**8. Success stories so far**

There are many instances where repurposed drugs have shown successful results in subclinical, preclinical, and clinical levels. Mice could be cured of both drug susceptible and MDR infection mice were given TDZ [79, 102] alone [103] and in combination with INH [104]. This study was extrapolated to non-responsive MDR-TB patients in a Buenos Aires hospital (Argentina) [105] and weeks later, patients got cured of TB. The protocol was then modified and included nonresponsive antibiotics, and out of 12 XDR-TB patients, 10 were pronounced cured of the infection [106]. TDZ was also used by *Udwadia* et al. for the therapy of XDR-TB patients in Mumbai (India) and it was found to improve significantly their quality of life. The subclinical and preclinical success of the drug TDZ, against MDR TB and XDR TB, led to a public call to consider TDZ for therapy of non-responsive MDR/XDR-TB under compassionate basis [107]. Meropenem, in combination with clavulanate, was adjusted with the drug regimen and approved by the European Medicines Agency and the US Food and Drug Administration (FDA) for curing TB in 8 months. FDA-approved antidiabetic drug metformin, was shown to enhance the efficacy of other anti-TB drugs against the drug-resistant tuberculosis [47]. *Reports from* Microbiology and Infectious Diseases at the National Institute of Allergy & Infectious Diseases (NIAID) and Stop TB Partnership new drug working group state that drug resistance has arisen against every currently available tuberculosis drug. Successful treatment for extensively drug-resistant (XDR) cases is less than half of that for drug-susceptible tuberculosis; this makes situation grave and urges for new antibiotics against the global killer. Many compounds in TB-advanced clinical trials were formerly used to treat other infectious diseases/TB, and now, they have been repurposed for the treatment of TB [108–110]. Revival of sulfamethoxazole (SMX) in TB occurred when it was first used to prevent the Pneumocystis jirovecii like infections in HIV/TB patients [111]. In a

#### *Antituberculosis Drug Repurposing: A New Hope for Tackling Multi-Challenging TB in Timely… DOI: http://dx.doi.org/10.5772/intechopen.101642*

Nigerian trial study on patients of HIV-MDR-TB co-infection, efficiency of MDR-TB treatment by TMP/SMX confirmed a significantly shorter time to sputum conversion in these patients [112]. Sulfadiazine, an antileprosy drug, was repurposed in the treatment of MDR-TB and XDR-TB [113] suggesting that sulfadiazine regimen is safe and effective against MDR-TB and TDR-TB treatment [113, 114]. Clofazimine (CZM), an old antileprosy drug, was repurposed for managing the treatment of MDR-TB [110]. CZM is now recommended as a second-line anti-TB drug and used in combination with other anti-TB drugs for the treatment of drug-resistant tuberculosis in 9–12 months. Previous published studies have reported that CZM has good quality efficacy and little toxicity against drug-resistant mycobacterial strains in animal models, which suggested, CZM as a promising anti-TB drug for the management of MDR-TB [111]. Linezolid, an oxazolidinone antibiotic used for the treatment of grampositive bacterial infections [115], is being potentially repurposed for the treatment of drug resistant TB (MDR-TB and XDR-TB) [116]. But it has been limited by various side effects such as neurotoxicity and hematologic toxicity [90]. Safety of bedaquiline and linezolid drug combination has been evaluated by a case study for XDR-TB and found to be safe in even the late third trimester of pregnancy or pregnant woman. Post-treatment, pregnant woman gave birth to a normal child who grew without fatalities [90]. Minocycline is another anti-leprotic drug [117], which was repurposed in 2008 for managing the treatment of XDR-TB patient in Japan [118]. *In vitro* activity of meropenem combined with clavulanate against XDR strains calls for repurposing the beta-lactams as new anti-TB drugs [119]. Carbapenems have been used successfully as part of salvage therapies for XDR patients, which have to be administered intravenously [120]. Recently, an early bactericidal activity-Phase II (EBA Phase II) clinical trial has validated the promising potential of a carbapenem combined with amoxicillin and clavulanic acid for TB treatment [121]. In a controlled clinical trial in tuberculosis, inhaled IFN-γ was effective. These experiences warrant the continued evaluation of inhaled IFN-γ in human clinical trials [47]. Certain clinical studies are exploring the potential of NSAIDs in TB treatment. NCT02060006 is a Phase 3 trial to identify meloxicam in preventing TB immune reconstitution inflammatory syndrome (IRIS), a serious clinical issue in HIV co-infected TB patients. Phase 2 clinical study (NCT02237365) of aspirin and ibuprofen is an adjunctive treatment for TB meningitis for the treatment of XDR-TB in addition to the standard therapy (NCT02781909). The immune-modulatory function of NSAIDs (etoricoxib) in increasing the protection offered when administered alongside a TB vaccine is being investigated in the trial NCT02503839. Other drug-screening study revealed carprofen, an NSAID, to selectively inhibit the growth of replicating, non-replicating, and MDR clinical isolates of *M. tuberculosis* at 40 mg/L [122, 123].

