Liposome formulations for treatment of active tuberculosis

Abstract

The present invention relates to the use of a therapeutic agent based on cell wall fragments of a virulent strain of Mycobacterium tuberculosis complex for the preparation of a medicament for the treatment of a patient suffering from active tuberculosis.

Description

Technical Field

[0001] The present invention relates to the use of therapeutic agents based on cell wall fragments of virulent strains of Mycobacterium tuberculosis-complex in the preparation of a medicament for treating patients with active tuberculosis. This can be a stand-alone treatment or concurrently with chemotherapy (e.g. antibiotic treatment). Background Art

[0002] Tuberculosis (TB) is a chronic infectious disease caused by Mycobacterium tuberculosis complex (MTB-C) bacteria, which include species such as M. tuberculosis, M. bovis, M. microti, and M. africanum. According to the World Health Organization (WHO), in 2019, approximately 10 million people developed TB and approximately 1.4 million died.

[0003] A widely used vaccine based on an attenuated variant of the bacterium Mycobacterium bovis is called BCG (Bacillus Calmette-Guérin). Other vaccines against TB based on cell wall fragments of virulent or non-virulent strains of Mycobacteria are described in the prior art.

[0004] The inventors of the present invention have previously developed particularly effective immunotherapeutic agents based on cell wall fragments of virulent strains of the Mycobacterium tuberculosis complex (EP1090318B1).

[0005] Treatment for patients with active TB typically involves long-term treatment with multiple drugs, such as isoniazid, rifampicin, pyrazinamide, and ethambutol (HRZE). Consequently, drug resistance is a growing problem, and cases of multidrug-resistant TB (MDR-TB) are increasing.

[0006] Therefore, there is an urgent need for more effective treatments for those with active TB. Summary of the Invention

[0007] The present inventors have discovered that an agent based on cell wall fragments of virulent strains of the Mycobacterium tuberculosis complex is highly effective in treating patients with active TB, exhibiting bactericidal activity both alone and in combination with chemotherapy. As a stand-alone treatment, the agent has similar efficacy to chemotherapy at the initiation of active TB treatment. When used in combination with chemotherapy, the agent provides a strong synergistic effect. Neither of these effects would have been predicted from the prior art, which strongly suggests that such agents should not be used in subjects with active TB due to severe side effects.

[0008] definition

[0009] "FCMtb" stands for fragments from the Mycobacterium tuberculosis complex (MTB-C) strain. This is the raw drug substance of the liposomal formulation (drug product) of the present invention.

[0010] If not otherwise specified, "particle size" refers to the diameter of the particle. If the particle size cannot be determined precisely, it refers to the approximate particle size.

[0011] The "z-average" is the average particle size, which can be determined as described in the Materials and Methods section.

[0012] abbreviation

[0013] BCG

[0014] CFU colony forming unit

[0015] CMCiB Catalan Center for Comparative Medicine and Bioimaging (Centre de Medicina ComparativaI Bioimatge de Catalunya)

[0016] DP drug products

[0017] DS API

[0018] ELISA enzyme-linked immunosorbent assay

[0019] ELISPOT enzyme-linked immunospot assay

[0020] EMEA European Medicines Agency

[0021] FCMtb Mycobacterium tuberculosis cell fragments

[0022] FIM is first reported in humans

[0023] Collection of HMtb Mtb expansion cultures

[0024] IFN-γ Interferon gamma

[0025] IGTP Germans Trias IPujol Institute

[0026] IMP Investigational Medicinal Products

[0027] IPC process control

[0028] LCS Liposome Concentrate Suspension

[0029] LS liposome suspension

[0030] LTBI Latent tuberculosis infection

[0031] LPS lipopolysaccharide

[0032] Mtb Mycobacterium tuberculosis

[0033] Mtb-C Mycobacterium tuberculosis complex

[0034] NZB New Zealand Black

[0035] NZW New Zealand White

[0036] PPD-purified protein derivatives

[0037] qs sufficient

[0038] TB Tuberculosis

[0039] TST Tuberculin skin test

[0040] UTE Tuberculosis Experimental Unit (Unitat de Tuberculosi Experimental)

[0041] WHO World Health Organization

[0042] w / v weight / volume

[0043] w / w weight / weight

[0044] WSL working seed batch BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1: Flowchart showing the upstream process of the bulk drug substance (FCMtb), including the materials and reagents involved in the process and appropriate process controls.

[0046] Figure 2: Flowchart showing the downstream processing of the bulk drug substance (FCMtb), including the materials and reagents involved in the process and appropriate process controls.

[0047] Figure 3: Protein characterization. Figure 3a: SDS-PAGE stained with Coomassie Brilliant Blue. Lane 1: Molecular weight. Lane 2: Reference FCMtb-81 batch. Lane 3: Reference FCMtb-81 batch. Lane 4: FCMtb-83 stability study batch for 12 months at -20°C. Lane 5: FCMtb-83 stability study batch for 12 months at -20°C. Lane 6: FCMtb-86 stability study batch for 1 month at room temperature (RT). Lane 7: FCMtb-86 stability study batch for 1 month at room temperature. Lane 8: FCMtb-86 stability study batch for 1 month at 40°C. Lane 9: FCMtb-86 stability study batch for 1 month at 40°C. Lane 10: Molecular weight. Figure 3b Silver-stained SDS-PAGE. Lane 1: Molecular weight. Lane 2: Reference FCMtb-81 batch. Lane 3: Reference FCMtb-81 batch. Lane 4: FCMtb-83 batch studied for 12-month stability at -20°C. Lane 5: FCMtb-83 batch studied for 12-month stability at -20°C. Lane 6: FCMtb-86 batch studied for 1-month stability at room temperature. Lane 7: FCMtb-86 batch studied for 1-month stability at room temperature. Lane 8: FCMtb-86 batch studied for 1-month stability at 40°C. Lane 9: FCMtb-86 batch studied for 1-month stability at 40°C. Lane 10: Molecular weight. Figure 3c Western blot analysis of HSP70, PstS1, 85 complex, and HSP16.3 antigens from Mycobacterium tuberculosis. Lane 1: Molecular weight. Lane 2: HSP16.3 antigen. Lane 3: 85A antigen. Lane 4: PstS1 antigen. Lane 5: HSP70 antigen. Lane 6: FCMtb-87. Lane 7: FCMtb-87 HMtb. Lane 8: FCMtb-86. Lane 9: FCMtb-86 HMtb. Lane 10: Molecular weight.

[0048] Figure 4: Lipid analysis. Figure 4a : TLC analysis of trehalose dimycolate (TDM). Lane 1: TDM standard. Lane 2: Reference FCMtb-81 batch. Lane 3: FCMtb-86 batch studied for 6-month stability at room temperature. Lane 4: FCMtb-86 batch studied for 6-month stability at 40°C. Lane 5: FCMtb-86 batch studied for 6-month stability at -20°C. Lane 6: TDM standard. Figure 4b: TLC analysis of mycolic acids (MA). Lane 1: MA standard. Lane 2: Reference FCMtb-81 batch. Lane 3: FCMtb-86 batch studied for 6 months of stability at room temperature. Lane 4: FCMtb-86 batch studied for 6 months of stability at room temperature. Lane 5: FCMtb-86 batch studied for 6 months of stability at 40°C. Lane 6: FCMtb-86 batch studied for 6 months of stability at 40°C. Lane 7: FCMtb-86 batch studied for 6 months of stability at -20°C. Lane 8: FCMtb-86 batch studied for 6 months of stability at -20°C. Lane 9: MA standard. Figure 4c Western blot analysis of lipoarabinomannan (LAM) from different FCMtb batches and their corresponding HMtb. Lane 1: Molecular weight. Lane 2: LAM antigen, 0.270 μg. Lane 3: LAM antigen, 0.068 μg. Lane 4: LAM antigen, 0.017 μg. Lane 5: FCMtb-83 batch. Lane 6: FCMtb-83 HMtb. Lane 7: FCMtb-86 batch. Lane 8: FCMtb-86 HMtb. Lane 9: Molecular weight. LM: Lipomannan.

[0049] Figure 5 : Freeze-fracturing preparations of liposome concentrate (LCS) bulk (electron microscopy).

[0050] Figure 6: Process flow diagram according to a preferred approach for the production of the drug product.

[0051] Figure 7: Exemplary time points for drug product (RUTI) administration in Example 9. RUTI was administered as a single dose containing 200 μg FCMtb at different time points after the start of chemotherapy treatment and sacrificed at several different time points (see Example 9 for details).

[0052] Figure 8: Effect of treatment on bacillary load in the lung (A) and spleen (B) relative to the standard chemotherapy (SCT) control group. Each point represents the CFU per animal. Mean values. Statistical significance was tested using an unpaired t-test, *p < 0.05.

[0053] Figure 9 : Effect of treatment on lung pathology; histological analysis. Each point represents the % lung lesion per animal. Mean values are shown. Statistical significance was tested using an unpaired t-test, *p < 0.05.

[0054] Figure 10 : Effect of treatment on T cell immune response. Splenocytes were obtained from each spleen and incubated in the presence of PPD for 24 h. Each point represents 10 6The number of IFN-γ spots per cell was averaged. Statistical significance was tested using an unpaired t-test, *p < 0.05.

[0055] Figure 11 : Effect of treatment on T cell immune response. Splenocytes were obtained from each spleen and incubated in the presence of ESAT-6 for 24 hours. Each point represents 10 6 The number of IFN-γ spots per cell was averaged. Statistical significance was tested using an unpaired t-test, *p < 0.05.

[0056] Figure 12 Effect of treatment on T cell immune responses. Splenocytes were obtained from each spleen and incubated in the presence of HSP16 for 24 hours. Each point represents the number of IFN-γ spots per E+06 cells. Mean values are shown. Statistical significance was tested using an unpaired t-test.

[0057] Figure 13: Figure 13a / b / c show different exemplary schedules of administration and sacrifice employed in Example 10.

[0058] Figure 14 Effect of treatment on bacillary load in the lungs. Each point represents the CFU per animal. Median ± interquartile range for each treatment group. Statistical significance was tested using the nonparametric Mann-Whitney test, *p < 0.05, **p < 0.01.

[0059] Figure 15 Effect of treatment on bacillary load in the spleen. Each point represents the CFU per animal. Median ± interquartile range for each treatment group. Statistical significance was tested using the nonparametric Mann-Whitney test; *p < 0.05, **p < 0.01.

[0060] Figure 16 Effects of treatment on lung pathology; histological analysis. Each point represents the percentage of lung lesions in each animal. Median ± interquartile range for each treatment group. Statistical significance was tested using the nonparametric Mann-Whitney test; *p < 0.05, **p < 0.01.

[0061] Figure 17: Effect of treatment on T cell immune responses. Splenocytes were harvested from each spleen and incubated for 24 hours in the presence of PPD or ESAT-6. Each dot represents the number of IFN-γ spots per E+06 cells. Median ± interquartile range for each treatment group. Statistical significance was tested using the nonparametric Mann-Whitney test, *p < 0.05, **p < 0.01.

[0062] Figure 18: Effect of treatment on T cell immune responses. Splenocytes were harvested from each spleen and incubated for 24 hours in the presence of HSP16 or PSTS1. Each dot represents the number of IFNγ spots per E+06 cells. Mean values were obtained. Statistical significance was tested using an unpaired t-test, *p > 0.05. DETAILED DESCRIPTION

[0063] The present inventors have surprisingly found that the administration of an immunotherapeutic agent based on cell wall fragments of a virulent strain of Mycobacterium tuberculosis complex, previously used exclusively for the prevention of TB (i.e., before a patient develops active TB, or after administration of chemoprophylaxis), is an effective treatment for patients with active TB who have not yet received chemotherapy.

[0064] The agent exhibits bactericidal effects as a stand-alone treatment and can synergistically exhibit bactericidal effects by improving the efficacy of standard TB chemotherapy when administered prior to or concurrently with chemotherapy.

[0065] When administered to experimental animals (mice) that had already developed active TB (and had not yet received chemotherapy), the agent immediately reduced bacterial loads by 15-fold within the first week as a standalone treatment, with an efficacy similar to standard chemotherapy. Furthermore, when administered to subjects infected with tuberculosis before or concurrently with chemotherapy, a significant synergistic effect was observed. In this case, bacterial loads were reduced by 225-fold within the first week, an order of magnitude greater than would be expected from a simple additive effect.

[0066] This novel use of the agent in TB treatment, for example, would allow for reduced use of chemotherapy and improved treatment of patients.

[0067] Such a solution could not be expected from the available prior art, primarily because it is well known that administration of such agents to subjects with active tuberculosis results in strong adverse side effects. This phenomenon has been particularly well studied in the context of tuberculin use without prior chemotherapy. In this context, the "Koch phenomenon" describes the appearance of a skin reaction within a few days at the site of inoculation with live tuberculosis bacilli or tuberculosis bacilli extracts (tuberculin) in guinea pigs with TB.

