WO2025017047A1

WO2025017047A1 - Combination products of at least three antibiotics comprising bedaquiline, pretomanid or delamanid, and a cytochrome bc1 inhibitor, and their use in the treatment of mycobacterial infections

Info

Publication number: WO2025017047A1
Application number: PCT/EP2024/070206
Authority: WO
Inventors: Dirk Antonie LAMPRECHT; Clara AGUILAR PÉREZ; Nacer Lounis; Alexander Stephen PYM
Original assignee: Janssen Pharmaceutica NV
Current assignee: Janssen Pharmaceutica NV
Priority date: 2023-07-18
Filing date: 2024-07-17
Publication date: 2025-01-23
Anticipated expiration: 2026-01-18

Abstract

The present invention relates to novel combinations, which are useful in the treatment of tuberculosis.

Description

COMBINATION PRODUCTS OF AT LEAST THREE ANTIBIOTICS COMPRISING BEDAQUILINE, PRETOMANID OR DELAMANID, AND A CYTOCHROME BC1 INHIBITOR, AND THEIR USE IN THE TREATMENT OF MYCOBACTERIAL INFECTIONS

The present invention relates to novel combinations. The invention also relates to such combinations for use as pharmaceuticals, for instance in the treatment of bacterial diseases, including diseases caused by pathogenic mycobacteria such as Mycobacterium tuberculosis. Such combinations may be advantageous in the treatment of tuberculosis.

BACKGROUND OF THE INVENTION

Mycobacterium tuberculosis is the causative agent of tuberculosis (TB), a serious and potentially fatal infection with a world-wide distribution. Estimates from the World Health Organization indicate that more than 8 million people contract TB each year, and 2 million people die from tuberculosis yearly. In the last decade, TB cases have grown 20% worldwide with the highest burden in the most impoverished communities. If these trends continue, TB incidence will increase by 41% in the next twenty years. Fifty years since the introduction of an effective chemotherapy, TB remains after AIDS, the leading infectious cause of adult mortality in the world. Complicating the TB epidemic is the rising tide of multi-drug-resistant strains, and the deadly symbiosis with HIV. People who are HIV-positive and infected with TB are 30 times more likely to develop active TB than people who are HIV-negative and TB is responsible for the death of one out of every three people with HIV/AIDS worldwide.

Existing approaches to treatment of tuberculosis all involve the combination of multiple agents. For example, the regimen recommended by the U.S. Public Health Service is a combination of isoniazid, rifampicin and pyrazinamide for two months, followed by isoniazid and rifampicin alone for a further four months. These drugs are continued for a further seven months in patients infected with HIV. For patients infected with multidrug resistant strains of AT. tuberculosis, agents such as ethambutol, streptomycin, kanamycin, amikacin, capreomycin, ethionamide, cycloserine, ciprofoxacin and ofloxacin are added to the combination therapies. There exists no single agent that is effective in the clinical treatment of tuberculosis, nor any combination of agents that offers the possibility of therapy of less than six months’ duration.

There is a high medical need for new drugs or new combinations of drugs that improve current treatment by enabling regimens that facilitate patient and provider compliance. Shorter regimens and those that require less supervision are the best way to achieve this. Most of the benefit from treatment comes in the first 2 months, during the intensive, or bactericidal, phase when four drugs are given together; the bacterial burden is greatly reduced, and patients become non-infectious. The 4- to 6-month continuation, or sterilizing, phase is required to eliminate persisting bacilli and to minimize the risk of relapse. Novel drugs or combinations that potentially shorten treatment to 2 months or less would be extremely beneficial. It would also be beneficial to reduce the number of drugs required. Facilitating compliance by requiring less intensive supervision may also be beneficial. Obviously, novel drugs or novel combinaions of drugs that reduce both the total length of treatment and the frequency of drug administration would provide the greatest benefit.

Complicating the TB epidemic is the increasing incidence of multi-drug-resistant strains or MDR-TB. Up to four percent of all cases worldwide are considered MDR-TB - those resistant to the most effective drugs of the four-drug standard, isoniazid and rifampin. MDR-TB is lethal when untreated and cannot be adequately treated through the standard therapy, so treatment requires up to 2 years of "second-line" drugs. These drugs are often toxic, expensive and marginally effective. In the absence of an effective therapy, infectious MDR-TB patients continue to spread the disease, producing new infections with MDR-TB strains. There is a high medical need for new therapies (e.g. combinations) likely to demonstrate activity against drug-resistant, in particular MDR strains.

The term “drug resistant” as used hereinbefore or hereinafter is a term well understood by the person skilled in microbiology. A drug resistant Mycobacterium is a Mycobacterium which is no longer susceptible to at least one previously effective drug; which has developed the ability to withstand antibiotic attack by at least one previously effective drug. A drug resistant strain may relay that ability to withstand to its progeny. Said resistance may be due to random genetic mutations in the bacterial cell that alters its sensitivity to a single drug or to different drugs.

MDR tuberculosis is a specific form of drug resistant tuberculosis due to a bacterium resistant to at least two previously effective drugs (for instance, as defined by the WHO, and can be isoniazid and rifampicin, with or without resistance to other drugs, which are at present the two most powerful anti-TB drugs). Thus, whenever used hereinbefore or hereinafter “drug resistant” includes multi drug resistant.

Another factor in the control of the TB epidemic is the problem of latent TB. In spite of decades of tuberculosis (TB) control programs, about 2 billion people are infected by M. tuberculosis, though asymptomatically. About 10% of these individuals are at risk of developing active TB during their lifespan. The global epidemic of TB is fuelled by infection of HIV patients with TB and rise of multi-drug resistant TB strains (MDR-TB). The reactivation of latent TB is a high risk factor for disease development and accounts for 32% deaths in HIV infected individuals. To control TB epidemic, the need is to discover new drugs that can kill dormant or latent bacilli. The dormant TB can get reactivated to cause disease by several factors like suppression of host immunity by use of immunosuppressive agents like antibodies against tumor necrosis factor a or interferon-y. In case of HIV positive patients, the only prophylactic treatment available for latent TB is two three-months regimens of rifampicin, pyrazinamide. The efficacy of the treatment regime is still not clear and furthermore the length of the treatments is an important constrain in resource-limited environments. Hence there is a drastic need to identify new drugs, which can act as chemoprophylatic agents for individuals harboring latent TB bacilli.

The tubercle bacilli enter healthy individuals by inhalation; they are phagocytosed by the alveolar macrophages of the lungs. This leads to potent immune response and formation of granulomas, which consist of macrophages infected with M. tuberculosis surrounded by T cells. After a period of 6-8 weeks the host immune response cause death of infected cells by necrosis and accumulation of caseous material with certain extracellular bacilli, surrounded by macrophages, epitheloid cells and layers of lymphoid tissue at the periphery. In case of healthy individuals, most of the mycobacteria are killed in these environments but a small proportion of bacilli still survive and are thought to exist in a non-replicating, hypometabolic state and are tolerant to killing by anti-TB drugs like isoniazid. These bacilli can remain in the altered physiological environments even for individual’s lifetime without showing any clinical symptoms of disease. However, in 10% of the cases these latent bacilli may reactivate to cause disease. One of the hypothesis about development of these persistent bacteria is patho-physiological environment in human lesions namely, reduced oxygen tension, nutrient limitation, and acidic pH. These factors have been postulated to render these bacteria phenotypically tolerant to major anti -mycobacterial drugs.

In addition to the management of the TB epidemic, there is the emerging problem of resistance to first-line antibiotic agents. Some important examples include penicillin- resistant Streptococcus pneumoniae, vancomycin-resistant enterococci, methicillin- resistant Staphylococcus aureus, multi-resistant salmonellae. The consequences of resistance to antibiotic agents are severe. Infections caused by resistant microbes fail to respond to treatment, resulting in prolonged illness and greater risk of death. Treatment failures also lead to longer periods of infectivity, which increase the numbers of infected people moving in the community and thus exposing the general population to the risk of contracting a resistant strain infection.

Hospitals are a critical component of the antimicrobial resistance problem worldwide. The combination of highly susceptible patients, intensive and prolonged antimicrobial use, and cross-infection has resulted in infections with highly resistant bacterial pathogens.

Self-medication with antimicrobials is another major factor contributing to resistance. Self-medicated antimicrobials may be unnecessary, are often inadequately dosed, or may not contain adequate amounts of active drug.

Patient compliance with recommended treatment is another major problem. Patients forget to take medication, interrupt their treatment when they begin to feel better, or may be unable to afford a full course, thereby creating an ideal environment for microbes to adapt rather than be killed.

Because of the emerging resistance to multiple antibiotics, physicians are confronted with infections for which there is no effective therapy. The morbidity, mortality, and financial costs of such infections impose an increasing burden for health care systems worldwide.

Therefore, there is a high need for new therapies to treat bacterial infections, especially mycobacterial infections.

As mentioned above, several drugs already exist for the treatment of tuberculosis, and there are a number of different combination regimens that are either approved by regulatory authorities or in the WHO Guidelines. Amongst the relatively established tuberculosis drugs, there is for instance, pyrazinamide (PZA), clofazimine (CFZ), isoniazid (INH), rifampicin (RIF) and fluoroquinolones. Other antibiotics include amoxicillin, ethionamide, ethambutol as well as certain macrocyclic molecules.

Various regimens exist for the treatment of drug-sensitive tuberculosis, including various combinations of established drugs for a 4- or 6-month treatment duration. There was a time when treatment of drug-resistant tuberculosis was up to two years. However, more recently approved tuberculosis drugs have reducted this 2 year treatment regime time period, advancing the field. The first of such more recent approvals was bedaquiline (marketed as Sirturo®), which in 2012 was the first new tuberculosis drug approved by the US FDA in 40 years. Since then, there have also been approvals for two nitroimidazole derivatives, namely delamanid and pretomanid. Further, oxazolidinone antibiotics, such as the approved linezolid (LZD) and sutezolid (SZD), which is in development and is also an antibiotic that has been tested against tuberculosis in certain models. The usage of LZD was curtailed by dose- and durationdependent toxicity. Among the more recently approved tuberculosis drugs, pretomanid was approved as a part of a regimen with bedaquiline and linezolid for the treatment of certain MDR-TB and XDR-TB (extensively drug-resistant tuberculosis) populations (pursuant to the Nix-TB clinical trial), and delamanid and bedaquiline were approved for certain MDR populations, where in all cases it was indicated that consideration should be given to official guidance on the appropriate use of antibacterial agents. For instance, certain regimens are also disclosed in WO 2017/066053.

