Evaluation of Collagen in Leukoplakia, Oral Submucous Fibrosis and Oral Squamous Cell Carcinomas Using Polarizing Microscopy and Immunohistochemistry

Objectives: Picrosirius red and MMP are capable of degrading extracellular matrix proteins, expressed in lesions such as squamous cell carcinomas. The present study was undertaken with an aim to analyze and compare changes in collagen using Picrosirius red staining under polarizing microscopy and immunohistochemical staining using anti MMP-13 in samples of oral leukoplakia, oral submucous fibrosis and oral squamous cell carcinoma. Materials and Methods: A total of 70 slides were prepared and divided into 3 groups. Group I comprised 10 slides of normal gingival tissue, Group II 40 slides of potentially malignant disorders and Group III 20 slides of well differentiated oral squamous cell carcinoma. Half the slides for each group were stained with Picrosirius red stain and the remainder with antibodies to MMP-13. Rerults: In Group II, MMP-13 connective tissue expression was greater in OSMF as compared to leukoplakia. Group III showed elevated expression among 70% of cases. Picrosirius red staining in Group II cases, showed higher staining Yellow-Orange andGreen-Yellow mature fibers in OSMF than leukoplakia cases while in Group III, 50% OSCC cases showed Green-yellow stained immature thin fibers. Conclusion: In future, therapeutic measures targeted against MMP-13 may inhibit collagenolysis to some extent and delay spread of tumors. An easy and reliable method to determine the state of the stroma in such cases may be Picrosirius red staining with polarizing microscopy.

NOS2-deficient mice with hypoxic necrotizing lung lesions predict outcomes of tuberculosis chemotherapy in humans.

During active TB in humans a spectrum of pulmonary granulomas with central necrosis and hypoxia exists. BALB/c mice, predominantly used in TB drug development, do not reproduce this complex pathology thereby inaccurately predicting clinical outcome. We found that Nos2 -/- mice incapable of NO-production in immune cells as microbial defence uniformly develop hypoxic necrotizing lung lesions, widely observed in human TB. To study the impact of hypoxic necrosis on the efficacy of antimycobacterials and drug candidates, we subjected Nos2 -/- mice with TB to monotherapy before or after establishment of human-like pathology. Isoniazid induced a drug-tolerant persister population only when necrotic lesions were present. Rifapentine was more potent than rifampin prior to development of human-like pathology and equally potent thereafter, in agreement with recent clinical trials. Pretomanid, delamanid and the pre-clinical candidate BTZ043 were bactericidal independent of pulmonary pathology. Linezolid was bacteriostatic in TB-infected Nos2 -/- mice but significantly improved lung pathology. Hypoxic necrotizing lesions rendered moxifloxacin less active. In conclusion, Nos2 -/- mice are a predictive TB drug development tool owing to their consistent development of human-like pathology.

Pharmacokinetics-pharmacodynamics analysis of bicyclic 4-nitroimidazole analogs in a murine model of tuberculosis.

PA-824 is a bicyclic 4-nitroimidazole, currently in phase II clinical trials for the treatment of tuberculosis. Dose fractionation pharmacokinetic-pharmacodynamic studies in mice indicated that the driver of PA-824 in vivo efficacy is the time during which the free drug concentrations in plasma are above the MIC (fT>MIC). In this study, a panel of closely related potent bicyclic 4-nitroimidazoles was profiled in both in vivo PK and efficacy studies. In an established murine TB model, the efficacy of diverse nitroimidazole analogs ranged between 0.5 and 2.3 log CFU reduction compared to untreated controls. Further, a retrospective analysis was performed for a set of seven nitroimidazole analogs to identify the PK parameters that correlate with in vivo efficacy. Our findings show that the in vivo efficacy of bicyclic 4-nitroimidazoles correlated better with lung PK than with plasma PK. Further, nitroimidazole analogs with moderate-to-high volume of distribution and Lung to plasma ratios of >2 showed good efficacy. Among all the PK-PD indices, total lung T>MIC correlated the best with in vivo efficacy (rs = 0.88) followed by lung Cmax/MIC and AUC/MIC. Thus, lung drug distribution studies could potentially be exploited to guide the selection of compounds for efficacy studies, thereby accelerating the drug discovery efforts in finding new nitroimidazole analogs.

