LC-MS/MS method for the quantitation of decitabine and venetoclax in rat plasma after SPE: Application to pharmacokinetic study.

This study developed a novel, sensitive and selective LC-MS/MS method for the concurrent determination of DCB and VTX in rat plasma using encorafenib as internal standard (IS). To identify DCB, VTX, and IS, the positive multiple reaction monitoring (MRM) mode was used. Chromatographic separation was carried out using a reversed-phase Agilent Eclipse plus C18 column (100 mm × 2.1 mm, 3.5 µm) and an isocratic mobile phase made up of water with 0.1% formic acid and acetonitrile (50:50, v/v, pH 3.2) at a flow rate of 0.30 mL/min for 3.0 min. Prior to analysis, the DCB and VTX with the IS were extracted from plasma using the solid-phase extraction (SPE) method. High recovery rates for DCB, VTX and IS were achieved using the C18 cartridge without interference from plasma endogenous. The developed method was validated as per the FDA guidelines over a linear concentration range in rat plasma from 5-3000 and 5-1000 ng/mL for DCB and VTX, respectively with r2 ≥ 0.998. For both drugs, the lower limits of detection (LLOD) were 2.0 ng/mL. After the HLOQ sample was injected, less than 20% of the LLOQ of DCB, VTX, and less than 5% of the IS carry-over in the blank sample was attained. The overall recoveries of DCB and VTX from rat plasma were in the range of 90.68-97.56%, and the mean RSD of accuracy and precision results was ≤6.84%. For the first time, the newly developed approach was effectively used in a pharmacokinetic study on the simultaneous oral administration of DCB and VTX in rats that received 15.0 mg/kg of DCB and 100.0 mg/kg of VTX.

A Liquid Chromatography Tandem Mass Spectrometry Method for the Simultaneous Estimation of the Dopamine Receptor Antagonist LE300 and Its N-methyl Metabolite in Plasma: Application to a Pharmacokinetic Study.

LE300 is a novel dopamine receptor antagonist used to treat cocaine addiction. In the current study, a sensitive and fast liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been established and validated for the simultaneous analysis of LE300 and its N-methyl metabolite, MLE300, in rat plasma with an application in a pharmacokinetic study. The chromatographic elution of LE300, MLE300, and Ponatinib (IS, internal standard), was carried out on a 50 mm C18 analytical column (ID: 2.1 mm and particle size: 1.8 µm) maintained at 22 ± 2 °C. The run time was 5 min at a flow rate of 0.3 mL/min. The mobile phase consisted of 42% aqueous solvent (10 mM ammonium formate, pH: 4.2 with formic acid) and 58% organic solvent (acetonitrile). Plasma samples were pretreated using protein precipitation with acetonitrile. The electrospray ionization (ESI) source was used to generate an ion-utilizing positive mode. A multiple reaction monitoring mass analyzer mode was utilized for the quantification of analytes. The linearity of the calibration curves in rat plasma ranged from 1 to 200 ng/mL (r2 = 0.9997) and from 2 to 200 ng/mL (r2 = 0.9984) for LE300 and MLE300, respectively. The lower limits of detection (LLOD) were 0.3 ng/mL and 0.7 ng/mL in rat plasma for LE300 and MLE300, respectively. Accuracy (RE%) ranged from -1.71% to -0.07% and -4.18% to -1.48% (inter-day), and from -3.3% to -1.47% and -4.89% to -2.15% (intra-day) for LE300 and MLE300, respectively. The precision (RSD%) was less than 2.43% and 1.77% for the inter-day, and 2.77% and 1.73% for intra-day of LE300 and MLE300, respectively. These results are in agreement with FDA guidelines. The developed LC-MS/MS method was applied in a pharmacokinetic study in Wistar rats. Tmax and Cmax were 2 h and 151.12 ± 12.5 ng/mL for LE300, and 3 h and 170.4 ± 23.3 ng/mL for MLE300.

A Rapid and Sensitive Liquid Chromatography-Tandem Mass Spectrometry Bioanalytical Method for the Quantification of Encorafenib and Binimetinib as a First-Line Treatment for Advanced (Unresectable or Metastatic) Melanoma-Application to a Pharmacokinetic Study.

