Novel Findings of Anti-Filarial Drug Target and Structure-Based Virtual Screening for Drug Discovery.

Lymphatic filariasis and onchocerciasis caused by filarial nematodes are important diseases leading to considerable morbidity throughout tropical countries. Diethylcarbamazine (DEC), albendazole (ALB), and ivermectin (IVM) used in massive drug administration are not highly effective in killing the long-lived adult worms, and there is demand for the development of novel macrofilaricidal drugs affecting new molecular targets. A Ca2+ binding protein, calumenin, was identified as a novel and nematode-specific drug target for filariasis, due to its involvement in fertility and cuticle development in nematodes. As sterilizing and killing effects of the adult worms are considered to be ideal profiles of new drugs, calumenin could be an eligible drug target. Indeed, the Caenorhabditis elegans mutant model of calumenin exhibited enhanced drug acceptability to both microfilaricidal drugs (ALB and IVM) even at the adult stage, proving the roles of the nematode cuticle in efficient drug entry. Molecular modeling revealed that structural features of calumenin were only conserved among nematodes (C. elegans, Brugia malayi, and Onchocerca volvulus). Structural conservation and the specificity of nematode calumenins enabled the development of drugs with good target selectivity between parasites and human hosts. Structure-based virtual screening resulted in the discovery of itraconazole (ITC), an inhibitor of sterol biosynthesis, as a nematode calumenin-targeting ligand. The inhibitory potential of ITC was tested using a nematode mutant model of calumenin.

Assessing endgame strategies for the elimination of lymphatic filariasis: A model-based evaluation of the impact of DEC-medicated salt.

Concern is growing regarding the prospects of achieving the global elimination of lymphatic filariasis (LF) by 2020. Apart from operational difficulties, evidence is emerging which points to unique challenges that could confound achieving LF elimination as extinction targets draw near. Diethylcarbamazine (DEC)-medicated salt may overcome these complex challenges posed by the endgame phase of parasite elimination. We calibrated LF transmission models using Bayesian data-model assimilation techniques to baseline and follow-up infection data from 11 communities that underwent DEC salt medication. The fitted models were used to assess the utility of DEC salt treatment for achieving LF elimination, in comparison with other current and proposed drug regimens, during the endgame phase. DEC-medicated salt consistently reduced microfilaria (mf) prevalence from 1% mf to site-specific elimination thresholds more quickly than the other investigated treatments. The application of DEC salt generally required less than one year to achieve site-specific LF elimination, while annual and biannual MDA options required significantly longer durations to achieve the same task. The use of DEC-medicated salt also lowered between-site variance in extinction timelines, especially when combined with vector control. These results indicate that the implementation of DEC-medicated salt, where feasible, can overcome endgame challenges facing LF elimination programs.

On the Thermal Stability of the Diethylcarbamazine-Fortified Table Salt Used in the Control of Lymphatic Filariasis.

Diethylcarbamazine, administered as a water-soluble citrate salt, has been used for more than 50 years as the first-line drug in the treatment of lymphatic filariasis. Mass drug administration programs have been successful in reducing microfilaremia and providing important collateral deworming benefits. One of these initiatives is based on the addition of diethylcarbamazine citrate to table salt. The fortified salt retaining the efficacy of the drug in reducing microfilaremia, but there is little information about its behavior above room temperature. In this study, the thermal stability of diethylcarbamazine, as a free base and a citrate salt, was investigated by differential scanning calorimetry and thermogravimetry under different conditions. Diethylcarbamazine does not release hazardous degradation substances above its melting point. It was also confirmed that this drug is stable at normal cooking temperatures, even when dry heat cooking methods, such as baking or grilling, are considered. However, if the drug is formulated as a salt, as in the case of the citrate, special attention needs to be given to the degradation substances of the counter ion.

Liquid chromatography-mass spectrometry analysis of diethylcarbamazine in human plasma for clinical pharmacokinetic studies.

A sensitive and selective liquid chromatographic method using mass spectrometric detection was developed for the determination of diethylcarbamazine (DEC) in human plasma. DEC and its stable isotope internal standard d3-DEC were extracted from 0.25mL of human plasma using solid phase extraction. Chromatography was performed using a Phenomenex Synergi 4µ Fusion-RP column (2mm×250mm) with gradient elution. The retention time was approximately 4.8min. The assay was linear from 4 to 2200ng/mL. Analysis of quality control samples at 12, 300, and 1700ng/mL (N=15) had interday coefficients of variation of 8.4%, 5.4%, and 6.2%, respectively (N=15). Interday bias results were -2.2%, 6.0%, and 0.8%, respectively. Recovery of DEC from plasma ranged from 84.2% to 90.1%. The method was successfully applied to clinical samples from patients with lymphatic filariasis from a drug-drug interaction study between DEC and albendazole and/or ivermectin.

Diethylcarbamazine attenuates the development of carrageenan-induced lung injury in mice.

