Methods for Treating Disease by Skewing Macrophage Phenotype Using Alternating Electric Fields

Abstract

Methods of treating disease and infection by applying an alternating electric field to tissue and then administering a treatment for the disease or infection are provided. In some cases, the alternating electric field may be applied at least until symptoms of the disease are sufficiently ameliorated. Exemplary methods can be used to treat kidney, heart, brain, and bone diseases and conditions.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 160,437, filed March 12, 2022, which is incorporated by reference in its entirety. [Background technology]

[0002] Tumor Treating Electric Fields (TTFields) are an effective anti-neoplastic treatment that involves the application of low intensity, intermediate frequency (e.g., 100-500 kHz), alternating electric fields to a target area.

[0003] In an in vivo setting, TTFields therapy can be delivered using a wearable and portable device (Optune®). The delivery system includes a field generator, four adhesive patches (non-invasive isolated transducer arrays), a rechargeable battery, and a carry case. The transducer arrays are applied to the skin and connected to the device and battery. The therapy is designed to be worn for as long as possible throughout the day and night. In a preclinical setting, TTFields can be applied in vitro, for example, using the Inovitro™ TTFields Lab Bench System. Inovitro™ includes a TTFields generator and a base plate with eight ceramic dishes per plate. Cells are seeded on a coverslip placed inside each dish. TTFields are applied in each dish using two orthogonal transducer array pairs insulated by a high-permittivity ceramic. In both in vivo and in vitro situations, the orientation of the TTFields switches by 90° every second, thus covering different directional axes of cell division.

[0004] Macrophages have important roles in many pathologies and can trigger various immune responses. Hirayama et al., The Phagocytic function of Macrophage-enforcing innate immunity and tissue homeostasis, Int J Mol Sci. January 2018, 19(1): 92. Mills et al., Macrophages at the fork in the road for disease or health, Front Immunol. 2015, 6: 59. Macrophages secrete various cytokines that can promote or inhibit inflammatory responses depending on the context. Wang et al., Molecular mechanisms that influence the macrophage m1-m2 polarization balance, Front Immunol. 2014 5:614. In addition, macrophages have a major role in wound healing and fibrosis. Gombozhapova et al., Macrophage activation and polarization in post-infraction remodeling, Journal of biomedical science (2017) 24:13, Feng et al., Wnt / β-Catenin promoted macrophage alternative activation contributes to kidney fibrosis, J Am Soc Nephrol, 2018 29(1):182~193.

[0005] Macrophages can differentiate into two classes of activated macrophages: M1 (M1-classically activated macrophages) and M2 (M2-alternatively activated macrophages). Yunna et al., Macrophage M1 / M2 polarization, Eur J Pharmacol, 15 Jun 2020, 877. M1 macrophages are typically associated with proinflammatory responses. M2 macrophages are associated with wound healing and typically secrete anti-inflammatory cytokines. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] U.S. Patent No. 7,565,205 [Non-patent literature]

[0007] [Non-Patent Document 1] Hirayama et al., The Phagocytic function of Macrophage-enforcing innate immunity and tissue homeostasis, Int J Mol Sci. January 2018, 19(1): 92 [Non-Patent Document 2] Mills et al., Macrophages at the fork in the road for disease or health, Front Immunol. 2015, 6: 59 [Non-Patent Document 3] Wang et al., Molecular mechanisms that influence the macrophage m1-m2 polarization balance, Front Immunol. 2014 5:614 [Non-Patent Document 4] Gombozhapova et al., Macrophage activation and polarization in post-infraction remodelling, Journal of biomedical science (2017) 24:13 [Non-Patent Document 5] Feng et al., Wnt / β-Catenin promoted macrophage alternative activation contributes to kidney fibrosis, J Am Soc Nephrol, 2018 29(1):182~193 [Non-Patent Document 6] Yunna et al., Macrophage M1 / M2 polarization, Eur J Pharmacol, June 15, 2020, 877 [Non-Patent Document 7] www.accessdata.fda.gov / drugsatfda_docs / label / 2011 / 016324s034s035lbl.pdf [Non-Patent Document 8] Edin S, The Distribution of Macrophages with a M1 or M2 Phenotype in Relation to Prognosis and the Molecular Characteristics of Colorectal Cancer. PLoS ONE 7(10)(2012) [Non-Patent Document 9] Sica et al., Macrophage polarization in pathology, Cell. Mol. Life Sci, 2015 Summary of the Invention [Means for solving the problem]

[0008] As described herein, the phenotype of macrophages in a target area (e.g., tissues and living subjects, or cells seeded on a coverslip) can be skewed by applying an AC electric field to cells in the target area. In some cases, the phenotype of macrophages can be skewed from an M0 or M2 phenotype to an M1 phenotype by applying an AC electric field to a target tissue, location, or cell type in a subject in need of treatment for a disease or condition before or during treatment of the subject with a drug or other treatment.