The combination regimen targeting BRAF and MEK inhibition, for instance, encorafenib (Braftovi™, ENF) plus binimetinib (Mektovi®, BNB), are now recommended as first-line treatment in patients with unresectable or metastatic melanoma with a BRAF V600-activating mutation. Patients treated with combination therapy of ENF and BNB demonstrated a delay in resistance development, increases in antitumor activity, and attenuation of toxicities compared with the activity of either agent alone. However, the pharmacokinetic profile of the FDA-approved ENF and BNB is still unclear. In this study, a rapid and sensitive LC-MS/MS bioanalytical method for simultaneous quantification of ENF and BNB in rat plasma was developed and validated. Chromatography was performed on an Agilent Eclipse plus C18 column (50 mm × 2.1 mm, 1.8 µm), with an isocratic mobile phase composed of 0.1% formic acid in water/acetonitrile (67:33, v/v, pH 3.2) at a flow rate of 0.35 mL/min. A positive multiple reaction monitoring (MRM) mode was chosen for detection and the process of analysis was run for 2 min. Plasma samples were pre-treated using protein precipitation with acetonitrile containing spebrutinib as the internal standard (IS). Method validation was assessed as per the FDA guidelines for the determination of ENF and BNB over concentration ranges of 0.5-3000 ng/mL (r2 ≥ 0.997) for each drug (plasma). The lower limits of detection (LLOD) for both drugs were 0.2 ng/mL. The mean relative standard deviation (RSD) of the results for accuracy and precision was ≤ 7.52%, and the overall recoveries of ENF and BNB from rat plasma were in the range of 92.88-102.28%. The newly developed approach is the first LC-MS/MS bioanalytical method that can perform simultaneous quantification of ENF and BNB in rat plasma and its application to a pharmacokinetic study. The mean result for Cmax for BNB and ENF was found to be 3.43 ± 0.46 and 16.42 ± 1.47 µg/mL achieved at 1.0 h for both drugs, respectively. The AUC0-∞ for BNB and ENF was found to be 18.16 ± 1.31 and 36.52 ± 3.92 µg/mL.h, respectively. On the other hand, the elimination half-life (t1/2kel) parameters for BNB and ENF in the rat plasma were found to be 3.39 ± 0.43 h and 2.48 ± 0.24 h, and these results are consistent with previously reported values.

S-substituted 2-mercaptoquinazolin-4(3H)-one and 4-ethylbenzensulfonamides act as potent and selective human carbonic anhydrase IX and XII inhibitors.

We evaluated the hCA (CA, EC 4.2.1.1) inhibitory activity of novel 4-(2-(2-substituted-thio-4-oxoquinazolin-3(4H)-yl)ethyl)benzenesulfonamides (compounds 2-20) towards the isoforms I, II, IX, and XII. hCA Isoforms were effectively inhibited by most of new compounds comparable to those of AAZ. Compounds 2 and 4 showed interestingly efficient and selective antitumor (hCA IX and hCA XII) inhibitor activities (KIs; 40.7, 13.0, and 8.0, 10.8 nM, respectively). Compounds 4 and 5 showed selective hCA IX inhibitory activity over hCA I (SI; 95 and 24), hCA IX/hCA II (SI; 23 and 5.8) and selective hCA XII inhibitory activity over hCA I (SI; 70 and 44), hCA XII/hCA II, (SI; 17 and 10) respectively compared to AAZ. Compounds 12-17, and 19-20 showed selective inhibitory activity towards hCA IX over hCA I and hCA II, with selectivity ranges of 27-195 and 3.2-19, respectively, while compounds 12, 14-17, and 19 exhibited selective inhibition towards hCA XII over hCA I and hCA II, with selectivity ratios of 48-158 and 5.4-31 respectively, compared to AAZ. Molecular docking analysis was carried out to investigate the selective interactions among the most active derivatives, 17 and 20 and hCAs isoenzymes. Compounds 17 and 20, which are highly selective CA IX and XII inhibitors, exhibited excellent interaction within the putative binding site of both enzymes, comparable to the co-crystallized inhibitors.HighlightsQuinazoline-linked ethylbenzenesulfonamides inhibiting CA were synthesised.The new molecules potently inhibited the hCA isoforms I, II, IV, and IX.Compounds 4 and 5 were found to be selective hCA IX/hCA I and hCA IX/hCA II inhibitors.Compounds 4 and 5 were found to be selective hCA XII/hCA I and hCA XII/hCA II inhibitors.Compounds 12-17, 19, and 20 were found to be selective hCA IX/hCA I and hCA IX/hCA II inhibitors.Compounds 12, 14-17, 19 were found to be selective hCA XII/hCA I and hCA XII/hCA II inhibitors.Compounds 4 and 5 are selective hCA IX and XII inhibitors over hCA I (selectivity ratios of 95, 23, and 24, 5.8, respectively) and hCA II (selectivity ratios of 70, 17, and 44, 10 respectively). Compounds 12-17, and 19-20 are selective hCA IX inhibitors over hCA I (selectivity ratios of 27-195) and hCA II (selectivity ratios of 3.2-19). Compounds 12, 14-17 and 19 are also selective hCA XII inhibitors over hCA I (selectivity ratios of 48-158) and hCA II (selectivity ratios of 5.4-31).

