Description:FIELD OF INVENTION
[0001] The present disclosure relates generally to the field of therapeutic combinations and compositions thereof. In particular, the present disclosure relates to therapeutic combinations and kits having Meclizine, methods, and applications thereof. 5
BACKGROUND OF INVENTION
[0002]
Infection with the human pathogen Mycobacterium tuberculosis (Mtb) constitutes one of the most complex inter-organismic interactions, characterized by remarkable phenotypic diversity within both the pathogen and host cells, such as macrophages. This heterogeneity manifests in various 10 forms, including differences in macrophage polarization states (M1/M2), metabolic pathways (glycolysis/fatty acid oxidation), and ontogenetic origins (alveolar macrophages/interstitial macrophages). Such phenotypic variability plays a pivotal role in the persistence of tuberculosis (TB), necessitating prolonged treatment regimens that often lead to patient non-compliance and 15 the emergence of drug-resistant strains.
[0003]
In vivo studies reveal that diverse functional states of macrophages create distinct cellular niches for Mtb, each influencing bacterial physiology and susceptibility to antimicrobial agents. Activation of macrophages via interferon-gamma (IFNγ) induces phenotypic drug tolerance by shifting Mtb 20 from an actively proliferating state to a metabolically quiescent, non-replicating form. Genome-wide screening in Mtb-infected macrophages has identified key factors such as magnesium transporter (MMGT1) and lipid droplets that facilitate the transition of bacteria into a stressed, persistent state. Notably, nitric oxide (NO) produced by activated macrophages inhibits 25 bacterial respiration, Fe-S cluster homeostasis, and central carbon metabolism, thereby fostering a state of metabolic quiescence associated with phenotypic drug tolerance. This highlights that the growth rate of Mtb varies significantly among individual macrophages, correlated with pre-existing differences in inducible nitric oxide synthase (iNOS) activity. 30 2
[0004]
Furthermore, extreme forms of drug tolerance are observed in non-replicating Mtb populations within cavity caseum derived from infected rabbits. Clinically relevant mutations in genes governing carbon metabolism—such as glycerol (glpK), acetate (icl1), and propionate (prpR)—alongside toxin-antitoxin modules, induce fitness defects and confer multi-5 drug tolerance both within macrophages and in animal models, potentially compromising antibiotic efficacy in humans. While metabolic quiescence induced by stress is a frequent contributor to drug tolerance, actively replicating Mtb within macrophages also exhibit tolerance mechanisms. Animal studies demonstrate that both proliferating and non-replicating 10 bacterial subpopulations can resume growth following drug withdrawal, indicating that growth arrest alone does not fully account for drug tolerance. Recent investigations involving alveolar macrophages from pulmonary TB patients further corroborate the coexistence of metabolically active and quiescent bacterial subpopulations displaying multi-drug tolerance. These 15 insights underscore the necessity of dissecting host and bacterial determinants underlying drug tolerance, aiming to unravel the complexities behind the protracted nature of TB therapy.
[0005]
Therefore, there is a need in the art to develop a targeted drug to overcome drug tolerance emerged in Mtb inside macrophage to improve 20 treatment efficacy during infection.
SUMMARY OF THE INVENTION
[0006]
In a first aspect of the present disclosure, there is provided a pharmaceutical composition comprising: (a) Meclizine; and (b) at least one anti-tuberculosis drug. 25
[0007]
In a second aspect of the present disclosure, there is provided Meclizine for use in enhancing efficacy of at least one anti-tuberculosis drug.
[0008]
In a third aspect of the present disclosure, there is provided Meclizine for use in treating tuberculosis, or symptoms thereof, in a subject, wherein said Meclizine is in combination with at least one anti-tuberculosis drug. 30 3
[0009]
In a fourth aspect of the present disclosure, there is provided a combination comprising Meclizine and at least one anti-tuberculosis drug.
[0010]
In a fifth aspect of the present disclosure, there is provided a method for reprogramming metabolism in Mycobacterium infected macrophage, the method comprising: contacting macrophages with Meclizine. 5
[0011]
In a sixth aspect of the present disclosure, there is provided a method of treating tuberculosis in a subject, the method comprising: administering to the subject a combination of a therapeutically effective amount of Meclizine and at least one anti-tuberculosis drug; or a therapeutically effective amount of the pharmaceutical composition as disclosed herein. 10
[0012]
In a seventh aspect of the present disclosure, there is provided a kit for treating tuberculosis, comprising: Meclizine; and at least one anti-tuberculosis drug.
[0013]
These and other features, aspects, and advantages of the present subject matter will be better understood with reference to the following 15 detailed description and appended claims. This summary is provided to introduce a selection of concepts in a simplified form. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter. 20
BRIEF DESCRIPTION OF DRAWINGS
[0014]
Fig. 1 depicts A) Schematic showing flow sorting–coupled RNA sequencing of macrophage subpopulations.; B) comparing infected and uninfected macrophages; C) PCA plot showing the subpopulations within the infected macrophages: ‘bystanders’, ‘oxidized’, ‘reduced’; D) Heat map of 25 genes differentially expressed between the subpopulations; genes clustered according to their involvement in pathways: E) Oxidative phosphorylation. F) Nrf2 regulon. G) Hippo signalling. Heatmaps are generated from three independent experiments with base mean >10, FDR < 0.1, and log2FC > 0.6, in accordance with an embodiment of the present disclosure. 30 4
[0015]
Fig. 2 depicts A) Workflow of the flow-sorting coupled seahorse extracellular flux analysis; B) and C) A modified mitostress test was performed to calculate mitochondrial parameters. BR- basal respiration, ATP- ATP production, H+-leak- proton leak; D) and E) ECAR test was performed to assess the parameters associated with glycolysis in the three sorted BMDM 5 subpopulations. Gly- glycolysis, GC- glycolytic capacity, NGA- non-glycolytic acidification; F) Mitochondrial ROS in Mtb-roGFP2 infected BMDMs. Antimycin A used as the positive control; G) Cellular ROS measured in Mtb-roGFP2 infected BMDMs, 100 μM menadione used as the control. MFI- median fluorescence intensity, a.u.-arbitrary units, in 10 accordance with an embodiment of the present disclosure.
[0016]
Fig. 3 depicts A) z-normalized RNA-Seq data of uninfected, bystanders, oxidized, and reduced populations where genes are clustered by expression levels. Only those genes are represented that satisfy two of the following criteria: belong to the Nrf2 pathway (red) and bear the Nrf2 binding 15 motif in the promoter region (green) or have an Nrf2 ChIP-seq (Chromatin immunoprecipitation followed by sequencing) peak detected near the promoter region (blue). Genes in black represent those that are significantly upregulated in the reduced subpopulation in comparison to the oxidized subpopulation (no gene upregulated in the oxidized subpopulation was 20 detected amongst the above genes); B) Mitochondrial ROS in BMDMs transfected either with scrambled siRNA (siSCR, yellow) or siRNA targeted against NRF2 (siNRF2, blue) at 24 h p.i. MFI- median fluorescence intensity, a.u.-arbitrary units; C) and D) Modified mitostress test of siSCR or siNRF2 BMDMs at 24 h p.i. infected with Mtb H37Rv at a moi of 2. BR- basal 25 respiration, ATP- ATP production, H+-leak- proton leak, nmOCR- non-mitochondrial respiration; E) Redox profile of Mtb-roGFP2 infected siSCR or siNRF2 BMDMs at 24 h p.i; F) Bar plot showing the percentage survival of Mtb in siSCR and siNRF2 BMDMs, treated with 3X MIC of INH (0.375 μg/ml) for 48 h. INH treatment was initiated at 24 h p.i. Percentage survival 30 is calculated compared to the untreated BMDMs; G) Mitochondrial ROS 5
assessed in infected BMDMs upon treatment SFN at the indicated concentrations at 24 h p.i.; H) Glycolytic function test showing the extracellular acidification rate (ECAR) upon treatment with 5 μM SFN at 24 h p.i.; I) Percentage distribution of redox-diverse fractions of Mtb-roGFP2 in BMDMs treated with the indicated concentrations of SFN at 24 h p.i.; J) 5 Antibiotic tolerance of intracellular Mtb assessed by CFUs. Percentage survival is compared to 0 h untreated group, in accordance with an embodiment of the present disclosure.
[0017]
Fig. 4 depicts A) Heat map showing expression of different subunits of the pyruvate dehydrogenase complex in the macrophage subpopulations: 10 uninfected (green), bystanders (grey), macrophages harboring EMSH-oxidized (yellow) or EMSH-reduced (blue) Mtb; B) Mechanism of action of UK5099, a mitochondrial pyruvate carrier (MPC) inhibitor; C) and D) Modified mitostress test of Mtb-infected BMDMs upon treatment with 10 μM UK5099 at 24 h p.i. BR- basal respiration, ATP- ATP production, H+-leak- proton 15 leak, nmOCR- non-mitochondrial respiration; E) Redox profile of intracellular Mtb in BMDMs 24h p.i. treated with indicated concentrations of UK5099; F) Antibiotic tolerance to 3X MIC of INH (MIC: 0.125 μg/ml) or moxifloxacin (MOXI; MIC: 0.25 μg/ml) with or without 10 μM UK5099. Percentage survival is compared to untreated cells at 0 h p.i.; G) Schematic 20 representation of cellular metabolic pathways in the presence of 10 mM glucose or 10 mM galactose as sole sugar sources; H) Experimental design to determine the effect of glucose and galactose on the redox poise and antibiotic tolerance of intracellular Mtb; I) to K) Extracellular flux analysis of Mtb-infected BMDMs at 24h p.i. to measure mitochondrial respiration (I), 25 glycolysis (J), and a ratio of OCR to ECAR (K); L) Redox profile of the intracellular Mtb in the presence of 10 mM glucose or 10 mM galactose as sole sugars at 24 h p.i.; M) Antibiotic tolerance to 3X MIC of INH in glucose- or galactose-containing medium. Percentage survival is compared to untreated cells after 48 h INH treatment. Data are expressed as mean ± S.D. 30 of three independent experiments. p-value determined using an unpaired t-
6
test with Welch’s correction. ns- non-significant, *p<0.05, **p<0.01, ***p<0.001, in accordance with an embodiment of the present disclosure.
