Chemotherapeutic Interventions Against Tuberculosis

Tuberculosis is the second leading cause of infectious deaths globally. Many effective conventional antimycobacterial drugs have been available, however, emergence of multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) has overshadowed the effectiveness of the current first and second line drugs. Further, currently available agents are complicated by serious side effects, drug interactions and long-term administration. This has prompted urgent research efforts in the discovery and development of new anti-tuberculosis agent(s). Several families of compounds are currently being explored for the treatment of tuberculosis. This review article presents an account of the existing chemotherapeutics and highlights the therapeutic potential of emerging molecules that are at different stages of development for the management of tuberculosis disease.


Introduction
In 1905, Robert Koch, a German physician, was awarded the Nobel Prize for his milestone discovery of Mycobacterium tuberculosis (Mtb), the bacillus of tuberculosis (TB). Despite his groundbreaking discovery, it took more than half a century to find a cure against the bacilli to save OPEN ACCESS millions of human lives. The death of Koch in 1910 prevented him from witnessing the life-saving consequences of his pioneering research. Among these was the first antibiotic for tuberculosis patients, streptomycin, a natural compound. However, the requirement of intravenous administration of streptomycin and development of resistance to it soon necessitated the need of next generation of antibiotics against Mtb. Many antimycobacterial drugs have been discovered since then, classified as first-line and second line drugs. Recent emergence of multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) [1], have seriously compromised the usefulness of these current first and second line drugs, once again creating an urgent need for newer, safer and more effective anti-tuberulosis treatments. Adding to this crisis is the limited use of second line drugs for MDR-TB and XDR-TB due to their toxicity and serious side effects. Moreover, recently, totally drug-resistant tuberculosis (TDR-TB) has emerged which is resistant to a wider range of drugs than XDR-TB. Cases of TDR-TB have been reported in several countries including Italy, Iran and India [2].
Bacillus Calmette Guerin (BCG) vaccine, an attenuated strain of M. bovis reliably protects only newborns against Mtb but is ineffective in adult pulmonary TB. The vaccination may also lead to TB-like infection in immunocompromised people [3].
The increasing incidence of MDR-TB, XDR-TB, and TB-HIV coinfection have raised the alarm for the discovery and development of novel anti-tuberculosis agent(s) that do not possess cross-resistance with current antimycobacterial drugs and have minimal toxicity. This article summarizes the features of current anti-tuberculosis drugs and the pharmacological properties of novel compounds that are in the process of development for antimycobacterial therapy.

First Line Drugs
First line anti-tuberculosis drugs include rifampicin, isoniazid, pyrazinamide and ethambutol ( Figure 1). This drug was discovered in 1966. It possesses very potent in vitro activity against Mtb with an MIC of 0.05-0.5 μg/mL. Rifampicin is highly active against Gram-positive bacteria including Mtb. Unlike many other antibiotics, it is lipid soluble, penetrates cell membranes and kills intracellular bacteria [4]. It acts by the inhibition of DNA-dependent RNA polymerase in bacterial cells by binding to its β-subunit, thus preventing transcription of RNA and subsequent translation of proteins [5,6]. A daily regimen of 10 mg/kg (up to 600 mg/day) orally or an intermittent regimen of 10 mg/kg (up to 600 mg/day) orally, are effective [7]. However, Mtb quickly develops resistance to rifampicin hence the drug is recommended to be used in combination with other antibiotics. Most of the Mtb clinical isolates resistant to rifampicin show mutations in the rpoB gene that encodes the β-subunit of RNA polymerase. These mutations cause conformational changes in the polymerase that result in a low affinity for the drug rendering it ineffective [8]. The side effects include hepatitis with elevation of bile and bilirubin, anaemia, leucopenia, thrombocytopenia, bleeding, fever, eosinophilia, leucopenia, thrombocytopenia, purpura, haemolysis and nephrotoxicity [9]. Interestingly, no serious side effects have been observed in breastfed infants during rifampicin therapy [10,11]. The drugs for possible interactions with rifampicin include 4-aminosalicylic acid (PAS), HIV protease inhibitors, warfarin, oral contraceptives, cyclosporine, itraconazole, digoxin, verapamil, nifedipine, simvastatin, midazolam, clarithromycin, lorazepam atorvastatin, antiretroviral agents, rosiglitazone/pioglitazone, celecoxib, caspofungin [12].

