INFECTIONS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to Indian Provisional Application No. 201841019182, entitled “COMPOSITION AND METHOD OF TREATMENT OF BACTERIAL INFECTIONS”, filed on May 22, 2018, the complete specification of which was filed on May 2, 2019, the full disclosures of which are incorporated herein by reference.

FIELD OF THE INVENTION

[0002] The present invention generally relates to bacterial infections and in particular to a composition, combination, preparation method and kit for treatment of mycobacterial infections.

DESCRIPTION OF THE RELATED ART

[0003] Peptidoglycan (PG) is an important cell wall component which provides structural integrity to the bacteria. Gram positive bacterial PG is composed of disaccharide pentapeptide monomer subunits. The disaccharide backbone is made up of alternating sugar N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc). The MurNAc unit is further linked to the stem peptide consisting of amino acid residues L-Alanine-D-Glutamine-/we.v diaminopimelic acid (/«DAP)-D-Alanine-D- Alanine and the stem peptides are highly cross linked which gives a mesh like structure. The cross linkage takes place between D-Alanine (D-Ala) and /«DAP of neighbouring stem peptides mediated by the enzyme D,D-transpeptidase, which is known as classical type of PG cross linking. Beta-lactam class of antibiotics (amoxicillin) inhibits D,D- transpeptidase enzyme activity, leading to cell lysis followed by bacterial death by increased osmotic pressure. To resist beta-lactams, the bacteria produce an enzyme called beta-lactamase which cleaves the beta-lactam ring of the antibiotics and makes it inactive. Beta-lactamase inhibitors such as clavulanic acid block beta-lactamase enzyme and make the bacteria susceptible to beta-lactam antibiotics. So, treating bacteria with a combination of beta-lactam antibiotics and beta-lactamase inhibitor has been suggested to lead to improved bacterial cell wall lysis and death. However, treating some bacteria such as Mycobacterium tuberculosis (Mtb), Mycobacterium abscessus, Mycobacterium bovis, Mycobacterium marinum, Mycobacterium leprae, Mycobacterium kansasii, etc. with conventional beta-lactams is not enough since they produce beta-lactamase as well as a second type of PG cross linkage between mDAP of neighbouring stem peptides. This type of PG cross-linking is called non-classical type mediated by an enzyme called L,D-transpeptidase (Ldt).

[0004] An increasing number of cases reporting infection with multi-drug resistant (MDR) and extensively drug-resistant (XDR) strains ofM tuberculosis have diminished our capability to respond effectively against this threat. Recent investigations show that, carbapenem class of injectable beta-lactams like meropenem, ertapenem etc. may be effective in killing Mtb by blocking both type of PG cross linkages (classical and non- classical), and may be comparatively resistant to the enzymatic degradation by beta- lactamase. The US patent application US20110190253 describes a method of treating TB using carbapenem class of antibiotics. The US patent document’9273341 describes a method of treatment of bacterial infections such as tuberculosis by inhibition of the Ldt. The US patent document’9499535 discloses compounds that inhibits kinases such as SYK (Spleen Tyrosine Kinase), LRRK2 (Leucine-rich repeat kinase 2) and/or MYLK (Myosin light chain kinase) or mutants thereof. The US patent document’9005626 discloses liquid compositions capable of including active agents and methods for making or developing same. The disclosed liquid compositions include foaming agents which enables foaming of the liquid compositions. The US patent document’8623419 discloses a method of preparation of a macromolecular microparticle for preparing pharmaceuticals of defined dimensions. The microspheres are prepared by contacting an aqueous solution of a protein or other macromolecule with an organic solvent and a counter ion, and chilling the solution. However, these antibiotics have very low oral bioavailabibty as compared to other beta-lactams. It is given intravenously and hence prolonged hospitalization of the patients is required and the treatment cost will be high. An effective and safe oral therapy for treating bacterial infections such as Mtb and targeting both types of PG cross-linking is needed.

[0005] The current treatment standard (WHO) is AMC (amoxicillin: clavulanic acid) along with the Meropenem (MEM) drug as add-on therapeutic options for the treatment of drug resistant TB, since clavulanic acid is only available in combination with amoxicillin (AMX). Recent studies demonstrated that carbapenem subclass of beta- lactam antibiotics are effective in killing MDR and XDR strains of Mtb. However, carbapenems are not hydrolytically stable, which limits its antibiotic administration to a controlled intravenous infusion and also requires prolonged hospitalization.

SUMMARY OF THE INVENTION

[0006] The present invention discloses compositions for treating bacterial infections and in particular to a combination for treatment of mycobacterial infections.

[0007] In various embodiments provided herein is a composition for treating mycobacterial infections in a subject in need thereof. The composition includes diosmin, diosmetin, or a pharmaceutically acceptable salt thereof and one or more second agents including beta-lactam antibiotic and/or beta-lactamase inhibitor. In one embodiment, the second agent is amoxicillin and clavulanic acid. In one embodiment, 1-3000 mg of diosmin, or diosmetin, or a pharmaceutically acceptable salt thereof, 1-1500 mg of amoxicillin and 1-375 mg of clavulanic acid is provided.

[0008] In one embodiment, the composition further includes one or more anti- mycobacterial drug selected from the group comprising of ethambutol, isoniazid, pyrazinamide, rifampicin, streptomycin, amikacin, kanamycin, capreomycin, viomycin, enviomycin, ciprofloxacin, levofloxacin, moxifloxacin, ethionamide, prothionamide, cycloserine, terizidone, clarithromycin, linezolid, thioacetazone and or thioridazine. In one embodiment, the mycobacterial infection is selected from an infection caused by Mycobacterium tuberculosis, Mycobacterium abscessus, Mycobacterium marinum, Mycobacterium smegmatis, Mycobacterium bovis, Mycobacterium africanum, Mycobacterium canetti, Mycobacterium caprae, Mycobacterium microti, Mycobacterium kansasii, Mycobacterium leprae, Mycobacterium avium, Mycobacterium paratuberculosis, Mycobacterium pinnipedii, and bacterial infection caused by Escherichia coli, Enterococcus faecalis, Enterococcus faecium, Klebsiella pnemoniae, Shigella sonnei, Salmonella enterica and or Yersinia enterocolitica. In one embodiment, the mycobacterial infection is drug resistant tuberculosis. In one embodiment, the beta- lactam antibiotic is selected from the group of benzylpenicilbn, phenoxymethyl penicillin, procaine penicillin, cloxacillin, dicloxacilbn, flucloxacilbn, methicillin, nafcilbn, oxacillin, temocillin, amoxicillin, ampicillin, mecillinam, carbenicilbn, ticarcilbn, azlocilbn, mezlocillin, piperacillin, cefazobn, cephalexin, cephalosporin, cephalothin, cefaclor, cefamandole, cefuroxime, cefotetan, cefoxitin, cefixime, cefotaxime, cefpodoxime, ceftazidime, ceftriaxone, cefepime, cefpirome, biapenem, doripenem, ertapenem, faropenem, imipenem, meropenem, panipenem, razupenem, tebipenem, thienamycin, aztreonam, tigemonam, nocardicin, and tabtoxinine. In one embodiment, the beta-lactamase inhibitor is selected from the group comprising of sulbactam, tazobactam, clavulanic acid, avibactam and vaborbactam. In one embodiment, the weight ratio of amoxicillin to clavulanic acid is in the range of 2: 1 to 16: 1. In one embodiment, the weight ratio of diosmin, diosmentin, or a pharmaceutically acceptable salt thereof to amoxicillin and clavulanic acid is 2: 1 or greater. In one embodiment, an oral dosage form including the composition of claim 1 is provided.

