-7 cells after impaired mitochondrial function This impairs the ability of the body to cope with such a stressful metabolic environment. Cancer cells are forced to shift to a glycolytic phenotype. After this metabolic shift, cancer cells High reliance on glucose to meet high energy demands. In this scenario, d‐T The anti-mitochondrial effect induced by PP treatment is due to an obligate and inflexible dependency on glycolysis. It acts as a functional metabolic synchronizer for the

[0049] d-TPP inhibits mitochondrial ATP production and shifts CSCs to a glycolytic state This capability opens the door to a variety of potential treatment strategies, especially therapeutic combinations. For example, d-TPP can be combined with a compound that inhibits glycolysis or OXPHOS. This timing of combination therapy allows the effects of d-TPP to occur first, followed by glycolysis and Alternatively, d-TPP can be used as a second As an example, the combination of d-TPP and vitamin C may be used in combination with Together, these studies provide a metabolic "two-hit" strategy to target the metabolic vulnerabilities of CSCs. d-TPP inhibits ATP production, shifting CSCs to a glycolytic profile. Vitamin C inhibits glycolysis, leaving CSCs with no metabolic options. Others include deoxyglucose, doxycycline, niclosamide, and berberine chloride. Inhibitors of may be used in combination with d-TPP.

[0050] To demonstrate the efficacy of this two-hit therapy approach, we performed a tumor formation assay and a cancer This is the dose of a drug that is selectively toxic to only the target cell but not to normal cells. Further evaluation was performed using 0.100 nM of d-TPP. Tumor mass assay results for 100 nM d-TPP in combination with various inhibitor compounds Starting from Figures 7A and 7B, the natural glycolysis inhibitor vitamin C and the synthetic glycolysis inhibitor The inhibitor 2-deoxyglucose (2-DG) was included along with d-TPP. As shown in Figure 7B, vitamin C and 2-DG have no effect on the effect of d-TPP on CSC activity. The inhibitory effect was enhanced. In particular, treatment with vitamin C in combination with 100 nM d-TPP When the 250 μM and 500 μM CSCs were Separately, the IC50 of vitamin C was As can be seen, d-TPP significantly inhibited the sensitivity of CSC to vitamin C. increased sexual activity by nearly four times.

[0051] The inhibitory activity of 2-DG was already observed at a concentration of 10 mM, which was greater than that of d-TPP alone. The inhibitory effect was even more dramatic at 20 mM, resulting in a two-fold increase in activity. SC activity was almost completely suppressed, with less than 10% of the residual tumor-like mass forming ability remaining.

[0052] Next, Figures 7C-7E show two FDA-approved compounds combined with d-TPP. doxycycline and niclosamide, as well as the natural compound berberine chloride. The results show that each of these compounds behaves as an OXPHOS inhibitor. This behavior is thought to be due to the additional metabolic pathway in cancer cells that were weakened by d-TPP treatment. Based on the understanding that stressors can help eradicate remaining CSC populations Doxycycline is known to impair mitochondrial biogenesis and function. As shown in Figure 7C, low doses of the antibiotic doxycycline (e.g., 10 d-TPP was sufficient to double the efficacy of d-TPP on CSC activity. was enhanced in the presence of 30 μM doxycycline. As shown in Figure 7D, Niclosamide, an anti-tapeworm drug that inhibits OXPHOS, has d-regulatory effects on CSC activity. The potency of TPP was increased by approximately 2-fold at 250 nM and approximately 3-fold at 500 nM. 7E is a natural compound and OXPHOS inhibitor, chloramphenicol, in combination with d-TPP. The results of berberine are shown below. Berberine chloride was found to be effective in preventing Coptidis rhizoma (Covid-19). ptis chinensis Franch) and Phellodendri cor Extracted from tex (Phellodendron amurense Ruprecht) It is the major alkaloid found in plants and is known for its antimalarial, anti-inflammatory, and antibiotic activities. The effect of berberine chloride was already evident at 1 μM, resulting in a 60% reduction At the highest concentration tested (10 μM), berberine chloride inhibited CSC formation by more than 80%. , increasing the efficacy of d-TPP by approximately five-fold.