[0018]
Fig. 5 depicts A) Bar plot showing the mitochondrial respiratory parameters determined by the modified mitostress test at 24 h p.i. upon treatment with 20 μM MEC or 0.2% DMSO (solvent control). BR- basal 5 respiration, ATP- ATP production, H+-leak- proton leak; B) Glycolytic stress test to assess the glycolytic levels in infected macrophages 24 h p.i. upon treatment with 20 μM MEC or 0.2% DMSO. Gly- glycolysis, GC- glycolytic capacity, NGA- non-glycolytic acidification; C) Mitochondrial ROS measured upon treatment with 20 μM MEC at 24 h p.i.; D) Mitochondrial 10 membrane polarization measured by JC-1 dye at 24 h p.i.. 50 μM carbonyl cyanide-p-trifluoromethoxy phenylhydrazone (FCCP) treatment used as positive control; UI: uninfected macrophages; E) Representative pseudo coloured 3D views of uninfected and mCherry expressing Mtb-infected BMDMs at 24 h p.i., with and without exposure to 20 μM Meclizine. 15 Mitochondria (amber), Mtb H37Rv (green), and nucleus (purple) are shown as surfaces. Plots show the surface area (μm2) and volume (μm3) of mitochondria in uninfected and infected BMDMs (n>500 from 6-8 cells per condition). Data are median with interquartile range. p-values calculated using a one-way ANOVA with Dunn’s multiple comparisons test. UI: 20 uninfected BMDMs, Inf: infected BMDMs; F) Oxidative phosphorylation gene expression between 0.2% DMSO-treated and MEC-treated BMDMs; G) net enrichment score (NES) calculated by gene set enrichment analysis (GSEA) for the differentially expressed genes between MEC- and DMSO-treated infected macrophages; H) Redox profile of the intracellular Mtb in the 25 presence of indicated concentrations of MEC at 24 h p.i.; I) Antibiotic tolerance in Mtb-BMDMs treated with 20 μM MEC and 0.5 mM 2-DG in the presence of 3X MIC of INH (0.375 μg/ml) or MOXI (0.75 μg/ml). Data are expressed as mean ± S.D. for three biological replicates done in triplicate. p-value determined using an unpaired t-test with Welch’s correction. Ns: non-30 7
significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, in accordance with an embodiment of the present disclosure.
[0019]
Fig. 6 depicts A) Strategy for investigating the efficacy of Meclizine (MEC) at reducing tolerance against INH in C3HeB/FeJ mice; B) Bacterial CFUs counted from lungs at the indicated time-points. N≥7 for the 10 weeks 5 p.i. timepoint. Data are expressed as mean ± S.D, and the p-value was determined by the Mann-Whitney test; C) Gross pathology of lungs of Mtb-infected mice at 10 weeks p.i across experimental groups; D) Hematoxylin and eosin–stained lung sections (after 6 weeks of treatment) from mice infected with Mtb for all experimental groups. Pathology sections show 10 granuloma (G), alveolar space (AS), collapsed parenchyma (CP), and necrotic area (N). All images were taken at 10X magnification; E) Granuloma score was calculated from the histopathological lung sections, F) AM and IM populations in the lungs of animals treated with Meclizine. For E. and F., data are expressed as mean ± S.D. and the p-value was determined by an unpaired 15 t-test with Welch’s correction. ns-non-significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, in accordance with an embodiment of the present disclosure.
[0020]
Fig. 7 depicts that Meclizine exhibits no adverse interaction with anti-TB drugs, wherein A) depicts three groups of treatment in BALB/c mice used 20 in the pharmacokinetic study: MEC alone, front-line anti-TB combination therapy (HREZ), and combination (MEC + HREZ); B)-F) depicts line plots indicate pharmacokinetic profiles of MEC and individual drugs of the anti-TB therapy regimen analysed individually and in the presence of each other in the plasma of animals. Differences were non-significant by the Mann-25 Whitney test (p > 0.05); G) depicts ratios of Cmax and AUClast of individual drugs or a combination with Meclizine to analyse drug-drug interactions. Doses used are the following: MEC, 25 mg/kg body weight, i.p.; H, 25 mg/kg body weight, p.o.; R, 10 mg/kg body weight, p.o.; E, 200 mg/kg body weight, p.o.; Z, 150 mg/kg body weight, orally; BDL, below the detection limit. All 30 data are means ± SD of concentrations at each time point of samples in 8
triplicates (n = 3 animals per group); and H) depicts Lung deposition of MEC alone and combined with anti-TB drugs at 6 h and 24 h post-intra-peritoneal administration of MEC, in accordance with an embodiment of the present disclosure.
DETAILED DESCRIPTION OF THE INVENTION 5
[0021]
Those skilled in the art will be aware that the present disclosure is subject to variations and modifications other than those specifically described. It is to be understood that the present disclosure includes all such variations and modifications. The disclosure also includes all such steps, features, compositions, and compounds referred to or indicated in this 10 specification, individually or collectively, and any and all combinations of any or more of such steps or features.
Definitions
[0022]
For convenience, before further description of the present disclosure, certain terms employed in the specification, and examples are delineated here. 15 These definitions should be read in light of the remainder of the disclosure and understood as by a person of skill in the art. The terms used herein have the meanings recognized and known to those of skill in the art, however, for convenience and completeness, particular terms and their meanings are set forth below. 20
[0023]
The articles “a”, “an” and “the” are used to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article.
[0024]
The terms “comprise” and “comprising” are used in the inclusive, open sense, meaning that additional elements may be included. It is not intended to be construed as “consists of only”. 25
[0025]
The term "at least one" is used to mean one or more and thus includes individual components as well as mixtures/combinations.
[0026]
The term “including” is used to mean “including but not limited to”. “including” and “including but not limited to” are used interchangeably. 9
[0027]
The term “pharmaceutical composition” as used herein, includes a combination of active pharmaceutical ingredients. The pharmaceutical compositions, according to embodiments herein, includes combinations wherein the active pharmaceutical ingredients may be administered simultaneously, separately, or sequentially. In an aspect of the present 5 disclosure, the pharmaceutical compositions includes combinations wherein the active pharmaceutical ingredients are meclizine and at least one anti-tuberculosis drug combined in a weight ratio range of 1:10 to 10:1.
[0028]
The term “Meclizine” refers to a compound having the name 1-((4-Chlorophenyl)(phenyl)methyl)-4-(3-methylbenzyl)piperazine, or derivatives 10 thereof. The term includes Meclizine base, pharmaceutically acceptable salts thereof including Meclizine HCl, 1- (4-Chlorobenzhydryl) -4- (3-methylbenzyl) -piperazine dihydrochloride, or a hydrate thereof including Meclizine 2HCl monohydrate. It is an antihistamine medication primarily used to prevent and treat nausea, vomiting, and dizziness caused by 15 motion sickness and vertigo. It belongs to the class of first-generation H1 histamine receptor antagonists and works by blocking the action of histamine in the brain, which helps reduce symptoms associated with motion-related disorders. In an aspect of present disclosure, Meclizine is used for modulating macrophage metabolism by affecting mitochondrial activity and cellular 20 bioenergetics, to influence macrophage polarization and immune responses.
[0029]
The term “anti-tuberculosis drug” refers to a medication specifically used to treat Tuberculosis (TB), a bacterial infection caused primarily by Mycobacterium tuberculosis (Mtb). These drugs work by targeting various aspects of the bacteria's cell structure, metabolism, or replication process to 25 eliminate the infection and prevent its spread. In an aspect of the present disclosure, anti-tuberculosis drug is selected from the group consisting of isoniazid, rifampicin, ethambutol, and pyrazinamide.
[0030]
The term "pharmaceutically acceptable" refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of 30 sound medical judgment, suitable for use in contact with the tissues of human 10
beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. Accordingly, the term “pharmaceutically acceptable carrier” refers to pharmaceutically acceptable substance or excipient that is used to facilitate the delivery of a drug. These carriers do not have any therapeutic effect 5 themselves. In an aspect of the present disclosure, pharmaceutically acceptable carrier comprises at least one of polymer, surfactant, solvent, or combinations thereof.
[0031]
The term “polymer” refers to large, chain-like molecule that is safe for use in pharmaceutical formulations and is utilized to deliver, stabilize, or 10 control the release of drugs within the body. These polymers are biocompatible, non-toxic, and non-immunogenic, which makes them suitable for applications such as drug delivery systems, controlled-release formulations, or as excipients in various pharmaceutical products. In an aspect of the present disclosure, the polymer is hydroxypropyl 15 methylcellulose.
[0032]
The term “surfactant” refers to a surface-active agent that reduces surface and interfacial tension between different phases, such as oil and water, thereby facilitating the emulsification, solubilization, or dispersion of drugs in pharmaceutical formulations. In an aspect of the present disclosure, the 20 surfactant is selected from polysorbate, polyethylene glycol, polyethoxylated castor oil, or combinations thereof.
[0033]
The term “solvent” refers to a liquid substance capable of dissolving or dispersing active pharmaceutical ingredients (APIs) in a pharmaceutical formulation, facilitating their administration and absorption in the body. 25 These solvents are non-toxic, safe for human use, and compatible with the drug formulation. In an aspect of the present disclosure, the solvent is dimethyl sulfoxide (DMSO).