There are several other known drugs that are used for treating tuberculosis, which may act via different mechanisms of action. There are numerous drugs in discovery and at various stages of development. For instance, Journal article Nature Medicine, 19, 1157-1160 (2013) by Pethe et al “Discovery of Q203, a potent clinical candidate for the treatment of tuberculosis” identifies a specific compound that was tested against M. tuberculosis. This compound Q203, now known as telacabec, is depicted below.

It is postulated that it acts by interfering with ATP synthase in AT. tuberculosis, and that the inhibition of cytochrome Z>ci activity is the primary mode of action. Cytochrome Z>ci is an essential component of the electron transport chain required for ATP synthesis. This clinical candidate is also discussed in journal article, J. Medicinal Chemistry, 2014, 57 (12), pp5293-5305. It is stated to have activity against MDR tuberculosis, and have activity against the strain M. tuberculosis H37Rv at a MICso of 0.28 nM inside macrophages. Positive control data (using known anti-TB compounds bedaquiline, isoniazid and moxifloxacin) are also reported. This document also suggests the mode of action, based on studies with mutants. It appeared that Q203 (telacabec) was highly active against both replicating and non-replicating bacteria. Certain combinaitons containing a cytochrome bcl inhibitor have also been disclosed, for instance in WO 2018/158280.

It is of great benefit to be able to discover new combinations and/or better combinations of known drugs given that: - combinations are likely to remain the treatment guidelines (e.g. given drug-resistant bacterial forms); and - access to the best combinations will ultimately advance patient outcomes.

SUMMARY OF THE INVENTION

It has been found that certain combinations, for use in the treatment of tuberculosis, have been found to be particularly effective (as described hereinafter in the biological results).

Thus, in an aspect of the invention, there is provided: a combination comprising:

(i) bedaquiline (or another ATP synthase inhibitor such as TBAJ-587 or TBAJ-

876), or a pharmaceutically acceptable salt thereof;

(ii) pretomanid or delamanid, or a pharmaceutically acceptable salt thereof; and

(iii) a cytochrome bc inhibitor, or a pharmaceutically acceptable salt thereof, which combination may be referred to as “combination of the invention”.

Given the results seen (see the examples hereinafter), there is also provided: a combination consisting of (e.g. consisting essentially of) the following active ingredients:

(i) bedaquiline, or another suitable ATP synthase inhibitor (e.g. TBAJ-587 or

TBAJ-876), or a pharmaceutically acceptable salt thereof;

(iii) a cytochrome bc inhibitor, or a pharmaceutically acceptable salt thereof, which combination may also be referred to as “combination of the invention”.

As mentioned hereinbefore, bedaquiline is a known ATP synthase inhibitor, marketed as the fumarate salt (Sirturo®). There are also other back-up ATP synthase inhibitors in the clinic known as TBAJ-587 and TBAJ-876:

Such compounds may be prepared in accordance with the procedures disclosed in WO 2017/155909 (followed by a separation to produce to enantiomers via chiral chromatography, e.g. using SFC), and in the context of the combinations of the invention, may replace bedaquiline.

Pretomanid and delaminid are both known antibacterials in the nitroimidazole class. As mentioned here, both have received marketing approval.

Such combinations of the invention are useful as medicaments. For instance, such combinations may in particular be useful in the treatment of a mycobacterial infection (especially Mycobacterium tuberculosis, which may also be referred to as “tuberculosis” herein). For the purposes of the invention, tuberculosis means any form of tuberculosis such as the active form or the latent form. The latent (or dormant) for is elaborated upon hereinafter. The form may also include a drug-resistant form of tuberculosis (e.g. a muti-drug resistant form, MDR form, which includes an extensively multi-drug resistant form). The combinations of the invention may be expected to be effective against MDR tuberculosis given that MDR refers to resistance due to a bacterium resistant to at least isoniazid and rifampicin (with or without resistance to other drugs), and hence the combinations of the invention, may still therefore be useful in the treatment of MDR tuberculosis. Combinations of the invention may, in an embodiment, be particularly be useful in the treatment of fluoro-quinolinone resistant strains (which strains may be resistant to other antibioitics too, e.g those mentioned above, such as rifampicin and/or isoniazid, etc).

However, although it is thought that three drugs of this combination may themselves be sufficient (e.g. potent enough), such combinations of the invention may further comprise additional antibacterial (e.g. anti-tuberculosis) drugs. For instance, any one or more (e.g. one or two) of the following antibacterial (e.g. anti-tuberculosis) agents (or pharmaceutically acceptable salts thereof) may be mentioned in addition to the essential two agents (so forming e.g. a quadruple combination, etc): other antibacterial agents known to interfere with the respiratory chain of Mycobacterium tuberculosis, including for example inhibitors of ndh2 (e.g. clofazimine); other antibacterial agents that may target the electon transport chain, e.g. that target the cytochrome bd oxidase (e.g. Aurachin D analogues); other mycobacterial agents for example rifampicin (=rifampin); isoniazid; pyrazinamide; amikacin; ethionamide; ethambutol; streptomycin; paraaminosalicylic acid; cycloserine; capreomycin; kanamycin; thioacetazone; PA- 824; delamanid; quinolones/fluoroquinolones (such as for example moxifloxacin, gatifloxacin, ofloxacin, ciprofloxacin, sparfloxacin); macrolides (such as for example clarithromycin, amoxycillin with clavulanic acid); rifamycins; rifabutin; rifapentin; as well as others, which are currently being developed (but may not yet be on the market or have only recently come onto the market; see e.g. http://www.newtbdrugs.org/pipeline.php), for instance delanamid, pretonamid and the like.

In a particular embodiment, combinations of the invention further comprise clofazimine.

A cytochrome bc\ inhibitor is referred to herein, and may more specifically be referred to as a compound that inhibits cytochrome bc\ in the ETC of Mycobacterium tuberculosis, thereby interfering with ATP synthesis resulting in preventing the bacterium from replicating, or killing it. In an embodiment, the cytochrome bc\ inhibiton of such a compound is its primary mode of action (against Mycobacterium tuberculosis'). By “inhibits” in this context, we mean that the compound is indicated as inhibiting (cytochrome bc or is known to inhibit, e.g. in a relevant test or assay, for instance as described hereinafter. For example, the compound may be tested for antibacterial activity in any one of Pharmacological Tests 1 to 4 described hereinbelow and, in an embodiment, is understood to fall within the scope of “inhibitor” in this context if anti-bacterial activity is measured, for instance, if the IC50 value is less than 10 pM (or if the pICso value is more than 5). In order to definitively determine whether a compound is a cytochrome bc inhibitor (acting primarily via that mode of action), generation of mutants resistant to the compound and further sequencing of the whole genome may be performed as was performed in the Nature Medicine journal article referenced herein (i.e. journal article Nature Medicine, 19, 1157-1160 (2013) by Pethe et al, the content of which is hereby incorporated by reference, in particular the detail provided around identifying a compound as being a cytochrome bc inhibitor). For instance, MIC50 values may be tested against mutant strains of Mycobaterium tuberculosis. Where mutants show an increase in MIC50 for the compound being tested (e.g. an increase of several orders of magnitude, such as 10-fold or 50-fold or, in an embodiment, 100-fold higher or more) but remain susceptible to other or standard antituberculosis drugs, then the compound is a “bci inhibitor” when the mutation is at the cytochrome b subunit (qcrB, also known as Rv2196 of the cytochrome bci complex). Sequence analysis (e.g. of qcrB) may also confirm that mutation of Thr313 to either alanine or isoleucine is associated with resistance to the tested compound, thereby also confirming that the tested compound is a “Z>cy inhibitor”. Further still, a re-introduction of mutation Ala313 by homologous recombination in parental Mycobacterium tuberculosis H37Rv may be tested to see if it confers resistance to the compound being tested, which may further demonstrate that the substitution is directly and specifically involved in the mechanism of resistance, also further confirming that the tested compound is a “Z>cy inhibitor”. Any compound targeting the respiratory chain may potentially inhibit the production of ATP - and hence a cytochrome bci inhibitor may also interfere with ATP synthesis, for instance causing a reduction in ATP levels (e.g. intracellular ATP). Hence, a suitable test may be conducted that measures intracellular ATP levels (to determine whether the test compound reduces ATP levels) and (a) further test(s) can be conducted thereafter to determine e.g. whether the relevant compound targets ATP synthase (e.g. bedaquiline is an ATP synthase inhibitor) or a cytochrome bc inhibitor, for instance conduting the mutation tests as specified above.

Currently, there is no enzymatic assay test for a “Z>ci inhibitor”, and the reason for this is that the bcc complex (more information around which can be found at https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4205543/) is suggested to interact directly with other enzymes (to achieve electron transfer) and functions as a supercomplex.

In an embodiment, particular bc\ inhibitors that may be mentioned include: JNJ-2901 (as described herein);

Q203 to telacabec (e.g. in a non-salt form); any of the compounds disclosed in international patent applications WO 2011/113606, WO 2015/014993, WO 2017/001660, WO 2017/001661, WO 2021/048342, WO 2022/194803, WO 2022/194905, WO 2022/194906, WO 2023/073090, etc all of which are hereby incorporated by reference.

In particular, the cytochrome bc inhibitor is a specific compound as defined herein, or a specific cytochrome bc inhibitor that receives a regulatory approval (e.g. before a stringent regulatory authority, such as the EMA and/or FDA, or a WHO-pre- qualification).