[0053] These results suggest that mitochondrial damaging agents such as d-TPP may disrupt the normal functioning of the cell's energy machinery. Impairing essential functions may affect the ability of CSCs to use alternative fuels and metabolic pathways. This effect has been demonstrated to affect the biology and proliferation of CSCs, as well as the proliferation and proliferation of cancer cells. This adversely affects CSC survival and proliferation. The cancer patients treated with d-TPP become more vulnerable to other cancer treatments, including radiation therapy. The CSCs become more sensitive to other chemotherapeutic agents, radiation, and phototherapy. Effective dose of d-TPP

[0054] The xCELLigence system was used to measure the effect of d on cell proliferation of adherent MCF-7 cells. The pharmacokinetics of the effect of TPP was analyzed. By measuring cell number-dependent electrical impedance, we can obtain information about cell health and behavior. Evaluate real-time label-free monitoring. Real-time cell analysis at 50n Treatment with d-TPP at concentrations of 100 nM, 100 nM, and 250 nM for 48 and 72 hours The control was vehicle only. xCELLigence analysis results The results for 48 hours of d-TPP treatment are shown in Figures 8A and 8B. Figures 8C and 8D show the results for 72 hours of d-TPP treatment. Thus, the effect of d-TPP on MCF-7 cells was dose- and time-dependent. There was a trend towards decreased cell numbers after 72 hours of treatment at the lowest dose tested (50 nM d-TPP). At higher doses (100 nM), a major cytostatic effect was detected. Toxicity was present at a d-TPP concentration of 250 nM.

[0055] Further evaluation of both efficacy and toxicity will be performed by Innovotion (Latronche, France). Using the CAM model available from The CAM assay was performed on human breast tumors initiated with d-TPP. For example, at a concentration of 25 μM, d-TPP inhibited both cell proliferation and metastasis. Treatment led to a ~40% inhibition of tumor growth and a ~75% inhibition of metastasis. For the assay, fertilized eggs were incubated at 37.5°C and 50% relative humidity for 9 days. The graft was dropped into each egg through a small hole drilled through the shell into the air chamber, above the CAM. 1cm on eggshell 2 This transfer process was repeated for each egg in the test group. Because this implantation process is an invasive surgical procedure, it was performed within a few hours after tumor implantation. It is expected that some mortality will occur. The data reported herein are At least 10 eggs per test group were successfully implanted.

[0056] MDA-MB-231 tumor cell line was cultured in 10% FBS and 1% penicillin / streptomycin (100 mM NaCl) medium. After a 9-day incubation period, the cells were cultured in DMEM medium supplemented with tomycin. The cells were detached with trypsin, washed with complete medium, and suspended in transplantation medium. 6 pcs An inoculum of cells was added to the CAM of each egg, after which the eggs were randomly divided into test groups. Tumors were detectable within one day of implantation. The negative control group was 0.125% DMSO, and d-TPP glutamate at concentrations of 6.25 μM, 25.0 mM, and 62.5 mM. Each egg in the group was treated 8 times per day. Afterwards, tumor growth was quantitatively evaluated. The top part of the CAM (with the tumor) was removed and buffered with PBS. After washing with buffer, the tumors were transferred directly into PFA for fixation for 48 hours. The AM tissue was carefully dissected and weighed. The mean tumor weight (mg) is shown in Figure 9. All quantitative data related to the evaluation of this egg were analyzed (post-hoc tests between groups). One-way analysis of variance (including the variance of the 1-way ANOVA) was performed using the dedicated computer software Prism (registered trademark All analyses were performed using GraphPad Software. Statistical differences between groups are indicated on the graphs by the presence of an asterisk, which has the following meaning: The results are visualized in Table 1: no asterisk means p>0.05; * means 0.5≥p>0. 01; ** means 0.01 ≥ p > 0.001; *** means 0.001 ≥ p As can be seen in Figure 9, d-TPP at a medium dose ( At a dose of 25.0 μM and a high dose of 62.5 μM, tumor growth was significantly inhibited. Consistent with the evaluation, the tumor growth inhibitory effect of d-TPP was dose-dependent.