[0034]
The term "therapeutically effective amount" refers to the quantity of a drug or therapeutic agent that is sufficient to produce a desired therapeutic 30 effect in a patient. This amount varies depending on factors such as the 11
condition being treated, the patient's age, weight, health status, and the specific characteristics of the drug. Essentially, it is the minimum dose needed to achieve the intended beneficial outcome without causing significant adverse effects.
[0035]
The term “macrophage” refers to a type of large, specialized white 5 blood cell that plays a key role in the body's immune system. Its primary functions include detecting, engulfing, and destroying pathogens such as bacteria, viruses, and fungi through a process called phagocytosis. Macrophages also help initiate and regulate immune responses by presenting antigens to other immune cells and releasing signalling molecules like 10 cytokines. They are found in various tissues throughout the body, where they contribute to immune defence, tissue repair, and clearance of cellular debris.
[0036]
The term “reprogramming” refers to the process of altering or modifying the metabolic pathways and functional state of macrophages to influence their behaviour and immune responses. This can involve shifting 15 macrophages from one metabolic profile to another—such as from a pro-inflammatory (M1-like) state, which relies heavily on glycolysis, to an anti-inflammatory (M2-like) state, which depends more on oxidative phosphorylation and fatty acid metabolism.
[0037]
The term “oxidative phosphorylation” refers to a metabolic process 20 that occurs in the mitochondria of cells, where energy from nutrients is used to produce adenosine triphosphate (ATP), the primary energy currency of the cell. During this process, electrons are transferred through a series of protein complexes known as the electron transport chain, which creates a proton gradient across the inner mitochondrial membrane. The flow of protons back 25 into the mitochondrial matrix through an enzyme called ATP synthase drives the synthesis of ATP from adenosine diphosphate (ADP) and inorganic phosphate.
[0038]
The term “glycolysis” refers to a metabolic pathway that breaks down glucose, a six-carbon sugar, into two molecules of pyruvate, a three-carbon 30 compound. This process occurs in the cytoplasm of cells and does not require 12
oxygen (it is anaerobic). During glycolysis, a small amount of energy is released and captured in the form of ATP and NADH, which are used as energy carriers.
[0039]
The term “tolerance” refers to the ability of Mtb (the bacteria that causes tuberculosis) to survive or withstand the effects of an anti-tuberculosis 5 drug, despite not necessarily being resistant in the strict sense. Specifically, drug tolerance means that the bacteria are less susceptible to being killed or inhibited by the medication, often leading to prolonged infection or treatment difficulty.
[0040]
The term “administering” refers to the act of giving or applying a 10 substance, such as Meclizine and at least one anti-tuberculosis drug, to a subject (e.g., a patient) in a controlled manner. It involves delivering these medications in a way that allows them to be absorbed into the body, such as by oral ingestion, injection, or other appropriate methods, with the goal of achieving their therapeutic effect against tuberculosis. 15
[0041]
The term “therapeutic dose” refers to the amount of a drug that is sufficient to produce the desired therapeutic effect or benefit in a patient without causing significant adverse effects. It is the dose that effectively treats or manages a condition, such as an infection or disease, while maintaining safety and minimizing toxicity. 20
[0042]
All percentages, parts and ratios are based upon the total weight of the compositions of the present disclosure unless otherwise indicated. Ratios, concentrations, amounts, and other numerical data may be presented herein in a range format. It is to be understood that such range format is used merely for convenience and brevity and should be interpreted flexibly to include not 25 only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. For example, a therapeutic dose in a range of 150 mg/kg to about 250 mg/kg per day should be interpreted to include not only the explicitly recited 30 limits of 150 mg/kg to about 250 mg/kg per day, but also to include sub-
13
ranges, such as 170 mg/kg to about 220 mg/kg per day, 180 mg/kg to about 210 mg/kg per day and so forth, as well as individual amounts, including fractional amounts, within the specified ranges, such as 175 mg/kg, 201 mg/kg for example.
[0043]
Unless defined otherwise, all technical and scientific terms used 5 herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the disclosure, the preferred methods and materials are now described. 10
[0044]
The present disclosure is not to be limited in scope by the specific embodiments described herein, which are intended for the purposes of exemplification only. Functionally equivalent products, compositions, and methods are clearly within the scope of the disclosure, as described herein.
[0045]
As discussed in the background, Mycobacterium tuberculosis (Mtb) 15 population exhibits the phenomenon of heterogeneity during infection, particularly how variations in the host macrophage environment contribute to redox diversity and consequently influence drug tolerance. A link between redox heterogeneity within replicating Mtb and differential susceptibility to anti-tuberculosis (TB) drugs was established, with certain bacterial 20 subpopulations displaying tolerance. However, the underlying mechanisms driving this heterogeneity remained unclear. Specifically, it was unknown which aspects of macrophage physiology or heterogeneity induce the redox diversification in Mtb and whether this heterogeneity is a cause or consequence of changes within the host cell environment. This unresolved 25 question hindered the development of strategies to effectively target drug-tolerant bacterial subpopulations, representing a significant obstacle in TB treatment efforts.
[0046]
Accordingly, the present disclosure provides a mechanistic dissection of how macrophage heterogeneity influences redox diversity in Mtb 30 populations during infection. By employing a combination of advanced 14
techniques such as redox biosensors, flow sorting, and RNA sequencing, the researchers identified that differences in gene expression among infected macrophages create metabolically distinct environments, which in turn lead to varied redox states and drug susceptibilities in Mtb. Notably, they uncovered that the transcriptional activity of the master regulator NRF2 5 orchestrates a cell-protective antioxidant program that modulates bioenergetic pathways, resulting in redox heterogeneity and drug tolerance. The present disclosure demonstrated that manipulating mitochondrial bioenergetics either genetically or pharmacologically can collapse this heterogeneity and reduce drug tolerance, both in cultured macrophages and 10 in mouse models. This offers a potential therapeutic strategy to target drug-tolerant Mtb subpopulations by reprogramming host cell metabolism to alter the redox environment within infected macrophages.
[0047]
In an embodiment of the present disclosure, there is provided a pharmaceutical composition comprising: (a) Meclizine; and (b) at least one 15 anti-tuberculosis drug.
[0048]
In an embodiment of the present disclosure, there is provided a pharmaceutical composition as disclosed herein, wherein the Meclizine and the at least one anti-tuberculosis drug are present in a weight ratio of from 1:10 to 10:1. In another embodiment of the present disclosure, the Meclizine 20 and the at least one anti-tuberculosis drug are present in a weight ratio of from 1.5:8.5 to 2:8. In yet another embodiment of the present disclosure, the Meclizine and the at least one anti-tuberculosis drug are present in a weight ratio of from 2.1:7.9 to 3:7.
[0049]
In an embodiment of the present disclosure, there is provided a 25 pharmaceutical composition as disclosed herein, wherein at least one anti-tuberculosis drug is selected from the group consisting of isoniazid, rifampicin, ethambutol, and pyrazinamide.
[0050]
In an embodiment of the present disclosure, there is provided a pharmaceutical composition as disclosed herein, wherein the composition 30 further comprises a pharmaceutically acceptable carrier. 15
[0051]
In an embodiment of the present disclosure, there is provided a pharmaceutical composition as disclosed herein, wherein the pharmaceutically acceptable carrier comprises at least one of polymer, surfactant, solvent or combinations thereof. In another embodiment of the present disclosure, the pharmaceutically acceptable carrier is solvent. 5
[0052]
In an embodiment of the present disclosure, there is provided a pharmaceutical composition as disclosed herein, wherein the polymer is hydroxypropyl methylcellulose, the surfactant is selected from polysorbate, polyethylene glycol, polyethoxylated castor oil or combinations thereof and the solvent is DMSO. In another embodiment of the present disclosure, the 10 polysorbate is Tween 80 and polyethoxylated castor oil is Cremophor EL.
[0053]
In an embodiment of the present disclosure, there is provided a pharmaceutical composition as disclosed herein, wherein at least one anti-tuberculosis drug is present in a therapeutically effective amount.
[0054]
In an embodiment of the present disclosure, there is provided 15 Meclizine for use in enhancing efficacy of at least one anti-tuberculosis drug.
[0055]
In an embodiment of the present disclosure, there is provided a use as disclosed herein, wherein the at least one anti-tuberculosis drug is selected from the group consisting of isoniazid, rifampicin, ethambutol, and pyrazinamide. 20
[0056]
In an embodiment of the present disclosure, there is provided a use as disclosed herein, wherein the Meclizine enhances efficacy of the at least one anti-tuberculosis drug by reprogramming macrophage metabolism.
[0057]
In an embodiment of the present disclosure, there is provided a use as disclosed herein, wherein the Meclizine shifts macrophage metabolism from 25 oxidative phosphorylation to glycolysis.
[0058]
In an embodiment of the present disclosure, there is provided a use as disclosed herein, wherein the Meclizine reduces tolerance of Mtb against the anti-tuberculosis drug.
[0059]
In an embodiment of the present disclosure, there is provided 30 Meclizine for use in treating Tuberculosis, or symptoms thereof, in a subject,
16
wherein said Meclizine is in combination with at least one anti-tuberculosis drug.
[0060]
In an embodiment of the present disclosure, there is provided a combination comprising Meclizine and at least one anti-tuberculosis drug.
[0061]
In an embodiment of the present disclosure, there is provided a 5 combination as disclosed herein, wherein the combination is for treating and/or alleviating Tuberculosis or symptoms thereof in a subject.
[0062]
In an embodiment of the present disclosure, there is provided a method for reprogramming metabolism in Mycobacterium infected macrophage, the method comprising: contacting macrophages with 10 Meclizine.
