Newer patents in antimycobacterial therapy.

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Newer patents in antimycobacterial therapy

Tuberculosis caused by Mycobacterium tuberculosis is a global health emergency. This deadly disease has far-reaching social and economic implications. Diseased individuals need prolonged polypharmacy which is not without ill effects. Treatment compliance is often compromised contributing to rising resistance. HIV co-infection has further worsened the scenario. On the other hand, no new anti-TB drug has hit the market in last 4–5 decades. After a long latency, only the last few years have witnessed growing research in this direction and a widening anti-TB drug clinical pipeline. The compounds in preclinical stage of development have also shown a heartening increase. The present review is an attempt to discuss novel promising patents in this field.

Mycobacteria occupy a significant position in the human infective flora. Tuberculosis (TB) caused by Mycobacteria tuberculosis is an airborne communicable disease which has plagued humanity since antiquity. Treatment has seen a shift from sanatorium to domiciliary care with discovery of antimycobacterial properties of the drug isoniazid in 1940s. As more anti-TB compounds were discovered, it was soon realized that combination of 2–4 anti-TB drugs for longer periods was essential to kill the heterogenous populations of bacteria and prevent resistance [1] . Though this effective and inexpensive chemotherapy led to TB decline for a while, but soon global TB resurgence was noticed. This was attributed to poor compliance (due to high pill count, prolonged duration of therapy and drug adverse effects), irrational prescriptions, substandard drugs, drug resistance and co-epidemic of HIV [1] . TB has now emerged as a global health crisis. A third of world population is infected with TB with an estimated 9 million new incident cases per year. TB claims 1.3–2 million lives annually. High TB prevalence is seen in developing countries with scarce resources. Resistant TB has reached alarming new dimensions affecting 50 million people worldwide with reported fatality of around 20–80% [1–3] .

10.4155/PPA.15.9 © 2015 Future Science Ltd

SK Shahid Shahid Clinic, 8-Jayanti, 353/21, RB Mehta Road, Ghatkopar (East), Mumbai 400 077, India Tel.: +91 986 903 6606; [email protected]

Unfortunately, new anti-TB drug discovery did not keep pace with this rise in resistant TB. The slow research into newer TB drugs was possibly due to low profit: investment ratio, and cumbersome and lengthy new drug development process. It is vital to find new treatment alternatives to control this fatal disease [4] . The present review is an attempt to look into the present status of research into antimycobacterial drugs. Microbiology & infection biology Mycobacterium tuberculosis is a rod-shaped, nonspore forming, aerobic, slow-growing acid-fast bacteria with a thick rigid waxy cell wall. The cell wall is made of complex and unique carbohydrates, lipids and proteins. Alpha, keto and methoxy mycolic acids are an important component of cell wall. Below the cell wall and over the plasma membrane is a layer of arabinogalactan and peptidoglycan. Porins are present in the cell walls which allow to and fro movements of substances. In total 4000 genes constitute the mycobacterial genome. Between 6 and 8% of these are devoted to fatty acid metabolism. The remaining encodes various enzymes and transcriptional regulators essential for bacteria’s growth and survival. These enzymes and regulators serve as mycobacterial

Pharm. Pat. Anal. (2015) 4(3), 219–238

part of

ISSN 2046-8954

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Patent Review Shahid

Key terms Porins: They are beta barrel transmembrane proteins that form a pore through which small molecule substances can diffuse. Central carbon metabolism: Metabolism wherein carbon is utilized by bacteria for its physiology by tricarboxylic acid (TCA), glycolysis, pentose phosphate shunt and gluconeogenesis. Glyoxylate shunt pathway: Pathway that metabolizes acetate or long-chain fatty acids for energy, especially seen in persistent mycobacteria. Mycobacterial persistence: Condition in which drugsusceptible bacilli persist in body in spite of chemotherapy. Multidrug resistant: This is said of bacilli resistant to isoniazid and rifampicin. Extensively drug resistant: Said of bacilli resistant to isoniazid, rifampicin and two more anti-TB drugs such as fluoroquinolones and aminoglycosides.

virulence factors and are being studied for their putative role as drug targets [5] . Protein kinases, histidine kinases, protein tyrosine phosphatases are vital mycobacterial signaling enzymes essential for survival. Mycobacteria require glycerol, citrate and asparagine for their growth. Glucose is not the primary mode of nutrition in them. Like other bacteria, central carbon metabolism is crucial for mycobacterial physiology, virulence, pathogenesis and survival [6] . They also require cholesterol for virulence. It is transported into cell by mammalian cell entry 4 (Mce4) transporter which is regulated by KstR and KstR2 genes. The cholesterol is used for energy production and as carbon source for manufacture of various metabolite intermediates. It is catabolized by oxygenases and such enzymes which degrade its alkyl side-chain via tricarboxylic acid (TCA) cycle. The steroid ring is also converted to alkene and used for energy. PropionylCoA-assimilating methylcitrate cycle (MCC) is also essential for growth on cholesterol [7] . Recent studies have demonstrated that activation of lipid peroxidation and reduction of antioxidation characterize TB infection [8] . Quorum sensing has been noticed in mycobacteria and aids in its persistence [9] . Isocitrate lyase and maltase synthase are major enzymes of glyoxylate shunt pathway which is a route of energy production during mycobacterial persistence [10] . Cyclopropane synthase plays a major role in cell wall modification, and bacterial virulence and persistence. The purine and pyrimidine salvage pathways in mycobacteria are beneficial during periods of stress and quiescent phase [5] . Mycobacteria tuberculosis is transmitted by inhaled route. Likelihood of infection is determined by infectivity of the droplet nuclei released by patient,

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proximity, frequency and duration of exposure, and susceptibility status of exposed person. Lungs are primary site of infection. Alveolar macrophages engulf these bacilli. Their receptors (toll-like receptors [TLR], nucleotide-binding oligomerization domain [NOD]-like receptors [NLRs], C-type lectins, complement, surfactant protein A, cholesterol) assist in this phagocytosis. Lipoproteins, glycolipoproteins, lipomannan, lipoarabinomannan (LAM) and mammalian cell entry (mce) proteins form the corresponding ligands [5] . Phagosomes containing the engulfed mycobacteria are formed within macrophages. Many of these ingested bacteria are destroyed by phagosome-lysosome fusion technique and by release of reactive oxygen and nitrogen intermediates, and antimicrobial peptides (AMPs). Macrophage also signals other immune cells such as dendritic cells, natural killer cells and T-lymphocytes. Inflammatory process is initiated which tries to limit the pathogen. Cytokine and various proteolytic enzymes are poured out. TNF-α, IFN-γ and interleukins are released. On the other hand, mycobacteria possess complex mechanisms which blunt host’s immune response and evade it [11] . They inhibit dendritic cells and modulate host signaling pathway. They diminish macrophage apoptosis (programmed cell death) by altering intracellular calcium levels and acting on ASK-1 (Apoptosis signal-regulating kinase-1), MAPK (mitogen-activated protein kinase), JAK/ STAT (Janus Kinase/Signal transducer and activator of transcription) pathways. Besides, they stimulate secretion of IL-10 which in turn suppresses TNF-α leading to fall in macrophage apoptosis. An optimal cytokine balance and CD4 + and CD8 + T cells are required for clearance of mycobacteria from host body [11] . Predominant cell-mediated immunity is seen in TB and the array of activated T-cells, macrophages and such immune cells lead to granuloma formation. Extensive tissue damage occurs and caseous necrosis is seen in center of this granuloma [5] . This necrosis is probably dead apoptosised macrophages. There is hypoxia, low pH and fewer nutrients in center of this granuloma. In this famished scenario, the mycobacteria enter a dormant phase of low metabolic rate with no colony formation. Latent or quiescent TBis said to have been established (Figure 1) . Some bacteria manage to survive, multiply and get released when the macrophage disintegrates. The organisms which escape the host’s local immunity undergo lymphatic and/or circulatory dissemination into bones, joints, liver, spleen, brain and other body organs. Proteolytic enzymes such as matrix metalloproteinases (MMP) degrade the matrix components and aid TB spread [12] .

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Around 5–10% of latent cases will progress to TB disease at some point in their lives. Extremes of age, malnutrition, diabetes and immunosuppressed states such as HIV positivity have high risk of TB progression. Also in infected individuals, despite chemotherapy, some organisms manage to remain and these are the phenotypic persisters (drug-susceptible which resist attack by surrounding drugs). These get reactivated with immune weakening due to age or disease. Status of present drug therapy & resistance Rapid replicators of bacteria are possibly eradicated rapidly and easily whereas persisters seem to be difficult to clear. Hence, lengthy polypharmacy is required for cure. Since the last 50 years, TB chemotherapeutic regimen has not shown much change. The regimen includes isoniazid (INH), rifampicin (RMP), pyrazinamide (PZA), ethambutol (ETH) or streptomycin for 2 months followed by 4 months of INH and RMP (Table 1) . Ninety percent (90%) drug susceptible active TB in non-HIV patients is cured with 6 months therapy with four drugs [13] . However, irrational prescribing practices, substandard drugs and nonadherence to complex regimens increase selection of drug-resistant strains of TB

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bacilli. This along with rise in HIV co-infection has undermined the efforts of chemotherapy. Increasing percentage of mycobacterial isolates are now resistant to INH and RMP (multidrug resistant TB, MDR-TB). Isolates with additional resistance to fluoroquinolones and second-line TB drugs are also being noted (extensively drug-resistant TB, XDR-TB). Besides, there are phenotypic persisters who evade the killing effects of chemotherapeutic drugs [14] . These resistant and persistent strains are difficult and expensive to treat. It seems a Herculean task to manage such highly fatal infections. Twenty or more months of treatment with less effective, more toxic and costlier drugs manages to cure only 60–75% of patients and this proportion is even lower in HIV-coinfected patients. Besides, HIV patients have added problem of drug interactions and more severe and unique adversities. It is imperative to have safer and cheaper alternatives with novel mechanism of action and eradication capability which could be effective even as monotherapy or dual therapy. Drugs with less frequent dosing and capability to shorten TB therapy duration would be acceptable. The new drug should also be active against resistant TB pathogens, especially in HIV patients and also in

TB bacilli with ligands

TLR2, TLR4, NOD-1, surfactant, complement, cholesterol receptors

Lungs

Alveolar macrophages

Inhalation Infective droplet nuclei

Signal

Dendritic cells and T- lymphocytes

Infected macrophages

NK cells, Th1 lymphocytes recruitment and activation

NF-κβ, TNF-α, IFN-γ, IL-1, IL-2, chemokines

Bacterial proliferation

ROI, RNI Phagolysosomes and bacterial kill

Immune cells fibroblasts Healing Granuloma formation

Latent infection Clearance of infection Progressive TB

Localized TB Disseminated TB

Figure 1. Pathogenesis of tuberculosis.


