Exploration of the Plausible Mechanism of Ethambutol Induced Ocular Toxicity by Using Proteomics Informed Physiologically Based Pharmacokinetic (PBPK) Modeling.

PURPOSE: Ethambutol (EMB) is a first-line anti-tubercular drug that is known to cause optic neuropathy. The exact mechanism of its eye toxicity is unknown; however, proposition is metal chelating effect of both EMB and its metabolite 2,2'-(ethylenediamino)-dibutyric acid (EDBA). The latter is formed by sequential metabolism of EMB by alcohol dehydrogenases (ADHs) and aldehyde dehydrogenases (ALDHs). The purpose of this study was to predict the levels of drug and EDBA in the eye using physiologically based pharmacokinetic (PBPK) modeling. METHODS: The PBPK model of EMB was developed using GastroPlus. The intrinsic hepatic clearance of ALDH, calculated by the model, was scaled down using proteomics data to estimate the rate of formation of EDBA in the eye. Additionally, the comparative permeability of EMB and EDBA was assessed by employing in silico and in vitro approaches. The rate of formation of EDBA in the eye and permeability data were then incorporated in a compartmental model to predict the ocular levels of EMB and EDBA. RESULTS: The simulation results of compartmental model highlighted that there was an on-site formation of EDBA upon metabolism of EMB. Furthermore, in silico and in vitro studies revealed that EDBA possessed much lower permeability than EMB. These observations meant that once EDBA was formed in the eye, it was not permeated out and hence achieved higher ocular concentration. CONCLUSION: The on-site formation of EDBA in the eye, its higher local concentration due to lower ocular clearance and its pre-known characteristic to chelate metal species better explains the ocular toxicity shown by EMB.

Eggshell derived brushite bone cement with minimal inflammatory response and higher osteoconductive potential.

Brushite cements are known for excellent osteoconductive and degradation properties, however, its widespread use is limited due to rapid setting time and poor mechanical properties. The eggshell derived calcium phosphates exhibits improved physical and biological properties due to the presence of biologically relevant ions. In this study, eggshell derived brushite cement (EB) was fabricated using ß-tricalcium phosphate synthesized from eggshells. The presence of trace elements in EB prolonged its setting time. The size of brushite crystals in EB was found to be smaller than the pure brushite cement (PB) leading to increased initial compressive strength and higher in vitro degradation rate. The L6 and MG63 cell lines exhibited good biocompatibility with the cement at the end 72 h. In vivo studies of the cements were performed in rat calvarial defect model. Micro CT analysis showed faster degradation and accelerated bone formation in EB filled defect. Histological studies revealed infiltration of inflammatory cells into the implant site for both the cements till 6th week. However, inflammation was found to be significantly reduced at the 12th week in EB compared to PB leading to complete bone bridge formation. Multi-ion substituted EB seems to be a potential bone substitute material with a reasonable setting time for ease of handling, higher mechanical strength, minimal inflammatory response and higher bone regeneration.

Development of Egg Shell Derived Carbonated Apatite Nanocarrier System for Drug Delivery.

Carbonated apatite has a chemical composition quite similar to biological apatite found in native bone. The incorporation of carbonate (CO2-3) ions groups into the apatitic crystal structure can tailor its crystallinity, solubility and biological activity that benefit the bone repair and regeneration. In this study, we report a simple and elegant method of synthesizing carbonated calcium deficient hydroxyapatite (ECCDHA) nanoparticles from egg shell wastes and its efficacy has been compared with synthetic calcium deficient hydroxyapatite (SCDHA) nanoparticles. Egg shell contains about 94% of calcium carbonate. Fourier transform infrared (FT-IR) spectroscopy results confirmed the carbonate substitution in the apatite as B-type and CHNS/O elemental analysis showed 6 wt.% of carbonate content in ECCDHA. Energy dispersive spectroscopy (EDS) analysis confirmed the presence of biologically relevant elements such as magnesium, strontium, fluoride, potassium etc., in ECCDHA inherited from the egg shell. In vitro cell culture studies confirmed that the ECCDHA is cellular compatible and it has enhanced cell adhesion and proliferation of L6 myoblast cells as compared to SCDHA. The potential of ECCDHA suitable for bone drug applications was tested with an antibiotic drug, doxycycline. The results showed higher drug loading and releasing for ECCDHA as compared to SCDHA during the period of study. Based on these results, the ECCDHA may be considered as a potential bone substitute and drug carrier system.

Hybrid drug combination: Anti-diabetic treatment of type 2 diabetic Wistar rats with combination of ellagic acid and pioglitazone.

BACKGROUND: Pioglitazone is an effective drug for the treatment of type 2 diabetes. The drug was suspended from Indian market in June 2013 due to the risk of bladder cancer but was reintroduced in July 2013 because its benefits outweighed the risks. The risks associated with pioglitazone can be minimized if its dose is reduced. HYPOTHESIS: Ellagic acid, a polyphenolic antioxidant, is reported to reduce blood sugar in diabetic rats. The mechanism of anti-diabetic action of ellagic acid is not known. Drugs with same pharmacological action but different mechanism may act in synergistic way when combined together. The combination of ellagic acid with pioglitazone could enhance its activity and hence reduce its dose. STUDY DESIGN AND METHODS: Diabetes was induced in Wistar rats by intraperitoneal administration of 175 mg of nicotinamide/kg body weight in combination with 65 mg of streptozotocin/kg body weight and then fed with high fat diet for 4 weeks (Group II-VII). Non-diabetic rats were fed with normal chow diet (Group I). Group I and II received vehicle only whereas group III to VII received ellagic acid, pioglitazone or their combination. The treatment was given orally once a day for 21 days. RESULTS: The induction of type 2 diabetes in rats caused increase in blood glucose, LDL, triglyceride, and cholesterol and decrease in HDL. The diabetic rats showed improvement in hyperglycemia, dyslipidemia, liver and kidney function markers after treatment with ellagic acid, pioglitazone or their combinations. A combination of 10 mg of ellagic acid/kg BW with 10 mg of pioglitazone/kg BW resulted in significant improvement in all the biochemical parameters when compared to any of the individual treatment. The treatment of diabetic rats with the same combination significantly increased the expression levels of GLUT4, PPAR-γ and adiponectin in skeletal muscle. CONCLUSION: The present study indicates that the dose of pioglitazone, required to achieve normoglycemia in diabetic rats, can be reduced by two folds by combining it with ellagic acid. The combinations reported here can be superior, since dose associated side effects and toxicity of the pioglitazone can be reduced.