[0009] As described herein, skewing or shifting a subject's macrophage phenotype from M0 (naive) or M2 (non-classically activated) to M1 (classically activated) induces a "pro-inflammatory" immune response, which makes subjects with certain pathologies more susceptible to treatment. Thus, prior to treatment of a disease or condition (e.g., bacterial infection, endocarditis), "nudging" macrophage subtypes toward the M1 phenotype using an AC electric field can increase the efficacy of the treatment or reduce the required treatment dose and avoid unnecessary side effects.

[0010] In one aspect, a first method is provided that includes treating a Tropheryma whipplei, Salmonella, or Mycobacterium infection in a subject's digestive system by applying an alternating electric field at a frequency between 50 and 1000 kHz (e.g., between 100 and 500 kHz) to the digestive system of the subject and administering to the subject a therapeutically effective amount of an agent for treating the Tropheryma whipplei, Salmonella, or Mycobacterium infection.

[0011] In another aspect, a second method is provided that includes treating a Mycobacterium infection in the lungs of a subject by applying an alternating electric field at a frequency between 50 and 1000 kHz (e.g., between 100 and 500 kHz) to the lungs of the subject and administering to the subject a therapeutically effective amount of an agent for treating the Mycobacterium infection.

[0012] In a further aspect, a third method is provided that includes treating a Mycobacterium bovis bacillus infection in the brain of a subject by applying an alternating electric field at a frequency between 50 and 1000 kHz (e.g., between 100 and 500 kHz) to the brain of the subject and administering to the subject a therapeutically effective amount of an agent for treating the Mycobacterium infection.

[0013] In yet another aspect, a fourth method is provided that includes treating a Mycobacterium bovis bacillus infection in a bone or joint of a subject by applying an alternating electric field at a frequency between 50 and 1000 kHz (e.g., between 100 and 500 kHz) to the bone or joint of the subject and administering to the subject a therapeutically effective amount of an agent for treating the Mycobacterium bovis bacillus infection.

[0014] In another aspect, a fifth method is provided that includes treating a Mycobacterium bovis bacillus infection in a subject's heart by applying an alternating electric field at a frequency between 50 and 1000 kHz (e.g., between 100 and 500 kHz) to the subject's heart and administering to the subject a therapeutically effective amount of an agent for treating the Mycobacterium bovis bacillus infection.

[0015] In a further aspect, a sixth method is provided that includes treating endocarditis caused by Mycobacterium bovis bacillus infection in the endocardium of the subject's heart by applying an alternating electric field at a frequency between 50 and 1000 kHz (e.g., between 100 and 500 kHz) to the subject's heart and administering to the subject a therapeutically effective amount of an agent for treating the Mycobacterium bovis bacillus infection. [Brief description of the drawings]

[0016] [Figure 1A] FIG. 1 shows a representative analysis of cytotoxicity (cell numbers expressed as percentage of control) of control M0 macrophages and TTFields-treated (24 h and 48 h) M0 macrophages. [Figure 1B] FIG. 1 shows a representative flow cytometry analysis of M0 macrophages polarized towards the pro-inflammatory M1 macrophage phenotype after no treatment (control) or 48 h of TTFields treatment. [Figure 1C]FIG. 1C is a representative bar graph showing the data from FIG. 1B, showing the results for the expression of CD80+ / MHCII high on the cell surface of control macrophages and M0 macrophages treated with an AC electric field, as well as the results by MFI (mean fluorescence intensity) for CD80+ cells and MHCII high cells. [Figure 1D] FIG. 13 shows a typical cytokine secretion profile of M0 macrophages treated with TTFields for 48 hours compared to control cells, showing the fold increase in expression (secretion profile) over control untreated M0 macrophages. [Figure 2A] FIG. 1 shows a representative analysis of cytotoxicity (cell numbers expressed as percentage of control) of polarized untreated M1 macrophages and TTFields-treated (24 h) polarized M1 macrophages. [Figure 2B] FIG. 13 shows a representative flow cytometry analysis of polarized M1 macrophages after no treatment (control) or 24 h of TTFields treatment. [Figure 2C] Representative bar graphs showing the data from FIG. 2B, showing the expression of CD80+ / MHCII high on the cell surface of control macrophages and polarized M1 macrophages treated with an AC electric field, the MFI (mean fluorescence intensity) of CD80+ and MHCII high cells, and the pro-inflammatory marker inducible nitric oxide synthase (iNOS). [Figure 2D] FIG. 1 shows a typical cytokine secretion profile of polarized M1 macrophages treated with TTFields for 24 hours compared to control cells, showing the fold increase in expression (secretion profile) over control untreated polarized M1 macrophages. [Figure 3A] FIG. 1 shows a representative analysis of cytotoxicity (cell numbers expressed as percentage of control) of control and TTFields-treated (24 h) polarized M2 macrophages. [Figure 3B]FIG. 13 shows a representative flow cytometry analysis of polarized M2 macrophages after no treatment (control) or 48 h of TTFields treatment. [Figure 3C] Representative bar graphs showing the data from FIG. 3B, showing the expression of CD80+ / MHCII high on the cell surface of control macrophages and polarized M2 macrophages treated with an AC electric field, the MFI (mean fluorescence intensity) results for CD80+ cells and MHCII high cells, and the inflammatory marker inducible nitric oxide synthase (iNOS). [Figure 3D] FIG. 1 shows a typical cytokine secretion profile of polarized M2 cells treated with TTFields for 48 hours compared to control cells. The fold increase in expression (secretion profile) is shown relative to control untreated polarized M2 cells. [Figure 4] FIG. 13 shows the effects of TTFields on M1 and M2 macrophages. [Diagram 5] FIG. 1 shows that TTFields polarized M2 macrophages towards a pro-inflammatory M1 phenotype and increased the pro-inflammatory properties of M1 macrophages. [Figure 6] FIG. 13 shows that TTFields altered macrophage cytokine secretion towards a pro-inflammatory pattern. [Figure 7] FIG. 13 shows the effect of TTFields on the expression of phenotypic markers in M1 and M2 macrophages. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] All references cited herein, including but not limited to patents and patent applications, are hereby incorporated by reference in their entirety.