[0009] In various embodiments, a method of inhibiting L,D transpeptidase 1 (Ldtl) and L,D transpeptidase 2 (Ldt2) activity in bacterial cells is provided. The method includes adding a composition comprising diosmin, or diosmetin, or a pharmaceutically acceptable salt thereof; amoxicillin; and clavulanic acid. The diosmin, diosmentin, or a pharmaceutically acceptable salt thereof binds to one or more residues of L,D transpeptidase 1 (Ldtl) and L,D transpeptidase 2 (Ldt2) thereby inhibiting their activity. This and other aspects are disclosed.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010] The invention has other advantages and features which will be more readily apparent from the following detailed description of the invention and the appended claims, when taken in conjunction with the accompanying drawings, in which:

[0011] FIG. 1 depicts a multiple sequence alignment of L,D transpeptidases of different mycobacterial species.

[0012] FIG. 2A depicts top overlapping hits selected from a virtual screening workflow towards both Ldt _Mti and Ldt _Mt 2 enzymes.

[0013] FIG. 2B depicts the structure of diosmin (DIO).

[0014] FIG. 3A depicts docked complex of Ldt _Mti with DIO.

[0015] FIG. 3B depicts molecular interactions of DIO towards the active site of Ldt _Mti · [0016] FIG. 3C depicts docked complex of Ldt _Mt 2 with DIO.

[0017] FIG. 3D depicts molecular interactions of DIO towards the active site of Ldt _Mt 2· [0018] FIG. 3E depicts MEP surface map of the Ldt _Mti - DIO active site regions.

[0019] FIG. 3F depicts MEP surface map of the Ldt _Mt 2 - DIO active site regions.

[0020] FIG. 4A depicts a root mean square deviation plot of Ldt _Mti and Ldt _Mti -DIO complex.

[0021] FIG. 4B depicts root mean square deviation plot of Ldt _Mti, Ldt _Mt 2-DIO docked complex and Ldt _Mt 2-Meropenem (MEM) crystal structure during 20 ns molecular dynamics simulation.

[0022] FIG. 5A depicts binding studies of DIO towards Ldt _Mti and Ldt _Mt 2 indicated that both DIO and DMT has affinity towards both the proteins.

[0023] FIG. 5B depicts binding studies of DMT towards Ldt _Mti and Ldt _Mt 2 indicated that both DIO and DMT has affinity towards both the proteins.

[0024] FIG. 5C depicts comparative in vitro anti-mycobacterial activity of Amoxicillin- clavulanate (AMC), DIO, Diosmetin (DMT), AMC-DIO and AMC-DMT.

[0025] FIG. 6A depicts Scanning electron micrography (SEM) images of M. marinum incubated without any drugs. [0026] FIG. 6B depicts Scanning electron micrography (SEM) images of M. marinum incubated with AMC (8 pg/ml).

[002h FIG. 6C depicts Scanning electron micrography (SEM) images of M. marinum incubated with DIO (500 pg/ml).

[0028] FIG. 6D shows Scanning electron micrography (SEM) images of M. marinum incubated with DMT (500 pg/ml).

[0029] FIG. 6E shows Scanning electron micrography (SEM) images of M. marinum incubated with AMC (8 pg/ml) - DIO (500 pg/ml).

[0030] FIG. 6F depicts Scanning electron micrography (SEM) images of M. marinum incubated with AMC (8 pg/ml) - DMT (500 pg/ml).

[0031] FIG. 7A depicts survival ofM marinum i n fected Drosophila melanogaster flies on treatment with (DIO (0.25, 0.5, 1.0, and 2.0 mg/ml).

[0032] FIG. 7B depicts survival of M. marinum infected Drosophila melanogaster flies on treatment with AMC (0.5 and 1.0 mg/ml).

[0033] FIG. 7C depicts survival ofM marinum infected Drosophila melanogaster flies on treatment with AMC - DIO combination.

[0034] FIG. 7D depicts survival of M. marinum infected Drosophila melanogaster flies on treatment with AMC- DMT combination.

[0035] FIG. 7E depicts survival of M. marinum infected Drosophila melanogaster flies on treatment with Rifampicin (RIF; 0.25 mg/ml) and RIF along with 1 and 2 mg/ml AUG and DIO drugs respectively.

[0036] FIG. 8A depicts a Q TOF LC-MS/MS analysis of DIO fed fly extract showing fragmentation pattern for DIO.

[003h FIG. 8B depicts a Q TOF LC-MS/MS analysis of DIO fed fly extract showing fragmentation pattern for DMT.

[0038] FIG. 9A depicts acid fast bacilli (AFB) staining of M. marinum infected flies after 9 days without any treatment [0039] FIG. 9B depicts acid fast bacilli (AFB) staining ofM marinum infected flies after 9 days of treatment with DIO.

[0040] FIG. 9C depicts acid fast bacilli (AFB) staining ofM marinum infected flies after 9 days with AMC.

[0041] FIG. 9D depicts acid fast bacilli (AFB) staining of M. marinum infected flies after 9 days after treatment with a combination of AMC-DIO.

[0042] FIG. 9E depicts acid fast bacilli (AFB) staining of M. marinum infected flies after 9 days with DMT.

[0043] FIG. 9F depicts acid fast bacilli (AFB) staining ofM marinum infected flies after 9 days of treatment with combination of AMC-DMT.

[0044] FIG. 9G depicts acid fast bacilli (AFB) staining of M. marinum infected flies after 9 days of treatment with combination of RIF.

[0045] FIG. 9H depicts acid fast bacilli (AFB) staining of M. marinum infected flies after 9 days after treatment with combination of RIF- AMC -DIO.