[0068] Without wishing to be bound by theory, Koch's phenomenon describes adverse reactions in subjects who already have TB and are vaccinated with agents based on, for example, cell wall fragments of virulent strains of the Mycobacterium tuberculosis complex.

[0069] In such cases, the inoculated area becomes hardened and darkened. This effect is not limited to the inoculation point, but extends to a diameter of 0.5-1 cm. The altered skin may also necrotize and slough off, leaving a flat ulcer that usually heals quickly and permanently without involving adjacent lymph nodes. This effect is caused not only by live tuberculosis bacteria, but also by inactivated tuberculosis bacteria or their fragments.

[0070] Therefore, the use of agents as described herein in patients with active TB without prior chemotherapy is strongly discouraged in the prior art due to the potential effects not only on the inoculation site but also on the damaged lungs.

[0071] In addition, the agents proposed in EP1090318B1 or EP2090318B1 are being developed and tested as prophylactic agents for people who have not yet been infected with TB or for administration to people with latent TB after standard chemoprophylaxis.

[0072] Therefore, it was not predicted that this agent would have such a strong effect on the bacterial load in subjects infected with active tuberculosis on its own, let alone such a strong synergistic effect with existing chemotherapy when administered prior to or concurrently with chemotherapy, especially in terms of efficacy, cost-effectiveness, reducing potential side effects of treatment, and most importantly, preventing the development of multidrug resistance, the latter being a key element for successful tuberculosis chemotherapy.

[0073] The present invention provides a therapeutic vaccine for use in a method of treating active tuberculosis. For example, this can be a medicament containing fragments from the Mycobacterium tuberculosis complex for use in a method of treating active tuberculosis, not limited to those specific embodiments disclosed herein.

[0074] More specifically, the present invention provides a liposome formulation (drug product) comprising fragments from Mycobacterium tuberculosis complex (MTB-C) strains (FCMtb, bulk drug substance) and a liposome-forming agent for use in a method of treating active tuberculosis.

[0075] In several examples of the present invention, the pharmaceutical product is referred to as "RUTI," which is a particularly preferred embodiment of the liposomal formulations described herein.

[0076] Unless expressly stated otherwise, the term "comprising" is used in the context of this application to indicate that other members may optionally be present in addition to the listed members introduced by "comprising".

[0077] However, specific embodiments of the present invention are envisaged, and the term "comprising" encompasses the possibility that no additional members are present, ie, for the purposes of this embodiment, "comprising" should be understood to have the meaning of "consisting of.

[0078] This detailed description discloses specific and / or preferred variations of each feature of the present invention. The present invention also considers as particularly preferred embodiments embodiments resulting from combining two or more specific and / or preferred variations described for two or more features of the present invention.

[0079] It is generally believed that the process of liposomation creates a lipid environment that promotes solubilization and results in a suspension of substances such as FCMtb (the bulk drug substance of the present invention).Liposomes within the meaning of the present invention may be unilamellar, multilamellar or a combination thereof.

[0080] FCMtb (raw material) can be any type of substance derived from an MTB-C strain, preferably a fragment derived from a protein and / or lipid. FCMtb within the meaning of the present application is typically a mixture of different protein antigens and lipids from MTB-C cells. Cell fragments can be obtained by any method known to those skilled in the art suitable for disrupting microbial or bacterial cells (e.g., specifically, MTB-C cells) (e.g., homogenization). Homogenization can be performed by ultrasonic treatment or by using small beads (e.g., silica or zirconium oxide / silica beads) with a diameter of about 0.1 mm in combination with a mechanical homogenizer.

[0081] A mechanical homogenizer that can be used is, for example, the BioSpec type. By this homogenization process, the MTB-C cells are disrupted, thereby obtaining small cell fragments, typically including nanometer cell wall fragments. A typical related feature of the production of cell fragments is the "detoxification" of the cell wall fragments by defatting, which is a process well known to those skilled in the art and allows the removal of endotoxin-like molecules. Thus, FCMtb is preferably detoxified, pasteurized and lyophilized. The final drug product (i.e., the liposomal formulation of the present invention, most preferably RUTI as described in the examples) is prepared as a liposomal formulation of FCMtb in sucrose, filtered through 0.22 nm. The final drug product, most preferably RUTI, can optionally be lyophilized to facilitate its storage. To this end, the final drug product, most preferably RUTI, can be dispensed into vials and lyophilized, for example at a temperature in the range of -45°C to 25°C and a pressure of 0.1 to 0.5 mbar (e.g., 0.150 mbar).

[0082] The liposomes according to the present invention typically have a size distribution in which at least 99.9% (by number) of the liposomes are smaller than 1 μm. In a particular embodiment, the z-average size of the particles, as determined by dynamic light scattering, is 120 nm or less, preferably 110 nm or less, more preferably 95 nm or less, and most preferably 80 nm or less. In dynamic light scattering, the z-average parameter is considered to be a stable and important number that can be obtained by this technique and is a size number that is preferably used for quality control purposes. Preferably, the liposomes of the formulation according to the present invention are monomodal, i.e. they show only one peak in the dynamic light scattering measurement. More preferably, the liposomes of the formulation according to the present invention are spherical, as can be tested by electron microscopy of a freeze-fracture preparation of the liposome formulation (drug product), as shown in Example 8. Thus, spherical means that for at least 90% of the liposome particles (by number), all surface points of a single particle are at similar or identical distances from the center of the liposome, i.e., the ratio of the minimum radius of such a particle to the maximum radius of the same particle is 0.6 or greater, 0.7 or greater, 0.8 or greater, or 0.9 or greater. The liposome formulation (pharmaceutical product) according to the present invention may comprise multilamellar or unilamellar liposomes, or mixtures thereof. In accordance with standard knowledge of those skilled in the art, dynamic light scattering measurements should be performed in a suitable buffer (i.e., a buffer that does not itself cause the rupture, disintegration or fusion of the liposomes or significantly destabilizes them physically in any other way). As a rule of thumb, any buffer may be suitable as long as the ionic strength and pH are comparable to those of the buffer in which the liposomes are formed. Preferably, a buffer similar or identical in composition to the buffer in which the liposomes are formed is used.

[0083] The liposomal formulation (drug product) further comprises 1 to 20% (w / v) sucrose, preferably 2 to 12% (w / v) sucrose, more preferably 3 to 8% (w / v) sucrose, most preferably 4 to 6% (w / v) sucrose. Approximately 5% sucrose is particularly preferred.

[0084] It is important to note that while each of these embodiments, one involving a specific particle size and the other involving the presence of sucrose, can be achieved individually, these embodiments should not be viewed as mutually exclusive and may well appear in combination.

[0085] In a specific embodiment, the above-mentioned liposome formulation has a z-average particle size in the range of 40 to 120 nm, preferably 50 to 100 nm, more preferably 55 to 95 nm, more preferably 55 to 80 nm. Thus, the z-average particle size is preferably measured by dynamic light scattering, as described in detail in the general description above and in the "Materials and Methods" section below.

[0086] In an alternative specific embodiment, the z-average particle size of the above liposomal formulation may be smaller, such that the liposomal formulation is an emulsion, ie in this specific embodiment the z-average size of the particles is preferably below 40 nm.

[0087] In a preferred embodiment of any one or more of the above embodiments, the liposomes of the formulation according to the present invention are also monodisperse, meaning that no significant width of the size distribution is observed. This is technically tested by a low polydispersity index (PDI) as determined by dynamic light scattering (e.g., 0.400 or less, preferably 0.300 or less). Thus, the liposome formulation is one in which the polydispersity index of the particles as determined by dynamic light scattering is 0.400 or less, preferably 0.300 or less, and most preferably 0.250 or less.

[0088] Fragments from Mycobacterium tuberculosis complex (MTB-C) strains can be obtained by a process comprising an upstream process and a downstream process. For illustrative purposes, the main steps are briefly described here and a specific mode of carrying out the process is given in Examples 2 and 3 below.

[0089] Upstream process (Example 2):

[0090] Step 1: Mtb-WSL culture

[0091] Step 2: Collection of Mtb-WSL and inoculation and growth of Mtb expansion cultures

[0092] Step 3: Collection of Mtb-expanded cultures (HMtb) and freezing

[0093] Downstream process (Example 3):

[0094] Step 4: Cell disruption and defatting (purification)

[0095] Step 5: Pasteurization

[0096] Step 6: Filter and Fill

[0097] Step 7: Freeze-drying, packaging, and labeling

[0098] In a more preferred embodiment of any of the above embodiments, the Mycobacterium tuberculosis complex (MTB-C) strain is a virulent strain of Mycobacterium tuberculosis complex (MTB-C). Virulent refers to the specific pathogenicity and / or ability of the bacillus to invade the host's tissues. The virulent strain can be any virulent strain of any species belonging to MTB-C, but preferably belongs to a strain of Mycobacterium tuberculosis. The MTB-C strain according to the present invention can be cultured by inoculation in a culture medium familiar to those skilled in the art, such as Middlebrook 7H10 or 7H11 agar, Sauton's medium or Proskauer-Beck medium. The virulent strain is preferably cultured for an extended period of time, such as a period of time equal to or greater than three weeks, preferably comprising 3 to 4 weeks. The culture temperature is preferably maintained at 34°C to 38°C. Once the culture is completed, the cells are collected and separated using techniques well known in the art, such as those described in patent application ES2231037-A1.

[0099] The liposome agent of the liposome preparation (drug product) is preferably a hydrogenated, partially hydrogenated or non-hydrogenated phospholipid. The phospholipid used can be or comprise, for example: phosphatidylcholine, phosphatidylserine and phosphatidylinositol. The most typical is phosphatidylcholine, which can be synthesized or isolated from various natural sources. Preferably, the liposome forming agent is or comprises phosphatidylcholine, which is selected from the group consisting of egg yolk phosphatidylcholine and soy phosphatidylcholine. Soy phosphatidylcholine is a complex mixture of various phospholipids, especially phosphatidylcholine, and is particularly preferred. Typical lipids that may also be included in the formulation (whether as liposome forming agents themselves or as other ingredients) are: dihexadecyl phosphate (DCP), dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol (DMPG), dioleoylphosphatidylcholine (DOPc), dioleoylphosphatidylethanolamine (DOPE), dioleoylphosphatidylserine (DOPS), dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylglycerol (DPPG), phosphatidylcholine (PC) and / or phosphatidylserine (PS), wherein the corresponding lipids may be hydrogenated, partially hydrogenated or non-hydrogenated. Liposomes may be formed using conventional helper lipids and techniques well known to those skilled in the art, such as those described in patent application ES2231037-A1.

[0100] It is also preferred that, in any of the above embodiments, the ratio of (a) fragments from the Mycobacterium tuberculosis complex (MTB-C) strain to (b) the liposome-forming agent is from 0.01:1 to 1:1, preferably from 0.06:1 to 0.1:1. In a more preferred embodiment of any of the above embodiments, the liposome formulation further comprises: (d) a tonicity active agent. Generally, all types of agents capable of altering surface tension values can be used as tonicity active agents within the meaning of the present invention, excluding compounds falling under the definition of liposome-forming agents described above. Various types of tonicity active agents are known to those skilled in the art and can be used in the liposome formulations according to the present invention. As known to those skilled in the art, tonicity active agents are generally chemical substances having a polar-nonpolar structure. Without wishing to be bound by any particular theory, tonicity active agents generally have a tendency to localize to the surface of the particles, thereby creating a monolayer at the interface that reduces the surface tension value. Tonicity active agents are also referred to as surfactants or active surface agents. In preferred embodiments of the liposome formulation containing a surfactant, the tonicity active agent is selected from sterols and their derivatives, such as cholesterol, and / or bile salts or their derivatives, such as bile salts. Particularly preferred embodiments are those wherein the tonicity active agent is selected from cholate, deoxycholate, cholesterol and cholesterol succinate monoester.A good but non-limiting way of practicing the invention is wherein the liposomes of the formulation comprise both soy derived lecithin and sodium cholate.

[0101] In an even more preferred embodiment, the liposome formulation comprising (d) a tonicity active agent is a liposome formulation wherein the ratio between (a) and (d) is from 0.05:1 to 3:5 (w / w). Various types of liposome forming agents can be used, as is well known to those skilled in the art.

[0102] The liposomes may optionally contain additives to improve their stability, such as vitamin E, which is known to act as a lipid antioxidant.

[0103] In a more preferred embodiment, the above-mentioned liposome preparation is a liposome preparation, wherein the fragments of MTB-C cells are or comprise cell wall fragments.