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Table 1. Tuberculosis chemotherapeutic drugs. Drug

Mechanism of action

Remarks

Isoniazid (INH)

Acts on cell wall

Effective against rapidly multiplying bacteria

Rifmapicin (RMP)

Inhibitsβ subunit of RNA polymerase and nucleic acid synthesis

Acts of bacteria with metabolic spurts

Pyrazinamide (PZA)

Depletes membrane energy

Acts in acidic media, bacteria with low metabolic rate are also killed

Ethambutol (ETH)

Acts on cell wall arabinogalactan via arabinosyltransferase

On rapidly proliferating bacteria, decreases INH resistance

Streptomycin

Decreases protein synthesis

Injectable, nephro- and ototoxicity

Kanamycin



Capreomycin (polypeptide antibiotic)



Flouroquinolones

Decreases DNA synthesis via DNA Resistance rising gyrase and/or topoisomerase IV inhibition

Ethionamide

Inhibits mycolic acid synthesis

Oral, thyroid hormone inhibition

Para-amino-salicylic acid

Folate synthesis inhibition

GI side effects, hepatitis, to be avoided in patients with G6PD deficiency, goitre

Cycloserine

Inhibits cell wall peptidoglycan biosynthesis via action on d -alanine racemase and ligase

–

First-line drugs

Second-line drugs

INH: Isoniazid.

latent TB. It should not only kill the rapidly multiplying bacteria but also sterilize the lesion by eradicating persisters. It would require rigorous research to find such a multifaceted drug. However, lack of sufficient investment return, improper study-animal models and long time needed for drug development has led to slow pace of research in this direction. Burden of TB is ironically high in the third world countries which lack adequate infrastructure, human and financial resources for research. Nonetheless, better insight into bacterial pathogenetic processes and survival could aid identification of newer and more effective drug targets. Complete mycobacterial genome-sequence decoding in 1998 has assisted in shedding light on the various regulatory mechanisms and metabolic pathways and thereby exposed new drug targets [5,15] .

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Considering the global threat of TB, various public, government and private initiatives, alliances and partnerships have been established in the last decade or so to strengthen the pipeline of anti-TB drugs. Clinical trial approvals have been made faster. Newer drug delivery systems are being exploited to enhance drug bioavailability and reduce ill effects [16] . The anti-TB horizon now appears promising with a number of drugs in the clinical pipeline. Older drugs have been repurposed for TB and improved analogues of existing antimycobacterial drugs are being manufactured. Besides, newer targets for anti-TB therapy are being explored [17] . Natural products with good antimycobacterial activity are being considered for supplementary therapy [18] . Various newer patents which seem to highlight the landscape of antimycobacterial therapy are being outlined below.



Improvement in current drug regimens Since new drug development is an arduous, time-consuming and expensive affair, efforts have been made to improve on present regimens: • Improvement of compliance – Adherence to therapy is an issue and could be bettered by close monitoring for adversities and their prompt management [19] . Drug interactions need to be anticipated especially with HIV co-infection and handled accordingly. Besides, combining 2–4 anti-TB drugs into one formulation is being attempted and studied for its capability to improve compliance; • Modification of present regimen – Addition of aminoglycosides or fluoroquinolones to current regimens has been fruitful. Fifty percentage augmentation in RMP dose was found to cutback therapy duration by a third [5] . However, beyond double dose or as intermittent therapy RMP was found not to be well-tolerated [5] ; • New drug delivery techniques – Anti-TB drug formulations with newly exploited drug delivery systems could be tapped for effective tissue reach. The present anti-TB drugs could be fabricated in a way so as to be able to reach the target tissue/ cell, or need lesser frequency of dosing and hence improve patient compliance. Aerosolized form of anti-TB drugs loaded in microspheres/nanoparticles can directly deliver the active ingredient to the lung. Global Alliance on TB (GATB) is currently assessing efficacy, advantages and safety of this drug delivery system in TB management [16,20] . Controlled release formulations of TB drugs are being tried. Liposomes and biodegradable polymer-based technologies are employed for in vivo extremely targeted sustained delivery of conventional anti-TB drugs. This leads to improved compliance and clinical outcome [20] . A claim has been filed for addition of silicondioxide in nanoparticle form to aminoglycoside or capreomycin in ratio of 1: 10–70. This facilitates intracellular drug delivery [21] . Multidrug preparations of antitubercular drugs providing differentiated gastrointestinal release of INH and RMP could supposedly enhance patient compliance and hence treatment outcomes [22] ; • Addition of drug resistance revertants – Since resistance is one of the major reasons for therapy failure and spread of the disease, addition of agents that could reverse this resistance to current anti-TB regimen would be beneficial. Resistant bacteria have an active efflux pump which clears


Patent Review

the antibiotic from the bacteria. Phenothiazine or thioxanthene derivatives are efllux pump blockers and, hence, MDR-revertant. A Danish chemist has recorded a patent for a benign thioridazine derivative which is the minus form of racemic thioridiazine (JEK 47). It has same effect with lesser side effects. Thus the same treatment which failed for MDR-TB could turn effective with this new cheap drug [23] . Besides, organosilicon compounds (SILA-409 and SILA-421) also possess efflux pump inhibitory action [24] . The antihypertensive drug verapamil could double up as an efflux inhibitor and, hence, when used with conventional anti-TB drugs could help overcome resistance [25] ; • Adjunct therapy – Immunomodulators administered along with routine anti-TB drugs could enhance efficacy [15] . Various cytokines such as IL-12, IFN-γ, TNF-α and antibody against IL-10 is being researched in TB. Analogs of adenine triphosphate (ATP) have been found to potentiate macrophage antimycobacterial activity [26] . Dominique Maes has claimed that a plant alkaloid, uleine could boost immunity by stimulating macrophages to produce nitric oxide. It is safe and could be an adjunct therapy with antitubercle medications [27] . Vitamin D also has immunealtering powers possibly via Toll-like receptor signaling or by stimulation of the antimicrobial peptide, cathelicidin. GSH, glutathione or its precursor N-acetyl cysteine has been shown to elevate local nitric oxide. Adding 5-lipoxygenase inhibitor and cyclooxygenase pathway product to anti-TB regimen alters the eicosanoid balance favoring an enhanced innate immune response. This immunotherapeutic aid reduces severity, mortality and decreases TB therapy duration. This is especially useful in immunosuppressed patients and in those with MDR-TB [28] . Human innate antimicrobial peptide system may also be boosted by butyric acid, phenylbutyric acid, their salts and specific iminosugars [29,30] . Anti-TB drugs with immunomodulator in suitable nanoparticles are also being tried for better bioavailability and solubility. Key term Latent TB: It is the phase of TB wherein TB bacilli escape host immune system and enter into a dormant or ‘zombie’ phase with low metabolic rate but no growth. They tend to persist in the body for years or even lifetime of host. The individual harbors the bacilli but has no clinical signs or symptoms of disease and is not infectious. Such latent cases are at risk of reactivation and progression when host immunity is disrupted.


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Patent Review Shahid Drug rescue or resurrection of previously rejected drugs Health and research authorities in the USA and UK have embarked on proposals and projects wherein previously abandoned compounds are being re-examined and retried for TB treatment. The drugs might have been tried in other conditions and could not reach approval phase due to ineffectiveness or toxicity. Such cast off existing molecules could be re-employed for TB trials with or without dose alteration [31] . Repurposing of old drugs Certain effective drugs used for other diseases have been studied and found to be antimycobacterial. They could be repurposed as anti-TB medications. Various newer and dual acting fluoroquinolones are being tested in combination with first-line and/or newer experimental anti-TB drugs for their efficacy in management of TB. Combination of moxifloxacin (fourth generation synthetic fluoroquinolone) with PA-824 (experimental drug) and pyrazinamide improved sterilising action in mice and was found to have comparable efficacy as the traditional HRZE regimen in clinical studies [4,32] . Moxifloxacin can cut down TB treatment duration. Murine studies have shown that 2RMZ+4RM is better than 2RHZ+4RH in terms of time to culture conversion [4] . The oxazolidinone antibiotic, linezolid has been successfully tried in resistant TB. It hinders early protein synthesis and has minimal risk of resistance development [15,33] . To reduce its ill effects and drug interactions, its dose could be halved after sputum conversion without compromising on its efficacy [33] . The antifungal azole group has also been seen to possess antimycobacterial activity. The drug acts by targeting cytochrome P450 homologs in the bacteria [15] . Antimycobacterial activity has also been noted in the topical antifungal pyrrolnitrin, isolated from Pseudomonas pyrrocinia. It functions via inhibition of protein kinase III which has a key role in osmosensory signal transduction system. The antipsychotic phenothiazines have been assessed and found to have considerable in vitro anti-TB activity against susceptible and resistant bacilli [15] . They can kill intracellular bacteria and act on multiple bacterial targets (cell wall and aerobic respiration) [34] . Safer phenothiazine and thioxanthene derivatives are being developed and patented for trials in control of TB [23,35] . They also have an added advantage of preventing and reversing resistance to other antibiotics [23,34] . Cipemastat (Ro 32–3555) and Batimastat (BB-94) are collegenase and matrix metalloproteinase (MMP) inhibitors used as antiarthritic agents. In TB, their action favors granuloma formation, and hence disease localisation. They have been repurposed as anti-TB drug candidate [36] .

