Methods of determining natural time progression of immune response and methods and compositions for altering intracellular pH to control disease

Abstract

The present invention provides a method of determining a stage in a natural time progression of an immune response in an individual. It describes a method for controlling an immune response by a modulation mechanism for differential operation in lymphocytes at different stages in the natural time progress of the immune response. Wherein one regulating mechanism is an energy metabolism mechanism for controlling intracellular pH (pHi) so as to control cell death and proliferation. Also described are combined methods of reducing the pHi of tumor cells to induce death thereof using compositions combined by energy metabolism modulators. Finally, the present invention provides methods of treating skin or mucosal infections or neoplasia by topical administration of a pH regulator that reduces pH.

Description

[0001] Related patent applications

[0002] This application claims the benefit of the following U.S. Provisional Patent Applications under 35 U.S.C. § 119(e): Application No. 63 / 416,614 (filed on October 17, 2022); Application No. 63 / 456,791 (filed on April 3, 2023); and Application No. 63 / 536,720 (filed on September 6, 2023). The disclosures of these provisional patent applications are hereby incorporated by reference in their entireties into this application.

[0003] Background

[0004] New therapies and strategies that achieve high therapeutic efficacy and minimize adverse side effects benefit patients suffering from various diseases, such as immune diseases and cancers, whose pathogenesis is attributed in part or in whole to cell death and / or proliferation.

[0005] Overview

[0006] The present invention describes a method or composition for controlling an immune response by controlling immune regulatory mechanisms operating in lymphocytes at different stages of the natural time course of an immune response, wherein the control and the natural time course of the immune response are guided and determined by the intracellular pH (pHi) and mitochondrial membrane potential (MMP) of lymphocytes.

[0007] In some embodiments, the immune regulatory mechanism includes an energy metabolism mechanism that affects the pHi of lymphocytes.

[0008] In some embodiments, the stages in the natural time course of an immune response are determined by dividing lymphocytes into relatively high and low pHi populations, which are respectively referred to as P and N lymphocytes or populations, and further by the values of pHi and MMP of P and N lymphocytes. Thus, the early stage is characterized by N lymphocytes as the major cell population, and there is an inverse relationship between MMP and pHi in all lymphocytes; the middle stage is determined by the emergence of P lymphocytes with a positive correlation between MMP and pHi, and N lymphocytes may still be the major lymphocyte population but may not be; the late stage is dominated by P lymphocytes with a positive correlation between MMP and pHi.

[0009] In the later stage, lymphocytes can be in one of four different energy states: the e1 state is characterized by the presence of a large number of cells called "n lymphocytes", which are N lymphocytes with low MMP and a weak or no negative correlation between MMP and pHi, and the MMP of the remaining N and P lymphocytes is also relatively low; the e2 state is characterized by the presence of a small number of n lymphocytes, and overall the remaining lymphocytes have a higher MMP than those in the e1 state; the e3 state is characterized by the absence of n lymphocytes and a strong positive or negative relationship between MMP and pHi; the e4 state is characterized by the predominance of n lymphocytes in the N population, and only a few (if any) N lymphocytes exhibit high MMP and an inverse relationship between MMP and pHi. Alternatively, the criterion for determining the stage of the natural time course of the immune response is the difference in the expression level and / or activity of molecules present in lymphocytes in different stages and energy states determined by the aforementioned method. The method for determining the natural time course of the immune response or the pHi and MMP of lymphocytes includes experimental techniques for simultaneously measuring cell surface molecules, pHi, MMP, and staining with Annexin V in one experimental step.

[0010] In some embodiments, a method of controlling a disease refers to a method for treating and / or preventing an immune disease.

[0011] In some embodiments, a disease is treated or prevented by regulating the energy metabolism of cells to lower the intracellular pH (pHi) to induce apoptosis, while increasing pHi promotes cell survival and proliferation. In some embodiments, lowering pHi is achieved by increasing the influx of pyruvate and / or fatty acids into the TCA cycle, inhibiting amino acid energy metabolism, inhibiting glycolysis, restricting the influx of amino acid carbon backbones and / or fatty acids into the TCA cycle to trigger an imbalance between ATP synthesis and hydrolysis, or various combinations of these methods of regulating energy metabolism. In some embodiments, increasing pHi is achieved by enhancing amino acid energy metabolism and / or glycolysis to generate energy, restricting the influx of pyruvate and / or fatty acids into the TCA cycle, restoring the balance between ATP synthesis and hydrolysis, or various combinations of these methods of regulating energy metabolism.

[0012] In some embodiments, an immune disease refers to a disease whose pathogenesis is partially or wholly attributed to an excessive immune response. They include, but are not limited to, autoimmune diseases, allergic diseases, and infectious diseases in which the immune response damages the structure and / or function of host tissues or organs. The treatment of such diseases includes reducing the pHi of lymphocytes to induce apoptosis according to the natural time course of the immune response or the pHi of lymphocytes and MMP or the relative ratio of P and N lymphocytes: in the early stage, mainly by increasing the influx of pyruvate and / or fatty acids into the TCA cycle, and by inhibiting amino acid energy metabolism and / or glycolysis, or various combinations of these methods of regulating energy metabolism; in the middle stage, mainly by inhibiting amino acid energy metabolism, increasing the influx of pyruvate and / or fatty acids into the TCA cycle, and selectively inhibiting glycolysis, by restricting the influx of amino acid carbon backbones and / or fatty acids into the TCA cycle to cause an imbalance between ATP synthesis and hydrolysis, or various combinations of these methods of regulating energy metabolism; in the late stage, mainly by restricting the influx of amino acid carbon backbones and / or fatty acids into the TCA cycle to cause an imbalance between ATP synthesis and hydrolysis, inhibiting glycolysis and / or amino acid energy metabolism, and selectively increasing the influx of pyruvate and / or fatty acids into the TCA cycle, or various combinations of these methods of regulating energy metabolism.

[0013] In some embodiments, an immune disease refers to a disease whose pathogenesis is partially or wholly attributed to an insufficient immune response to an infection, vaccine, or cancer cells, and is treated or prevented by increasing the pHi of lymphocytes to promote the survival and proliferation of lymphocytes.

[0014] In some embodiments, a method or composition for controlling a disease is used to reduce intracellular pH (pHi) to induce apoptosis in tumor cells. Such a method includes: constructing a set of single energy metabolism regulators at low doses or concentrations and a composition composed of these regulators to reduce the pHi of tumor cells; screening a series of such compositions according to their ability to inhibit the growth and / or survival of tumor cells; and simultaneously or non-simultaneously screening the ability of this series of such compositions to inhibit the growth or survival of normal cells; selecting a composition that exhibits a relatively strong inhibitory effect on tumor cells, with or without considering its relatively weak or no inhibitory effect on normal cells, and further adjusting the dose of one or more energy metabolism regulators in the selected composition to achieve optimal inhibition of tumor cells while minimizing inhibition of normal cells. The energy metabolism regulators used for this purpose are selected from those that can increase the influx of pyruvate and / or fatty acids into the TCA cycle, inhibit amino acid energy metabolism, inhibit glycolysis, and cause an imbalance between ATP synthesis and hydrolysis by restricting the influx of the amino acid carbon backbone and / or fatty acids into the TCA cycle. In some embodiments, this series of compositions includes the compositions summarized in Table 1 of this application and compositions composed of metabolic regulators with similar or identical activities. In some embodiments, a composition containing energy metabolism regulators identical or similar to those of Compositions 8', 12', 15, 16', 19, 21', 8''', 12''' and 16''' described in Table 1 and Example K is a potential anti-cancer compound.

[0015] In some embodiments, Ki-67 is used as a biomarker to predict an individual's responsiveness to low-pHi-based cancer treatment, where the expression level of Ki-67 in cancer cells or the percentage of cancer cells expressing high levels of Ki-67 serves as a predictor of an individual's response to cancer treatment using a pH regulator that reduces pHi.

[0016] In some embodiments, a skin or mucosal infection or tumor can be treated by directly applying a pH regulator that can reduce the pH value to the affected area.

[0017] This application further provides a method or composition for treating or preventing a disease by regulating the energy metabolism of cells to reduce intracellular pH (pHi) to induce apoptosis or increase pHi to promote cell survival and proliferation, wherein,

[0018] Reducing pHi is achieved by increasing the influx of pyruvate and / or fatty acids into the TCA cycle, inhibiting amino acid energy metabolism, inhibiting glycolysis, inducing an imbalance between ATP synthesis and hydrolysis by restricting the influx of the amino acid carbon backbone and / or fatty acids into the TCA cycle, or various combinations of these energy metabolism regulatory pathways;

[0019] Elevating pHi is achieved by enhancing amino acid energy metabolism and / or glycolysis, restricting the influx of pyruvate and / or fatty acids into the TCA cycle, restoring the balance between ATP synthesis and hydrolysis, or a combination thereof; or by various combinations of these pathways that regulate energy metabolism; and

[0020] Regulating energy metabolism is achieved by administering a composition comprising an energy metabolism regulator.

[0021] While the subject matter disclosed herein is susceptible to various modifications and alternative forms, specific embodiments thereof have been shown by way of example in the drawings and will be described in detail herein. It should be understood, however, that the drawings and detailed description presented herein are not intended to limit the subject matter disclosed herein to the particular embodiments shown, but on the contrary, the intention is to cover all modifications, equivalents, and alternative forms falling within the spirit and scope of the subject matter as defined by the appended claims.

[0022] Other features and advantages of the present invention will become apparent from the following detailed description of the preferred embodiments of the invention, taken in conjunction with the accompanying drawings.

[0023] Brief Description of the Drawings

[0024] The drawings are not drawn to scale. In the drawings, each identical or nearly identical component that is illustrated in various drawings is represented by a like numeral. For clarity, not every component in every drawing is labeled. In the drawings:

[0025] Figure 1A - 1D Shows the evolution of P (high pHi) and N (low pHi) cell populations in specified live lymphocytes in mouse MLN after secondary immunization with OVA or in mouse DLN after immunization with MOG over time during the immune response. Figure 1A - 1C Shows the early ( Figure 1A )、mid ( Figure 1B ) and late ( Figure 1C ) phases of the natural time course of the immune response against OVA. Figure 1D Shows examples of the phases of the natural time course of the immune response against MOG. Lym: all live lymphocytes, CD4: live CD4 T cells, CD8: live CD8 T cells, CD19: live B cells.

[0026] Figure 2A - 2E Shows the inhibition of lymphocytes in the MLN of mice after secondary immunization with OVA at the mid - stage of the natural time course of the immune response by a metabolic regulator. Figure 2AShown are reduced pHi values in different live lymphocyte populations (bottom panels) in mice treated with inhibitors of glycolysis (GSK) or glutaminolysis (CB-839) (top panels), or with promoters of pyruvate (DCA) or fatty acid (C75) entry into the TCA cycle. Figure 2B is a dot plot of flow cytometry showing Figure 2A pHi and MMP, and P and N cell populations of the same lymphocytes in Figure 2C is reflective of Figure 2B the data shown in TM bar graphs of the % of P and N lymphocytes, fold change in absolute number of live cells, and mean fluorescence intensity of pHrodo Figure 2D is a dot plot showing early apoptotic cells (Annexin V - ) in the indicated live lymphocyte populations and treatment groups. Figure 2E is a bar graph showing Figure 2D the percentage of early apoptotic cells in

[0027] Figure 3A - 3C shows the inhibition of metabolic modulators on lymphocytes in the Early stage mesenteric lymph nodes (MLN) of mice after secondary immunization with OVA at the natural time course of the immune response. Figure 3A is a dot plot of flow cytometry showing the pHi and MMP status of lymphocytes, and P and N cell populations in mice treated with vehicle control, CB-839, or DCA. Figure 3B is a dot plot of early apoptotic cells (Annexin V - ) in the indicated live lymphocyte populations and treatment groups. Figure 3C is a bar graph representing Figure 3B the data in

[0028] Figure 4A - 4E shows the inhibition of metabolic modulators on lymphocytes in the Late e1 state mesenteric lymph nodes (MLN) of mice after secondary immunization with OVA at the natural time course of the immune response. Figure 4A is a dot plot of flow cytometry showing the pHi and MMP status of lymphocytes, and P and N cell populations in mice treated with the indicated different metabolic modulators. The n cell population within N lymphocytes has a lower MMP, and there is a weak or no correlation between MMP and pHi. CPI-613 is a metabolic modulator that blocks the entry of amino acid carbon skeletons into the TCA cycle. Etomoxir (Etom) is a pharmacological metabolic modulator that inhibits fatty acid influx into the TCA cycle. Figure 4Bis a bar graph presenting data in numerical form Figure 4A showing the percentages of P and N lymphocytes, the fold change in their absolute numbers, and the fold change in the total live lymphocyte population shown. Figure 4C is a dot plot showing the early apoptotic cells (Annexin V - ) of the live lymphocyte population shown and the treatment groups.

[0029] Figure 4D is a bar graph showing the percentages (upper) of early apoptotic cells and the pHi of N lymphocytes (lower) in the lymphocyte population shown and the treatment groups. Figure 4E is a set of dot plots comparing the pHi and MMP status of the total live lymphocytes in the treatment groups shown.

