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•New 1,3,5-triazinyl chalcone hybrids were designed and synthesized.•Cisplatin with hybrids 10 and 12 resulted in inhibition of A549 cells viability.•Analytical and apoptosis ...induction assays for DNA binding for 10 and 12.•Molecular docking revealed the binding mode of 10 and 12 to DNA dodecamer.•10 and 12 potentiated the anticancer effect of cisplatin against A549 cells.
In the pursuit of new compounds for co-treatment to enhance the anticancer efficacy of cisplatin against lung adenocarcinoma, a series of chalcone-tethered 1,3,5-triazines was designed and synthesized. MTT assay was used to evaluate the anticancer activity of the combinations in which two hybrids 10 and 12 were found to significantly inhibit A549 cancer cells viability and their IC50 values were 24.5 and 17 µM, respectively in reference to cisplatin (IC50 = 21.5 µM). The combined effect of cisplatin with each of 10 and 12 was analyzed according to Chou-Talalay method against both A549 and normal human fibroblast cells. Mechanistic studies employing MALDI-TOF MS and fluorescence spectroscopy using Evagreen probe inferred that 10 and 12 induced DNA double strand breaks in contrast to cisplatin which induces DNA interstrand cross-links. Also, DNA damage kinetics study demonstrated the difference in the rate of DNA damage induced by both 10 and 12 alone and in combination with cisplatin. Further Annexin V-FITC/propidium iodide dual staining assay provided evidence that 10 and 12 induced apoptosis via different pattern to cisplatin and their combination with cisplatin promoted more cells to enter late apoptosis and necrosis. Molecular docking of 10 and 12 in the active pocket of DNA dodecamer displayed their binding modes with higher number of stable hydrogen bond donor as well as π-H interactions in reference to the original ligand.
•A simple, inexpensive, rapid and eco-friendly HPTLC method was developed for the simultaneous analysis of sofosbuvir and ledipasvir.•Determination of sofosbuvir and ledipasvir in pharmaceutical ...dosage form and biological fluids, including plasma and urine samples.•Determination of sofosbuvir and ledipasvir in the presence of their degradation products after performing forced degradation studies.•A degradation pathway of sofosbuvir and ledipasvir was suggested under acidic, alkaline and oxidative conditions.•Advantages of the proposed method over previously published methods are discussed.
Recently, many single-pill combinations are used as a promising choice in hepatitis C treatment. However, this trend made a serious challenge to drug analysts due to difficulty of analysis of two or more drugs simultaneously. In this study, sofosbuvir (SF) and ledipasvir (LD) were simultaneously analyzed by developing a simple, inexpensive, rapid and eco-friendly high-performance thin layer chromatographic (HPTLC) method with densitometric detection. Separation was carried out using aluminum HPTLC plates silica gel 60 F254 as the stationary phase developed using the mobile phase ethyl acetate: methanol: water: glacial acetic acid (30: 1.5: 1: 0.2% v/v). Development was done in a twin-trough chamber followed by densitometric detection of SF and LD at 260 and 320 nm, respectively. The calibration curves for standard solutions showed a rectilinear relationship for SF and LD in the range of 2 –12 and 0.45–6.0 μg band−1, respectively with correlation coefficients > 0.9995 and limits of detection of 0.6 and 0.1 μg band−1 for SF and LD, respectively. While, calibration curves for biological samples showed linear relationships for SF and LD in the range of 1.0–20 and 0.2–6.0 μg mL−1, respectively with correlation coefficients > 0.99 and limits of detection of 0.21 and 0.27 μg mL−1 for SF in plasma and urine samples, respectively and 0.05 and 0.03 μg mL−1 for LD in plasma and urine samples, respectively. Also, it was effectively used in the analysis of the investigated drugs within their pharmaceutical form and biological fluids without any matrix interference with high precision (% RSD < 2%) and good accuracy with% Er value < 2%. Moreover, SF and LD were analyzed along with their degradation products after performing forced degradation studies.
•A simple, inexpensive, eco-friendly universal HPLC method was developed for the simultaneous analysis of sofosbuvir, ledipasvir, daclatasvir, velpatasvir.•Determination of the four studied drugs in ...pharmaceutical dosage form and biological fluids.•Eco-scale and GAPI evaluation proved that the proposed method is an excellent green analytical method.•Advantages of the proposed method over previously published methods are discussed.