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Columbia University has patented these and claim that these drugs could lessen pulmonary tissue destruction in TB. The MMP inhibitors could also take the form of an antibody, a fusion protein with soluble portion of MMP receptor, or an RNA molecule. These could prevent binding of a natural ligand to MMP [36] . The alcohol withdrawal assisting drug, Disulfiram has been found to inhibit mycobacterial growth by unknown mechanism. It is effective against intracellular as well as resistant mycobacteria [37] . Cornell University has filed a patent for use of proteasome inhibitors in TB. Proteasome is an enzyme complex responsible for damaged and unwanted protein degradation. This complex in mycobacteria could be targeted selectively by oxathiozol-2-one. Hence, used with standard therapy, they could be beneficial in eradication of persisters [38] . A nanostructured ionic complex of carbohydrates, proteins and/or polypeptides, salts, metals and intercalated with iodine has been found to have good anti-TB activity. The unique fabrication penetrates cells easily and is also immunogenic stimulating monocytes, macrophages and cytotoxic T-lymphocytes [39] . Pleuromutilin derivatives, Tiamulin and Valnemulin have been found to possess good antimycobacterial properties [40] . Re-engineering of existing antitubercular drugs Analogues of current anti-TB drugs could be manufactured to increase efficacy, safety and overcome shortcomings of parent compounds. Longer acting, more effective and safer derivatives of rifamycins have been synthesized which are in advanced clinical phase of study. These include rifapentine, rifabutin and rifalazil (KRM-1618). [5,13,15] . They have been studied in combination with moxifloxacin or existing anti-TB drugs with good outcomes. Rifapentine combination with isoniazid for latent TB is being developed by Sanofi. The less water-soluble rifalazil is difficult to combine in one composition with other anti-TB drugs. Hence, adding a surfactant and a lipophilic antioxidant is being trialed to make it possible for unit dose preparation of rifalazil with other anti-TB medications [41] . Activbiotics Pharma Llc. has patented rifalazil for pulmonary administration which tends to lessen the required quantity and systemic toxicity [42] . Modification of INH by linkages with different anilines via carbonyl group was found to enhance its antimycobacterial activity. Longer alkyl substituents fared better due to the favored affinity and interaction with enoyl reductase (InhA) [43] . Preclinical trials of pentacyano(isoniazid)ferrate (II) compound (IQG-607) has shown that it is effective, safe, orally active with intracellular killing power [44] . SQ109 (a diamine related to ETH) acts on MmpL3 (essential membrane mycolic acid transporter) and



Patent Review

inhibits cell wall synthesis [5,45] . It has synergistic action with INH, RMP and newer experimental drugs such as TMC-207. It is bactericidal against susceptible, resistant and intracellular pathogens [5] . It has advantage of less frequent dosing and faster clearance. It can shorten therapy time by 30% [14] . SQ73 is another potent ethylene diamine developed by Sequella. Some PZA analogues have been shown to act on replicating and persistent TB bacilli [46] . Studies on various riminophenazine analogs are being carried out in Beijing, China. TBI166, a clofazimine with C2-pyridylamino substitution has superior safety profile compared with the parent compound clofazimine. Other analogues with heteroaromatic substitution also have been found to be effective and safe [47,48] . Tetramethylpiperidine-substituted phenazines B4169 and B4128 (TMP phenazines) are more potent against tubercle bacilli. Hybrids of phenazines with phthalimido and naphthalimido moieties have also shown some promise in this field [49] .

for resistant TB. Preclinical studies of this improved second-generation version of PA-824 have been fruitful. Its favorable pharmacokinetics and good oral bioavailability makes it a great candidate for new antiTB drug. Diarylquinolines belonging to quinoline group have also been found to possess antimycobacterial potency. The most promising drug in this group is TMC207 [R207910, compound J, bedaquiline, Sirturo]. It is 1-(6-Bromo-2-methoxy-quinolin-3-yl)4-dimethylamino-2-naphthalen-1-yl-1-phenyl-butan2-ol) with molecular formula C32H31BrN2O2 [52] . Since 2012, it is available outside clinical trials and has been approved for specific indications. TMC207 acts on proton pump of mycobacterial ATP synthase enzyme [15] . It is effective with first-line drugs in susceptible, resistant and latent TB. Chances of cross resistance and drug interactions are negligible. It could be used safely in once weekly dosing [5] , though its activity may be delayed to 4–7 days after dosing [14] . Various pyrrole derivatives such as LL3858 (Sudoterb) New antimycobacterial molecules and BM212 are being studied by Lupin for efficacy in Countless molecules have been screened by newer tech- combination with standard first-line drugs. BM212 niques, identified and tested against tubercle bacilli. possibly targets MmpL3. It is effective also against Many have passed the in vitro and preclinical phase resistant and intracellular strains [5,53,54] . An improved but only some have been able to prove themselves in BM212 analogue is underway. Linezolid, an oxaclinical trials (Table 2) . zolidinone is being tested for treatment of extensively drug-resistant TB in Korea [33] . Toxicity concerns have Promising molecules in clinical study phase prompted further research into this group and now its PA-824 [pretomanid, {(6S)-2-nitro-6-([4-{trifluoro analogues are being synthesised and trialed. U-10040 methoxy)benzyl}oxy}- 6, 7-dihydro-5H-imidazo[2,1- (sutezolid) is found to be effective but its clinical trial b][1,3]oxazine}, C14H12F3N3O5] and OPC-67683 has been thwarted due to its tardy pace. A nonoxa[delamanid,[(2R )-2-Methyl-6-nitro-2-[(4-{4-[4- zolidinone analogue of sutezolid with good antituber(trifluoromethoxy)phenoxy]-1-piperidinyl}phenoxy) cular activity was obtained by substituting acetamimethyl]-2,3-dihydroimidazo[2,1-b][1,3]oxazole, domethyloxazolidinone with a biphenylmethyl group. C25H25F3N4O6] are nitroimidazopyrans which are AZD5847 (posizolid, previously called as AZD2563 currently in advanced stage clinical trials as anti-TB with molecular formula C21H21F2N3O7) is modified drugs [13] . They act on cell wall of drug susceptible as analogue of linezolid developed by AstraZeneca. It has well as resistant bacteria [50,51] . Mycobacterial respira- generated immense interest and is in clinical trials in tory poisoning is also noticed with PA-824. Risk of South Africa [13,55] . It is more effective and has action cross resistance and drug interactions is minimal. They against intracellular bacilli as well. It has an additive achieve high intracellular concentrations and hence are effect with other anti-TB drugs [55] . Tedizolid phosof use in latent TB. They hold promise to shorten dura- phate (TR-701) is a next generation oxazolidinone with tion of TB regimens to 4 months [5] . Low-dose aero- superior efficacy, pharmacokinetics and safety profile zolized PA-824 has been tried in animals with good compared with linezolid. It acts on intracellular and outcomes [14] . PA-824 improves eradication and suc- resistant mycobacteria [56,57] . Radezolid (RX-1741), an cess rate of conventional therapy. The ambitious Phase orally active investigational biaryl oxazolidinone from II trial of NC-002 (New combination 2) of TB Alliance was launched in 2012 and consists of investigation Key term of combination of PA-824, moxifloxacin and pyrazinQuinolines: Also known as benzopyridine, these are amide in drug-sensitive and resistant TB patients [32] . heterocyclic aromatic organic compounds with molecular Besides, NC-003 trial consists of efficacy and safety formula C9H7N. They consist of a benzene and a pyridine of PA-824 with bedaquiline (a diarylquinoline), cloring which are fused at two adjacent carbon atoms. They are the parent compound base for various alkaloids and fazimine and PZA in TB. New Zealand scientists have antimicrobial agents. developed a new molecule of nitroimidazole (TBA-354)



225

226


Pyrrole derivative, BM212 Oxazolidinone, AZD5847 Oxazolidinones, tedizolid, radezolid Oxazolidinone Ranbezolid Other substituted oxazolidinones Perchlozone

US7691837

US2035219

US0102523

WO6043121

US7691889

US0052265

Caprazene nucleoside, CPZEN-45 Capuramycin analogue, SQ-641 8-methoxy fluoroquinolone with 3-aminopyrrolidyl substitution Isothiazoloquinolone, ACH-702 Spectinamides Cyclic peptides from Nonomuraea sp. Anthraquinones from marine fungus, Nigrospora sp.

US0005371

US0281054

US8476429

US0114601

US0249155

WO2144790

CN102603525

Substituted 1,2,4 triazoles Substituted nitazoxanide Substituted 4-arylthiazole and other derivatives 4-quinoline methanol derivative

US8865910

US0317070

WO2164572

WO2068560

Derivatives of other chemotherapeutic drugs

Pyridomycin derivative

WO4040709

Antibiotic derivatives

Molecules in preclinical study phase

Diarylquinoline, TMC207

US7498343

Promising molecules in clinical study phase

Compound

Table 2. New compounds against Tubercolosis bacilli.

Patent

Jenrin Discovery, Wilmington, DE, USA

Indian Council of Scientific and Industrial Research, India

University of Virginia Patent Foundation, USA

Council of Scientific and Industrial Research, India

Ocean university of China, China

B&C Biopharm, South Korea

University of Tennessee Research Foundation, USA

Achillion Pharmaceuticals Inc., New Haven, Connecticut, USA

Daiichi Sankyo Company Limited, Japan

Sequella Inc., Rockville, USA

Meiji Seika Kaisha Ltd, Zaidan Hojin Biseibutsu Kagaku Kenkyu Kai, Japan

ETH Zurich, Switzerland

Joint Stock Company ‘Pharmasyntez’, Russia

Lupin Limited, India

Ranbaxy Lab Ltd, India

Trius Therapeutics Inc., California, USA

Astrazeneca Ab, Sweden

Lupin Limited, India

Janssen Pharmaceutica N.V., Raritan, NJ, USA

Assignee

2012

2012

2013

2012

2012

2012

2014

2012

2013

2009

2014

2014

2013

2010

2006

2013

2012

2010

2009

Year

[75]

[74]

[73]

[72]

[71]

[70]

[69]

[66]

[65]

[63]

[62]

[61]

[60]

[59]

[58]

[57]

[55]

[54]

[52]

Ref.




proline aminoacids containing quinazolinones Ruthenium complexes Iminosugar derivative 3-aminopyrazole derivative Thiazolamines Thiadiazolamines Xanthone analogue Substituted chromenone derivative Chloropyrimidine Nitrobenzothiazole derivative benzothiazoninethione derivatives Serine protease antagonist, alpha-1antitrypsin Serine hydrolase inhibitors C1pP1P2 protease inhibitors

IP254676

WO1137503

WO4143999

WO4017915

US0288133

US0203802

US0095064

WO3036207

WO3049567

WO1019405

US0245008

WO2162912

EP2520654

US0190234

US0243255

4-quinolylhydrazones Bifunctional molecules of rifamycin+quinolone/oxazolidinone Multifunctional molecule

WO1047814

US0143373

WO3148174

Bi/multifunctional molecules

US0099614

Cationic α-helical antimicrobial peptides

2-substituted benzimidazole

US8232410

Antimicrobial peptides

Didiperidine derivative, SQ609

Compound

US8198303

Other novel molecules

Patent

Table 2. New compounds against Tubercolosis bacilli (cont.).