[0030] Figure 5A - 5B Shows Late e2 the imbalance between ATP synthesis and hydrolysis in lymphocytes in the state shown. Figure 5A is a bar graph showing the relative numbers of ATP within live lymphocytes in the murine MLN of the treatment groups shown. Figure 5B Shows the expression of intracellular ATP and Ki-67 in the sorted lymphocyte population. The sorting gate is shown on the left.

[0031] Figure 6A - 6B Shows the inhibition of metabolic modulators on lymphocytes in the murine MLN of mice receiving a second immunization with OVA Late e1 in the state of the natural time course of the immune response. Figure 6A is a set of bar graphs showing the fold change in the absolute numbers of live cells and the percentages of early apoptotic cells in the live lymphocyte population shown and the treatment groups. Figure 6B is a set of dot plots comparing the distribution of the pHi and MMP (upper graph) and early apoptotic cells (lower graph) of the total live lymphocytes in mice treated with vehicle or DCA. The numbers in large font represent the percentages of N lymphocytes and early apoptotic cells with low pHi.

[0032] Figure 7 Shows the inhibition of metabolic modulators on live lymphocytes in the murine MLN of mice receiving a second immunization with OVA Late e3 in the state of the natural time course of the immune response. The upper and lower graphs show the fold change in the absolute numbers of live cells in the lymphocyte population shown and the treatment groups.

[0033] Figure 8 Shows the inhibition of metabolic modulators on live lymphocytes in the murine MLN of mice receiving a second immunization with OVA Late e4 in the state of the natural time course of the immune response. The bar graphs shown show the fold change in the absolute numbers of cells, pHi, and percentages of early apoptotic cells in the lymphocyte population shown and the treatment groups.

[0034] Figure 9A - 9B Shows the inhibition of the immune response of mice after 2 rounds of OVA immunization by a metabolic regulator. Figure 9A Is a bar graph of the fold change in the absolute cell numbers of lymphocyte populations MLN and treatment regimens in the indicated MLN described in Example I. Figure 9B Shows the respiratory resistance of mice in the indicated treatment groups.

[0035] Figure 10A - 10B Shows the inhibition of metabolic regulators on EAE model Live lymphocytes in the DLN and NDLN of mice immunized with MOG peptide in. Figure 10A Are two sets of dot plots showing the pHi and MMP status (left panel) and early apoptotic cells (right panel) of total live lymphocytes or CD4 T cells in the DLN or NDLN of mice in the indicated treatment groups. Figure 10B Is a set of bar graphs showing the percentages of N lymphocytes, pHi, and early apoptotic cells in the indicated live lymphocyte populations and treatment groups.

[0036] Figure 11A - 11D Shows screening for Metabolic regulator composition for cancer treatment .

[0037] Figure 12 Shows the correlation between Ki-67 levels and the sensitivity of Raji tumor cells to death induced by low pHi. The upper left panel shows that treatment with HOAc but not NaOH decreased the pHi of Raji cells. The upper right panel shows that early apoptosis occurred in Raji cells with low pHi. The lower left panel is an overlaid histogram showing the mean fluorescence intensity (MFI) of Ki-67 in different subsets of Raji cells expressing high, low, or low to negative levels of Ki-67. The lower right panel shows the fold change in live cells of different subsets of Raji after treatment with HOAc relative to live cells after treatment with saline.

[0038] Figure 13A - 13C Shows the treatment of skin or mucosal infections using HOAc. Figure 13A Is a copy of a medical record showing Figure 13B The clinical diagnosis of onychomycosis of the left great toe. Figure 13B Are photographs of toenails infected with onychomycosis before and 1.5 months after treatment with a composition containing HOAc and water. Figure 13C Are photographs showing two infected lesions (indicated by arrows) on the upper lip mucosa before treatment (D0) and after treatment for one day (D1) and two days (D3) with a composition containing HOAc and water.

[0039] Figure 14These are two photographs showing two benign tumor lesions (indicated by arrows) on the facial skin before treatment (D0) with a composition containing HOAc and water and 20 days later.

[0040] Definitions

[0041] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the subject matter of this invention pertains. According to long-standing patent law convention, the articles "a" and "the" when used in the specification (including the claims) refer to "one or more". For example, reference to "a cell" can include a plurality of such cells, and so on.

[0042] Unless otherwise indicated, all numbers expressing quantities of ingredients, conditions, etc. used in the specification and claims are to be understood as being modified by the term "about". Accordingly, unless indicated to the contrary, the numerical values set forth in this specification and the appended claims are approximations that may vary depending upon the properties sought to be obtained by the presently disclosed subject matter.

[0043] As used in this invention, when referring to values or amounts of mass, weight, time, volume, concentration, dose, and / or percentage, the term "about" can encompass variations relative to a particular amount, which in some embodiments can be + / −0.01 - 50%, such variations being appropriate in the disclosed packages and methods.

[0044] The term "administer" or "administration" as used in this invention refers to delivering a composition to the interior or exterior of an individual's body, systemically or locally to a particular anatomical location, by any of a variety of routes and methods of administration. Suitable routes and methods of administration include (but are not limited to) intravenous (i.v.) injection / infusion, intraperitoneal (i.p.) injection, intratracheal (i.t.) injection, instillation or spraying, inhalation of an aerosol or vapor, topical application, intranasal spraying, intradermal injection, subcutaneous injection, and rectal delivery. Methods of administration also include the rate of administration, i.e., the amount of the composition delivered to the individual per unit time. Administration to a particular anatomical location can be with or without the aid of guiding / navigating techniques (such as imaging techniques). The choice of pharmaceutically acceptable routes and modes of administration depends on the nature of the disease, the tissue and / or organ affected by the disease, and should be determined by the individual's attending healthcare provider within the scope of reasonable medical judgment.

[0045] "Allergic disease" as used in this invention refers to a disease caused by an inflammatory and / or immune response resulting from exposure to an allergen.

[0046] The term "anatomical location" refers to any spatial site within or on an individual's body. For example, an anatomical location can be an interstitial space, an organ lumen, a body cavity, an intravascular space, a mucosal or cutaneous location.

[0047] The term "aqueous environment" refers to extracellular fluid and / or intracellular fluid at any anatomical location within or on the surface of a subject, intracellular fluid of in vitro cultured cells, cell culture medium, and any aqueous solution prepared in vitro. Examples of extracellular fluid include (but are not limited to) interstitial fluid, plasma, lymph, mucus, bronchoalveolar fluid, cerebrospinal fluid, synovial fluid, thoracic and abdominal fluids.

[0048] The terms "by way of example", "for example", and "such as" used in the present invention are not restrictive and do not exclude other applications similar to the examples.

[0049] The "autoimmune disease" used in the present invention refers to a disease characterized by an immune and inflammatory response to one or more components of an individual's own tissues, cells, and / or body fluids.

[0050] The "composition" used in the present invention refers to a pharmaceutical preparation formulated from a certain amount of one or more pharmaceutically acceptable compounds with or without one or more other drugs and one or more pharmaceutically acceptable solvents, solutions, excipients, and / or carriers. While maintaining the therapeutic or prophylactic efficacy of the composition, the concentrations of pH regulators and / or cell proliferation inhibitors must be set at levels that meet the "pharmaceutically acceptable" standard. The composition can take various physical forms, including (but not limited to) liquids, aerosols, vapors, creams, gels, capsules, tablets, powders, granules, etc. All or part of the components of the composition can be pre-mixed or provided separately and mixed before use. It should be understood that the composition to be used for a specific individual at a specific time will be determined by the individual's attending healthcare provider within the scope of reasonable medical judgment.

[0051] The "immune response" used in the present invention refers to the activation, proliferation, migration, and functional execution of innate, adaptive, or innate and adaptive immune cells in specialized lymphoid organs / tissues such as lymph nodes, spleen, and Peyer's patches, as well as at the anatomical location where the antigen is present, and its consequences at the cellular and molecular levels. Innate immune cells are activated by receptors other than antigen receptors. The activation of adaptive immune cells (mainly B cells, T cells, and NKT cells) is mainly caused by the binding of antigen receptors (BCR or TCR) to antigens or superantigens, but may also be caused by the stimulation of B cell or T cell mitogens (such as lipopolysaccharide, concanavalin A), and also includes bystander activation stimulated by cytokines.

[0052] The term "infectious disease" refers to a disease that can be transmitted between individuals and is caused by a microbial pathogen (e.g., pneumonia). Microorganisms can be bacteria, viruses, fungi, or parasites. The microorganism itself not only directly harms the infected individual, but the excessive inflammatory and / or immune responses to the microorganism can also lead to pathological and pathophysiological consequences, while appropriate inflammatory and / or immune responses to the microorganism are necessary for clearing the infection.

[0053] The terms "inflammatory cell" and "immune cell" used in the present invention are used interchangeably and refer to innate immune cells and adaptive immune cells, including cells of the lymphoid and myeloid lineages. Inflammatory cells can circulate through the blood and lymphatic systems and can migrate to and settle in specific tissues or anatomical locations.

[0054] The term "inhibitor" refers to a molecule or a mixture of different molecules that can directly or indirectly alter, interfere with, reduce, downregulate, block, inhibit, abolish, or degrade the expression, amount, or activity of an enzyme, membrane transport protein, or ion channel or any other molecule that plays a role in a biochemical or biological process.

[0055] The present invention uses "tumor" or "tumorous" to describe a dysregulated benign or malignant cell proliferation. Solid and non-solid tumors are tumorous diseases. Malignant solid and non-solid tumors are synonymous with cancer.

[0056] In the present invention, the term "normal cell" refers to a cell that does not play a pathogenic or adverse role in a disease and can be a cell that protects or prevents an individual against a disease or a cell that is unrelated to the disease.

[0057] The present invention uses "excessive" to describe an inflammatory and / or immune response that damages the structure and / or function of an individual's tissue and / or organ. The immune and / or inflammatory responses to allergens and autoantigens in allergic and autoimmune diseases are generally considered to be excessive immune and / or inflammatory responses. Such a response can also be excessive when the immune and / or inflammatory response to an infection damages normal tissue or organ.

[0058] The "pH regulator" used in the present invention refers to any pharmaceutically acceptable compound that can change or help resist changes in the pH value of an aqueous environment.

[0059] The term "pharmaceutically acceptable" is used to describe the properties of any molecule and / or compound or a mixture of different molecules or compounds that, within the scope of reasonable medical judgment and on the premise of a reasonable risk / benefit ratio, is suitable for use in an individual without causing excessive toxicity, allergic reactions, irritation, and / or other problems or complications.

[0060] The "proliferation" used in the present invention refers to an increase in the number of cells through cell division and / or the survival of dividing cells.

[0061] As used herein, the term "individual" refers to a human or non-human animal (e.g., mouse, dog, cat, cow, horse). An individual seeking diagnosis, treatment, and / or prevention of a disease is a patient.

[0062] The "activity" or "activities" of a molecule refers to the biochemical or biological consequences that occur when the molecule is introduced into or present in an intracellular or extracellular environment.

[0063] As used in this application, "amino acid energy metabolism" refers to any biochemical reaction and molecular event that results in the breakdown of arginine, glutamine, histidine, or proline to produce ammonia or urea and glutamate; the export of glutamate from the cell; or the participation of glutamate in the malate-aspartate shuttle; or the further metabolism of glutamate to produce malate and aspartate and the participation of malate and aspartate in the malate-aspartate shuttle; and subsequent catabolic processes or reactions that further convert malate to lactate and secrete lactate into the extracellular space; when the term is used with other amino acids, it specifically refers to the reaction that removes the amino group from the amino acid to produce ammonia. The term "ammonia" as used in this definition includes the interconversion of ammonia and ammonium, such that the nitrogen atom from the amino group of the amino acid can exist in equilibrium between ammonia and ammonium. Glutamine breakdown is a specific type of amino acid energy metabolism that breaks down glutamine.

[0064] When used to describe a molecule or a portion of a molecule on or within a lymphocyte, "differential expression or presence" refers to a qualitative (presence or absence) or quantitative difference in one or more molecules or portions of a molecule or their activities at early, middle, and late stages of the natural time course of an immune response, or in different lymphocytes that can be distinguished based on pHi and MMP characteristics.

[0065] "Entry of amino acid carbon skeletons into the TCA cycle" refers to the biochemical reaction steps in which an amino acid is converted to α-ketoglutarate, succinyl coenzyme A, fumarate, or oxaloacetate, and the catabolic reactions that occur immediately after these metabolites enter the TCA cycle, such as the reaction catalyzed by α-ketoglutarate dehydrogenase in the TCA cycle; and the biochemical reaction steps that convert an amino acid to pyruvate.

[0066] As used in this application, "glycolytic energy supply" or simply "glycolysis" refers to the biochemical reactions that occur when glucose enters the cell and in the cytosol, which produce a net of 2 ATP and 2 lactate per glucose molecule; and the excretion of lactate from the cell.

[0067] "The influx of fatty acids into the TCA cycle" refers to the process in which fatty acids enter cells, are converted into fatty acyl-CoA, and then the fatty acyl-CoA is transported into mitochondria, as well as the process of β-oxidatively decomposing the long carbon chains of fatty acyl-CoA to generate acetyl-CoA. Activators and inhibitors of the activity or expression of proteins or enzymes involved in or regulating these processes, particularly but not limited to proteins or enzymes in the rate-limiting steps such as carnitine palmitoyltransferase 1 (CPT-1), are part of the energy metabolism regulators referred to in this application.