[0104] Any strain belonging to MTBC can be used, preferably any strain belonging to Mycobacterium tuberculosis. In another more preferred embodiment, the above-mentioned liposomal formulation comprises fragments of MTB-C strain NCTC13536, which was preserved in NCTC (National Type Culture Collection) in London, England in 2010 (Example 1). Another strain that can be used and its fragments (therefore can be included in the liposomal formulation) is called H37Rv, which is available, for example, from the National Type Culture Collection (NCTC) in London, England (preservation number NC007416) and is often used by researchers in this field. More than one strain can also be used, that is, the liposomal formulation comprises fragments of multiple strains (e.g., two, three or more strains).

[0105] Considering the approximately 4000 putative antigens of Mycobacterium tuberculosis, it is impossible to analyze the bulk drug substance of all these proteins. However, it has been shown that certain MTB-C proteins are relevant for the desired immune response. These are five protein bands with sizes of approximately 6, 10, 16, 30, 38 and 70 kDa (Renshaw et al., 2005, EMBO Journal 24, 2491-2498; Singh et al., 2005, Clin.Diagn.Lab.Immunol.12(2), 354-358; Rodriguez-Hernandez et al., 2020, Biomed and Biotechnol 21(11): 856-870; Meier et al., 2018, Frontiers in Immunology Vol 9 Article 2476). Therefore, in an even more preferred embodiment, the above-mentioned liposomal formulation comprises at least two, preferably three, more preferably four, more preferably five, and most preferably all of the following:

[0106] (i) a first polypeptide having a molecular weight of about 70 kDa as measured after electrophoresis on a sodium dodecyl sulfate (SDS) polyacrylamide gel, wherein the first polypeptide has a mass fingerprint similar to the mass fingerprint of Mycobacterium tuberculosis HSP70 protein (Rv0350),

[0107] (ii) a second polypeptide having a molecular weight of about 38 kDa as measured after electrophoresis on a sodium dodecyl sulfate (SDS) polyacrylamide gel, wherein the second polypeptide has a mass fingerprint similar to the mass fingerprint of the Mycobacterium tuberculosis PsTS1 protein (Rv0934),

[0108] (iii) a third polypeptide having a molecular weight of about 30-34 kDa as measured after electrophoresis on a sodium dodecyl sulfate (SDS) polyacrylamide gel, wherein the third polypeptide has a mass fingerprint similar to the mass fingerprint of the Mycobacterium tuberculosis Ag85 complex, comprising Ag85A and Ag85B proteins (Rv3804c and Rv1866c, respectively),

[0109] (iv) a fourth polypeptide having a molecular weight of about 16 kDa as measured after electrophoresis on a sodium dodecyl sulfate (SDS) polyacrylamide gel, wherein the fourth polypeptide has a mass fingerprint similar to that of the Mycobacterium tuberculosis HSP16 protein (Rv2031c),

[0110] (iv) a fifth polypeptide having a molecular weight of about 10 kDa as measured after electrophoresis on a sodium dodecyl sulfate (SDS) polyacrylamide gel, wherein the fifth polypeptide has a mass fingerprint similar to the mass fingerprint of the Mycobacterium tuberculosis CFP10 protein (Rv3874), and

[0111] (v) a sixth polypeptide having a molecular weight of about 6 kDa as measured after electrophoresis on a sodium dodecyl sulfate (SDS) polyacrylamide gel, wherein the sixth polypeptide has a mass fingerprint similar to that of the Mycobacterium tuberculosis ESAT-6 protein (Rv3875).

[0112] In a more preferred embodiment, the liposomal formulation further comprises a lipopolypeptide having a molecular weight of approximately 19 kDa as measured after electrophoresis on a sodium dodecyl sulfate (SDS) polyacrylamide gel, wherein the lipopolypeptide has a mass fingerprint similar to that of the 19 kDa lipoprotein antigen precursor of Mycobacterium tuberculosis, LpqH (Rv3763). The corresponding bands can be visualized by methods known in the art (e.g., silver staining). The present researchers were surprised to find that this polypeptide induced a high total IgG humoral response, which was possibly the highest humoral response of all the antigens in the formulation.

[0113] In Example 4 an example of how to identify a polypeptide or lipopolypeptide is given.

[0114] Even more preferably, the liposomal formulation is further characterized by the presence of at least one of the following Mycobacterium tuberculosis antigens, or fragments thereof: HSP70, PsTS1, B5 complex, HSP16, and most preferably the presence of at least one of HSP70, PsTS1, B5 complex, and HSP16. Within this meaning, a fragment is any portion of any of these polypeptides, such as a degradation product. Various methods are possible for obtaining such fragments, such as chemical or enzymatic hydrolysis, and it is not relevant whether fragmentation has been intentionally performed before or after liposome formation. Preferably, corresponding fragments can be assigned to their respective sources, for example by substantial overlap in amino acid sequence, such as at least 5, at least 10, or at least 20 consecutive amino acids.

[0115] The bulk drug substance (FCMtb) is produced by growing bacilli under stress conditions of starvation, low pO2 and low pH, conditions gradually achieved by culturing on solid media and leading to stationary growth, wherein the slow metabolism in stationary growth makes the bacilli more resistant to stress. The polyantigenicity of DS (polyantigenic protein mixture plus lipids, rather than purified antigens alone) appears to be an advantage, and in this sense, the disruption process was chosen to allow optimal presentation of this cell antigen mixture.

[0116] Another relevant feature of the manufacture of DS is the defatting of cell wall fragments, which allows the removal of endotoxin-like molecules.

[0117] Under such conditions, cultured bacilli primarily consist of non-replicating bacilli. Therefore, antigens present in FCMtb are expected to trigger a broad multi-antigenic response against both viable and non-replicating bacilli.

[0118] More details on the production process can be found in the Examples.

[0119] Mycobacterium glycolipids have long been considered to have immunomodulatory activity, and it is noteworthy that granulomatous reactions are induced and a potent adjuvant-like effect is exerted. Therefore, in a preferred embodiment, the above-mentioned liposome formulation contains lipids or derivatives thereof typically found in Mycobacterium tuberculosis, such as conjugated products (such as sugar-conjugated lipids). Several immunogenic lipid components (Brennan, Tuberculosis (Edinburgh), 2003, 83 (1-3), 91-97 have been identified in Mycobacterium tuberculosis samples; Chouldhary et al., 2018, Journal Immunology, 200: 3053-3066), and analytical methods (electrophoresis, SDS-PAGE, thin-layer chromatography, protein blotting) for its determination have been developed. Although the separation of each lipid component will require such intense processing that it is difficult to obtain quantitative data or percentages of each component detectable in MTB-C extracts or liposome formulations, qualitative characterization can be applied to characterize further preferred embodiments of the present invention. According to this further preferred embodiment, one or more mycolic acids are included, preferably belonging to any one or more of types I, III or IV. Alternatively or additionally, a glycoconjugated mycolic acid, preferably trehalose bimycolate, may be included in the formulation. Alternatively or additionally, a glycolipid, lipoarabinomannan (LAM), may be included in the formulation. Furthermore, the polyantigenicity of the fragments (a polyantigenic protein mixture plus lipids, rather than purified antigens alone) is considered an advantage, so that the skilled artisan can adjust the cell disruption process to allow for an optimal mixture of cell antigens.

[0120] Homogenization of the MTB-C cells is performed in the presence of one or more surfactants, preferably nonionic surfactants. Thus, the liposomal formulation (pharmaceutical product, preferably RUTI) may additionally contain one or more such surfactants. Many such surfactants are within the standard knowledge of those skilled in the art. Preferably, the nonionic surfactant used is selected from the group consisting of alkylphenol ethoxylates and sorbitan ester ethoxylates. More preferably, the nonionic surfactant is selected from the group consisting of octylphenol ethoxylates. Even more preferably, octylphenol ethoxylates having an ethylene oxide content of 7 to 8 moles are used, which can be found commercially under the name Triton X-100. The homogenate containing cell wall fragments is subjected to conventional treatment to separate and remove unbroken cells and dissolved components. For example, centrifugation at different speeds and washing with a buffer solution as described in patent application ES2231037-A1 can be used. After performing the purification process, a precipitate containing cell wall fragments is obtained. The precipitate is dispersed in phosphate buffered saline (PBS) buffer and subjected to conventional treatment to ensure complete inactivation of any MTB-C cells that may still survive the disruption and purification process. The treatment can be chemical, such as by formaldehyde treatment, or physical, such as by autoclaving or pasteurization. Examples of lipid characterization are given in Example 5.

[0121] In a further preferred embodiment, the above liposome formulation further comprises one or more salts or solutions thereof, wherein the preferred salt is sodium chloride.

[0122] In an even more preferred embodiment of any of the above, the liposome formulation is freeze-dried. The liposomes can be subjected to freeze-drying to obtain an immunotherapeutic agent in the form of freeze-dried liposomes. For this purpose, the dispersion can be dispensed into vials and freeze-dried at low temperatures, for example at a temperature in the range of -45°C to 25°C and a pressure of 0.1 to 0.5 mbar (e.g. 0.150 mbar). The vials obtained after freeze-drying contain a liposome formulation suitable as an immunotherapeutic agent, and they are preferably stored at very low temperatures, for example 5°C.

[0123] The present invention also provides a suspension in which the liposomal formulation (pharmaceutical product, RUTI) of any of the above claims is reconstituted in a solvent. In a preferred embodiment, the solvent of the suspension is aqueous, more preferably and most preferably is or comprises physiological serum. Methods for suspending liposomal formulations in solvents are well known to those skilled in the art. A particularly advantageous property of the formulation according to the present invention is that it can be suspended more rapidly than conventional liposomal formulations containing MTB-C fragments.

[0124] One object of the present invention is to provide a pharmaceutical composition comprising cell wall fragments of an MTB-C strain, wherein the pharmaceutical composition is or comprises a liposome formulation as described in any of the above embodiments, or a combination thereof. The primary objective of the pharmaceutical / galenic formulation of the bulk drug substance is to obtain an effective and stable suspension that is well recognized by cells and has the potential to elicit a relevant cellular immune response in humans or animals. To this end, the present invention also provides a pharmaceutical composition comprising the liposome formulation or suspension as described in any one or more of the above embodiments and a pharmaceutically acceptable carrier, excipient, or diluent. Various such carriers, excipients, and diluents are known to those skilled in the art and are not limited by the present application. Rather, any substance suitable as a carrier, excipient, or diluent may be used. In a preferred embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable adjuvant. Thus, an adjuvant is understood to include a substance included in this embodiment of the invention, wherein the adjuvant is a substance capable of stimulating the immune system in response to a target antigen when applied to a human or animal body, wherein the adjuvant itself does not confer immunity. Without wishing to be limited to any particular adjuvant substance, preferred embodiments are those in which the adjuvant is an aluminium salt (e.g. aluminium chloride), or mineral oil or a composition comprising mineral oil (e.g. Freund's incomplete adjuvant (IFA) or Freund's complete adjuvant (CFA)), or an ammonium halide (e.g. an alkylated ammonium bromide, such as dimethyldioctadecyl ammonium bromide).

[0125] The present invention also provides a product for use in a method of treating a human or animal body by therapy. That is, it provides a liposomal formulation according to any one or more of the above embodiments, a suspension according to any one or more of the above embodiments, or a pharmaceutical composition according to any one or more of the above embodiments, for use in a method of treating a human or animal body by therapy.

[0126] The drug can be administered to a mucosa (e.g., ocular mucosa, intranasal mucosa, oral mucosa, gastric mucosa, intestinal mucosa, vaginal mucosa, or urethral mucosa), or parenterally (e.g., subcutaneously, intradermally, intramuscularly, intravenously, or intraperitoneally). Parenteral administration is preferred. In a specific embodiment, the present invention provides the liposomal formulation, suspension, or pharmaceutical composition for injection.

[0127] In another embodiment, the present invention provides a liposomal formulation, suspension, or pharmaceutical composition for use in a method of treating active tuberculosis. The inventors of this study have found that the formulation according to the present invention is highly effective in treating patients with active TB, both as a standalone treatment and in combination with known chemotherapy. As a standalone treatment, the formulation has similar efficacy to chemotherapy. When combined with chemotherapy, the formulation provides a strong synergistic effect.

[0128] In the context of treating active TB, "chemotherapy" refers to any treatment recommended by the WHO. Those skilled in the art will know which treatments the WHO recommends at present. In particular, this includes but is not limited to various antibiotic therapies. For example, chemotherapy can include any of the following: isoniazid, rifampicin, rifapentine, rifabutin, levofloxacin, moxifloxacin, gatifloxacin, ofloxacin, ciprofloxacin, sparfloxacin, bedaquiline, putomanib, linezolid, clofazimine, cycloserine, terizidone, ethambutol, delamanid, pyrazinamide, thiamin, cilastatin, meropenem, etapenem, amikacin, streptomycin, ethionamide, prothionamide, para-aminosalicylic acid, kanamycin, capreomycin, amoxicillin / clavulanic acid, ampicillin / clavulanic acid, gentamicin, tobramycin, clarithromycin, azithromycin, thiosemicarbazide.