Active ingredients (e.g. the essential ATP synthase inhibitor, pretomanid/delaminid and cytochrome bc inhibitor, and/or the optional further antibacterial agents) of the combinations of the invention may also be in the form of a pharmaceutically acceptable salt. Pharmaceutically-acceptable salts include acid addition salts and base addition salts. Such salts may be formed by conventional means, for example by reaction of a free acid or a free base form of the relevant active ingredient (e.g. the ATP synthase inhibitor, pretomanid/delaminid, cytochrome bc\ inhibitor, and/or the optional further antibacterial agents) with one or more equivalents of an appropriate acid or base, optionally in a solvent, or in a medium in which the salt is insoluble, followed by removal of said solvent, or said medium, using standard techniques (e.g. in vacuo, by freeze-drying or by filtration). Salts may also be prepared by exchanging a counter-ion of a compound of the invention in the form of a salt with another counter-ion, for example using a suitable ion exchange resin.

The pharmaceutically acceptable acid addition salts as mentioned hereinabove are meant to comprise the therapeutically active non-toxic acid addition salt forms that the relevant active ingredient (e.g. the essential ATP synthase inhibitor, pretomanid/delaminid and cytochrome bc inhibitor, and/or the optional further antibacterial agents) are able to form. These pharmaceutically acceptable acid addition salts can conveniently be obtained by treating the base form with such appropriate acid. Appropriate acids comprise, for example, inorganic acids such as hydrohalic acids, e.g. hydrochloric or hydrobromic acid, sulfuric, nitric, phosphoric and the like acids; or organic acids such as, for example, acetic, propanoic, hydroxyacetic, lactic, pyruvic, oxalic (i.e. ethanedioic), malonic, succinic (i.e. butanedioic acid), maleic, fumaric, malic, tartaric, citric, methanesulfonic, ethanesulfonic, benzenesulfonic, /?-toluenesulfonic, cyclamic, salicylic, /?-aminosalicyclic, pamoic and the like acids. For the purposes of this invention solvates, prodrugs, N-oxides and stereoisomers of the relevant active ingredient (e.g. the essential ATP synthase inhibitor, pretomanid/delaminid and cytochrome bc inhibitor, and/or the optional further antibacterial agents) are also included within the scope of the invention.

The term “prodrug” of a relevant compound of the invention includes any compound that, following oral or parenteral administration, is metabolised in vivo to form that compound in an experimentally-detectable amount, and within a predetermined time (e.g. within a dosing interval of between 6 and 24 hours (i.e. once to four times daily)). For the avoidance of doubt, the term “parenteral” administration includes all forms of administration other than oral administration.

Prodrugs of compounds mentioned herein may be prepared by modifying functional groups present on the compound in such a way that the modifications are cleaved, in vivo when such prodrug is administered to a mammalian subject. The modifications typically are achieved by synthesising the parent compound with a prodrug substituent. Prodrugs include compounds mentioned herein wherein a hydroxyl, amino, sulfhydryl, carboxy or carbonyl group in that compound is bonded to any group that may be cleaved in vivo to regenerate the free hydroxyl, amino, sulfhydryl, carboxy or carbonyl group, respectively.

Examples of prodrugs include, but are not limited to, esters and carbamates of hydroxy functional groups, esters groups of carboxyl functional groups, N-acyl derivatives and N-Mannich bases. General information on prodrugs may be found e.g. in Bundegaard, H. “Design of Prodrugs” p. 1-92, Elesevier, New York-Oxford (1985).

The skilled person will appreciate that compounds that are a part of the combinations of the invention include those that are stable. That is, they are sufficiently robust to survive isolation from e.g. a reaction mixture to a useful degree of purity and/or also that such combinations of the invention may be substantially stable, e.g. to chemical degradation. In an embodiment, the compounds of the combinations of the invention are sufficiently stable from a shelf life perspective, and contact of one with the other does not result in accerlerated chemical degradation of any one.

The combinations of the invention may be useful in the treatment of active tuberculosis and may also be useful in the treatment of latent or dormant tuberculosis. The combinations may be effective by having a bacteriostatic effect, but may also have a bacteriocidal effect. It is indicated that they may also be useful in the treatment of latent tuberculosis because the combinations (or any one of the essential components of the combination, e.g. the ATP synthase inhibitor, the bc\ inhibitor) may act by interfering with ATP synthase, which may also impact on the latent tuberculosis bacilli. It is an advantage to have combinations that are effective against active tuberculosis and also against latent tuberculosis, for instance that can have an impact on or kill latent tuberculosis bacilli. In order to control the tuberculosis epidemic, this is important as the latent tuberculosis can get reactivated to cause active tuberculosis, and several factors can influence this happening, e.g. suppression of host immunity by use of immunosuppressive agents (such as antibodies against tumour necrosis factor a or interferon-y). Doses of the combination (and each active ingredient of the combination) may be influenced if it is being used to treat active or latent tuberculosis.

The quantity of each drug should be an effective amount to elicit a biological or medicinal response. The daily dose of the drug may of course vary depending on factors such as: - already approved (e.g. by an appropriate regulatory body such as EMA or the US FDA) recommended daily doses; - efficacy of doses lower than those already approved (or being studied in clinical trials); - patient tolerability; - the daily dose of the other drug (or drugs) forming part of the relevant combination; - any synergistic effects between the components of the combination; - the mode of administration.

Regarding doses, in general, satisfactory results will be obtained when the relevant compound of the combination of the invention is administered at a daily dosage not exceeding 1 or 2 grams, e.g. in the range from 1 to 50 mg/kg or from 10 to 50 mg/kg body weight. However, doses may be adjusted depending on response rates.

In an embodiment, bedaquiline (or the alternative ATP synthase inhibitor) is administered at a dosage of 200 to 400 mg daily (qd). In a further embodiment, bedaquiline is administered at 400 mg daily (qd) for two weeks followed by 200 mg three times a week (tiw).

In an embodiment pertomanid (or delamanid) is administered at a dosage of 100 to 200 mg daily (qd).

Daily doses for the cytochrome bc\ inhibitor (e.g. Q203, or a pharmaceutically acceptable salt thereof) may, for instance be 1.5 to 15 mg/kg (up to 1g). Hence daily doses may for instance be between 50 mg and 1000 mg and, in one embodiment, may be between 50 mg and 250 mg (e.g. about 50, 75, 100, 150 or 200 mg) or in another embodiment may be between 50 mg and 800 mg (e.g. between 100 mg and 800 mg, for instance about 100, 200, 300, 400, 500, 600, 700 or 800 mg).

Optional further antibacterial drugs that may be included in the combinations of the invention may be administered at daily doses recommended by a regulatory body (when e.g. approved in combination with other antibacterial agents), and are preferably administered at a daily dosage not exceeding 1 or 2 grams, e.g. in the range from 1 to 50 mg/kg body weight (for instance, in the range from 1 to 25 mg/kg, from 1.5 to 25 mg/kg, or from 2 to 15 mg/kg body weight).

For instance, the optional pyrazinamide: daily doses for PZA (or a pharmaceutically acceptable salt thereof) may, for instance, be 15 to 30 mg/kg (up to 2g), or, an alternative dosing regimen of 50 to 75 mg/kg (up to 3g) twice a week. Hence, daily doses may be between for instance 500 mg and 2000 mg (e.g. about 1000, about 1500 or about 2000 mg).

Given that combinations of the invention are seen to be advantageous (e.g. synergistic, as exemplified in the examples section), then such combinations are envisioned to have, in one embodiment, a possible advantage that fewer (or no) other antibacterial (anti-tuberculosis) drugs are required in the treatment phase, and/or, in another embodiment, a possible advantage that the doses (e.g. daily doses) of either one of the two components of the combination (ATP synthase inhibitor, pretomanid/delaminid or the cytochrome bc inhibitor) and/or any additional optional antibacterial agent (as defined herein) may be less than expected (for example, less than may be recommended by a regulatory body, when labelled for use in combination with other antibacterials such as rifampin/isoniazid and/or ethambutol, or less than that tested in clinical trials). Hence, the expected daily doses of the cytochrome bc inhibitor (or a pharmaceutically acceptable salt thereof) may be 0.75 to 7.5 mg/kg (up to 500 mg). Hence daily doses may for instance be between 25 mg and 500 mg and, in an embodiment, may be between 25 mg and 125 mg (e.g. about 25, 50, 75 or 100 mg).

All amounts mentioned in this disclosure refer to the free form (i.e. non-salt form). The values given below represent free-form equivalents, i.e., quantities as if the free form would be administered. If salts are administered the amounts need to be calculated in function of the molecular weight ratio between the salt and the free form. The doses (e.g. daily doses) described herein are calculated for an average body weight specified, and should be recalculated in case of paediatric applications, or when used with patients with a substantially diverging body weight.

The treatment duration for tuberculosis can be more than a year. However, it is envisioned that treatment duration may be reduced using the combinations of the invention. For instance, treatment duration may be 36 weeks or less, for instance 24 weeks or less. In certain embodiments, the treatment duration may be less than 20 weeks, for instance 16 weeks or less, or, 12 weeks or less.

In aspects of the invention, there is provided combinations of the invention, as described herein, for use as medicaments or pharmaceuticals. Such combinations may be useful in the treatment of a disease caused by Mycobacterial tuberculosis (e.g. in the treatment of tuberculosis).

Hence, there is also provided a pharmaceutical composition (or formulation) comprising a pharmaceutically acceptable carrier and, as active ingredient, a therapeutically effective amount of a combination of the invention. Such combinations may be formulated into pharmaceutical compositions as described hereinafter.

Accordingly, in another aspect of the invention, there is provided a method of treating a patient suffering from, or at risk of, a disease caused by Mycobacterial tuberculosis (tuberculosis), which method comprises administering a therapeutically effective amount of a combination of the invention or a pharmaceutical composition of the invention. In an embodiment, the patient is human.

In further embodiments, there is provided a method of treatment as defined herein wherein the method further comprises a treatment duration period as defined herein (e.g. a treatment duration of 36 weeks or less, 24 weeks or less or, in a particular embodiment, a treatement period of 16 weeks or less or 12 weeks or less). Alternatively, there is provided a combination for use as described herein, wherein the use is for a certain duration period (e.g. a treatment duration of 36 weeks or less, 24 weeks or less or, in a particular embodiment, a treatement period of 16 weeks or less or 12 weeks or less).