[0063]
In an embodiment of the present disclosure, there is provided a method as disclosed herein, wherein the method comprises contacting the macrophages with the pharmaceutical composition as disclosed herein.
[0064]
In an embodiment of the present disclosure, there is provided a 15 method as disclosed herein, wherein the reprogramming comprises shifting macrophage metabolism from oxidative phosphorylation to glycolysis.
[0065]
In an embodiment of the present disclosure, there is provided a method as disclosed herein, wherein the reprogramming reduces drug tolerance of Mtb in the macrophages. 20
[0066]
In an embodiment of the present disclosure, there is provided a method as disclosed herein, wherein the method enhances efficacy of at least one anti-tuberculosis drug against Mtb in the macrophages.
[0067]
In an embodiment of the present disclosure, there is provided a method of treating tuberculosis in a subject, the method comprising: 25 administering to the subject a combination of a therapeutically effective amount of Meclizine and at least one anti-tuberculosis drug; or a therapeutically effective amount of the pharmaceutical composition as disclosed herein. 17
[0068]
In an embodiment of the present disclosure, there is provided a method of treating tuberculosis as disclosed herein, wherein said method enhances efficacy of the at least one anti-tuberculosis drug against Mtb.
[0069]
In an embodiment of the present disclosure, there is provided a method of treating tuberculosis as disclosed herein, wherein said method 5 reduces drug tolerance of Mtb.
[0070]
In an embodiment of the present disclosure, there is provided a method of treating tuberculosis as disclosed herein, wherein said method reprograms macrophage metabolism in the subject.
[0071]
In an embodiment of the present disclosure, there is provided a 10 method of treating tuberculosis as disclosed herein, wherein the Meclizine and the at least one anti-tuberculosis drug are administered simultaneously, separately, or sequentially.
[0072]
In an embodiment of the present disclosure, there is provided a method of treating tuberculosis as disclosed herein, wherein the Meclizine is 15 administered at a dose of about 10 mg to about 100 mg/kg per day, preferably 25 mg to about 50 mg/kg per day.
[0073]
In an embodiment of the present disclosure, there is provided a method of treating tuberculosis as disclosed herein, wherein the at least one anti-tuberculosis drug is administered at a standard therapeutic dose. 20
[0074]
In an embodiment of the present disclosure, there is provided a method of treating tuberculosis as disclosed herein, wherein the standard therapeutic dose comprises: isoniazid at about 5 mg/kg to about 10 mg/kg per day; rifampicin at about 10 mg/kg to about 20 mg/kg per day; ethambutol at about 150 mg/kg to about 250 mg/kg per day; or pyrazinamide at about 150 25 mg/kg to about 250 mg/kg per day.
[0075]
Embodiments herein achieve compositions/combination and kits having Meclizine for use as immunomodulator in improving Tuberculosis therapy.
[0076]
Embodiments herein provide a kit for treating tuberculosis, for 30 reprogramming metabolism of macrophages infected with Mtb, for shifting
18
macrophage metabolism from oxidative phosphorylation to glycolysis, for reducing tolerance of Mtb against the anti-tuberculosis drug, for enhancing efficacy of at least one anti-tuberculosis drug, and/ or for treating and/or alleviating Tuberculosis or symptoms thereof in a subject.
[0077]
In an embodiment of the present disclosure, there is provided a kit 5 comprising: (a) Meclizine; and (b) at least one anti-tuberculosis drug. In an embodiment of the present disclosure, there is provided a kit as disclosed herein, wherein the Meclizine and the at least one anti-tuberculosis drug are present in a weight ratio of from 1:10 to 10:1. In another embodiment of the present disclosure, the Meclizine and the at least one anti-tuberculosis drug 10 are present in a weight ratio of from 1.5:8.5 to 2:8. In yet another embodiment of the present disclosure, the Meclizine and the at least one anti-tuberculosis drug are present in a weight ratio of from 2.1:7.9 to 3:7.
[0078]
In an embodiment of the present disclosure, there is provided a kit as disclosed herein, wherein the kit comprises at least one dose of the at least 15 one anti-tuberculosis drug, wherein the dose comprises: isoniazid at about 100 mg to 300 mg; rifampicin at 150 mg to 300 mg; ethambutol at 100 mg to 400 mg; or pyrazinamide at 150 mg to 400 mg. In an embodiment of the present disclosure, the kit comprises the at least one anti-tuberculosis drug in the form of a tablet. 20
[0079]
In an embodiment of the present disclosure, there is provided a kit as disclosed herein, wherein the kit comprises Meclizine at a dose of 25 mg to 100 mg. In an embodiment of the present disclosure, the kit comprises the Meclizine in the form of a tablet.
[0080]
Although the present disclosure has been described in considerable 25 detail with reference to certain embodiments and implementations thereof, other embodiments are possible to cover the modifications and variations of the present disclosure.
EXAMPLES 19
[0081]
The disclosure will now be illustrated with working examples, which is intended to illustrate the working of disclosure and not intended to take restrictively to imply any limitations on the scope of the present disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art 5 to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, the exemplary methods, devices, and materials are described herein. It is to be understood that this disclosure is not limited to particular methods, and experimental conditions described, as such 10 methods and conditions may apply.
Materials and Methods:
[0082]
For the purpose of the present disclosure, Meclizine, Isoniazid, Rifampicin, and Bone marrow-derived macrophages (BMDMs) were procured from Sigma Aldrich. 15
EXAMPLE 1
Transcriptional profiling of macrophage sub-populations
[0083]
A primary mouse bone marrow-derived macrophages (BMDMs) were infected with an Mtb strain carrying an established redox biosensor (Mtb-roGFP2) plasmid that reports EMSH (mycothiol redox potential) of Mtb. 20 Biosensor expression led to ratiometric changes in the fluorescence excitation at 405 and 488 nm with a uniform emission at 510 nm in response to redox changes in Mtb. Under oxidative and reductive conditions, the fluorescence ratio of 405/488 was observed to increase and decrease, respectively. Flow sorting pipeline that averages the median fluorescence ratio (405/488) of the 25 biosensor expressed by intraphagosomal Mtb-roGFP2 to gate and sort BMDMs into subsets enriched with either EMSH-reduced, EMSH-basal, or EMSH-oxidized bacteria was used. EMSH-reduced bacteria inside THP-1 macrophages were replicative and tolerant to isoniazid (INH) and rifampicin
20
(RIF), whereas EMSH-basal and -oxidized Mtb showed drug sensitivity. Using the biosensor-based gating strategy, BMDMs infected with Mtb-roGFP2 were sorted at 24 h post-infection (p.i,), treated them with anti-TB drugs (isoniazid, rifampicin, moxifloxacin, and bedaquiline at 3X the in vitro minimum inhibitory concentration (MIC)) for 48 h, and confirmed that the EMSH-5 reduced fraction is uniformly more tolerant to multiple anti-TB drugs than the EMSH-oxidized fraction. In subsequent experiments, BMDMs harboring EMSH-reduced or EMSH-oxidized bacteria were focussed, as these fractions represent drug-tolerant or drug-sensitive phenotypes of Mtb, respectively.
[0084]
Mtb-roGFP2-infected BMDMs 24 h p.i. were sorted and subjected to 10 RNA-sequencing (RNA-seq) (Fig. 1, wherein A) depicts schematic showing flow sorting–coupled RNA sequencing of macrophage subpopulations: uninfected (green), bystanders (grey), macrophages harboring EMSH-oxidized (yellow, here on labelled as ‘oxidized’) or EMSH-reduced (blue, here on labelled as ‘reduced’) Mtb) to acquire a mechanistic understanding of why 15 macrophage subsets harbor redox diverse Mtb populations. Four macrophage fractions were sorted for analysis by RNA-seq: BMDMs-predominantly harboring (i) EMSH-reduced bacilli and, (ii) EMSH-oxidized bacilli, (iii) bystander BMDMs, and (iv) uninfected (Fig. 1, wherein A) depicts schematic showing flow sorting–coupled RNA sequencing of macrophage 20 subpopulations). Differentially expressed genes were identified (DEG) [base mean > 10, fold change > 1.5, false discovery rate (FDR)< 0.1] between Mtb-infected BMDM subsets, bystanders, and uninfected BMDMs. Principal components analysis (PCA) showed that samples clustered with their biological replicates with clear segregation between uninfected and Mtb-25 infected BMDMs (Fig. 1, wherein B depicts principal component analysis (PCA) plot). An additional PCA on all infected BMDMs consistently revealed clear segregation of two populations of cells containing drug-tolerant (EMSH-reduced) or -sensitive (EMSH-oxidized) Mtb, indicating differences in the transcriptome signatures (Fig. 1, wherein C depicts PCA 30 plot). Compared to the uninfected control, the expression of 6460 and 6225 21
genes was affected in the BMDMs harboring EMSH-reduced and EMSH-oxidized populations, respectively (Fig. 1, wherein D depicts heat map). Direct comparison of transcriptomes revealed that 825 genes were differentially regulated between EMSH-oxidized and EMSH-reduced fractions (374 and 367 genes were upregulated in EMSH-oxidized and EMSH-reduced 5 fractions, respectively) (Fig. 1, wherein D depicts heat map). Expression of 6005 genes was deregulated between bystanders and uninfected BMDMs. The bystander transcriptome overlapped with both the infected subpopulations (fold change > 2.0, false discovery rate [FDR]< 0.1); the extent of overlap was ~ 2-fold more with the EMSH-oxidized fraction (160 vs. 10 84 genes). The uniquely overlapping genes between bystander BMDMs and EMSH-oxidized BMDMs were 212 compared to 99 for the bystander vs EMSH-reduced fractions. Consistent with this result, flow sorting of bystander BMDMs and subsequent infection with Mtb-roGFP2 uniformly shifted the EMSH of Mtb towards the oxidative state. 15
[0085]
The transcriptome of EMSH-oxidised and EMSH-reduced BMDMs overlapped considerably with that of previously reported transcriptional data of diverse macrophage populations infected with Mtb ex vivo and in vivo (BMDMs, AM, and IM). Overlap analysis between 825 DEGs of oxidized vs reduced fractions with the DEGs between Mtb-infected AM or IM showed a 20 marginally higher overlap of EMSH-oxidized BMDMs with IM (17%, Fisher exact test: p<0.0001) compared to the EMSH-reduced BMDMs (11%, Fisher exact test: p<0.0001). DEGs between oxidized and reduced fractions significantly overlapped with Mtb-infected AMs isolated from the bronchoalveolar lavage (BAL) fluid of mice during the early stages of 25 infection (day 10 p.i.), confirming the uniformity in the primary response of macrophages to infection ex vivo and in vivo. However, among the overlapping genes, the early transcriptome of bronchoalveolar lavage (BAL) fluid-isolated murine AMs showed higher similarity with BMDMs containing EMSH-reduced than EMSH-oxidized bacteria (47 vs 15 respectively), suggesting 30 the macrophages harboring EMSH-reduced Mtb are AM-like in their
22
transcriptional response. There was no significant overlap with the transcriptomes of M1 and M2 macrophages, which suggests that these macrophage subpopulations do not segregate along the M1-M2 axis.