Mutation at G103 of MtbFtsZ Altered their Sensitivity to Coumarins.

Coumarins are natural polyphenol lactones comprising of fused rings of benzene and α-pyrone. The current study demonstrates the inhibitory effect of coumarins with various substitutions on Mycobacterium smegmatis mc2 155. We also demonstrate the effect of pomegranate (Punica granatum) extract containing ellagic acid, on M. smegmatis as well as their affect on MtbFtsZ (FtsZ from Mycobacterium tuberculosis). The ellagic acid extracts from pomegranate peels inhibit mycobacteria with a MIC of 25 µM and 0.3 to 3.5 mg/mL, respectively, but failed to inhibit the polymerization of MtbFtsZ. However, the coumarins were shown to inhibit the polymerization and GTPase activity of the protein as well as have an inhibitory affect on M. smegmatis mc2 155. Docking of the most active coumarin (7-Dimethyl-4-methyl coumarin with MIC of 38.7 µM) to the GTP binding site suggests that it interacted with the G103 residue. Based on the docking results two mutants of varying activity (G103S and G103A) were constructed to elucidate the interaction of MtbFtsZ and coumarins. Mutation of G103 with Serine (a bulky group) results in an inactive mutant and substitution with alanine produces a variant that retains most of the activity of the wild type. There is a disruption of the protofilament formation of the MtbFtsZ upon interaction with coumarins as demonstrated by TEM. The coumarins increase the length of Mycobacteria five times and MtbFtsZ localization is disturbed. The mutant proteins altered the GTPase and polymerization activity of coumarins as compared to wild type protein. The results here support that coumarins inhibit proliferation of Mycobacteria by targeting the assembly of MtbFtsZ and provide the possible binding site of coumarins on MtbFtsZ. This study may aid in the design of natural products as anti-mycobacterial agents. The currently reported GTP analogs for FtsZ are toxic to the human cell lines; natural coumarins targeting the GTP binding site of MtbFtsZ may hold promise as an important drug lead for tuberculosis treatment.

FtsZ inhibition and redox modulation with one chemical scaffold: Potential use of dihydroquinolines against mycobacteria.

The dual effect of FtsZ inhibition and oxidative stress by a group of 1,2-dihydroquinolines that culminate in bactericidal effect on mycobacterium strains is demonstrated. They inhibited the non-pathogenic Mycobacterium smegmatis mc(2) 155 with MIC as low as 0.9 µg/mL and induced filamentation. Detailed studies revealed their ability to inhibit polymerization and GTPase activity of MtbFtsZ (Mycobacterial filamentous temperature sensitive Z) with an IC50 value of ∼40 µM. In addition to such target specific effects, these compounds exerted a global cellular effect by causing redox-imbalance that was evident from overproduction of ROS in treated cells. Such multi-targeting effect with one chemical scaffold has considerable significance in this era of emerging drug resistance and could offer promise in the development of new therapeutic agents against tuberculosis.

Phytochemicals as inhibitors of bacterial cell division protein FtsZ: coumarins are promising candidates.

Naturally occurring phytochemicals with reported antibacterial activity were screened for their ability to inhibit the bacterial cell division protein Escherichia coli FtsZ. Among the representative compounds, coumarins inhibit the GTPase and polymerization activities of this protein effectively. Further screening with ten coumarin analogs we identified two promising candidates, scopoletin and daphnetin. The former is found to inhibit the GTPase activity of the protein in a noncompetitive manner. Docking of these coumarins with the modeled protein indicate that they bind to T7 loop, which is different from the GTP-binding site (active site), thereby supporting the experimental data. Lowest binding energy is obtained with scopoletin. 3D QSAR indicates the need for groups such as hydroxyl, diethyl, or dimethyl amino in the 7th carbon for enhanced activity. None of the coumarins exhibited cytotoxicity against NIH/3T3 and human embryonic kidney cell lines. The length of Bacillus subtilis increases in the presence of these compounds probably due to the lack of septum formation. Results of this study indicate the role of coumarins in halting the first step of bacterial cell division process.

Non-peptidyl insulin mimetics as a potential antidiabetic agent.

Insulin has an important role in the maintenance of blood sugar. It is the only available therapeutic agent for the treatment of type 1 diabetes mellitus and there is a dire need for an oral substitute. Different categories of compounds including mono and di substituted benzoquinones, vanadium based compounds and natural products have been reported to cause insulin-like effects either by increasing phosphorylation of insulin receptor (IR) or inhibiting the protein tyrosine phosphatases. This review summarizes the development of various insulin mimetics with special emphasis on their structure-activity relationships and various biological actions they produce.