The components or antibacterial drugs of the combinations of the invention (including the two essential antibacterial drugs of the combination and the further optional drugs) may be formulated separately (e.g. as defined herein) or may be formulated together so forming for example a fixed dose formulation. The latter may have advantages in terms of compliance. In some embodiments, the two (or optionally more) antibacterial drugs of the combinations of the invention can be co-administered, in other embodiments the antibacterial drugs (of the combinations) may be sequentially administered, while in still other embodiments they can be administered substantially simultaneously. In some of the latter embodiments, administration entails taking such antibacterial drugs within 30 minutes or less of each other, in some embodiments 15 minutes or less of each other. In some embodiments, the antibacterial drugs are administered once per day, at approximately the same time each day. For example, the antibacterial drugs are administered within a time range of 4 hours of the original time of administration on the first day, that is, ± 2 hours, or ± 1 hour, or in still other embodiments ± 30 minutes of the time on the original administration day.

In some embodiments, the antibacterial drugs of the invention are administered as separate oral capsules or oral tablets. Other formulations may include solid dispersions.

Hence, when a combination is referred to herein, such a combination may be a single formulation comprising all antibacterial drugs of the combinations of the invention (i.e. the two essential ones mentioned herein and, optionally, one or more further antibacterials) or it may be a combination product (such a kit of parts) where each of the antibacterial drugs of the combinations of the invention may be packaged together either as separate forms (each comprising one of the antibacterial drugs) or as two or more forms (depending on the total number of antibacterial drugs in the combination of the invention). In an embodiment, each antibacterial drug of the combination of the invention is formulated separately and/or is also packaged separately but may be labelled for use in combination with one or more of the other antibacterial drugs of the combinations of the invention. The antibacterial drugs of the combination (as described herein) may be co-administered, sequentially administered, or administered substantially simultaneously. Hence the individual dosage forms of each of the antibacterial drugs can be administered as separate forms (e.g., as separate tablets or capsules) as described herein or, in other embodiments, may be administered as a single form containing all three active substances or as two forms (one containing any two of the active substances and the other containing the remaining active substance).

The antibacterial drugs of the combinations of the invention may be formulated into various pharmaceutical forms for administration purposes. As mentioned herein, this formulating may be done on an individual antibacterial drug or a combination of antibacterial drugs that form part of the combinations of the invention. As appropriate, compositions may include those usually employed for systemically administering drugs. To prepare the pharmaceutical compositions the relevant antibacterial drug (or combination of relevant antibacterial drugs) is combined in intimate admixture with a pharmaceutically acceptable carrier, which carrier may take a wide variety of forms depending on the form of preparation desired for administration. These pharmaceutical compositions are desirable in unitary dosage form suitable, in particular, for administration orally or by parenteral injection. For example, in preparing the compositions in oral dosage form, any of the usual pharmaceutical media may be employed such as, for example, water, glycols, oils, alcohols and the like in the case of oral liquid preparations such as suspensions, syrups, elixirs, emulsions and solutions; or solid carriers such as starches, sugars, kaolin, diluents, lubricants, binders, disintegrating agents and the like in the case of powders, pills, capsules and tablets. Because of their ease in administration, tablets and capsules represent the most advantageous oral dosage unit forms in which case solid pharmaceutical carriers are obviously employed. For parenteral compositions, the carrier will usually comprise sterile water, at least in large part, though other ingredients, for example, to aid solubility, may be included. Injectable solutions, for example, may be prepared in which the carrier comprises saline solution, glucose solution or a mixture of saline and glucose solution. Injectable suspensions may also be prepared in which case appropriate liquid carriers, suspending agents and the like may be employed. Also included are solid form preparations which are intended to be converted, shortly before use, to liquid form preparations.

Depending on the mode of administration, the pharmaceutical composition will preferably comprise from 0.05 to 99 % by weight, more preferably from 0.1 to 70 % by weight, even more preferably from 0.1 to 50 % by weight of the active ingredient(s), and, from 1 to 99.95 % by weight, more preferably from 30 to 99.9 % by weight, even more preferably from 50 to 99.9 % by weight of a pharmaceutically acceptable carrier, all percentages being based on the total weight of the composition.

Any pharmaceutical composition mentioned herein (e.g. a pharmaceutical composition comprising one antibacterial drug or a combination of antibacterial drugs of the combination of the invention) may additionally contain various other ingredients known in the art, for example, a lubricant, stabilising agent, buffering agent, emulsifying agent, viscosity-regulating agent, surfactant, preservative, flavouring or colorant. It is especially advantageous to formulate the aforementioned pharmaceutical compositions in unit dosage form for ease of administration and uniformity of dosage. Unit dosage form as used herein refers to physically discrete units suitable as unitary dosages, each unit containing a predetermined quantity of active ingredient calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. Examples of such unit dosage forms are tablets (including scored or coated tablets), capsules, pills, powder packets, wafers, suppositories, injectable solutions or suspensions and the like, and segregated multiples thereof.

As mentioned hereion, the combination of antibacterial drugs as described herein may be co-administered, sequentially administered, or administered substantially simultaneously (as described herein). Hence the individual dosage forms of each of the antibacterial drugs can be administered as separate forms (e.g. as separate tablets or capsules) as described herein or, in an alternative embodiment, may be administered as a single form containing all actives or as two or more forms (e.g. where there are three antibacterial drugs, one containing any two and the other containing the remaining one).

There is also provided a process for preparing a pharmaceutical formulation as defined herein comprising bringing into association any one (or more, e.g. the two essential active ingredients and, optionally, further antibacterials as defined herein) of the active ingredients of the combination of the invention, with one (or more) pharmaceutically acceptable excipient or carrier.

There is also provided a process for preparing a combination product as defined herein comprising:

- bringing into association each of the components (e.g. as separate pharmaceutical formulations) of the combination product and co-packaging (e.g. as a kit of parts) or indicated that the intended use is in combination (with the other components); and/or

- bringing into association each of the components in the preparation of a pharmaceutical formulation comprising such components.

In this respect, there is also provided a use of any individual component of the combination of the invention, labelled for use with the other components. In this regard, there is also provided a method of medical treatment (e.g. for the treatment of tuberculosis, as defined herein, including DR-TB) comprising adminstation of any one of the components of the invention, wherein there are instructions for co-administration with the other components.

More specifically, there is provided and one of the following:

- Bedaquiline (or another ATP synthase inhibitor as defined herein), or a pharmaceutically acceptable salt thereof, for use in a combination regimen as defined herein (for instance labelled for use in combination with pretomanid or delamanid, and a cytochrome bcl inhibitor as defined herein, and one or more optional further antibacterials as defined herein, e.g. clofazimine), for coadministration in the treatment of a mycobacterial infection as defined herein (e.g. tuberculosis, including DR-TB);

- Pretomanid or delaminid, or a pharmaceutically acceptable salt thereof, for use in a combination regimen as defined herein (for instance labelled for use in combination with an ATP synthase inhibitor as defined herein, and a cytochrome bcl inhibitor as defined herein, and one or more optional further antibacterials as defined herein, e.g. clofazimine), for co-administration in the treatment of a mycobacterial infection as defined herein (e.g. tuberculosis, including DR-TB);

A cytochrome bcl inhibitor, as defined herein, or a pharmaceutically acceptable salt thereof, for use in a combination regimen as defined herein (for instance labelled for use in combination with pretomanid or delamanid, and an ATP synthase inhibitor as defined herein, and one or more optional further antibacterials as defined herein, e.g. clofazimine), for co-administration in the treatment of a mycobacterial infection as defined herein (e.g. tuberculosis, including DR-TB);

A method of medical treatment (e.g. for the treatment of tuberculosis, as defined herein, including DR-TB) comprising adminstation of bedaquiline (or another ATP synthase inhibitor as defined herein), or a pharmaceutically acceptable salt thereof, wherein there are instructions for co-administration with pretomanid or delamanid, and a cytochrome bcl inhibitor as defined herein, and one or more optional further antibacterials as defined herein, e.g. clofazimine

A method of medical treatment (e.g. for the treatment of tuberculosis, as defined herein, including DR-TB) comprising adminstation of pretomanid or delaminid, or a pharmaceutically acceptable salt thereof, wherein there are instructions for co-administration with an ATP synthase inhibitor as defined herein, and a cytochrome bcl inhibitor as defined herein, and one or more optional further antibacterials as defined herein, e.g. clofazimine; and/or A method of medical treatment (e.g. for the treatment of tuberculosis, as defined herein, including DR-TB) comprising adminstation of a cytochrome bcl inhibitor, as defined herein, or a pharmaceutically acceptable salt thereof, wherein there are instructions for co-administration with pretomanid or delamanid, and an ATP synthase inhibitor as defined herein, and one or more optional further antibacterials as defined herein, e.g. clofazimine.

GENERAL PREPARATION

The compounds according to the invention can generally be prepared by a succession of steps, each of which may be known to the skilled person or described herein.

EXPERIMENTAL PART

Q203 (telacabec) may be prepared in accordance with the methods described in the documents mentioned hereinbefore, e.g. patent document WO 2011/113606 and/or journal articles J. Medicinal Chemistry, 2014, 57 (12), pp5293-5305 o Nature Medicine, 19, 1157-1160 (2013) by Pethe et al “Discovery of Q203, a potent clinical candidate for the treatment of tuberculosis”. For example, in WO 2011/113606, compound (289) at page 126 provides characterising data for Q203, and preparation methods are described at pages 17-30, in Nature Medicine, the synthesis of the compound is described in the accompanying “Online Methods” as well as in the J. Medicinal Chemistry article in the experimental sections.

Other inibitors of cytochrome bcl activity, may be those disclosed (and prepared using methods disclosed) in international patent applications such as WO 2017/001660, WO 2017/001661, WO 2017/001660, WO 2021/048342, WO 2022/194803, WO 2022/194905, WO 2022/194906, WO 2023/073090, etc all of which are hereby incorporated by reference.

The invention is described herein, also with reference to the Figures:

FIGURE 1: Measurement of Lung CFUs in mouse study groups after 2 and 6 weeks in a chronic infection model

FIGURE 2: represents testing of various combinations in a further chronic mouse model, where: B represents bedaquiline; R represents rifampicin; J represents JNJ- 2901; Z represents pyrazinamide; H represenst isoniazid; C represents clofazmine FIGURE 3: Measurement of lung CFUs in certain combinations in the relapse mouse model Compounds - cytochrome bcl inibitors - these are as described in the literature (including the patent applications mentioned herein) and prepared according to the procedures described therein.