[0129] In the context of the present invention, antibiotic therapy is preferred. In a preferred embodiment, the antibiotic therapy includes at least one of ethambutol, isoniazid, pyrazinamide, rifampicin, streptomycin, amikacin, kanamycin, capreomycin, puromycin, enviromycin, ciprofloxacin, levofloxacin, moxifloxacin, ethionamide, prothionamide or cycloserine terizidone. In a particularly preferred embodiment, the antibiotic therapy includes RIMSTAR / HRZE.

[0130] "Active TB" is active TB in the lungs and / or active TB outside the lungs. Preferably, active TB is active TB in the lungs. In particular, active TB includes, but is not limited to, drug-susceptible TB, rifampicin-resistant TB, multidrug-resistant TB, or extensively drug-resistant TB.

[0131] Without wishing to be bound by theory, the most relevant difference between latent and active tuberculosis is the expected bacillary load, which in latent tuberculosis is at most about 10 5 CFU. Any higher bacillary load can be considered active tuberculosis. The bacillary load can be as much as x10,000 or x100,000 times higher, i.e., up to 10 9 / 10 10 CFU. The highest number of bacilli can be found during the exponential phase.

[0132] Furthermore, those skilled in the art are aware of other experimental methods to determine whether a patient has active TB, such as a positive smear test (due to high bacillary counts in the lungs) and / or TB lesions in the lungs, eg, visible on X-ray.

[0133] The appropriate dosage of the liposomal formulation, suspension, or pharmaceutical composition described above for use in a method of treating the human body by therapy depends on several parameters, including the method of administration and the subject to be treated. In a preferred embodiment, it is for administration to a human. In a preferred embodiment, this occurs at a dosage comprising 1 to 1000 μg FCMtb / dose, preferably 3 to 250 μg FCMtb / dose. In humans, 25 μg FCMtb / dose is particularly preferred.

[0134] In two more specific embodiments, the liposomal formulation, suspension or pharmaceutical composition described above with respect to its use in a method for treating active tuberculosis is: (a) used in the absence of chemotherapy or (b) used in the presence of chemotherapy. In particular, the liposomal formulation can assist chemotherapy by increasing the efficacy of chemotherapy, which can shorten the duration of treatment.

[0135] The liposomal formulation, suspension or pharmaceutical composition for use in a method for treating active tuberculosis can be administered in the form of a single dose or several (e.g., two, three, four, five or more) doses, by repeated administration at certain time intervals, whether in the presence or absence of chemotherapy. Most preferably, the liposomal formulation, suspension or pharmaceutical composition for use in a method for treating active tuberculosis can be administered once or twice.

[0136] In a particularly preferred embodiment, the liposomal formulation, suspension or pharmaceutical composition is administered to a human once during chemotherapy as a single dose of 25 μg FCMtb. This can be done at any time during chemotherapy for active TB.

[0137] Typically, the intensive chemotherapy phase of treatment of active TB may vary between different types of active TB (e.g., drug-sensitive TB, rifampicin-resistant TB, multidrug-resistant TB, or extensively drug-resistant TB), and the liposomal formulation may be administered at any point during this chemotherapy.

[0138] In another particularly preferred embodiment, the liposomal formulation is administered as a stand-alone therapy.

[0139] Examples 9 and 10 are examples of how the liposomal formulations according to the present invention can be used to treat active tuberculosis in the presence or absence of chemotherapy and in different dosing schedules.

[0140] Together, these examples clearly demonstrate that bacillary loads are reduced when the liposomal formulation (drug product, RUTI) is administered alone. This effect is comparable to chemotherapy during the first week. Without wishing to be bound by theory, the rapid effect suggests a mechanism involving both adaptive and innate immune responses. In addition, an even more pronounced reduction in bacillary load can be observed when combined with chemotherapy. This reduction is clearly synergistic.

[0141] At the same time, no pathological observations attributable to Koch's phenomenon were observed in any of the experiments (see parameter % lung lesion area). In contrast, both the formulation alone and in combination with chemotherapy (RIMSTAR) showed an improvement in % lung lesions compared to the control group.

[0142] Example 11 also demonstrates that no significant safety issues were observed when RUTI was administered to patients with MDR-TB or DS-TB together with standard antibiotic treatment (either 1 week or 1 month after starting antibiotic therapy).

[0143] A preferred embodiment of the use of the liposomal formulation, suspension, or pharmaceutical composition in treating active TB is in combination or adjunctive therapy. Medical practitioners often use adjunctive therapy to achieve better cure rates or a faster response to primary treatment. Combination therapy or adjunctive therapy involves the use of more than one drug to treat the human or animal body through therapy.

[0144] A preferred embodiment of the combination therapy is one in which the combination therapy comprises one or more of an antibiotic, preferably isoniazid and ansamycin, wherein the ansamycin is most preferably rifampicin.

[0145] A particularly preferred embodiment is one in which the combination therapy comprises oral administration of RIMSTAR (HRZE) at a single dose of 5 mg rifampicin / mL; 2.5 mg isoniazid / ml; 13.3 mg pyrazinamide; 9.2 mg ethambutol / ml. One skilled in the art will appreciate the standard duration and dosage of these chemotherapies in drug-susceptible TB (e.g., antibiotic therapy, see Examples 9 and 10).

[0146] In combination therapy or adjuvant therapy, administration of the two (or more) substances may be simultaneous, or may be accomplished in two (or more) inoculations separated in time.

[0147] Materials and methods

[0148] Reference Materials

[0149] a) Monoclonal antibodies: Specific monoclonal antibodies (anti-HSP70, anti-PSTS1, anti-HSP-16.3 (from Lionex Diagnostic GmbH, Braunschweig, Germany)) were used to identify the protein profile of the FCMtb batch.

[0150] b) Albumin standard: This standard is used for the determination of protein content and consists of bovine albumin in 0.9% saline solution (2 mg / ml) and is stored in sodium azide (manufacturer: Pierce).

[0151] c) Trehalose 6,6'-dimycolate (TDM) standard from Mycobacterium tuberculosis: Commercially available TDM (Sigma) was used to authenticate the FCMtb batch of TDM.

[0152] d) Mycolic acid standards from Mycobacterium tuberculosis: Commercially available mycolic acids (Sigma) were used to authenticate the mycolic acids of the FCMtb batch.

[0153] e) Molecular weight marker: A commercially available molecular weight marker, called SeeBlue Plus Prestained Standard (Invitrogen), was used.

[0154] Determination of parameters

[0155] a) pH

[0156] The pH of the reconstituted suspension of the bulk drug substance (FCMtb) (20 mg / ml) was determined potentiometrically according to Ph. Eur. 2.2.3 and USP <791>.

[0157] b) Moisture content

[0158] The test for the determination of residual moisture of the freeze-dried FCMtb was performed using Coulometric Karl Fisher equipment and following the general instructions for moisture determination of Ph. Eur. Method 2.5.12 and USP <921>.

[0159] c) Determination of total protein content

[0160] Tests for determination of the total protein content of FCMtb were performed using a commercial kit (BCA kit, Pierce) and following Ph. Eur. Method 2.5.33, Method 4 (bicinchoninic acid or BCA assay) and USP <1057>.

[0161] d) Protein identification by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)

[0162] The assay was performed according to Ph. Eur. method 2.2.31 and USP <726>; protein detection in gels was performed by modified Coomassie staining or silver staining. Test sample: FCMtb reconstituted in purified water at a concentration of 40 or 20 mg / ml. Reference solution: molecular weight markers, purified antigen, FCMtb reference standard.

[0163] Table 1: Reference Antigens

[0164] Purified antigen Mycobacterium tuberculosis HSP70 protein (Rv0350) Mycobacterium tuberculosis PsTS1 protein (Rv0934) Mycobacterium tuberculosis Ag85 complex (Ag85B and Ag85B) proteins (Rv1886c and Rv3804c) Mycobacterium tuberculosis HSP-16.3 protein (Rv2031c) Mycobacterium tuberculosis 19kDa protein (Rv3763) Mycobacterium tuberculosis CFP10 protein (Rv3874) Mycobacterium tuberculosis ESAT6 protein (Rv3875)

[0165] For Coomassie staining, Gel-Code blue staining reagent solution (Pierce) was used according to the manufacturer's instructions. For silver staining, Invitrogen's PROTSIL1 test kit was used according to the manufacturer's instructions. For Western blot analysis, proteins were separated by SDS PAGE according to standard methods known in the art, and then electrophoretically transferred to a PVDF membrane for immunoassay using specific monoclonal antibodies. The interaction of the antigen-antibody was visualized by incubation with an anti-antibody that triggered a chemiluminescent reaction. Antigens (mycobacterium tuberculosis HSP70 protein (70 kDa), mycobacterium tuberculosis PsTS1 protein (38 kDa), mycobacterium tuberculosis 85 complex (30-34 kDa)) and specific monoclonal antibodies anti-HSP70, anti-PsTS1, anti-HSP-16.3, and anti-Ag85B from Lionex (Braunschweig, Germany) were used.

[0166] e) Identification of mycolic acids

[0167] Mycolic acids in FCMtb were examined by one-dimensional TLC following Ph. Eur. method 2.2.27. Test sample: lyophilized FCMtb, 20 mg. Reference solution: mycolic acid standard (Sigma).

[0168] program:

[0169] a) Extraction process: The samples were extracted with chloroform:methanol (1:1) and then incubated overnight. The supernatant fraction was removed.

[0170] b) Mycobacterial Acid Esterification: Add 2 mL of methanol:toluene:sulfuric acid (30:15:1; v / v) to each tube and allow esterification to complete overnight. Then, add 2 mL of n-hexane. Recycle the supernatant into a new tube, and add another 2 mL of n-hexane. Dry under a stream of nitrogen and resuspend in 500 μL of hexane.

[0171] c) TLC: 20 μL of each sample was applied to a line parallel to the edge of a plate (Silica gel 60 (20×20 cm) (Merck)). Chromatography was performed three times in a saturated tank using a mobile phase (diethyl ether:n-hexane (15:85, v / v)). The plate was then allowed to air dry.

[0172] d) Mycolic acids were visualized by spraying the plates with a solution of phosphomolybdic acid in 96° ethanol and heating at 120° C. for 10 minutes.

[0173] Mycolic acids in FCMtb samples were determined by spot comparison with commercial mycolic acid standards. Results were expressed as qualitative data (presence (positive) / absence (negative) of assessed mycolic acids).

[0174] f) Identification of Trehalose 6,6'-dimycolate (TDM):

[0175] Test sample: lyophilized FCMtb, 20-40 mg. Reference solution: TDM standard (Sigma).

[0176] program:

[0177] (i) Extraction process: Samples were extracted with chloroform:methanol (1:1; v / v) and then incubated overnight. The supernatant fraction was dried under a stream of nitrogen and weighed. Finally, the dried sample was resuspended in chloroform at a final concentration of 40 mg / mL.

[0178] (ii) TLC: 10 ml of each sample was applied to a line parallel to the edge of a plate (Silica Gel 60 (20×20 cm) (Merck)). Chromatography was performed in a saturated tank using a mobile phase (chloroform:methanol:water (60:12:1; v / v)). The plate was then allowed to air dry.

[0179] (iii) Detection: TDM was visualized by spraying the plates with a 1% anthrone solution in sulfuric acid and heating at 120°C for 5 minutes.

[0180] (iv) Identification: TDM in FCMtb samples was determined by comparison with commercially available TDM used to generate standard spots. Results were expressed as qualitative data, ie, presence (positive) / absence (negative) of the assessed TDM.

[0181] g) Identification of Lipoarabinomannan (LAM):

[0182] For western blot analysis of LAM, compounds were separated by SDS PAGE according to standard methods and then electrophoretically transferred to nitrocellulose membranes for immunodetection using the specific antibody CS35. Antigen-antibody interactions were visualized by incubation with an anti-antibody (goat anti-mouse IgG IR dye 800CW) that triggered an alkaline phosphatase reaction.

[0183] h) Sterility

[0184] According to the knowledge of the skilled person, all processes requiring sterility are performed under aseptic conditions; this also applies if sterility is not explicitly mentioned for any given step requiring the same. Sterility tests are evaluated according to the provisions of Ph. Eur. 2.6.1 (USP <71>).

[0185] i) Mycobacterial inactivation

[0186] The inactivation of mycobacteria was assessed according to Ph. Eur. 2.6.2.