University of Georgia Research Foundation Inc., USA

Cumbre Pharmaceuticals Inc., Dallas, TX

Forschungzentrum Borstel, Christian-Albrechts-Universitat Zu Kiel, Germany

Hodges RS, Ziqing J, USA

President and Fellows of Harvard College, USA

Cornell University, USA

The Regents of the University of Colorado, USA

Sichuan University, China

Sanofi, Paris, France

Vertex Pharmaceuticals Incorporated, USA

The Broad Institute Inc. and Massachusetts General Hospital, USA

Agency for Science, Technology and Research, Singapore Health Services Pte Ltd, Singapore

Glaxo Group Limited, UK

Glaxo Group Limited, UK

GlaxoSmithKline, Brentford, GB

Universiteit Utrecht Holding B.V., Stichting Voor De Technische Wetenschappen, Netherlands

Unither Virology, Llc., USA

Universidade Estadual Paulista Julio De Mesquita FilhoUNESP, Sao Paulo, Brazil

Meyyanathan SN, India

The Research Foundation of State University of New York, USA

Sequella Inc., Rockville, USA

Assignee

2013

2009

2011

2010

2014

2011

2012

2012

2013

2011

2013

2013

2012

2013

2014

2014

2014

2011

2012

2012

2012

Year

[101]

[100]

[99]

[97]

[96]

[95]

[94]

[91]

[90]

[88]

[87]

[86]

[85]

[84]

[83]

[82]

[81]

[80]

[79]

[78]

[76]

Ref.


Patent Review

227

Patent Review Shahid Rib-X Pharmaceuticals has completed some Phase II trials. They do not suffer from ribosome-based linezolid resistance [57] . Ranbezolid (RBx-7644), a novel oxazolidinone from Ranbaxy competitively inhibits monoamine oxidase-A (MAO-A) and kills resistant mycobacteria and those within macrophages [58] . Lupin is also investigating into antimycobacterial properties of specific substituted oxazolidinones [59] . 4-thioureido-iminomethylpyridinium perchlorate/perchlozone has selective antimycobacterial activity against susceptible and resistant strains. It shows synergism with firstline drugs, is stable and is well-tolerated [60] . It has been approved in Russia for MDR-TB in 2012 at a dose of 9.5–12.5 mg/kg for 6 months. It accelerates sputum conversion in MDR-TB and minimises tissue damage. It could replace fluoroquinolones as second-line for MDR-TB. However it has come under scanner since the trial data has not been rigorously peer-reviewed. Molecules in preclinical study phase

• Antibiotic derivatives – Various old antibiotics have been re-engineered to form effective and safe antimycobacterial drugs. The old bacteria-derived antibiotic pyridomycin (C27H32N4O8) paralyses an important step in cell wall synthesis. However, its drawback is its instability. On the basis of this antibiotic, Altmann and his colleagues at ETH Zurich were successful in developing a basic molecule structure which could form the foundation of several new anti-TB drugs. The lead compound is stable and easily manufactured. These would be especially useful for resistant TB [61] . A patent has been filed to that effect recently [61] . Caprazamycin-B (CPZ-B), a liponucleoside antibiotic derived from Streptomyces sp. MK730–62F2 has specificity against mycobacterial species. Its derivative CPZEN-45 or Caprazene nucleoside is under preclinical trial in Japan [62] . It acts on first step of cell wall synthesis and decreases lipid I formation via translocase 1 inhibition. Animal studies have revealed dose dependent efficacy and safety in lung TB [15] . Capuramycin is another naturally occurring nucleoside-based compound from Streptomyces Griseus. A preclinical study is in progress with a semisynthetic capuramycin analogue (SQ-641) which has poor intracellular penetration but nonetheless possesses substantial postantibiotic effect. In view of its low aqueous solubility, it is incorporated in TPGS-micelles (α-tocopheryl polyethylene glycol succinate) or administered as a nanoemulsion formulation. It too targets translocase I (TL-I or MurX/MraY) hampering cell wall synthesis [63] . Lesser potent capuramycin analogues include SQ997 and SQ922. Another capuramycin ana-

228


logue, UT-01320 inhibiting RNA polymerase was found to be effective against dormant mycobacteria and showed synergism with SQ-641 [64] . Quinolones with varied substitutions are being developed and tested against TB bacilli. DC-159a is a new 8-methoxy fluoroquinolone with 3-aminopyrrolidyl substitution. It acts on both DNA gyrase as well as Topoisomerase IV enzymes. This dual targeting is more balanced than that of moxifloxacin or gatifloxacin and makes it more potent with less chances of resistance. It is capable of shortening therapy duration and has superior pharmacokinetic profile compared with previous quinolones [65] . Achillon Pharmaceuticals is working at preclinical stage on a new subclass of quinolones, isothiazoloquinolone such as ACH-702 for their role on TB bacilli. They have been found to eradicate intracellular bacteria effectively [56,66] . 3, 9-disubstituted-6-oxo-6, 9-dihydro3H- [1–3] -triazolo [4, 5—h, g] quinolonecarboxylic acids and their esters have potent and selective antimycobacterial activity. They are not cytotoxic and are active also against resistant strains [67] . TBK-613 and TBK-544, another set of promising quinolone analogues, are under evaluation in Korea [5,68] . Toluidine derivatives and salts also act selectively against mycobacteria. Their cheapness and easy to manufacture are added qualities [53] . Spectinomycin per se has no antimycobacterial action. Its amide analogues, however, were found to safely decrease mycobacterial load and improve survival in murine models. They inhibit ribosomes and overcome native drug efflux. Hence, they can are effective against resistant mycobacteria [69] . Novel cyclic peptides segregated from Nonomuraea sp. MJM5123 can act on replicating/ nonreplicating sensitive and resistant bacteria [70] . 4-Deoxybostrycin is an anthraquinone derived from Mangrove marine endophytic fungus Nigrospora sp. from the South China Sea. Nigrosporin is deoxy form of 4-deoxybostrycin. They are more potent with efficacy against resistant TB strains [71] ; • Derivatives of other chemotherapeutic drugs: The antifungal 1,2,4 triazoles have been variously substituted to develop new anti-TB drugs [72] . Adding or replacing lipophilic cores in antiprotozoal nitazoxanide has been demonstrated to enhance its killing action on replicating and dormant TB bacilli [73] . Substituted 4-aryl-thiazoles and other derivatives have been put on record as potential antiTB candidate [73,74] . These analogs inhibit pyruvate ferredoxin oxidoreductase (PFOR) enzyme. Substituted piperidine derivative of mefloquine (4-quinoline methanol) is found to possess superior efficacy and safety as antituberculer drug [75] ;



• Other novel molecules: Sequella is experimenting preclinically with an orally active small molecule containing didiperidine pharmacophore (SQ609) which inhibits mycobacterial cell wall synthesis [76] . It is selective, effective against bacilli within macrophages and safe with good aqueous solubility. Q203 is an imidazopyridine amide which affects respiratory cytochrome bc1 complex in Mycobacterium tuberculosis. It is orally active, has immunostimulating properties and presently is in advanced preclinical development phase for resistant TB [77] . Novel 2-substituted benzimidazole derivatives have been synthesized and are being tested against TB bacilli. Halogen substituted derivatives seem to possess better efficacy [78] . The alkyl-mercaptan containing benzimidazole/benzothiazole compounds also have good antitubercular action with low toxicity [53] . Quinazolinones containing proline aminoacids instead of amino compounds are being patented for synthesis and use as antimycobacterial drug [79] . They are claimed to be superior to INH. Ruthenium (II)-centered coordination complexes such as with phosphine/diimine/picolinate possess high, selective and safe antitubercular activity [80] . Research is ongoing on an interesting class of compounds called iminosugars (iminosaccharides) wherin O in sugars is replaced by N. These compounds are stable containing a carbohydrate moiety and enter cells by targeting the carbohydrate receptors. Their unique mechanism of action kills resistant and dormant TB bacilli. These novel chemicals and their derivatives are early stage TB drug candidates under development [81,82] . InhAinhibiting 3-aminopyrazole derivatives have been patented by GlaxoSmithKline for TB [83] . Unlike INH which needs prior activation to inhibit InhA, derivatives of 3-aminopyrazole, thiazolamines and thiadiazolamines can directly act on InhA [84,85] ; • Other molecules which have been proven to demonstrate good antimycobacterial activity include derivatives of fluorobenzyl, alditol, 1, 3-thiazine, α,β-unsaturated acyclic sugar ketones, some 1,2,4,5-tetraoxacycloalkanes, benzoxazine-diones, benzothiophenes and benzoquinoxalines [53] . Small molecules such as xanthone analogues and quinoxaline 1-oxides possess good antimycobacterial capability, favorable pharmacokinetics and ease of synthesis [53,86] . In most of these, the mechanism of action is unknown. Arabinofuranosyl transferaseinhibiting C-phosphonate analogs are also being studied in TB. Substituted chromenone derivatives in TB are patented by The Broad Institute Inc. and Massachusetts General Hospital [87] . Excellent and


Patent Review

specific antimycobacterial activity has also been noticed in some chloropyrimidine derivatives [88] . They possibly inhibit protein kinase. They are also easily manufactured and safe for human use. Substituted indigoids also perform via inhibition of protein kinase [89] . Nitrobenzothiazole and benzothiazoninethione derivatives possess antitubercular activity and are being tried in combination with other anti-TB drugs [90,91] . Lilienkampf et al. are studying antitubercular activity of derivatives of 3-isoxazolecarboxylic acid esters [92] . Nitrofuranylamides is another group of antimycobacterials with similar potency as ETH. They probably inhibit mycobacterial UDP-Gal mutase and another unknown cellular target [49] . Pentacyclo-undecane derived cyclic tetra-amines have been studied and were found to have good potency against mycobacteria [93] . Protease inhibitors are also being variously studied for their role in mycobacterial inhibition. In vitro studies of alpha-1-antitrypsin, a serine protease antagonist have shown that it blocks internalization of TB bacilli into macrophages [94] . Also serine hydrolase inhibitors caused disruption of intrabacterial pH homeostasis, thereby killing the bacteria [95] . President and Fellows of Harvard College have claimed that C1pP1P2 protease is essential for mycobacterial growth and virulence. Hence inhibiting this could assist in bacillary control [96] . All the above molecules have contributed to a robust preclinical anti-TB drug pipeline; • Antimicrobial peptides – Synthetic cationic α-helical antimicrobial peptides (AMPs) derivatives were investigated for possible antitubercular activity [97] . Adding methionine residues was found to improve pharmacokinetics and antimycobacterial efficacy against susceptible as well as resistant TB strains [98] . The AMP, M(LLKK)2M lysed mycobacterial cell wall with no resistance development even with multiple exposures. Synergism with rifampicin was observed [98] ; • Bi/multifunctional molecules – Bimolecular drugs are also being trialed against tubercle bacilli. 4-quinolylhydrazones, the structural hybrids of isoniazid and quinolones, possess potent antitubercular activity against resistant strains but their high clogP and hence poor permeability has limited further clinical studies [53,99] . Bifunctional molecules containing rifamycin with quinolone or oxazolidinone moieties has been patented claiming efficacy and safety in TB. They synergise with less risk of resistance [100] . University of Georgia Research Foundation Inc. have designed multifunctional molecules