Hurdles in TB drug repurposing: Upon entering and infecting the host, *M.tb* spreads to different micro-niches and evolves as heterogeneous population. To eliminate each physiological state of the bug, any new anti-TB needs to be active under these conditions [124]. Development of *in vitro* models for non-replicating and replicating Bacilli has not been successfully achieved. Subpopulations of nonreplicating bacilli have present inside host arise need for the lengthy anti TB drug therapy and turn out to be reservoir from which drug-resistant bacteria emerge [125]. A multi-stress model of non-replication has been put forward [94]. But a disadvantage of this type of model is the need for a recovery or outgrowth phase that implies bacilli being replicated, which makes interpretation more difficult [33]. A rapid method has been developed to measure bactericidal activity against nonreplicating *M. tuberculosis*, without requirement of the outgrowth period, and easily detecting luminescence of viable *M. tuberculosis* strain constitutively expressing luciferase [95]. Compounds with bactericidal activity against non-replicating bacteria were identified employing a pH-sensitive green fluorescence protein screening

approach devised to identify compounds that disrupt the ability of *M. tuberculosis* to maintain its internal pH in an acidic environment [126]. Since TB is a complex disease, no *in vitro* model has been till date established to predict *in vivo* efficacy [127]. Tuberculosis is the leading cause of death from infectious disease. Current drug therapy requires a combination of antibiotics taken over >6 months. An urgent need for new agents that can shorten therapy is required. To develop new drugs, simple *in vitro* assays are required that can identify efficacious compounds rapidly and predict *in vivo* activity in the human. Areas covered: This review focusses on the most relevant *in vitro* assays that can be utilized in a drug discovery program, which mimics different aspects of infection or disease. The focus is largely on assays used to test >1000s of compounds reliably and robustly. However, some assays used for 10s to 100 s of compounds are included where the utility outweighs the low capacity. Literature searches for high-throughput screening, models, and *in vitro* assays were undertaken. Expert opinion: drug discovery and development in tuberculosis is extremely challenging due to the requirement for predicting drug efficacy in a disease with complex pathology in which bacteria exist in heterogeneous states in inaccessible locations. A combination of assays can be used to determine profiles against replicating, non-replicating, intracellular, and tolerant bacteria [127]. To establish best representative model of the real metabolic state, either replicating or non-replicating bug in various environments inside human host is a challenge. Screening models fail to fulfill requirements of extensive follow-on experiments in the human host to ascertain efficacy, pharmacokinetics, pharmacodynamics, and toxicity, and thus hamper the optimization for improvement of repurposed drug efficacy using medicinal chemistry approach. In addition, safety concern of the drug and its interaction with other antimicrobial agents are the critical issues to be addressed as the treatment duration of the disease is long. Ideally, the new drugs are expected to decrease required treatment durations hence improving patient compliance and treatment outcomes. The co-existence of HIV and TB emphasizes that new lead must be compatible with antiretroviral therapy as well as active against resistant forms of TB [96]. Targeting the drug to the site of infection is very long and eventful process, which often makes the compound unable to reach its target in active state. A drug molecule has to travel from the blood circulation to nonvascularized pulmonary lesions wherefrom it shall diffuse into necrotic foci and the caseum of granuloma and then permeate the lipid-rich cell envelope of bacilli at the requisite MIC value for the pathogen [97]. Common challenges of drug repurposing also affect drug reuse against TB. Optimizing selection criteria of target population to evaluate the expected outcome of the drug is one of major challenges. Any error in subject selection can give unexpected adverse results of drug. For example, Thalidomide when prescribed for pregnant women in first trimester for managing morning sickness resulted in amelia and phocomelia [100]. Repurposing of old drug for new indication needs addressing the dosing regimen and route of administration to yield the considerable benefits against new target. Patient-specific repurposing of drug shall be aimed to evade the adverse events, which might occur due to differential response of different patients to the repurposed drug. Moreover, prerequisite data on drug-drug interactions, pharmacodynamics, and pharmacokinetics of the drug shall be keenly studied prior to further studies of the drug.