[0187] j) Bacterial endotoxins

[0188] Bacterial endotoxin testing (LAL test, Limulus Amebocyte Lysate) was performed according to the general instructions of Ph. Eur. method 2.6.14, following method D (chromogenic kinetic method) and USP <85>.

[0189] Bacillus disruption

[0190] The choice of broken bacteria is believed to allow for optimal presentation of cellular antigens, in particular cell wall antigens. The fragmentation of FCMtb (raw drug substance) was determined by both dynamic light scattering (as described below) and laser diffraction methods. Laser diffraction allows fragmentation to be measured in the range of 0.04 μm to 2000 μm. The assay was performed at the Scientific and Technical Service Center (Servicios Científico-Técnicos) of the University of Barcelona, Spain. Instrument: Coulter LS13320, equipped with a Universal Liquid Module (ULM). Solvent: purified water / mineral oil. Results: plotted as a histogram, representing the relative frequency (%) of the number of particles before the particle diameter (0.04 μm-2000 μm).

[0191] Determination of z-average particle size and polydispersity index

[0192] The average particle size as described in this document is determined by dynamic light scattering (DLS), which is based on the physical concept of Brownian motion of particles, defined in the Stokes-Einstein equation:

[0193]

[0194] in:

[0195] d(H) = hydrodynamic diameter

[0196] D = translational diffusion coefficient

[0197] k = Boltzmann constant

[0198] T = absolute temperature

[0199] η = viscosity

[0200] Without wishing to be bound by any particular theory, the Stokes-Einstein equations state that particles suspended in a liquid medium are in constant and random motion, the speed of which depends on their size: the larger the particle, the slower the Brownian motion.

[0201] In dynamic light scattering measurements, a sample containing the particles to be measured is illuminated with a monochromatic light source (preferably a laser), and the intensity of the scattered light fluctuates over time in a correlation function. For example, if large particles are being measured, the scattered light intensity fluctuates slowly due to their slow movement, and the correlation takes a long time to decay. On the other hand, if small particles are being measured, the scattered light intensity fluctuates rapidly due to their fast movement, and the correlation of the signal decays more quickly. According to the present invention, the particles are preferably measured using the following instrument: the Zetasizer nano zs (Malvern Instruments), using purified water / mineral oil as solvent.

[0202] Unless otherwise indicated, the instrument was used and, if applicable, adjusted and calibrated in accordance with the manufacturer's instructions.

[0203] The dimensions are calculated from the correlation function using various algorithms. In this case, the "cumulant analysis" defined in ISO 13321 Part 8 is applied. The correlation function fits the results to a single exponential curve that allows the calculation of the following parameters:

[0204] - Average size or z-average diameter of the particle distribution. This average size is an intensive average.

[0205] - Polydispersity index (PDI), corresponding to the breadth of the particle size distribution.

[0206] The results are typically plotted as a histogram showing the relative frequency (%) of the number of particles with respect to the particle diameter, which can be any diameter comprised in the range from 1 nm to 3 μm.

[0207] Diagnosis of active tuberculosis

[0208] The method for diagnosing active tuberculosis is well known to those skilled in the art. It is based on the detection of symptoms, such as cough, chest pain, hemoptysis, etc. that continue for three or more weeks. Systemic symptoms include low-grade fever, chills, night sweats, loss of appetite or easy fatigue. The diagnosis of tuberculosis is based on the detection of Mycobacterium tuberculosis bacilli in patient samples. Presumption is from the detection of acid-fast bacilli, which can be confirmed by its culture or by the detection of Mycobacterium tuberculosis nucleic acid.

[0209] Example

[0210] The present invention is illustrated below by way of examples. These examples are for illustrative purposes and should in no way be construed as limiting the scope of the present invention.

[0211] Example 1: Isolation of strain Mycobacterium tuberculosis NCTC 13536

[0212] The starting material for producing FCMtb (bulk drug substance) is an inoculum of strain NCTC 13536, synonymously referred to as 511 or Mycobacterium tuberculosis NCTC 13536 or Mycobacterium tuberculosis strain RUTI, which is a Mycobacterium tuberculosis strain isolated from an immunocompetent patient diagnosed with pulmonary tuberculosis in Barcelona, Spain. This strain was deposited in 2010 at the NCTC in London, UK, an official depository under the Budapest Treaty. This strain is also deposited at the Strain Collection of the Microbiology Service of the Sant Pau Hospital in Barcelona, Spain.

[0213] Two passages of the original strain were performed in 1995 and 1996, respectively. MSL PB#1 corresponds to the second passage of the original strain of Mycobacterium tuberculosis NCTC 13536, performed in October 1996, and the resulting 100 vials (3 ml sterile glass vials) were stored at -70 ± 5° C. This strain has low genetic polymorphism, as determined by standard methods in the art.

[0214] Example 2: Upstream process for producing bulk drug substance: production of MTB-C cells

[0215] The process flow diagram is given in Figure 1. The starting material for producing FCMtb (raw material) is an inoculum of strain Mycobacterium tuberculosis NCTC 13536 (Example 1). In order to ensure a continuous supply of this starting material, a seed batch system is preferably used. Therefore, a working seed batch (WSL) derived from a master seed batch (MSL) is used to produce FCMtb.

[0216] Table 2: Current process control (IPC) performed in upstream processes

[0217]

[0218] (1) Cultivation of Mtb WSL

[0219] DS production begins with thawing a vial of WSL. Each vial of WSL contains 0.5 mL and is used to generate two WSL cultures run in parallel. To this end, 0.2 mL of WSL is plated onto a 7H11 agar plate for each WSL culture and incubated at 37 ± 1°C for 15-16 days. A visual inspection for growth and contamination (IPC1) is performed after 9 ± 1 days of incubation.

[0220] (2) Collection of Mtb WSL and expansion of Mtb culture

[0221] After 15-16 days of incubation of Mtb WSL, the completeness of the incubation period was recorded (IPC2) and a visual inspection of the growth was performed to check the appearance and color of the bacterial culture (IPC3). The temperature register was controlled to confirm that the incubation temperature was maintained at 37±1°C throughout the incubation period (IPC4).

[0222] The colony from the Mtb WSL culture was then transferred to a test tube containing a small amount of glass beads, and water for injection was added until the final concentration of Mtb was 9-10 mg / mL to obtain an Mtb inoculum for culture expansion. After mixing the bacterial suspension, 200-1008 7H11 agar plates were inoculated with the Mtb inoculum using a sterile cotton swab soaked with the bacterial suspension to obtain a confluent culture. The plates were incubated at 37 ± 1 ° C under atmospheric control for 21 ± 1 days. At this point, two IPCs were performed on the final inoculum suspension (inoculum after plate inoculation): the final inoculum viable concentration (CFU / mL) (IPC5) and the final inoculum sterility test (IPC6).

[0223] During incubation of the inoculated plates at 37±1°C, visual inspections for growth and absence of contamination were performed before 10 days (IPC7) and after 14±2 days (IPC8) of incubation.

[0224] (3) Collection and freezing of Mtb

[0225] After incubation at 37 ± 1 ° C for 21 ± 1 days and before collection, the temperature register is checked to confirm that the incubation temperature is maintained at 37 ± 1 ° C throughout the incubation period until collection (IPC9). Then, the bacterial growth is collected from the agar plate and transferred to a sterile test tube to obtain a collection of Mtb (HMtb). A visual inspection is performed during the collection to check the purity of the appropriate Mtb growth and bacterial culture, and a final registration is performed once the collection is completed (IPC10). The weight of the HMtb should be within the range of 72-400 g. The HMtb is then frozen and stored at -80 ° C ± 5 ° C. The sterility test of the HMtb is performed on the first collected plate (IPC11).

[0226] Example 3: Downstream Process for Producing Bulk Drug Substances

[0227] The flow chart of the process is shown in Figure 2.

[0228] Table 3: Current process control (IPC) performed in downstream processes

[0229]

[0230] (4) Cell disruption and defatting (purification)

[0231] Frozen HMtb (Example 2) was thawed by thawing ramp (step 1: 10 hours at 10°C (0.1°C / min); step 2: 6 hours at 4°C (0.1°C / min); and step 3: ∞ hours at 8°C (0.1°C / min). Sterile PBS buffer (pH 7.0-7.7, see IPC12) containing 4% triton-X100 was then added and subsequently transferred to a sterile disruption tank containing sterile silica-zirconia beads. Cell disruption was then performed in a bead mill at 4500 rpm for 45 minutes at 20±2°C.

[0232] Once the process is complete, the cell-disrupted fraction is separated from the beads by continuous washing (repeated shaking and sedimentation cycles) in sterile PBS buffer (pH 7.0-7.7) containing 4% triton-X100. At this point, the pH of the cell debris (supernatant) is controlled (IPC13). A final centrifugation at 845 g for 30-45 minutes at 4°C is then performed to separate the bacterial debris from the intact bacilli and beads. The supernatant is collected.

[0233] In order to remove the cytoplasmic fraction and obtain a suspension rich in cell debris, the supernatant was centrifuged twice at high speed (27,000g) at 4°C for approximately 60 minutes. After the first centrifugation, the yellow supernatant (rich in soluble proteins and lipids) was discarded and the precipitate was resuspended in PBS and further centrifuged under the same conditions as above. Afterwards, the appearance (IPC14) of the discarded supernatant must be clear and colorless. The resulting precipitate (>10 g) was weighed and resuspended with an appropriate volume of sterile PBS (45-150 mL) to obtain a maximum concentration of 0.2 mg / mL (DS or FCMtb suspension).

[0234] In addition, the sterility of PBS used for cell debris purification and pellet resuspension was tested (IPC15).

[0235] (5) Pasteurization

[0236] To inactivate any residual bacilli, the entire bulk DS, FCMtb (raw drug substance) suspension was pasteurized at 65 ± 2°C for 60-65 minutes. However, prior to pasteurization, an aliquot of the FCMtb suspension was removed to test for cell viability (CFU / mL) (IPC16). Once the material was pasteurized, it was physically isolated from untreated material, i.e., the space used prior to pasteurization was clearly separated from the space used in the subsequent filling process.

[0237] (6) Filter and fill 3

[0238] After pasteurization, the bulk DS exits the BSL3 chamber and enters a positive pressure sterile room (Class B / A-1). Residual beads are removed by filtration (40 μm) and sterile, depyrogenated vials are filled with 0.5-2 ml of FCMtb suspension. Four filled vials are used for sterility testing (IPC17). Finally, the filled vials are freeze-dried.

[0239] (7) Freeze drying, packaging and labeling

[0240] All vials were lyophilized at a temperature of about -45°C to 30°C and a pressure of 0.310 mbar for about 18 hours (0.5 ml volume per vial) and under an N2 atmosphere.

[0241] Once lyophilized, the vials are sealed, labeled, and capped using aseptic technique. This is a continuous process: the filled vials are visually inspected and, if they are correct (good appearance of lumps, correct dosage, unbroken vials), they are first capped with aluminum caps and then labeled as follows (if the visual inspection of the capped vials is correct):

[0242] ■Vial number,

[0243] ■Product name,

[0244] ■Batch number,

[0245] ■Date of manufacture,

[0246] ■Storage conditions,

[0247] ■Shelf life.

[0248] In general, vials were visually inspected after freeze-drying, during capping, and during labeling.

[0249] The packaged DS were then stored at -20°C ± 5°C.

[0250] Example 4: Protein Characterization of Drug Substances

[0251] Based on the literature (Andersen P., 1997, Sc and J. Immunol.; 45(2): 115-31; Geisel et al., 2005, J. Immunol.; 174(8): 5007-15; Stewart et al., 2005, Infect. Immun., 73(10): 6831-7., Wang et al., 2007, J. Mol. Biol., 366(2): 375-81; Rodriguez-Hernandez et al., 2020, Biomed and Biotechnol 21(11): 856-870; Meier et al., 2018, Frontiers in Immunology Vol. 9Article 2476), some protein bands were selected as representatives for protein spectrum evaluation: heat shock protein (HSP) 70 protein (Rv0350); phosphate binding (PsTS1) (38kDa) protein (Rv0934); outer cell wall antigen 85 complex (30-34kDa) (Rv3804c & Rv1866c); heat shock HSP16 (16kDa) protein (Rv2031c); 19kDa protein (Rv3763), CFP10 protein (Rv3874) and ESAT-6 protein (Rv3875).

[0252] (A) Determination of total protein content: Total protein levels in FCMtb were quantified using the bicinchoninic acid (BCA) assay. Total protein constitutes approximately 15% (w / w) of the FCMtb content. Reference standards FCMtb-81, FCMtb-83, FCMtb-86, and FCMtb-87 contain 189, 187, 150, and 160 μg protein / mg FCMtb, respectively.