[0057] Metastatic invasion was also quantitatively evaluated. 2 Collect the pieces and put them in groups. The number of metastatic cells was evaluated in eight samples per group (n=8). Human DNA was extracted and analyzed by qPCR using primers specific for human Alu sequences. Calculations of Cq for each sample, average Cq, and relative amount of metastasis for each group were performed using B The data was directly managed by the io-Rad® CFX Maestro software. l-Time PCR Data Markup Language (RDML) data According to the standard (http: / / www.rdml.org), Cq is the curvature of the amplification curve. Cq is defined as the cycle at which the PCR reaction reaches its maximum (fractional PCR cycle). The CR curve is obtained during the exponential phase of amplification, where it is linear. The basic results of qPCR are as follows: The lower the Cq value, the higher the initial number of copies of the target gene. , the difference in one cycle between the two reactions is: The lower Cq value indicates that the gene in the reaction had twice as many copies. The results of metastatic invasion measured by qPCR for Alu sequences in the negative control and In comparison, regression of metastases can be observed after treatment with moderate doses of d-TPP. For the treated group, statistical evaluation was performed on the only three surviving specimens in the group. This affected the statistical analysis.

[0058] Embryonic tolerance of d-TPP treatment was also quantitatively evaluated. Embryonic survival rates after transfer and during treatment were The number of dead embryos was also counted on the final day to evaluate the embryotoxicity induced by the treatment. FIG. 11 shows the percentage of live and dead embryos in each group described herein. Figure 12 shows the percentage of couplers showing survival (from the day of implantation to the day of harvest). The results are shown in Fig. 1. d-TPP showed no toxicity at medium and low doses. (However, as expected, some mortality occurred in the negative control.) However, the higher the dose, the more deaths there were. Toxicity studies are required to establish dosing regimens. It should be understood by a person of ordinary skill in the art that the above analysis is necessary. However, the above results suggest that the tumor growth inhibition and metastasis inhibition effects of d-TPP are not related to toxicity. It has been demonstrated that it is present even at concentrations far below the threshold value.

[0059] The data presented here further validate the use of a metabolic "two-hit" approach. The above approach aims to manipulate cancer metabolism to increase the metabolic flexibility of CSCs and to develop more efficient cell proliferation and cell death. This leads to absolute metabolic inflexibility that is used to allow efficient CSC suppression. The potential of d-TPP to act as a safe driver of such metabolic shifts paves the way for further investigation of the role of this compound in cancer biology and energetics. in combination with other metabolic inhibitors as an adjunct to conventional chemotherapy. Further research is needed to support the use of TPP-based compounds. Similar results were obtained for other A-TPP compounds with saturated linear alkyl chains containing eight carbon atoms. It should be understood that the effects are expected.

[0060] The active compound is an AT having a saturated linear alkyl chain containing from about 9 to about 18 carbons. PP compounds, such as d-TPP, have been shown to target cancer cells, including CSCs and circulating tumor cells. and thereby treat and eradicate cancer, prevent or reduce the likelihood of tumor recurrence, and reduce metastasis. In addition, d-TP can be used as a therapeutic agent to prevent or reduce the likelihood of P shifts cancer cells into a glycolytic state, making them more resistant to chemotherapy, radiation, and phototherapy. To sensitize cancer cells and reduce their resistance to other medicines, such as doxycycline can be used as an active compound.