[0046] FIG. 91 shows AFB grading of the M. marinum infected flies during the drug treatment (3, 5, 7 and 9 days) showed that no bacilli were found in infected flies after 9 days of treatment with AMC-DIO, AMC-DMT and RIF-AMC-DIO drugs

[004h FIG. 10A depicts Drug susceptibility testing of AMC-DIO combination against Mycobacterium tuberculosis (Mtb) H37Ra.

[0048] FIG. 10B depicts the susceptibility of multi-drug resistant Mtb clinical strain towards AMC, DIO, and AMC-DIO using MGIT 960 system, indicating a synergistic anti-tubercular effect of the drug combination, whereas the drugs AMC or DIO alone not showed any significant anti-tubercular activity. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0049] While the invention has been disclosed with reference to certain embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the scope of the invention. In addition, many modifications may be made to adapt to a particular situation or material to the teachings of the invention without departing from its scope.

[0050] Throughout the specification and claims, the following terms take the meanings explicitly associated herein unless the context clearly dictates otherwise. The meaning of "a", "an", and "the" include plural references. The meaning of "in" includes "in" and "on." Referring to the drawings, like numbers indicate like parts throughout the views. Additionally, a reference to the singular includes a reference to the plural unless otherwise stated or inconsistent with the disclosure herein.

[0051] The terms “treat”, “treating”, and “treatment” refer to a method of alleviating or abrogating a disease and/or its attendant symptoms. The terms“prevent”, “preventing”, and“prevention” refer to a method of preventing the onset of a disease and/or its attendant symptoms or barring a subject from acquiring a disease. As used herein,“prevent”,“preventing”, and“prevention” also include delaying the onset of a disease and/or its attendant symptoms and reducing a subject’s risk of acquiring a disease. The phrase“therapeutically effective amount” or“pharmaceutically effective amount” means a compound, or a pharmaceutically acceptable salt thereof, sufficient to prevent the development of or to alleviate to some extent one or more of the symptoms of the condition or disorder being treated when administered alone or in conjunction with another therapeutic agent or treatment in a particular subject or subject population. For example in a human or other mammal , a therapeutically effective amount can be determined experimentally in a laboratory or clinical setting , or may be the amount required by the guidelines of the United States Food and Drug Administration , or equivalent foreign agency , for the particular disease and subject being treated. [0052] The present invention in its various embodiments relates to a composition, an oral dosage form, kits, method of preparation thereof and method for treatment of bacterial infections including mycobacterial infections such as tuberculosis (TB).

[0053] In various embodiments, provided herein is a composition for treatment of bacterial infections. The composition includes a therapeutically effective amount of a first agent including one or more of diosmin, diosmetin, or a pharmaceutically acceptable salt thereof. The composition additionally includes a second agent that may include at least one beta-lactam antibiotic and/or at least one beta-lactamase inhibitor. In one embodiment, the second agent is a combination of amoxicillin and clavulanic acid (or AMC).

[0054] The first agent such as diosmin, and second agent such as amoxicillin and clavulanic acid combination may be provided at a dosage of 10, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000 mg, or any number therebetween. In one embodiment, the concentration of the first agent is in the range of 10- 3000 mg. In one embodiment, the concentration of the second agent is in the range of 10- 2000 mg. In one embodiment, diosmin, diosmentin or pharmaceutically acceptable salt thereof may be present in any therapeutically acceptable concentration, typically between 1-3000 mg, 1-3000 mg/day, or 0.1 to 30 mg/ml. In one embodiment, amoxicillin or pharmaceutically acceptable salt thereof may be present in any therapeutically acceptable concentration, typically between 1-2000 mg, 1-2000 mg/day, or 0.1 to 20 mg/ml. In one embodiment, clavulanic acid or pharmaceutically acceptable salt thereof may be present in a therapeutically acceptable concentration, typically between 1-500 mg, 1-500 mg/day, or 0.1 to 5 mg/ml.

[0055] In one embodiment, diosmin, diosmentin or a pharmaceutically acceptable thereof to other active agents such as AMC is 2: 1, 3: 1, 4: 1, 5 : 1, 6: 1, 7: 1, 8: 1, 9: 1, 10: 1, 20: 1, 50: 1, 100: 1, 200: 1, or greater. In one embodiment, the ratio of beta-lactam to beta-lactamase inhibitor is varied between 16: 1 to 1: 8, more typically about 4: 1. In one embodiment, the ratio of amoxicillin to clavulanic acid is 2: 1, 3: 1, 4: 1, 5: 1, 6: 1, 9: 1, 10: 1, 11 : 1, 12: 1, 13: 1, 14: 1, 15: 1, 16: 1, or any number therebetween.

[0056] In some embodiments, the beta-lactam is selected from the group consisting of benzylpenicillin, phenoxymethyl penicillin, procaine penicillin, cloxacillin, did oxacillin, flucloxacillin, methicillin, nafcillin, oxacillin, temocillin, amoxicillin, ampicillin, mecillinam, carbenicillin, ticarcillin, azlocillin, mezlocillin, piperacillin, cefazolin, cephalexin, cephalosporin, cephalothin, cefaclor, cefamandole, cefuroxime, cefotetan, cefoxitin, cefixime, cefotaxime, cefpodoxime, ceftazidime, ceftriaxone, cefepime, cefpirome, biapenem, doripenem, ertapenem, faropenem, imipenem, meropenem, panipenem, razupenem, tebipenem, thienamycin, aztreonam, tigemonam, nocardicin, and tabtoxinine.

[005h In some embodiments, the beta-lactamase inhibitor is selected from the group of sulbactam, tazobactam, clavulanic acid, avibactam and vaborbactam.

[0058] In various embodiments, the composition additionally includes at least one antimycobacterial drug to treat certain infections such as standard drugs to treat drug- resistant tuberculosis. The standard TB drugs may include one or more of rifampicin, isoniazid, ethambutol, pyrazinamide, rifampicin, streptomycin, amikacin, kanamycin, capreomycin, viomycin, enviomycin, ciprofloxacin, levofloxacin, moxifloxacin, ethionamide, prothionamide, cycloserine, terizidone, clarithromycin, linezolid, thioacetazone or thioridazine. In some embodiments, the composition is an effective and alternative treatment of TB in a shorter period of time. The anti-mycobacterial drug may be present at a therapeutically suitable concentration, typically about 8-12 mg/kg for rifampicin, 4-6 mg/kg for isoniazid, 20-30 mg/kg for pyrazinamide, and/or 15-25 mg/kg for ethambutol. [0059] In some embodiments, the composition includes one or more additional agents such as diluents, excipients and/or pharmaceutically acceptable carriers. In some embodiments, the compound may include its isotopically labeled form. In some embodiments, composition is conjugated with one or more additional agents such as a small molecule drug or antibody. In some embodiments, the composition is used in combination therapy with other agents for an effective therapy. The combinations may be materials or particles or other molecules either conjugated with the drug or free standing in the solution. In various embodiments, derivatives of the composition may be used to improve the absorption and retention characteristics of the composition. In accordance with another aspect of the invention there is also provided a process for the preparation of a pharmaceutical formulation including admixing the first and second agents with one or more pharmaceutically acceptable carriers, diluents, or excipients.