Synthesis, anti-inflammatory, cytotoxic, and COX-1/2 inhibitory activities of cyclic imides bearing 3-benzenesulfonamide, oxime, and ß-phenylalanine scaffolds: a molecular docking study.

Cyclic imides containing 3-benzenesulfonamide, oxime, and ß-phenylalanine derivatives were synthesised and evaluated to elucidate their in vivo anti-inflammatory and ulcerogenic activity and in vitro cytotoxic effects. Most active anti-inflammatory agents were subjected to in vitro COX-1/2 inhibition assay. 3-Benzenesulfonamides (2-4, and 9), oximes (11-13), and ß-phenylalanine derivative (18) showed potential anti-inflammatory activities with 71.2-82.9% oedema inhibition relative to celecoxib and diclofenac (85.6 and 83.4%, respectively). Most active cyclic imides 4, 9, 12, 13, and 18 possessed ED50 of 35.4-45.3 mg kg-1 relative to that of celecoxib (34.1 mg kg-1). For the cytotoxic evaluation, the selected derivatives 2-6 and 8 exhibited weak positive cytotoxic effects (PCE = 2/59-5/59) at 10 µM compared to the standard drug, imatinib (PCE = 20/59). Cyclic imides bearing 3-benzenesulfonamide (2-5, and 9), acetophenone oxime (11-14, 18, and 19) exhibited high selectivity against COX-2 with SI > 55.6-333.3 relative to that for celecoxib [SI > 387.6]. ß-Phenylalanine derivatives 21-24 and 28 were non-selective towards COX-1/2 isozymes as indicated by their SI of 0.46-0.68.

Synthesis of benzensulfonamides linked to quinazoline scaffolds as novel carbonic anhydrase inhibitors.

Carbonic anhydrase (CA) inhibitory activities of newly synthesized quinazoline-linked benzensulfonamides 10-29, 31, 32, 35, 36, and 45-51 against human CA (hCA) isoforms I, II, IX, and XII were measured and compared to that of acetazolamide (AAZ) as a standard inhibitor. Potent selective inhibitory activity against hCA I was exerted by compounds 14, 15, 17, 19, 20, 21, 24, 25, 28, 29, 31, 35, 45, 47, 49, and 51 with inhibition constant (KIs) values of 39.4-354.7 nM that were nearly equivalent or even greater than that of AAZ (KI, 250.0 nM). Compounds 15, 20, 24, 28, 29, 45 and 47 proved to have inhibitory activities against hCA II with (KIs, 0.73-16.5 nM) that were similar or improved to that of AAZ (KI, 12.0 nM). Compounds 13-29, 31-32, and 45-51 displayed potent hCA IX inhibitory activities (KIs, 1.6-32.2 nM) that were more effective than or nearly equal to AAZ (KI, 25.0 nM). Compounds 14, 15, 20, 21, 26, 45, and 47 exerted potent hCA XII inhibitory activities (KIs, 5.2-9.2 nM), indicating similar CAI activities as compared to that of AAZ (KI, 5.7 nM).

Synthesis of hapten, generation of specific polyclonal antibody and development of ELISA with high sensitivity for therapeutic monitoring of crizotinib.

Crizotinib (CZT) is a potent drug used for treatment of non-small cell lung cancer (NSCLC); however, its circulating concentration variability has been associated with acquired resistance and toxicity, restricting the success of cancer treatment. As such, the development of an assay that monitors CZT plasma concentrations in patients is a valuable tool in cancer treatment. In this study, a hapten of CZT was synthesized by introducing the acetohydrazide moiety as a spacer into the chemical structure of CZT. The chemical structure of the CZT acetohydrazide (hapten) was confirmed by mass, 1H-, and 13C-NMR spectrometric techniques. The hapten was coupled to each of bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH) proteins by ethyl-3-(3-dimethylaminopropyl) carbodiimide as a coupling reagent. CZT-KLH conjugate was used for immunization and generation of a polyclonal antibody recognizing CZT with high affinity (IC50 = 0.5 ng/mL). The polyclonal antibody was used in the development of an ELISA for determination of CZT. The ELISA involved a competitive binding reaction between CZT, in its samples, and immobilized CZT-BSA conjugate for the binding sites on a limited amount of the anti-CZT antibody. The assay limit of detection was 0.03 ng/mL and the working range was 0.05 - 24 ng/mL. Analytical recovery of CZT from spiked plasma was 101.98 ± 2.99%. The precisions of the assay were satisfactory; RSD was 3.2 - 6.5% and 4.8 - 8.2%, for the intra- and inter-assay precision, respectively. The assay is superior to all the existing chromatographic methods for CZT in terms of its procedure simplicity, convenience, and does not require treatment of plasma samples prior to the analysis. The proposed ELISA is anticipated to effectively contribute to the therapeutic monitoring of CZT in clinical settings.