5

For instance, the following compound, also known as “JNJ-2901” (or “JNJ901” or “2901” or “901”) may be prepared (Compound 154 from WO 2017/001660):

Synthesis

Synthesis of Compound 154

15

Preparation of intermediate FD

Accordingly, intermediate FD was prepared according to the scheme above starting from intermediate F yielding 1.29 g as a white solid, 81%

20 Preparation of intermediate FE

To a solution of 6-chloro-2-ethylimidazo[l,2-a]pyridine-3 -carboxylic acid (CAS [1216142-18-5], 0.117 g, 0.504 mmol) in DCM (5.1 mL) and tri ethylamine (0.18 mL) were added EDCI (145 mg, 0.756 mmol) and HOBt (103 mg, 0.760 mmol) and the mixture was stirred at room temperature for 30 min. Intermediate FD (0.162 g,

25 0.536 mmol) was added and the mixture was stirred at room temperature for 4 h. The mixture was washed with water (2x). The organic layer was dried over MgSC , filtered and evaporated to dryness to give 0.293 g of intermediate FE as colourless oil (quant.), used as such in the next step.

Preparation of intermediate FF

To a solution of intermediate FE (0.291 g, 0.572 mmol) in methanol (5.9 mL) was added trimethylchlorosilane (0.37 mL, 2.94 mmol) and the mixture was stirred at room temperature for 16 h. The mixture was evaporated to dryness to give 0.304 g of intermediate FF as a pale yellow foam (quant.).

Preparation of Compound 154

Trifluoromethanesulfonic anhydride (0.12 mL, 0.696 mmol) was added to a solution of intermediate FF (155 mg, 0.348 mmol) and DMAP (2.13 mg, 17.4 pmol) in triethylamine (0.39 mL, 2.78 mmol) and DCM (5.3 mL) at 0 °C. The resulting mixture was stirred at 0 °C for 6 h. Water was added and the organic layer was washed with water, dried over MgSCU, filtered and evaporated to dryness. The crude product was purified by preparative LC (irregular SiOH, 15-40 pm, 40 g, Grace, dry loading (silica), mobile phase gradient Heptane/EtOAc from 90/10 to 10/90) to obtain 186 mg of a pale yellow solid, which was triturated in heptane and purified by preparative LC (spherical Cl 8 25 pm, 40 g YMC-ODS-25, dry loading (Celite®), mobile phase gradient: 0.2% aq. NFLHCCL/MeCN from 90/10 to 0/100) to give 0.112 g of Compound 154 as a white solid (59%).

1H NMR (400 MHz, DMSO-d6) 5 ppm 9.07 (s, 1 H), 8.47 (br s, 1 H), 7.67 (d, J = 8.1 Hz, 1 H), 7.46 (br d, J = 9.1 Hz, 1 H), 7.30 (br d, J = 8.1 Hz, 2 H), 7.20 (br d, J = 7.6 Hz, 2 H), 4.49 (br d, J = 5.1 Hz, 2 H), 4.41 (s, 2 H), 4.18 (s, 2 H), 3.39-3.31 (m, 1 H), 2.98 (q, J = 7.4 Hz, 2 H), 2.63 - 2.58 (m, 2 H), 2.34-2.29 (m, 2 H), 1.26 (br t, J = 7.3 Hz, 3 H)

Further cytochrome bcl inhibitors

As mentioned above, compounds are known or may be prepared in accordance with those documents already published, including international patent application WO 2023/073090 (the disclosures of which are hereby incorporated by reference), which discloses a number of compounds, including those within the following scope: A compound according to formula (IX)

wherein

X¹ represents =N- or =CH-;

X², X⁵ and X⁶ each independently represent =N-, =CH- or =C(CH3)- (and in one embodiment, X² represents =CH-, in a further embodiment X⁵ represents =CH- and in yet a further embodiment, X⁶ represents =N-);

R¹ and R² each independently represent a substituent selected from hydrogen, -CH3, -F, -Cl, -OCH3, -NH2, - CH2NH2 (and in an embodiment each independently represent H); R³ represents a substituent selected from H, -CF3, -CHF2, -CH3, -CH2CH3, and cyclopropyl (and in an embodiment represents -CH2CH3);

R⁴ represents a substituent selected from H, F and -CH3 (and in an embodiment represents H);

R⁵ represents H, -CH₃, -CH2CH3, -CH2CH2CH3, cyclopropyl, -OH, -OCH3, -OCF3, -OCH2CH2OCH3, -CF₃, -CHF₂, -CF2CH3, -NH₂, -NH(SO₂)CF₃, -N(CH₃)(SO₂)CF3, or -SO2CF3 (and in an embodiment represents -CF3), or a pharmaceutically-acceptable salt thereof.

For instance, the following Compound X (also referred to as Compound 177) has been prepared (and may be used in the combination studies mentioned herein):

Intermediate 11-23

Boron trifluoride diethyl etherate [109-63-7] (approx. 0.1 molar equiv) may be added dropwise to a solution of 2-aminopyrimidine [109-12-6], (approx. 1 equiv; e.g. 5 g, 52.6 mmol), ethyl propionyl acetate [4949-44-4] (approx. 1.5 molar equiv) and (di acetoxy iodo)benzene [3240-34-4] (approx. 1.5 molar equiv) in dry 2- methyltetrahydrofuran, in a 2-neck round bottom flask equipped with a condenser, at rt under N2. The mixture may be stirred at 60 °C for 16 h. A saturated NaHCOs aqueous solution may be added, and the mixture extracted with EtOAc. The organic layer may be separated, dried (MgSCU), filtered and the solvents evaporated in vacuo. The crude product may be purified by flash column chromatography (e.g. silica; EtOAc in heptane 0/100 to 40/60). The desired fractions may collected and concentrated in vacuo to yield intermediate 11-23 as an orange solid (e.g. 8.7 g, 74.5%).

Synthesis of intermediate 11-83

Intermediate 11-83 (22 g, 99.9%, pale yellow solid) was prepared according to a standard procedure for converting the ester to a -COOH moiety, for instance adding a solution of Intermediate 11-23 to a mixture of ethanol and water, bringing to pH 7 by a IM HC1 aqueous solution addition and concentrated in vacuo.

Synthesis of intermediate 11-55

Intermediate 11-55 was prepared by converting the -COOH moiety of Intermediate II- 83 to a -COCI moiety, for instance by reaction with thionyl chloride, to yield Intermediate 11-55 as a dark brown solid.

Synthesis of intermediate 1-43

Intermediate 1-43 was prepared according to a procedure using 2-amino-5- (trifluoromethyl)pyridine and:

Synthesis of intermediate 1-44

Intermediate 1-44 was prepared according to a procedure using 4-cyanobenzoyl chloride and intermediate 1-43.

Synthesis of intermediate 1-45

Intermediate 1-45 was prepared according to a procedure using intermediate 1-44, sodium borohydride [16940-66-2], nickel (II) chloride hexahydrate [7791-20-0] and ditertbutyl dicarbonate [24424-99-5], for instance in dry methanol at -5 °C under N2.

Synthesis of intermediate 1-46

Intermediate 1-46 was prepared according to a hydrogenation procedure using intermediate 1-45.

Synthesis of intermediate 1-47

Intermediate 1-47 was prepared according to a procedure using intermediate 1-46 (and removing the protecting group). Synthesis of Compounds 177 (Compound X) and 178

Isomers 177 (Compound X) and 178 were synthesized from intermediates 11-55 and I- 47, after SFC separation (Jasco SFC prep system, i-cellulose column (Phenomenex) 250*30mm, 5mm particle size, isocratic mode at 100 ml/min of CO2 (40%) / MeOH (60%) / diethylamine (0.1%) at 30 °C, 120 bars), to yield 177 (386.1 mg, 48% yield) and 178 (335.8 mg, 42% yield) as white solids.

Compound 177 (Compound X): ’H NMR (400 MHz, DMSO) 5 9.33 (dd, J = 6.9, 2.0 Hz, 1H), 8.63 (dd, J = 4.2, 2.0 Hz, 1H), 8.54 (t, J = 5.9 Hz, 1H), 7.74 (d, J = 8.3 Hz, 2H), 7.39 (d, J = 8.3 Hz, 2H), 7.17 (dt, J = 12.4, 6.2 Hz, 1H), 6.53 (s, 1H), 4.55 (d, J = 5.9 Hz, 2H), 4.31 (dd, J = 12.9, 3.5 Hz, 1H), 4.12 (td, J = 12.2, 4.8 Hz, 1H), 3.14 (dd, J = 15.8, 3.8 Hz, 1H), 3.04 (q, J = 7.5 Hz, 2H), 2.80 (dd, J = 15.8, 1 l.fl Hz, 1H), 2.28 (dd, J = 13.3, 2.3 Hz, 1H), 2.04 (ddd, J = 25.0, 11.9, 5.8 Hz, 1H), 1.30 (t, J = 7.5 Hz, 3H).

Compound 178: ’H NMR (400 MHz, DMSO) 5 9.33 (dd, J = 6.9, 2.0 Hz, 1H), 8.63 (dd, J = 4.2, 2.0 Hz, 1H), 8.54 (t, J = 5.9 Hz, 1H), 7.74 (d, J = 8.3 Hz, 2H), 7.39 (d, J = 8.3 Hz, 2H), 7.17 (dd, J = 6.9, 4.2 Hz, 1H), 6.53 (s, 1H), 4.55 (d, J = 5.9 Hz, 2H), 4.31 (dd, J = 12.7, 3.4 Hz, 1H), 4.12 (td, J = 12.3, 4.8 Hz, 1H), 3.13 (dt, J = 16.0, 7.9 Hz, 1H), 3.04 (q, J = 7.5 Hz, 2H), 2.80 (dd, J = 15.8, 11.2 Hz, 1H), 2.28 (dd, J = 13.4, 2.3 Hz, 1H), 2.04 (ddd, J = 24.9, 11.8, 5.7 Hz, 1H), 1.30 (t, J = 7.5 Hz, 3H).