[0060] Pharmaceutical formulations may be presented in unit dose forms containing a predetermined amount of active ingredient per unit dose. Such a unit may contain, for example, 0.1 mg to 3000 mg, typically 1 mg to 2000 mg, more preferably 50 mg to 1000 mg of the agents, depending on the condition being treated, the severity of the condition, the time of administration, the route of administration, the rate of excretion of the compound employed, the duration of treatment, and the age, gender, weight, and condition of the patient, or pharmaceutical formulations may be presented in unit dose forms containing a predetermined amount of an active ingredient per dose. Preferred unit dosage formulations are those containing a daily dose or sub-dose, as herein above recited, or an appropriate fraction thereof, of an active ingredient. Size of the dose that accompany the first and second agents is determined by the existence, nature and extent of any adverse side-effects in a subject. Furthermore, such pharmaceutical formulations may be prepared by any of the methods well known in the pharmacy art.

[0061] Pharmaceutical formulations may be adapted for administration by any appropriate route, for example by the oral (including buccal or sublingual), rectal, nasal, topical (including buccal, sublingual, or transdermal), peritoneal, vaginal, or parenteral (including subcutaneous, intramuscular, intravenous, or intradermal) route. Such formulations may be prepared by any method known in the art of pharmacy, for example by bringing into association the active ingredient with the carrier(s) or excipient(s). In addition, compounds of the present invention can be administered using conventional drug delivery technology, for example, intra-arterial stents.

[0062] Pharmaceutical formulations adapted for oral administration may be presented as discrete units such as capsules or tablets; powders or granules; solutions or suspensions in aqueous or non-aqueous liquids; edible foams or whips; or oil-in-water liquid emulsions or water-in-oil emulsions. Pharmaceutically acceptable carrier used herein, maybe a non-toxic, inert solid, semi-solid, or liquid filler, diluent, encapsulating material, or formulations auxiliary of any type. For instance, for oral administration in the form of a tablet or capsule, the active drug component can be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like. Powders are prepared by comminuting the compound to a suitable fine size and mixing with a similarly comminuted pharmaceutical carrier such as an edible carbohydrate, as, for example, starch or mannitol. Flavoring, preservative, dispersing, and coloring agent can also be present.

[0063] Capsules are made by preparing a powder mixture, as described above, and filling formed gelatin sheaths. Glidants and lubricants such as colloidal silica, talc, magnesium stearate, calcium stearate, or solid polyethylene glycol can be added to the powder mixture before the filling operation. A disintegrating or solubilizing agent such as agar-agar, calcium carbonate, or sodium carbonate can also be added to improve the availability of the medicament when the capsule is ingested.

[0064] Moreover, when desired or necessary, suitable binders, lubricants, disintegrating agents, and coloring agents can also be incorporated into the mixture. Suitable binders include starch, gelatin, natural sugars such as glucose or beta-lactose, com sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes, and the like. Lubricants used in these dosage forms include sodium oleate, sodium chloride, and the like. Disintegrators include, without limitation, starch, methyl cellulose, agar, bentonite, xanthan gum, and the like. Tablets are formulated, for example, by preparing a powder mixture, granulating or slugging, adding a lubricant and disintegrant, and pressing into tablets. A powder mixture is prepared by mixing the compound, suitable comminuted, with a diluent or base as described above, and optionally, with a binder such as carboxymethylcellulose, an alginate, gelating, or polyvinyl pyrrolidone, a solution retardant such as paraffin, a resorption accelerator such as a quaternary salt and/or and absorption agent such as bentonite, kaolin, or dicalcium phosphate. The powder mixture can be granulated by wetting with a binder such as syrup, starch paste, acacia mucilage, or solutions of cellulosic or polymeric materials and forcing through a screen. As an alternative to granulating, the powder mixture can be run through the tablet machine and the result is imperfectly formed slugs broken into granules. The granules can be lubricated to prevent sticking to the tablet forming dies by means of the addition of stearic acid, a stearate salt, talc, or mineral oil. The lubricated mixture is then compressed into tablets. The compounds of the present invention can also be combined with a free flowing inert carrier and compressed into tablets directly without going through the granulating or slugging steps. A clear or opaque protective coating consisting of a sealing coat of shellac, a coating of sugar or polymeric material, and a polish coating of wax can be provided. Dyestuffs can be added to these coatings to distinguish different unit dosages.

[0065] Oral fluids such as solutions, syrups, and elixirs can be prepared in dosage unit form so that a given quantity contains a predetermined amount of the compound. Syrups can be prepared by dissolving the compound in a suitably flavored aqueous solution, while elixirs are prepared through the use of a non-toxic vehicle. Solubilizers and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, polyethylene glycols, tetrahydrofurfuryl alcohol, ethoxylatedisostearyl alcohols, polyoxyethylene sorbitol and sorbitol ethers, preservatives, flavor additive such as peppermint oil or natural sweeteners, or saccharin or other artificial sweeteners, and the like can also be added.

[0066] Where appropriate, dosage unit formulations for oral administration can be microencapsulated. The formulation can also be prepared to prolong or sustain the release as for example by coating or embedding particulate material in polymers, wax, or the like.

[006h The composition may also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles, and multilamellar vesicles. Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine, or phosphatidylcholines.

[0068] The composition may also be delivered by the use of monoclonal antibodies as individual carriers to which the compound molecules are coupled. The compounds may also be coupled with soluble polymers as targetable drug carriers. Such polymers can include polyvinylpyrrolidone, pyran copolymer, polyhydroxypropylmethacrylamidephenol, polyhydroxyethylaspartami dephenol, or polyethyleneoxidepolylysine substituted with palmitoyl residues. Furthermore, the compounds may be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, polyepsiloncaprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates, and cross-linked or amphipathic block copolymers of hydrogels.

[0069] Pharmaceutical formulations adapted for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats, and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets.

[0070] It should be understood that in addition to the ingredients particularly mentioned above, the formulations may include other agents conventional in the art having regard to the type of formulation in question, for example those suitable for oral administration may include flavoring agents.

[0071] A therapeutically effective amount of a compound of the present invention will depend upon a number of factors including, for example, the age and weight of the animal, the precise condition requiring treatment and its severity, the nature of the formulation, and the route of administration, and will ultimately be at the discretion of the attendant physician or veterinarian. However, an effective amount of a compound of for treatment of mycobacterial infections, for example TB, will generally be in the range of 0.1 to 100 mg/kg body weight of recipient (mammal) per day and more usually in the range of 1 to 20 mg/kg body weight per day.