Intra-oral schwannoma- a case report.

Neurilemmoma (Schwannoma) is a benign tumour of neuroectodermal origin. It usually occurs as a asymptomatic, solitary, smooth-surfaced and slow growing lesion, emerging at any age, with as such, no gender prelidiction. Occurring as a common tumour in the head and neck region, its intraoral presentation is very rare. Here, we are reporting a rare case of intraoral schwannoma of the posterior palate which occurred in a 34-year-old female patient who had chief complaint of a painless, slow growing swelling on posterior palate.

Structure of Ddn, the deazaflavin-dependent nitroreductase from Mycobacterium tuberculosis involved in bioreductive activation of PA-824.

Tuberculosis continues to be a global health threat, making bicyclic nitroimidazoles an important new class of therapeutics. A deazaflavin-dependent nitroreductase (Ddn) from Mycobacterium tuberculosis catalyzes the reduction of nitroimidazoles such as PA-824, resulting in intracellular release of lethal reactive nitrogen species. The N-terminal 30 residues of Ddn are functionally important but are flexible or access multiple conformations, preventing structural characterization of the full-length, enzymatically active enzyme. Several structures were determined of a truncated, inactive Ddn protein core with and without bound F(420) deazaflavin coenzyme as well as of a catalytically competent homolog from Nocardia farcinica. Mutagenesis studies based on these structures identified residues important for binding of F(420) and PA-824. The proposed orientation of the tail of PA-824 toward the N terminus of Ddn is consistent with current structure-activity relationship data.

Substrate specificity of the deazaflavin-dependent nitroreductase from Mycobacterium tuberculosis responsible for the bioreductive activation of bicyclic nitroimidazoles.

The bicyclic 4-nitroimidazoles PA-824 and OPC-67683 represent a promising novel class of therapeutics for tuberculosis and are currently in phase II clinical development. Both compounds are pro-drugs that are reductively activated by a deazaflavin (F(420)) dependent nitroreductase (Ddn). Herein we describe the biochemical properties of Ddn including the optimal enzymatic turnover conditions and substrate specificity. The preference of the enzyme for the (S) isomer of PA-824 over the (R) isomer is directed by the presence of a long hydrophobic tail. Nitroimidazo-oxazoles bearing only short alkyl substituents at the C-7 position of the oxazole were reduced by Ddn without any stereochemical preference. However, with bulkier substitutions on the tail of the oxazole, Ddn displayed stereospecificity. Ddn mediated metabolism of PA-824 results in the release of reactive nitrogen species. We have employed a direct chemiluminescence based nitric oxide (NO) detection assay to measure the kinetics of NO production by Ddn. Binding affinity of PA-824 to Ddn was monitored through intrinsic fluorescence quenching of the protein facilitating a turnover-independent assessment of affinity. Our results indicate that (R)-PA-824, despite not being turned over by Ddn, binds to the enzyme with the same affinity as the active (S) isomer. This result, in combination with docking studies in the active site, suggests that the (R) isomer probably has a different binding mode than the (S) with the C-3 of the imidazole ring orienting in a non-productive position with respect to the incoming hydride from F(420). The results presented provide insight into the biochemical mechanism of reduction and elucidate structural features important for understanding substrate binding.

Structure-activity relationships of antitubercular nitroimidazoles. 3. Exploration of the linker and lipophilic tail of ((s)-2-nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazin-6-yl)-(4-trifluoromethoxybenzyl)amine (6-amino PA-824).

The (S)-2-nitro-6-(4-(trifluoromethoxy)benzyloxy)-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine named PA-824 (1) has demonstrated antitubercular activity in vitro and in animal models and is currently in clinical trials. We synthesized derivatives at three positions of the 4-(trifluoromethoxy)benzylamino tail, and these were tested for whole-cell activity against both replicating and nonreplicating Mycobacterium tuberculosis (Mtb). In addition, we determined their kinetic parameters as substrates of the deazaflavin-dependent nitroreductase (Ddn) from Mtb that reductively activates these pro-drugs. These studies yielded multiple compounds with 40 nM aerobic whole cell activity and 1.6 µM anaerobic whole cell activity: 10-fold improvements over both characteristics from the parent molecule. Some of these compounds exhibited enhanced solubility with acceptable stability to microsomal and in vivo metabolism. Analysis of the conformational preferences of these analogues using quantum chemistry suggests a preference for a pseudoequatorial orientation of the linker and lipophilic tail.