Isoniazid
Isoniazid (INH) was discovered in 1952. It acts as a bactericidal agent with an MIC of 0.01-0.2 μg/mL for fast replicating mycobacteria [13]. It is bacteriostatic to slow-growing or non-dividing mycobacteria like Mtb and therefore, is used to treat latent tuberculosis. Isoniazid is a actually a prodrug and is activated by the mycobacterial enzyme catalase-peroxidase (KatG), which catalyzes the formation of the isonicotinic acyl-NADH complex. Subsequently, this complex binds to the enoyl-acyl carrier protein reductase InhA, and then blocks the natural substrate enoyl-AcpM and fatty acid synthase. This results in inhibition of mycolic acid synthesis which is an essential component in the formation of the mycobacterial cell wall [14,15]. Resistance to isoniazid occurs due to mutations in several genes, including katG, ahpC, inhA, kasA and ndh. In adults, the recommended daily dose of INH is 5 mg/kg/day (max 300 mg daily). For intermittent dosing (twice or three times/week), 19-15 mg/kg/day (max 900 mg/day) is used. The recommended dose for children is 8 to 12 mg/kg/day [7,16]. INH is metabolized in the liver and its metabolites are excreted in the urine [17]. INH chronic toxicity affects the liver, haematologic-and peripheral nervous systems resulting in acute hepatitis, peripheral neuropathy and haemolytic anaemia [18].

Pyrazinamide
Pyrazinamide (PZA) was discovered in 1952. It is mainly bacteriostatic but can be bactericidal for replicating Mtb. It possesses an MIC of 20-100 μg/mL. When used as part of combination therapy, PZA accelerates the sterilizing effect of INH and rifampin [19]. This has enabled reductions in the duration of treatment for susceptible M. tuberculosis isolates from nine to six months and for this reason is used in the first two months of treatment [20]. PZA is also effective for the treatment of tuberculous meningitis [21]. Like isoniazid, PZA is a prodrug. In acidic conditions, the enzyme pyrazinamidase (present in Mtb), converts it to the active form, pyrazinoic acid which subsequently inhibits the enzyme fatty acid synthase (FAS) I, required by the bacterium to synthesize fatty acids [22][23][24]. Mutations of the pyrazinamidase gene (pncA) are responsible for PZA resistance in Mtb [25]. Most alterations occur in a 561 bp region of the open reading frame or in an 82 bp region of its putative promoter [26,27].

Ethambutol
This drug was discovered in 1961. Ethambutol (EMB) is a bacteriostatic drug against actively growing mycobacteria. It blocks formation of the cell wall of Mtb by inhibiting the enzyme arabinosyl transferase involved in the synthesis of arabinogalactan. Arabinogalactan is an essential component in the formation of the mycolyl-arabinogalactan-peptidoglycan complex of the Mtb cell wall [30,31]. Mutation in gene embB is responsible for resistance to ethambutol [30].
Ethambutol is well absorbed in the gastrointestinal tract, and is efficiently distributed in body tissues and fluids. Fifty percent of the given dose is excreted unchanged in urine [32]. Ethambutol is used at 15-25 mg/Kg once daily dose for 6-8 weeks concurrent with isoniazid therapy [33]. Adverse effects of EMB include peripheral neuropathy, red-green color blindness, arthralgia, hyperuricaemia and optic neuritis [34].

Drug Discovery Program
This section includes early stage drug discovery, molecules in development, molecules at the pre-clinical stage, molecules in phase I trials, molecules in phase II trials and molecules in phase III trials.

Early Stage Drug Discovery
After a long pause, the last decade proved to be a golden era in the hunt for new tuberculosis drug(s). Tremendous efforts and high priority research are underway for finding better drugs to combat wild-type and drug-resistant Mtb. Since the last decade, the private sector and government agencies have participated in the fight against this devastating disease. Apart from major financial contributions by corporations, basic and semi-applied researchers are also continuing to make significant progress, despite facing financial constraints. The following section describes some representative examples of different classes of molecules from early stage screening studies carried out in the last decade by these groups.