[0086]
To understand the basis of drug tolerance exhibited by EMSH-reduced bacilli in BMDMs, pathway analysis using the gene ontology tool (ShinyGO 5 0.80) and pathway enrichment tool (Enrichr) was performed. It was found that genes associated with mitochondrial respiration (oxidative phosphorylation [OXPHOS]), central carbon metabolism (TCA cycle), Hippo signalling, and cell protective antioxidant responses (Nrf2- regulon) were induced more in BMDMs containing EMSH-reduced bacilli than in -oxidized 10 bacilli (Fig. 1, wherein D-G shows heat maps). NRF2 is an expert regulator of a cell-protective antioxidant transcriptional signature, including antioxidant production (Nqo1, Cat, Txnrd1), iron metabolism (Slc7a11, Slc7a2, Clec4e, Fbxl5), and cytoplasmic thiol production (Gsta3, Gclc, Gstm1 and Gstm2). These genes were induced in macrophages with EMSH-15 reduced fraction versus -oxidized fraction (Fig. 1, wherein F shows heat maps). Consistent with this, using a transcription factor enrichment tool (ChEA3), it was found that the genes upregulated in BMDMs harboring EMSH-reduced bacteria contained an Nrf2-binding motif. Enrichment of Nrf2-specific genes was further substantiated by a significant commonality 20 (enrichment FDR: 1.0E-05) with the genes downregulated in the transcriptome of lung tissues of Nrf2 knock out (Nrf2-/-) transgenic mice. Another transcription factor, Bach2, negatively regulates the antioxidant response by suppressing Nrf2 transcriptional activity. As expected, genes upregulated in lung macrophages of Bach2 knock out (Bach2-/-) mice 25 overlapped significantly with genes induced in BMDMs harboring EMSH-reduced bacteria (enrichment FDR: 3.0E-24). Ontology-based pathway enrichment further established the upregulation of antioxidant pathways in the macrophages harboring EMSH-reduced Mtb. The oxidized fraction of infected BMDMs showed enrichment of genes involved in cell cycle, DNA 30 23
damage response (base-excision, DNA mismatch repair), and complement activation pathways.
[0087]
Activation of the Nrf2 and hippo pathways, which respond to redox and energetic changes, along with upregulation of TCA cycle and OXPHOS genes in BMDMs containing EMSH-reduced bacteria, suggest that host energy 5 homeostasis could be an important physiological parameter required for mediating redox-dependent drug tolerance in Mtb populations.
EXAMPLE 2
Nrf2-dependent changes in mitochondrial bioenergetics
[0088]
Transcriptomic data suggest a role for mechanisms controlling host 10 redox balance and mitochondrial bioenergetics in supporting the emergence of a drug-tolerant EMSH-reduced population during infection. To clarify this link, BMDMs enriched with EMSH-reduced or EMSH-oxidized bacteria were sorted and applied an extracellular flux analyzer to measure oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) (Fig. 2, 15 wherein A) shows workflow). Mammalian cells rely on cytosolic glycolysis and mitochondrial oxidative phosphorylation (OXPHOS) to generate energy for cellular functions. The OCR quantifies OXPHOS, whereas ECAR quantifies acidification due to lactate export during glycolytic flux. Consistent with RNA-seq data, BMDMs harboring EMSH-reduced Mtb exhibited higher 20 basal respiration and ATP-linked OCR than EMSH-oxidized fraction (Fig. 2, B and C modified mitostress test). Conversely, glycolytic parameters, such as glucose metabolism and glycolytic capacities, were significantly higher in EMSH-oxidized BMDMs than in the EMSH-reduced fraction (Fig. 2, wherein D and E show ECAR test). 25
[0089]
The drop in mitochondrial OXPHOS in BMDMs containing EMSH-oxidized bacilli was accompanied by higher mitochondrial reactive oxygen species (mitoROS, a hallmark of mitochondrial stress) compared to BMDMs harboring EMSH-reduced bacteria (Fig. 2, wherein F) depicts mitochondrial 24
ROS). MitoROS is a significant contributor to cellular ROS. In agreement with this, the accumulation of cellular ROS was ~3-fold higher in BMDMs containing EMSH-oxidized Mtb than in the EMSH-reduced fraction (Fig. 2, G) Cellular ROS, wherein data are expressed as mean ± S.D representative of three independent experiments. p-value determined using an unpaired t-test 5 with Welch’s correction. ns- non-significant, *p<0.05, **p<0.01). The generation of ROS was dependent on the electron transport chain (ETC) during forward electron transport (FET), as well as when electrons flow backwards (reverse electron transport or RET) through complex I. To establish the source of ROS (FET or RET) within the ETC of BMDMs, 10 mitoROS in BMDMs treated with rotenone was measured. In FET, rotenone facilitates ROS generation at complex I. In contrast, rotenone reduces ROS by blocking complex I during RET. The addition of rotenone uniformly reduces mitoROS in all the subsets of BMDMs infected with Mtb, consistent with earlier reports of RET as the primary contributor of mitoROS during Mtb 15 infection. Consistent with the RNA-seq data, the bystander BMDMs exhibited respiratory and redox changes similar to BMDMs containing EMSH-oxidized Mtb (Fig. 2). These data demonstrated that BMDMs containing drug-tolerant EMSH-reduced bacilli maintain mitochondrial function but restrained glycolytic flux, whereas the EMSH-oxidized fraction of BMDMs 20 experience glycolytic shift and mitochondrial stress.
[0090]
Nrf2 is a prominent factor that sustains the structural and functional integrity of mitochondria (e.g., ATP synthesis, membrane potential, mitoROS, and substrate availability) and that is overexpressed in alveolar macrophages during early stages of Mtb replication in mice. Since an Nrf2-25 driven transcriptional response was featured in RNA-Seq analysis, it was hypothesized that Nrf2 could link macrophage bioenergetics to redox diversity in Mtb. Supporting this, it was found that several genes upregulated in macrophages containing EMSH-reduced Mtb had an NRF2 ChIP-Seq peak, while none of the genes upregulated in the macrophages harboring EMSH-30 oxidized Mtb showed these peaks (Fig. 3, wherein A) depicts z-normalized
25
RNA-Seq data, wherein data are expressed as mean ± S.D. of three independent experiments. p-value determined using an unpaired t-test with Welch’s correction. ns- non-significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). On this basis, expression of Nrf2 in BMDMs was reduced using siRNA (siNRF2-BMDMs), which resulted in a significant down-5 regulation of Nrf2 and its dependent genes. As expected, the knockdown of Nrf2 increased mitoROS more than the scrambled siRNA (siSCR-BMDMs) control upon Mtb infection (Fig. 3, wherein B) depicts Mitochondrial ROS). Next, the effect of Nrf2 knockdown on mitochondrial respiration, EMSH of Mtb, and INH tolerance was investigated. Basal OCR and ATP-linked OCR 10 were dramatically reduced in siNRF2-BMDMs compared to siSCR-BMDMs at 24 h p.i., indicating decreased mitochondria respiration (Fig. 3, C and D Modified mitostress test). Nrf2 knockdown did not increase glycolysis to compensate for the decreased mitochondrial respiration, which could be due to partial silencing of Nrf2. Diminished mitochondrial respiration and 15 enhanced mitoROS correlated with an increased fraction of siNRF2-BMDMs harboring EMSH-oxidized Mtb relative to siSCR-BMDMs 24 h p.i.(Fig. 3, wherein E) depicts redox profile). It was also determined whether the more significant fraction of siNrf2-BMDMs harboring EMSH-oxidized Mtb reduces INH tolerance: survival of Mtb was comparable in siNRF2 and siSCR 20 BMDMs at 72 h p.i. It was observed that Mtb growing in siNRF2 BMDMs were significantly more sensitive to INH than those in siSCR BMDMs (percentage survival in siNRF2: 19 % ± 1 %, siSCR: 39 % ± 3 %) (Fig. 3, F) Bar plot).