[0018] An alternating electric field can be applied to an area of ​​a subject's body to stimulate the immune system, for example, by shifting or skewing the phenotype of macrophages to the "inflammatory" M1 phenotype. Skewing the macrophage phenotype to the M1 phenotype can make the subject more susceptible to treatment of a disease or condition, such as an infection, because the subject's immune system has been stimulated. As described herein, an alternating electric field can be applied to an area of ​​the body before or during treatment of a disease or condition.

[0019] The AC electric fields discussed herein are similar to Tumor Treating Electric Fields (TTFields) and can be applied at the same 200 kHz frequency used by Optune, or at different frequencies (e.g., 50 to 1000 kHz or 100 to 500 kHz) using hardware similar to Novocure's Optune® device. The size and shape of the transducer array used to apply the AC electric field to the subject's body will vary depending on the anatomical location to which the AC field is applied. The term "TTFields" as used herein and in the drawings is synonymous with the term "AC electric field."

[0020] In one aspect, a first method is provided that includes treating a Tropheryma whipperi, Salmonella, or Mycobacterium infection in a subject's digestive system by applying an alternating electric field at a frequency between 50 and 1000 kHz (e.g., between 100 and 500 kHz) to the subject's digestive system and administering to the subject a therapeutically effective amount of an agent for treating the Tropheryma whipperi, Salmonella, or Mycobacterium infection.

[0021] In some cases of the first method, the antibiotic is selected from the group consisting of one or more of penicillin, streptomycin, tetracycline, ceftriaxone, meropenem, cotrimoxazole, doxycycline, hydroxychloroquine, fluoroquinolone, and azithromycin.

[0022] In some cases of the first method, the alternating electric field is applied for at least 24 hours or at least 48 hours. In some cases of the first method, the frequency is between 125 and 175 kHz. In some cases of the first method, the alternating electric field is applied until the subject's digestive system is free of Mycobacterium infection. The alternating electric field can be applied before, during, or after standard treatment or other treatment for the disease or condition.

[0023] The term "therapeutically effective amount" as used herein refers to an amount of an agent or a dosage of an agent sufficient to achieve its intended purpose of ameliorating, preventing, treating, or curing a specified disease or condition. A therapeutically effective amount of an agent can be determined, for example, by one of skill in the art from the drug or product label or from the results of experiments or clinical trials designed to determine the therapeutically effective dosage, pharmacokinetics, or other characteristics of the agent.

[0024] For example, one of skill in the art can determine a therapeutically effective dose of azathioprine by consulting the drug label approved by the U.S. Food and Drug Administration (FDA) for azathioprine, available at www.accessdata.fda.gov / drugsatfda_docs / label / 2011 / 016324s034s035lbl.pdf. One of skill in the art can consult the product label approved by the FDA or other regulatory agency for any approved drug or treatment referenced herein. The term "treatment" as used herein refers to prescribing, administering, or instructing another person to administer a drug or treatment to a subject.

[0025] The skewing or shift of macrophage phenotype can be determined, for example, by determining the ratio of M1 to M2 macrophage phenotype using a suitable method disclosed herein (e.g., flow cytometry, immunohistochemical staining of biopsies), or by other suitable methods (see, for example, Edin S, The Distribution of Macrophages with a M1 or M2 Phenotype in Relation to Prognosis and the Molecular Characteristics of Colorectal Cancer. PLoS ONE 7(10)(2012); Sica et al., Macrophage polarization in pathology, Cell. Mol. Life Sci, 2015).

