Tuberculosis (TB) is a respiratory transmitted disease affecting nearly 32 per cent of the world’s population, more than any other infectious disease. Among the infected individuals, approximately eight million develop active TB and almost two million of these die from this disease per year. Of new TB cases, 95 per cent occur in developing countries every year and approximately one million young women per year are victimized with this disease in the developing world.
Nowadays, the treatment of infectious diseases is facing a serious problem worldwide, as microorganisms become resistant to multiple antimicrobial agents, which leads to increase morbidity, mortality and healthcare costs. Apart from this, tuberculosis is again becoming a serious worldwide problem due to its association with the human immunodeficiency virus (HIV).
Current drugs regimen for TB
One of the most effective first-line anti-TB drugs is isoniazid (INH). The common therapeutics consist of a six months regimen, using streptomycin/ethambutol in combination with INH, rifampicin, and pyrazinamide for two months followed by rifampicin and INH biweekly for four months. INH-resistant strains have becomea severe setback to early therapeutic success and life-threatening complication. The treatment of MDR-TB patients thus requires the administration of second line drugs (amikacin, kanamycin, capreomycin, cycloserine, para-aminosalicylic acid, ethionamide, and fluoroquinolones). However, these drugs are more toxic and less efficientand have a longer regiment’s time. The cost of the treatment of MDR-TB is also 100 times higher than that of the basic six monthd short-course chemotherapy regiments.
Drug resistance - a major challenge
Multidrug-resistance is commonly defined as the ability of an organism, such as the tuberculosis bacillus to demonstrate resistance against one or several drugs making the treatment inactive or less effective. Multi-drug resistant MDR-TB, which is defined as resistance to at least rifampicin and isoniazid, two current firs t-line drugs, is increasing each year. It is estimated that at least 4 per cent of all worldwide TB patients have strains that are resistant to at least one of the current first-line drugs and over 400,000 new cases are detected each year. The most problematic issue with the current first-line TB regimen is that inadequate adherence to the treatment course, due to its length, complexity and associated adverse effects, is driving selection of much more difficult and expensive-to-treat multi-drug-resistant tuberculosis (MDR-TB) strains. Inadequate adherence to treatment occurs despite extensive global efforts by the World Health Organization (WHO), ministries of health, and others to implement the highly labour intensive TB treatment programme known as Directly Observed Therapy Short-course (DOTS), which includes direct observation of treatment by public health workers. The WHO has estimated that in 2004 there were 424,203 cases of MDR-TB globally; 181,408 occurred in patients who had already been treated with standard (first-line) therapy. Treatment for MDR-TB typically requires 18–24 months of combination therapy with second-line drugs that are less efficacious, more toxic and much more expensive than the four first-line drugs. Recently, a subset of MDR-TB strains has been identified as extensively (or extremely) drug-resistant (XDR-TB).
Poor management of chemotherapy has contributed to the emergence of drug resistance, and this is particularly relevant in TB. The complex nature and length of the treatment, side effects, various socioeconomic factors, and the tendency for patients to feel well long before safe completion of the prescribed course promote non-adherence. An unacceptable degree of non-adherence prompted the development of DOTS in which therapy is directly monitored. Initially, this acronym described only the intensively managed chemotherapy regime but has since become used to describe a broader public health strategy that has been adopted in over 150 countries.
New drugs for TB - still a distant dream
Researchers have known that TB treatment must be delivered in multi-drug regimens to prevent the development of drug resistance. Traditionally, when developing a new drug, researchers would replace an existing drug within the regimen or add the new candidate to the existing standard treatment. Each of these trials could last six years or longer, which means that a novel TB regimen could take decades to develop using this model. With more than 9 million new cases of TB each year and growing numbers of people resistant to the current treatment; such a long wait could be fatal. Although much information about TB has been gained over the last decade, including the complete sequencing of its genome, it is not understood how this relates towards developing new, faster-acting TB drugs. The drug discovery process is also complex and expensive.
Shortcoming, challenges of TB drug development
Identification of drugs that will shorten treatment and thus improve adherence is key to radically improving active TB treatment, decreasing demands on national TB control programmes, and preventing further selection of resistant strains. In an ideal scenario, searching such drugs would be based on knowledge of the underlying mechanisms of mycobacterial persistence, leading to identification of crucial targets. Presently, a clear understanding of persistence mechanisms and fully validated animal models that can reliably predict human treatment duration are lacking, and thus it seems to be an efficient path to developing drugs for shortening treatment. Though the mouse model appears to reflect human treatment results in many cases, it still lacks adequate data for considering as a validated one. In the absence of basic biological understanding of persistence, shortening therapy of active disease to days rather than months is still a dream. Realistically, current animal model evidence and clinical data indicate that shortening treatment to three to four months should, however, be achievable even with combinations of current and new drugs already in the pipeline.
A second challenge for TB drug R&D is the long timeline of clinical trials. Phase 2 studies for TB drugs typically require at least two years, and pivotal trials a minimum of three years from beginning patient enrolment to finalized study reports. These relatively long periods result from a number of factors. The requirement for multidrug therapy represents one of the crucial challenges for TB drug R&D, as it has several repercussions that affect the R&D process. First, because as little as a few weeks of monotherapy may lead to the development of drug resistance, it is not ethical to test single drugs beyond the stage of Early Bactericidal Activity (EBA) studies (which have maximally involved 14 days of treatment).
The third challenge in TB drug development is presented by the very high efficacy of the current standard regimen for active, drug-sensitive disease - routinely over 95 per cent under trial conditions. As a result, a new regimen must be tested for noninferiority rather than superiority compared to a standard control arm to avoid impractically large patient numbers and the very small ‘window’ in which a new regimen could possibly be shown to be ‘superior’ to standard treatment. However, superiority of a new TB regimen may be demonstrated by providing convincing data that clinically significant shortening of treatment duration with the new drug combination is ‘noninferior’ to standard therapy.