[0091]
As an additional verification of a functional linkage between Nrf2 and 25 mycobacterial redox, sorafenib, a pharmacological inhibitor of the Nrf2-Keap1 axis was used. Sorafenib attenuates the nuclear translocation of Nrf2 and suppresses the transcriptional expression of Nrf2-dependent antioxidant and respiratory genes, resulting in elevated mitochondrial stress. Consistent with this observation, BMDMs treated with non-toxic concentrations of 30 sorafenib (SFN, 2.5-5 μM) led to suppressed OCR, a compensatory increase 26
in glycolytic rate, and enhanced mitoROS (Fig. 3, wherein G-H depicts mitochondrial ROS- Glycolytic function test). Importantly, SFN treatment of BMDMs diminished redox heterogeneity in intra-phagosomal Mtb, with most of the BMDMs harboring bacteria in the EMSH-basal or -oxidized states (Fig. 3, wherein I) depicts percentage distribution of redox-diverse fractions). 5 Lastly, ~ 50% of Mtb survived INH treatment in BMDMs, whereas survival of Mtb reduced to 24% upon exposure to SFN (5 μM) with INH (Fig. 3, wherein J) depicts antibiotic tolerance of intracellular Mtb). It was confirmed that 5 μM of SFN did not affect Mtb survival in the 7H9 growth medium, macrophage viability and the MIC of SFN against Mtb is 20 μM, suggesting 10 that SFN lowers INH tolerance of intra-phagosomal Mtb by modulating host pathways. These data suggested that the differential induction of Nrf2 was one of the mechanisms that account for the observed bioenergetic differences in macrophages resulting in redox heterogeneity and drug tolerance of Mtb.
EXAMPLE 3 15
Metabolic activity of macrophages
[0092]
Data indicated a preferential engagement of mitochondrial OXPHOS by BMDMs harboring drug-tolerant EMSH-reduced bacteria, whereas drug-sensitive EMSH-oxidized Mtb resides in BMDMs committed to glycolysis. These observations motivated to determine whether shifting metabolic 20 reliance interchangeably between mitochondrial respiration and glycolysis modulates redox heterogeneity and drug tolerance in Mtb. To do this, first the energy source driving mitochondrial respiration and the emergence of drug-tolerant, EMSH-reduced Mtb in BMDMs were examined.
[0093]
Mitochondria utilize glucose, glutamine (Gln), and fatty acids to 25 generate ATP by OXPHOS. The preference of BMDMs harboring redox-diverse Mtb to use glucose, Gln, or fatty acids as energy sources in the mitochondrial stress test were assessed. To test the oxidation of endogenous fatty acids, BMDMs containing EMSH-reduced and EMSH-oxidized Mtb was flow-sorted. OCR after treatment with the inhibitor of fatty acid oxidation, 30 27
etomoxir (Eto) was measured. Both basal OCR and ATP-linked OCR were significantly reduced upon Eto treatment of BMDMs containing EMSH-reduced bacteria. However, Eto treatment also suppresses the OCR of BMDMs harboring EMSH-oxidized and bystander BMDMs. Moreover, Eto treatment did not affect the redox heterogeneity displayed by intra-5 phagosomal Mtb. Like Eto, trimetazidine, which inhibits the 3-keto acyl CoA thiolase step in the β-oxidation of fatty acids, had no influence on redox heterogeneity exhibited by intra-phagosomal Mtb. These findings agree with fatty acids being a principal energy source for Mtb-infected macrophages. However, the fatty acid-dependent changes in the metabolic state of infected 10 macrophages are unlikely to cause redox variations in the Mtb population.
[0094]
Inhibition of Gln oxidation by bis-2-(5-phenylacetamido-1,3,4-thiadia-zol-2-yl) ethyl sulphide (BPTES) did not affect the OCR of bystander or BMDMs containing redox diverse Mtb. As expected, BPTES treatment did not influence the redox heterogeneity of Mtb. Inspection of RNA-seq data 15 revealed enhanced expression of mitochondrially located pyruvate dehydrogenase complex (PDC) in BMDMs harboring EMSH-reduced compared to EMSH-oxidized fraction (Fig. 4, wherein A) depicts heat map). It was hypothesized that the EMSH-reduced fraction utilizes pyruvate for mitochondrial OXPHOS rather than lactic acid production. Fig. 2 (B and C 20 depicting modified mitostress test) demonstrating higher OCR and lower ECAR in EMSH-reduced fraction of infected BMDMs was consistent with a preferential glucose flux that supports mitochondrial respiration over glycolysis. Inhibition of mitochondrial oxidation of glucose upon treatment with UK5099, which blocks pyruvate transport from the cytoplasm into 25 mitochondria, resulted in a significant reduction in basal and ATP-linked OCR of BMDMs (Fig. 4, wherein B depicts mechanism of action of UK5099- C-D depicts modified mitostress test). Importantly, and in contrast to Eto and BPTES, UK5099-mediated inhibition of OCR correlated with a decrease in EMSH-reduced bacteria with a concomitant increase in EMSH-oxidized and 30 basal fractions in a concentration-dependent manner (Fig. 4, E) Redox 28
profile). Consistent with this, drug tolerance assays suggested that UK5099 uniformly weakens the ability of Mtb to tolerate INH and MOXI (Fig. 4, F) Antibiotic tolerance; survival in INH: 32% ± 3%, INH+UK5099: 18% ± 1%; MOXI: 52% ± 3%, MOXI+UK5099: 34% ± 4%).
[0095]
Efforts to confirm the contribution of pyruvate to redox-dependent 5 drug tolerance of Mtb was complicated by the fact that multiple mitochondrial enzymes (e.g., glutamate-pyruvate transaminase 2 or alanine aminotransferase 2, serine: pyruvate aminotransferase, L-cysteine to pyruvate conversion pathway) directly contributed to pyruvate flux for OXPHOS without the need for transporting cytosolic pyruvate. To circumvent this issue, 10 cellular energy metabolism was shifted by selectively culturing BMDMs on glucose or galactose as the sole sugar source. Production of pyruvate due to glycolysis yielded two net ATP, while pyruvate yield, via galactose metabolism, generates no net ATP. Therefore, using galactose as the sole sugar source coerces mammalian cells to generate ATP by utilizing pyruvate 15 to fuel mitochondrial OXPHOS (Fig. 4, wherein G) depicts schematic representation of cellular metabolic pathways and H) depicts experimental design). In agreement with this idea, when Mtb-infected BMDMs were grown in galactose, they exhibited a reduction in ECAR, reflecting decreased glycolysis and increased OCR, resulting in a ~5-fold increase in the 20 OCR/ECAR ratio compared to glucose as the carbon source (Fig. 4, wherein I-K depicts extracellular flux analysis). These data were consistent with a switch to pyruvate oxidation by mitochondria when BMDMs use galactose as an energy source.
[0096]
If a shift in metabolic reliance to galactose-linked mitochondrial 25 OXPHOS induces a reductive shift in the EMSH of Mtb, leading to increased drug tolerance was studied. Compared to glucose, culturing in galactose resulted in a higher fraction of BMDMs harboring EMSH-reduced Mtb (Fig. 4, wherein L) depicts redox profile), the increased fraction of EMSH-reduced Mtb resulted in 63% of bacteria surviving INH in galactose-grown conditions 30 compared to 30% in glucose-grown conditions (Fig. 4, wherein M) depicts
29
Antibiotic tolerance). Taken together, the data showed that inherent metabolic plasticity displayed by macrophages in sugar utilization promoted redox diversity in Mtb to tolerate antibiotic pressure. Overall, these results indicated that while both fatty acids and glucose support bioenergetics in infected BMDMs, the flux of glucose via pyruvate for OXPHOS is necessary to induce 5 a reductive shift in the EMSH of Mtb. In contrast, glycolysis likely plays a prominent role in maintaining bioenergetics in BMDMs containing EMSH-oxidized Mtb.
EXAMPLE 4
Reprogramming macrophage metabolism 10
[0097]
Given that macrophages’ intrinsic ability to shift their reliance on mitochondrial OXPHOS relative to glycolysis, thereby promoting redox-dependent drug tolerance, it was hypothesized that targeting such a shift could have the potential for treating TB. However, pharmacological agents that can safely redirect energy metabolism from OXPHOS to glycolysis without 15 compromising therapeutic value are limited in general and completely absent from the TB field. The deployment of a nutrient-sensitized screening strategy has identified an FDA-approved drug, Meclizine, which shifts cellular energy metabolism from mitochondrial respiration to glycolysis in a variety of mammalian cells. Meclizine was available without prescription for the 20 treatment of nausea and vomiting, crosses the blood-brain barrier, and previously has not been explored as a treatment for TB. Based on these findings, the mechanism and potential therapeutic utility of Meclizine in targeting redox-driven drug tolerance in Mtb was investigated.