[0068] "The influx of pyruvate into the TCA cycle" in this application refers to the process in which pyruvate enters mitochondria, is catabolized to generate acetyl-CoA, or directly converts pyruvate into oxaloacetate to participate in the TCA cycle; and the processes regulating these events, such as the inactivation of pyruvate dehydrogenase (PDH) by phosphorylation by PDH kinase. Inhibitors and activators of the activity or expression of proteins or enzymes in these processes are part of the energy metabolism regulators referred to in this application.

[0069] When "low dose" is used to describe an energy metabolism regulator, it refers to a dose that, when administered to an individual alone or in combination with other energy metabolism regulators or other compounds, does not cause more severe adverse side effects than those generally considered acceptable in the medical community.

[0070] "Lymphocytes" as used in this application can be all lymphocytes, or lymphocyte subsets associated with the pathogenesis of specific diseases, such as CD4 T cells in multiple sclerosis and its animal model experimental autoimmune encephalomyelitis (EAE), or B cells in lupus. Normal lymphocytes are non-tumor lymphocytes from an individual or a healthy donor.

[0071] "Mitochondrial membrane potential" refers to the inner mitochondrial membrane potential generated by the proton gradient between the mitochondrial matrix and the intermembrane space. The mitochondrial membrane potential can be measured by various methods, for example, staining cells with MitoSpy fluorescent dye (Biolegend) and then detecting the fluorescence in the cells by flow cytometry.

[0072] "Energy metabolism regulator / agent" and "metabolism regulator / agent" are used interchangeably in this disclosure and refer to activators or inhibitors of the activity or expression of proteins or enzymes involved in or regulating the biochemical reactions that break down glucose, fatty acids, and amino acids to produce ATP. The terms activator, stimulator, accelerator, and promoter are used interchangeably in this context.

[0073] "Significant expression" is used to describe the expression of a protein or a protein gene, which refers to the presence of specific positive signals in the test results as compared with the negative control. For example, positive signals appear when specific rather than non-specific antibodies are used in flow cytometry or Western blotting, or when specific rather than non-specific primers for the gene of the protein are used in RT-PCR. In some cases, in order to select effective protein or enzyme targets for a specific type of cancer, the expression of a protein or an enzyme in such cancer may be compared with that in a group of other cancers or normal tissues or cells (such as normal lymphocytes). Relatively stronger (not necessarily higher than the control) expression in that specific type of cancer will form the basis for selecting the protein or enzyme as a potential effective target.

[0074] Detailed description

[0075] The present disclosure is intended to provide sufficient details for understanding one or more specific embodiments of the invention in a broader sense. These descriptions elaborate and illustrate the features of these embodiments, but do not limit the subject matter of the invention to the specifically described embodiments and features. Thoughts inspired by these descriptions may result in more and similar embodiments and features, but this does not lead to going beyond the scope of the presently disclosed subject matter. For example, using different compounds with similar or identical activities as those described in the embodiments and examples.

[0076] The immune response can be beneficial or harmful to an individual's health. An immune response against an infectious organism or cancer cells protects the individual, while an immune response against an allergen or autoantigen leads to allergic diseases and autoimmune diseases, respectively. In fact, even an immune response to an infection, such as leishmaniasis, can result in immunopathology in the host. Therefore, medical methods that regulate the immune response can benefit patients suffering from various diseases. The immune response is a dynamic process. A typical adaptive immune response to an infection lasts for 2 - 3 weeks and ceases after the infection is eliminated (1). The entire immune response to an allergen or autoantigen may last longer due to repeated stimulation by the antigen, but the lifespan of an individual lymphocyte is similar. In this regard, lymphocytes in the immune response initially undergo clonal expansion or proliferation, but at the same time, they are constantly dying by apoptosis. Eventually, most of the expanded lymphocytes die by apoptosis, while a few of them become memory cells. (21,22). Therefore, manipulating lymphocyte death and proliferation can not only control the intensity of the ongoing immune response but also affect the formation of immune memory by influencing the fate of memory cell precursors, which will determine the future response to the same antigen. The mechanisms that regulate lymphocyte death, proliferation, migration, and function may change with the natural time course of the immune response. To effectively control the immune response, treatment must target the mechanisms that are operative in lymphocytes at the time of medical intervention. However, this concept has not been fully appreciated, and in general, current treatments for immune diseases do not take into account the kinetic changes in immune regulation.

[0077] This may be because most immunological studies are conducted in a laboratory setting where the time of contact with the antigen is known, and thus researchers can empirically know when to collect samples for their specific studies. In contrast, under clinical conditions, it is usually not known when an individual first contacts the antigen or how long their immune response has been ongoing when they seek medical treatment. Therefore, in order to intervene in the immune response according to its natural time course, there must be a method to determine the natural time course or the stage in that time course of the immune response. However, no such method currently exists.

[0078] Energy metabolism plays an important role in immune responses. Early studies found that, similar to the situation known as the Warburg effect observed in tumor cells, proliferating lymphocytes tend to utilize glycolysis to generate energy (23). Recent studies have shown that the choice of energy metabolism also plays a role in lymphocyte subset differentiation (24). In tumor cells, the contribution of glycolysis to total energy production ranges from less than 5% to over 50% (2). The relative contribution of glycolysis to lymphocyte energy production is unclear. A large number of previous studies have also found that the PI3K / AKT / mTOR pathway is a mechanism that promotes the Warburg effect (25). In addition to the Warburg effect, glutaminolysis is another characteristic of highly proliferating cells and is also considered another hallmark of cancer cells (3). In fact, 20% of cancer patients ultimately die from cachexia, which results from the breakdown of adipose tissue and muscle to replenish glutamine in the blood for consumption by cancer cells (4).

[0079] In the Warburg effect, the waste product lactate produced by glycolysis is excreted extracellularly, thereby creating an acidic extracellular tumor microenvironment (TME). Research on the biological consequences of the Warburg effect has mainly focused on this acidic TME. It has been reported that the acidic TME can promote the invasion of tumor cells into surrounding tissues (5). It also negatively regulates the functions of innate immune cells and the activities of cytotoxic T cells (6-10). However, little is known about how the Warburg effect and glutaminolysis regulate the survival and proliferation rates of highly proliferating cells themselves. A notable feature of the Warburg effect is its low energy production efficiency, as it yields only 2 ATPs per glucose molecule decomposed, while up to 34 ATPs can be produced through mitochondrial respiration (11). Similarly, although glutamine is highly catabolized in tumor cells, at least half of its carbon atoms are not oxidized to produce ATP but are secreted by tumor cells in the form of lactate (12). Thus, the tendency of proliferating cells to utilize the Warburg effect and glutaminolysis for energy metabolism creates a contradiction between the high energy demand of proliferating cells and the low efficiency of energy production by these two pathways. This paradox, particularly how it affects the fate of proliferating cells, has not been explained. The inventors of the present invention found in a previously pending patent application (International Application No. PCT / US22 / 47932, the entire content of which is incorporated herein by reference) that low intracellular pH (pHi) induces apoptosis, while high pHi allows cells to proliferate at a high rate. These results were obtained in cells that had not been subjected to any treatment, so these cells retained intact mitochondria, which is different from earlier studies that described cytosolic acidification due to redistribution of mitochondrial contents caused by experimental treatment that damaged mitochondria (13-16). The data disclosed in the present application further indicate that different metabolic pathways regulate pHi in different ways. Glutaminolysis and glycolysis play an indispensable role in increasing pHi or maintaining a high pHi, thereby avoiding cell death caused by low pHi resulting from energy production by other pathways.

[0080] Given the importance of energy metabolism in immune responses and tumorigenesis, treating immune diseases and cancers by manipulating energy metabolism is an attractive idea. For immune diseases, such efforts are still in their infancy. In contrast, the development of cancer therapies targeting metabolic pathways has a rather long history. Unfortunately, such efforts have not been very successful. The major hurdle is the conflict between achieving a therapeutic effect and avoiding adverse side effects (17). Metabolic pathways preferentially used in cancer are typically selected as targets. To achieve a high therapeutic effect, such a pathway must be blocked nearly completely. However, key enzymes in such a pathway, such as lactate dehydrogenase in the Warburg effect, are usually elevated in cancer cells (18). Thus, to effectively block the pathway, high doses of inhibitors must be used. Since most metabolic pathways are shared by cancer cells and normal cells, high doses of inhibitors also cause severe adverse side effects. This application solves this problem by adopting a new strategy, namely, reducing the pHi of cancer cells to induce apoptosis of cancer cells by simultaneously partially manipulating (in some cases by partially blocking and partially enhancing) multiple pathways. Since no single pathway is blocked nearly completely or enhanced to the extreme, this strategy is unlikely to cause severe side effects. At the same time, relatively small alterations in multiple pathways can jointly reduce the pHi to a level sufficient to induce apoptosis of cancer cells as well as adverse pathological immune cells.

[0081] Pharmacological regulation of energy metabolism can be regarded as a special type of chemotherapy. For chemotherapy as well as many other types of treatment, it is very important to have biomarkers that can predict the cancer's response to treatment. However, predictive biomarkers for most of the available chemotherapeutic drugs are still elusive (19). Ki-67 is a widely used and reliable marker for proliferating cells, and its level is positively correlated with the levels of rRNA and DNA synthesis (20). However, Ki-67 is not a reliable predictive biomarker for existing chemotherapeutic drugs (19). Instead, the data disclosed in this application indicate that it can be a predictive biomarker for low pHi cancer treatment.

[0082] Methods for determining the natural time course of immune responses

[0083] When an individual with an immune disease is presented to a healthcare provider, it is often difficult to accurately determine when the lymphocytes in that individual were exposed to a pathological antigen (e.g., a microbial product, an allergen, or an autoantigen). As a result, it is not possible to determine at what point in the natural time course of the immune response the individual presented for medical treatment. Understanding or determining the natural time course of the immune response is important for effectively modulating the immune response because, as disclosed herein, the mechanisms of energy production in lymphocytes are different at different time points during the immune response. This difference is important for controlling lymphocyte death and proliferation. Other mechanisms that control lymphocyte death, survival, proliferation, and function may also be different when lymphocytes are in the early, middle, or late stages of the immune response. Thus, a method that enables medical professionals to determine the natural time course of the immune response can also be applied to other therapies targeting multiple immune regulatory mechanisms to improve targeting precision and efficacy. However, this problem has not been fully recognized and studied by the clinical and biomedical research communities, and there is no available method to determine the natural time course of the immune response.

[0084] This patent application provides such a method by measuring the pHi and MMP of lymphocytes. In this method, lymphocytes are divided into two populations, one with a relatively high pHi and the other with a relatively low pHi, referred to as P and N lymphocytes or populations, respectively. The early stage of the immune response is dominated by N lymphocytes, with P lymphocytes being in the minority, but all lymphocytes at this stage show an inverse relationship between their MMP and pHi. The middle stage is marked by the appearance of P lymphocytes with a positive correlation between MMP and pHi, while N lymphocytes continue to show a negative correlation between their pHi and MMP. At this stage, N lymphocytes may or may not continue to be the majority lymphocytes. As the immune response progresses further, the percentage of P lymphocytes increases, and they maintain a positive correlation between their MMP and pHi. When P lymphocytes become the dominant lymphocytes, the immune response enters the late stage.

[0085] Late-stage lymphocytes can be in any of four energy states: -e1, -e2, -e3, and -e4. The e1 state is characterized by the presence of a large number of "n lymphocytes", which are N lymphocytes but have a low MMP, and there is a weak or no negative correlation between MMP and pHi. Overall, the MMP of the remaining N and P lymphocytes is also relatively low. The e2 state is characterized by the presence of a small number of n lymphocytes, and overall, the remaining lymphocytes have a higher MMP than those in the e1 state. The e3 state is characterized by the absence of n lymphocytes, and there is a strong positive correlation and a negative correlation between MMP and pHi in P and N lymphocytes, respectively. Finally, the e4 state is characterized by the predominance of n lymphocytes in the N population, and only a few (if any) N lymphocytes have a high MMP and a strong inverse relationship between MMP and pHi.

[0086] However, in some cases, the immune response may not go through all the different stages. Depending on the history and degree of exposure to environmental antigens, the immune response may start at a stage later than the early stage. In one embodiment of the EAE model, the lymphocytes of non-immunized littermate mice showed an MMP and pHi profile similar to that of the late e2 state. This indicates that an active immune response already existed in the mice before they were immunized with a specific antigen. Nevertheless, after immunization, the number of N lymphocytes in the lymphocytes of the draining lymph nodes (DLN) gradually decreased, although they skipped the early and middle stages. Although the dynamic changes in lymphocyte pHi and MMP do reflect the natural time course of the immune response, for practical purposes, treatment strategies can be designed based only on the pHi and MMP of lymphocytes without having to refer to the concept of the natural time course of the immune response. The use of this different narrative should not be construed as a substantial difference from this disclosure.

