Use of Structure-And Ligand-Based Drug Design Tools for the Discovery of Small Molecule Inhibitors of Cysteine Proteases for the Treatment of Malaria and Sars Infection

A wide array of molecular modeling tools were utilized to design and develop inhibitors against cysteine protease of P. Falciparum Malaria and Severe Acute Respiratory Syndrome (SARS). A number of potent inhibitors were developed against cysteine protease and hemoglobinase of P. falciparum , referred as Falcipains (FPs), by the structure-based virtual screening of the focused libraries enriched in soft-electrophiles containing compounds. Twenty one diverse, non-peptidic, low micromolar hits were identified. A combined data mining and combinatorial library synthesis approach was performed to discover analogs of virtual screening hits and establish the structure-activity relationships (SAR). However, the resulting SAR of the identified hits was unusually steep in some cases and could not be explained by a traditional analysis of the interactions (electrostatics, van der Waals or H-bond). To gain insights, a statistical thermodynamic analysis of explicit solvent in the ligand binding domain of FP-2 and FP-3 was performed that explained some of the complex trends in the SAR. Furthermore, the moderate potency of a subset of FP-2 hits was elucidated using quantum mechanics calculations that shoreduced reactivity of the electrophilic center of these hits. In addition, solvent thermodynamics and reactivity analysis also helped to elucidate the complex trends in SAR of peptidomimetic inhibitors of FP-2 and FP-3 synthesized in our laboratory. Multi nanosecond explicit solvent molecular dynamics simulations were carried out using the docking poses of the known inhibitors in the binding site of SARS-3CLpro, a cysteine protease important for replication of SARS virus, to study the overall stability of the binding site interactions as well as identify important changes in the interaction profile that were not apparent from the docking study. Analysis of the simulation studies led to the identification of certain protein-ligand interaction patterns which would be useful in further structure based design efforts against cysteine protease (3CLpro) of SARS

Quantification of cinnarizine and dimenhydrinate in tablet dosage form by simultaneous equation spectrophotometric method

Simple, accurate, precise, reproducible, requiring no prior separation and economical procedures for simultaneous estimation of Cinnarizine(CNZ) and Dimenhydrinate(DMH) in tablet dosage form have been developed. Method employs formation and solving of simultaneous equation using 250 nm and 277 nm as two analytical wavelengths for both drugs in methanol. CNZ and DMH at their respective ? max 250 nm and 277 nm shows linearity in a concentration range of 2-12 ?g /ml and 10-35 ?g /ml. Recovery studies for CNZ 98.9-100.75% and 96.16-100.69% for DMH in case of simultaneous equation method confirming the accuracy of the proposed method. The proposed method is recommended for routine analysis since it is rapid, simple, accurate and also sensitive and specific

Expression in Yeast Links Field Polymorphisms in PfATP6 to in Vitro Artemisinin Resistance and Identifies New Inhibitor Classes

Background. The mechanism of action of artemisinins against malaria is unclear, despite their widespread use in combination therapies and the emergence of resistance. Results. Here, we report expression of PfATP6 (a SERCA pump) in yeast and demonstrate its inhibition by artemisinins. Mutations in PfATP6 identified in field isolates (such as S769N) and in laboratory clones (such as L263E) decrease susceptibility to artemisinins, whereas they increase susceptibility to unrelated inhibitors such as cyclopiazonic acid. As predicted from the yeast model, Plasmodium falciparum with the L263E mutation is also more susceptible to cyclopiazonic acid. An inability to knockout parasite SERCA pumps provides genetic evidence that they are essential in asexual stages of development. Thaperoxides are a new class of potent antimalarial designed to act by inhibiting PfATP6. Results in yeast confirm this inhibition. Conclusions. The identification of inhibitors effective against mutated PfATP6 suggests ways in which artemisinin resistance may be overcom

Analysis of Pfizer Compounds in EPA’s ToxCast Chemicals-Assay Space

The U.S. Environmental Protection Agency (EPA) launched the ToxCast program in 2007 with the goal of evaluating high-throughput <i>in vitro</i> assays to prioritize chemicals that need toxicity testing. Their goal was to develop predictive bioactivity signatures for toxic compounds using a set of <i>in vitro</i> assays and/or <i>in silico</i> properties. In 2009, Pfizer joined the ToxCast initiative by contributing 52 compounds with preclinical and clinical data for profiling across the multiple assay platforms available. Here, we describe the initial analysis of the Pfizer subset of compounds within the ToxCast chemical (<i>n</i> = 1814) and <i>in vitro</i> assay (<i>n</i> = 486) space. An analysis of the hit rate of Pfizer compounds in the ToxCast assay panel allowed us to focus our mining of assays potentially most relevant to the attrition of our compounds. We compared the bioactivity profile of Pfizer compounds to other compounds in the ToxCast chemical space to gain insights into common toxicity pathways. Additionally, we explored the similarity in the chemical and biological spaces between drug-like compounds and environmental chemicals in ToxCast and compared the <i>in vivo</i> profiles of a subset of failed pharmaceuticals having high similarity in both spaces. We found differences in the chemical and biological spaces of pharmaceuticals compared to environmental chemicals, which may question the applicability of bioactivity signatures developed exclusively based on the latter to drug-like compounds if used without prior validation with the ToxCast Phase-II chemicals. Finally, our analysis has allowed us to identify novel interactions for our compounds in particular with multiple nuclear receptors that were previously not known. This insight may help us to identify potential liabilities with future novel compounds