[0098]
Whether Meclizine induces a reduction in OCR concomitant with an 25 increase in ECAR in Mtb-infected BMDMs was determined. Treatment with 20 μM of Meclizine reduced mitochondrial OCR and increased ECAR in Mtb-infected BMDMs (Fig. 5, wherein A) depicts bar plot showing the mitochondrial respiratory parameters, and B) depicts glycolytic stress test). Upon Meclizine treatment, it was also observed that mitoROS, and 30 mitochondrial depolarization increased (Fig. 5, wherein C) depicts 30
mitochondrial ROS and D) depicts mitochondrial membrane polarization). A change in cellular metabolism was often associated with mitochondrial remodelling. It was assessed whether Meclizine treatment affects the mitochondrial architecture by imaging mitochondria in Mtb-infected macrophages treated with Meclizine and observed that the surface area and 5 the volume of mitochondria reduce significantly upon treatment (Fig. 5, wherein E) illustrates representative pseudo coloured 3D views of uninfected and mCherry expressing Mtb-infected BMDMs). These findings suggested that mitochondrial fragmentation increased upon Meclizine treatment, which could lead to a metabolic shift from OXPHOS towards glycolysis. 10
[0099]
However, these changes in mitochondrial function by 20 μM Meclizine did not result in the killing of BMDMs infected with Mtb, likely due to the redirection of metabolism to glycolysis. To interrogate signalling pathways that could explain the metabolic switchover induced by Meclizine during infection, global RNA-seq of Mtb-infected BMDMs treated with 15 Meclizine and untreated control (treated with 0.2% dimethyl sulfoxide [DMSO]) for 24 h were performed. Mtb-infected BMDMs treated with Meclizine showed differential regulation of 1088 genes compared to the DMSO control (log2-fold change [FC] >0.6, false discovery rate [FDR] < 0.1). Remarkably, the transcriptional response of Meclizine-treated BMDMs 20 reversed the expression of pathways elevated in BMDMs harboring EMSH-reduced Mtb (Fig. 5, wherein F) depict oxidative phosphorylation gene expression and G) depicts net enrichment score (NES)). For example, Meclizine treatment suppressed the Nrf2 regulon, hippo signalling, and OXPHOS genes (Fig. 5). Down-regulation of a significant repertoire of genes 25 associated with OXPHOS explained the suppression of mitochondrial respiration in Mtb-infected BMDMs upon Meclizine treatment (Fig 5, wherein A) depicts bar plot showing the mitochondrial respiratory parameters and F) depicts oxidative phosphorylation gene expression). The lack of deregulation of glycolytic genes suggested that increased glycolysis 30 compensates for diminished mitochondrial respiration. Additionally,
31
Meclizine treatment downregulates the expression of the aminoacyl-tRNA biosynthesis pathway. One likely possibility is that inhibition of OXPHOS results in ATP limitation, which is critical for tRNA charging. Aminoacyl-tRNA synthetases (ARS) were involved in various cellular processes, such as immune and inflammatory response, angiogenesis, and apoptosis, which can 5 affect Mtb’s pathophysiology during infection. Data indicated that a Meclizine-induced metabolic shift was associated with significant changes in the expression of genes controlled by regulators of energy metabolism and redox stress, Nrf2 and HIPPO signalling.
EXAMPLE 5 10
Shifting of macrophage metabolism
[0100]
Whether Meclizine-mediated reshaping of macrophage metabolism reverses the drug tolerance and redox heterogeneity displayed by intraphagosomal Mtb were determined. Pre-treatment with Meclizine abolished the fraction of intra-phagosomal Mtb displaying reductive-EMSH in 15 a concentration-dependent manner (Fig. 5, wherein H) shows redox profile). Next, it was examined whether Meclizine reduced drug tolerance during infection. BMDMs with and without Meclizine were infected with Mtb for 24 h and exposed to 3X MIC of INH and Mox for an additional 48 h before lysis and enumeration of viable counts. It was found that the addition of Meclizine 20 reduced INH tolerance significantly (Fig. 5, I) showing antibiotic tolerance; INH: 39 % ± 4 %, INH+MEC: 17 % ± 2 %). A similar decrease in tolerance was observed upon substitution of INH with MOXI (MOXI: 36 % ± 7 %, MOXI+MEC: 13 % ± 4 %) (Fig. 5, I) showing antibiotic tolerance). To determine whether the increased ability of INH and MOXI to kill Mtb in 25 macrophages is linked to elevated glycolysis by Meclizine, glycolysis was poisoned using an inhibitor of the first hexokinase-mediated step in glycolysis, 2-deoxyglucose (2DG). Inhibition of glycolysis enhanced bacterial survival in BMDMs and abrogated the potentiating effect of Meclizine on lethality induced by INH and MOXI (Fig. 5, I) showing 30 antibiotic tolerance). The impact of Meclizine (20 μM) was mediated through 32
changes in macrophage metabolism, as extracellular Mtb remains viable even at extremely high Meclizine concentrations (320 μM). Findings implied that increased OXPHOS and suppressed glycolytic flux in Mtb-infected BMDMs induce drug tolerance, which Meclizine reverses.
[0101]
Next, Mtb’s response to INH in the presence of Meclizine in a murine 5 model of chronic infection was tested. Because the pathophysiology of human TB and tolerance to anti-TB drugs were closely recapitulated in C3HeB/FeJ mice, this mouse was aerosol-infected line with Mtb. At two weeks p.i, animals were treated with Meclizine for two weeks, followed by treatment with INH, Meclizine, INH plus Meclizine for an additional six weeks, and 10 then the lung bacillary load was measured (Fig. 6, wherein A) depicts strategy for investigating the efficacy of Meclizine (MEC) at reducing tolerance against INH). Meclizine dose (25 mg/kg/body weight) was based on mouse experiments conducted previously. As reported earlier, INH monotherapy reduced the bacterial burden from ~106 to ~104 per lung at six weeks 15 (p=0.0002) of treatment (Fig. 6, wherein B) depicts bacterial CFUs counted from lungs). Meclizine alone showed a marginal (~2.5-fold decrease) effect on bacterial viability over time (Fig. 6, wherein B) depicts bacterial CFUs counted from lungs). Relative to the control regimen (INH alone), the addition of Meclizine decreased lung CFU by ~20-fold after six weeks (p= 20 0.0001) of treatment (Fig. 6, wherein B) depicts bacterial CFUs counted from lungs). The gross and histopathological changes observed in the lungs after six weeks of therapy were correlated to the observed bacillary load (Fig. 6, wherein C) depicts gross pathology of lungs of Mtb-infected mice and D) depicts hematoxylin and eosin–stained lung sections). The extent of lung 25 damage was greater in the untreated (score = 37.5) and Meclizine-treated animals (score = 35), intermediate in INH-treated animals (score = 17.5), and lowest in the case of the Meclizine plus INH-treated animals (score = 2.5) (Fig 6, wherein E) depicts granuloma score).
[0102]
Since in vitro studies indicated that Meclizine reduces drug tolerance 30 of intracellular Mtb by enhancing glycolysis and suppressing OXPHOS, we 33
also asked whether Meclizine stimulates a similar effect in vivo. The proportion of alveolar (AM) and interstitial macrophages (IM) upon Meclizine treatment was examined. During lung infection in mice, Mtb resides in glycolytically active IMs and mitochondrially respiring AMs. Expectedly, treatment of infected mice with the glycolytic inhibitor 2-DG 5 decreased the number of glycolytically active IMs. In line with vitro findings, the proportion of glycolytically active IMs increased and AMs decreased at six weeks post-Meclizine treatment (Fig. 6, wherein F) depicts AM and IM populations in the lungs of animals treated with Meclizine). Together, these results confirmed that adjunct therapy with Meclizine counteracts drug 10 tolerance by reshaping the metabolism of infected macrophages.
EXAMPLE 6
Interaction of Meclizine with anti-TB drugs
[0103]
The pharmacological compatibility of Meclizine was assessed by measuring its interaction with clinically relevant, first-line anti-TB drugs 15 (isoniazid or H, rifampicin or R, ethambutol or E, and pyrazinamide or Z) given as a combination. A single-dose pharmacokinetic interaction was also performed by administering HREZ orally at the human equivalent doses with and without Meclizine (25 mg/kg/body weight, intraperitoneally [i.p]) in mice. An additional group of mice that was dosed with Meclizine (25 20 mg/kg/body weight, intraperitoneally [i.p]) was included to compare the PK profile of Meclizine in the presence of an HREZ combination (Fig. 7, wherein A) depicts the three groups of treatment used in the pharmacokinetic study). Plasma and lung homogenates were analysed for individual drugs using liquid chromatography-mass spectrometry, and parameters such as maximum 25 plasma concentration (Cmax) and area under the plasma concentration-time curve (AUClast) were quantified as a ratio for single-treatment groups vs. the combination (Fig. 7, wherein B)-F) depicts line plots indicate pharmacokinetic profiles). PK profiles revealed no to moderate drug-drug interaction when Meclizine was administered with HREZ. Cmax and AUClast 30 for Meclizine, when administered alone, were 0.31 ug/ml and 1.04 ug/ml, 34
respectively, which increased to 0.733 ug/ml and 2.36 ug/ml, respectively when administered along with HREZ. Similarly, Cmax and AUClast for H/R/E/Z administered with Meclizine were 5.25 ug/ml and 13.8 ug/ml*h for H, 8.8 ug/ml and 130.18 ug/ml*h for R, 1.92 ug/ml and 9.69 ug/ml*h for E and 63.1 ug/ml and 216.99 ug/ml*h for Z, respectively (Fig 7, wherein B)-F) 5 depicts line plots indicating pharmacokinetic profiles and G depicts ratios of Cmax and AUClast). The plasma PK profiles of HREZ remained unchanged in the presence of Meclizine. Comparative ratios of Cmax and AUClast for H, R, and Z with and without Meclizine were close to 1 except for E, which showed minor interaction (Cmax ratio: 1.41 and AUClast: 1.22) (Fig 7, wherein G) 10 depicts ratios of Cmax and AUClast). The average concentration of drug permeation in the lungs was determined at 6 and 24 h post-treatment, and the lung accumulation of HREZ in the presence or absence of Meclizine remained comparable (Fig 7, wherein H) depicts lung deposition of MEC alone and combined with anti-TB drugs). Similarly, HREZ did not affected 15 lung deposition of Meclizine at 6 and 24 h post-treatment (Fig 7, wherein H) depicts lung deposition of MEC alone and combined with anti-TB drugs). Overall, the PK results suggested no adverse interactions between the HREZ plus Meclizine combination vs. HREZ alone. Taken together, our study demonstrated an effect of Meclizine on drug tolerance, no significant drug-20 drug interaction with HREZ, and a potentiating effect of Meclizine on isoniazid.