[0087] This application extends the claims of the method for determining the natural time course of an immune response to include determining the stage in the natural course of an immune response using molecules and / or the activities of molecules that differ in lymphocytes at different stages defined by pHi and MMP profiles. This is a logical extension since it is clear that differences in molecules and / or the activities of molecules should be as effective as pHi and MMP in determining the stages of the natural time course of an immune response. The method can also be used in combination with other methods for defining different lymphocyte subsets, for example, one or more of the methods of using MHC tetramers to identify antigen-specific lymphocytes, detecting surface markers of memory cells, etc. The lymphocytes in the method can be derived from tissues or body fluids such as lymph nodes, spleen, mucosa, blood, cerebrospinal fluid, bronchoalveolar lavage fluid, etc. Although all lymphocytes can be analyzed, in cases where a particular lymphocyte subset is most relevant to the pathogenesis of a disease, such as CD4 T cells in multiple sclerosis and EAE, and B cells in lupus, analysis can be specifically directed to such cell populations. If MHC tetramers are available for determining antigen-specific T cells, the analysis can be focused on such T cells.

[0088] For the convenience of the clinical application of the method, a kit containing all the necessary reagents will be provided to medical or veterinary professionals, such as reagents for detecting pHi, MMP, molecules and / or their activities that are differentially present / expressed in lymphocytes at the early, middle and late stages of the natural time course of an immune response, as well as a detailed description of the method for determining the stage in the natural time course of the immune response in a subject. Equipment and laboratory tools can also be provided as part of the kit.

[0089] A technique for one-step staining of cell surface markers, pHi, MMP and Annexin V

[0090] In this application, pHi, MMP and early apoptosis of cells, i.e., positive staining of Annexin V, are detected by flow cytometry. Since different buffers and temperatures are required, the experimental procedure for preparing cells for flow cytometry analysis for this purpose usually includes at least four steps: staining the cell surface markers at 4 °C, staining the cells with a pH indicator at 37 °C, staining the cells with MitoSpy at 37 °C to measure MMP, and finally staining the cells in Annexin binding buffer at room temperature to detect early apoptosis of the cells. This long process is not only laborious but also increases the risk of introducing artifacts. For example, multiple steps of incubating cells in serum-free buffer at 37 °C or room temperature can lead to cell death. To overcome these problems and obtain experimental results that best reflect the in vivo immune response, the present invention has designed a one-step technique at 37 °C and in the presence of Ca 2+Cells are simultaneously and briefly stained for cell surface markers, pHi, MMP, and annexin V in a buffer. The buffer containing Ca 2+ can be, but is not limited to, annexin V binding buffer or live cell imaging solution (LCIS) (Life Technology, Grand Island, NY) supplemented or not supplemented with Ca 2+ In one embodiment, 2x10 6 lymph node cells are incubated with 100 μl of LCIS supplemented with 0.7 mM CaCl TM containing BV421-conjugated anti-mouse CD4, APC-Fire 750-conjugated anti-mouse CD8, APC-conjugated anti-mouse CD19 antibodies diluted at a predetermined dilution of 1:500, and pH indicator pHrodo 2 Green AM and MitoSpy Orange at a final concentration of 100 nM for 20 minutes at 37°C. In another embodiment, Raji and Jurkat cells are stained in the same buffer for 15 minutes at 37°C and cooled in ice water for 20 minutes.

[0091] Regulating the immune response according to its natural time course

[0092] The regulatory mechanisms of the immune response, namely the control mechanisms of lymphocyte death, proliferation, function, migratory behavior, and metabolism, etc., may vary at different stages of the natural time course of the immune response. Therefore, in order to achieve effective treatment or prevention of immune diseases, one must specifically or preferentially target these mechanisms that act at the stage of the natural time course of the immune response in the individual's body when seeking medical services. Although it is not excluded that this strategy can be applied to other mechanisms of controlling the immune response, the present invention focuses on how to regulate lymphocyte pHi and thereby control lymphocyte death and / or proliferation by targeting the energy generation mechanism operating in lymphocytes at different stages of the natural time course of the immune response. Or, it can also be said that the present invention provides methods and compositions for directionally controlling lymphocyte energy metabolism based on lymphocyte pHi and MMP. In some embodiments, the treatment aims to reduce pHi to inhibit excessive immune responses, which are considered the root cause of immune diseases, including but not limited to allergic diseases, autoimmune diseases, and infectious diseases in which the immune response damages the structure and / or function of normal tissues or cells. In some embodiments, the treatment aims to increase the pHi of lymphocytes to promote lymphocyte survival and / or proliferation, thereby promoting the immune response to infection, cancer, or vaccines.

[0093] Some embodiments target lymphocytes at an early stage of the natural time course to reduce their pHi to inhibit an excessive immune response. At this stage, the lymphocytes are mainly N lymphocytes with high MMP, and there is a strong inverse relationship between MMP and pHi. Since the strong inverse relationship indicates that the oxidation of carbon from pyruvate or fatty acids is the main contributor to MMP, an effective strategy for early reduction of lymphocyte pHi is to increase the influx of pyruvate and / or fatty acids into the TCA cycle, further increasing MMP, which in turn drives the reduction of pHi to induce apoptosis. Thus, in some embodiments, increasing the influx of pyruvate into the TCA cycle can be achieved by administering to an individual a pyruvate dehydrogenase kinase inhibitor such as dichloroacetic acid (DCA), for example, by intraperitoneal injection or intravenous drip of a saline composition containing 11.01 nM to 11.01 M DCA into the individual at a dose of 0.01 ml to 10 ml / kg body weight. In some embodiments, increasing the influx of fatty acids into the TCA cycle can be achieved by administering to an individual a carnitine palmitoyltransferase-1 (CPT-1) activator (e.g., C75), for example, by administering to the individual a composition containing 1.1 nM to 1.1 M C75 in 5% DMSO and 95% saline at a dose of 0.01 ml to 100 ml / kg body weight.

[0094] Amino acid energy metabolism, particularly glutaminolysis and glycolysis as energy metabolism, also contributes to energy production at this stage, and may contribute more in the minority of P lymphocytes than in N lymphocytes, but even in P lymphocytes, it does not reach the level of changing the inverse relationship between MMP and pHi. It is expected that glycolysis itself does not increase protons in the cell, but reduces the cell's dependence on mitochondrial ATP production, thereby indirectly weakening the pressure of pHi decline in early lymphocytes. In contrast, it is expected that amino acid deamination, the output of deaminated carboxylic acids, and the participation of amino acid downstream metabolites in the malate-aspartate shuttle can remove protons and thus increase pHi. Therefore, inhibiting these pathways also helps to reduce pHi. Accordingly, in some embodiments, inhibitors of amino acid energy metabolism and / or glycolysis are used alone or in combination with energy metabolism regulators that increase the influx of pyruvate and / or fatty acids into the TCA cycle to reduce pHi. In some embodiments, a glutaminase inhibitor such as CB-839 is administered to an individual by intraperitoneal injection or intravenous infusion at a dose of 0.01 ml to 100 ml / kg body weight with a composition of 5% DMSO and 95% saline containing 11 nM to 11 μM CB-839 to inhibit glutaminolysis. In some embodiments, a lactate dehydrogenase inhibitor such as GSK2837808A (abbreviated as GSK) is administered to an individual by intraperitoneal injection or intravenous infusion with a composition of 5% DMSO and 95% saline containing 52.8 pM to 52.8 mM GSK at a dose of 0.01 ml to 100 ml / kg body weight to inhibit glycolysis. In some embodiments, a glutaminolysis inhibitor or other inhibitor of amino acid energy metabolism and a glycolysis inhibitor can be administered together to an individual. In some embodiments, an inhibitor of amino acid energy metabolism and / or glycolysis can be administered together with a promoter of the influx of pyruvate and / or fatty acids into the TCA cycle.

[0095] In some embodiments, the aim of treatment is to elevate the pHi of lymphocytes at an early stage of the natural time course of the immune response to promote the survival and proliferation of lymphocytes against infection or cancer. In some embodiments, pHi is elevated by promoting amino acid energy metabolism such as glutaminolysis and / or glycolysis. In some embodiments, the influx of pyruvate and / or fatty acids into the TCA cycle is restricted. In some examples, a combination of these two strategies is employed.

[0096] The intermediate stage of the natural time course of the immune response is marked by a transition to P lymphocytes with a positive correlation between MMP and pHi. Such a transitional state is expected to be well-suited for manipulation of lymphocyte metabolism. Importantly, the positive correlation between MMP and pHi indicates that catabolism of the amino acid carbon backbone in the TCA cycle (e.g., α-ketoglutarate derived from glutamine) has become the major source of lymphocyte MMP. Since the overall effect of breaking down amino acids for energy is expected to reduce protons in the cell, higher MMP no longer equals more proton accumulation. Thus, in some embodiments, the transition to P lymphocytes with a positive correlation between MMP and pHi is attenuated by exploiting inhibitors of amino acid energy metabolism, thereby efficiently achieving inhibition of excessive immune responses. In some embodiments, the inhibitor of amino acid energy metabolism is the glutaminase inhibitor CB-839. CB-839 is administered to an individual at a dose of 0.01 ml to 100 ml / kg body weight by intraperitoneal injection or intravenous infusion as a composition comprising 11 nM to 11 μM CB-839 in 5% DMSO and 95% saline to inhibit glutamine breakdown. In some embodiments, entry of the amino acid carbon backbone into the TCA cycle is restricted or blocked. To this end, in some embodiments, the α-ketoglutarate dehydrogenase inhibitor CIP-613 is administered to an individual at a dose of 0.01 ml to 100 ml / kg body weight as a composition comprising 0.11 nM to 0.11 M CIP-613 in 5% DMSO and 95% saline. Glycolysis is another way to generate ATP during the intermediate stage of the natural time course of the immune response. Thus, in some embodiments, a glycolysis inhibitor such as GSK is administered to an individual at a dose of 0.01 ml to 100 ml / kg body weight by intraperitoneal injection or intravenous infusion as a composition comprising 52.8 pM to 52.8 mM GSK, 5% DMSO and 95% saline to reduce pHi.

[0097] In addition, the influx of pyruvate and fatty acids into the TCA cycle will also continue to make a significant contribution to MMP. Thus, in some embodiments, promoters that utilize the influx of pyruvate and / or fatty acids into the TCA cycle can also effectively achieve the inhibition of excessive immune responses during the intermediate stage. However, unlike inhibiting amino acid energy metabolism, increasing the influx of pyruvate or fatty acids into the TCA does not attenuate the transition to P lymphocytes that have a positive correlation between MMP and pHi. In some embodiments, the promoter for the influx of pyruvate into the TCA cycle is DCA, and a physiological saline composition containing 11.01 nM to 11.01 M DCA is administered to an individual by intraperitoneal injection or intravenous drip at a dose of 0.01 ml to 10 ml / kg body weight. In some embodiments, the promoter for the influx of fatty acids into the TCA cycle is C75. C75 is administered to an individual by intraperitoneal injection or intravenous drip at a dose of 0.01 ml to 10 ml / kg body weight in a composition of 5% DMSO and 95% saline containing 1.1 nM to 1.1 M C75.

[0098] In some embodiments, various combinations of the above compounds can be administered to an individual to inhibit excessive immune responses. In some embodiments, stimulating amino acid energy metabolism such as glutaminolysis to promote the transition to P lymphocytes that have a positive correlation between MMP and pHi and / or elevating pHi to reduce apoptosis or promote lymphocyte survival, thereby enhancing the immune response to infection or cancer.

[0099] In the later stage of the natural time course of the immune response, most lymphocytes have been transformed into P lymphocytes that have a positive correlation between MMP and pHi. Thus, P lymphocytes will be the main target for manipulating the immune response at this stage. As described above, the positive correlation between MMP and pHi is expected to indicate that the oxidation of the amino acid carbon backbone is the main contributor to MMP or ATP in P lymphocytes. In addition, the oxidation of carbon derived from fatty acids, or rather, the influx of fatty acids into the TCA cycle, is also a major contributor to the production of MMP and ATP in P lymphocytes in the later stage.

[0100] Thus, in a later stage, in some embodiments, an inhibitor of amino acid energy metabolism and / or glycolysis (such as CB-839 and / or GSK) is administered to a subject with the above-described composition and dosage to achieve the purpose of inhibiting an excessive immune response. In some embodiments, inhibiting the immune response is achieved with an inhibitor of the entry of the amino acid carbon backbone and / or the influx of fatty acids into the TCA cycle. Without wishing to be bound by any particular theory, the data disclosed herein indicate that such treatment induces an imbalance between ATP synthesis and hydrolysis. In some embodiments, the inhibitor of the entry of the amino acid carbon backbone into the TCA cycle is CIP-613 and is administered to an individual with the above-described composition and dosage. In some embodiments, the inhibitor of the influx of fatty acids into the TCA cycle is Etomoxir (Etom). It is administered to an individual in the form of a composition of 10% DMSO and 90% saline containing 0.55 μM to 0.55 M Etom at a dosage of 0.01 to 100 ml / kg body weight. In some embodiments, particularly but not exclusively in cases where a majority of the minority N lymphocytes have a high MMP and there is a strong inverse relationship between MMP and pHi (such as in the late stage of e2 or e3), a promoter of the influx of pyruvate and / or fatty acids into the TCA cycle (such as DCA and C75) is administered to an individual to inhibit an excessive immune response. DCA and / or C75 are administered with the above-described composition and dosage. In some embodiments, various combinations of the energy metabolism regulators described in this paragraph are administered to an individual to inhibit an excessive immune response. In addition, the presence of late-stage small n lymphocytes indicates that ATP synthesis by mitochondrial respiration is insufficient to power the high proliferation rate of these cells. In the late e1 and e2 states, n lymphocytes remain highly responsive to treatment with a promoter of the influx of pyruvate and / or fatty acids into the TCA cycle, and thus their MMP, as well as the MMP of other lymphocytes, is increased by such treatment. Therefore, in some embodiments, although other cases are not excluded, in the case where n lymphocytes are detected, treatment with an inhibitor of amino acid energy metabolism and / or glycolysis, and / or treatment with an inhibitor of the entry of the amino acid carbon backbone and / or the influx of fatty acids into the TCA cycle, is combined with a promoter of the influx of pyruvate and / or fatty acids into the TCA cycle to inhibit an excessive immune response.

