Asha Bhagat

In silico identification of viper phospholipaseA2 inhibitors: validation by in vitro, in vivo studies

AbstractSnake venom, particularly of vipers from the Indian subcontinent, contains Phospholipase A2 (PLA2) as one its constituents which is widely implicated in hemorrhagic, cardiac arrest and death. Development of inhibitors of the protein can facilitate the weakening or annihilation of the venom toxicity and save many human lives. In the present communication, our studies relate to the design and development of structure-based ligands as inhibitors of PLA2 of Viper venom. The study involves the computational approach towards evaluating a library of molecules comprising of natural products, and synthetic molecules through docking studies on the venom protein PDB ID: 1OXL (a dimer, available in the literature). In silico experiments have resulted in the identification of several of them as PLA2 inhibitors. The inhibitory effect of PLA2 by these compounds is attributed to a great extent to their interaction with the residues Phe 46 and Val47 of chain B of the target protein and hence these two residues are identified as the key contributor for the said activity. In order to validate the in silico findings, a selected panel of compounds have been tested by in vitro and in vivo experiments against the venom, which has led to the observance of significant corroboration between the wet lab and in silico findings, validating thereby the in silico approach used in the present study. FigureInteraction of the potent inhibitor with PLA2

Synthesis and Structure-Activity Relationships of Vasicine Analogues as Bronchodilatory Agents

The series of vasicine (1) analogues, an alkaloid from Adhatoda vasica Nees., were synthesized with changes in A, B or C rings. Compounds 13-19 were evaluated for in vitro bronchodilatory activity using isolated guinea pig tracheal chain. Compounds 3-8 were also synthesized in good yields using microwave-mediated synthesis under solvent free conditions. Compounds 5 and 8 with seven-member C ring were more active than etofylline and caused 100% relaxation of both the histamine and acetycholine pre-contracted guinea pig tracheal chain. The structure-activity relationship studies showed that the quinazoline and oxo functionalities were essential for activity. The compounds without C ring and instead having aliphatic and phenyl substitutions in B ring showed relaxation against histamine pre-contracted tracheal chain only, 2-methyl substituted analogues, 12 and 13, being most active with 100% relaxation effect.

Analogues of boswellic acids as inhibitors of pro-inflammatory cytokines TNF-α and IL-6

A library of boswellic acid analogues were synthesized and tested for their anti-inflammatory potential on key inflammatory mediators, TNF-α and IL-6. The study led to the identification of lead compounds showing significant inhibition of the cytokines, TNF-α and IL-6 both in vitro and in vivo.

Anti-inflammatory potential of hentriacontane in LPS stimulated RAW 264.7 cells and mice model

Hentriacontane, has various pharmacological effects including anti-inflammatory, antitumor and antimicrobial activities. Its anti-inflammatory potential has been demonstrated in peritoneal macrophages. However detailed studies on other models elucidating the mechanistic description of the mode of action has not been done. Hence, the aim of the present study is to evaluate the anti-inflammatory potential of hentriacontane both in-vivo (Balb/c mice) and in-vitro (RAW 264.7 cells). Cytokine inhibition of both pro-inflammatory (TNF-α, IL-6, MCP-1 and IL-1β) and anti-inflammatory (IL-10) cytokines was studied in RAW 264.7 cells and Balb/c mice. Suppressive potential of hentriacontane on NO, PGE2, LTB4 and on LPS induced translocation of NF-κB in RAW 264.7 cells was studied. Further investigations on the effect of hentriacontane on phagocytic index, carrageenan induced paw oedema in mice and on organ weight were done. It was found that hentriacontane significantly reduced all the parameters of inflammation in the experiments under study at all the concentrations, 10μM, 5μM and 1μM (in-vitro) and 5mg/kg, 2mg/kg and 1mg/kg (in-vivo). The highest concentration used in the two models presented the most significant results. The results indicate that hentriacontane is a potent suppressor of inflammatory cytokines and other mediators. Moreover it also has regulatory effect on NF-κB. Hence, hentriacontane is a potential candidate for investigations to develop anti-inflammatory drug.