Nucleosides
The nucleoside class of compounds are well known for their antiviral and anticancer properties. They can be classified as pyrimidine or purine nucleosides.

Pyrimidine Nucleosides
In early 2000s, Vanheusden et al. reported modified nucleoside and nucleotide derivatives as inhibitors of a mycobacterial enzyme thymidine monophosphate kinase (TMPKmt). In 2004, they reported a series of bicyclic analogues of thymidine [45] where compound 1 ( Figure 2) demonstrated a Ki of 3.5 μM for TMPKmt with a good selectivity index (SI 200) over its human counterpart TMPKh. In these studies, however, only enzyme inhibition was reported and inhibition of mycobacterial replication was not demonstrated.
The complete genome sequence of Mycobacterium tuberculosis has been determined [46], which identified many of the genes required for encoding enzymes involved in nucleic acid synthesis, and pyrimidine and purine biosynthesis. We hypothesized that modified nucleosides could target several enzymes involved in RNA and DNA metabolism and were the first to investigate and demonstrate potent antimycobacterial activity of 5-substituted pyrimidine nucleoside analogs [47]. The microplate alamar blue assay (MABA) [48] was used to evaluate the antimycobacterial activity of test nucleosides. We observed that the most potent TMPKmt inhibitors reported earlier [49][50][51] did not show antituberculosis activity against mycobacterial replication as determined by MABA assay [47].
In the same year our group reported antimycobacterial effects of several 5-alkyl-and 5-alkynylfuranopyrimidines and related 2'-deoxynucleosides. Compounds with 5-arylalkynyl substituents displayed potent in vitro antitubercular activity against M. bovis and Mtb (MIC 0.5-5 μg/mL). We selected compound 12 to test its potency in a mouse model (BALB/c) of Mtb (H37Ra) infection. At a dose of 50 mg/kg for 5 weeks, statistically significant reduction in mycobacterial load was observed in lungs, livers and spleens of the treated mice. This is the first evidence of antimycobacterial potential of 5-substituted pyrimidine nucleosides in an animal model as a potential new class of antituberculosis agents [58].

Carbohydrates
Carbohydrates have been evaluated as antituberculosis agents for a long time. Some selected reports are summarized here. In 2005 bis-glycosylated diamino alcohols were reported by Tripathi et al., where their compound, 17, showed moderate activity against Mtb H37Ra and against Mtb (H37Rv). This compound was also active against an MDR strain and showed mild protection in mice at 25 mg/Kg dose [63].
Derivatives of stachyose were reported by Chiba et al. The most active compound in the series against Mtb H37Rv was 18 (OCT359, MIC 3.13 μg/mL) which was also evaluated against various drug-sensitive and -resistant clinical isolates of Mtb. Interestingly, 25 clinical isolates of drug-resistant Mtb and 19 drug-sensitive Mtb were sensitive to OCT359 (MICs ranging from 3.13 to 25 μg/mL) [64].
Recently in this class, compound 19 (OCT313HK, Glc-NAc-PDTC) showed potent anti-tuberculosis activity against wild-type, and clinical isolates of Mtb, including MDR and XDR strains at similar concentrations (MIC 6.25-12.5 μg/mL) [65] (Figure 6).               TLM is an inhibitor of the β-ketoacyl-acyl carrier protein synthase (KAS) enzymes, which are part of the bacterial fatty acid synthase pathway. TLM has MIC of 62.5 μM against Mtb [87,88]. TLM also inhibits human FAS-I enzyme [89], however, its lower affinity (IC 50 100 μM) for this enzyme can make it worthy as a selective anti-tuberculosis agent [90] (Figure 18). It is also effective against both drug sensitive and extremely drug resistant (XDR) Mtb in a mouse model of acute tuberculosis with 1-1.5 log CFU reduction in the lungs. Its mode of action is not specified [93] (Figure 19).     Tryptanthrin (indolo [2,1-b]quinazolin-6,12-dione), is a natural product that was obtained from a Chinese plant, Strobilanthes cusia. It has broad-spectrum biological activities including anti-tuberculosis property. Tryptanthrin demonstrated MIC of 1 μg/mL against Mtb in BACTEC assay. It showed MIC values of 0.5-1.0 μg/mL against MDR-TB strains [95]. Preclinical evaluation of tryptanthrin has been conducted [96] (Figure 24).