[0061] With respect to the active compounds, empirical second inhibitor compounds are known in the art in various forms. In the case of A-TPP compounds such as d-TPP, the active compound is available in the form of a solid or In some embodiments, d-TPP can be administered orally as a liquid. The emulsion can be administered intramuscularly, intravenously, or by inhalation. In the form of liposomal d-TPP (including, for the avoidance of doubt, its salts) As a suspension, it can be administered by inhalation, intravenously, or intramuscularly. In this case, the active compound or a salt thereof may be, for example, about 0.001, 0.01, 0.1, or 0.5 millimolar. micrometers to about 5, 10, 20 micrometers or more, and optionally from about 1 to about In the form of a plurality of solid particles or droplets having any desired particle size of 2 micrometers. It is understood that the particular mode of administration may vary and is within the scope of this disclosure. The general parameters of d-TPP (e.g., manufacturing, transportation, storage, shelf life, etc.) It is understood that form and concentration may be determinative.

[0062] The pharmaceutical composition of the present approach comprises d-TPP (including its salts) as the active compound. in any pharma- ceutically acceptable carrier. If a solution is desired, water is used as a water-soluble For water solubility, glycerol may be the carrier of choice for the compound or its salt. glycol, propylene glycol, polyethylene glycol, or mixtures thereof. Additionally, methods for improving water solubility may be used in conjunction with the approach of the present invention. In the latter case, the organic vehicle may contain a significant amount of water. In either case, the solution can be dissolved in water by any suitable means known in the art, e.g. Sterilization can be achieved by filtration, for example through a 0.22 micrometer filter. After sterilization, The solution can be dispensed into suitable containers, such as pyrogen-free glass vials. This is done in an aseptic manner. A sterile closure device can then be placed onto the vial, and If necessary, the contents of the vial can be lyophilized. Glycolysis inhibitors or OXPHOS inhibitors, etc. The embodiments including the second inhibitor compound of the present invention include those of the second inhibitor compounds available in the art. The approach of the present invention is to administer the same or similar forms simultaneously, unless otherwise specified. It is not intended to be limited to the mode of administration.

[0063] In addition to one or more active compounds, the pharmaceutical formulations of the present approach may include any of the compounds known in the art. For example, some embodiments may include an acid (e.g., hydrochloric acid) and a base. or buffer solutions (e.g., sodium acetate, sodium borate, sodium citrate, gluconate, etc.) may contain pH adjusters such as sodium phosphate, sodium lactate, sodium phosphate Some embodiments include methylparaben, propylparaben, and benzyl alcohol. Antimicrobial preservatives are often used to preserve the formulation when it is designed for multiple doses. The pharmaceutical formulations described herein are intended to be used in a pharmaceutical composition that is suitable for use ... The mixture can be freeze-dried using techniques such as

[0064] In embodiments involving oral administration of the active compound, the pharmaceutical composition may be in the form of a capsule, tablet, pill, powder, or the like. The formulation may be in the form of a drug, solution, suspension, etc. Sodium citrate, calcium carbonate, Tablets containing various excipients such as calcium phosphate, polyvinylpyrrolidone, sucrose, Starch (e.g. potato or tapioca) combined with a binder such as gelatin or acacia It can be used with a variety of disintegrants, such as starch, calcium carbonate, and certain complex silicates. Lubricants such as magnesium tearate, sodium lauryl sulfate, and talc are added for tableting purposes. Solid compositions of a similar type may also be included as target substances in soft and hard filled gelatin capsules. Related materials include lactose and high molecular weight polyethylene. When aqueous suspensions and / or elixirs are desired for oral administration, The compounds of the presently disclosed subject matter can be mixed with various sweetening, flavoring, coloring, emulsifying, and / or suspending agents. , as well as water, ethanol, propylene glycol, glycerin, and various similar The d-TPP can be combined with a diluent such as a combination of In embodiments including the compound, the second inhibitor compound is limited to the form of a d-TPP compound. The pharmaceutical composition may be administered in separate forms without being packaged together.