## **9. Status of TB drug repurposing and its future perspective**

Drug repurposing is undoubtedly an alluring strategy to develop a new treatment regimen for tuberculosis within a short span of time and also to treat and curb drugresistant pathogens [128]. Few of the repurposed drugs have shown great potential

#### *Antituberculosis Drug Repurposing: A New Hope for Tackling Multi-Challenging TB in Timely… DOI: http://dx.doi.org/10.5772/intechopen.101642*

for future treatment of TB and have been extensively studied. Nevertheless, the incidence of resistance in the *M.tb* population is occurring at a very fast rate and therefore, we urgently need a new improved treatment regime via repurposing many drugs using various approaches such as experimental and computational biology [129, 130] to scrutinize the potential of already existing thousands of drugs to minimize the time required for novel drug discovery. Organization of such studies is on the human cohorts, as the influence of the host-protective immune system continues to gain attention in the advancement of host-directed therapies, so effect of repurposed drugs on the balance of the host immune system, infection, and inflammation shall be explored. This will update concepts to design combinational therapies to shorten the treatment regime and preventing drug resistance while being cost effective and safe for general masses [131]. Repurposing drugs assuredly provide an appealing strategy in the process of modern drug development and exceptionally/especially against tuberculosis, which already have numerous engrossing old drugs with *in vitro* growth inhibitory activities. Using different methods for whole-cell evaluations such as HT-SPOTi [132, 133] and micro-plate Alamar blue assays (MABA) [134] has turn out to be crucial for the expeditious detection of various old drugs that have promising potential in drug repurposing. Many of the potential anti-TB drugs were identified through serendipity, and amalgamating the various assays with systems biology will in turn provide a reasonable approach in the identification of these drugs [135]. TB drug discovery paradigm converses from the conventional one-target one-drug to a multi-target multidrug scheme, and various potential drugs for repurposing are being recognized and put forth into the advanced phases of clinical trials. As an alternative in the treatment of drug-resistance, repurposed drugs have already proven their potential and effectiveness. Endeavor to repurpose inexpensive, safe and universally available drugs should continue to deliver the anti-TB therapies required by many who would not otherwise have access to a cure [128]. On one side, it becomes imperious to find new candidate drugs to control TB, and on the other side, it is also important to continuously redefine, revise, reclassify, and perhaps, repurpose drugs that are already in use. The drug repurposing offers manifold advantages. It is therefore pivotal to understand their secondary targets and various endogenous molecular mechanisms of action and its translation into a multidrug combinatorial treatment regimen. Identification of mechanism of action of these repurposed drugs will definitely strengthen their inclusion in clinical trials and gravel the way for designing more targeted drugs. As antimicrobial resistance deepens, the search to find novel drugs and to evaluate the mechanism of resistance would widen our search to novel concepts as well to find a better cure to curb TB than what already exists. Repositioning of pre-existing drugs seems to be a strategy to avoid enormous investment in funds and time. Drugs with already known toxicity and safety profiles have been screened against the TB pathogen and found to be effective against various physiological states of pathogen. The endogenous targets of these drugs against *M.tb* are likely to be novel; thus, minimal chances of resistance arise. Moreover, few of these drugs may have multiple targets, which indicate minimal development of resistance. Thus, repurposing the pre-existing molecules offers colossal/enormous potential to tackle extensively drug-resistant TB infections. Fluoroquinolones prevent DNA replication by inhibiting topoisomerase II and IV; two examples *viz.* gatifloxacin and moxifloxacin are active against *M. tuberculosis* both *in vitro* and *in vivo* conditions [136, 137] and thus used as second-line drugs against TB [138]. Moxifloxacin was advanced to phase III clinical trials to evaluate its potential to shorten the duration of conventional TB therapy (**Figure 5**) [139, 140].