[0101] Reducing pHi for cancer treatment by a combination method

[0102] Metabolic plasticity provides cancer cells with an advantage for survival and growth. This also poses a great challenge to cancer therapy targeting a single metabolic pathway. Cancer cells can adopt alternative metabolic pathways to render such therapy ineffective. Even disregarding this, therapy targeting a single pathway requires near-complete blockade of that pathway to achieve high therapeutic efficacy. This would require very high doses of inhibitors because cancer cells typically elevate the expression of enzymes required for their preferred energy-producing pathways. Since most metabolic pathways are shared by cancer and normal cells, near-complete blockade of a pathway with high doses of inhibitors may lead to severe adverse side effects. The data disclosed in the present application indicate that there are multiple ways to reduce pHi to induce apoptosis. This provides an opportunity to reduce the pHi of cancer cells by incompletely modulating multiple pathways. While partial modulation of any single pathway may not be sufficient to induce apoptosis, partial modulation of multiple pathways can together reduce the pHi of cancer cells to a level sufficient to induce apoptosis. Since no single pathway is near-complete blocked or over-driven, this strategy is unlikely to cause adverse side effects yet can achieve high therapeutic efficacy.

[0103] Based on this hypothesis, the present invention discloses the establishment of a set of compositions, which includes a mixture (referred to as a combinatorial composition) consisting of a small number of various compounds that each regulate different metabolic pathways, and this set of compositions may or may not include the individual components that make up these combinatorial compositions. This set of compositions is screened according to the ability to inhibit the growth (increase in cell number) and / or survival of cancer cells required for treatment. This screening may or may not be combined with the screening for inhibiting the growth and / or survival of normal cells. Thus, compositions that show strong inhibitory effects on cancer cells while showing relatively weak or no inhibitory effects on normal cells (compared to the average value of all compositions and the vehicle-treated control) are screened out, and the screened compositions are provided in the amounts required by the recipient. In some embodiments, the normal cells used in this screening process are peripheral white blood cells, such as white blood cells from the peripheral blood of a subject or a healthy donor, which are stimulated with concanavalin A (ConA) or phytohemagglutinin (PHA) and / or lipopolysaccharide (LPS) with or without interleukin-2 and / or interleukin-4. In some embodiments, the normal cells are a mixture of allergic white blood cells. In some embodiments, the pathways targeted by this set of compositions are the influx of pyruvate into the TCA cycle, the influx of fatty acids into the TCA cycle, the entry of the carbon backbone of amino acids into the TCA cycle, amino acid energy metabolism, energy-producing glycolysis, etc. The data in the present invention demonstrate for the first time that manipulating these pathways alone or in combination can reduce the pHi of cancer cells to induce apoptosis. In some embodiments, the compounds targeting the above pathways and used to prepare the combinatorial composition are lactate dehydrogenase inhibitors, malate-aspartate shuttle inhibitors, glutaminase inhibitors, pyruvate dehydrogenase kinase inhibitors, α-ketoglutarate dehydrogenase inhibitors, carnitine palmitoyltransferase inhibitors, carnitine palmitoyltransferase activators. In some embodiments, the compositions for cancer treatment are compositions 8', 12', 15, 16', 19, and 21' listed in Table 1, and their respective components and exemplary concentrations that can be adjusted up and down are also provided in Table 1.

[0104] For convenient clinical application, the individual compounds and combinatorial compositions that make up the combinatorial composition, together with other necessary reagents, such as reagents for measuring cell viability, growth / proliferation, pHi, and MMP, as well as instructions for use, can be provided to healthcare professionals as a kit, with or without related equipment and laboratory tools.

[0105] Ki-67 as a predictive marker for the responsiveness of cancer treatment based on low pHi

[0106] For most cancers, biomarkers predictive of responsiveness to treatment remain elusive. Ki-67 is a reliable marker of proliferating cells, and its expression level is positively correlated with rRNA and DNA synthesis, but it is not a biomarker with a clear predictive ability for responsiveness to existing chemotherapeutic drugs. However, in the present invention, Ki-67 will be used as a biomarker to predict an individual's responsiveness to a new low-pHi-based cancer therapy. Low-pHi-based cancer therapy refers to a cancer therapy that induces cancer cell death by reducing the pHi of cancer cells. High levels of Ki-67 and / or a high percentage of Ki-67+ cancer cells predict a positive response to low-pHi-based cancer therapy. In some embodiments, the expression of Ki-67 is detected and measured in cancer cells that have been intracellularly stained for Ki-67 by flow cytometry. Cancer cells can also be stained with markers to help distinguish normal cells from cancer cells. In some embodiments, immunohistochemical staining of tumor tissue is used to detect Ki-67 and markers that identify tumor cells. In some embodiments, techniques such as real-time RT-PCR are used to measure the transcript of Ki-67. For ease of clinical application, all detection reagents and instructions can be provided to healthcare professionals as a kit.

[0107] Treatment of skin and mucosal infections with a pH regulator

[0108] Some skin and mucosal infections, such as fungal infections of the nails, are very difficult to treat. The present invention provides a solution to this problem. In some embodiments, microbial infections of the skin (including skin appendages such as nails) and mucosa are treated by topically applying a composition comprising a pH regulator that reduces the pH. Skin and mucosal infections include, but are not limited to, onychomycosis, tinea pedis, impetigo, cellulitis, gonorrhea, etc. In some embodiments, onychomycosis is treated by topically applying a pH regulator composition that reduces the pH of the infected area and the surrounding area. In some embodiments, the pH regulator that reduces the pH is HOAc, which is included in a composition of 0.01 to 100% HOAc by volume in water. In some embodiments, tinea pedis is treated by topically applying a composition comprising 0.01% to 100% HOAc by volume in water, and in some embodiments, the infected area is immersed in this composition diluted with water to treat the infection. In some embodiments, mucosal infections caused by known or unknown pathogens are treated by topically applying a composition of water comprising 0.01 to 100% HOAc by volume. The above compositions can be diluted according to an individual's preference.

[0109] Treatment of skin tumors with a pH regulator

[0110] Skin tumors (such as basal cell carcinoma) are typically treated by surgery and radiotherapy. These treatments not only put stress on the patient, but in cases where the tumor occurs on the facial skin, the patient may also be reluctant to undergo certain therapies, such as surgery. Here, the present invention provides an alternative that is substantially stress-free and has little impact on the patient's appearance. Thus, in some embodiments, skin tumor formation such as basal cell carcinoma is treated by topically applying a composition comprising a pH regulator that reduces the pH in an aqueous environment. In some examples, the pH regulator is HOAc, which is contained in a composition containing 0.01% to 100% HOAc by volume in water. The composition can be diluted according to the patient's preference. In some embodiments, the pH regulator can be injected subcutaneously to make it easier to reach the tumor cells. In some embodiments, a superficial incision is made at the affected area prior to topical application to facilitate delivery of the pH regulator to the tumor cells.

[0111] Specific examples

[0112] Materials and methods

[0113] Mice and disease animal models

[0114] Balb / c and C57BL / 6 mice were purchased from The Jackson Laboratory (Bar Harbor, ME, USA) and were housed and bred in the animal facilities of Therazwimm. Animal studies were conducted according to protocols approved by the Therazwimm Institutional Animal Care and Use Committee. Asthma induction and analysis were performed essentially as described previously (26). For sensitization, grade II ovalbumin (OVA) (Sigma-Aldrich, St. Louis, MO, USA) and alum adjuvant (Thermo Scientific, Rockford, IL, USA) were freshly mixed in PBS to make a sensitization solution containing 200 μg / ml OVA and 33.4% (v / v) alum. Adult mice received an intraperitoneal injection of 100 μl of the sensitization solution (20 μg OVA / mouse) on day 0. On day 13, each mouse was re-sensitized by an intraperitoneal injection of 500 μl of freshly prepared sensitization solution (100 μg OVA / mouse). Unless otherwise noted, on days 13 and 14 after the second sensitization, the mice were challenged with antigen by an intratracheal injection of OVA (100 μg / mouse) in 80 μl of saline. Mediastinal and parathymic lymph nodes draining the lungs (collectively referred to as mediastinal lymph nodes or MLN in this application) (27) were collected as draining lymph nodes (DLN), and cervical, facial, and / or inguinal lymph nodes were collected as non-draining lymph nodes (NDLN).

[0115] The method for inducing experimental autoimmune encephalomyelitis (EAE) in C57BL / 6 mice was as described previously (28). Myelin oligodendrocyte glycoprotein peptide MOG35-55 (Abbreviation: MOG) (G.L. Biochem, Shanghai, China), heat-killed Mycobacterium tuberculosis H37Ra (Difco Laboratories, Detroit, MI, USA), and incomplete Freund's adjuvant (IFA) (Difco Laboratories) were mixed in PBS to prepare an antigen emulsion. The final antigen emulsion contained 1.5 mg / ml of MOG, 189 μg / ml of Mycobacterium tuberculosis H37Ra, and 50% (by volume) of IFA. On day 0, mice were immunized by subcutaneous injection with the antigen emulsion (100 μl / site) at two sites, one in front of and one behind the dorsal area. Two hours later, the mice were injected intraperitoneally with pertussis toxin freshly prepared in ice-cold PBS (List Biological Laboratory, Campbell, CA, USA) (200 ng dissolved in 100 μl PBS / mouse). On day 1, the mice received a second intraperitoneal injection of the same dose of pertussis toxin. Starting on day 10, when no disease symptoms were yet present, the clinical symptoms of the mice were scored as previously described (28). Wet feed was provided to mice with a clinical score of 3 or higher.

[0116] Measurement of airway hyperresponsiveness (AHR)

[0117] The method for measuring AHR was as described previously (26). Briefly, 4 days after the last OVA challenge, individual mice were anesthetized and a small cannula was inserted into the trachea. The mice were mechanically ventilated using a Buxco Elan series RC instrument (200 breaths per minute; 0.2 ml tidal volume), and challenged by inhalation of an aerosolized methacholine solution (10 μl) of increasing concentration (6.25, 12.5, 25, 50 mg / ml, with each challenge lasting 3 minutes). The mice were treated with a composition of metabolic regulators

[0118] At the time specified in each experiment, the mice received an intraperitoneal injection of 200 μl of the composition listed in Table 1. For this purpose, the day on which the mice received the first antigen challenge in a round of experiments in which they were treated or from which tissues were obtained was designated as day 0. The mice were typically sacrificed 20 to 24 hours after the last injection (of the composition).

[0119] Culture of tumor cells

[0120] Jurkat and Raji tumor cell lines were cultured at 37 °C and 5% CO 2Maintained in complete RPMI-1640 medium (RPMI-1640 plus 1x GlutaMAX, 100 U / ml Pen-Strep (Gibco Life Technologies, Grand Island, NY, USA) and 5% heat-inactivated fetal bovine serum (FBS) (Atlanta Biologicals, Flowery Branch, GA, USA)). To analyze the effect of HOAc treatment on pHi, tumor cells (2 x 10 6 cells / ml) were incubated in FBS containing 10% saline or saline with 87.5 mM HOAc or NaOH in a 37 °C water bath for 20 minutes. To study the inhibition of tumor cell growth, cells in the exponential growth phase were collected and seeded at 2.5 x 10 5 cells / ml in complete RPMI-1640 medium, and the metabolic regulator compositions listed in Table 1 were added at a 1:10 dilution. After 2 days of culture, half of the medium was replaced with fresh medium with the same concentration of metabolic regulator. One day later, the viable cell count, pHi, MMP, and apoptosis of the cells were analyzed by flow cytometry.

[0121] In vitro culture and treatment of lymphocytes

[0122] Lymph node cells from non-immunized mice were cultured in flat-bottom 96-well plates at a concentration of 2 x 10 6 cells / ml in complete RPMI-1640 medium containing ConA (2.5 μg / ml), LPS (5 μg / ml), IL-2 (20 units / ml), and IL-4 (10 ng / ml). The compositions listed in Table 1 were added to the medium at a 1:10 dilution. On the 3rd day after stimulation, the cells were harvested and the viable cell count, pHi, MMP, and apoptosis were analyzed by flow cytometry.