[0072] The compounds of the present invention and therapeutically acceptable salts thereof, may be employed alone or in combination with other therapeutic agents for the treatment of the above-mentioned conditions. The composition may be administered together or separately and when administered separately this may occur simultaneously or sequentially in any order. The amounts of the compound(s) of formula (I) and the other pharmaceutically active agent(s) and the relative timings of administration will be selected in order to achieve the desired combined therapeutic effect.

[0073] In one embodiment, the composition is formulated as an oral dosage form.

[0074] In some embodiments, the bacterial infection is selected from the group selected from those caused by: Mycobacterium tuberculosis, Mycobacterium abscessus, Mycobacterium marinum, Mycobacterium smegmatis, Mycobacterium bovis, Mycobacterium africanum, Mycobacterium canetti, Mycobacterium caprae, Mycobacterium microti, Mycobacterium kansasii, Mycobacterium leprae, Mycobacterium avium, Mycobacterium paratuberculosis, Mycobacterium pinnipedii, Escherichia coli, Enterococcus faecalis, Enterococcus faecium, Klebsiella pnemoniae, shigella sonnei, Salmonella enterica or Yersinia enterocolitica.

[0075] In some embodiments, the mycobacteria is selected from the groups consisting of Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium africanum, Mycobacterium canetti, Mycobacterium caprae, Mycobacterium microti, Mycobacterium leprae, Mycobacterium avium, Mycobacterium paratuberculosis and Mycobacterium pinnipedii. In various embodiments, the aforementioned composition is effective in curing tuberculosis.

[0076] In some embodiments, provided herein is a method of treatment for treatment of bacterial infections, the method including administering a composition of diosmin, diosmentin, or a pharmaceutically acceptable salt thereof, at least one beta-lactam antibiotic and at least one beta-lactamase inhibitor. In other embodiments, the method includes administering a combination of diosmin, diosmentin, or a pharmaceutically acceptable thereof, amoxicillin and clavulanic acid. In some embodiments, the method additionally includes administering an antimycobacterial drug together or separately. In some embodiments, the diosmin is administered at a concentration of about 1000-3000 mg/day, the beta-lactam administered at a concentration of about 1000-1500 mg/day, the beta-lactamase inhibitor is administered at a concentration of about 250-375 mg/day, or the anti-mycobacterial drug is administered at a concentration of about 8-12 mg/kg for rifampicin, 4-6 mg/kg for isoniazid, 20-30 mg/kg for pyrazinamide, and 15 - 25 mg/kg for ethambutol. In some embodiments, one or more addition active ingredient is administered together or separately. In some embodiments, one or more of the standard TB drugs, including but not limited to rifampicin, isoniazid, ethambutol and pyrazinamide, are used for the combination treatment. In some embodiments, the combination treatment is an effective and alternative treatment of TB in a shorter period of time.

[0077] The benefits of the claimed composition, dosage form and method of treatment to patients include, oral administration instead of intravenous, increased bioavailability and economic viability. In some embodiments, the combination therapy is effective for treatment of drug resistant TB patients. In some embodiments, the composition sensitizes drug resistant TB patients to one or more standard TB drugs including but not limited to rifampicin, isoniazid, ethambutol and pyrazinamide. In some embodiments, the combination shows a similar or equivalent toxicity profile to that of the individual drugs. In some embodiments, the combination is well tolerated over the administration period in patients in need thereof.

[0078] In various embodiments, a method of inhibiting Ldt(Mtl) and Ldt(Mt2) in bacterial cells is provided. The method includes adding diosmin, diosmentin or a pharmaceutically acceptable salt thereof, a beta-lactam and a beta-lactamase inhibitor to inhibit Ldt(Mtl) and Ldt(Mt2) in bacterial cells. In some embodiments, one or more anti- mycobacterial drugs are additionally added. In some embodiments, diosmin binds to one or more residues of Ldt _Mt 2 selected from Asn 356, Thr 320, Ile 302, His 336, Ser 351, Trp 340, His 352, and Met 303. In some embodiments, diosmin binds to one more residues of Ldt _Mti selected from His 224, Asn 228, Thr 193, Thr 194, Ser 223, Cys 226, Trp 212, and His 224. [0079] Without being bound to any particular theory, it is suggested herein that diosmin efficiently binds to two functional mycobacterial L,D- transpeptidase enzyme (Ldt _Mti and Ldt _Mt 2) active sites responsible for the non-classical type (meso- dioaminopimebc acid (mDAP) - mDAP) peptidoglycan (PG) cross linkages. Further, the combination of diosmin with beta-lactam antibiotics such as amoxicillin and beta-lactamase inhibitor such as clavulanic acid has superior and synergistic in vitro and in vivo anti- mycobacterial activity.

[0080] While the invention has been disclosed with reference to certain embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the scope of the invention. In addition, many modifications may be made to adapt to a particular situation or material the teachings of the invention without departing from its scope. Further, the examples to follow are not to be construed as limiting the scope of the invention which will be as delineated in the claims appended hereto.

[0081] EXAMPLES

[0082] Example 1: Virtual Screening of FDA approved drugs against mycobacterial L,D transpeptidases

[0083] Emerging antibiotic resistance is a major concern in current TB chemotherapy. Simultaneous inhibition of D,D-transpeptidase and Ldt enzymes prevents the formation of both PG crosslinking, which is a key molecular mechanism for Mtb growth, survival and drug resistance. We adopted an in silico strategy to identify drugs that may be useful in addressing this transpeptidase molecular mechanism. The unexpected and significant anti-mycobacterial activity was then validated for the selected drugs using both in vitro and in vivo model systems.

[0084] Computational drug repurposing strategy was adapted to repurpose FDA approved drugs for using as an anti-TB agent. The protein target of interest in the present study is L,Dtranspeptidase (Ldt) enzymes (Ldt-land Ldt-2) which are responsible for non-classical cell wall (PG) cross linking. The crystal structure of Mycobacterium tuberculosis Ldt-land Ldt-2 (Ldt _Mti and Ldt _M c) was retrieved from the protein databank (PDB). The active site of both the mycobacterial Ldt enzymes were reported consisting of a His, Ser and Cys residues acting as catalytic triad. Crystal structure studies had revealed key catalytic triad residues Cys 226, His 208, Ser 209 of Ldt _Mti and Cys 354, His 336, Ser 337 of Ldt _M c that are directly involved in non-classical PG cross-linking. Further, previous studies had shown that Mtb gene knockout encoding for Ldt _Mt 2 (Rv25l8c) resulted in altered colony morphology and loss of virulence.