[0026] In another aspect, a second method is provided that includes treating a Mycobacterium infection in the lungs of a subject by applying an alternating electric field at a frequency between 50 and 1000 kHz (e.g., between 100 and 500 kHz) to the lungs of the subject and administering to the subject a therapeutically effective amount of an agent for treating the Mycobacterium infection.

[0027] In some cases of the second method, the antibiotic is selected from the group consisting of one or more of clarithromycin, azithromycin, rifampin, rifabutin, ethambutol, streptomycin, and amikacin.

[0028] In some cases of the second method, the alternating electric field is applied for at least 24 hours or at least 48 hours. In some cases of the second method, the frequency is between 125 and 175 kHz. In some cases of the second method, the alternating electric field is applied until the subject's lungs are free of Mycobacterium infection. The alternating electric field can be applied before, during, or after standard treatment or other treatment for the disease or condition.

[0029] In a further aspect, a third method is provided that includes treating a Mycobacterium bovis bacillus infection in the brain of a subject by applying an alternating electric field at a frequency between 50 and 1000 kHz (e.g., between 100 and 500 kHz) to the brain of the subject and administering to the subject a therapeutically effective amount of an agent for treating the Mycobacterium bovis bacillus infection.

[0030] In some cases of the third method, the antibiotic is selected from the group consisting of one or more of isoniazid, rifampicin, ethambutol, moxifloxacin, pyrazinamide, and streptomycin.

[0031] In some instances, the third method further comprises administering to the subject a therapeutically effective amount of a steroid. The steroid can be selected from the group consisting of one or more of prednisone and dexamethasone.

[0032] In some cases of the third method, the alternating electric field is applied for at least 24 hours or at least 48 hours. In some cases of the third method, the frequency is between 125 and 175 kHz. In some cases of the third method, the alternating electric field is applied until the subject's brain is free of Mycobacterium bovis bacilli. The alternating electric field can be applied before, during, or after standard treatment or other treatment for the disease or condition.

[0033] In yet another aspect, a fourth method is provided that includes treating a Mycobacterium bovis bacillus infection in a bone or joint of a subject by applying an alternating electric field at a frequency between 50 and 1000 kHz (e.g., between 100 and 500 kHz) to the bone or joint of the subject and administering to the subject a therapeutically effective amount of an agent for treating the Mycobacterium bovis bacillus infection.

[0034] In some cases of the fourth method, the antibiotic is selected from the group consisting of one or more of isoniazid, rifampicin, ethambutol, moxifloxacin, pyrazinamide, and streptomycin.

[0035] In some instances, the fourth method further comprises administering to the subject a therapeutically effective amount of a steroid. The steroid can be selected from the group consisting of one or more of prednisone and dexamethasone.

[0036] In some cases of the fourth method, the alternating electric field is applied for at least 24 hours or at least 48 hours. In some cases of the fourth method, the frequency is between 125 and 175 kHz. In some cases of the fourth method, the alternating electric field is applied until the subject's bone or joint is free of Mycobacterium bovis bacilli infection. The alternating electric field can be applied before, during, or after standard treatment or other treatment for the disease or condition.

[0037] In another aspect, a fifth method is provided that includes treating a Mycobacterium bovis bacillus infection in a subject's heart by applying an alternating electric field at a frequency between 100 and 500 kHz to the subject's heart and administering to the subject a therapeutically effective amount of an agent for treating the Mycobacterium bovis bacillus infection.

[0038] In some cases of the fifth method, the antibiotic is selected from the group consisting of one or more of isoniazid, rifampicin, ethambutol, moxifloxacin, pyrazinamide, and streptomycin.

[0039] In some instances, the fifth method further comprises administering to the subject a therapeutically effective amount of a steroid. The steroid can be selected from the group consisting of one or more of prednisone and dexamethasone.

[0040] In some cases of the fifth method, the alternating electric field is applied for at least 24 hours or at least 48 hours. In some cases of the fifth method, the frequency is between 125 and 175 kHz. In some cases of the fifth method, the alternating electric field is applied until the subject's heart is free of Mycobacterium bovis bacilli. The alternating electric field can be applied before, during, or after standard treatment or other treatment for the disease or condition.

[0041] In a further aspect, a sixth method is provided comprising treating endocarditis caused by infection of the Mycobacterium bovis bacillus in the endocardium of the subject's heart by applying an alternating electric field at a frequency between 100 and 500 kHz to the subject's heart and administering to the subject a therapeutically effective amount of an agent for treating the infection with Mycobacterium bovis bacillus.

[0042] In some cases of the sixth method, the antibiotic is selected from the group consisting of one or more of isoniazid, rifampicin, ethambutol, moxifloxacin, pyrazinamide, and streptomycin.

[0043] In some instances, the sixth method further comprises administering to the subject a therapeutically effective amount of a steroid. The steroid can be selected from the group consisting of one or more of prednisone and dexamethasone.