Diethylcarbamazine (DEC) is an antifilarial drug with potent anti-inflammatory properties as a result of its interference with the metabolism of arachidonic acid. The aim of the present study was to evaluate the anti-inflammatory activity of DEC in a mouse model of acute inflammation (carrageenan-induced pleurisy). The injection of carrageenan into the pleural cavity induced the accumulation of fluid containing a large number of polymorphonuclear cells (PMNs) as well as infiltration of PMNs in lung tissues and increased production of nitrite and tumor necrosis factor-α and increased expression of interleukin-1ß, cyclooxygenase (COX-2), and inducible nitric oxide synthase. Carrageenan also induced the expression of nuclear factor-κB. The oral administration of DEC (50 mg/Kg) three days prior to the carrageenan challenge led to a significant reduction in all inflammation markers. The present findings demonstrate that DEC is a potential drug for the treatment of acute lung inflammation.

Floating elementary osmotic pump tablet (FEOPT) for controlled delivery of diethylcarbamazine citrate: a water-soluble drug.

The present work investigates the feasibility of the design of a novel floating elementary osmotic pump tablet (FEOPT) to prolong the gastric residence of a highly water-soluble drug. Diethylcarbamazine citrate (DEC) was chosen as a model drug. The FEOPT consisted of an osmotic core (DEC, mannitol, and hydrophilic polymers) coated with a semipermeable layer (cellulose acetate) and a gas-generating gelling layer (sodium bicarbonate, hydrophilic polymers) followed by a polymeric film (Eudragit RL 30D). The effect of formulation variables such as concentration of polymers, types of diluent, and coat thickness of semipermeable membrane was evaluated in terms of physical parameters, floating lag time, duration of floatation, and in vitro drug release. The Fourier transform infrared and X-ray diffraction analysis were carried out to study the physicochemical changes in the drug excipients powder blend. The integrity of the orifice and polymeric film layer was confirmed from scanning electron microscopy image. All the developed FEOPT showed floating lag time of less than 8 min and floating duration of 24 h. A zero-order drug release could be attained for DEC. The formulations were found to be stable up to 3 months of stability testing at 40°C/75% relative humidity.

Rural-urban population movement, community context and impact on DEC fortified salt program to eliminate lymphatic filariasis.

Diethylcarbamazinecitrate (DEC) salt in conjunction with annual single-dose mass drug administration (MDA) with DEC tablets can be considered as potential option to hasten the process of Lymphatic Filariasis (LF) elimination. Consumption of DEC tablet/salt by at least 80% of the endemic population is crucial in achieving elimination in five years. This study examines the determinants of rural-urban population movement and its implication on DEC fortified salt program to control LF. Data was collected through questionnaire from 150 each movers and non-movers from 10 randomly selected villages and also using Key informant (KI) interviews in Villupuram district in Tamil Nadu. Households with at least one family member engaged in movement at any point of time in the previous year, range from 24 - 43% in different villages. Knowledge on cause, control, ongoing LF elimination programs and compliance with DEC tablets (28.7%) and salt (30%) were significantly higher (p < 0.05) among non-movers than movers (4.7% and 3.3% respectively). In order to achieve the goal of elimination of LF by 2020, measures need to be undertaken to ensure that the social mobilization activities and LF intervention programs need to cover the 24-43% of mobile population.

Fluorescent diethylcarbamazine analogues: sites of accumulation in Brugia malayi.

New fluorescein and rhodamine B-labeled antifilarial drug DEC analogues for use in drug localization studies with confocal microscopy have been prepared by a high-yield three-step synthesis. The resulting beta-amine-substituted DEC analogue has a single ethyl substituent which is beta-aminated to accommodate the fluorophore of either fluorescein isothiocyananate or rhodamine B. Confocal microscopy is used to show that the drug accumulates in the adult filarial worms in the pharynx, esophagus, and near the nerve ring of all adults, as well as in the uteri and vulva and the testes of the females and males.

Reaction crystallization of pharmaceutical molecular complexes.

A mechanism for cocrystal synthesis is reported whereby nucleation and growth of cocrystals are directed by the effect of the cocrystal components on reducing the solubility of the molecular complex to be crystallized. The carbamazepine:nicotinamide cocrystal (CBZ:NCT) was chosen as a model system to study the reaction cocrystallization pathways and kinetics in aqueous and organic solvents. Fiber optic Raman spectroscopy and Raman microscopy were used for in situ monitoring of the cocrystallization in macroscopic and microscopic scales in solutions, suspensions, slurries, and wet solid phases of cocrystal components. This study demonstrates the advantages of reaction cocrystallization methods to develop rational approaches for high-throughput screening of cocrystals that can be transferable to control batch and continuous cocrystallization processes.