[0085] Initially, we performed MSA of Ldts of Mtb, M. marinum, and M. smegmatis, in order to understand evolutionary sequence conservation. Interestingly, Mtb Ldt _Mti shares a sequence identity of 81.7% and 56.9% towards Ldt _Mmi and Ldt _Msi respectively. Similarly, Mtb Ldt _M c shares a sequence identity of 82.2% and 67.3% towards Ldt _Mmi and Ldt _Ms 2 respectively, and is presented in FIG. 1, the sequence alignment of the catalytic region of L,D transpeptidases of Mtb (Ldt _Mti & Ldt _Mt 2), M. marinum (Ldt _Mmi & LdtM _{m )} and M. smegmatis (Ldt _MSi & Ldt _MS 2)· The three important and conserved catalytic residues Cys (C), His (H) and Ser (S) for all of them are boxed. The rectangular boxes and asterisks indicate the reported key residues involved in the inhibitor binding of Ldt _Mti and Ldt _Mt 2 respectively. The primary sequence analysis suggests that the Ldt enzymes of all these three microorganisms (Mtb, M. marinum and M. smegmatis) share high sequence identity, suggesting its similar 3D functional fold. Recent studies showed that carbapenems MEM, ETP, Tebipenem etc. covalently bind to both these Ldt enzymes via active site Cys residue. X-ray crystallographic and kinetic studies revealed Met 175/303, Tyr 190/318, His 224/352, Gly 225/353, and Asn 228/356 residues of Ldt- l/Ldt-2 forming key non-covalent interactions with the inhibitor and most of these residues are well conserved in our multiple sequence analysis. The present studies validate inhibition of both Ldt _Mti and Ldt _Mt 2 enzymes based on successful prediction of the conserved Ldt _Mti and Ldt _Mt 2 enzymes. [0086] FDA approved drugs from DrugBank database was virtual screened (VS) using molecular docking technique against Ldt _Mti and Ldt _Mt 2· Best 10% of Glide high scoring drugs in both VS hits were identified and two drugs were further selected among the top hits against both Ldt _Mti and Ldt _M c enzymes on the basis of docking pose and binding affinity (kcal/mol) (Table 1, FIG. 2A). Acarbose, was obtained as best VS hit with a docking score of -10.01 and -13.16 kcal/mol for Ldt _Mti and Ldt _Mt 2 enzymes respectively. Acarbose drug has low bioavailability and was used for non-insulin dependent diabetes mellitus therapeutically. It was reported earlier that acarbose binds to trehalose synthase of M. smegmatis. Diosmin (DIO) is the second highest ranked VS hit with binding affinity of -9.37 and -10.55 kcal/mol towards active sites of Ldt _Mti and Ldt _Mt 2 (Table 1). DIO a bioflavonoid compound is used to treat venous disorders and also act as dietary supplement. It also possesses anti-inflammatory and anti-oxidant activities. Since Acarbose has very low bioavailability (~2%), we further selected DIO for the in vitro and in vivo studies.

[0087] Diosmin is a semisynthetic flavonoid molecule, which is approved in various places for use as a nutrient supplement and for the treatment of venous diseases, such as, chronic venous insufficiency (CVI), leg swelling, stasis dermatitis and venous ulcers. Diosmin is composed of a flavanoid moiety which is linked to a carbohydrate moiety via O-glycosidic bond as shown in FIG. 2B. The molecular mechanism of DIO interaction with Ldt _Mti residues - His 208, His 224, Val 222, Asn 228, Thr 193, Thr 194, and Ser 223 via hydrogen bonding, sulfur atom of Cys 226 form a p-bond with the flavonoid moiety of DIO; His 224 and Trp 213 are making p-p stacking interactions with DIO. Further, interaction of DIO towards Ldt _Mt 2 showed Asp 304, Gln 327, Tyr 330, Ser 331, Tyr 308, and Thr 320 residues making hydrogen bonding interactions; p-p stacking interactions with Met 303, His 352, Tyr 330 and Tyr 318, shown in Table 1 and FIG. 3A- 3F. Experimentally reported key residues- His 224, Cys 226, Asn 228 of Ldt _Mti and Ser 331, Tyr 308, Thr 320, His 352, Met 303, Tyr 318 of Ldt _Mt2 10,16,19,20,24 required for binding carbapenems to address molecular mechanism of inhibition process. Thus, DIO compound adopted a similar mode of binding to the active site of both Ldt enzymes. Residues in bold take part in hydrogen bonding interaction and others are involved in hydrophobic interactions with the Ldt enzyme (Table 1). Experimentally reported amino acid residues involved in PG binding mechanisms are underlined.

Table 1: The highest ranked overlapping hits towards both Ldt _Mti and Ldt _Mt 2 enzymes obtained after virtual screening and the molecular interactions of Ldt _Mti and Ldt _Mt 2 towards Diosmin.

[0088] DIO is hydrolyzed into its aglycone form Diosmetin (DMT) by intestinal microflora enzymes. The binding mode of DMT towards both Ldt _Mti and Ldt _Mt 2 with respect to DIO was analyzed for understanding the molecular mechanism of interactions. The molecular docking and electrostatic potential surface map of these complexes showed flavonoid moiety of DIO is occupied in the active site tunnel of Ldts and its carbohydrate moiety lie outside its active site regions. This implies flavonoid moiety of DIO contribute for Ldts enzyme inhibition. Further, binding mode of DMT at the active sites of Ldt _Mti and Ldt _Mt 2 adopts similar orientation and position as flavonoid moiety of DIO, with a binding affinity of -6.48 kcal/mol and -6.23 kcal/mol respectively (not shown).

[0089] To effectively block PG biosynthesis of Mtb, both classical and non-classical cross linkages mediated by D,D-transpeptidase and Ldt enzymes needs simultaneous inhibition. In the present example, DIO (FDA approved drug) or DMT was repurposed for tuberculosis (TB) along with AMC (amoxicillin: clavulanic acid - 4: 1). Beta-lactam antibiotic, AMX is specific for D,D-transpeptidase enzyme. Glide molecular docking of AMX at active site of D,D transpeptidase (PDB ID: 5CXW) enzyme showed binding energy of -5.64 kcal/mol (not shown). Our results elaborated below suggest strong binding of DIO with Mtb-Ldt enzymes and AMX with D,D-transpeptidase enzyme active sites, leading to a synergistic/combined molecular mechanism of Mtb growth inhibition.