Development and validation of a highly sensitive LC–MS/MS-ESI method for quantification of IIIM-019—A novel nitroimidazole derivative with promising action against Tuberculosis: Application to drug development

The study aims to illustrate an analytical validation of a rapid and sensitive liquid chromatography (LC) coupled to tandem mass spectrometry (MS-MS) and electrospray ionization (ESI) method for quantification of IIIM-019 (a novel nitroimidazole derivative with potential activity against Tuberculosis) in mice plasma. The extraction of the analyte and the internal standard (Tolbutamide) from the plasma samples involves protein precipitation using acetonitrile. The chromatographic separation was accomplished using a gradient mode and the mobile phase comprised of acetonitrile and 0.1% formic acid in water. The flow rate used was 0.7 ml/min on a C18e high performance Chromolith column. IIIM-019 and Tolbutamide (IS) were analyzed by combined reversed-phase LC/MS-MS with positive ion electrospray ionization. The MS-MS ion transitions used were 533>170.1, 533>198 for IIIM-019 and 271>74, 271>155 for internal standard (IS) respectively. The method was linear over a concentration range of 0.5-1000 ng/ml and the lower limit of quantification was 0.50 ng/ml. The entire study was validated for accuracy, precision, linearity, range, selectivity, lower limit of quantification (LLOQ), recovery, and matrix effect in accordance with the FDA guidelines of method validation. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. The intra and inter-day precisions were in the range of 0.51-11.18% and 0.51-7.55%. The pharmacokinetics was performed on male Balb/c mice by oral (2.5mg/kg), intraperitoneal (2.5mg/kg) and intravenous (1mg/kg) routes. The oral bioavailability of IIIM-019 was 51.6%. The method was also applied successfully in determining microsomal stability wherein the compound was found to be very slightly metabolized by rat liver microsomes.

Kaempferol-3-o-β-d-glucuronate exhibit potential anti-inflammatory effect in LPS stimulated RAW 264.7 cells and mice model

&NA; Kaempferol‐3‐O‐&bgr;‐d‐glucuronide (K3G) having various pharmacological effects was explored for its anti‐inflammatory effect in LPS induced RAW 264.7 cells and mice model. K3G significantly inhibited various pro‐inflammatory mediators like IL‐1&bgr;, NO, PGE2, and LTB4. It upregulated the secretion of anti‐inflammatory cytokine IL‐10. K3G is found to reduce inflammation when studied for parameters like phagocytic index, carrageenan induced paw edema in mice and organ weight. It reduced inflammation in a dose dependent manner both in‐vitro and in‐vivo. Further molecular insights into the study reveal that K3G blocks the phosphorylation of NF‐kB which is key regulator of inflammation, thereby exhibiting anti‐inflammatory potential. Hence, this study permits further investigation to develop K3G as anti‐inflammatory drug. Graphical abstract Figure. No caption available. HighlightsK3G inhibited pro‐inflammatory mediators IL‐1&bgr;, NO, PGE2, LTB4 and upregulated anti‐inflammatory cytokine IL‐10.K3G reduced inflammation when studied for parameters like phagocytic index and carrageenan induced paw edema in mice.K3G exerts its effect by NF‐kB suppressionAnti‐inflammatory effect has been proved both in‐vitro and in‐vivo.All this data permit K3G to be established as an anti‐inflammatory drug.

Murrayanine Attenuates Lipopolysaccharide-induced Inflammation and Protects Mice from Sepsis-Associated Organ Failure

Murrayanine (MK) is the main compound isolated from Murraya koenigii, an aromatic plant belonging to the Rutaceae family, also known as curry leaf tree. Murrayanine was reported to possess potential antioxidant, antimycobacterial and antifungal effects. However, its effect in sepsis remains unclear. This study was designed to investigate the anti‐inflammatory effect of MK using both in vitro and in vivo assay. Results of this study indicated that MK decreased NO, TNF‐α and IL‐6 production in both lipopolysaccharide (LPS)‐stimulated RAW 264.7 cells and murine peritoneal macrophages. Moreover, iNOS and COX‐2 protein expression as well as their downstream product, PGE2, was also decreased effectively in RAW 264.7 cells. Furthermore, MK decreased the phosphorylation of IKB and repressed NF‐kB activity in LPS‐activated RAW 264.7 cells. Additionally, we evaluated MK efficacy in vivo using LPS‐induced sepsis, a systemic inflammation model in mice. Administration of MK inhibits pro‐inflammatory cytokines (TNF‐α and IL‐6) secretion; decreases AST, ALT, BUN and CRE level in mouse sera; mitigates lung, liver and kidney injuries; and also increases LPS‐challenged mice survival rate. Collectively, our results suggest that MK exerts potential as a new anti‐inflammatory and immunosuppressive drug in sepsis treatment.