[0044] In some cases of the sixth method, the alternating electric field is applied for at least 24 hours or at least 48 hours. In some cases of the sixth method, the frequency is between 125 and 175 kHz. In some cases of the sixth method, the alternating electric field is applied until the endothelium of the heart is free of Mycobacterium bovis bacilli. The alternating electric field can be applied before, during, or after standard treatment or other treatment for the disease or condition.

[0045] In yet another aspect, a method is provided that includes treating lupus nephritis in a kidney of a subject by applying an alternating electric field at a frequency between 50 and 1000 kHz (e.g., between 100 and 500 kHz) to the kidney of the subject and administering to the subject a therapeutically effective amount of an agent for treating lupus nephritis.

[0046] In some cases of the method of treating lupus nephritis in the kidney of a subject, the alternating electric field is applied for at least 24 hours or at least 48 hours. In some cases, the frequency is between 125 and 175 kHz. In some cases, the alternating electric field is applied until the subject is no longer diagnosed with lupus nephritis. The alternating electric field can be applied before, during, or after standard treatment or other treatment for the disease or condition.

[0047] In some cases of the methods of treating lupus nephritis in the kidney of a subject, the agent for treating lupus nephritis is selected from the group consisting of one or more of azathioprine, cyclophosphamide, prednisone, prednisolone, voclosporin, tacrolimus, mycophenolic acid, and rituximab.

[0048] In some cases, the method of treating lupus nephritis in the kidney of a subject further comprises administering to the subject a therapeutically effective amount of an angiotensin-converting enzyme (ACE) inhibitor or an angiotensin II receptor blocker (ARB).

[0049] The ACE inhibitor may be selected from the group consisting of one or more of benazepril, captopril, enalapril, fosinopril, lisinopril, moexipril, perindopril, quinapril, ramipril, and trandolapril, and the ARB is selected from the group consisting of one or more of azilsartan, candesartan, eprosartan, irbesartan, losartan, olmesartan, telmisartan, and valsartan.

[0050] In a typical experiment described herein, bone marrow cells were flushed from the femurs and tibias of 5-8 week old Balb / C mice to generate bone marrow derived macrophages (BMDMs) and differentiated for 7 days with GM-CSF. To examine the response of individual macrophages to TTFields, macrophages were stimulated with LPS+IFN-γ (M1 polarization) or IL-4 (M2 polarization) for 24 h and then treated with TTFields (150 kHz) for 24 h and profiled. M1 and M2 polarization resulted in cell death after 48 h of exposure to TTFields. Therefore, experiments using M1 and M2 polarized macrophages, as described below, were evaluated at 24 h rather than 48 h.

[0051] Flow cytometry was used to assess surface expression of F4 / 80 (macrophage biomarker) and activation markers including major histocompatibility complex class II (MHC II), CD80, and inducible nitric oxide synthase (iNOS).

[0052] TTFields were applied using the Inovitro™ System. In these examples, cells were treated with TTFields at an intensity of 1.75 V / cm (RMS) and a frequency of 150 kHz for 24 or 48 hours (h).

[0053] Flow cytometry (MACSQuant Analyzer 10, Miltenyi Biotec, Bergisch Gladbach, Germany) was used to assess surface expression of F4 / 80 (a pan-macrophage biomarker) and activation markers including major histocompatibility complex class II (MHC II), CD80, and inducible nitric oxide synthase (iNOS). Viobility 405 / 452 Fixable Dye (Viobility Dye) was used to distinguish dead cells. Cytotoxicity was analyzed based on the percentage of F4 / 80 positive cells from single cell counts.

[0054] A multiplex secretion assay was used to measure the heterogeneity of stimulated macrophages (LEGENDplex™ Mouse Macrophage / Microglia Panel (13-plex) Cat. No. 740846, Biolegend). The fold increase or decrease in secretion of 13 different cytokines (CXCL1 (KC), IL-18, IL-23, IL-12p70, IL6, TNF-α, IL-12p40, free active TGF-β1, CCL22 (MDC), IL-10, IL-6, G-CSF, CCL17 (TARC), and IL-1β) following exposure to TTFields was measured. The levels of cytokine secretion were normalized to controls.

[0055] Data are presented as mean ± SEM. Statistical significance of differences was assessed using GraphPad Prism 9 software (GraphPad Software, La Jolla, CA). Differences between groups were considered to be >0.05. * p>0.01, ** p<0.01, and *** A value of p<0.001 was considered significant.

[0056] Examples 1 and 2 below provide experimental data showing that application of TTFields to BMDMs increased the pro-inflammatory phenotype of M1 polarized BMDMs and induced phenotypic skewing of M2 polarized BMDMs towards an M1 phenotype. These results elucidate a novel immunomodulatory role for TTFields on macrophage polarization. EXAMPLES

[0057] Example 1 Figures 1A-D show that M0 macrophages are polarized by TTFields to a pro-inflammatory M1 phenotype.