229

Patent Review Shahid containing following structural components: dibenzylpyridinone basic body, a piperazine carboxamide unit with aromatic or substituted aromatic group on second piperazine nitrogen and a diketo-enolic functionality. They act on multidrug resistant TB bacilli, have low toxicity, lesser likelihood of metabolic degradation, favorable hydrophobicity and drug–drug interaction profile [101] . Emerging new targets In view of rising resistance, it is necessary to discover new drug targets which could help eradicate resistant, dormant and persisting TB bacilli. With elucidation and deeper understanding of mycobacterial metabolism and means of survival and virulence, several newer targets have unfolded (Figure 2) . Attempts to tackle

these processes and pathways have led to discovery of specific molecules to counteract them (Table 3) . • Bacterial targets – Various bacterial targets have been newly found. Their inhibitors could restrain mycobacterial proliferation and endanger their survival. These targets include various vital enzymes, genes controlling these enzymes and other proteins, and mycobacterial virulence factors. Glycolytic or Embden-Meyerhof pathway, electron transport chains and fermentation pathways hold promise as targets for anti-TB drugs. Sigma factors which regulate the mycobacterial regulators during periods of environmental stress could be inhibited in order to exterminate the persisters [102] . PhoPR is a two component virulence regulation system found in myco-

Cell wall, arabinogalactan, peptidoglycan, transmembrane proteins

Ribosomes Cytosol

InhA, Translocase I, arabinofuranosyl transferase, β-KAS, mycoyl transferase, type I polyketide synthase, Type 1 and 2 fatty acid synthase, PptT, MmpL3, MurD ligase, MurB, D-alanine racemase, DprE1, Dxr

Peptide deformylase, acetolactate synthase ribosome 30 S and 50 S

Siderophores

Isocitrate lyase, maltase synthase, peroxidationanti-oxidation system, ATP synthase, MAO, respira tory cytochrome bc1 complex, PFOR

DNA/genes Matrix metalloproteases FtsZ, purine and pyrimidine salvage pathways, DNA gyrase/Topoisomerase IV, Oligopeptides, PhoPR, RNA polymerase

ADP

ATP

Secreted proteins

microRNA, protein kinase enzymes, tyrosine phosphatase

IL-1, IL-12, IFN-γ, TNF-α T-lymphocytes p38, ERK-1/2 Host signalling pathway, kinase/phosphatase system GPR109A Protein DRAM-1 miRNA

Macrophages

Figure 2. Mycobacterial and host therapeutic drug targets.

230




Patent Review

Table 3. New bacterial and host targets and their patents. Patent

Compound

Assignee

Year

Ref.

WO2143522

THPP Tetrahydropyrazolo[1,5-a] pyrimidines

GlaxoSmithKline, UK

2012

[105]

WO4037900

Indole carboxamide

Novartis Ag, Basel, Switzerland

2014

[106]

US0286130

2-amino substituted 1, 3-benzothiazin-4-one, BTZ043

Leibniz Inst Naturstoff Forsch, Germany

2010

[109]

WO0245007

Piperazine substitution of benzothiazinone, PBTZ169

Ecole Polytechnique Federale de Lausanne (Epfl), Switzerland

2013

[110]

WO3006444

1-deoxy- d -xylulose-5-phosphate reductoisomerase (Dxr) inhibitor

George Washington University et al., USA

2013

[116]

WO2149049

Tyrosine phosphatase B inhibitors

Indiana University Research and Technology 2012 Corporation, USA

[118]

US0135568

ATP synthase inhibitor

FASgen Inc., The John Hopkins University, USA

2006

[119]

US0024611

5’-O-[N-(salicyl)sulfamoyl] adenosine, siderophore biosynthesis inhibitors

Cornell Research Foundation Inc., New York, Sloan-Kettering Institute for Cancer Research

2014

[120]

US6310058

Siderophore analogs

University of Notre Dame Du Lac,Indiana

2001

[121]

US8664255

Malate synthase inhibitors

The Texas A and M university system, Texas

2014

[122]

US0113429

FtsZ inhibitor

Lucile WE, Reynolds RC, Suling WJ, USA

2010

[123]

US0331460

FtsZ inhibitor, Chrysopaentin

University of Pittsburgh of the Commonwealth system of Higher Education, USA

2013

[124]

US7427624

Purine nucleoside Analogs

Gilead Sciences Inc., Foster City, CA

2008

[125]

US0070860

Uti Limited Partnership. Canada

2008

[126]

WO3134416

Autophagy promoters

University of Pittsburgh of the Commonwealth system of Higher Education, USA

2013

[129]

Cell wall targets

Cytoplasmic targets

Nuclear targets

bacteria and this system is vital for bacterial virulence and long-term survival in host [103] . Targeting this enzyme could substantially reduce treatment period. • Cell wall targets – The mycobacterial cell wall is a viable drug target. β-ketoacyl synthase (KAS) is a unique mycobacterial accessory fatty acid synthase which also plays an important role in cell wall biosynthesis. It is potentially inhibited by certain alkylsulfinyl amides and thiolactomycin analogues. The ease of synthesis and low cost of these small-sized molecules makes them an attractive compound for use as anti-TB drugs [49] . Mycoyl transferase in mycolic acid synthesis could be targeted by various 6, 6′-diamino-6, 6′-dideoxytrehalosebased derivatives with good outcomes [53] . Type 1 polyketide synthase, and type 1 and 2 fatty acid


synthase needed for bacterial cell-wall components are activated by 4’-phosphopantethienyl transferase (PptT). PptT transfers 4’phosphopantethiene moiety from coenzyme A, which then gets covalently attach to the synthase and activates it. This target could be utilised for development of inhibitors which could thus block cell wall biosynthesis [104] . Another much studied target is mycolic acid transporter, MmpL3. Its blockage negatively affects cell wall biosynthesis and thus nullifies mycobacterial existence. Besides SQ109 and BM212, THPP Tetrahydropyrazolo[1,5-a]pyrimidines, urea derivative, AU-1235 {1-(2-adamantyl)-3-(2,3,4-trifluorphenyl)urea}, C215 (N-(2,4-dichlorobenzyl)1-propy l-1H-b en z o [ d ] i m id a z ol-5 - a m i ne ) , indoleamide and indole carboxamide target MmpL3 [53,105–107] . MurD ligase and MurB


231

Patent Review Shahid enzyme are essential for peptidoglycan biosynthesis and could be targeted with specific inhibitors [108] . MurB enzyme is decreased by 4-thiazolidinone derivatives [53] . d-alanine required for peptidoglycan synthesis is synthesized by transformation of l-alanine by cytoplasmic enzyme, d-alanine racemase. 5-amino-furanoside derivatives can inhibit this enzyme and thus aid in inhibiting mycobacteria [53] . Decaprenylphosphoryl-d-ribofuranose2’-epimerase (DprE1) enzyme required for cell wall arabinan biosynthesis is being studied as a possible drug target. BTZ043 (C17H16F3N3O5S, UNIIG55ZH52P5], a specific 2-amino substituted 1, 3-benzothiazin-4-one and dinitrobenzamides inhibit DprE1 [109] . Piperazine substitution of benzothiazinone (2-piperazine-1-yl-4H-1, 3-benzothiazin-4-one or PBTZ169) enhances its efficacy, safety and aqueous solubility since the piperazine moiety acts as a hydrophilic agent to increase water solubility of the antimycobacterial compound [110] . Its combination with bedaquiline and PZA yielded better results than conventional TB therapy [53] . Clinical trials on this preclinical drug candidate would be initiated in 2015. Small molecule 1,4 azaindole and pyrazolopyridones cause noncovalent inhibition of DprE1 [111,112] . Nitroquinoxalines and 377790 triazole compounds also inhibit DprE1 enzyme [53] . Terpenoid metabolites are vital for cell wall integrity, physiology and pathogenesis in TB [113] . Homologues of these metabolites such as asperterpenoid could inhibit this system thereby jeopardizing survival and growth of TB bacilli [114] . Synthesis of these metabolites is catalyzed by 1-deoxy-d-xylulose-5-phosphate reductoisomerase (Dxr) via nonmevalonate/MEP pathway. Fosmidomycin and FR900098 can inhibit this enzyme but are too polar to penetrate mycobacterial cell wall [115] . Lipophilic group containing inhibitors of this enzyme can enter the bacteria and prevent conversion of 1-deoxy-d-xylulose 5 phosphate (Dxp) to 2-C-methyl-d-eryhtritol-4-phosphate (MEP) [116] . Bacterial viability thus runs into peril. Butylacetylphosphonate also inhibits this enzyme and does not require the glycerol-3-phosphate transporter (glpT) for intracellular entry. This compound displays synergism with fosmidomycin which acts on second stage of MEP pathway namely on MEP synthase [117] . Benzofurans, salicylic acid and certain indole derivatives inhibit tyrosine phosphatase B enzyme which has an important role in TB pathology [118] . This enzyme is secreted into the macrophages and hence the inhibitors do not need to cross the thick mycobacterial cell wall for their action;