Development and validation of an ELISA with high sensitivity for therapeutic monitoring of afatinib.

AIM: To support the therapeutic drug monitoring of afatinib (AFT), an ELISA was required. RESULTS: A hapten for AFT was prepared and linked to each of BSA and KLH proteins by diazotization/coupling reaction. A polyclonal antibody recognizing AFT with high affinity (IC50 = 40 ng ml-1) was generated and used in the development of a competitive ELISA for quantitation of AFT in plasma samples. The assay limit of detection was 2 ng ml-1. The assay accuracy and precision were proved. CONCLUSION: The assay is an appropriate alternative to the existing LC-MS/MS assays for AFT and it is anticipated to effectively contribute to the therapeutic drug monitoring of AFT in clinical settings.

Synthesis, antitumour activities and molecular docking of thiocarboxylic acid ester-based NSAID scaffolds: COX-2 inhibition and mechanistic studies.

A new series of NSAID thioesters were synthesized and evaluated for their in vitro antitumor effects against a panel of four human tumor cell lines, namely: HepG2, MCF-7, HCT-116 and Caco-2, using the MTT assay. Compared to the reference drugs 5-FU, afatinib and celecoxib, compounds 2b, 3b, 6a, 7a, 7b and 8a showed potent broad-spectrum antitumor activity against the selected tumour cell lines. Accordingly, these compounds were selected for mechanistic studies about COX inhibition and kinase assays. In vitro COX-1/COX-2 enzyme inhibition assay results indicated that compounds 2b, 3b, 6a, 7a, 7b, 8a and 8 b selectively inhibited the COX-2 enzyme (IC50 = ∼0.20-0.69 µM), with SI values of (>72.5-250) compared with celecoxib (IC50 = 0.16 µM, COX-2 SI: > 312.5); however, all the tested compounds did not inhibit the COX-1 enzyme (IC50 > 50 µM). On the other hand, EGFR, HER2, HER4 and cSrc kinase inhibition assays were evaluated at a 10 µM concentration. The selected candidates displayed limited activities against the various tested kinases; the compounds 2a, 3b, 6a, 7a, 7b and 8a showed no activity to weak activity (% inhibition = ∼0-10%). The molecular docking study revealed the importance of the thioester moiety for the interaction of the drugs with the amino acids in the active sites of COX-2. The aforementioned results indicated that thioester based on NSAID scaffolds derivatives may serve as new antitumor compounds.

Novel spectrophotometric method for determination of cinacalcet hydrochloride in its tablets via derivatization with 1,2-naphthoquinone-4-sulphonate.

This study represents the first report on the development of a novel spectrophotometric method for determination of cinacalcet hydrochloride (CIN) in its tablet dosage forms. Studies were carried out to investigate the reaction between CIN and 1,2-naphthoquinone-4-sulphonate (NQS) reagent. In alkaline medium (pH 8.5), an orange red-colored product exhibiting maximum absorption peak (λmax) at 490 nm was produced. The stoichiometry and kinetic of the reaction were investigated and the reaction mechanism was postulated. This color-developing reaction was employed in the development of a simple and rapid visible-spectrophotometric method for determination of CIN in its tablets. Under the optimized reaction conditions, Beer's law correlating the absorbance with CIN concentration was obeyed in the range of 3 - 100 µg/ml with good correlation coefficient (0.9993). The molar absorptivity (ε) was 4.2 × 105 l/mol/cm. The limits of detection and quantification were 1.9 and 5.7 µg/ml, respectively. The precision of the method was satisfactory; the values of relative standard deviations (RSD) did not exceed 2%. No interference was observed from the excipients that are present in the tablets. The proposed method was applied successfully for the determination of CIN in its pharmaceutical tablets with good accuracy and precisions; the label claim percentage was 100.80 - 102.23 ± 1.27 - 1.62%. The results were compared favorably with those of a reference pre-validated method. The method is practical and valuable in terms of its routine application in quality control laboratories.