Comparison of efficacy and safety of fixed dose combination of rosuvastatin and choline fenofibrate to fixed dose combination of rosuvastatin and fenofibrate in patients of mixed dyslipidemia: A randomized, open-label, multicentre clinical trial in Indian population

Introduction: This study was conducted to evaluate the safety and efficacy of fixed-dose combination (FDC) of rosuvastatin and choline fenofibrate in comparison to rosuvastatin and fenofibrate FDC among Indian patients of mixed dyslipidemia. This would be a first study evaluating FDC of rosuvastatin and choline fenofibrate in Indian population. Methods: A multicenter, open-label, randomized, active controlled, comparative, parallel-design study was conducted at 12 centers spread all across India. Mixed dyslipidemic patients aged 18–70 years were randomized to FDC of rosuvastatin 10 mg and choline fenofibrate 135 mg (RCF group) and FDC of rosuvastatin 10 mg and fenofibrate 160 mg (RF group) once daily for approximately 180 days. The primary endpoint of study was percentage change in serum triglyceride level at the end of study from baseline. Results: Of 290 patients screened, 240 patients were enrolled in this study (120 patients in each group). At the end of 180 days, there was a significant reduction in triglyceride level in both the groups (−37.7% in RCF group and −37.8% reduction in RF group; P < 0.0001 for both); however, the difference between both the groups was not statistically significant (P = 0.94). Similarly, there was significant increase (P < 0.0001 for both) in high-density lipoprotein cholesterol (HDL-C) in both groups (+17.8% in RCF group and +14.9% in rosuvastatin fenofibrate RF group). Low-density lipoprotein cholesterol (LDL-C), very low-LDL (VLDL-C), and total cholesterol were also reduced significantly in both groups (P < 0.0001). However, the difference between two groups for increase in HDL-C and decrease in LDL-C, VLDL-C, and total cholesterol was not significant. Both the treatments were safe and well tolerated. Conclusion: Overall, FDC of rosuvastatin and choline fenofibrate is as safe and effective as rosuvastatin and fenofibrate combination in Indian patients with mixed dyslipidemia with added advantage improved patient compliance as it can be taken irrespective of intake of food

Quality assessment of different marketed brands of Dasamoolaristam, an Ayurvedic formulation

Arista is a classical Ayurvedic preparation that is typically used as a digestive and cardiotonic. The present Investigation evaluated five different brands of Dasamoolaristam available in the market as per WHO and Indian Pharmacopoeial specifications. Various physicochemical parameters such as alcohol-soluble extractive, water-soluble extractive, total ash, acid-insoluble ash, total solid, and alcohol content were determined. The present investigation reveals that all the preparations contain acceptable levels of alcohol (less than 12% v/v). However, the preparations were found to contain unacceptable limits of microbial load although all showed the absence of Escherichia coli, Salmonella species, and Staphylococcus aureus

Analytical Rp-hplc Method for Development and Validation of Citicoline Sodium and Methylcobalamin in Combined Tablet Formulation

Reverse Phase-High Performance Liquid Chromatography (RP-HPLC) method have been developed and validated for theestimation of Citicoline Sodium and Methylcobalaminin Bulk drug and Pharmaceutical dosage form. The developedmethod is rapid, accurate, precise, simple and economical than the previous methods. The separation was carried outusingPhenomenex Luna , C18, column having 250mm X 4.6mm, 5?m particle size, in isocratic mode, with mobile phase containing Acetonitrile: 0.02M KH2PO4 [60: 40, v/v]. The flow rate is 1 ml / min and effluents are monitored at 554 nm. Chromatogram showed peak at a retention time of 3.8 min for Citicoline sodium and 2.3 min for Methylcobalamin . The method is validated for system suitability, linearity, precision, accuracy specificity, ruggedness, robustness, LOD and LOQ. Recovery of Citicoline Sodium and Methylcobalamin is found to be in the range of 99.76 - 101.79 % and 110.92% and 111.79%, respectively. The LOD and LOQ for estimation of Citicoline Sodium and Methylcobalamin are found to be 0.003 ?g / ml, 0.0.01 ?g / ml, and 0.01?g / ml, 0.05 ?g / ml respectively. Proposed method can be successfully applied for the quantitative determination of Citicoline Sodiumand Methylcobalamin in Bulk drug and Pharmaceutical dosage form