[0058] Figure 1A shows that application of TTFields for 24 and 48 hours results in minimal cytotoxicity to M0 macrophages.

[0059] Figure 1B shows representative flow cytometry plots showing that expression of CD80 and MHC-II markers increased from 21.1 to 44.4 percent after 48 hours of exposure to TTFields. These results demonstrate a skewing from an M0 to an M1 macrophage phenotype.

[0060] FIG. 1C shows a bar graph quantifying the flow cytometry results of FIG. 1B and shows increased expression of proinflammatory CD80 and MHC-II markers in conjunction with increased fluorescence intensity of CD80 and MHC-II markers.

[0061] Figure ID shows the fold increase in secretion of pro-inflammatory cytokines following application of TTFields for 48 hours. Table 1 below shows the measured pro- and anti-inflammatory cytokines. The "secretion profile", represented on the y-axis of Figure ID, shows the fold increase in cytokine levels compared to control macrophages not exposed to TTFields. The increased expression of most pro-inflammatory cytokines (e.g., CXCL1, TNFα, IL1β) further indicates a shift from M0 macrophages to a pro-inflammatory M1 macrophage phenotype.

[0062] [Table 1]

[0063] Taken together, the results in Figures 1A-1D demonstrate a significant shift from an M0 macrophage to an M1 phenotype induced by application of TTFields.

[0064] Figures 2A-D demonstrate that TTFields further promote the pro-inflammatory phenotype of M1 macrophages already polarized to the M1 phenotype by IFN-γ and LPS. As mentioned above, stimulation of macrophages with IFN-γ and LPS can lead to cell death after 48 hours. Therefore, 24 hours of TTFields exposure was used in the exemplary experiment of Figures 2A-D.

[0065] Figure 2A shows minimal cytotoxicity resulting from application of TTFields to M1 polarized macrophages for 24 hours.

[0066] Figure 2B shows representative flow cytometry plots demonstrating that expression of CD80 and MHC-II markers increased from 58.5 to 73.3 percent after 24-hour exposure of M1 macrophages already stimulated toward an M1 phenotype by IFN-γ and LPS. These results demonstrate further skewing of M1 polarized macrophages toward a more pro-inflammatory M1 macrophage phenotype.

[0067] FIG. 2C shows a bar graph quantifying the flow cytometry results of FIG. 2B and shows increased expression of pro-inflammatory CD80 and MHC-II markers, increased fluorescence intensity of CD80 and MHC-II markers, and increased percentage of pro-inflammatory iNOS marker.

[0068] Figure 2D shows the fold increase in proinflammatory cytokine secretion in M1 polarized macrophages following application of TTFields for 24 hours. The increased expression of most proinflammatory cytokines (e.g., CXCL1, IL-6, TNFα) further indicates a shift from polarized M1 macrophages to a more proinflammatory M1 macrophage phenotype.

[0069] Taken together, the results in Figures 2A-D demonstrate a significant shift from polarized M1 macrophages to a more pro-inflammatory M1 phenotype induced by application of TTFields.

[0070] Figures 3A-3D show that TTFields mediate the expression of CD80+ cells and MHC-II 高 We demonstrate that IL-4 induces a phenotypic skewing from polarized M2 macrophages to an M1 phenotype, as shown by an increased percentage of cells. As mentioned above, stimulation of macrophages with IL-4 can result in cell death after 48 hours. Therefore, 24 hours of TTFields exposure was used in the exemplary experiments in Figures 3A-D.

[0071] Figure 3A shows minimal cytotoxicity produced by application of TTFields to IL-4 polarized M2 macrophages for 24 hours.

[0072] Figure 3B shows representative flow cytometry plots demonstrating that expression of CD80 and MHC-II markers further increased from 34.2 to 52.2 percent in IL-4 polarized M2 macrophages after 24 hours of exposure to TTFields. These results demonstrate the skewing of IL-4 polarized M2 macrophages towards an M1 macrophage phenotype.

[0073] Figure 3C shows a bar graph quantifying the flow cytometry results of Figure 3B and shows increased expression of the proinflammatory CD80 marker and increased fluorescence intensity of the CD80 marker. No significant changes were seen in the expression levels of MHCII or iNOS.

[0074] Figure 3D shows the fold increase in secretion of proinflammatory cytokines following application of TTFields for 24 hours. The increased expression of most proinflammatory cytokines (e.g., CXCL1, IL-6) further indicates a shift from a polarized M2 macrophage to an M1 macrophage phenotype.

[0075] Example 2 Figure 4 shows that TTFields induce morphological changes in M2 macrophages, converting them to a dendritic-like M1 phenotype, while M1 macrophages remain unchanged.

[0076] FIG. 5 shows that TTFields polarized M2 macrophages towards a pro-inflammatory M1 phenotype and increased the pro-inflammatory properties of M1 macrophages as indicated by increased expression of CD80 and MHC-II.