This article presents the simultaneous determination of multiple recently approved direct-acting antiviral (DAA) regimens using a sensitive, robust method. Reversed-phase high-performance liquid chromatographic method (RP-HPLC) has been developed to represent a green alternative of other reported methods for the simultaneous determination of the most common four DAAs used for chronic hepatitis C treatment, including sofosbuvir (SF), ledipasvir (LD), daclatasvir (DC), velpatasvir (VP) and tenofovir (TO) as internal standard (IS) in bulk, pharmaceutical dosage form and plasma samples. This method was performed with an environment-friendly mobile phase with the least amount of energy consumption and waste generation. Chromatographic conditions have been optimized in order to obtain well-resolved symmetrical peaks within a reasonable elution time. Separation was achieved using Waters XBridge BEH C18 Column (4.6 × 150 mm) using isocratic elution mode at a flow rate 0.8 mL.min−1 with the mobile phase 0.1% (v/v) triethylamine acidified water with ortho-phosphoric acid (OPA) pH 2.53 and acetonitrile in the ratio (70:30 v/v). Sample extraction was done using diethyl ether for the analyte and IS extraction from the plasma samples. The suggested method was validated within the concentration range (0.4–20 μg.mL−1) for all the studied drugs according to the ICH guidelines. Calibration plots showed a linear relationship for SF, LD, DC and VP with correlation coefficients ≥ 0.9995 and acceptable recoveries. The method has been applied for the analysis of the studied drugs in plasma samples and pharmaceutical dosage forms without any matrix interference and with % RSD less than 2%, indicating the method to be precise. The suggested method was also assessed using the analytical Eco-scale tool and Green Analytical Procedure Index (GAPI) by comparing the method with other reported methods in terms of sample preparation, consuming hazardous reagents, instrument energy consumption and waste generation. Eco-scale and GAPI results proved the greenness of our proposed method. Moreover, the proposed method has the advantage of analyzing several pharmacologically related drugs in the same run simultaneously, which allows the analysis of alternative therapeutic formulations under the same running conditions. Thus, much effort and time is saved for developing a specific method for every pharmaceutical mixture. This promotes the proposed method to be used for both quality control analysis and any bioavailability study for the simultaneous determination of any of the four antiviral drugs in the pharmaceutical formulation or plasma samples without any harmful effect on the environment or human health.
Green analytical chemistry is one of the newest trends in analytical chemistry nowadays targeting the concept of green laboratory practices on chemists and environment. In this text, green practices ...are proposed in this work for the determination of sofosbuvir (SF) and velpatasvir (VP) in their pharmaceutical formulation. The analysis of SF in a binary mixture with VP represents an analytical challenge due to the complete overlapping of the UV spectrum of SF by that of VP. Therefore, the direct absorbance and derivative measurements cannot resolve such interference and failed to determine SF. In this paper, three direct and simple methods were developed for the analysis of SF without any interference from VP without sample pre-treatment. The proposed methods include measuring the second derivative amplitude of the ratio spectrum of the mixture using VP as a divisor, measuring the absorbance difference of the mixture in NaOH solution against its HCl solution, and using the derivative compensation technique. On the other hand, VP was determined specifically in presence of SF by two methods. Firstly, by its reaction with 4-chloro-7-nitrobenzofurazan (NBD-Cl) where the reaction product was measured spectrophotometrically and spectrofluorometrically and secondly through the reaction of VP with 3-methyl-2-benzothiazolinone hydrazone hydrochloride (MBTH). The calibration curves showed good correlation coefficient (
r
2
> 0.999). The developed methods were highly precise with RSD% values less than 2%. The method greenness profile was compared with other published methods by applying the eco-scale protocol. Assessment results proved that our analytical procedure is greener than other reported methods. Moreover, upon comparison with other methods, the proposed methods showed better or comparable sensitivity in addition to being inexpensive and ecofriendly. Accordingly, these methods could be readily applied for quality control purposes as an eco-friendly, simple and efficient analytical tool.
Some receptors and signaling molecules, such as Rho-kinase (ROCK), localize in caveolae. We asked whether the function of histamine receptors (H.sub.1) and 5-hydroxytryptamine (serotonin) receptors ...(5-HT.sub.2A) in bovine tracheal smooth muscle are modified after caveolae disruption and if so, whether the altered ROCK activity plays a role in this modification. Methyl-beta-cyclodextrin (MbetaCD), used to deplete membrane cholesterol, was shown to disrupt caveolae and diminish sustained contractions to histamine (~80%), 5-HT (100%), a-methyl-5-HT (100%), and KCl (~30%). Cholesterol-loaded MbetaCD (CL-MbetaCD) restored the responses to KCl and partially restored the responses to agonists. ROCK inhibition by Y-27632 diminished contractions to histamine (~85%) and 5-HT (~59%). 5-HT or histamine stimulation augmented ROCK activity. These increases were reduced by MbetaCD and partially reestablished by CL-MbetaCD. The increase in intracellular Ca.sup.2+ that was induced by both agonists was reduced by MbetaCD. The presence of caveolin-1 (Cav-1), H.sub.1, 5-HT.sub.2A, and ROCK' was corroborated by immunoblotting of membrane fractions from sucrose gradients and by confocal microscopy. H.sub.1 receptors coimmunoprecipitated with Cav-1 in caveolar and noncaveolar membrane fractions, whereas 5- HT.sub.2A receptors appeared to be restricted to noncaveolar membrane fractions. We conclude that caveolar and cholesterol integrity are indispensable for the proper functionality of the H.sub.1 and 5-HT.sub.2A receptors through their Rho/ROCK signaling.
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