Cell wall permeability assisted virtual screening to identify potential direct InhA inhibitors of Mycobacterium tuberculosis and their biological evaluation.

The arising cases of isoniazid-resistance have motivated research interests toward new class of molecules known as direct InhA inhibitors. Here, a combine approach of shape-based pharmacophore and descriptor-based 2D QSAR was used to identify the potential direct InhA inhibitors. The approach is duly assisted with in vitro testing and molecular dynamics simulations. A combination of empirical parameters was derived to use as a filter for cell wall permeability while 2D QSAR was used as another filter to predict the biological activity. Both filters were applied to prioritize the molecules for biological evaluation against anti-TB activity. It led to 6 potential molecules which showed > 90% inhibition of H37Rv strain of Mycobacterium tuberculosis in BACTEC assay. Further, MMGBSA binding free energy of identified molecules was compared with available highly potent molecule, 5-hexyl-2-(2-methylphenoxy) phenol (IC50 = 5nM) using molecular dynamics simulations. It showed two molecules with comparatively higher affinity toward InhA as compared to potent molecule. It indicated the candidature of identified molecules to be further considered in anti-TB drug development pipeline.

In Vitro-In Vivo Evaluation of Novel Co-spray Dried Rifampicin Phospholipid Lipospheres for Oral Delivery.

The objective of this study comprises of developing novel co-spray dried rifampicin phospholipid lipospheres (SDRPL) to investigate its influence on rifampicin solubility and oral bioavailability. Solid-state techniques were employed to characterize the liposphere formulation. SDRPL solubility was determined in distilled water. BACTEC 460TB System was employed to evaluate SDRPL antimycobacterial activity. The oral bioavailability of the lipospheres was evaluated in Sprague Dawley rats. Lipospheres exhibited amorphous, smooth spherical morphology with a significant increase (p < 0.001) in solubility of SDRPL (2:1), 350.9 ± 23 versus 105.1 ± 12 µg/ml and SDRPL (1:1) 306.4 ± 20 versus 105.1 ± 12 µg/ml in comparison to rifampicin (RMP). SDRPL exhibited enhanced activity against Mycobacterium tuberculosis, H37Rv strain, with over twofolds less minimum inhibitory concentration (MIC) than the free drug. Lipospheres exhibited higher peak plasma concentration (109.92 ± 25 versus 54.31 ± 18 µg/ml), faster T max (two versus four hours), and enhanced area under the curve (AUC0-∞) (406.92 ± 18 versus 147.72 ± 15 µg h/L) in comparison to pure RMP. Thus, SDRPL represents a promising carrier system exhibiting enhanced antimycobacterial activity and oral bioavailability of rifampicin.

Determination of the activity of standard anti-tuberculosis drugs against intramacrophage Mycobacterium tuberculosis, in vitro: MGIT 960 as a viable alternative for BACTEC 460.

BACTEC 460 has now been phased out, so the search for an alternative is imperative. We have determined the activity of standard anti-tuberculosis drugs against intramacrophage Mycobacterium tuberculosis, in vitro, by using BACTEC 460 and MGIT 960 methods. The minimum inhibitory concentrations of isoniazid, rifampicin, ethambutol and streptomycin against intracellular M. tuberculosis H37Rv were found to be 0.2, 0.8, 8.0, and 5.0 µg/mL, respectively, by both methods. These results show a significant (p<0.001) concordance between minimum inhibitory concentrations obtained by these two different methods. MGIT 960 system uses a robust florescence quenching-based oxygen sensor, requires no radioisotope, is safe, and relatively easy to operate. Apparently, this is the first report wherein MGIT 960 has been validated for anti-tubercular susceptibility testing against intracellular M. tuberculosis H37Rv. Our preliminary data thus clearly demonstrate that the MGIT 960 method can be considered as a promising alternative to BACTEC 460 method.

Models of latent tuberculosis: their salient features, limitations, and development.