Other antibacterials, such as bedaquiline (BDQ), pretomanid (Pa), delamanid, linezolid (L), sutezolid, clofazimine, etc may be available on the market.

BIOLOGICAL EXAMPLE 1

Pharmacological examples

MIC determination for testing compounds against M. tuberculosis.

TEST 1

Appropriate solutions of experimental and reference compounds are made in 96 well plates with 7H9 medium. Samples of Mycobacterium tuberculosis strain H37Rv are taken from cultures in logarithmic growth phase. These are first diluted to obtain an optical density of 0.3 at 600 nm wavelength and then diluted 1/100, resulting in an inoculum of approximately 5x10 exp5 colony forming units per well. Plates are incubated at 37°C in plastic bags to prevent evaporation. After 7 days, resazurin is added to all wells. Two days later, fluorescence is measured on a Gemini EM Microplate Reader with 543 excitation and 590 nm emission wavelengths and MICso and/or pICso values (or the like, e.g. IC50, IC90, PIC90, etc) are (or were) calculated.

TEST 2

Round-bottom, sterile 96-well plastic microtiter plates are filled with 100 pl of Middlebrook (lx) 7H9 broth medium. Subsequently, an extra 100 pl medium is added to column 2. Stock solutions (200 x final test concentration) of compounds are added in 2 pl volumes to a series of duplicate wells in column 2 so as to allow evaluation of their effects on bacterial growth. Serial 2-fold dilutions are made directly in the microtiter plates from column 2 to 11 using a multipipette. Pipette tips are changed after every 3 dilutions to minimize pipetting errors with high hydrophobic compounds. Untreated control samples with (column 1) and without (column 12) inoculum are included in each microtiter plate. Approximately 10000 CFU per well of Mycobacterium tuberculosis (strain H37RV), in a volume of 100 pl in Middlebrook (lx) 7H9 broth medium, is added to the rows A to H, except column 12. The same volume of broth medium without inoculum is added to column 12 in row A to H. The cultures are incubated at 37°C for 7 days in a humidified atmosphere (incubator with open air valve and continuous ventilation). On day 7 the bacterial growth is checked visually.

The 90 % minimal inhibitory concentration (MIC90) is determined as the concentration with no visual bacterial growth.

TEST 3: Time kill assays

Bactericidal or bacteriostatic activity of the compounds can be determined in a time kill assay using the broth dilution method. In a time kill assay on Mycobacterium tuberculosis (strain H37RV), the starting inoculum of AT. tuberculosis is 10⁶ CFU / ml in Middlebrook (lx) 7H9 broth. The antibacterial compounds are used at the concentration of 0.1 to 10 times the MIC90. Tubes receiving no antibacterial agent constitute the culture growth control. The tubes containing the microorganism and the test compounds are incubated at 37 °C. After 0, 1, 4, 7, 14 and 21 days of incubation samples are removed for determination of viable counts by serial dilution (10'¹ to 10'⁶) in Middlebrook 7H9 medium and plating (100 pl) on Middlebrook 7H11 agar. The plates are incubated at 37 °C for 21 days and the number of colonies are determined. Killing curves can be constructed by plotting the logioCFU per ml versus time. A bactericidal effect is commonly defined as 3-logio decrease in number of CFU per ml as compared to untreated inoculum. The potential carryover effect of the drugs is removed by serial dilutions and counting the colonies at highest dilution used for plating.

TEST 4 (see also test 1 above; in this test a different strain of Mycobacterium tuberculosis strain is employed)

Appropriate solutions of experimental and reference compounds are made in 96 well plates with 7H9 medium. Samples of Mycobacterium tuberculosis strain EH 4.0 (361.269) are taken from cultures in stationary growth phase. These are first diluted to obtain an optical density of 0.3 at 600 nm wavelength and then diluted 1/100, resulting in an inoculum of approximately 5x10 exp5 colony forming units per well. Plates are incubated at 37°C in plastic bags to prevent evaporation. After 7 days, resazurin is added to all wells. Two days later, fluorescence is measured on a Gemini EM Microplate Reader with 543 nm excitation and 590 nm emission wavelengths and MIC50 and/or pIC50 values (or the like, e.g. IC50, IC90, pIC90, etc) are (or were) calculated. pICso values may be recorded below in pg/mL.

RESULTS

Compounds are / were tested in Test 1, 2, 3 and/or 4 described above (in section “Pharmacological Examples”).

For instance, JNJ-2901 was tested in Test 4 resulting in an IC50 of 0.002 pg/ml. Evidence of its cytochrome bcl inhibition was done by testing with an MTB strain carrying a mutation in qcrB (A937G, 313 Thr->Ala), resistant to cytochrome be inhibition. Other antibiotics are roughly equally resistant to the strain with and without the mutation, but JNJ-2901 had about a 50-fold decrease in activity when tested against the strain with the mutation.

For instance, Compound X was tested in Test 1 or a similar assay and found to have antibacterial activity, for insance as described in WO 2023/073090 where a pMICso of 7.85 or MIC90 (pm) of 0.056 may be observed. Further Biological Examples - IN VIVO COMBINATION STUDIES

BIOLOGICAL EXAMPLE 1A - Chronic Mouse model

Background and Aim

Aim:

The goal of this study is to evaluate the efficacy of the Cytochrome be inhibitor 901 alone and in combination with bedaquiline (BDQ), clofazimine (CFZ), pretomanid (Pa), and linezolid (LZD) in a high dose aerosol Balb/c mouse model of tuberculosis. Treatment is started 10 days after AT. tuberculosis Erdman infection, and lasts for 6 weeks with 5/7 dosing. Enumeration of bacterial loads in lungs (and spleens) is determined after 2 and 6 weeks of treatment.

Method description:

This experimental model seeks to evaluate efficacy against actively replicating intracellular AT. tuberculosis in a lethal infection model. Treatments were initiated 10 days after a high-dose aerosol infection with the AT. tuberculosis Erdman strain, and was continued for 6 weeks, 5 days per week. The high-dose aerosol Balb/c mouse model is generally employed to assess activity of potent compounds and/or drug combinations against primarily actively replicating, intracellular AT. tuberculosis. Bacterial loads in lungs are high at the start of treatment (generally between 10⁷ or 10⁸) in this lethal infection model, in order to ensure a sufficiently large efficacy window for potent combinations. The model is also suited for relapse studies to evaluate the ability of test agents or drug regimens to achieve a durable cure.

Reference for the high dose aerosol model:

De Groote MA, Gilliland JC, Wells CL, Brooks EJ, Woolhiser LK, Gruppo V, Peloquin CA, Orme IA, Lenaerts AJ. Comparative Studies Evaluating Mouse Models Used for Efficacy Testing of Experimental Drugs against Mycobacterium tuberculosis. Antimicrob. Agents Chemother. 2011. 55(3): 1237-1247

Protocol

1. Six to eight week old Balb/c female mice were ordered from Jackson Laboratories. Mice were rested at least one week after delivery before infection. For efficacy time points, a minimum of 6 mice were used per treatment group.

For relapse time points, 15 mice per group are required. Inoculum preparation for the high dose aerosol starts from a fresh culture. Two and a half weeks prior to infection, a 1 mL of frozen seed stock (lot 11-14-12 VG at 3.151xl0e7 CFU/mL) was used to inoculate 9 mL of 7H9-glycerol-ADC media containing 0.05% Tween 80 (7H9-P80). The culture was incubated at 37°C with stirring until the culture became turbid (~ 6 days). The turbid culture was then diluted 1 :5 to yield two 25 mL 7H9-P80 cultures (5 mL of the culture + 20 mL fresh media) and incubated at 37°C with stirring for 7 days. A final sub-culture was prepared using various dilutions of the bacterial culture that resulted in six 15 mL cultures of 7H9-P80 with starting OD at 600 of 0.15, 0.2, 0.25, 0.3, 0.35, 0.4. These were further incubated for three more days (up to the day of mouse infection). Ten mL of the culture with a final OD600 in the range of 0.76 to 0.80 was then used for each aerosol infection. For two consecutive aerosols, two final 15 mL cultures with OD600s as close to 0.8 as possible can be combined, and subsequently diluted in sterile water in order to achieve a final OD600 of 0.76 to 0.80. Sterility testing on TSA plates was performed from the passaged stock at each stage to ensure the culture was free of fast growing contaminates. The mice were infected by high-dose aerosol using a Glas-Col Inhalation Exposure System. The glas-col nebulization was performed with a 13-17 SCFH compressed air setting, and a 80 SCFH main (negative air) setting; using a 15 minute preheat cycle, a 60 minute nebulization cycle, a 40 minute cloud decay cycle, followed by a 15 minute decontamination cycle. Six mice per aerosol run were sacrificed post-infection to determine bacterial uptake. Whole lungs were aseptically harvested in Precellys tubes (Bertin cat# KT03961-1-396.7) and homogenized in 4 mL of IX PBS using a Precellys tissue homogenizer. Five-fold serial dilutions (0-7) were plated on 7H11-0 ADC agar. The remaining homogenate was stored by mixing 2 x 1.0 mL of the homogenates 1 : 1 with 40% glycerol+0.1% tween 80, stored at -80° should re-plating be required. All remaining mice were then randomly distributed into group cages, such that each group roughly had an equal number of mice from both the first (AM) and second (PM) aerosol runs, in case two aerosol runs were performed. At Day 10 post aerosol, 6 mice were sacrificed to determine the bacterial load in the lungs and spleens at the start of therapy. Mice were weighed prior to sacrifice. Gross pathology observations of the lungs and spleens were made. Lungs (were divided into three vials; left lobe, two upper right [cranial] and lower right lobes [caudal]), and spleens were aseptically harvested and frozen at -80°C.

6. Therapy was administered once per day (QD), at 5 days per week, via oral gavage (at 200 pL per mouse) and was started on day 10. For drug combinations, the dosing was separated by at least one hour (LZD is given at least 4 hours after the first doses of BDQ and Pa).

7. At intermittent time points, mice were sacrificed for the enumeration of the bacterial load._After 2 and 6 week of treatment, mice were sacrificed 5 days after dosing (E.g. the last day of dosing on Fri, and sacrifice on a Wed). Mice were weighed prior to sacrifice. Gross pathology observations of the lungs and spleens were made. Whole lungs and spleens were aseptically harvested and frozen at - 80°C. Previously frozen tissues were recovered and homogenized in either IX PBS or 10% Bovine Serum Albumin (BSA) in IX PBS.