Schematic illustration of the *Mycobacterium tuberculosis* cell membrane includes the electron transport chain (ETC), efflux pumps (EPs) and the site of action of

#### **Figure 5.**

*Mechanism of action of new anti-TB agents in different stages of clinical drug development pipeline for tuberculosis.*

several antituberculosis drugs. By damaging the cell membrane, the lipophilic drugs will affect the activity of several membrane enzymes such as those involved in the ETC and efflux pumps responsible for the extrusion of several compounds from the cell. The inhibition of any component of the ETC reduces energy production and disrupts membrane potential. Consequently, the disruption of the PMF reduces the activity of the efflux pumps. SQ-109 has been reported to act by inhibiting the mycobacterial trehalose monomycolate transporter MmpL3, involved in cell wall biosynthesis [141]. PA-824 is effective not only toward the actively replicating but also against the non-replicating bacteria. They inhibit the synthesis of mycolic acids and induce respiratory poisoning [142] Q-203 targets the Cytochrome b subunit (QcrB) of the cytochrome bc1 complex (complex III), which is an essential component of the *M. tuberculosis* respiratory electron transport chain, forcing *M. tuberculosis* to use the cytochrome bd, a terminal oxidase energetically less efficient [143]. Q-203 causes a rapid depletion of the intracellular ATP levels at 1.1 nM and is able to interfere with ATP homeostasis in nonreplicating *M. tuberculosis* at concentrations of <10 nM, suggesting the inhibition of cytochrome bc1 activity as its primary mode of action [144]. Diarylquinolines target subunit c of mycobacterial ATP synthase [145]. Mycobacterial membrane protein large (MmpL) proteins, which belong to the resistance, nodulation and cell division (RND) superfamily of transporters, play a central role in shuttling lipid components to the cell wall. These transporters work with accessory proteins to translocate virulence-associated envelope lipids and siderophores across the inner membrane [146]. Capuramycin and its analogs are strong translocase I (MurX/MraY) inhibitors [147]. Oxazolidinones inhibit the initiation of protein synthesis by preventing the formation of the tRNAfMet-mRNA-70S (or 30S) subunit ternary complex [148].

It is under evaluation in a TB Alliance phase III clinical trial with pretomanid and pyrazinamide (PaMZ). Mycobacterial resistance to fluoroquinolones is evident [149] caused by stepwise mutations in the target genes such as gyrA and gyrB [150]. There is no visible cross-resistance observed with the other first-line drugs [151], but there is cross-resistance within this group of molecules. Indeed, this cross-resistance is not universal [152], and newer fluoroquinolones such as TBK613 will still be effective