232


• Cytoplasmic targets – Therapies could also be directed at the various cytosolic enzymes and pathways. Peptide deformylase, a metalloprotease enzyme, is being studied as new anti-TB drug target. Its inhibition would prevent polypeptide maturation and hence result in bacterial death. Lack of human counterpart of this enzyme makes peptide deformylase inhibitors such as BB3497 safe in humans. But these inhibitors are unable to eradicate slow growing bacteria [15] . FASgen Inc. has come out with a small molecule ATP synthase inhibitor, FAS20013. It is a β-sulfonylacetamides (3-sulfonyltridecanamide) which acts intracellularly to eradicate the latent forms which are at high risk of activation in the future. This orally active compound has good activity against resistant TB strains and can kill them in a short time [119] . The siderophores (carboxymycobactins and mycobactins) are high affinity iron chelating compounds present in TB bacteria that help them to acquire iron from the iron deficit environment of the host. 5’-O-[N-(salicyl)sulfamoyl] adenosine reportedly blocks their biosynthesis thereby halting bacterial multiplication [120] . Siderophore analogs are also being researched into as antimycobacterial drugs [121] . Isocitrate lyase inhibitors such as salicylanilide benzoates, pyrazine-2-carboxylates and nitropropionamide were tried for antimycobacterial role but research on this front was fruitless [15,53] . Malate synthase is another glyoxylate shunt enzyme and a promising target for new drug. Efforts are being concentrated on developing its inhibitors for further studies [53,122] . Certain monoamine oxidase (MAO) inhibitors such as pyradazinoindoles have good antimycobacterial activity and are being studied for their possible role as antitubercular therapies [53] . The lipid peroxidation-antioxidation imbalance of pulmonary TB could also constitute a viable target and antioxidant and membranes stabilizing interventions may be considered, especially in the relapse form of TB [8] . Aromatic amino acid synthesis pathway such as the shikimate pathway, nonaromatic amino acid synthesis pathway and various other bacterial metabolic pathways are being evaluated in detail for their putative usefulness in TB treatment [15] ; • Nuclear targets – FtsZ (Filamenting temperaturesensitive mutant Z, a bacterial tubulin polymerase homolog) inhibitors include deazapteridine compounds such as SRI-3072, which have shown usefulness for inhibiting mycobacteria [53,123,124] . They act on cellular division. Purine salvage pathway is essential for growth and survival in mycobacteria. Purine nucleoside analogs target purine nucleosidase enzyme of this pathway to produce anti-TB



actions [125,126] . The potent and easily synthesizable 9-benzylpurines and arylpurines act on intracellular TB bacilli. 9-Sulfonylated or sulfenylated 6-mercaptopurines have also been found to be potent and selective against mycobacteria [49] ; • Host therapeutic targets – Role of host cells in pathogenesis of TB could be exploited for TB therapy [127] . Host signaling pathway could be modified so as to prevent mycobacterial growth and survival. Activating MAPK pathway by external means is one possibility. Host protein DRAM1 (DNA damage-regulated autophagy modulator-1) could be set as target to be activated which would trigger the immune response against Mycobacteria tuberculosis [128,129] . TNF-α increase and macrophage autophagy has been noticed with the selective serotonin reuptake inhibitor, fluoxetine [130] . Inhibition of p38, ERK-1/2 (extracellular signal-related kinase) has been tried and p38 MAPK inhibition could be achieved by Gefitinib via Epidermal Growth Factor Receptor (EGFR) inhibition. This in turn induces autophagy and causes bacterial growth restriction. [130] . Elevated G-protein coupled receptor 109A (GPR109A) has been seen in TB infection [130] . Mepenzolate bromide (SPR-10199) can inhibit GPR109A. Its use could help in early phase of mycobacterial invasion and in TB therapy. Macrophage and animal studies in mice have suggested its usefulness in this direction [130] . The studies are still preliminary and more needs to be done before the drug could enter clinical trial phase for TB. Lung damage in TB could be minimised by use of inhibitors of specific host enzymes [36] . Various receptors, ion channel transport system, anti-inflammatory mediators, microRNA and kinase enzymes could be variably targeted to assist host-immune response. Genetic engineering There have been attempts to inactivate mycobacterial genes, or those host genes which assist mycobacteria in

Patent Review

Key term Autophagy: An adaptive response to stressful events in which unwanted or dysfunctional cellular components are degraded by means of lysosomes and recycled.

localisation and infection. Avi Biopharma has invented an oligonucleotide analog which is antisense to mycobacterial gene or rRNA or the assisting host gene. They conjugated the antisense oligonucleotide with cell penetrating arginine-rich 6–14 aminoacid long polypeptide [131] . Oligopeptides targeted against virulencemediating protein may also prove useful in treatment of TB [132] . Presently, research on this front is limited. Immunization Due to inability of pharmacotherapy alone to control TB, interest has been rekindled in building effective immunotherapeutic vaccines. Traditional BCG immunisation protects only against disseminated TB in younger children. Hence, improved vaccines are required. Oral and injected heat-killed Mycobacterium vaccae when tested with chemotherapy yielded enhanced results [133] . Newer attenuated strains of other M. tuberculosis mutants are also being investigated as immunoprotective means. Vaccine comprising of M. tuberculosis genes in recombinant M. bovis BCG holds promise for enhanced efficacy with safety [134] . Novel recombinant technology-derived novel lectin-liked secretory protein (Smtl-13) is being patented for use as vaccine as well as immunodiagnostic agent [135] . Isis Innovation has claimed that parenteral administration of first antigen or its encoding polynucleotide and pulmonary administration of second antigen or its encoding polynucleotide provides enhanced protection against mycobacteria. The two administrations may be sequential less than 4 weeks apart or simultaneous [136] . Chimeric porin polypeptides or their encoding nucleic acids could be delivered via carrier proteins or virus-like particles into host. This has been claimed to enhance immune response and hence, could serve as prophylactic or supplementary therapeutic agent [137] . Various expression or delivery vectors tend to simplify

Table 4. Herbal antimycobacterial patents. Patent

Plant

Assignee

Year

Ref.

US7321002

Allium sativum

Billings Pharmaceuticals Inc., USA

2008

[141]

US0171498

Leucas stelligera, aerial portions


2014

[143]

US0040007

Byttneria herbecea


2013

[146]

US0101688

PHY906 consisting of Scutellaria baicalensis, Glycyrrhiza uralensis, Ziziphus jujuba and Paeonia lactiflora

Yale University, USA

2013

[147]



233

Patent Review Shahid vaccine administration and improve outcomes [133] . With better insights into tubercle virulence, subunit, multivalent and DNA TB vaccines are being constructed for further protective studies. Claim has been made of use of passive immunotherapy to shorten duration of TB chemotherapy. Specific IgA1 anti-TB antibodies/expression vectors comprising of expression genes for these antibodies administered via the airways in HIV-coinfected TB patients or in those with MDR TB is being studied. Concurrent inoculation with IFN-gamma or anti-IL-4 monoclonal antibody has been said to prolong protective phase [138] . Plant chloroplasts expressing MTB ESAT-6 and M72 protein fused to cholera toxin B subunit is being patented claiming efficacy as a TB vaccine [139] . Herbal supplements Phytotherapeutics have intrigued humans since centuries and its antimicrobial use has been exploited worldwide. It is cheap, devoid of ill effects with less chances of resistance. A number of plants from Indian, African and South American subcontinent have been evaluated for their anti-TB actions. Cyclopeptide alkaloids from root of Ziziphus Mauritiana (Indian jujube or Ber or Bor) have a novel mode of action against TB bacteria [140] . This botanical extract could increase therapeutic index of routine anti-TB drugs. Allium sativum (garlic) has been known to be antimicrobial and immunomodulating [141] . It contains allicin which inhibits glutathione peroxidise and, hence, bacterial replication is curtailed. Its ajoene hampers cell wall formation. Both of these garlic compounds also interfere with quorum sensing (Table 4) . Besides, extracts of Acalypha indica, Adhatoda vasica, Allium cepa, Aloe vera and Alstonia scholaris were found to inhibit mycobacteria [68,142] . Extracts of Calpurnia aurea (root), Ocimum basilicum/sanctum or Indian Tulsi (seeds), Artemisia abyssinica (leaves), Croton macrostachyus (leaves), Eucalyptus camaldulensis (leaves), Antidesma membranaceum, Crassocephalum manii, Entada abyssinica, Croton dichogamus and Rubia cordifolia also demonstrated significant antimycobacterial activity [142] . Acetone extracts of pulverised aerial portions of Leucas stelligera were found to contain labdane diterpenes and flavones with antimycobacterial potency [143] . Similarly, methanolic extracts of leaves and branches of the Amazon forest plant Duroia macrophylla were rich with terpenes and flavonoids and had antimycobcaterial efficacy [144] . Juniperus Communis and Terminalia Avicennioides also possess antimycobacterial terpenoids [145] . Byttneria herbecea extract can inhibit glutamine synthetase and act on both growing as well as dormant bacilli [146] . Herbal composition PHY906 consisting of Scutellaria baicalensis, Glycyrrhiza uralensis, Ziziphus jujuba and Paeonia lactiflora has been patented for use in increasing therapeu-

234


tic index of chemotherapy [147] . Tryptanthrin, a novel weakly basic indoloquinazolinone alkaloid obtained from indigo plant is chemosensitising and has shown efficacy against MDR strains of M. tuberculosis [49] . Conclusion TB is a serious threat to humankind with diagnostic, therapeutic and prophylactic limitations. The recent upsurge in resistant cases necessitates novel therapies. But the clinical drug pipeline is still narrow. Last decade has witnessed increasing coordination in research on TB drug development. Obstacles and delays in drug approvals have seen some improvements. Regulations on governing drug trials in TB have been simplified. There has been an impetus in TB research and many new promising small molecules are in early stages of development and provide some ray of hope for future. Genomic and proteomic insights and advanced screening technologies have aided this development. The preclinical pipeline for TB has become robust. Use of surrogate molecular markers in TB seems to have lessened time required to know new drug response. Future perspective Future for novel TB drugs now does not appear bleak. Coming years would witness more studies with novel molecules. Commercial benefits to researching companies and longer patent protection would facilitate more research on this front. Bifunctional and multifunctional molecules with action against two or more targets are being developed which would improve compliance and lessen resistance. Studies on targeted drug delivery, bacteriophage remedy, new sources and forms of antimicrobial peptides, M1 and M2 macrophage modulation, quorum sensing inhibitors are ongoing and should bear fruit soon. Research in high burden communities and countries, in HIV patients, in resistant cases and children is being emphasized into. Attempts to better social infrastructure and patient treatment adherence need to be carried out on a war footing. All this and more, with proper application, would in the long run lead to a TB-free dawn. Financial & competing interests disclosure The author has no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.

Key term Bacteriophage: A DNA or RNA virus which infects and replicates within bacteria.