[0065] Further embodiments provided herein include the activator disclosed herein. The techniques for forming liposomal suspensions are well known in the art. When the compound is a water-soluble salt, it can be delivered using conventional liposome technology. It can be incorporated into lipid vesicles. In such cases, the water solubility of the active compound allows The active compound can be substantially contained within the hydrophilic center or core of the liposome. The lipid layer that is applied can be of any conventional composition and can contain no cholesterol. The active compound of interest may be cholesterol-free or cholesterol-free. If water insoluble, the salt can be incorporated into the liposomes, again using conventional liposome formation techniques. In either case, the carboxylate can be substantially packed into a hydrophobic lipid bilayer forming a structure of In this case, the liposomes produced were stable when standard sonication and homogenization techniques were used. The active compounds disclosed herein can be reduced in size in the same manner as described above. The liposome preparation containing the liposome can be freeze-dried to produce a freeze-dried product, which can then be dissolved in a pharma- ceutical acceptable solvent such as water. The liposome suspension can be reconstituted with an acceptable carrier to regenerate the liposome suspension.

[0066] With respect to pharmaceutical compositions, a pharma- ceutical effective amount of the active compounds described herein may be administered by a medical professional. The dosage is determined by the patient's condition, size and age, and depends on the route of delivery. In a limited embodiment, a dosage of about 0.1 to about 200 mg / kg is therapeutically effective, The weight ratio is the weight of the active compound, including salts, relative to the subject's body weight. In some embodiments, the dosage is up to about 1 to 5, 10, 20, 30, or 40 can be the amount of active compound required to provide a serum concentration of the active compound in μM. In some embodiments, the dose is from about 1 mg / kg to about 10 mg / kg, and in some embodiments, the dose is from about 1 mg / kg to about 10 mg / kg. In some embodiments, a dosage of about 10 mg / kg to about 50 mg / kg can be used for oral administration. Typically, a dosage of about 0.5 mg / kg to 5 mg / kg can be used for intramuscular injection. In some embodiments, the dosage is about 1 μmol for intravenous or oral administration. / kg to about 50 μmol / kg, or optionally from about 22 μmol / kg to about 33 μmol / kg g of the compound. Oral dosage forms can be, for example, 5 mg per tablet or other solid dosage form. It may contain any suitable amount of active ingredient, including up to 50, 100, 200, or 500 mg. This can be done.

[0067] The following paragraphs describe the materials and methods used in the experiments and data generation described above. d-TPP bromide salt, doxycycline, ascorbic acid, 2-deoxy-D- Glucose (2‐DG), berberine chloride, and niclosamide were purchased from Sigma Aldric. Ascorbic acid and 2-deoxy-D-glucose were dissolved in the cell culture medium. All compounds were dissolved in DMSO, except for glucose (2-DG).

[0068] With respect to the cell cultures referred to herein, MCF7 and MDA-MB-231 breast cancer Cells were obtained from ATCC. Human immortalized fibroblasts (hTERT-BJ1) were obtained from Clo The cells were cultured in a humidified atmosphere containing 5% CO2 at 37°C. 10% FBS (fetal bovine serum), 2 Dulbecco's modified EGFR supplemented with 1 mM GlutaMAX and 1% Pen-Strep. Dulbecco's modified Eagle's medium:D The cells were cultured in MEM.

[0069] The tumor mass assay was performed as follows: MCF-7 or MDA-MB-231 cells Single cell suspensions were subjected to enzymatic lysis (1× trypsin EDTA, Sigma Aldrich ), and manual disaggregation (25 gauge needle). The cells were then cultured in the presence of treatment ( 2-Hydroxyethyl methacrylate) (PolyHEMA, Sigma Aldrich) The tumor-like mass medium (DMEM-F12 / B) under non-adherent conditions was cultured in a culture dish coated with 100 μg / mL of ... 27 / 20‐ng / ml EGF / PenStrep), 500 cells / cm 2 With a density of The cells were grown for 5 days in a humidified incubator at 37 °C, atmospheric pressure, and 5% (v After 5 days of culture, spheres >50 μm were placed in the eyepiece. The cells were counted using a scale and the percentage of plated cells that formed spheres was calculated. This is referred to as percentage tumor formation. The tumor assay was performed in triplicate. Repeated three times independently.