#### *Antituberculosis Drug Repurposing: A New Hope for Tackling Multi-Challenging TB in Timely… DOI: http://dx.doi.org/10.5772/intechopen.101642*

against fluoroquinolone-resistant strains. This demonstrates the coherent nature of the development of novel drug and drug repositioning, and structure-activity relationship of a repurposed drug enables the design of novel molecules with higher potency. Nitroimidazopyrans, resembling the antibiotic metronidazole, is active against actively growing and dormant *M. tuberculosis* [33, 153]. The novel chemical entities (NCEs) OPC-67683 and PA-824 are currently in clinical trials [154]. Metronidazole is also highly active against *M. tuberculosis* [33] and has been reported to prevent the reactivation of dormant bacilli in macaque infection models [155]. Clavulanate, a β-lactamase inhibitor, in concurrence with carbapenems showed killing of *M. tuberculosis in vitro* [156] and in a murine TB model [157]. β-lactam tebipenem, originally developed to tackle respiratory and otolaryngological infections in pediatric patients [158], is to be the most potent anti-TB oral carbapenem in combination with clavulanic acid, and clinical trials may start soon. Clofazimine, the antileprosy drug with promising candidate to get repurposed in treating incidences of multidrug-resistant (MDR)- and XDR-TB, is listed as a World Health Organization recommended second-line drug. Members of the avermectin family, traditionally used as antihelminthic agents, have been found to inhibit the growth of even MDR strains of *M. tuberculosis in vitro* [159]. Nitazoxanide has been found to inhibit both replicating and non-replicating forms of *M. tuberculosis* [160, 161]. Disulfiram inhibited *M. tuberculosis* H37Rv growth at a concentration of 5.26 mM [162]. Disulfarim showed the same level of inhibition against clinical isolates and MDR and XDR strains, and an *in vivo* experiment on guinea pigs demonstrated astonishing bactericidal activity [162]. Non-steroidal anti-inflammatory drugs (NSAIDs), oxyphenbutazone [72], and carprofen [163] inhibited the growth of *M. tuberculosis* H37Rv at micromolar concentrations. To develop novel TB treatments, drug repurposing has procured acceptance and has gained pace, with various drugs that are already at different phases of preclinical and clinical trials (**Table 1**) (**Figure 6**) [123].

The drugs and their targets are highlighted in lighter and darker shaded boxes, respectively. The anagram MAGP is used to indicate the "mycolic acid–arabinogalactan–peptidoglycan" layer of the mycobacterial cell wall and PBP refers to the penicillin-binding proteins responsible for the maturation of the cell wall peptidoglycan [177] inhibition of efflux pumps by Thioridazine [178] Fluoroquinolones (moxifloxacin, gatifloxacin), with target of gyrase, are among the drugs used to treat tuberculosis [179]. Oxazolidinones: (Linezolid) kills *Mycobacterium tuberculosis* by binding and blocking tRNA in the peptidyltransferase center (PTC) on the 50Sribosomal subunit, which includes the 5SrRNA and 23S rRNA [180]. Nitroimidazole derivatives: (Metronidazole) with lower reduction potential can selectively tap into the redox system of the microbe (as opposed to mammals) and produce bactericidal activity specific to the microbe [154]. The combination of clavulanate with β-lactams, especially meropenem, was also tested for the ability to inhibit the growth of extensively drug-resistant (XDR) clinical strains of *M. tuberculosis* [119]. *Ibuprofen (IBF) and carprofen, two non-steroidal anti-inflammatory drugs currently used as pain relievers in humans and animals, respectively, displayed specific growth inhibitory properties against the M. tuberculosis complex.* IBP showed antitubercular properties, while carprofen was the most potent among the 2-arylpropanoic class. On the basis of the human targets of the 2-arylpropanoic analgesics, the protein initiation factor infB (Rv2839c) of *M tuberculosis* was proposed as a potential molecular target [163].

Entacapone and tolcapone inhibit enoyl-acyl carrier protein reductase (InhA) [71], which is important component in the synthesis of long-chain mycolic acids. Entacapone and tolcapone are not prodrugs like isoniazid and do not require enzymatic activation. Thus, the primary mutations in enzyme causing resistance


**Name Class Current use** *In vitro* **MIC against H37Rv Stage of repurposing References** Thioridazine Phenothiazine Antipsychotic 27 μM Anti-TB property detected *in vitro* by BACTEC 460-TB [79] Tolcapone Nitrocatechol Anti-Parkinson's drug 457 μM Anti-TB property predicted by system biology [174]