[0123] Flow cytometry analysis

[0124] Fluorescent-conjugated antibodies against mouse CD4, CD8, CD19, Ki-67; Zombie-Green and Zombie-Violet fixable viability kits; MitoSpy Orange, fluorescent or biotin-conjugated Annexin V, 7AAD, fluorescent-conjugated streptavidin, and Foxp3 staining buffer sets were purchased from Biolegend (San Diego, CA, USA). The PE-conjugated anti-human Ki-67 antibody was purchased from eBioscience (San Diego, CA, USA). pHrodoTM Green AM, pHrodoTM Red AM, and PowerLoad were purchased from ThermoFisher Scientific (Waltham, MA).

[0125] For live cell counting, lymph node or tumor cells were prepared as single cell suspensions in the same volume of buffer. Aliquots of the same volume of cells from different samples were stained with 7AAD with or without additional staining. Cells were analyzed by flow cytometry with the same acquisition volume, flow rate, and acquisition time.

[0126] For lymphocyte staining of pHi, MMP, Annexin V, and surface markers CD4, CD8, and CD19, cells were washed once with serum-free Annexin V binding buffer and then incubated in 100 μl of Annexin V binding buffer or live cell imaging solution (LCIS) supplemented with 0.7 mM CaCl 2 (ThermoFisher Scientific, Waltham, MA) containing fluorescent-conjugated antibodies, Annexin V, pHrodoTM Green, and MitoSpy Orange diluted to the predetermined dilutions in a 37°C water bath for 20 minutes. After washing with ice-cold Annexin V binding buffer plus 1% FBS, cells were resuspended in 100 μl of Annexin V binding buffer or LCIS supplemented with Ca 2+ 7AAD was added prior to flow cytometry analysis. For Ki-67 staining, sorted live lymphocytes were fixed and permeabilized using the Foxp3 staining buffer kit and then stained with the fluorescent-conjugated anti-mouse Ki-67 antibody. (Biolegend, San Diego, CA, USA).

[0127] Flow cytometry data acquisition was performed using an AttuneTM cytometer (Invitrogen, Carlsbad, CA). Data analysis was performed using FlowJo X.

[0128] Intracellular ATP counting

[0129] Wash lymphocytes twice with cold normal PBS and freeze-thaw twice in 150 μl PBS to release intracellular ATP. Briefly centrifuge the lysate in a microcentrifuge at 10,000 rpm. Recover the supernatant, and 50 μl of it is used for luciferase assay using RealTime-Glo™ Extracellular ATP Assay Reagent (Promega, Madison, Wisconsin, USA).

[0130] Example A. Determining the natural time course of the immune response

[0131] In an asthma mouse model, after intratracheal challenge with OVA, the pH and MMP of lymphocytes in non-draining lymph nodes (NDLN) and mediastinal lymph nodes (MLN) draining the lungs were analyzed over time. Lymphocytes were divided into P and N populations, with high pHi and low pHi, respectively ( Figure 1A - 1C ). At the early stage, all lymphocytes, whether P or N populations, showed a negative correlation between MMP and pHi, which in turn was negatively correlated with the fluorescence intensity of the pHi indicator pHrodo TM Green. The percentage of the N population was lower compared to non-immunized mice (without OVA sensitization and challenge). In the same immunized mice, the N population in MLN was further reduced compared to that in NDLN. Nevertheless, the N population was the major lymphocyte population. Thus, the early stage was defined by the majority of the N population and the inverse relationship between MMP and pHi in all lymphocytes ( Figure 1A ).

[0132] Over time, the immune response in MLNs entered the mid-stage ( Figure 1B ). At this stage, lymphocytes in the P population showed a positive correlation between MMP and pHi. However, the N population may or may not still be the major lymphocyte population, and a large proportion of them had high MMP. It must also be noted that not all lymphocyte subsets entered the intermediate stage simultaneously. In the asthma model, at the time points analyzed, CD4 and CD8 T cells in MLNs had entered the mid-stage, while B cells were still in the early stage ( Figure 1B ). In the second mid-stage, T cells in the P population differentiated into two populations with relatively high and low MMP and pHi, respectively ( Figure 1B ).

[0133] After the mid-stage, the proportion of the N cell population further decreased, and the P cell population became the overwhelmingly dominant cell population. In such a late stage, the N cell population may continue to decline. The late-stage lymphocytes detected in the asthma model were in one of four different energy states, called late e1, e2, e3, and e4 ( Figure 1C)。The characteristics of the late e1 state are that a significant number of N lymphocytes have low MMP, and the correlation between MMP and pHi is weak or non-existent. These cells are called "n" cells. Nevertheless, there are still some N lymphocytes with medium levels of MMP, and there is an inverse relationship between their MMP and pHi. In the late e1 state, P lymphocytes show low to medium levels of MMP( Figure 1C )。In the late e2 state, n cells can still be detected in the N cell population, but they are not as prominent as in the late e1 state. Importantly, compared with the late e1 state, the MMP in both P and N lymphocytes has increased( Figure 1C )。In contrast, n cells are basically absent in the late e3 state, and P and N lymphocytes show a steep (strong) positive correlation and negative correlation between MMP and pHi, respectively( Figure 1C )。High and low MMP P lymphocytes can be detected in large numbers in both the late e2 and e3 states( Figure 1C )。In the late e4 state, almost all N lymphocytes have transformed into n cells, and few (if any) show a strong correlation between MMP and pHi. Similarly, the MMP levels of most P lymphocytes are comparable to those of n cells rather than being divided into different populations( Figure 1C )。

[0134] However, the immunological response characteristics based on lymphocyte pHi and MMP should not be understood to mean that every immunological response must go through all different stages. Instead, depending on the history and degree of exposure to environmental antigens, the immunological response may start at a stage after the above-mentioned early stages. For example, in a study of an EAE model, the lymphocytes of non-immunized littermate mice showed an MMP and pHi profile similar to that of the late e2 state described in the asthma model( Figure 1D )。This indicates that mice have an active ongoing immunological response before being immunized with a specific antigen. Nevertheless, after immunization, the N cell population of lymphocytes in the draining lymph nodes (DLN) gradually decreases, although they skip the early and middle stages( Figure 1D )。

[0135] Example B. Controlling the immunological response in the mid-stage of an asthma model

[0136] Although the dynamic changes of pHi and MMP in lymphocytes reflect the natural course of the immunological response, for practical purposes, one may not have to rely on the concept of the natural course of the immunological response, but rather on the situation of lymphocyte pHi and MMP to design treatment strategies. This different narrative should not be considered essentially different from the content disclosed in the present invention. Essentially, the content disclosed in the present invention emphasizes using the situation of lymphocyte pHi and MMP to guide the treatment of immune diseases.

[0137] In the intermediate stage, during the process of lymphocytes transforming into P lymphocytes where MMP is positively correlated with pHi. Two strategies were designed to reduce the pHi of lymphocytes so as to induce lymphocyte apoptosis and thus inhibit the immune response to the experimental allergen OVA. One strategy was to inhibit glycolysis or amino acid energy metabolism, such as glutaminolysis. Glycolysis was inhibited by intraperitoneal injection of the lactate dehydrogenase inhibitor GSK-2837808A (abbreviated as GSK), while glutaminolysis was inhibited by intraperitoneal injection of the glutaminase inhibitor CB-839. The other strategy was to increase the influx of pyruvate and / or fatty acids into the TCA cycle. The increased influx of pyruvate was achieved by intraperitoneal injection of the pyruvate dehydrogenase kinase inhibitor dichloroacetate (DCA). The increased influx of fatty acids was achieved by intraperitoneal injection of the carnitine palmitoyltransferase-1 (CPT-1) inhibitor C75. Neither of these two strategies had been proven to reduce pHi to induce lymphocyte apoptosis. As Figure 2A shown, both strategies were able to effectively reduce the pHi of lymphocytes in the MLN of mice sensitized and stimulated with OVA. One might speculate that these compounds regulate mitochondrial respiration, but it should be noted that not any compound that regulates mitochondrial respiration can have the same or similar effects (data not shown).

[0138] The effects of these compounds on the intensity of the immune response were evaluated by directly counting the number of live lymphocytes in the MLN and measuring the percentage of early apoptotic cells. The latter was used to represent apoptosis because the cell membrane leakage of apoptotic (dead) cells made their pHi unable to be measured. However, the percentage of early apoptotic cells might underestimate the effect of the compounds because dead cells were quickly cleared in vivo (29) and might disintegrate during the experimental procedures in vitro and thus could not be detected in such tests. Therefore, live cell counting was crucial and had to be considered when interpreting the early apoptosis data.

[0139] The effects of the compounds on the P and N populations are shown in Figure 2B and Figure 2B the same data in numerical form are also shown in Figure 2C In this study, at the respective doses used, the percentage and absolute number of live P lymphocytes were greatly reduced by the inhibition of glutaminolysis and also reduced by the inhibition of glycolysis but to a lesser extent( Figure 2C , upper and middle panels). Therefore, the transformation into P lymphocytes and their acquisition of the positive correlation between MMP and pHi largely depend on glutaminolysis. In the N population, the inhibition of glycolysis or glutaminolysis had only a small effect on CD4 T cells, but the inhibition of glutaminolysis greatly reduced the absolute number of live CD8 T and B (CD19+) cells. Treatment with DCA or C75 significantly reduced both P and N lymphocytes( Figure 2C, (in the middle figure). Early apoptotic cells are Figure 2D shown in Figure 2E and represented numerically in Figure 2D . Notably, early apoptotic cells were concentrated in cells with low pHi ( Figure 2E ). Although all compounds significantly reduced viable lymphocytes, an increase in early apoptotic cells was only detected in some treatment groups ( Figure 2C ), indicating that the assay underestimated the effect of the compounds. Increasing pyruvate influx into the TCA cycle significantly reduced the pHi of B cells in population N, which, even in the vehicle control, had a higher MMP than T cells. This could explain why increasing pyruvate influx into the TCA cycle led to a more significant reduction in B cells in population N than any other treatment (

[0140] Example C. Controlling the early immune response in an asthma model

[0141] Example B showed that, at the doses used, the glutaminolysis inhibitor CB-839 and the pyruvate influx enhancer DCA were the most effective in reducing the immune response to OVA stimulation. In this example, the effects of these two compounds on the early stage of the immune response were tested. Both compounds caused a significant decrease in pHi, resulting in an increase in the total number of viable lymphocytes and the percentage of N cell population in T and B cells ( Figure 3A ). Early apoptosis was again shown to be associated with low pHi ( Figure 3B ). CB-839 increased the percentage of early apoptotic cells in all lymphocyte populations, while DCA increased early apoptosis in T cells but not in B cells. The changes in early apoptosis were correlated with the changes in the pHi value of N lymphocytes ( Figure 3C ).

[0142] Example D. Controlling lymphocyte populations in the late e2 state in an asthma model

[0143] The first set of studies focused on the late e2 state because various lymphocyte subsets exist in this state, including lymphocytes with high MMP, where MMP is negatively or positively correlated with pHi, and the n lymphocyte subset ( Figure 1C ). In addition to DCA and C75, CPI-613 and Etomoxir (abbreviated as Etom) were also studied. CPI-613 is an inhibitor of α-ketoglutarate dehydrogenase (α-KGDH) and pyruvate dehydrogenase. However, since pyruvate can enter the TCA cycle through the pyruvate carboxylase bypass pathway, CPI-613 is expected to mainly block the entry of the carbon backbone of amino acids (such as glutamine) using α-ketoglutarate as the entry point into the TCA cycle. Etom is an inhibitor of CPT-1 and is therefore an inhibitor of fatty acid influx into the TCA cycle.

[0144] The effects of CPI-613 and Etom alone and in combination, and the combination of DCA and C75 on the P and N population distributions are shown in Figure 4A . Figure 4B The percentages of the P and N populations are shown. CPI-613 and Etom alone or in combination decreased the percentage and absolute number of all lymphocytes in the P population ( Figure 4B , upper and middle panels). This result illustrates the non-redundancy between the amino acid backbone and fatty acids for energy production in P lymphocytes. In contrast, these two compounds alone or in combination increased the percentage and absolute number of the N population. ( Figure 4B , upper and middle panels). Thus, a reduction in carbon oxidation of amino acids and / or fatty acids in the TCA cycle may have converted P lymphocytes into N lymphocytes, and P and N lymphocytes respond to this treatment in opposite ways.

[0145] In contrast, the combination of DCA and C75 showed a small decrease in P lymphocytes and a slight increase in CD8 T cells and B cells in the N population. When analyzing total live lymphocytes, T, and B cells, the combination of CPI-613 and Etom maximally reduced the numbers of CD4 and CD8 T cells, while all treatments reduced live B cells to a similar extent. A substantial reduction in the absolute numbers of live T cells and total lymphocytes was also observed in all other treatment groups. ( Figure 4B , lower panel). Since the combination of DCA and C75 increased the absolute number of N lymphocytes, the reduction in the total number of live lymphocytes caused by this combination may be the result of the reduction in P lymphocytes.