Novel drug targets in TB treatment
Following are some of the novel targets, which are being exploited in search of the newer molecules to manage TB.
Nitrate reductase
M. tuberculosis was originally thought to be an obligate aerobe, but there are numerous experimental indicators that the bacterium can grow in microareophilic environments, especially during the later stages of infection, e.g., in lung granulomas. Wild type M. tuberculosis has been shown to possess an inducible nitrate reductase which allows respiration using NO3 as a final electron acceptor. If anaerobic or microareophilic growth is an important feature of M. tuberculosis physiology during infection, the existence of nitrate reductase could be a significant factor in sustaining growth under these conditions.Thus this enzyme can be a possible target for development of new drugs. Nitrate reductase activity of M. tuberculosis was sensitive to inhibition by both tungstate and azide, suggesting that this enzyme is a membrane-bound molybdenum-containing complex similar to the corresponding narGHJI of E. coli.
Glutamine synthetase
The enzyme is not secreted into culture filtrates during growth but results from cell leakage and lysis. GlnA1 mutations have not been made in M. tuberculosis, but the specific glutamine synthase inhibitor, L-methionine-SR-sulfoximine (MSO), inhibits the growth of Mycobacterium tuberculosis in vitro and in macrophages but has no effect on non-pathogenic mycobacterium. This enzyme essentially plays an important role in nitrogen metabolism, and is involved in the synthesis of a poly-L-glutamate-glutamine cell wall component found in pathogenic mycobacteria. These findings have led to the suggestion that this enzyme could be a possible target for the development of new drugs.
Shikimate kinase
Shikimate kinase is a very attractive target as it is vital for the survival of M.tuberculosis but is absent in mammals. Hence, inhibitors designed against Shikimate kinase will be very specific against the pathogen and will be least harmful to the host. Till date, no drug candidates are available against this target. The crystal structure of M. tuberculosis Shikimate kinase complexed with shikimate has been used to identify a dipeptide inhibitor using in-silico structure-based design approach. The designated peptidic inhibitor has a predicted binding affinity of 5.5 nm which is 8000 times better than substrate shikimate and 10 times greater than the best suggested inhibitor. As small peptides are known to be non-toxic, this inhibitor could be a lead compound in the development of anti-tuberculosis drugs.
Alpha galactosylceramide
Recent studies have shown that invariant natural killer T(iNKT), which are a unique subset of T cells can protect mice against M.tuberculosis infection. Pharmacological activation on iNKT cells by α-galactosylceramide (a-GalCer) could be used to treat tuberculosis. The ability of a-GalCer activated iNKT cells to suppress M. tuberculosis replication was evaluated using an in vitro coculture system. Results showed that a-GalCer plus isoniazid controls bacterial growth better than a-GalCer or INH alone.
Mycolyltransferase 85A
The enzymes of the antigen 85 complex (Ag85A, B and C) possess mycolyltransferase activity and catalyse the synthesis of the most abundant glycolipid of the mycobacterial cell wall, the cord factor. The cord factor (trehalose 6,6’-dimycolate,TDM) is essential for the integrity of the mycobacterial cell wall and pathogenesis of the bacillus. Thus, TDM biosynthesis is regarded as apotential drug target for the control of M.tuberculosis infections.
Recent drug discovery, development in anti-TB treatment
The worldwide problem caused by TB and the lack of new drugs in the market makes it imperative to have new drugs to fight efficiently against the rapid spread of multidrug resistant TB strain against all major antituberculer drugs in the market. In this context, there is an urgent need for TB drugs with fewer toxic side effects, improved pharmacokinetics properties, extensive and potent activity against Gram-positive and Gram-negative bacteria, including resistant strains and drugs able to reduce the total duration of treatment.
Coelho T.S. et al recently reported N-acylhydrazone derivatives which they have evaluated against INH-susceptible as well as INH-resistance strains of M. tuberculosis. The results showed that these drugs were inactive against the resistant strains, due to the ability inhagene to disturb the binding the ligands to the target protein. Adamantylureas were previously identi?ed as a group of compounds active against Mycobacterium tuberculosis in culture with minimum inhibitor concentrations (MICs) below 0.1µg/ml. These compounds have been shown to target MmpL3, a protein involved in secretion of trehalose mono-mycolate. They also inhibit both human soluble epoxide hydrolase (hsEH) and M. tuberculosis epoxide hydrolases. Scherman M.S et al have screened 1600 derivatives of Adamantylureas and tested against Mycobacterium tuberculosis.
Would drug re-positioning help?
Development of the current clinical portfolio, in part, is focused on the repurposing of existing antibiotics alongside current TB drugs as part of new multidrug regimes. Gatifloxacin (GAT), moxifloxacin (MXF), levofloxacin (LVF), linezolid, and metronidazole are being trialed by multi-partnership consortia which include the TB Alliance, The Bill and Melinda Gates Foundation, WHO, National Institute of Allergy and Infective Disease, National Institutes of Health, USA (NIAID/NIH), Wellcome Trust, Lupin, Bayer Pharmaceuticals, Centres for Disease Control (CDC), Research and Training in Tropical Diseases (TDR), and their partner academic or research institutions.
One must hope that these united efforts would certainly bring out a novel regimen for TB patients which could effectively reduce the duration of treatment and fight resistance of all sorts.
(The authors are associated with Dept. of Pharmaceutical Chemistry, VES College of Pharmacy, Mumbai-400 074)