[0090] The structural stability of both Ldt enzymes in complex with DIO was analysed for 20 ns molecular dynamics (MD) simulations. Since, Meropenem (MEM) is reported as a potent Ldt inhibitor; Ldt _Mt 2:MEM crystal structure (PDB ID: 4GSU) was used as a control for our MD simulations. Trajectories after MD simulations was analysed to check stability of complexes, Ldt _Mt 2:MEM, Ldt _Mti DIO, and Ldt _Mt 2:DIO. Good structural stability was observed for DIO in complex with Ldt _Mti and Ldt _Mt 2, as shown in FIG. 4A and 4B. Ldt _Mt 2:MEM was unstable throughout MD simulations, as shown by high backbone fluctuations with an average root mean square deviation (RMSD) of 2.34 A (FIG. 4B), whereas the average RMSD of Ldt _Mti : DIO and Ldt _Mt 2:DIO complexes was 1.5 A and 1.3 A. Using MM-GBSA method, (AG) values for Ldt _Mt 2:MEM complexes was -15.10 kcal/mol; a two fold increase in AG was observed for docked complexes of Ldt _M u :DIO and Ldt _Mt 2:DIO as -30.61 and -30.13 kcal/mol (not shown). Our in silico results thus broadly indicate that DIO predicted a better binding affinity towards both Ldt enzymes in comparison to that of MEM. The root mean square deviation plot of (a) Ldt _Mti -DIO complex, (b) Ldt _Mt 2-DIO docked complex and Ldt _Mt 2-Meropenem (MEM) crystal structure during 20 ns MD simulations. [0091] Example 2: Experimental binding studies of DIO and DMT and Mycobacterial Ldt enzymes

[0092] All the drugs and strains used here were obtained commercially. The binding of Ldt _Mti and Ldt _M c with DIO was studied using bioassay method. The mycobacterial Ldt enzymes were allowed to bind to the Ni-NTA column and DIO (or DMT) were allowed to interact with the column bound Ldt enzymes. The complexes of Ldt-DIO/(or Ldt- DMT) were eluted, and DIO (or DMT) were separated from the Ldt proteins by ultrafiltration technique. We measured the optical density (OD) at 263.5 nm of the filtrate. DIO is found to be interacting with both the Ldt enzymes and the OD values of DIO in Ldt _Mt 2-DIO fractions was 0.245 and for Ldt _Mti -DIO fractions was 0.15, shown in FIG. 5A. This result suggests that DIO has more binding affinity towards Ldt _Mt 2 in comparison to that of Ldt _Mti enzyme. Our molecular docking results also showed a similar trend of binding affinity when DIO was docked at the active site of Ldt _Mt 2 and Ldt _Mti , as described earlier (Table 1). Similarly, we analysed the binding of DMT towards both the mycobacterial Ldt enzymes. The peak at 344 nm indicated the presence of DMT in the eluted fractions. The OD values of DMT in the eluted fraction of both Ldt _Mti and Ldt _Mt 2 enzymes are almost similar as shown in FIG. 5B. Thus, DMT might have a similar binding affinity towards both the enzymes and is also in good agreement with our molecular docking studies.

[0093] Example 3: In vitro anti-mycobacterial activity studies of different drugs towards M. marinum

[0094] MDR clinical isolate of Mtb was identified by BACTEC MGIT 960 as Mtb, resistant to all the first line anti-tubercular drugs (INH, RIF, Pyrazinamide, and Ethambutol) and second line quinolones (Ciprofloxacin, Ofloxacin, Levofloxacin). In vitro anti-mycobacterial activity of AMC, DIO, DMT alone and their combinations are tested against M. marinum. No significant growth inhibition was observed when M. marinum cultured in the presence of increasing concentrations of 100, 250 or 500 pg/ml DIO or DMT. Minimum bactericidal concentration (MBC) of AMC against M. marinum obtained in our study was 8 pg/ml. MBC was defined as the lowest concentration of the drugs that kills 99% of the mycobacteria. Next, increasing amount of AMC (0.5 pg/ml to 4 pg/ml) was added to the M. marinum cultures along with the fixed concentration 500 pg/ml of DIO or DMT, which showed a gradual decrease in CFU as compared to AMC alone (FIG. 5C). The MBC of AMC was reduced to 4 pg/ml from 8 pg/ml, as shown in Table 2 when used with DIO (500 pg/ml). Further, AMC-DMT combination of drugs exhibited higher anti-mycobacterial activity as compared to AMC-DIO, as indicated by the reduction in the colony forming unit (CFU), shown in FIG. 5C. The present results indicated that AMC has synergistic anti-mycobacterial activity in combination with DIO or DMT.

TABLE 2: The minimum bactericidal concentration (MBC) of augmentin alone and in combination with DIO (500 pg/ml) against M marinum andM smegmatis.

[0095] Example 4: M. marinum treated with AMC-DIO (or DMT) combination of drugs severely altered its cell surface morphology

[0096] Scanning electron microscopy (SEM) was performed for M. marinum incubated with drugs. Commercially acquired M. marinum was grown for 2 days in middlebroke 7H9 medium supplemented with 0.05% Tween 80 and albumin-dextrose complex (ADC) enrichment at 29°C under 120 rpm shaking. Then AMC (8 pg/ml), DIO (500 pg/ml), DMT (500 pg/ml), AMC (8 pg/ml)- DIO (500 pg/ml), and AMC (8 pg/ml)- DIO (500 pg/ml) combination were added into separate culture tubes and incubated for 48 hrs. Untreated M marinum culture was used as the control. [009h The PG layer of mycobacteria provides structural integrity and is essential for its growth and survival. M. marinum without any drug treatment (FIG. 6A) or AMC (8 pg/ml) (FIG. 6B), DIO (500 pg/ml) (FIG. 6C), DMT(500 pg/ml) (FIG. 6D), AMC (8 pg/ml) - DIO (500 pg/ml) (FIG. 6E) and AMC (8 pg/ml) - DMT (500 pg/ml were examined. M. marinum treated with DIO (or DMT) alone revealed a smooth cell surface as depicted in FIG. 6C and FIG. 6D. AMC treated M. marinum exhibited slightly roughened cell surface with dents (FIG. 6B). However, M. marinum upon treatment with AMC-DIO or AMC-DMT combination of drugs showed dramatic changes in their cellular surface morphology and consisted of unrecognizable large amorphous mass of cell debris (FIG. 6E and 6F). Degraded cells appeared shrunk and corrugated with evident atrophy. In addition, bulges were present all over mycobacterial cell surface. It is well known that Mtb cell wall has both DD-transpepetidase and Ldt enzymes, which play a key role in PG cross-linking, and severe cellular atrophy seen on AMC-DIO drug combination due to synergistic inhibition of PG cross-linking enzymes. The present combination of drugs AMC-DIO (or AMC-DMT) may be administered orally as a single formulation or separately. It showed high in vitro anti-mycobacterial activity and is suitable for TB therapy.