Latent tuberculosis is a subclinical condition caused by Mycobacterium tuberculosis, which affects about one-third of the population across the world. To abridge the chemotherapy of tuberculosis, it is necessary to have active drugs against latent form of M. tuberculosis. Therefore, it is imperative to devise in vitro and models of latent tuberculosis to explore potential drugs. In vitro models such as hypoxia, nutrient starvation, and multiple stresses are based on adverse conditions encountered by bacilli in granuloma. Bacilli experience oxygen depletion condition in hypoxia model, whereas the nutrient starvation model is based on deprivation of total nutrients from a culture medium. In the multiple stress model dormancy is induced by more than one type of stress. In silico mathematical models have also been developed to predict the interactions of bacilli with the host immune system and to propose structures for potential anti tuberculosis compounds. Besides these in vitro and in silico models, there are a number of in vivo animal models like mouse, guinea pig, rabbit, etc. Although they simulate human latent tuberculosis up to a certain extent but do not truly replicate human infection. All these models have their inherent merits and demerits. However, there is no perfect model for latent tuberculosis. Therefore, it is imperative to upgrade and refine existing models or develop a new model. However, battery of models will always be a better alternative to any single model as they will complement each other by overcoming their limitations.

A short-term model for preliminary screening of potential anti-tubercular compounds.

Screening of new agents for anti-tubercular activity is a challenging task due to the virulent and slow growing nature of mycobacteria. In this study, we explored the use of Mycobacterium smegmatis as an alternate model to virulent strains. We observed that the effect of standard anti-tubercular drugs was similar in mice infected with M. smegmatis and Mycobacterium tuberculosis. The total duration of the experiment, including incubation time, was 10 days in the M. smegmatis-infected mice model compared with 2 months in M. tuberculosis-infected mice. This model of anti-tubercular screening is a simple, easy to carry out, less time-consuming, safer and economical alternative for preliminary in vivo screening of potential anti-tubercular agents.

Evaluation of BACTEC 460 TB system for rapid in vitro screening of drugs against latent state Mycobacterium tuberculosis H37Rv under hypoxia conditions.

Mycobacterium tuberculosis exhibits latent state due to its ability to survive for extended periods under oxygen depletion conditions. The conventional plating method employed for in vitro screening of antitubercular agents against latent state M. tuberculosis is laborious and time consuming exercise. Towards this end, BACTEC 460 TB system of antitubercular screening was evaluated for testing efficacy of antimicrobial agents in hypoxia induced model of latent tuberculosis, in vitro. In this study, drugs like isoniazid, metronidazole and rifampin were tested at concentrations- 2, 10 and 50 microg/ml for their antilatency activity by using BACTEC and conventional methods. Results obtained from both the methods were comparable (P>0.05) and a good correlation was observed between colony forming units and growth index values. Further, time to determine mycobacterial growth was significantly reduced (P

Effect of morphine on Mycobacterium smegmatis infection in mice and macrophages.

The immunomodulatory effects of opioids are known in various infections. However, little is known about the effects of opioids in tuberculosis (TB). In the present study, we report the effects of morphine in Mycobacterium smegmatis infection in mice and macrophages. Morphine exerted a dose-dependent suppression of infection in vivo: 50 and 100 mg/kg morphine exerted significant (P<0.05) suppression whereas 5 mg/kg morphine showed no effect. Analogous to the in vivo effects, incubation of M. smegmatis-infected mouse peritoneal macrophages with morphine (100 µM) showed significant reduction in intramacrophage CFU counts. However, morphine did not show any direct antimycobacterial activity in broth dilution assay upto 100 µM concentration. Further, morphine-induced intramacrophage killing of M. smegmatis was abrogated by naloxone and aminoguanidine indicating the involvement of opioid-receptor activation and nitric oxide production in protective effects of morphine. In conclusion, morphine suppressed the progression of experimental TB in both mice and macrophage models.