[0070] Extracellular Acidification Rate (ECAR) and Real-Time Oxygen Consumption Rate for Metabolic Flux Analysis The OCR assay was performed using Seahorse Extracellular Flux (X The measurements were performed using a Fe-96 analyzer (Seahorse Bioscience). Briefly, 15,000 MCF-7 cells per well were cultured in XFe-96 The cells were seeded onto 2-well cell culture plates and incubated overnight to allow attachment. Treatment with increasing concentrations of d-TPP (50 nM ÷ 500 nM) for 4 hours Vehicle only (DMSO) control cells were treated in parallel. The cells were then incubated in pre-warmed X-rays. F assay medium (or for OCR measurements, 10 mM glucose, 1 mM pyruvate XF assay medium supplemented with 1 mM riboflavin, 2 mM L-glutamine, and adjusted to pH 7.4) ), and the cells were incubated at 37°C in a non-CO2 incubator for 1 hour. The XF assay was maintained at 175 μL / well for 80 min during the incubation period. 5 μL of M glucose, 9 μM oligomycin, and (for ECAR measurements) 1 M 2-deoxyglucose or (for OCR measurements) 10 μM oligomycin , 9 μM FCCP, 10 μM rotenone, 10 μM antimycin A, XF assay The protein was placed in agar medium and inserted into the injection port of the XFe-96 sensor cartridge. After normalizing the measurements by content, the data sets were analyzed with XFe-96 software ( SRB). All experiments were repeated three times independently.

[0071] Based on measurements of cellular protein content, MCF-7 and hTERT-BJ1 cells Cell viability was evaluated using sulforhodamine B (SRB) The assay was performed using d-TPP (50 nM ÷ 1 μM) for 24, 48, or 72 hours. After treatment with 10% trichloroacetic acid, the cells were incubated in a cold room for 1 h. The sections were fixed with acetic acid (TCA) for 1 hour and then dried at room temperature overnight. The cells were then incubated with SRB for 15 min, washed twice with 1% acetic acid, and incubated for at least 1 h. The protein-bound dye was then dried in air for 1 h. Finally, the protein-bound dye was diluted with 10 mM Tris pH 8. The solution was dissolved in 0.8 and read at 540 nm using a plate reader.

[0072] xCELLigence real-time cell analysis The Real-Time Analysis (RTCA) system is provided by ACEA Biosciences Inc. The xCELLigence RTCA system is supplied by Adherent cells by measuring electrical impedance expressed as index (CI) values This provides a useful approach for real-time monitoring of the biological status of 00 MCF-7 cells were seeded in a 16-well plate (E plate). After a period of time, cells were incubated with vehicle or increasing concentrations of d-TPP (50 nM to 250 nM ) for an additional 48 or 72 hours. RTCA was induced by cells This was done by measuring the electrical impedance values ​​obtained over a 96-hour period. This approach allows for the determination of the initiation and kinetics of cellular responses. The experiment was performed three times independently with quadruplicate samples for each condition. It was returned.

[0073] Unless otherwise stated, the data disclosed herein are based on 3 replicates per experiment. The mean ± standard deviation obtained over three or more independent experiments with at least three technical replicates. The mean mean error of the mean (SEM) is expressed. Statistical significance was determined using t-test. p≦0.0 A value of 5 was considered significant.

[0074] The terminology used in the above description of embodiments of the inventive approach is not intended to be limiting. The present description and the embodiments are for illustrative purposes only and are not intended to be limiting. As used in the appended claims, the singular forms "a," "an," and "the above" are intended to be used interchangeably. "(the)" includes the plural as well, unless the context clearly indicates otherwise. The approach of the present invention is described in detail below in the detailed description of the invention. As will be apparent from the discussion, numerous alternatives, modifications, and equivalents are encompassed.