8. After homogenization, lung and spleen homogenates were plated on 7H11 agar or charcoal* containing 7H11 quad plates, and serially diluted in IX PBS or 10% BSA. Enumeration of CFU occurred after 3-5 weeks incubation at 37°C in a dryair incubator.

9. In case compounds are highly protein bound, have a long half-life and/or are lipophilic, the following precautions are taken to prevent drug carry-over: Organs are homogenized in sterile 10% bovine serum albumin (BSA) (Sigma A-2153) in IX PBS. Organ homogenates are serially diluted in 10% BSA in PBS, and plated onto 7H11/OADC + 0.4% [w:v] activated charcoal (Sigma C-9157). Colonies are allowed to grow for 5 weeks before counting.

10. Statistical analysis for bactericidal readouts: The viable counts were converted to logarithms, which were then evaluated by a one-way ANOVA followed by a pairwise multiple comparison using the Dunnett’s test or Tukey’s test [or by Kruskal -Wallis One Way Analysis of Variance on Ranks if data fail normality or equal variance tests] (SigmaStat software program). Differences were considered significant at the 95% level of confidence.

11. PK plasma collection: During week 2 and week 6 of dosing, timed dosing/bleeding of the mice was conducted in order to collect blood at the steady- state Cmin (24 hours post dose) and Cmax (7 hours post dose for 901). Blood was collected from all dosing groups (including control groups). Blood was collected via sub-mandibular vein puncture into K3EDTA tubes and centrifuged to separate the plasma. Aliquots of plasma were then frozen at -80°C. The plasma was then thawed and chemically sterilized prior to sending for analysis.

12.

Design of the study

* Untreated mice were not expected to live because the high-dose aerosol produces a lethal infection. Results of this chronic infection mouse study

Amongt other test hereinbelow (and Figure 1) shows the results of the bacterial loads in lungs, measured in LogioCFU

2 Week Lungs (2 Weeks Post Treatment Start) - measured in LogioCFU

6 Week Lungs (2 Weeks Post Treatment Start) - measured in LogioCFU

It can be seen from the results that the combination of cytochrome bcl inhibitor (JNJ- 2901) and bedaquiline (BDQ) and pretomanid (BPa901) suggest a slight antagonism in comparison with bedaquiline plus pretomanid (BPa) alone. In this chronic infection mouse model, BPa901 also exhibits less CFU reduction compared with BPaL. Due to the antagonism between the ATP synthase inhibitor (bedaquiline in this case) and the cytochrome bcl inhibitor (JNJ-2901 in this case), it was not expected, based on this chronic mouse model, that this would be a successful combination.

Figure 2 represents testing of various combinations in a further chronic mouse model, where:

B represents bedaquiline R represents rifampicin J represents JNJ-2901 Z represents pyrazinamide H represenst isoniazid C represents clofazmine

BIOLOGICAL EXAMPLE IB - Relapse Mouse model - Studies A, B and C

Methods

All treatment regimens were evaluated in three studies (Studies A-C), in a relapsing mouse infection model (see e.g. Li, S.Y., et al. Evaluation of moxifloxacin-containing regimens in pathologically distinct murine tuberculosis models. Antimicrob. Agents Chemother. 59, 4026-4030 (2015)).

Study design

In Studies A and B, mice were infected by intranasal inoculation of 50 pL of M. tuberculosis H37Rv at inoculum level of 4.5 logic CFU/mouse. Treatment was initiated 2 weeks pi, when the bacterial burden in the lungs was >7.19 logic CFU. The mice were treated five days per week by oral gavage (at 10 mL/kg), for 4, 8, and 12 weeks and received different treatment combinations (Table 1) containing bedaquiline (25 mg/kg), pretomanid (40 mg/kg), linezolid (100 mg/kg), clofazimine (20 mg/kg), and Be inhibitor (5 mg/kg) (where the be inhibitor in all these studies was JNJ-2901). For all groups, the first two drugs were administered in the morning and the last two drugs in the afternoon, with around 2 hours between the administration of each drug. The bacterial load in lungs (CFU/lung) was assessed at the end of each treatment, after a standard washout period of 3 days, in five animals/group. An untreated control group consisted of 10 mice (five at D-13 to act as infection control and five at DO to determine the infection level at treatment start). At the end of 8 or 12 weeks of treatment, 15 animals per group were held without treatment for 12 weeks to determine the proportion of mice with relapse. The total number of mice was 160 in Study A and 255 in Study B. Other study endpoints included animal weights weekly prior to treatment and post treatment during the relapse period and 3 times weekly during treatment phase, lung weight one day and 14 days pi and at the end of each period of treatment and relapse period, and observation of treatment-emergent adverse effects. In Study C, 4-6 weeks old mice were infected by high-dose aerosol with M. tuberculosis H37Rv (~7 logic CFU/mL). Treatment started at 2 weeks pi (when the bacterial burden was >7 logic CFU) and was administered by oral gavage, once daily, five days per week. Mice received different treatment combinations (two or three drugs; Table 2) containing bedaquiline (25 mg/kg), pretomanid (40 mg/kg), linezolid (100 mg/kg) and Be inhibitor (5 mg/kg) (as mentioned in all these studies, JNJ-901). The untreated control group consisted of 26 mice (eight at D-13, eight at DO, and five at each 2 and 4 weeks pi). Lung CFU was assessed at the end of each treatment and monthly during the relapse period in five animals/group, as well as at the end of the relapse period (15 mice/group).

Enumeration of CFU from lung

In studies A and B, samples in Gentle Macs tubes were homogenized in phosphate- buffered saline (PBS) + 10% bovine serum albumin (BSA). Homogenates were plated pure 500 pl and 50 pl or serial diluted (50pl) by 1/5 or 1/10 in PBS-BSA 10%. Dilutions were plated on 7H11-0 ADC plates containing 0.4% activated charcoal and incubated at 37°C for CFU quantification using an automatic SCAN- 1200 counter (Interscience). For relapse assessments, the total lung homogenate was plated undiluted. Reads were performed after 4 and 6 weeks of incubation at 37°C. For each sample, remaining volumes were stored in case additional plating should be required.

Statistical analysis

In the relapsing mouse infection model, a sample size of 15 mice per group for relapse assessments is typically used to achieve sufficient statistical power (Li, S. Y., et al. Evaluation of moxifloxacin-containing regimens in pathologically distinct murine tuberculosis models. Antimicrob. Agents Chemother. 59, 4026-4030 (2015)) Mean CFU/lung was log-transformed before analysis and were evaluated by one-way analysis of variance (ANOVA), followed by a pairwise multiple comparison using Dunnett’s test or Tukey’s test. The Kruskal -Wallis one-way ANOVA on ranks or equal variance tests were used if data failed normality. The proportions of mice relapsing were compared using Fisher’s exact test. Differences were considered significant at the 95% level of confidence.

Analyses can be carried out using GraphPad Prism software program. Results of these relapse mouse model studies

Also, depicted in Figures 3, and 4, the following Table 1 and 2 show the key results (where B = bedaquiline; Pa = pretomanid; L = linezolid; Be = cytochrome bcl inhibitor (in these studies, JNJ-2901); C = clofazimine), and so BPaL = bedaquiline + pretomanid + linezolid, etc.

Table 1: Lung bacterial burden and percentage of relapse in M. tuberculosis- infected mice (Studies A and B)

Balb/c female were infected intranasally with a high inoculum (4.5 logic CFU) of with M. tuberculosis H37Rv. Treatment was administered from 2 weeks post-infection (pi) 5 days per week for 4, 8 and 12 weeks, 5 days per week. Lung bacterial burden was assessed after 4 and 8 weeks of treatment (n=5 mice/group). Twelve weeks after treatment cessation, the proportion of relapse was calculated (n=15 mice/group and n=30 mice/group [pooled data]). D-13 = 1 day pi; DO = day of treatment initiation, 2 weeks pi. Mice received the different treatments combinations containing bedaquiline (B; 25 mg/kg), pretomanid (Pa; 40 mg/kg), linezolid (L; 100 mg/kg), clofazimine (C; 20 mg/kg) and cytochrome Be inhibitor (Be; 5mg/kg) (in this case JNJ-2901). *The proportion of number of mice exhibiting CFU to total number of mice analysed was determined and expressed as % of relapse. **Data are from two studies. CFU, colony forming unit; D, day; ND, not determined; SD, standard deviation; Tx, treatment; wks, weeks. Figure 3 shows the measure of lung CFUs in various combinations (where B = bedaquiline; Pa = pretomanid; L= linezolid; C = clofazimine; and 901 = the Be inhibitor JNJ-2901 as described herein) after 8 week treatment. It can also be seen from the table above that after 8 weeks of treatment, then 12 weeks of relapse, the BPaC901 was the combination with the significantly reduced relapse rates compared with the other combinations (even compared with BPaL).

The above (8-week treatment results), and Figure 3 show that addition of a cytochrome be inhibitor (in this case JNJ-2901) to the 3 -drug BPaC regimen (bedaquiline, pretomanid and clofazimine) provided for a 4-drug regimen (BPaCBc - i.e bedaquiline, pretomanid, clofazimine and in the case JNJ-2901) that was 2 times more sterilizing in comparison. Such a BPaCBc 4-drug regimen also displayed a similar fold better sterilizing effect compared with BPaL.

Such a regimen (or such combinations of the invention) could therefore be useful in treatment of human tuberculosis, including drug-resistant tuberculosis as described herein (e.g. in an embodiment, such drug-resistant strain may be a fluoroquinolone resistant strain that may be further resistant to other antibacterials, for instance as described herein).

Further results in this chronic mouse model show the following results after 8- and 12- week treatment, and then results showing relapse rates after a futher 12 weeks: The following logiolCFU/lung results were seen after 8 weeks:

BPaL after 8-week treatment - 1.59

BPa901 after 8-week treatment - 4.20

And, after 12-weeks, the following logiolCFU/lungs results: BPaL after 12-week treatment - 0.54

BPa901 after 12-week treatment 0.29

Further after a further 12-week period following the 12 week treatment, the same number of mice relapsed in both the 3 -drug BPaL and the 3 -drug BPa901 regimens (in each case 4 out of 15).