[0098] Example 5: Synergistic effects of AMC and DIO (or DMT) drug combinations in M. marinum infected Drosophila melanogaster fly model

[0099] The in vivo efficacy of the AMC-DIO and AMC-DMT combination of drugs were tested in M. marinum infected D. melanogaster fly models. The fly model was previously validated by other researchers for anti-mycobacterial activity assessment of various drugs. Wild type D. melanogaster were fed and raised at 29°C on banana media. Following 6-7 hours of starvation, the flies were given 60 pl of 500 pg/ml cyclophosphomide. After 24 hrs the flies were infected with M. marinum. M. marinum (ATCC 927) was grown in middlebrook 7H9 broth supplemented with 0.05% Tween 80 and albumin-dextrose complex (ADC) enrichment for 5 days at 29°C under 120 rpm shaking. For the infection the flies were anesthetized on ice and the culture (OD ₆ oo - 3.0) was injected into the junction area between the ventral and dorsal cuticles of the flies. Following the infection the flies were incubated in banana medium containing drugs AMC (0.5 and 1 mg/ml), DIO (0.25, 0.50, 1 and 2 mg/ml) and Rifampicin (0.25 mg/ml) and their combinations. Flies were transferred to fresh vials supplemented with the same drug concentrations every other day. 20 flies per condition were used for the experiment and an equal number was used as controls. The number of dead flies was monitored daily and survival plot was generated.

[00100] A 100% mortality was seen in M marinum infected flies within 9-10 days as shown in FIG. 7A. Further, using mass spectrometry (MS) analysis, it was confirmed that diosmin (DIO) was getting converted to diosmetin (DMT) in the gut, similar to humans, as shown in FIG. 8A and 8B. Treatment of infected flies with different drug concentrations of either AMC or DIO individually does not show any marked fly survival (FIG. 7A and FIG. 7B). However, the survivability of the flies was significantly improved when treated with increasing concentration of AMC and DIO combination of drugs. Maximum fly survival was observed at higher concentration of AMC (1 mg/ml) and DIO (2 mg/ml) treated flies, which further showed 66.67% of survival, revealing the synergistic effect of drug combination (FIG. 7C). Further, we analyzed the efficacy of DMT (2 mg/ml) at the same concentration of DIO in combination with AMC (1 mg/ml) in infected flies. AMC -DMT combination of drugs also exhibited a synergistic activity with 60.0 % survival of infected flies, shown in FIG. 7D. Thus, the present MS analysis and fly survival studies indicated that DIO after oral administration is converted into DMT, and also the therapeutic effect of either AMC-DIO or AMC-DMT combination of drugs is similar inM marinum infected /) melanogaster fly model.

[00101] Additionally, the efficacy of AMC-DIO combination of drugs was checked along with a standard TB drug, RIF. When infected flies were fed with a lower concentration of RIF (0.25 mg/ml), no significant fly survival was observed. Interestingly, a better fly survival of 83.33% was observed when infected flies were treated with a combination of AMC (1 mg/ml), DIO (2 mg/ml) and RIF (0.25 mg/ml) drugs, shown in FIG. 7E. The statistical significance was analysed using ANOVA (***, P< 0.001; **, P <0.01; ns, not significant.

[00102] Further, bacterial load in the infected flies on 3, 5, 7 and 9 days of drug treatments were determined by AFB (Acid Fast Bacilli) staining shown in FIG. 9A-I. The number of bacteria present in 100 fields was counted and graded according to WHO/IUATLD Quantification scale.

[00103] Progressive increase in the severity of M. marinum infection was seen in flies treated with AMC, DIO and DMT drug individually, presented in Figure 9B, 9C, and 9E. The corresponding AFB smear grading of these drugs escalated to 3+ within five days of treatment shown in FIG. 91. However, a reduction in the bacterial load was observed with AMC-DIO or AMC-DMT combination of drug treatments. The flies were completely free from M. marinum infection as indicated by the absence of bacilli in the smears on the 9th day of treatment with AMC-DIO or AMC-DMT combination of drugs (FIG. 9D, 9F). This in vivo observation strongly supports our earlier fly survival studies and supports the synergistic activity of the drug combinations. Further, we checked the bacterial load in infected flies treated with RIF drug alone and its combination with AMC-DIO drugs. In RIF treated fly group, only a few bacilli were present in the smear samples after 9 days of treatment (FIG. 9G). However, the survival of infected flies was only -20% as mentioned above. Further, RIF-AMC-DIO combination of drug treatment was very effective and no bacilli were seen in fly smear from 7th day onwards presented in FIG. 9H and 91. In summary, treatment of M. marinum infected flies with individual drugs did not show any marked improvement in their fly survival, while the combination of AMC-DIO or AMC-DMT or AMC-DIO-RIF drugs showed better survival of infected flies.

[00104] Example 6: In vitro anti-mycobacterial activity of AMC-DIO combination of drugs against Mtb and MDR clinical strain.

[00105] Anti-mycobacterial activity of various concentrations of AMC, DIO and their combinations were tested against Mtb H37Ra strain using an automated MGIT 960 system. No mycobacterial growth inhibition was seen when AMC or DIO alone at any concentrations were tested. Interestingly, AMC at 12 pg/ml concentration showed a gradual reduction in the mycobacterial growth when added in combination with 500 pg/ml of DIO, as shown in FIG. 11A. Further, a complete inhibition of the growth oiMtb H37Ra strain was observed at concentrations of 20 pg/ml of AMC and 500 pg/ml of DIO combination of drugs (FIG. 11 A).

[00106] Resistance to different antibiotics is of major concern in the current TB treatment regimen. Thus, we tested the efficacy of individual AMC and DIO as well as an AMC-DIO combination of drugs at different concentrations, against MDR-Mtb clinical strain, presented in FIG. 11B. In this case as well the individual drugs (AMC or DIO) were not able to inhibit the bacterial growth of MDR clinical strain in consonance with that of the Mtb H37Ra strain. Interestingly, AMC starting from 12 pg/ml concentration showed a gradual reduction in the growth of MDR strain when added along with 500 pg/ml of DIO combination of drugs and a complete Mtb growth inhibition was seen when 20 pg/ml of AMC and 500 pg/ml of DIO combination was added, as shown in FIG. 10A. Further, a gradual reduction in the growth of MDR clinical strain was observed from 16 pg/ml of AMC and 500 pg/ml of DIO combination and a complete sensitivity of MDR strain was observed for the combination of AMC and DIO drugs at concentrations of 24 pg/ml and 500 pg/ml respectively as shown in FIG. 10B. Thus, the present data clearly suggest a synergistic inhibitory activity of the AMC-DIO combination of drugs against Mtb.