ADVANTAGES OF THE PRESENT DISCLOSURE
[0104]
The present disclosure provides a synergistic composition for treating tuberculosis. It achieves reprogramming macrophage metabolism to reduce 25 drug tolerance in Mtb infection. The present disclosure archives a method of enhancing efficacy of anti-tuberculosis drug by using Meclizine in combination with the anti-tuberculosis drug. 35
I/We Claim:
1.
A pharmaceutical composition comprising:
a)
Meclizine; and
b)
at least one anti-tuberculosis drug.
2.
The pharmaceutical composition as claimed in claim 1, wherein the 5 Meclizine and the at least one anti-tuberculosis drug are present in a weight ratio range of 1:10 to 10:1.
3.
The pharmaceutical composition as claimed in claim 1, wherein the at least one anti-tuberculosis drug is selected from the group consisting of isoniazid, rifampicin, ethambutol, and pyrazinamide. 10
4.
The pharmaceutical composition as claimed in claim 1, wherein the composition further comprises a pharmaceutically acceptable carrier.
5.
The pharmaceutical composition as claimed in claim 4, wherein the pharmaceutically acceptable carrier comprises at least one of polymer, surfactant, solvent, or combinations thereof. 15
6.
The pharmaceutical composition as claimed in claim 5, wherein the polymer is hydroxypropyl methylcellulose, the surfactant is selected from polysorbate, polyethylene glycol, polyethoxylated castor oil, or combinations thereof, and the solvent is DMSO.
7.
The pharmaceutical composition as claimed in claim 1, wherein the at 20 least one anti-tuberculosis drug is present in a therapeutically effective amount.
8.
Meclizine for use in enhancing efficacy of at least one anti-tuberculosis drug.
9.
Meclizine for use as claimed in claim 8, wherein the at least one anti-25 tuberculosis drug is selected from the group consisting of isoniazid, rifampicin, ethambutol, and pyrazinamide.
10.
Meclizine for use as claimed in claim 8, wherein the Meclizine enhances efficacy of the at least one anti-tuberculosis drug by reprogramming macrophage metabolism. 30 36
11.
Meclizine for use as claimed in claim 8, wherein the Meclizine shifts macrophage metabolism from oxidative phosphorylation to glycolysis.
12.
Meclizine for use as claimed in claim 8, wherein the Meclizine reduces tolerance of Mtb against the anti-tuberculosis drug. 5
13.
Meclizine for use in treating tuberculosis, or symptoms thereof, in a subject, wherein said Meclizine is in combination with at least one anti-tuberculosis drug.
14.
A combination comprising Meclizine and at least one anti-tuberculosis drug. 10
15.
The combination as claimed in claim 14, wherein the combination is for treating and/or alleviating Tuberculosis or symptoms thereof in a subject.
16.
A method for reprogramming metabolism in Mycobacterium infected macrophage, the method comprising: 15
contacting macrophages with the pharmaceutical composition as claimed in claim 1.
17.
The method as claimed in claim 16, wherein the reprogramming comprises shifting macrophage metabolism from oxidative phosphorylation to glycolysis, wherein the reprogramming reduces 20 drug tolerance of Mtb in the macrophages, and/or wherein the method enhances efficacy of at least one anti-tuberculosis drug against Mtb in the macrophages.
18.
A method of treating tuberculosis in a subject, the method comprising:
administering to the subject 25
(a)
a combination of a therapeutically effective amount of Meclizine and at least one anti-tuberculosis drug; or
(b)
a therapeutically effective amount of the pharmaceutical composition as claimed in claim 1. 37
19.
The method as claimed in claim 18, wherein said method enhances efficacy of the at least one anti-tuberculosis drug against Mtb.
20.
The method as claimed in claim 18, wherein said method reduces drug tolerance of Mtb, and/or wherein said method reprograms macrophage metabolism in the subject. 5
21.
The method as claimed in claim 18, wherein the Meclizine and the at least one anti-tuberculosis drug are administered simultaneously, separately, or sequentially.
22.
The method as claimed in claim 18, wherein the Meclizine is administered at a dose of about 10 mg to about 100 mg/kg per day, 10 preferably 25 mg to about 50 mg/kg per day.
23.
The method as claimed in claim 18, wherein the at least one anti-tuberculosis drug is administered at a standard therapeutic dose, wherein the standard therapeutic dose comprises:
isoniazid at about 5 mg/kg to about 10 mg/kg per day; 15
rifampicin at about 10 mg/kg to about 20 mg/kg per day;
ethambutol at about 150 mg/kg to about 250 mg/kg per day; or
pyrazinamide at about 150 mg/kg to about 250 mg/kg per day.
24.
A kit for treating tuberculosis, comprising: Meclizine; and at least one anti-tuberculosis drug. 20
38
ABSTRACT
A PHARMACEUTICAL COMPOSITION HAVING MECLIZINE FOR TREATING TUBERCULOSIS, AND METHODS THEREOF
The present disclosure provides a pharmaceutical composition comprising: 5 (a) Meclizine; and (b) at least one anti-tuberculosis drug. The present disclosure also provides Meclizine for use in treating Tuberculosis, or symptoms thereof, in a subject. Further, the present disclosure provides a combination comprising Meclizine and at least one anti-tuberculosis drug. Furthermore, the present disclosure also provides a method for 10 reprogramming metabolism in Mycobacterium infected macrophage, the method comprising: contacting macrophages with the pharmaceutical composition as disclosed herein. Additionally, the present disclosure provides a method of treating tuberculosis and a kit thereof. 39 , Claims:I/We Claim:
1.
A pharmaceutical composition comprising:
a)
Meclizine; and
b)
at least one anti-tuberculosis drug.
2.
The pharmaceutical composition as claimed in claim 1, wherein the 5 Meclizine and the at least one anti-tuberculosis drug are present in a weight ratio range of 1:10 to 10:1.
3.
The pharmaceutical composition as claimed in claim 1, wherein the at least one anti-tuberculosis drug is selected from the group consisting of isoniazid, rifampicin, ethambutol, and pyrazinamide. 10
4.
The pharmaceutical composition as claimed in claim 1, wherein the composition further comprises a pharmaceutically acceptable carrier.
5.
The pharmaceutical composition as claimed in claim 4, wherein the pharmaceutically acceptable carrier comprises at least one of polymer, surfactant, solvent, or combinations thereof. 15
6.
The pharmaceutical composition as claimed in claim 5, wherein the polymer is hydroxypropyl methylcellulose, the surfactant is selected from polysorbate, polyethylene glycol, polyethoxylated castor oil, or combinations thereof, and the solvent is DMSO.
7.
The pharmaceutical composition as claimed in claim 1, wherein the at 20 least one anti-tuberculosis drug is present in a therapeutically effective amount.
8.
Meclizine for use in enhancing efficacy of at least one anti-tuberculosis drug.
9.
Meclizine for use as claimed in claim 8, wherein the at least one anti-25 tuberculosis drug is selected from the group consisting of isoniazid, rifampicin, ethambutol, and pyrazinamide.
10.
Meclizine for use as claimed in claim 8, wherein the Meclizine enhances efficacy of the at least one anti-tuberculosis drug by reprogramming macrophage metabolism. 30 36
11.
Meclizine for use as claimed in claim 8, wherein the Meclizine shifts macrophage metabolism from oxidative phosphorylation to glycolysis.
12.
Meclizine for use as claimed in claim 8, wherein the Meclizine reduces tolerance of Mtb against the anti-tuberculosis drug. 5
13.
Meclizine for use in treating tuberculosis, or symptoms thereof, in a subject, wherein said Meclizine is in combination with at least one anti-tuberculosis drug.
14.
A combination comprising Meclizine and at least one anti-tuberculosis drug. 10
15.
The combination as claimed in claim 14, wherein the combination is for treating and/or alleviating Tuberculosis or symptoms thereof in a subject.
16.
A method for reprogramming metabolism in Mycobacterium infected macrophage, the method comprising: 15
contacting macrophages with the pharmaceutical composition as claimed in claim 1.
17.
The method as claimed in claim 16, wherein the reprogramming comprises shifting macrophage metabolism from oxidative phosphorylation to glycolysis, wherein the reprogramming reduces 20 drug tolerance of Mtb in the macrophages, and/or wherein the method enhances efficacy of at least one anti-tuberculosis drug against Mtb in the macrophages.
18.
A method of treating tuberculosis in a subject, the method comprising:
administering to the subject 25
(a)
a combination of a therapeutically effective amount of Meclizine and at least one anti-tuberculosis drug; or
(b)
a therapeutically effective amount of the pharmaceutical composition as claimed in claim 1. 37
19.
The method as claimed in claim 18, wherein said method enhances efficacy of the at least one anti-tuberculosis drug against Mtb.
20.
The method as claimed in claim 18, wherein said method reduces drug tolerance of Mtb, and/or wherein said method reprograms macrophage metabolism in the subject. 5
21.
The method as claimed in claim 18, wherein the Meclizine and the at least one anti-tuberculosis drug are administered simultaneously, separately, or sequentially.
22.
The method as claimed in claim 18, wherein the Meclizine is administered at a dose of about 10 mg to about 100 mg/kg per day, 10 preferably 25 mg to about 50 mg/kg per day.
23.
The method as claimed in claim 18, wherein the at least one anti-tuberculosis drug is administered at a standard therapeutic dose, wherein the standard therapeutic dose comprises:
isoniazid at about 5 mg/kg to about 10 mg/kg per day; 15
rifampicin at about 10 mg/kg to about 20 mg/kg per day;
ethambutol at about 150 mg/kg to about 250 mg/kg per day; or
pyrazinamide at about 150 mg/kg to about 250 mg/kg per day.
24.
A kit for treating tuberculosis, comprising: Meclizine; and at least one anti-tuberculosis drug.