Biopharmaceutic parameters, pharmacokinetics, transport and CYP-mediated drug interactions of IIIM-017: A novel nitroimidazooxazole analogue with anti-tuberculosis activity

Abstract Nitroimidazoles are emerging as a new class of therapeutic agents with potent anti‐tubercular activity. CSIR‐IIIM has synthesized a novel nitrohydroimidazooxazole (NHIO) analogue, IIIM‐017 with a MIC of 0.37 &mgr;g/ml (against H37Rv). Here, we aim at further exploration of physicochemical properties and preclinical absorption, metabolism, disposition and pharmacokinetics of IIIM‐017. In this study, in silico physicochemical parameters, lipophilicity, permeability, transport, hepatotoxicity, CYP mediated drug interactions and pharmacokinetics of IIIM‐017 were investigated. The results demonstrated that IIIM‐017 exhibited good physicochemical properties, comparable to PA‐824 and OPC‐67683. Caco‐2 transport studies revealed that the compound was highly permeable with Papp of 8.85 × 10− 6 (A–B) and 27.69 × 10− 6 (B–A) cm/s. Caco‐2 cells were also used to study P‐gp mediated transport and inhibition. IIM‐017 exhibited very low intrinsic clearance and no substantial hepatotoxicity in vitro. The compound did not have any inhibitory effect on human CYPs 1A2, 2C9, 2D6, 3A4 and 2C19 up to concentration of 30 &mgr;M. In vivo pharmacokinetics was performed on balb/c mice at 5 mg/kg (p.o) and 2.5 mg/kg (i.v.) and plasma drug concentrations were determined by LC‐MS/MS. The compound showed satisfactory PK parameters in mice. The results insinuate that IIIM‐017 should undergo further development as a potential treatment for tuberculosis. Graphical abstract Figure. No Caption available.

The amino analogue of β-boswellic acid efficiently attenuates the release of pro-inflammatory mediators than its parent compound through the suppression of NF-κB/IκBα signalling axis

Graphical abstract Figure. No Caption available. HighlightsComparative study of &bgr;‐boswellic acid and its analogue, BA‐25 for anti‐inflammatory potential.BA‐25 strongly inhibits the expression iNOS, COX‐2 in LPS‐evoked RAW 264.7 cells.Also suppresses the expression of TNF&agr;, IL‐1&bgr;, IL‐6, PGE2 & LTB4 efficiently.Checks inflammatory mediators via ROS‐dependent activation of NF‐&kgr;B/I&kgr;B&agr; axis.BA‐25 also shows modulatory potential in paw oedema modelled BALB/c mice. &NA; Natural product derivatives have proven to be cutting edge window for drug discovery and development. BA‐25 (3‐&agr;‐o‐acetoxy‐4&bgr;‐amino‐11‐oxo‐24‐norurs‐12‐ene) an amino analogue of &bgr;‐boswellic acid exhibited inhibition of TNF‐&agr; and IL‐6 in THP‐1 cells as demonstrated previously, however, the effect on principal inflammatory mediators such as cyclooxygenase‐2 (COX‐2), inducible nitric oxide synthase (iNOS) and the pathways that mediate this function remains unknown. This study was designed to examine the comparative anti‐inflammatory activity of BA‐25 with its parent compound, &bgr; boswellic acid both in vitro and in vivo. The effect of BA and BA‐25 on suppression of NO, PGE2, LTB4, COX‐2 in LPS‐stimulated RAW 264.7 cells was determined by ELISA, RT‐PCR and ROS by flow cytometry. Phosphorylation of NF‐kBp65, IKB degradation was determined by western blotting and also the nuclear localization of NF‐kBp65 was assessed by immunofluorescence. Furthermore, this study was extended on Carrageenan induced paw oedema modelled BALB/c mice. A novel derivative BA‐25, reported first time notably decreased the LPS (1 &mgr;g/mL) induced upregulation in the transcription of TNF‐&agr;, IL‐6, iNOS and COX‐2. Also the protein expression of iNOS and COX‐2 as well as their downstream products NO and PGE2 respectively, were also decreased efficiently at a concentration of 10 &mgr;M than BA. Moreover, LPS upregulated NF‐kB p65 expression and I&kgr;B degradation was significantly decreased after BA‐25 treatment. In addition, the treatment of BA‐25 also restored the paw oedema and decreased the magnitude of histopathological alterations. Our data together suggested that BA‐25 might be regarded as prospective therapeutic anti‐inflammatory alternative and demands further investigation in pharmacological studies.