Thin-layer chromatography method for the simultaneous quantification and stability testing of alprazolam and mebeverine in their combined pharmaceutical dosage form

A sensitive, selective and precise high-performance thin-layer chromatographic method was developed and validated for the simultaneous determination of alprazolam and mebeverine, both as bulk drugs and in formulations. The method employed HPTLC aluminium plates that had been pre-coated with silica gel 60F-254 as the stationary phase, while the solvent system was acetone:methanol:acetic acid (6:4:0.1, v/v/v). The Rf values of alprazolam and mebeverine were observed to be 0.80 ± 0.08 and 0.60 ± 0.05, respectively. The densitometric analysis was carried out in absorbance mode at 225 nm. The linear regression analysis for the calibration plots showed a good linear relationship for alprazolam and mebeverine over concentration ranges of 600 to 3600 ng/spot and 1000 to 6000 ng/spot, respectively. The limit of detection and the limit of quantification for alprazolam (mebeverine) were determined to be 63.97 (11.35) ng/spot and 193.85 (34.40) ng/spot, respectively. Alprazolam and mebeverine stock solutions were subjected to acid and alkali hydrolysis, chemical oxidation, dry heat degradation and photo-degradation. Both drugs were found to be susceptible to acid and alkali hydrolysis, chemical oxidation and photo-degradation, whereas both were found to be stable towards dry heat. The degraded product peaks were well resolved from the pure drug peak and displayed a significant difference in their Rf values. Stressed samples were assayed using the developed HPTLC method. The proposed method was validated with respect to linearity, accuracy, precision and robustness. The method was successfully applied to the estimation of alprazolam and mebeverine in marketed formulations. Statistical analysis showed that the method is repeatable, selective, and precise

Evaluating the Role of Multidrug Resistance Protein 3 (MDR3) Inhibition in Predicting Drug-Induced Liver Injury Using 125 Pharmaceuticals

The role of bile salt export protein (BSEP) inhibition in drug-induced liver injury (DILI) has been investigated widely, while inhibition of the canalicular multidrug resistant protein 3 (MDR3) has received less attention. This transporter plays a pivotal role in secretion of phospholipids into bile and functions coordinately with BSEP to mediate the formation of bile acid-containing biliary micelles. Therefore, inhibition of MDR3 in human hepatocytes was examined across 125 drugs (70 of Most-DILI-concern and 55 of No-DILI-concern). Of these tested, 41% of Most-DILI-concern and 47% of No-DILI-concern drugs had MDR3 IC₅₀ values of <50 μM. A better distinction across DILI classifications occurred when systemic exposure was considered where safety margins of 50-fold had low sensitivity (0.29), but high specificity (0.96). Analysis of physical chemical property space showed that basic compounds were twice as likely to be MDR3 inhibitors as acids, neutrals, and zwitterions and that inhibitors were more likely to have polar surface area (PSA) values of <100 Ų and cPFLogD values between 1.5 and 5. These descriptors, with different cutoffs, also highlighted a group of compounds that shared dual potency as MDR3 and BSEP inhibitors. Nine drugs classified as Most-DILI-concern compounds (four withdrawn, four boxed warning, and one liver injury warning in their approved label) had intrinsic potency features of <20 μM in both assays, thereby reinforcing the notion that multiple inhibitory mechanisms governing bile formation (bile acid and phospholipid efflux) may confer additional risk factors that play into more severe forms of DILI as shown by others for BSEP inhibitors combined with multidrug resistance-associated protein (MRP2, MRP3, MRP4) inhibitory properties. Avoiding physical property descriptors that highlight dual BSEP and MDR3 inhibition or testing drug candidates for inhibition of multiple efflux transporters (e.g., BSEP, MDR3, and MRPs) may be an effective strategy for prioritizing drug candidates with less likelihood of causing clinical DILI

Expression in Yeast Links Field Polymorphisms in PfATP6 to in Vitro Artemisinin Resistance and Identifies New Inhibitor Classes

Background. The mechanism of action of artemisinins against malaria is unclear, despite their widespread use in combination therapies and the emergence of resistance. Results. Here, we report expression of PfATP6 (a SERCA pump) in yeast and demonstrate its inhibition by artemisinins. Mutations in PfATP6 identified in field isolates (such as S769N) and in laboratory clones (such as L263E) decrease susceptibility to artemisinins, whereas they increase susceptibility to unrelated inhibitors such as cyclopiazonic acid. As predicted from the yeast model, Plasmodium falciparum with the L263E mutation is also more susceptible to cyclopiazonic acid. An inability to knockout parasite SERCA pumps provides genetic evidence that they are essential in asexual stages of development. Thaperoxides are a new class of potent antimalarial designed to act by inhibiting PfATP6. Results in yeast confirm this inhibition. Conclusions. The identification of inhibitors effective against mutated PfATP6 suggests ways in which artemisinin resistance may be overcome