These further results show that after 8 weeks of treatment BPaL shows a better bactericidal effect. But after 12 weeks of treatment, sterilizing activity of BPa901 is equal to BPaL. The same number of mice relapsed after 12 weeks of treatment (4/15). These results show that a cytochrome bcl inhibitor could be a replacement for linezolid. There may be several reasons to avoid using linezolid as a treatment regimen, and this provides an alternative.

Given the results in the chronic mouse model described above in Biological Example 1 A, the results from Studies A and B in Biological Example IB above were surprising, as it would have been expected that such as an addition of the cytochrome bcl inhibitor (in this case JNJ-2901) to a bedaquiline-containing (2- or 3-drug) regimen would have led to a less favourable effect, in view of the previous antagonism seen. Hence, this result was surprising, and displayed clear advantages, as evidenced in this replase mouse model setting, as follows:

- when a BC inhibitor (e.g. JNJ-2901) is added (for instance as described above) to an existing regimen (e.g. to a 2-drug or 3-drug regimen), then the sterilizing effect of the combination regimen (of the invention) is enhanced (e.g. about more than 1.5x, for instance by about 2x) - for instance in an 8-week or a 12- week treatment period

- when a BC inhibitor (e.g. JNJ-2901) is added to a 3-drug regimen (e.g. BPaC), then the resulting regimen (or combination of the invention) has an improved sterilizing effect (for instance about 2x improved) compared with the BPaL regimen (e.g. in an 8-week or a 12-week treatment period)

- when a BC inhibitor (e.g. JNJ-2901) is added to a 2-drug regimen (e.g. BPa), then the resulting regimen (or combination of the invention) has a roughly equal sterilizing effect compared with the BPaL regimen, when the treatment period is 12 weeks (this provides evidence that the cytochrome be inhibitor may replace linezolid in the clinic)

- when a BC inhibitor (e.g. JNJ-2901) is added to a 3-drug regimen (e.g. BPaC), then the resulting regimen (or combination of the invention), for instance, in an 8-week (or 12-week) treatment setting has significantly improved replase rates (after a 12-week period), for instance compared with BPaL

- when a BC inhibitor (e.g. JNJ-2901) is added to a 2-drug regimen (e.g. BPa), then the resulting regimen (or combination of the invention), for instance in a 12-week treatment setting, has roughly similar relapse rates compared with the BPaL regimen (also providing evidence that the cytochrome be inhibitor may replace linezolid in the clinic) Table 2: Lung bacterial during treatment and relapse period in M. tuberculosis- infected mice (Study C)

Four to 6 weeks old Balb/c female mice were infected by high-dose aerosol with M. tuberculosis H37Rv. Treatment was administered from 2 weeks post-infection (pi) 5 days per week for 2 to 4 months (2 months initial phase/2 months continuation phase). Mice were held for up to 8 months after treatment initiation to determine the proportion of relapse after treatment cessation. Mice received the different treatments combinations containing bedaquiline (B; 25mg/Kg), pretomanid (Pa; 40mg/Kg), linezolid (L; lOOmg/Kg) and Be inhibitor (Be; 5mg/Kg). * For the BPaL group, the relapse period differs (8 wks + 4 wks, 8weks + 8 wks etc.) In this case the Be inhibitor that was used was JNJ-2901.

**relative relapse proportion for the same number of mice as the other groups. N, total number of mice/group; n, number of mice with CFU at plating. CFU, colony forming unit; D, day; SD, standard deviation; wks, weeks.

These results show that the cytochrome be inhibitor JNJ-2901 is sterilizing in the companion phase when added to BDQ for 2 months. This was surprising given the results in the chronic mouse model study, and this strategy can be used as a PoC for pill burden reduction of the current SOC for treatment of tuberculosis (e.g. DR-TB / MDR- TB).

There is therefore provided for the addition of a cytochrome bcl inhibitor (e.g. JNJ- 2901) to a bedaquiline-containing (or another suitable ATP synthase inhibitorcontaining, such as TBAJ-587 or TBAJ-876-containing) regimen to: (i) enhance sterilizing effect

(ii) reduce the ATP-synthase (e.g. bedaquiline) pill burden, in a regimen for the treatment of tuberculosis (e.g. drug-resistant tuberculosis).

Biological EXAMPLE 1C

In the Balb/c RMM, mice are infected by high-dose aerosol using Mtb Erdman. Therapy consisting of 3-4 drug combinations is initiated around 11 days following aerosol infection. Groups of mice are euthanized one day after the last day of treatment typically with < 2- months of therapy to study bactericidal activity in lungs over time. Relapse rates are measured for all treatment groups in additional companion mice 3 months (12 weeks) after cessation of drug therapy. Detection of a single CFU is an indication of treatment failure or relapse. Here we tested a revised study design with more frequent sampling and smaller numbers of mice. This is the so-called Erasmus- Cognigen design allowing regression analysis and modelling of the resultant data to better calculate true relapse probability and time profiles.

These studies will employ the minimum number of animals suitable to derive a statistically valid comparison between study groups based on ribosomal synthesis ratios (RS ratio™) and conventional CFU-based endpoints.

References:

Walter ND et al Mycobacterium tuberculosis precursor rRNA as a measure of treatment-shortening activity of drugs and regimens. Nat Commun. 2021 May 18; 12(1):2899. doi: 10.1038/s41467-021-22833-6. PMID: 34006838.

Wynn EA et al.,. Transcriptional adaptation of Mycobacterium tuberculosis that survives prolonged multi-drug treatment in mice. mBio. 2023 Oct 31;14(6):e0236323. doi: 10.1128/mbio.02363-23. Epub ahead of print. PMID: 37905920. Mouse Metrics and Groups

B or BDQ = Bedaquiline (free form; but the fumarate salt form may also be used) Pa = pretomanid

L = linezolid

X = Compound X as described above (Compodun 177) 229 total Balb/c mice used for aerosol in 2 separate aerosol runs (with 114 and 115 in runs 1 and 2, respectively). Six mice are used for day 1 sacrifice (3 mice per aerosol); 9 mice are used for the start of treatment (Rx) Abbreviations and doses of drugs

Preliminary Bactericidal Results

Claims

1. A combination (e.g. combination regimen) comprising:

(i) bedaquiline, or a pharmaceutically acceptable salt thereof;

(iii) a cytochrome bc inhibitor, or a pharmaceutically acceptable salt thereof.

2. A combination as claimed in Claim 1, further comprising one or more antibacterials.

3. A combination as claimed in Claim 2, wherein the one or more antibacterials is clofazimine.

4. A combination (e.g. combination regimen) consisting essentially of:

(i) bedaquiline, or a pharmaceutically acceptable salt thereof;

(iii) a cytochrome bc inhibitor, or a pharmaceutically acceptable salt thereof.

5. A combination as claimed in any one of Claims 1 to 4, wherein the cytochrome bc inhibitor is JNJ-2901 or Compound X, or a pharmaceutically acceptable salt thereof.

6. A combination as claimed in any of the proceeding claims, which displays in vivo efficacy, for instance in a relapse mouse model (as described herein).

7. A combination as claimed in any one of Claim 1 to 3, or Claim 5 or 6 (as dependent on Claims 1 to 3), wherein the combination is at least a 4-drug regimen, and wherein the sterilizing effect is increased by about 2 times, compared either with the corresponding regimen without the cytochrome bcl inhibitor, or compared with BPaL (a regimen consisting of bedaquiline, pretomanid and linezolid).

8. A combination as claimed in Claim 7, wherein the treatment period is 8-12 weeks

(and wherein the sterilizing effect may be measured in a relapse mouse model, for instance as described herein).

9. A combination as claimed in any one of Claim 1 to 3, or Claim 5 or 6 (as dependent on Claims 1 to 3), wherein the combination is at least a 4-drug regimen, and in an 8-week (or 12-week) treatment setting in a relapse mouse model (as described herein), such combination has significantly improved replase rates (after a 12- week period), for instance compared with BPaL.

10. A combination as claimed in Claim 4 or Claim 5 and 6 (as dependent on Claim 4), wherein the combination is a 3-drug regimen, and in a 12-week treatment setting in a relapse mouse model (as described herein), such combination has either: (i) roughly equal sterilizing effect; and/or (ii) has roughly similar relapse rates, each of which are compared with the BPaL regimen.

11. A pharmaceutical formulation comprising a combination as claimed in any one of the preceding claims, and a pharmaceutically acceptable excipient or diluent.

12. A combination as claimed in any one of Claims 1 to 10, for use as a medicament.

13. A combination as claimed in any one of Claims 1 to 10, for use in the treatment of a mycobacterial infection (especially Mycobacterium tuberculosis).

14. A use of the combination as claimed in any one of Claims 1 to 10, in the manufacture of a medicament in the treatment of a mycobacterial infection (especially Mycobacterium tuberculosis).

15. A method of treating a mycobacterial infection (especially Mycobacterium tuberculosis) in a patient comprising administering an effective amount of a combination as claimed in any one of Claim 1 to 10.

16. A combination, use or method as claimed in any one of Claims 13 to 15, wherein the tuberculosis is latent tuberculosis.

17. A process for preparing a pharmaceutical formulation as defined in Claim 12 comprising bringing into association any one (or more, e.g. the essential active ingredients and, optionally, further antibacterials as defined herein) of the active ingredients of the combination of the invention, with one (or more) pharmaceutically acceptable excipient or carrier.

18. A process for preparing a combination product as defined herein comprising:

- bringing into association each of the components (e.g. as separate pharmaceutical formulations) of the combination product as claimed in any one of Claims 1 to 10 and co-packaging (e.g. as a kit of parts) or indicated that the intended use is in combination (with the other components); and/or

- bringing into association each of the components of the combination product as claimed in any one of Claims 1 to 10 in the preparation of a pharmaceutical formulation comprising such components.

19. A combination product as claimed in any one of Claims 1 to 10, as a combined preparation for simultaneous, separate or sequential use in the treatment of a bacterial infection.