Patent Review

Executive summary Background • TB is a global disease with significant morbidity and mortality. • A major concern in management is poor compliance with the prolonged and ill-tolerated polypharmacy. • Resistance to currently used drugs is also mounting rapidly. • Co-infection with HIV has further worsened the scenario leading to increase in TB prevalence, drug resistance, side effects of medications, drug interactions and fatalities.

Improvement in present regimen • Traditional anti-TB regimen could be improved with prompt and better management of ill effects, use of more superior drugs, targeted and controlled release formulations and drug delivery devices, addition of drug resistance reversing compounds, and supplementation with immunomodulators, innate immunity boosters and such medications.

Reuse of old drug molecules • Old molecules which were disposed due to clinical inefficacy or toxicity are being reconsidered for TB with dose modifications. Also drugs useful for other diseases are being assessed for their antimycobacterial activity and repurposed as anti-TB drugs.

Drug re-engineering • Present anti-TB medications could be structurally modified to improve on activity, pharmacokinetics or safety profile. • Analogues and derivatives of antibiotics and other drugs are been screened for their antitubercle efficacy.

Novel targets • Newer targets of bacteria and host are being exposed and studied in order to shorten therapy and sterilize TB lesions. Methods to overcome latent stage of TB are also been probed into. Genetic techniques are being employed to exterminate the TB bacilli.

Vaccination • Various whole, recombinant, multivalent, subunit, DNA, peptide-based and vector-based vaccines are being constructed and tested for immunoprotection.

Herbal • Natural and plant product extracts are being tried as supplementary aids in TB.

Others • Partnerships and alliances are being formed worldwide to share information about research in this direction for human benefit. All this would aid in making our vision of having a TB-free world by 2050 a reality.

References

6

Rhee KY, de Carvalho LPS, Bryk R et al. Central carbon metabolism in Mycobacterium tuberculosis: an unexpected frontier. Trends Microbiol. 19(7), 307–314 (2011).

7

Griffin JE, Pandey AK, Gilmore SA et al. Cholesterol metabolism in Mycobacterium tuberculosis requires transcriptional and metabolic adaptations. Chem. Biol. 19(2), 218–227 (2012).

8

Butov DO, Yanovsky FG, Kuzhko MM et al. Dynamics of oxidant-antioxidant system in patients with multidrugresistant tuberculosis receiving anti-mycobacterial therapy. J. Pulm. Respir. Med. 3(5), 161 (2013).

9

Bharati BK, Chatterji D. Quorum sensing and pathogenesis: role of small signalling molecules in bacterial persistence. Curr. Sci. 105(5), 643–656 (2013).

10

Kumar R. Glyoxylate shunt: combating mycobacteria at forefront. Int. J. Integr. Biol. 7(2), 69–72 (2009).

11

Jozefowski S, Sobota A, Kwiatkowska K. How Mycobacterium tuberculosis subverts host immune responses. BioEssays 30, 943–954 (2008).

12

Elkington PT, Ugrate-Gil CA, Friedland JS. Matrix metalloproteinases in tuberculosis. Eur. Respir. J. 38(2), 456–464 (2011).

Papers of special note have been highlighted as: • of interest 1

Keshavjee S, Farmer PE. 200th anniversary article: tuberculosis, drug resistance and the history of modern medicine. N. Engl. J. Med. 367, 931–936 (2012).

2

Zumla A, Raviglione M, Hafner R, Fordham von Reyn C. Current concepts: tuberculosis. N. Engl. J. Med. 368, 745–755 (2013).

3

World Health Organization. WHO Report 2014: Global Tuberculosis Control. WHO/HTM/TB/2014.19. www.who.int/tb/publications/global_report/en/

4

van den Boogaard J, Kibiki GS, Kisanga ER, Boeree MJ, Aarnoutse RE. New drugs against tuberculosis: problems, progress, and evaluation of agents in clinical development. Antimicrob. Agents Chemother. 53(3), 849–862 (2009).

•

Detailed discussion on some new antituberculosis agents

5

Dasgupta SB, Pieters J. Striking the right balance determines TB or not TB. Front. Immunol. 5, 455–463 (2014).

•

Vivid description of the pathogenesis, virulence factors and host immunity.



235

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•

Elaborate description about novel antimycobacterial drugs and their future.

14

Tam C-M, Yew WW, Yuen K-Y. Treatment of multidrugresistant and extensively drug-resistant tuberculosis: current status and future prospects. Expert Rev. Clin. Pharmacol. 2(4), 405–421 (2009).

15

Sarkar S, Mavanur RS. An overview of tuberculosis chemotherapy-a literature review. J. Pharm. Pharm. Sci. 14(2), 148–161 (2011).

16

Kaur M, Garg T, Rath G, Goyal AK. Current nanotechnological strategies for effective delivery of bioactive drug molecules in the treatment of tuberculosis. Crit. Rev. Ther. Drug Carrier Syst. 31(1), 49–88 (2014).

17

Tomioka H. Current status and perspective on drug targets in tubercle bacilli and drug design of antituberculous agents based on structure-activity relationship. Curr. Pharm. Des. 20(27), 4305–4306 (2014).

18

19

Ganihigama DU et al. Antimycobacterial activity of natural products and synthetic agents: pyrroloquinolines and vermelhotin as anti-tubercular leads against clinical multidrug resistant isolates of Mycobacterium tuberculosis. Eur. J. Med. Chem. 89, 1–12 (2015). Lai HM, Mazlan NA, Yusoff MS, Harun SN, Wee LJ, Thambrin MF. Management of side effects and drug interactions of anti-mycobacterial in tuberculosis. WebmedCentral INFECTIOUS DISEASES 2(12), WMC002749 (2011).

29

The International Centre for Diarrhoeal Disease Research: WO2140504 (2012).

30

Summit Corp Plc.: WO8009894 (2008).

31

Hemphill TA. The NIH promotes drug repurposing and rescue. Research-Technology Management 55(5), September/ October (2012).

32

Diacon AH et al. 14-day bactericidal activity of PA-824, bedaquiline, pyrazinamide, and moxifloxacin combinations: a randomised trial. Lancet 380(9846), 986–993 (2012).

33

Lee M, Lee J, Carroll MW et al. Linezolid for treatment of chronic extensively drug resistant tuberculosis. N. Engl. J. Med. 367, 1508–1518 (2012).

34

van Ingen J. The broad-spectrum antimycobacterial activities of phenothiazines, In vitro: somewhere in all of this there may be patentable potentials. Recent Pat Antiinfect Drug Discov 6(2), 104–109 (2011).

35

Bkg Pharma Aps. US0066451 (2014).

36

The Trustees of Columbia University in the city of New York: US0199289 (2014).

37

Horita Y, Takii T, Yagi T et al. Anti-tubercular activity of Disulfiram, an anti-alcoholism drug, against multi-drug and extensively drug-resistant Mycobacterium tuberculosis isolates. Antimicrob. Agents Chemother. 56(8), 4140–4145 (2012).

38

Cornell Research Foundation Inc. US0118274 (2011).

39

Kazakhstan Enterprises Research Centre: WO2091534 (2012).

40

Nabriva Theraprutics Ag. US8088823 (2012).

41

Activbiotics Pharma Llc. WO2103119 (2012).

42

Activbiotics Pharma Llc. WO2203116 (2012).

43

Rychtarčíková Z, Krátký M, Gazvoda M et al. Nsubstituted 2-isonicotinoylhydrazinecarboxamides-new antimycobacterial active molecules. Molecules 19, 3851–3868 (2014).

44

Campos MM. Preclinical evaluation of novel antituberculosis molecules. BMC Proc. 8(Suppl 4), O17 (2014).

45

Sequella Inc. US US7884097 (2011).

46

Shirude PS, Madhavapeddi P, Tucker JA et al. Aminopyrazinamides: novel and specific GyrB inhibitors that kill replicating and nonreplicating Mycobacterium Tuberculosis. ACS Chem. Biol. 8(3), 519–523 (2013).

20

Vora C, Patadia R, Mittal K, Mashru R. Recent patents and advances on anti-tuberculosis drug delivery and formulations. Recent Pat. Drug Deliv. Formul. 20137(2), 138–149 (2013).

•

Detailed account about newer drug delivery technologies and their application to tuberculosis.

21

Limonov VL. WO2154077 (2012).

22

du Toit LC, Danckwerts MP, Pillay V, Cooppan S, Choonara YE. US0179170 (2010).

23

Noa Sic Aps: US8623864 (2014).

24

Simons SO, Kristiansen JE, Hajos G et al. Activity of the efflux pump inhibitor SILA 421 against drug-resistant tuberculosis. Int. J. Antimicrob. Agents 41(5), 488–489 (2013).

47

Adams KN, Szumowski JD, Ramakrishnan L. Verapamil and its metabolite norverapamil inhibit macrophage-induced, bacterial efflux-pump-mediated tolerance to multiple antitubercular drugs. J. Infect. Dis. 210(3), 456–466 (2014).

Lu Y, Zheng M, Wang B et al. Clofazimine analogs with efficacy against experimental tuberculosis and reduced potential for accumulation Antimicrob. Agents Chemother. 55(11), 5185–5193 (2011).

48

Institute Materia Medica/Beijing Tuberculosis and Thoracic Tumor Research Institute. US0243327 (2014).

49

Kamal A, Azeeza S, Malik MS, Shaik AA, Rao MV. Efforts towards the development of new antitubercular agents: potential for thiolactomycin based compounds. J. Pharm. Pharm. Sci. 11(2), 56s–80s (2008).

•

Discussion about current and newer anti-TB drugs and detailed narration on thiolactomycin and its derivatives.

50

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25

26

236

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Placido R, Auricchio G, Falzoni S et al. P2X(7) purinergic receptors and extracellular ATP mediated apoptosis of human monocytes/macrophages infected with Mycobacterium tuberculosis reducing the intracellular bacterial viability. Cell. Immunol. 244(1), 10–18 (2006).

27

Parabolic Biologicals SPRL: WO1160684 (2011).

28

US Department of Health and Human Services: WO3019926 (2013).




51

Rawat B, Rawat DS. Antituberculosis drug research: a critical overview. Med. Res. Rev. 33, 693–764 (2013).

78

The Research Foundation of State University of New York. US8232410 (2012).

52

Janssen Pharmaceutica N.V. US7498343 (2009).

79

Meyyanathan SN. IP254676 (2012).

53

Lechartier B, Rybniker J, Zumla A, Cole ST. Tuberculosis drug discovery in the post-post genomic era. EMBO Mol. Med. 6(2): 158–168 (2014).