[0075] The terms "first," "second," and "third" "a"), "b"), "c") etc. are used herein to refer to various approaches of the invention. These terms may be used to describe various elements, and the claims may be interpreted in terms of these terms. It will be understood that these terms are not limiting. Therefore, the first element described below is used only to distinguish one element from another. The first element is a departure from the teachings of the present approach. It can also be called an element aspect, It may be referred to as a third element as well. Hence, the terms "first," "second," "third," and "third" are used interchangeably. The terms "a)", "b)", "c)" etc. are used to indicate order or precedence over the associated elements. It is not intended to imply any other hierarchical relationship but is used solely for identification purposes. The order of operations (or steps) is not limited to the order presented in the claims.

[0076] Unless otherwise defined, as used herein (including technical and scientific terms) All terms have the same meaning as commonly understood by one of ordinary skill in the art. Terms as defined in the dictionary are intended to provide an understanding of the meaning of such terms in the context of this application and the related art. The term "common name" shall be construed as having a meaning consistent with the meanings set forth herein and as expressly defined herein. It is understood that unless otherwise specified, the terms should not be interpreted in an idealized or overly formal sense. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the event of a conflict in terminology, the present specification becomes dominant.

[0077] Also, as used herein, "and / or" refers to any combination of Any and all possible combinations and alternatives of one or more of the listed items. When "or" is used, it refers to the absence of a combination and is inclusive. do.

[0078] Unless the context clearly indicates otherwise, the approaches of the invention described herein It is specifically contemplated that the various features of the present invention may be used in any combination. Furthermore, the inventive approach, in some embodiments, It is contemplated that any described feature or combination of features may be excluded or omitted. .

[0079] As used herein, the transitional phrase "consisting essentially of "(and grammatical variations thereof)" means the material or The steps do not substantially affect one or more of the basic novel features of the claims. things that do not materially affect "his basic and novel characteristic(s)" Therefore, as used herein, the term "essentially "Consisting of..." shall be interpreted as equivalent to "including, comprising..." It should not be.

[0080] As used herein to refer to a measurable quantity, e.g., amount or concentration, The word "about" means a range of from ±20%, ±10%, ±5%, ±1%, ± It is intended to encompass variations of 0.5%, or even as little as ±0.1%. Any ranges provided herein for specific values ​​are to be construed as including any other ranges and / or It may contain individual values.

[0081] Thus, specific embodiments of the inventive approach have been described, as claimed below. Many obvious variations of these embodiments may be made without departing from the spirit or scope of the invention. Since various modifications are possible, the appended claims may be modified to include specific details set forth in the above description. It should be understood that the present invention is not limited by the above.

Claims

1. Alkyltriphenylphosphite having a saturated linear alkyl chain containing 9 to 18 carbon atoms A composition for cancer therapy comprising an agonist (ATP) compound.

2. The composition of claim 1, wherein the A-TPP compound comprises dodecyl TPP (d-TPP). thing.

3. a second inhibitor compound selected from a glycolysis inhibitor compound and an OXPHOS inhibitor compound; The composition of claim 2 further comprising:

4. The composition of claim 3 , wherein the second inhibitor compound comprises Vitamin C.

5. 4. The composition of claim 3, wherein the second inhibitor compound comprises 2-deoxy-glucose. 。

6. The composition of claim 4 , wherein the second inhibitor compound comprises doxycycline.

7. 6. The composition of claim 5, wherein the second inhibitor compound comprises niclosamide.

8. 7. The composition of claim 6, wherein the second inhibitor compound comprises berberine chloride.

9. A pharmaceutical composition comprising a pharma- ceutical effective amount of d-TPP or a pharma- ceutical acceptable salt thereof.