[0253] (B) Protein profile identification by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE): The protein profile of the raw drug substance FCMtb was determined by comparison with reference antigens from Mycobacterium tuberculosis corresponding to: ESAT6 (6 kDa) (7); CFP10 (10 kDa) (6); HSP16 (16 kDa), Ag85 complex (30-34 kDa) (1); PsTS1 (38 kDa) (2); HSP70 (70 kDa) (4) and molecular weight marker MW (5), as shown in Figure 3. The protein profile of the raw drug substance FCMtb was determined by Coomassie staining and the bands (approximately 70 kDa, 38 kDa, 30-34 kDa and 16 kDa) were identified. The 19 kDa (lipopolypeptide), 10 kDa and 6 kDa bands were identified by silver staining.

[0254] (C) Protein profile identification by Western blotting using specific monoclonal antibodies: The protein profile of the drug substance FCMtb was determined by Western blotting using the following monoclonal antibodies (mAbs): anti-HSP70 (70 kDa), anti-PsTS1 (38 kDa), anti-Ag85B (85 complex 30-34 kD), and anti-HSP16.3 (16 kDa) (see Figure 3c ).

[0255] FCMtb-81 is a reference batch of FCMtb (bulk drug substance) according to a preferred mode of carrying out the present invention.

[0256] Example 5: Lipid Characterization of Bulk Drug Substance

[0257] The characterization of the lipid profile of FCMtb (raw drug substance) consisted of a fractionation process based on chloroform:methanol (1:1) extraction. The fractionation process performed in these studies was based on the procedure described by Delmas et al., 1997, Glycobiology 7(6), 811-7. Thin layer chromatography (TLC) is a method used to analyze the lipid and glycolipid content present in FCMtb. Specifically, polyacyltrehalose (PT), trehalose dimycolate (TDM), diacyltrehalose (DAT), sulfatides (SF) and other phospholipids as well as mycolic acids were identified in different FCMtb batches by thin layer chromatography (TLC). See Figure 4aThe supernatant was analyzed for its trehalose 6,6'-dimycolate (TDM) content by TLC. The mycolic acid content was determined in the precipitate using the TLC method. Although there are no known quantitative data on the lipid profile of MTB-C, the qualitative lipid profile established in the study is consistent with current scientific knowledge and allows for a standard characterization of the immunogenic lipids known to date. Overall, it has been demonstrated that the lipid content is consistent in different batches of liposomes containing FCMtb according to the invention. Figure 4c Identification of LAM and LM is shown.

[0258] Example 6: Characterization of disrupted cell material

[0259] Preliminary results using both methods indicate that the fragment size of FCMtb (raw drug substance) is mainly below 1 μm (99% < 1 μm), which is confirmed by electron microscopy examination of FCMtb: the fragment size is mainly in the range of 100 nm to 300 nm.

[0260] The level of residual DNA after extraction with a phenol / chloroform mixture was investigated by absorbance at 260 nm (detection limit: 0.2 μg DNA / mg FCMtb). Typical results obtained so far are below 10 μg DNA / mg FCMtb.

[0261] The consistency of the manufacturing process of the drug substance was demonstrated by reproducible lipid and protein profiles for different FCMtb batches.

[0262] Example 7: Effect of sucrose

[0263] In initial tests, one of the following excipients, (a) 1.5% glycine and (b) 5% sucrose, was optionally introduced into liposome formulations containing fragments of MTB-C strain NCTC 13536. Subsequently, the physicochemical properties and biological activities associated with the two formulations were comparatively evaluated.

[0264] The results of the measurements obtained after reconstitution of the lyophilized liposome compositions and after testing according to the current batch release specification parameters are shown in Table 4.

[0265] Table 4: Strength results for three different formulations measured after reconstitution of the lyophilized liposome compositions.

[0266]

[0267] The researchers of this study surprisingly found that the 5% sucrose formulation offers the advantage of better physicochemical results, such as moisture content or reconstitution time, respectively. But the most important fact is the significant reduction in liposome aggregation (particle size, z-average) shown with the formulation containing sucrose compared to the other two formulations. Dynamic light scattering analysis has shown that the z-average of the liposomes containing sucrose is 75 ± 20 nm (polydispersity index ≤ 0.350). Electron microscopy of freeze-fracture preparations of the liposome formulation containing sucrose showed a mixture of multilamellar and unilamellar liposomes with a size of 40 to 100 nm ( Figure 5 ).

[0268] Due to this improved parameter and the lower moisture levels observed for the 5% sucrose formulation (≤ 2%), it was expected that the stability results for this formulation would be improved.

[0269] Example 8: Manufacturing process of freeze-dried liposome preparation (drug product)

[0270] One embodiment of a process for manufacturing a pharmaceutical composition comprising a liposome formulation according to the present invention is shown in FIG6 .

[0271] The manufacturing process of a drug product (most preferably RUTI) can be divided into the following major steps:

[0272] -Step 1: Preparation of LCS bulk components

[0273] -Step 2: Preparation of LCS bulk

[0274] -Step 3: Dilution of LCS bulk to obtain LS bulk and terminal sterilization by filtration

[0275] -Step 4: Filling

[0276] -Step 5: Freeze-drying, encapsulation, labeling and packaging

[0277] (1) Preparation of LCS bulk components

[0278] The lipid phase of the liposomes consisted of a soy lecithin solution, a sodium cholate solution, and DS (FCMtb, see Examples 1-7). Soy lecithin was dissolved in anhydrous GR ethanol (1:1; w / w), and sodium cholate was dissolved in WFI (1:5; w / w). The solution was sterilized by filtration through a 0.2 μm membrane filter. The lipid phase was prepared in a Class C chamber.

[0279] The aqueous phase consisted of 0.9% sterile saline solution (NaCl) diluted with sterile WFI to give a final saline solution of 0.34%.The saline solution was prepared in a clean Class A / B room.

[0280] (2) Preparation of LCS bulk

[0281] To prepare LCS bulk, mix sodium lecithin solution and sodium cholate solution; then add lyophilized FCMtb with stirring. The ratio of the components is 0.03:0.2:0.7 (FCMtb (active drug substance): sodium cholate: soy lecithin; w / w / w).

[0282] The aqueous phase was transferred to a sterile stainless steel blender. The lipid phase (containing soy lecithin, sodium cholate, and DS FCMtb) was then added at a ratio of 2.7:1 (aqueous phase: lipid phase, w / w). These phases were mixed at 22,000 rpm for 3 minutes for homogenization and liposome formation.

[0283] After homogenization, the LCS bulk was transferred to another container and allowed to settle for at least 5 minutes.In-process control of particle size was performed on the LCS bulk.

[0284] (3) Dilute the LCS bulk to obtain LS bulk and sterilize by filtration

[0285] A 6.0% (w / w) sucrose solution was prepared in WFI and sterilized by filtration (0.2 μm membrane filter). The sucrose solution was then mixed with sterile WFI and LCS bulk in appropriate proportions to obtain a final LS bulk consisting of 10.5 mg LCS / mL in 5% sucrose solution (1.2 L), which was sterilized by filtration (0.2 μm membrane filter).

[0286] Perform filter integrity control before and after filter use. Perform PC before and after sterile filtration: visual inspection (IPC2), contamination detection (IPC3), particle size (IPC4), and sterility (IPC5).

[0287] (4) Filling

[0288] Vials were filled with 0.4 mL of LS bulk (under continuous stirring) and partially sealed for lyophilization.

[0289] (5) Freeze-drying, packaging and labeling

[0290] The freeze-drying process was carried out at a temperature ranging from -45°C to 25°C and at 0.150 mbars. The process lasted 27 hours.

[0291] At the end of lyophilization, the vials were completely stoppered under an N2 atmosphere. The vials were then sealed, labeled, and stored at 5°C ± 3°C. Visual inspections were performed on each vial throughout the process, after lyophilization, after sealing, and after labeling.

[0292] The label contains the following information:

[0293] - Vial number

[0294] -Product Name

[0295] -batch number

[0296] -Date of manufacture

[0297] -Storage conditions

[0298] -Shelf life

[0299] - "Experimental use".

[0300] For clinical batches, the labeling contains the requirements described in GMP.

[0301] Example 9: Efficacy of a liposomal formulation (drug product) containing fragments from Mycobacterium tuberculosis complex (bulk drug substance, FCMtb) in the C3Heb / FeJ mouse model of active TB, 200 μg dose

[0302] The liposomal formulation (drug product, Example 8) containing fragments from the Mycobacterium tuberculosis complex (bulk drug substance, Examples 1-7) used in this example is the most preferred embodiment: RUTI. The following examples, including all relevant figures, are labeled accordingly. Dosages refer to the bulk drug substance FCMtb.

[0303] Purpose

[0304] The aim of these experiments was to evaluate the efficacy of a liposomal formulation (drug product, RUTI) containing fragments from the Mycobacterium tuberculosis complex (bulk drug substance, FCMtb) in the C3Heb / FeJ model of active M. tuberculosis infection by reducing bacillary load and improving lung injury and T cell immune responses under chemotherapy (RIMSTAR, HRZE).

[0305] Experimental design

[0306] A study was conducted to evaluate the effect of liposomal formulations containing fragments from the Mycobacterium tuberculosis complex in reducing bacillary load, improving lung injury and cellular immunological responses in mice infected with Mycobacterium tuberculosis (C3HeB / FeJ) under chemotherapy (RIMSTAR, HRZE). The liposomal formulations were administered at a single dose of 200 μg FCMtb at weeks 0, 1, 2, and 3 after the start of chemotherapy treatment, and sacrificed starting one week after RUTI vaccination (Table 5 and Figure 7).

[0307] Table 5: Time of Example 9, ATB antibiotic (HRZE).

[0308]

[0309]

[0310] The results presented are from a single study using five female C3HeB / FeJ mice per group. Mice were infected intravenously via the tail vein with 1.0E+04 Mycobacterium tuberculosis H37Rv Pasteur strain (from batch 10 of the UTE stock IGTP). RIMSTAR (HRZE) chemotherapy was administered orally as a 0.2 mL dose (5 mg rifampicin / mL; 2.5 mg isoniazide / mL; 13.3 mg pyrazinamide; 9.2 mg ethambutol / mL).

[0311] RUTI / sham (saline control) groups were administered subcutaneously at weeks 0, 1, 2, and 3 after the start of chemotherapy. The RUTI dose was 200 μg FCMtb in 0.2 mL (see Table 5). Animals were sacrificed at various time points (Figure 7, Table 5). Following sacrifice, lungs and spleens were removed to assess bacillary load (CFU), lung injury, and T cell immune responses (from splenocytes).

[0312] The effects of RUTI on reducing bacillary load, improving lung injury and cellular immune response (ELispot) were investigated.

[0313] All procedures were performed in a BLS3 safety facility (CMCiB) according to experimental protocol DMAH6119, which was reviewed by the Animal Experimentation Ethics Committee of the Germans Trias i Pujol University Hospital (registration number B9900005) and approved by the Department of Agriculture, Livestock, Fisheries, Food and Natural Environment of the Government of Catalonia (Dept d'Agricultura, Ramaderia, Pesca, Alimentació i MediNatural), in accordance with current national and EU legislation on the protection of experimental animals (Catalan Government Act 1997; Spanish Royal Decree 1201 / 2005; and European legislation 86 / 609 / EEC; 91 / 628 / EEC; 92 / 65 / EEC and 90 / 425 / EEC).

[0314] Detailed experiments

[0315] Bacillus load (CFU / mL):

[0316] Lung lobe samples from each animal were collected, homogenized and several dilutions were plated on nutrient Middlebrook 7H11 agar (BD Diagnostics, USA). The number of CFU was counted after 21 days of incubation at 37°C and the results were expressed as CFU / mL.

[0317] Lung pathology:

[0318] The right lower lobe samples were fixed in 10% buffered formalin, embedded in paraffin, and stained with hematoxylin-eosin. 5 μm sections were used for microscopic observation, and the damaged area was analyzed using the NIS Elements D version 3.0x software package (Nikon Instruments Inc., Tokyo, Japan). The percentage of damaged area to total lung area was determined using four resections of the blocks containing samples from all groups.