[0077] Figure 6 shows that TTFields altered macrophage cytokine secretion to a pro-inflammatory pattern, accompanied by a significant increase in CXCL1 in M2.

[0078] FIG. 7 shows that TTFields attenuated the expression of M2 phenotype markers CD206 and ARG-1 in both M1 and M2 macrophages, but did not affect the expression of the M1 phenotype marker iNOS.

[0079] In some embodiments, the application of the electric field may be interrupted by pauses. For example, six sessions of 12 hours duration each, with a two-hour pause between sessions. In another embodiment, the step of applying the electric field has a duration of at least 3 hours. In some cases, the alternating electric field can be applied at 150 kHz and a strength of 175 V / m (RMS) for 24 hours or 48 hours.

[0080] In yet another embodiment, the frequency of the alternating electric field is between 50 and 1 MHz, between 100 and 500 kHz, between 125 and 175 kHz, or 150 kHz. In another embodiment, the agent is delivered to a tissue, location, or cell at a therapeutically effective concentration and the alternating electric field has a field strength of at least 1 V / cm in at least a portion of the tissue, location, or cell.

[0081] In yet other embodiments, the alternating electric field has a field strength of 0.1 to 10 V / cm, 1.30 to 4.0 V / cm, 1.50 to 2.0 V / cm, 1.70 to 1.80 V / cm (RMS). In certain embodiments, the alternating electric field has a field strength of 1.75 V / cm (RMS).

[0082] In yet another aspect, at least a portion of the applying step occurs simultaneously with at least a portion of the administering step.

[0083] The in vitro experiments described herein were performed using Novocure's Inovitro™ system. In these experiments, the direction of the AC electric field was switched between two perpendicular directions at 1 second intervals. However, in alternative embodiments, the direction of the AC electric field can be switched at a faster rate (e.g., at intervals between 1 and 1000 ms) or at a slower rate (e.g., at intervals between 1 and 100 seconds).

[0084] In the in vitro experiments described herein, the direction of the AC electric field was switched between two orthogonal directions by applying AC voltages in alternating order to two pairs of electrodes placed 90° apart from each other in 2D space. However, in alternative embodiments, the direction of the AC electric field may be switched between two non-orthogonal directions, or between three or more directions (if additional electrode pairs are provided), by repositioning the electrode pairs. For example, the direction of the AC electric field may be switched between three directions, each of which is determined by placing its own electrode pair. In some cases, these three electrode pairs may be placed such that the resulting electric fields are located 90° apart from each other in 3D space. In other alternative embodiments, the electrodes need not be arranged in pairs. See, for example, the electrode positioning described in U.S. Pat. No. 7,565,205, which is incorporated herein by reference. In other alternative embodiments, the direction of the electric field remains constant.

[0085] In the in vitro experiments using the Inovitro™ system described herein, the Inovitro™ system used conductive electrodes placed on the outer surface of the sidewall of the dish, and the electric field was capacitively coupled to the culture because the ceramic material of the sidewall acts as a dielectric. However, in alternative embodiments, the electric field may be applied directly to the cells without capacitive coupling (e.g., by changing the configuration of the Inovitro™ system so that the conductive electrodes are placed on the inner surface of the sidewall instead of the outer surface of the sidewall).

[0086] The methods described herein can also be applied in an in vivo context by applying an alternating electric field to a target region of a living subject's body (e.g., using Novocure's Optune® system). This can be done, for example, by positioning electrodes on or under the subject's skin such that application of an AC voltage between a selected subset of these electrodes provides an alternating electric field at the target region of the subject's body.

[0087] For example, in situations where the relevant cells are in the subject's brain, a pair of electrodes can be positioned on the front and back of the subject's head, and a second pair of electrodes can be positioned on the right and left sides of the subject's head. In some embodiments, the electrodes are capacitively coupled to the subject's body (e.g., by using an electrode that includes a conductive plate and has a dielectric layer placed between the conductive plate and the subject's body). However, in alternative embodiments, the dielectric layer can be omitted, in which case the conductive plate will be in direct contact with the subject's body. In another embodiment, the electrodes can be inserted subcutaneously under the patient's skin. An AC voltage generator applies an AC voltage of a selected frequency (e.g., 200 kHz) between the right and left electrodes for a first time period (e.g., 1 second), which induces an alternating electric field whose most dominant component of the field lines is parallel to the transverse axis of the subject's body.