[0146] Further analysis of early apoptosis again showed the correlation between early apoptosis and low pHi ( Figure 4C ). These treatments increased the percentage of early apoptotic cells, and the degree of increase was consistent with the decrease in pHi in the N population ( Figure 4D ). The present invention also studied the effect of the GSK+CB-839 combination, which inhibits glycolysis and glutaminolysis, on lymphocyte populations in the late e2 state. This treatment decreased the percentage of P lymphocytes, with a greater decrease in P lymphocytes with low MMP than those with high MMP, while it increased the percentage of N lymphocytes, with a greater increase in N lymphocytes with low MMP than those with high MMP. On the other hand, the DCA+C75 combination treatment increased the MMP of P and N lymphocytes. ( Figure 4E ).

[0147] Example E. Decreasing pHi by ATP synthesis and hydrolysis imbalance

[0148] Lymphocytes in the MLN of the asthma model in the late e2 state were also used to study the relationship between intracellular ATP count and pHi, cell death, and proliferation, as they contain different lymphocyte subsets, including a high MMP subset with a strong correlation between MMP and pHi and a low MMP subset with a weak or no correlation between MMP and pHi (n subset within the N group) (see Figure 1C ). Lymphocytes treated with vehicle, Etom, CPI-613 + Etom, and DCA + C75 were frozen and thawed to release intracellular ATP, and ATP count was measured by luciferase activity dependent on ATP concentration. The results showed that treatment with Etom or Etom + CPI-613 decreased intracellular ATP, while treatment with DCA + C75 increased intracellular ATP( Figure 5A ). Therefore, the high percentage of early apoptotic cells in the MLN of mice treated with Etom and (Etom + CPI-613) Figure 4D ) may be due to the reduction in ATP production in these lymphocytes, which would lead to the rate of ATP hydrolysis exceeding the rate of ATP synthesis, thereby decreasing pHi and inducing apoptosis.

[0149] Further studies aimed to explore the complex relationship between intracellular ATP count and cell death and proliferation. To this end, MLN lymphocytes from mice untreated with compounds were divided into four populations, P4, P5, P6, and P7, based on their pHi and MMP profiles( Figure 5B left), where the P7 population corresponded to the Figure 1C n subpopulation. Although having a lower MMP, P4 lymphocytes had the highest intracellular ATP count( Figure 5B middle), indicating that P4 cells may use mechanisms other than mitochondrial respiration (such as glycolysis) to supplement the deficiency in mitochondrial energy production.

[0150] In contrast, P6 cells had a similar MMP to P4 cells but the lowest intracellular ATP count( Figure 5B middle). However, counterintuitively, P6 cells had the highest proliferation rate as judged by their having the highest level of Ki-67( Figure 5B right). These results suggest that P6 cells are unable to power their high proliferation rate due to insufficient ATP supply and are thus prone to apoptosis. This would shift the balance between ATP synthesis and hydrolysis towards hydrolysis, thereby decreasing pHi. This situation is further supported by the fact that increasing pyruvate and fatty acid influx (TCA cycle) by treatment with DCA + C75 increases intracellular ATP( Figure 5A ).

[0151] Example F. Controlling lymphocyte populations in the late e1 state of the asthma model

[0152] In this example, mice that received OVA sensitization and challenge were treated with compounds that enhance (DCA, C75) pyruvate or fatty acid influx into the TCA cycle or limit amino acid carbon backbone (CPI-613) or fatty acid (Etom) influx into the TCA cycle, or combinations of these compounds ( Figure 6A ). DCA, C75, DCA + C75, and Etom decreased the total number of live CD4 T cells in the MLN. Given that CD4 T cells are most relevant to the pathogenesis of asthma, these results suggest that these compositions may be clinically useful for treating asthma. C75 or C75 + DCA decreased the total number of live CD8 T cells, while DCA or CPI-613 moderately increased CD8 T cells. C75, CPI-613, or CPI-613 + Etom decreased the total number of live B cells ( Figure 6A ). More details and data regarding early apoptosis and differential effects on P and N lymphocytes are presented in Figure 6A . Importantly, N lymphocytes in the e1 state are highly sensitive to DCA treatment that increases MMP and early apoptotic cells ( Figure 6B ).

[0153] Example G. Controlling lymphocyte populations in the late e3 state of the asthma model

[0154] Similar to the late e1 state but with further decreased potency, DCA + C75 caused a moderate reduction in the total number of live CD4, CD8 T cells, and B cells, with the strongest effect on CD4 T cells ( Figure 7 ). In contrast, treatment with the GSK + CB-839 combination that inhibits glycolysis and glutaminolysis reduced the total number of live CD4 and CD8 T cells to below 5% and 10% of the vehicle control, respectively, and B cells to below 14% ( Figure 7 ). This may be because the effect of C75 is opposite to that of Etom or DCA.

[0155] Example H. Controlling lymphocyte populations in the late e4 state of the asthma model

[0156] In the late e4 state, lymphocytes retained significant responsiveness to CB-839 but had mild responsiveness to GSK. CB-839 caused a substantial reduction in CD4 and CD8 T cells but not B cells. However, the combination of GSK + CB839 caused a moderate reduction in all lymphocyte populations ( Figure 8 ).

[0157] Example I. Controlling the immune response to two rounds of antigen challenge in the asthma model

[0158] In this example, OVA-sensitive mice received the first round of OVA stimulation and treatment with energy metabolism regulators. Twenty-eight days after the first OVA stimulation in the first round, the mice received the second round of OVA challenge and treatment with energy metabolism regulators. The day on which the mice received the first OVA stimulation in both rounds was defined as day 0. Three treatment regimens were employed: (a) On days 2, 3, and 4 of the first round, the mice received an intraperitoneal injection of the CPI-613+Etom combination, and in the second round of stimulation, the mice received the same treatment on days 3 and 4; (c) On days 4, 5, 6, 7, 8, 9, and 10 of the first round, an intraperitoneal injection of the GSK+CB-839 combination was given, and the same treatment was received on day 5 of the second round; (d) On days 2, 3, and 4 of the first round, the mice received an intraperitoneal injection of the DCA+C75 combination, and on days 4, 5, 6, 7, 8, 9, and 10, an intraperitoneal injection of the GSK+CB-839 combination was given, and in the second round, an intraperitoneal injection of the DCA+C75 combination was given on days 3 and 4, and an intraperitoneal injection of the GSK+CB-839 combination was given on day 5; in addition, one group of mice received an intraperitoneal injection of HOAc on days 2, 4, 6, 8, and 10 of the first round and on days 3 and 4 of the second round. On day 5 of the second round, airway hyperresponsiveness (AHR) (an important pathophysiological symptom of asthma) was measured in all compound-treated and vehicle-treated mice, and the MLNs were harvested for immunological analysis.

[0159] In the MLNs, all treatment regimens reduced CD4 T cells (thought to be most relevant to the pathogenesis of asthma) and B cells. All treatment regimens except regimen (a) also reduced CD8 T cells. ( Figure 9A ). In diseases such as asthma, the immune response to antigen stimulation is considered a key mechanism in the pathogenesis, and controlling this immune response is expected to relieve or cure the clinical symptoms of patients. Although it is not possible to measure all the clinical symptoms of the disease in a mouse model, as an example, the effect of the treatment on airway hyperresponsiveness (AHR) (an important pathophysiological symptom of asthma) was measured. Compared with the vehicle control, all treatment regimens reduced the AHR of the mice ( Figure 9B ).

[0160] Example J. Controlling the immune response in EAE

[0161] EAE is a mouse model of the human disease multiple sclerosis and is induced by subcutaneous immunization of mice with myelin oligodendrocyte glycoprotein (MOG) peptide. It is generally believed that the response of CD4 T cells to the MOG peptide is a key mechanism in the pathogenesis. In the first set of experiments in this example, as Figure 10AMice were treated with different modulators, and then the draining lymph nodes (DLN) and non-draining lymph nodes (NDLN) of the mice were analyzed. The pHi and MMP profiles showed that lymphocytes in the DLN were in the late e1 state, with a considerable number of N lymphocytes having low MMP and a weak negative correlation between MMP and pHi, but some N lymphocytes having high MMP and a strong negative correlation between MMP and pHi. In the NDLN, lymphocytes were in the intermediate stage, with N lymphocytes being the dominant population, but P lymphocytes were positively correlated with MMP and pHi( Figure 10A ). Treatment with GSK, CB-839, DCA, C75, or a combination of all 4 compounds increased the N cell population and decreased the total viable lymphocyte count and the pHi of key viable CD4 T cells. Consistent with the view that lower pHi induces apoptosis, the percentage of early apoptotic cells in CD4 T cells also increased in mice treated with the compounds( Figure 10B ). It is also worth noting that an increase in early apoptosis of CD4, CD8 T cells, and B cells was not observed in the NDLN of mice treated with GSK, CB-839, and a combination of all four compounds.( Figure 10B , bottom right). This finding suggests that such treatments are unlikely to cause non-specific depletion of lymphocytes, thereby impairing the patient's ability to respond to infection. In a separate experiment, mice were treated with a vehicle or a combination of GSK and CB-839, and the clinical symptoms of EAE were monitored. During the course of about 4 weeks after immunization, 75% of the vehicle control mice developed EAE, while none of the mice treated with the GSK+CB-839 combination developed the disease.

[0162] Example K. Combination methods for reducing pHi for cancer treatment

[0163] As described above, studies on normal lymphocytes have shown that energy metabolism can be manipulated in various ways to reduce pHi to induce apoptosis, namely by enhancing the influx of pyruvate and / or fatty acids into the TCA cycle; inhibiting amino acid energy metabolism such as glutaminolysis and / or glycolysis; triggering an imbalance between ATP synthesis and hydrolysis, etc. Therefore, the present invention hypothesizes that partial intervention in multiple energy metabolic pathways can jointly reduce the pHi of tumor cells to a level sufficient to induce apoptosis. Since this strategy does not completely block or over-drive any one pathway, it is unlikely to cause serious adverse side effects, while improving the therapeutic effect compared to strategies targeting only a single pathway. Based on this hypothesis, 9 compositions containing multiple compounds were designed, each compound targeting a different metabolic pathway, and these pathways have been shown herein to alter pHi. The components of these compositions and the combinations composed of their single components

[0164] They are numerically coded and summarized in Table 1. Assuming that the total blood volume of a mouse is about 2 ml, (30, 31), when injected into a mouse, the maximum blood concentration of a single compound will not exceed the published IC50. For the compositions labeled with an apostrophe ('), the maximum blood concentration of one of its components is expected not to exceed 1 / 10 of the IC50.

[0165] Table 1. Combinations composed of energy metabolism regulators (The concentrations in this table are the concentrations of the compounds in physiological saline. When used in mice, 200 μl of the composition is injected into each mouse; when used in cell culture, the composition is added to the medium at a 1:10 dilution.)

[0166]

[0167]

[0168]

[0169] These combinatorial compositions and the compositions composed of their individual components were screened according to their ability to inhibit the growth of human tumor cell lines Raji and Jurkat in the medium. Raji is a B-cell lymphoma cell line, and Jurkat is a T-cell leukemia cell line. The inhibitory effects of these compositions on the growth of normal lymphocytes activated by concanavalin A (ConA) and lipopolysaccharide (LPS) were also tested as an approximate indicator of the potential adverse effects of the treatment on an individual's ability to mount an immune response to infection. After culturing with the composition or vehicle control, 7AAD was used to distinguish live cells from dead cells and the tumor cells or lymphocytes were counted by flow cytometry. The results of the first screening showed that the combinatorial compositions 8', 19, 12', 16' and 21' strongly inhibited the growth of Raji cells, while 15, 8', 19' and 12' strongly inhibited the growth of Jurkat cells. In contrast, the individual components of these combinatorial compositions showed no or only slightly inhibitory effects on the tumor cells. For in vitro-activated normal lymphocytes, the combinatorial compositions 20' and 21' had a strong inhibitory effect, suggesting that these two compositions may cause potential adverse side effects. ( Figure 11A )。

[0170] Once the first screening result is obtained, the concentration or dosage of one or more components of the effective composition can be adjusted to enhance its efficacy. As an example of this strategy, the combined compositions 8', 12', and 16' effective against Raji and the combined composition 12' effective against Jurkat were further studied. As shown in Table 1, the concentration of components with an expected maximum plasma drug concentration not exceeding 1 / 10 of the IC50 (encoded with numbers and single apostrophes) was increased to three times the concentration in the original composition, and this change was denoted with triple apostrophes (”'). The results showed that the increase in the concentration of individual components in the composition enhanced the inhibitory potency of all combined compositions. Under the treatment of composition 16”', only a small number of Raji cells survived during the treatment. The individual components in these combined compositions again showed much less or no effect on inhibiting tumor cell growth.( Figure 11B )。

[0171] To demonstrate that the inhibition of tumor growth is associated with a decrease in pHi and the induction of apoptosis, the pHi of tumor cells after treatment with the composition was analyzed. In most cases, compositions composed of single components, except for compositions 14' and 14”', did not decrease or only slightly decreased the pHi of Raji cells, while the combined compositions significantly decreased the pHi. The same enhanced effect was also observed in Jurkat cells.( Figure 11C )。The early apoptosis of treated tumor cells was also analyzed. As mentioned before, early cell apoptosis or apoptosis assays tend to underestimate the effect of treatment on cell death because dead cells may disintegrate during in vitro experimental procedures. Nevertheless, compared with single-component treatment, the combined composition 16' increased the early apoptosis of Raji cells. However, the early cell apoptosis assay particularly underestimated the inhibitory effect of 16”' on Raji cells because although the results were lower than those of 16' or 14”', the number of live Raji cells after 16”' treatment was much less than that after 16' or 14”' treatment.