[0319] T cell immune response (Elispot technology):

[0320] Mechanically destroy spleen and filter through 40 μ m cell strainer (BD Diagnostic, the U.S.), make erythrocyte in lysis buffer (Tris 17mM, NH4Cl 0.14M) incubate 8 minutes.In RPMI 1640 (10% fetal bovine serum, streptomycin 100 μ g / ml, penicillin 100U / ml, 2-mercaptoethanol 0.025mM, sodium pyruvate 2mM) supplemented in 96-well plates (ELISpot plates) at 37 ℃ and 5% CO2, with or without stimulant (PPD, ESAT-5, HSP16.3 and PsTS1) carry out cell culture.ELISPOT is based on cytokine production and measures T cell.Cytokine secreted by activated T cell alone is as discrete spot on plastic plate, and it is counted to give the quantity of activated T cell.Each well contains 125,000 cells. The final concentration of PPD was 30 μg / ml well, and the final concentration of ESAT-6, HSP16.3, and 85B antigens was 10 μg / ml well. Stimuli were from Staten Serum Institute Denmarx (PPD) and Lionex Diagnostic and Therapeutics (ESAT-6, HSP16.3, and 85B antigens) in Germany. ELISPOT was performed after 24 hours of incubation with the stimuli to assess the number of spots induced under each condition. Spots were counted using an ELISPOT reader (IGTP).

[0321] Medicinal products containing Mtb cell wall fragments:

[0322] The raw drug substance (DS) consists of purified cell wall fragments of Mtb, called FCMtb. The preparation process of FCMtb is obtained by cell disruption of bacterial cultures of collected Mtb strains (see previous examples). The fragmented cell walls are then purified in the presence of Triton X100 to remove soluble components and endotoxin-like molecules. The structure of DS is closely related to the properties of the Mtb cell wall. Therefore, DS is mainly composed of a heterogeneous range of proteins and lipids from Mtb strains. RUTI, or IMP (drug product), is a liposome suspension of DS containing charged excipients. It is presented as a dry powder (for reconstitution with water for injection) and is stable at 5°C for at least 12 months.

[0323] Table 6: Quantitative and qualitative composition of each vial of RUTI.

[0324]

[0325]

[0326] 1 Contains phosphatidylcholine (not less than [NLT] 94.0%)

[0327] 2 Add as a 0.9% NaCl solution

[0328] 3 Disappears during processing

[0329] For administration to mice, the investigational medicinal product (IMP, medicinal product, RUTI) should be reconstituted with 0.266 ml of water for injection to yield a solution containing 1002.1 μg / ml FCMtb (266.7 μg / vial of FCMtb). The placebo used in this nonclinical trial consisted of normal saline (0.9% NaCl). A total volume of 0.200 ml of the reconstituted RUTI vaccine was administered subcutaneously in the neck area.

[0330] Data Analysis-Statistical Methods:

[0331] Graphical and statistical analyses were performed using GraphPad Prism version 8.0 for Windows (GraphPad Software, San Diego, California, USA), with differences of p < 0.05 considered statistically significant. Data are presented as the mean of each treatment group. Statistical analysis was performed using an unpaired t-test to find any differences between experimental groups.

[0332] result

[0333] Bacillary burden in lung and spleen tissues

[0334] RUTI was administered subcutaneously to infected C3HeB / FeJ mice to test its effect in an active TB infection model. The lung bacterial load and lung injury after RUTI vaccine administration at different time points after the start of ATB were lower and better than those in the control group (Figure 8, Figure 9 ).

[0335] RUTI also showed efficacy in a humanized TB model of Mtb-infected C3HeB / FeJ mice. Thus, when administered at different time points during ATB1 (e.g., day 0, 1 or 2 weeks), a single dose of RUTI reduced bacillary loads in the lungs and spleen (see Figure 8).

[0336] Pathology in lung tissue

[0337] The data showed that when RUTI was combined with chemotherapy at a dose of 200 μg FCMtb, lung lesions were significantly reduced. Thus, a single dose of RUTI reduced the area of lung lesions when administered at different time points after the start of ATB (see Figure 9 No safety issues were observed in mice at any time point following vaccination.

[0338] Cellular immune response (ELIspot)

[0339] The data showed that T cell immune responses against PPD, HSP-16.3 (16 KDa), PsTS1, and ESAT-6 were slightly induced in RUTI-vaccinated animals compared with the ATB group ( Figure 10 、 Figure 11 and Figure 12 ).

[0340] in conclusion

[0341] Using the active TB model in C3HeB / FeJ (highly similar to the liquefied and cavitary lesions of human TB), administration of RUTI (a preferred embodiment of the liposomal formulation described herein) resulted in a significant reduction in bacillary load and lung lesion area at various times after the start of chemotherapy. The RUTI-administered group showed induction of T cell immune responses against structural and latent antigens, albeit with mild effects. Thus, these data demonstrate for the first time the efficacy and safety of a therapeutic vaccine administered after induction of active TB and concurrently with standard chemotherapy.

[0342] Traditionally, therapeutic vaccination has received much attention due to its potential toxicity, a concept summarized in the so-called "Koch's phenomenon". The experimental TB mouse model in the C3HeB / FeJ strain provides a unique opportunity to test it because it develops "human-like" lesions with liquefaction, which have many similarities with human TB. Surprisingly, the administration of RUTI has been shown to improve the efficacy of chemotherapy by reducing the bacillary load and lesion area in the lungs when administered as early as day 0 of chemotherapy initiation, showing a significant improvement in lesion area and a trend towards a decrease in bacillary load (which became significant at week 3).

[0343] Regarding its mechanism of action, RUTI is designed to induce a multi-antigen response, enabling the detection of dormant bacilli. Even when the immune responses in splenocytes obtained with different Mtb antigens were not significantly different from controls, it showed a trend toward increased Th1 responses against HSP16.3 and PPD. With ESAT-6 and 85B antigens, there was an overall decrease, corresponding to a decrease in bacillary load over time. These data are intended to test immunotherapeutic vaccination in patients with active TB.

[0344] In summary, nonclinical results showed that RUTI had efficacy (bacillary load and lung lesion area) independent of when it was administered during standard chemotherapy. The results support the selection of a vaccination time point of 0 days from the start of chemotherapy. In addition, the reduction in bacillary load compared to chemotherapy was maintained over time.

[0345] Example 10: Efficacy of a liposomal formulation (drug product) containing fragments from Mycobacterium tuberculosis complex (bulk drug substance, FCMtb) in the C3Heb / FeJ mouse model of active TB, 200 μg and 25 μg doses

[0346] The liposomal formulation (drug product, Example 8) containing fragments from the Mycobacterium tuberculosis complex (bulk drug substance, Examples 1-7) used in this example is the most preferred embodiment: RUTI. The following examples, including all relevant figures, are labeled accordingly. Dosages refer to the bulk drug substance FCMtb.

[0347] Purpose

[0348] RUTI is administered in different schedules to evaluate its activity alone and in combination with chemotherapy. If chemotherapy is administered, different time periods are studied, such as 4 days or 25 days.

[0349] Male / female (1:1) C3HeB / FeJ animals (5-7 weeks old) were obtained from Jackson Laboratories (Harbor, Maine, USA).

[0350] Each mouse (IGTP-name-year) was identified by microarray (Anibio). Mice were monitored daily, weighed, and, if necessary, euthanized with isoflurane (inhalation overdose) according to a strict experimental protocol to ensure animal welfare.

[0351] The results presented are from a single study using 6 (3 female and 3 male) C3HeB / FeJ mice per group.

[0352] Mice were infected intravenously via the tail vein with 4.0E+04 Mycobacterium tuberculosis H37Rv Pasteur strain (batch 10 from UTE stock IGTP).

[0353] RIMSTAR (HRZE) chemotherapy was administered orally as a 0.2 mL dose (5 mg rifampicin / mL; 2.5 mg isoniazid / mL; 13.3 mg pyrazinamide; 9.2 mg ethambutol / mL).

[0354] In one experiment, 0 weeks after the start of chemotherapy (0d) and at the end of chemotherapy (see Figure 13c ), RUTI / sham (saline control) group was administered subcutaneously. In another experiment, when the shorter chemotherapy treatment time point 0 ( Figure 13a ), RUTI / sham (saline control) group. In further experiments, only RUTI / sham (saline control) and no chemotherapy ( Figure 13b ).

[0355] The animals were sacrificed at various time points during the experiment, for example 1 week and / or 6 weeks after RUTI administration (see Figure 13a After sacrifice, lungs and spleens were removed to assess bacillary load (CFU), lung injury, and T cell immune response (from splenocytes). Blood was recollected and serum was obtained and stored at -80°C.

[0356] The effects of RUTI on reducing bacillary load, improving lung injury, and cellular immune responses (ELispot) were investigated. All procedures were performed in a BLS3 safety facility (CMCiB) according to experimental protocol DMAH9559, which was reviewed by the Ethical Committee for Animal Experimentation of the Instituto Germans Triasi Pujol (IGTP) (registration number B9900005) and approved by the Department of Agriculture, Livestock, Fisheries, Food and Natural Environment (Dept d'Agricultura, Ramaderia, Pesca, Alimentació i MediNatural) of the Government of Catalonia in accordance with current national and EU legislation on the protection of experimental animals (Catalan Government Act 1997; Spanish Royal Decree 1201 / 2005; and European legislation 86 / 609 / EEC; 91 / 628 / EEC; 92 / 65 / EEC and 90 / 425 / EEC).

[0357] Detailed experiments

[0358] For bacillary load (CFU / mL), lung pathology, T cell immune response (Elispot technology) and data analysis - statistical methods, see Example 9.

[0359] Products containing Mtb cell wall fragments:

[0360] The drug product RUTI is a vaccine presented as a dry powder for reconstitution, containing 266.7 μg or 33.3 μg of FCMtb (the active pharmaceutical ingredient, disrupted cells of Mtb) per vial. It is supplied in amber glass vials that meet the current European Pharmacopoeia (Ph.Eur.) and United States Pharmacopoeia (USP) requirements for glass containers for pharmaceutical use.

[0361] The raw drug substance (DS) is composed of purified cell wall fragments of Mtb, referred to as FCMtb. The preparation process for FCMtb is obtained by collecting Mtb bacterial cultures by cell disruption (Examples 1-7). The fragmented cell walls are then purified in the presence of Triton X100 to remove soluble components and endotoxin-like molecules. The structure of DS is closely related to the properties of the Mtb cell wall. Therefore, DS is mainly composed of a heterogeneous range of proteins and lipids from Mtb strains. RUTI, i.e., the drug product (a preferred embodiment of the liposome formulation described in the present invention), is a DS liposome suspension containing a charged excipient. It is presented as a dry powder (for reconstitution with water for injection) and is stable at 5°C for at least 12 months.

[0362] Table 7: Quantitative and qualitative composition of each vial of RUTI vaccine

[0363]

[0364] 1 Contains phosphatidylcholine (not less than [NLT] 94.0%)

[0365] 2 Add as a 0.9% NaCl solution

[0366] 3 Disappears during processing

[0367] For administration to mice, the investigational drug product should be reconstituted with 0.266 ml of water for injection to yield a solution containing 1002.1 μg / mL FCMtb (FCMtb 266.7 μg / vial) and 125.3 μg / mL FCMtb (RUTI 33.3 μg / vial). The composition of the placebo used in this nonclinical trial was normal saline (0.9% NaCl).

[0368] The reconstituted RUTI vaccine will be administered subcutaneously in the neck area in a total volume of 0.200 ml.

[0369] result

[0370] RUTI is administered subcutaneously to infected C3HeB / FeJ mice to test its effect in the active TB infection model. A volume of 0.2 mL (containing 4.0E+04 Mtb CFU) is administered intravenously to infect animals. After 5 weeks, an injection of RUTI of 200 μg FCMtb or 25 μg FCMtb is administered individually and in combination with chemotherapy at time point 0 (S1w); or false administration (normal saline). In some experiments, a second injection of RUTI of 200 μg FCMtb or 25 μg FCMtb is administered at the end of chemotherapy (28d); or false administration (salt water). In some experiments, animals are killed one week after first RUTI administration / false administration or 2 weeks after second RUTI administration / false administration.

[0371] The efficacy of RUTI treatment was investigated by measuring bacillary loads in lung and spleen tissues, while safety was assessed by histopathology as the percentage of lung lesions. T immune responses were evaluated using ELISpot technology.

[0372] Bacillary burden in lung and spleen tissues

[0373] The data showed that bacillary loads were significantly reduced when RUTI was administered alone or in combination with chemotherapy at time point 0. RUTI doses of 200 μg FCMtb and 25 μg FCMtb showed similar CFU reductions (see Figure 14 ).

[0374] In the spleen, the data showed that when RUTI was administered alone at time point 0, the bacillary load was significantly reduced ( Figure 15 ).

[0375] Pathology in lung tissue

[0376] The data showed that lung injury was significantly reduced when RUTI was administered alone or in combination with chemotherapy at time point 0. RUTI at doses of 200 μg FCMtb and 25 μg FCMtb showed similar reductions in lung injury ( Figure 16 ).