Estudio de preformulación del citrato de dietilcarbamazina / Study of diethycarbamazine citrate preformulation

Se realizó el estudio de preformulación del citrato de dietilcarbamazina con el objetivo de conocer las características físico-químicas y tecnológicas de este fármaco para el posterior desarrollo de una tableta de 50 mg de dosis, el cual incluyó la determinación del tamaño de partícula micronizada y sin micronizar según el método de microscopia óptica, de manera que quedó demostrado la influencia que presenta éste en la disolución del fármaco in vitro. Se efectuó el estudio de incompatibilidad principio activo-excipientes mediante el método de calorimetría diferencial de barrido; no se encontró ninguna interacción entre éstos, en las condiciones de trabajo experimentales. Se estudiaron otras propiedades físico-químicas y tecnológicas del fármaco (AU)

Estudio de preformulación del citrato de dietilcarbamazina / Study of diethycarbamazine citrate preformulation

Se realizó el estudio de preformulación del citrato de dietilcarbamazina con el objetivo de conocer las características físico-químicas y tecnológicas de este fármaco para el posterior desarrollo de una tableta de 50 mg de dosis, el cual incluyó la determinación del tamaño de partícula micronizada y sin micronizar según el método de microscopia óptica, de manera que quedó demostrado la influencia que presenta éste en la disolución del fármaco in vitro. Se efectuó el estudio de incompatibilidad principio activo-excipientes mediante el método de calorimetría diferencial de barrido; no se encontró ninguna interacción entre éstos, en las condiciones de trabajo experimentales. Se estudiaron otras propiedades físico-químicas y tecnológicas del fármaco

Modes of action of anthelmintic drugs.

Modes of action of anthelmintic drugs are described. Some anthelmintic drugs act rapidly and selectively on neuromuscular transmission of nematodes. Levamisole, pyrantel and morantel are agonists at nicotinic acetylcholine receptors of nematode muscle and cause spastic paralysis. Dichlorvos and haloxon are organophosphorus cholinesterase antagonists. Piperazine is a GABA (gamma-amino-butyric acid) agonist at receptors on nematode muscles and causes flaccid paralysis. The avermectins increase the opening of glutamate-gated chloride (GluCl) channels and produce paralysis of pharyngeal pumping. Praziquantel has a selective effect on the tegument of trematodes and increases permeability of calcium. Other anthelmintics have a biochemical mode of action. The benzimidazole drugs bind selectively to beta-tubulin of nematodes, cestodes and fluke, and inhibit microtubule formation. The salicylanilides: rafoxanide, oxyclozanide, brotianide and closantel and the substituted phenol, nitroxynil, are proton ionophores. Clorsulon is a selective antagonist of fluke phosphoglycerate kinase and mutase. Diethylcarbamazine blocks host, and possibly parasite, enzymes involved in arachidonic acid metabolism, and enhances the innate, nonspecific immune system.

Specific gas chromatographic analysis of diethylcarbamazine in human plasma using solid-phase extraction.

Diethylcarbamazine (DEC, 1-diethylcarbamyl-4-methylpiperazine) is an antiparasitic piperazine derivative used in the treatment of lymphatic filariasis. DEC-N-oxide is a major metabolite in humans which has antifilarial activity. Gas chromatographic analysis of DEC in plasma can be complicated by the presence of the metabolite, since the thermally unstable DEC-N-oxide is converted to a material which coelutes with DEC under the conditions of the analysis. We now report a method to separate DEC-N-oxide from DEC in plasma using solid-phase extraction with subsequent gas chromatographic analysis using a nitrogen specific detector. 1-Diethylcarbamyl-4-ethylpiperazine (E-DEC) was the internal standard. The standard curve of DEC is linear in the range of 10 to 200 ng/ml. The limit of detection is 4 ng/ml. Reproducibility at 10, 100 and 200 ng/ml concentration points of the standard curve gives coefficients of variation of 6.1%, 7.8% and 1.6%, respectively. Recovery following solid-phase extraction is 99.3% for DEC and 94.8% for the internal standard. This sensitive and specific analytical method is suitable for pharmacokinetic studies of DEC.

Development of a competitive enzyme-linked immunosorbent assay for diethylcarbamazine.

A sensitive and reproducible competitive enzyme-linked immunosorbent assay (ELISA) for the determination of the concentration of diethylcarbamazine (DEC) in biological fluids was developed. Since DEC has no functional group to conjugate with bovine serum albumin (BSA), N-(2-aminoethyl)-N-ethyl-4-methyl-1-piperazinecarboxamide (DEC-NH2) was first synthesized. This compound was then converted to carboxyl DEC (DEC-COOH) and conjugated to BSA and to poly-L-lysine for use as immunogen and solid-phase marker, respectively. The competitive ELISA was conducted by simultaneously incubating DEC with mouse anti-DEC antiserum over DEC-poly-L-lysine solid phase. Subsequently, the binding of anti-DEC antibody was detected by using sheep anti-mouse IgG peroxidase conjugate as a tracer. The reliability, determined by the coefficient of variation for inter and intra-assay, was satisfactory. The cross-reactivities of anti-DEC antibodies with DEC metabolites, related compounds and ivermectin were negligible. Using this assay, DEC levels were easily determined in serum of Mongolian jirds (Meriones unguiculatus) up to 4 hours following a single dose of DEC citrate base (100 mg/kg of body weight) via intraperitoneal route.