10. a pharma- tically effective amount of a second compound selected from a glycolysis inhibitor compound and an OXPHOS inhibitor compound; 10. The pharmaceutical composition of claim 9 further comprising an inhibitor compound.

11. The second inhibitor compound comprises one of vitamin C and 2-deoxy-glucose. The pharmaceutical composition of claim 10 comprising:

12. The second inhibitor compound is selected from the group consisting of doxycycline, tetracycline, chlortetracycline, and the like.

11. The method of claim 10, comprising administering to the patient one of the following: cyclosporine, minocycline, and tigecycline. Pharmaceutical compositions.

13. The second inhibitor compound is selected from the group consisting of azithromycin, erythromycin, and telithromycin. , clarithromycin, and roxithromycin. A pharmaceutical composition comprising:

14. 11. The pharmaceutical composition of claim 10, wherein the second inhibitor compound comprises niclosamide.

15. 11. The pharmaceutical composition of claim 10, wherein the second inhibitor compound comprises berberine chloride.

16. The pharmaceutical composition is useful for treating cancer, eradicating cancer stem cells, eradicating circulating tumor cells, and preventing tumor recurrence. or reducing the possibility of metastasis, prevention of metastasis or reducing the possibility of metastasis, and chemotherapy, radiation therapy and for sensitizing the cancer to one of phototherapy. The pharmaceutical composition according to any one of claims 9 to 15.

17. A method for treating or preventing cancer, the method comprising administering to a subject a pharma- tically effective amount of a d-TPP-linked or a pharma- ceutically acceptable salt thereof.

18. 18. The method of claim 17, wherein the cancer is breast cancer.

19. a pharma- tically effective amount of a second inhibitor compound selected from a glycolysis inhibitor and an OXPHOS inhibitor; 20. The method of claim 17, further comprising administering

20. The second inhibitor compound is selected from the group consisting of vitamin C, 2-deoxy-glucose, doxycycline, 20. The method of claim 19, wherein the active ingredient is one of benzodiazepine, niclosamide, and berberine chloride.

21. The d-TPP compound or a pharma- ceutical acceptable salt thereof is administered in the presence of the second inhibitor compound.

20. The method of claim 19, wherein the administration is prior to administration.

22. 1. A method for treating or preventing one of tumor recurrence and metastasis of cancer, the method comprising administering to a subject administering a pharma- ceutical effective amount of a d-TPP compound or a pharma- ceutical acceptable salt thereof to the subject. A method comprising:

23. 23. The method of claim 22, wherein the cancer is breast cancer.

24. a pharma- tically effective amount of a second inhibitor compound selected from a glycolysis inhibitor and an OXPHOS inhibitor; 23. The method of claim 22, further comprising administering:

25. The second inhibitor compound is selected from the group consisting of vitamin C, 2-deoxy-glucose, doxycycline, 25. The method of claim 24, wherein the active ingredient is one of benzodiazepine, niclosamide, and berberine chloride.

26. The d-TPP compound or a pharma- ceutical acceptable salt thereof is administered in the presence of the second inhibitor compound.

25. The method of claim 24, wherein the administration is prior to administration.

27. 1. A method of shifting a cancer to a glycolytic state, the method comprising administering to the cancer a pharmacologic effective amount of A method comprising the step of administering a d-TPP compound or a pharma- ceutically acceptable salt thereof.

28. A method for increasing the efficacy of cancer therapy, the method comprising administering to a subject a pharma- tically effective amount of d- A method comprising the step of administering TPP or a pharma- ceutically acceptable salt thereof.

29. 30. The method of claim 28, wherein the cancer therapy comprises chemotherapy.

30. A method for eradicating CSCs and circulating tumor cells in a subject, the method comprising: To the examiner: a pharma- ceutical effective amount of a d-TPP compound or a pharma- ceutical acceptable salt thereof; and a pharma- tically effective amount of a second inhibitor compound selected from a glycolysis inhibitor and an OXPHOS inhibitor; The method of claim 1, further comprising administering

Images