A comparison of conventional and radiometric methods for the assessment of anti-tubercular activity of drugs against Mycobacterium tuberculosis in mice and macrophage models.

BACKGROUND: Presently, in vitro and in vivo screening of anti-tubercular drugs is a time-consuming exercise. Therefore, it is important to develop faster methods. MATERIAL AND METHODS: Towards this end, conventional plating and radiometric BACTEC methods of anti-tubercular screening were compared to determine the efficacy of anti-tubercular drugs (isoniazid and rifampicin) and morphine in Mycobacterium tuberculosis H37Rv-infected mice and macrophages. RESULTS: A linear correlation (R2 = 0.95) was observed between number of colony forming units (CFUs) and growth index (GI) values. BACTEC method was found to be faster and sensitive as compared to plating method. Further, BACTEC method, being a closed system, appeared to be less susceptible to microbial contamination and poses less biohazard. CONCLUSION: We conclude that BACTEC method can be employed for easy, precise, and rapid screening of anti-tubercular compounds and morphine in mice and macrophage models.

Effects of morphine during Mycobacterium tuberculosis H37Rv infection in mice.

The effects of opiates in various infections are well known; however, very little is known about tuberculosis infection. Therefore, in the present study, we report for the first time, the effects of morphine during murine tuberculosis. Mice were infected intravenously with Mycobacterium tuberculosis H37Rv, administered morphine (0.1-100 mg/kg subcutaneously on day 0 and day +15) and sacrificed on day +30 for CFU enumeration in lungs and spleen. Morphine exerted maximum suppression of infection at 5 mg/kg, and sometimes completes elimination of infection; naloxone, silica and aminoguanidine blocked the protective effect of morphine. In vitro, morphine lacked direct antimycobacterial activity up to 1x10(-4) M concentration, as assessed by radiometric BACTEC method. In macrophage model of infection, morphine showed maximal killing at 1x10(-7) M concentration, the activity was blocked by naloxone and aminoguanidine. These observations suggest that morphine exerts a dose-dependent effect in murine tuberculosis, the protective effect being naloxone-reversible and may involve macrophage-mediated protective mechanisms. These results may be helpful in developing new opioid-like chemical entities against tuberculosis infection.

Ring-substituted quinolines. Part 2: Synthesis and antimycobacterial activities of ring-substituted quinolinecarbohydrazide and ring-substituted quinolinecarboxamide analogues.

Additional structural modifications of the new chemical entity, 2,8-dicyclopentyl-4-methylquinoline (DCMQ; MIC=6.25 microg/mL, M. tuberculosis H37Rv) resulted in the synthesis of four new series of the ring-substituted quinolinecarbohydrazides (series 1-4) constituting 22 analogues. All new derivatives were evaluated for in vitro antimycobacterial activities against drug-sensitive M. tuberculosis H37Rv strain. Certain ring-substituted-2-quinolinecarbohydrazide analogues described herein showed good inhibitory activity. In particular, analogues 4-(1-adamantyl)-2-quinolinecarbohydrazide (2d), 4,5-dicyclopentyl-2-quinolinecarbohydrazide (2e), 4,8-dicyclopentyl-2-quinolinecarbohydrazide (2f), and 4,5-dicyclohexyl-2-quinolinecarbohydrazide (2g) have exhibited the MIC value of 6.25 microg/mL. Further investigation of the most suitable lead prototype, 4-(1-adamantyl)-2-quinolinecarbohydrazide (2d, series 1) led to the synthesis of N2-alkyl/N2,N2-dialkyl/N2-aryl-4-(1-adamantyl)-2-quinolinecarboxamides (series 5) consisting of 13 analogues. Some of the synthesized carboxamides 7a, 7h, and 7m reported herein have exhibited excellent antimycobacterial activities in the range of 6.25-3.125 microg/mL against drug-sensitive and drug-resistant M. tuberculosis H37Rv strains.