[0088] The AC voltage generator then applies an AC voltage of the same frequency (or a different frequency) between the anterior and posterior electrodes for a second time (e.g., 1 second), which induces an alternating electric field with the most dominant component of the field lines parallel to the sagittal axis of the subject's body. This two-step sequence is then repeated for the duration of the treatment. Optionally, a thermal sensor may be included in the electrode, and the AC voltage generator may be configured to reduce the amplitude of the AC voltage applied to the electrode if the temperature sensed at the electrode becomes too high. In some embodiments, one or more additional electrode pairs may be added and included in the sequence. In an alternative embodiment, only a single electrode pair is used, in which case the direction of the field lines is not switched. It should be noted that any of the parameters in this in vivo embodiment (e.g., frequency, field strength, time, speed of switching direction, and electrode placement) may be varied as described above in connection with the in vitro embodiment. However, in an in vivo context, care should be taken to ensure that the electric field remains safe for the subject at all times.

[0089] It should be noted that in the experiments described herein, the AC electric field was applied for an uninterrupted time interval (e.g., 24 hours, 48 ​​hours). However, in alternative embodiments, the application of the AC electric field may be interrupted, preferably by short breaks. For example, a 24-hour time interval may consist of the application of the AC electric field for six 4-hour blocks, with a 1-hour or 2-hour break between each of these blocks.

[0090] Although the present invention has been disclosed with reference to certain specific embodiments, numerous modifications, alterations, and variations to the described embodiments are possible without departing from the sphere and scope of the invention as defined in the appended claims. Thus, the present invention is not limited to the described embodiments, but has the full scope defined by the following claims and their equivalents.

[0091] [References] TIFF2024509964000003.tif35170

Claims

1. 1. A medicament for treating an infection in a subject, the medicament comprising a therapeutically effective amount of an antibiotic to treat the infection, the treatment comprising: applying an alternating electric field to the digestive system at a frequency between 50 and 1,000 kHz; and administering a therapeutically effective amount of an antibiotic to the subject. Including, Infection is Tropheryma whippelii, Salmonella, or Mycobacterium infections in the digestive system, Mycobacterium infection in the lungs, Mycobacterium bovis infection in the brain, Mycobacterium bovis bacillus infection in bones or joints, Mycobacterium bovis infection in the heart, or Mycobacterium bovis bacillus infection of the endothelium of the heart That is, drugs.

2. 2. The method of claim 1, wherein the infection is a Tropheryma whippelii, Salmonella, or Mycobacterium infection in the subject's digestive system and the antibiotic is selected from the group consisting of one or more of penicillin, streptomycin, tetracycline, ceftriaxone, meropenem, cotrimoxazole, doxycycline, hydroxychloroquine, fluoroquinolone, and azithromycin.

3. The method of claim 2, wherein the frequency is between 125 and 175 kHz.

4. The method of claim 2, wherein the alternating electric field is applied for at least 24 hours.

5. 3. The method of claim 2, wherein the alternating electric field is applied until the subject's digestive system is free of Tropheryma whippelii, Salmonella, or Mycobacterium infection.

6. 2. The method of claim 1, wherein the infection is a Mycobacterium infection in the lungs of the subject and the antibiotic is selected from the group consisting of one or more of clarithromycin, azithromycin, rifampin, rifabutin, ethambutol, streptomycin, and amikacin.

7. The method of claim 6, wherein the frequency is between 125 and 175 kHz.

8. The method of claim 6, wherein the alternating electric field is applied for at least 24 hours.

9. The method of claim 6, wherein the alternating electric field is applied until the subject's lungs are free of Mycobacterium infection.

10. 2. The method of claim 1, wherein the infection is a Mycobacterium bovis bacillus infection in the subject's brain or in a bone or joint or in the heart, and the antibiotic is selected from the group consisting of one or more of isoniazid, rifampicin, ethambutol, moxifloxacin, pyrazinamide, and streptomycin.

11. The method of claim 10, wherein the treatment further comprises administering to the subject a therapeutically effective amount of a steroid.

12. 12. The method of claim 11, wherein the steroid is selected from the group consisting of one or more of prednisone and dexamethasone.

13. The method of claim 10, wherein the frequency is between 125 and 175 kHz.

14. The method of claim 10, wherein the alternating electric field is applied for at least 24 hours.

15. 11. The method of claim 10, wherein the alternating electric field is applied until the subject's brain, bones or joints, or heart are free of Mycobacterium bovis bacilli infection.

16. 2. The method of claim 1, wherein the infection is a Mycobacterium bovis bacillus infection in the endothelium of the subject's heart and the antibiotic is selected from the group consisting of one or more of isoniazid, rifampicin, ethambutol, moxifloxacin, pyrazinamide, and streptomycin.

17. The method of claim 16, wherein the treatment further comprises administering to the subject a therapeutically effective amount of a steroid.

18. 18. The method of claim 17, wherein the steroid is selected from the group consisting of one or more of prednisone and dexamethasone.

19. 17. The method of claim 16, wherein the frequency is between 125 and 175 kHz.

20. The method of claim 16, wherein the alternating electric field is applied for at least 24 hours.

21. 17. The method of claim 16, wherein the alternating electric field is applied until the subject's heart is free of Mycobacterium bovis bacilli infection.

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