PA-824
PA-824 possesses an MIC in the range of 0.015 to 0.25 μg/ml and also retains activity against resistant isolates. It acts by inhibiting the synthesis of protein and cell wall lipids [102]. In a mouse model PA-824 was highly active for latent TB in combination with moxifloxacin [103]. Its minimum bactericidal dose (to reduce the lung CFU count by 99%) was found to be 100 mg/kg/day in murine studies. It is also effective against MDR strains and Mtb grown under oxygen depletion [104,105] ( Figure 28). OPC-67683 exhibited an MIC of 0.006 μg/mL [106]. In a mouse model, its efficacy was reported to be superior to existing anti-tuberculosis drugs without any evidence of cross-resistance. The mechanism of action of OPC-67683 is suggested to be similar to PA-824 [107] (Figure 29). The MIC value of TMC-207 ranges from 0.002 to 0.06 µg/mL for drug susceptible and drug resistant (INH, RMP, streptomycin, EMB, PZA and moxifloxacin) strains. It works on the proton pump of ATP synthase [108,109]. In mice, a single dose had bactericidal potency for about eight days. When used as monotherapy, a single dose of TMC-207 was as potent as the triple combination of RMP, INH, and PZA and was more active than RMP alone ( Figure 30).

Linezolid for the Treatment of MDR-Tuberculosis
Linezolid is an approved antibacterial drug (MIC 90 1-2 μg/mL) but has not been approved for TB [110]. One of the major concerns for its use as an anti-TB drug is the lack of information on its efficacy [111]. Its long-term use indicated thrombocytopenia, neuropathy and haematopoietic suppression [112] (Figure 31). Rifapentine is a derivative of rifampicin with an MIC of 0.03 μg/mL [113]. Its mode of action is similar to that of rifampicin [114]. Rifapentine can be used to treat latent TB in combination with either moxifloxacin or INH [103] (Figure 32).  Moxifloxacin, a broad-spectrum antibiotic (400 mg/day dose, MIC of 0.5 μg/mL), is active against both gram-positive and gram-negative bacteria. It displayed early bactericidal activity comparable to INH and rifampin in humans [115,116]. It binds to DNA gyrase and topisomerase IV, which are involved in bacterial replication. Moxifloxacin has no cross-resistance to other antituberculosis drug classes and has been shown to display good activity against MDR strains [117]. However, it has CNS side effects and drug interactions with other fluoroquinolones. Moxifloxacin has not been reported to be safe or effective in children younger than 18 year or in pregnant or lactating women [118]. Nuermberger et al. found that substituting moxifloxacin for INH shortens the duration of therapy for active disease much better than does substituting moxifloxacin for EMB [119] (Figure 33).

Gatifloxacin
Gatifloxacin is also a broad-spectrum antibiotic (dosage of 400 mg/day) and has the same mechanism of action as moxifloxacin. It is active against occasionally dividing Mtb, but not for dormant bacteria [120]. However, it can cause CNS toxicity and has been associated with increases in insulin levels among diabetics. Like moxifloxacin, it has also not been shown to be safe or effective in children younger than 18 years or in pregnant or lactating women.

Conclusions
Drug resistance is a critical issue in the treatment of TB. Combined and intensive efforts are required to discover new classes of anti-tuberculosis drugs, otherwise TB could become untreatable in the near future. Currently, several groups/institutions are working together to achieve this goal. These efforts should be continued and intensified to fight this ancient but re-emerging disease. To augment and bolster the development of new drugs for TB, government, private and public authorities need to enhance financial support for research at all levels, and modify regulations to ease the process of evaluation, validation and approval of new drugs. In addition, education and awareness by government, public and private agencies must contribute to preventing the spread of TB and drug resistant MDR or XDR TB.