[0377] Cellular immune response (ELIspot)

[0378] The data showed that the reduction of CFU resulted in a reduction of T cell immune responses against ESAT-6, whereas the administration of RUTI did not specifically induce ESAT-6 responses (see FIG17 ).

[0379] The data showed that administration of RUTI induced T cell immune responses against HSP16.3 and PsTS1 in a dose-dependent manner after a single injection ( FIG. 18 ).

[0380] in conclusion

[0381] Traditionally, therapeutic vaccination has received considerable attention due to its potential toxicity, a concept summarized in the so-called "Koch reaction." Briefly, this concept stems from Robert Koch's experience with Mtb-infected guinea pigs, where tuberculin inoculation induced a necrotic reaction within established granulomas of infection. This reaction has since become the basis for the pathological responses to therapeutic vaccination in patients with active TB. The experimental TB mouse model of the C3HeB / FeJ strain offers a unique opportunity to test this approach, as it develops "humanoid" lesions with liquefaction, which bear many similarities to human TB.

[0382] Using the active TB model in C3HeB / FeJ (highly similar to human TB liquefaction lesions), administration of RUTI alone or at time 0 of chemotherapy resulted in a reduction in bacillary load and a very significant reduction in the area of lung lesions. The RUTI-administered group showed induction of T cell immune responses against HSP-16.3 and PsTS1 (latent antigens) but not against PPD and ESAT-6.

[0383] Surprisingly, RUTI administered alone has demonstrated the ability to mimic chemotherapy's effects by reducing bacillary load and the area of lung lesions within the first week of treatment. Regarding its mechanism of action, RUTI is designed to induce a multi-antigen response, but no induction of T cell responses against PPD, ESAT-6, HSP16-3, or PsTS1 was observed. In the case of ESAT-6, there was an overall reduction, corresponding to a decrease in bacillary load over time. The lack of an increased immune response when RUTI was administered alone could be explained by the already increased response in infected mice. It is hypothesized that RUTI injection allows the recruitment of additional specific lymphocytes to halt the progression of endogenous reactivation, and the technique used was not sensitive enough to register a difference. This is in contrast to what we see with chemotherapy, which abruptly halts the immune response by killing the bacilli and halting the positive feedback loop with lymph nodes that stimulates lymphocyte proliferation. In this case, the mobilization induced by RUTI can be detected and generate a difference.

[0384] Additionally, the data confirmed the efficacy and safety of a therapeutic vaccine in combination with chemotherapy after induction of active TB. The effect of RUTI was observed in a dose-dependent manner (200 μg and 25 μg doses) after a single injection at time point 0 of standard chemotherapy. Regarding its mechanism of action, RUTI is designed to induce a multi-antigen response, enabling the detection of dormant bacilli. It demonstrated a statistically significant increase in Th1 responses against the HSP16.3 antigen and PsTS1. In the case of ESAT-6, there was an overall decrease, corresponding to a decrease in bacillary load over time.

[0385] It is also important to note the lack of effect after the second RUTI administration at the end of chemotherapy. This may be due to low bacillary loads. In this case, it would be more practical to wait at least six weeks to examine the effect of the RUTI vaccine against reactivation, as has been demonstrated. This "Cornell-like" approach is commonly used to test therapeutic vaccines. Therefore, the data obtained simply confirm previous data.

[0386] In summary, nonclinical results showed that RUTI was effective (bacillary load and lung lesion area) regardless of when it was administered alone or in combination with standard chemotherapy at the start of treatment. The results support the selected vaccination time point of day 0 from the start of chemotherapy. In addition, the reduction in bacillary load compared to chemotherapy was maintained over time.

[0387] Example 11: Phase IIb in patients with MDR-TB and DS-TB

[0388] As an adjunct to antibiotic treatment of TB, Vaccines stimulate host immune effectors to achieve elimination of actively replicating and persistent bacilli. The first step is to ensure The vaccine is safe and immunogenic and shortens the time to sputum culture conversion in patients with DS-TB and MDR-TB.

[0389] Based on the available data, a Phase IIb study is underway in India. The study is titled “Study of a novel chemotherapeutic agent for tuberculosis”. Double-Blind, Randomized, Placebo-Controlled Phase IIb Clinical Trial to Investigate the Efficacy of Therapeutic Vaccination Therapeutic Vaccination as adjuvant of Tuberculosis chemotherapy)” and was approved by the Drug Controller General of India (DCGI) on January 20, 2020.

[0390] The main objectives of the study are to:

[0391] 1. Evaluation Sputum culture conversion time of the vaccine (25 μg FCMtb) in patients with DS-TB and MDR-TB who responded well to standard TB treatment, as confirmed by clinical response.

[0392] 2. Explore Safety and immunogenicity of the vaccine (25 μg FCMtb) in patients with DS-TB and MDR-TB who have responded well to standard TB treatment.

[0393] This clinical trial was designed as a prospective, randomized, double-blind, multicenter, placebo-controlled Phase IIb clinical trial to evaluate Vaccine efficacy in patients with DS-TB and MDR-TB who have responded well to standard MDR-TB treatment. Vaccination will begin one week after completing standard DS-TB treatment (Cohort A), while another cohort will be vaccinated one month after completing standard MDR-TB treatment (Cohort B). All patients will be followed until completion of treatment.

[0394] The study plans to recruit 140 adult TB patients (aged > 18 years) with culture-confirmed DS-TB (90 patients) and MDR-TB (50 patients) who do not have any medical conditions that could impair the assessment of response to vaccination or increase the risk of adverse events. Currently, all 90 DS-TB patients and 20 MDR-TB patients have been enrolled and randomly assigned in a 1:1 ratio to receive 25 μg of dapoxetine 1 week or 4 weeks after starting standard MDR-TB treatment. The vaccine or placebo was administered subcutaneously as a single injection. No significant safety issues were reported, with only some local reactions observed.

[0395] Two further studies are underway investigating the administration of 25 μg of Safety of the vaccine relative to placebo: No significant safety concerns were identified.

[0396] For example, a previous study explored the Efficacy and safety of immunotherapy administered concomitantly with standard of care (CONSTAN).

[0397] The main objectives of the study are to:

[0398] 1. Evaluate the effect of RUTI on the administration of RUTI at the start of ATB treatment (day 0). Early bactericidal activity (from day 0 to day 14) of the vaccine (25 μg FCMtb) in DS-TB patients.

[0399] 2. Evaluate the effect of RUTI on the initiation of ATB treatment (day 0). Safety and tolerability of the vaccine (25 μg FCMtb) in patients with DS-TB.

[0400] References

[0401] Andersen P.,1997,Scand J.Immunol.;45(2):115-31

[0402] Brennan,Tuberculosis(Edinburgh),2003,83(1-3),91-97

[0403] Chouldhary et al., 2018, Journal Immunology, 200:3053-3066

[0404] Geisel et al., 2005, J. Immunol.; 174(8):5007-15

[0405] Meier et al., 2018, Frontiers in Immunology Vol9 Article 2476

[0406] Renshaw et al., 2005, EMBO Journal 24, 2491-2498

[0407] Rodriguez-Hernandez et al., 2020, Biomed and Biotechnol 21(11):856-870

[0408] Singh et al., 2005, Clin. Diagn. Lab. Immunol. 12(2), 354-358

[0409] Stewart et al., 2005, Infect. Immun., 73(10): 6831-7

[0410] Wang et al., 2007, J. Mol. Biol., 366(2):375-81.

Claims

1. A liposome preparation comprising: (a) Fragments from Mycobacterium tuberculosis-complex (MTB-C) strains, (b) a liposome-forming agent, (c) 1 to 20% (w / v) sucrose, wherein the z-average size of the particles as determined by dynamic light scattering is 150 nm or less, and the polydispersity index of the particles is 0.400 or less, and the liposomal formulation is for use in a method of treating active tuberculosis.

2. The liposomal formulation for use in a therapeutic method according to claim 1, wherein the Mycobacterium tuberculosis complex (MTB-C) strain is a virulent Mycobacterium tuberculosis complex (MTB-C) strain, preferably the MTB-C strain NCTC 13536, which was deposited at NCTC in London, UK in 2010.

3. The liposome formulation for use in a therapeutic method according to any one of claims 1 or 2, further comprising: (a) a tonicity active agent, and / or (b) one or more nonionic surfactants.

4. The liposome formulation for use in a therapeutic method according to any one of the preceding claims, wherein the liposome forming agent is a hydrogenated, partially hydrogenated or non-hydrogenated phospholipid, preferably, wherein the liposome forming agent is soy lecithin.

5. The liposomal formulation for use in a method of treatment according to any one of the preceding claims, comprising at least two of the following: (a) a first polypeptide having a molecular weight of about 70 kDa as measured after electrophoresis on a sodium dodecyl sulfate (SDS) polyacrylamide gel, wherein the first polypeptide has a mass fingerprint similar to the mass fingerprint of Mycobacterium tuberculosis HSP70 protein (Rv0350), (b) a second polypeptide having a molecular weight of about 38 kDa as measured after electrophoresis on a sodium dodecyl sulfate (SDS) polyacrylamide gel, wherein the second polypeptide has a mass fingerprint similar to the mass fingerprint of a 38 kDa protein (Rv0934) of Mycobacterium tuberculosis, (c) a third polypeptide having a molecular weight of about 30-34 kDa as measured after electrophoresis on a sodium dodecyl sulfate (SDS) polyacrylamide gel, wherein the third polypeptide has a mass fingerprint similar to the mass fingerprint of a Mycobacterium tuberculosis Ag85 complex protein (Rv1866c-Rv3804c), (d) a fourth polypeptide having a molecular weight of about 16 kDa as measured after electrophoresis on a sodium dodecyl sulfate (SDS) polyacrylamide gel, wherein the fourth polypeptide has a mass fingerprint similar to the mass fingerprint of the Mycobacterium tuberculosis HSP16.3 protein (Rv2031c), (d) a fifth polypeptide having a molecular weight of about 10 kDa as measured after electrophoresis on a sodium dodecyl sulfate (SDS) polyacrylamide gel, wherein the fifth polypeptide has a mass fingerprint similar to the mass fingerprint of Mycobacterium tuberculosis CFP10 protein (Rv3874), and (e) a sixth polypeptide having a molecular weight of about 6 kDa as measured after electrophoresis on a sodium dodecyl sulfate (SDS) polyacrylamide gel, wherein the sixth polypeptide has a mass fingerprint similar to the mass fingerprint of the Mycobacterium tuberculosis ESAT-6 protein (Rv3875).

6. A pharmaceutical composition comprising the liposome preparation according to any one of claims 1 to 5 and a pharmaceutically acceptable carrier or diluent and / or a pharmaceutically acceptable adjuvant, wherein the pharmaceutical composition is used for treating active tuberculosis.

7. The liposomal formulation or pharmaceutical composition for use in a therapeutic method according to any one of the preceding claims, wherein the active tuberculosis is drug-sensitive tuberculosis, rifampicin-resistant tuberculosis, multidrug-resistant tuberculosis or extensively drug-resistant tuberculosis.

8. The liposomal formulation or pharmaceutical composition for use in a method of treatment according to any one of the preceding claims, wherein the liposomal formulation is administered once or twice.

9. The liposomal formulation or pharmaceutical composition for use in a method of treatment according to any one of the preceding claims, wherein the liposomal formulation is administered in the absence of chemotherapy.

10. The liposomal formulation or pharmaceutical composition for use in a therapeutic method according to claims 1-8, wherein the liposomal formulation is administered before or simultaneously with chemotherapy.

11. The liposomal formulation or pharmaceutical composition for use in a method of treatment according to claim 10, wherein the chemotherapy lasts for 4 weeks.

12. The liposomal formulation or pharmaceutical composition for use in a method of treatment according to claim 10 or 11, wherein the first dose of the liposomal formulation or pharmaceutical composition is administered within four weeks of the first dose of the chemotherapy, optionally wherein the first dose of the liposomal formulation or pharmaceutical composition is administered together with the first dose of the chemotherapy.

13. The liposomal formulation or pharmaceutical composition for use in a therapeutic method according to claims 10-12, wherein the chemotherapy is an antibiotic treatment.

14. The liposomal formulation or pharmaceutical composition for use in a method of treatment according to claim 13, wherein the antibiotic treatment comprises at least one of ethambutol, isoniazid, pyrazinamide, rifampicin, streptomycin, amikacin, kanamycin, capreomycin, viomycin, enviromycin, ciprofloxacin, levofloxacin, moxifloxacin, ethionamide, prothionamide, or cycloserine terizidone, optionally wherein the antibiotic treatment comprises RIMSTAR.

15. The liposomal formulation or pharmaceutical composition for use in a method of treatment according to any one of claims 1 to 14, wherein the liposomal formulation or pharmaceutical composition is administered at a dose of 5-200 μg FCMtb per dose.

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