80

Universidade Estadual Paulista Julio De Mesquita FilhoUNESP. WO1137503 (2011).

81

Unither Virology, Llc. WO4143999 (2014).

•

Special notes on new antimycobacterial molecules and drug targets.

82

Universiteit Utrecht Holding B.V., Stichting Voor De Technische Wetenschappen. WO4017915 (2014).

54

Lupin Limited: US7691837 (2010).

83

GlaxoSmithKline. US0288133 (2014).

55

Astrazeneca Ab: US2035219 (2012).

84

Glaxo Group Limited. US0203802 (2013).

56

Molina-Torres CA, Barba-Marines A, Valles-Guerra O et al. Intracellular activity of tedizolid phosphate and ACH-702 versus Mycobacterium tuberculosis infected macrophages. Annals of Clinical Mircobiology and Antimicrobials 13(1), 13–17 (2014).

85

Glaxo Group Limited. US0095064 (2012).

86

Agency for Science, Technology and Research, Singapore Health Services Pte Ltd.: WO3036207 (2013).

87

The Broad Institute Inc. and Massachusetts General Hospital. WO3049567 (2013).

88

Vertex Pharmaceuticals Incorporated. WO1019405 (2011).

89

Klein LL, Petukhova V, Wan B et al. A novel indigoid antituberculosis agent. Bioorg. Med. Chem. Lett. 24(1), 268–270 (2014).

57

Trius Therapeutics Inc. US0102523 (2013).

58

Ranbaxy Lab Ltd. WO6043121 (2006).

59

Lupin Limited. US7691889 (2010).

60

Joint Stock Company ‘Pharmasyntez’. US0052265 (2013).

61

ETH Zurich. WO4040709 (2014).

90

Sanofi US0245008 (2013).

62

Meiji Seika Kaisha Ltd, Zaidan Hojin Biseibutsu Kagaku Kenkyu Kai. US0005371 (2014).

91

Sichuan University. WO2162912 (2012).

92

Lilienkampf A, Pieroni M, Franzblau SG, Bishai WR, Kozikowski AP. Derivatives of 3-isoxazolecarboxylic acid esters: a potent and selective compound class against replicating and nonreplicating Mycobacterium tuberculosis. Curr. Top. Med. Chem. 12, 729–734 (2012).

93

Onajole OK, Govender K, Govender P et al. Pentacycloundecane derived cyclic tetra-amines: synthesis and evaluation as potent anti-tuberculosis agents. Eur. J. Med. Chem. 44(11), 4297–4305 (2009).

94

The Regents of the University of Colorado. EP2520654 (2012).

95

Cornell University. US0190234 (2011).

96

President and Fellows of Harvard College. US0243255 (2014).

97

Hodges RS, Ziqing J. US0099614 (2010).

98

Khara JS, Wang Y, Ke XY et al. Anti-mycobacterial activities of synthetic cationic α-helical peptides and their synergism with rifampicin. Biomaterials 35(6), 2032–2038 (2014).

99

Forchungzentrum Borstel, Christian-Albrechts-Universitat Zu Kiel. WO1047814 (2011).

63

Sequella Inc. US0281054 (2009).

64

Siricilla S, Mitachi K, Wan B, Franzblau SG, Kurosu M. Discovery of a capuramycin analog that kills nonreplicating Mycobacterium tuberculosis and its synergistic effects with translocase I inhibitors. J. Antibiot. (Tokyo) doi:10.1038/ja.2014.133 (2014) (Epub ahead of print).

65

Daiichi Sankyo Company Limited US8476429 (2013).

66

Achillon Pharmaceuticals Inc. US0114601 (2012).

67

Briguglio I, Piras S, Corona P, Antonietta Pirisi M, Jabes D, Carta A. SAR and anti-mycobacterial activity of quinolones and triazoloquinolones: an update. Anti-Infect. Agents 11(1), 75–89 (2013).

68

Garg HK, Shrivastava A. Evaluation and impact of anti-tuberculosis drug: a review. Int. J. Sci. Res. 2(7), doi:10.15373/22778179/July2013/8 (2013).

69

University of Tennessee Research Foundation. US0249155 (2014).

70

B & C Biopharm.: WO2144790 (2012).

71

Ocean university of China. CN102603525 (2012).

100 Cumbre Pharmaceuticals Inc. US0143373 (2009).

72

Council of Scientific and Industrial Research. US8865910 (2012).

101 University of Georgia Research Foundation Inc. WO3148174

73

University of Virginia Patent Foundation. US0317070 (2013).

102 Sachdeva P, Misra R, Tyagi AK, Singh Y. The sigma factors

74

Indian Council of Scientific and Industrial Research. WO2164572 (2012).

75

Jenrin Discovery. WO2068560 (2012).

76

Sequella Inc.: US8198303 (2012).

77

Pethe K, Bifani P, Jang J et al. Discovery of Q203, a potent clinical candidate for the treatment of tuberculosis. Nat. Med. 19(9), 1157–1160 (2013).


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Mycobacterium tuberculosis virulence. Trends Microbiol. 16, 528–534 (2008). 104 Leblanc C, Prudhomme T, Tabouret G et al.

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126 Uti Limuted Partnership. US0070860 (2008).

106 Novartis Ag. WO4037900 (2014).

127 Hawn TR, Matheson AI, Maley SN, Vandal O. Host-

107 Grzegorzewicz AE, Pham H, Gundi VA et al. Inhibition of

mycolic acid transport across the Mycobacterium tuberculosis plasma membrane. Nat. Chem. Biol. 8, 334–341 (2012). 108 Arvind A, Kumar V, Saravanan P, Mohan CG. Homology

modelling, molecular dynamics and inhibitor binding study on MurD ligase of Mycobacterium tuberculosis. Interdiscip. Sci. 4(3), 223–238 (2012). 109 Leibniz Inst Naturstoff Forsch. US0286130 (2010). 110 Ecole Polytechnique Federale de Lausanne (Epfl).

WO0245007 (2013). 111 Chatterji M, Shandil R, Manjunatha MR et al. 1,4-azaindole,

a potential drug candidate for treatment of tuberculosis. Antimicrob. Agents Chemother. 58(9), 5325–5331 (2014). 112 Panda M, Ramachandran S, Ramachandran V et al.

Discovery of pyrazolopyridones as a novel class of noncovalent DprE1 inhibitor with potent anti-mycobacterial activity. J. Med. Chem. 57(11), 4761–4771 (2014). 113 Mann FM, Xu M, Deavenport EK, Peters RJ. Functional

characterization and evolution of the isotuberculosinol operon in mycobacterium tuberculosis and related mycobacteria. Front. Microbiol. 3, 368 (2012). 114 Huang X, Huang H, Li H et al. Asperterpenoid A, a new

sesterterpenoid as an inhibitor of Mycobacterium tuberculosis protein tyrosine phosphatase B from the culture of Aspergillus sp. 16–5c. Org. Lett. 15(4), 721–723 (2013). 115 Mckenny ES, Sargent M, Khan H et al. Lipophilic prodrugs

of FR900098 are antimicrobial against Francisella novicida in vivo and in vitro and show GlpT independent efficacy. PLoS ONE 7(10), E38167 (2012).

128 van der Vaart M, Korbee CJ, Lamers GE et al. The DNA

damage-regulated autophagy modulator DRAM1 links mycobacterial recognition via TLP-MYD88 to autophagic defense. Cell host microbe 15(6), 753–767 (2014). 129 University of Pittsburgh of the Commonwealth system of

Higher Education: WO3134416 (2013). 130 Stanley SA, Barczak AK, Silvis MR et al. Identification

of host-targeted small molecules that restrict intracellular Mycobacterium tuberculosis growth. PLoS Pathog. 10(2): e1003946 (2014). 131 Avi Biopharma. WO2064991 (2012). 132 Rath M. WO2177520 (2012). 133 Orme IM. Vaccine development for tuberculosis: current

progress. Drugs 73, 1015–1024 (2013). 134 The Regents of the University of California. US8163294

(2012). 135 Universidade Federal de Minas Gerais. WO2088577 (2012). 136 Isis Innovation Limited. WO2052748 (2012). 137 Uab Research Foundation. WO3033363 (2013). 138 King’s College of London, St. George’s University of London,

University of Dundee. WO2076868 (2012). 139 University of Central Florida Research Foundation Inc.

EP2771469 (2013). 140 Panseeta P, Lomchoey K, Prabpai S et al. Antiplasmodial and

antimycobacterial cyclopeptide alkaloids from the root of Ziziphus mauritiana. Phytochemistry 72(9), 909–915 (2011).

116 George Washington University et al: WO3006444 (2013).

141 Billings Pharmaceuticals Inc. US7321002 (2008).

117 Smith JM, Warringtom NV, Vierling RJ et al. Targeting

142 Samad A, Sultana Y, Akhter MS, Aqil M. Treatment of

DXP synthase in human pathogens: enzyme inhibition and antimicrobial activity of butylacetylphosphonate. J. Antibiot. 67, 77–83 (2014). 118 Indiana University Research and Technology Corporation.

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(2006). 120 Cornell Research Foundation Inc., Sloan-Kettering Institute

for Cancer Research. US0024611 (2014). 121 University of Notre Dame Du Lac. US6310058 (2001). 122 The Texas A and M university system. US8664255 (2014). 123 Lucile WE, Reynolds RC, Suling WJ. US0113429 (2010). 124 University of Pittsburgh of the Commonwealth system of

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Effects of broad-spectrum antimycobacterial therapy on chronic pulmonary sarcoidosis.

Limited resources lead to newer approach in jaundice therapy.

Prophetic patents in biotechnology.

Gene patents.

AZT patents.

Piecemeal patents.

Shock avoidance and the newer tachycardia therapy algorithms.

Newer gene editing technologies toward HIV gene therapy.

Crohn disease. Newer aspects of etiology, diagnosis and therapy.

Cytokine-modulating strategies and newer cytokine targets for arthritis therapy.

Product patents.

Patents in relation to microbiology.

Decoding gene patents in Australia.

Patents and literature.

Patents for genomes?

Patents, injunctions, and sutures.

Shortages of antimycobacterial drugs.

Newer fashions in illegitimacy.

Newer developments in pacemakers.

Differentiating stem cell patents.

Patents and literature.

Cetus retains PCR patents.

Controlled trial of antimycobacterial therapy in Crohn's disease. Clofazimine versus placebo.

Need for change in patents for drugs.