Synthesis, anti-tuberculosis activity and 3D-QSAR study of amino acid conjugates of 4-(adamantan-1-yl) group containing quinolines.

The synthesis, antimycobacterial activity and 3D-QSAR study of two series of 4-(adamantan-1-yl) group containing quinolines conjugated to amino acids are described. The most potent analogs displayed in vitro antimycobacterial activity ranging between 1.00 and 3.125 microg/mL. To understand the relationship between structure and activity, a 3D-QSAR analysis has been carried out by Comparative Molecular Field Analysis (CoMFA). The activities of molecules in the test sets were nicely predicted by the CoMFA model generated with field alignment. The best model was obtained using atom-fit alignment. Based on the molecular fields the relationships between structure and activity were easily rationalized.

Synthesis, anti-tuberculosis activity, and 3D-QSAR study of 4-(adamantan-1-yl)-2-substituted quinolines.

Structural optimization of the previously identified 4-(adamantan-1-yl)-2-quinolinecarbohydrazide (AQCH, MIC=6.25 microg/mL, 99% inhibition, Mycobacterium tuberculosis H37Rv) has led to two series of 4-(adamantan-1-yl)-2-substituted quinolines (Series 1-2). All new derivatives were evaluated in vitro for antimycobacterial activities against drug-sensitive M. tuberculosis H37Rv strain. Several 4-adamantan-1-yl-quinoline-2-carboxylic acid N'-alkylhydrazides (Series 1) described herein showed promising inhibitory activity. In particular, analogs 7, 9, 20, and 21 displayed MIC of 3.125 microg/mL. Further investigation of AQCH by its reaction with various aliphatic, aromatic, and heteroaromatic aldehydes led to the synthesis of 4-adamantan-1-yl-quinoline-2-carboxylic acid alkylidene hydrazides (Series 2). Analogs 42-44 and 48 have produced promising antimycobacterial activities (99% inhibition) at 3.125 microg/mL against drug-sensitive M. tuberculosis H37Rv strain. The most potent analog 35 of the series produced 99% inhibition at 1.00 microg/mL against drug-sensitive strain, and MIC of 3.125 microg/mL against isoniazid-resistant TB strain. To understand the relationship between structure and activity, a 3D-QSAR analysis has been carried out by three methods-comparative molecular field analysis (CoMFA), CoMFA with inclusion of a hydropathy field (HINT), and comparative molecular similarity indices analysis (CoMSIA). Several statistically significant CoMFA, CoMFA with HINT, and CoMSIA models were generated. Prediction of the activity of a test set of molecules was the best for the CoMFA model generated with database alignment. Based on the CoMFA contours, we have tried to explain the structure-activity relationships of the compounds reported herein.

Synthesis, anti-tuberculosis activity, and 3D-QSAR study of ring-substituted-2/4-quinolinecarbaldehyde derivatives.

We have previously identified ring-substituted quinolines as a new structural class of anti-tuberculosis agents. In our ongoing efforts at structural optimization of this class, four series of ring-substituted-2/4-quinolinecarbaldehyde derivatives were synthesized. All twenty-four compounds were synthesized using short and convenient one to two high yielding steps. The newly synthesized compounds were tested in vitro against drug-sensitive Mycobacterium tuberculosis H37Hv strain. Several derivatives were found to be promising inhibitors of M. tuberculosis. For example, derivatives 4a-c (Series 2), 7a-d (Series 3), and 8a-b (Series 4) displayed >90% inhibition at 6.25 microg/mL in the primary assay. The most active compounds, N-(2-fluorophenyl)-N'-quinolin-2-ylmethylene-hydrazine (4a), N-(2-adamantan-1-yl-quinolin-4-ylmethylene)-N'-(4-fluorophenyl)hydrazine (7c), and N-(2-cyclohexyl-quinolin-4-ylmethylene)-N'-(2-fluorophenyl)hydrazine (8a), exhibited 99% inhibition at the lowest tested concentration of 3.125 microg/mL against drug-sensitive M. tuberculosis H37Rv strain. The similarity index based on steric and electrostatic features of the molecules was used, in conjunction with principal component analysis and linear discriminant analysis, successively to classify the molecules based on their activity into two classes. This classification method gives us confidence in predicting the activity class of any new unsynthesized molecule belonging to these series.