[0172] Example L. Ki-67 level is related to susceptibility to death caused by low pHi

[0173] For most cancers, biomarkers that can predict the response to treatment are still difficult to determine. Ki-67 is a reliable marker for proliferating cells, and its expression level is positively correlated with rRNA and DNA synthesis, but it is not a factor that can clearly predict the reactivity to existing chemotherapeutic drugs. Contrary to this view, this example shows that the Ki-67 level is in fact a clear predictor of the reactivity of Raji cells to low pHi.

[0174] Experimental findings showed that treating Raji cells with HOAc decreased the pHi of tumor cells( Figure 12 ,upper left), and low pHi induced apoptosis(Figure 12 , upper right). In further studies, Raji cells with high, medium, low, or near-negative (lo / neg) Ki-67 staining were treated with saline or saline plus HOAc ( Figure 12 , lower left). Raji cells with high levels of Ki-67 that survived HOAc treatment accounted for only about 10% of the viable Raji cells treated with saline. In contrast, Raji cells with medium / low or near-negative expression of Ki-67 that survived HOAc treatment were approximately 60% of those treated with saline. ( Figure 12 , upper right). Thus, high levels of Ki-67 may be an excellent predictive biomarker for cancer treatment based on low pHi.

[0175] Example M. Treatment of skin and mucosal infections based on low pH

[0176] Toe fungal infection occurred on the right big toe of an individual and was diagnosed as onychomycosis ( Figure 13A ). Treatment with over-the-counter medications initially showed no improvement. Subsequently, treatment with a prescription compound ketoconazole 2% cream once daily for 1.5 months also showed no significant improvement. Then ketoconazole treatment was stopped, and the individual started self-treatment by topically applying an aqueous solution of HOAc to the infected toenail and the surrounding area. After polishing the toenail to remove excess toenail material in the infected area, 12% HOAc was applied twice a day, in the morning and before bedtime, for the first 3 days. Thereafter, 6% HOAc was applied once in the morning and 3% HOAc was applied once before bedtime daily. After about 2 weeks of treatment, significant improvement was observed, but the treatment continued for a total of 1.5 months. This therapy inhibited fungal growth and greatly improved the appearance of the toenail. ( Figure 13B ). To treat mucosal infection, there were two infected lesions on the inner side of the upper lip of the individual due to accidental biting. The individual self-treated the lesions by topically applying a 5% aqueous solution of HOAc 4 times a day (approximately every 4 hours). After two days of treatment, both lesions disappeared. ( Figure 13C ).

[0177] Example N. Treatment of skin tumors with pH regulators

[0178] Two blemishes on an individual's face were clinically diagnosed as benign tumors. The subject self-treated the tumor lesions by topically applying a 12.5% aqueous solution of HOAc 3 times a day (in the morning, at noon, and before bedtime) for 3 days, and then topically applying a 5% aqueous solution of HOAc 3 times a day (in the morning, at noon, and before bedtime) for 17 days. At the end of the treatment, both tumor lesions became negligible. ( Figure 14 ).

[0179] References

[0180] 1. p 447, Janeway’s Immunobiology, 8th ed. Garland Science, New York.

[0181] 2. Biochem Biophys Res Commun 313:459 - 65.

[0182] 3. Semin Cancer Biol 15:300 - 8.

[0183] 4. Medicina(Kaunas) 58.

[0184] 5. Cancer Res 73:1524 - 35.

[0185] 6. Front Immunol 8:184.

[0186] 7. Eur J Immunol 31:2829 - 38.

[0187] 8. Nature 513:559 - 63.

[0188] 9. J Immunother 23:196 - 207.

[0189] 10. Blood 53:1133 - 9.

[0190] 11. p 621 - 623, Lehninger Principles of Biochemistry. W.H. Freeman and Company, New York.

[0191] 12. Proc Natl Acad Sci U S A 104:19345 - 50.

[0192] 13. Biochem Biophys Res Commun 186:782 - 9.

[0193] 14. Proc Natl Acad Sci U S A 93:654 - 8.

[0194] 15. J Biol Chem 275:9244 - 50.

[0195] 16. Nat Cell Biol 2:318 - 25.

[0196] 17. Nat Rev Compound Discov 21:141 - 162.

[0197] 18. Cell 163:712 - 23.

[0198] 19. Front Oncol 12:960317.

[0199] 20. Oncotarget 6:11735 - 50.

[0200] 21. Curr Opin Immunol 14:354 - 9.

[0201] 22. Cell Death Differ 25:46 - 55.

[0202] 23. Nature 261:702 - 705.

[0203] 24. Immunol Rev 249:43 - 58.

[0204] 25. Cell 168:960 - 976.

[0205] 26. J Allergy Clin Immunol 139:508 - 518e4.

[0206] 27. Lab Anim 21:31 - 4.

[0207] 28. J Immunol 183:3583 - 90.

[0208] 29. Annu Rev Immunol 36:489 - 517.

[0209] 30. p 372. In Harkness J, Wagner J (ed), The biology and medicine of rabbits and rodents, 3rd ed. Lea&Febiger, Philadelphia.

[0210] 31. Mitruka B, Rawnsley H. 1981. Clinical, biochemical and hematological reference values in normal experimental animals and normal humans. Masson Publishing, New York.

Claims

1. A method or composition for controlling an immune response, wherein the method or composition controls the immune response by targeting the immunoregulatory mechanisms operating in lymphocytes at different stages of the natural time course of the immune response, wherein the natural time course of the immune response is guided and determined by the conditions of the intracellular pH (pHi) and mitochondrial membrane potential (MMP) of the lymphocytes.

2. The method or composition of claim 1, wherein the immunomodulatory mechanism comprises an energy metabolism mechanism that affects the pHi of lymphocytes.

3. The method or composition of claim 1 or 2, wherein the stage in the natural time course of the immune response is determined by dividing lymphocytes into relatively high and low pHi populations, referred to as P and N lymphocytes or populations, respectively, and further by the pHi and MMP profiles of the P and N lymphocytes, thereby Early on, it is determined by the predominance of N lymphocytes, and all lymphocytes show an inverse relationship between MMP and pHi; The middle stage is determined by the appearance of P lymphocytes with a positive correlation between MMP and pHi, and N lymphocytes may or may not still be the majority lymphocytes; The late phase is determined by the predominance of P lymphocytes with a positive correlation between MMP and pHi, and in the late phase lymphocytes can be in one of four different energy states: The e1 state is characterized by the presence of a significant number of cells referred to herein as "n lymphocytes", which are N lymphocytes with low MMP and a weak or no inverse relationship between MMP and pHi, and the remaining N and P lymphocytes also have relatively low MMP; The e2 state is characterized by the presence of a small number of n lymphocytes, and overall the remaining lymphocytes have higher MMP than the lymphocytes in the e1 state; The e3 state is characterized by the absence of n lymphocytes, with strong positive and negative correlations between MMP and pHi in P and N lymphocytes, respectively; The e4 state is characterized by the majority of n lymphocytes in the N population, with only a few, if any, N lymphocytes having high MMP and an inverse relationship between their MMP and pHi; Alternatively, criteria for determining the phase in the natural time course of an immune response are the differential patterns of molecules present or expressed in lymphocytes of different phases and energy states and / or the activity of such molecules as determined by the aforementioned methods.

4. The method or composition according to any one of claims 1 to 3, wherein the method or composition is used to treat and / or prevent immune diseases.

5. The method or composition according to any one of claims 1 to 4, wherein the method comprises lowering pHi by regulating the energy metabolism of cells to induce cell apoptosis or promoting cell survival and proliferation by increasing pHi to achieve the purpose of treating or preventing diseases, thereby, Lowering pHi is achieved by increasing the influx of pyruvate and / or fatty acids into the TCA cycle, inhibiting amino acid energy metabolism, inhibiting energy-generating glycolysis, inducing an imbalance between ATP synthesis and hydrolysis by limiting the influx of amino acid carbon backbones and / or fatty acids into the TCA cycle, or various combinations of the above pathways; Elevating pHi is achieved by enhancing amino acid energy metabolism and / or energy-producing glycolysis, restricting the influx of pyruvate and / or fatty acids into the TCA cycle, restoring the balance between ATP synthesis and hydrolysis, or various combinations of the above pathways; and Regulating energy metabolism is achieved by administering a composition comprising an energy metabolism regulator.

6. The method or composition according to claim 4 or 5, wherein the immune disease is a disease in which the pathogenesis is partly or wholly attributable to an excessive immune response, including but not limited to autoimmune diseases, allergic diseases, and infectious diseases in which the immune response damages the structure or function of host tissues or organs, wherein the treatment comprises reducing the pHi of lymphocytes to induce apoptosis according to the natural time course of the immune response or the pHi and MMP conditions of lymphocytes or the relative proportion of P and N lymphocytes. Wherein, In the early stage, it is mainly achieved by increasing the influx of pyruvate and / or fatty acids into the TCA cycle, selectively inhibiting amino acid energy metabolism and / or glycolytic energy metabolism, or various combinations of these pathways; In the middle stage, it is mainly achieved by inhibiting amino acid energy metabolism, increasing the influx of pyruvate and / or fatty acids into the TCA cycle, selectively inducing an imbalance between ATP synthesis and hydrolysis by inhibiting glycolytic energy metabolism, restricting the entry of amino acid carbon backbones and / or fatty acid influx into the TCA cycle, or various combinations of these pathways; In the late stage, it is mainly achieved by restricting the entry of amino acid carbon backbones and / or fatty acid influx into the TCA cycle to induce an imbalance between ATP synthesis and hydrolysis, inhibiting energy-producing glycolysis and / or amino acid energy metabolism, selectively increasing the influx of pyruvate and / or fatty acids into the TCA cycle, or various combinations of these pathways.

7. The method or composition according to claim 4 or 5, wherein the immune disease is a disease in which the pathogenesis is partly or completely attributable to an insufficient immune response to infection, vaccine, or cancer, and these diseases are treated or prevented by elevating the pHi of lymphocytes to promote the survival and proliferation of lymphocytes.

8. A method or composition for reducing intracellular pH (pHi) in a combined manner to induce apoptosis of tumor cells, wherein the method comprises: Constructing a set of compositions consisting of low-dose or low-concentration single energy metabolism regulators and various combined compositions formed by mixing these single energy metabolism regulators to reduce the pHi of tumor cells; and Screening this set of compositions according to the ability to inhibit the growth and / or survival of tumor cells; and simultaneously with or without screening whether this set of compositions inhibits the growth or survival of normal cells, thereby selecting a composition that exhibits a relatively strong inhibitory effect on tumor cells, taking into account or not taking into account the relatively weak or no inhibitory effect of the composition on normal cells, and further adjusting the dose of one or more energy metabolism regulators in the selected composition to achieve optimal inhibition of tumor cells while minimizing inhibition of normal cells.

9. The method or composition according to claim 8, wherein the energy metabolism regulator used to construct the composition group is selected from those energy metabolism regulators that can increase the influx of pyruvate and / or fatty acids into the TCA cycle, inhibit amino acid energy metabolism, inhibit energy-producing glycolysis, and cause an imbalance between ATP synthesis and hydrolysis by restricting the entry of the amino acid carbon backbone into and / or the influx of fatty acids into the TCA cycle.

10. The method or composition according to claim 8 or 9, wherein the composition group comprises the composition summarized in Table 1 of this application and a composition composed of a metabolic regulator having similar or the same activity.

11. The method or composition according to claim 10, wherein the composition group comprising a composition with the same or similar energy metabolism regulator as the compositions 8', 12', 15, 16', 19, 21', 8''', 12''' and 16''' described in Table 1 and Example K is an anticancer compound.

12. A method of using Ki-67 as a biomarker to predict the responsiveness of an individual to cancer treatment based on low pHi, wherein the expression level of Ki-67 in cancer cells or the percentage of cancer cells expressing high levels of Ki-67 serves as a predictive indicator of whether the individual will respond to cancer treatment using a pH regulator that reduces pHi.

13. The method or composition according to any one of claims 1-4, wherein the method comprises an experimental technique for staining cell surface molecules, pHi, MMP, and annexin V in a single step.

14. A method or composition for treating skin or mucosal infections or neoplasia by topically applying a pH regulator that reduces pH.

15. A method or composition for treating or preventing diseases by modulating the energy metabolism of cells to lower pHi to induce apoptosis or raise pHi to promote cell survival and proliferation, wherein, lowering pHi is achieved by increasing the influx of pyruvate and / or fatty acids into the TCA cycle, inhibiting amino acid energy metabolism, inhibiting energy-producing glycolysis, inducing an imbalance between ATP synthesis and hydrolysis by restricting the entry of the amino acid carbon backbone into and / or the influx of fatty acids into the TCA cycle, or various combinations of these pathways; raising pHi is achieved by enhancing amino acid energy metabolism and / or energy-producing glycolysis, restricting the influx of pyruvate and / or fatty acids into the TCA cycle, restoring the balance between ATP synthesis and hydrolysis, or various combinations of these pathways; and modulating energy metabolism is achieved by administering a composition comprising an energy metabolism regulator.

Images