3D-QSAR study of ring-substituted quinoline class of anti-tuberculosis agents.

A 3D-QSAR analysis of a new class of ring-substituted quinolines with anti-tuberculosis activity has been carried out by three methods-Comparative Molecular Field Analysis (CoMFA), CoMFA with inclusion of a hydropathy field (HINT), and Comparative Molecular Similarity Indices Analysis (CoMSIA). The conformation of the molecules was generated using a simulated annealing protocol and they were superimposed using features common to the set with database alignment (SYBYL) and field fit methods. Several statistically significant CoMFA, CoMFA with HINT, and CoMSIA models were generated. Prediction of the activity of a set of test molecules was the best for the CoMFA model generated with database alignment. Based upon the information contained in the CoMFA model, we have identified some novel features that can be incorporated into the quinoline framework to improve the activity.

Recent advances in new structural classes of anti-tuberculosis agents.

Tuberculosis (TB) is one of the most devastating diseases primarily due to several decades of neglect, and presents a global health threat of escalating proportions. TB is the second leading infectious cause of mortality today behind only HIV/AIDS. The impetus for developing new structural classes of anti-tuberculosis drugs comes from the emergence of multi-drug resistant (MDR) strains to commonly used drugs, substantially longer durations of therapy that are needed as a result of resistance, and the resurgence of disease in immuno-compromised patients. Recent years have witnessed emergence of many new structural classes of anti-TB agents, which have exhibited promising activities against drug-sensitive and drug-resistant strains of the causative organism Mycobacterium tuberculosis. These analogs ideally should decrease the overall duration of therapy with improved efficacy, and exhibit mechanisms of action different from those of existing drugs to counter the resistant strains of M. tuberculosis. This review provides a comprehensive literature compilation on advances in the new structural classes of anti-TB analogs reported during the past five years. Our discussion and observations are concentrated on chemotherapeutic potential of alphabetically listed twenty-seven new structural classes of anti-tuberculosis agents that include:- acetamides, 5-arylidene-2-thiohydantoins, benzoxazoles and benzothiazoles, benzoic acid hydrazones, benzoxazines, carbohydrates, chalcones, coumarins, deazapteridines, imidazoles, indoloquinazolinones, isothiosemicarbazones, mycobactins, 1,8-naphthyridines, phenazines, purines, pyridines, N-pyridinylsalicylamides, pyrimidines and thymidines, pyrroles, quinolines, quinoxalines, terpenes, thiadiazine thiones, thiolactomycines, toludines, and triazoles.

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.

Synthesis and antimycobacterial activities of ring-substituted quinolinecarboxylic acid/ester analogues. Part 1.

Structural optimization of recently discovered 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 ring-substituted quinolinecarboxylic acids/esters constituting 45 analogues. All new derivatives were evaluated for in vitro antimycobacterial activities against M. tuberculosis H37Rv. Certain ring-substituted-2-quinolinecarboxylic acid ester and ring-substituted-2-quinoline acetic acid ester analogues described herein showed moderate to good inhibitory activity. In particular, three analogues methyl 4,5-dicyclopentyl-2-quinolinecarboxylate (3b), methyl 4,8-dicyclopentyl-2-quinolinecarboxylate (3c) and ethyl 2-(2,8-dicyclopentyl-4-quinolyl)acetate (14g) exhibited excellent MIC values of 1.00, 2.00 and 4.00microg/mL, respectively. Results obtained indicate that substitution of the quinoline ring with dicyclopentyl substituent presumably enhances the antimycobacterial activities in the quinoline analogues described herein.

Substituted 4-methylquinolines as a new class of anti-tuberculosis agents.

We report synthesis and anti-tuberculosis activities of a series of novel ring-substituted quinolines. The most effective compound of the series 3d (MIC=6.25 microg/mL, Mycobacterium tuberculosis H37Rv strain) was synthesized in one step; thus is an attractive lead molecule for anti-tuberculosis drug development. The results of this study represent the discovery of ring-substituted 4-methylquinolines as new class of potential anti-tuberculosis agents.