Innovative use of σ and π electron acceptors in the development of three high throughput 96-microwell spectrophotometric assays for crizotinib.

Crizotinib (CZT) is a potent and selective tyrosine kinase inhibitor used for treatment of non-small cell lung cancer (NSCLC). The development of high-throughput assays for its quality control (QC) is very essential to assure its therapeutic benefits. CZT molecule has multiple electron-donating atoms that can contribute to the formation of colored charge-transfer (CT) complex with iodine as σ-electron acceptor and with 2,5-dichloro-3,6-dihydroxy-1,4-benzoquinone (CHBQ) and 7,7,8,8-tetracyanoquinodimethane (TCNQ) as π-electron acceptors. These reactions were prospective basis for development of three innovative 96-microwell-based spectrophotometric assays for CZT. The reactions of CZT with iodine, CHBQ and TCNQ were performed in 96-microwell assay plates and absorbances of the CT complexes were measured by microwell absorbance reader at their corresponding maximum absorption peaks. The measured absorbances were correlated with the CZT concentrations in its sample solutions. Beer's law was obeyed with excellent correlation coefficients in the range of 0.5-30, 2-500, and 5-500 µg mL-1 for assays using iodine, CHBQ and TCNQ, respectively. The limits of detection were 2.17, 0.85 and 6.23 µg mL-1 for assays using iodine, CHBQ and TCNQ, respectively. The validation studies confirmed the accuracy and precision of all the proposed assays. The assays were successfully applied in the determination of CZT in Xalkori capsules. The proposed assays have very simple procedures to run in QC laboratories. Also, both assays enable analyst to process large number of samples and use of very small volumes of the organic solvent (ecofriendly and inexpensive).

Development of two different formats of heterogeneous fluorescence immunoassay for bioanalysis of afatinib by employing fluorescence plate reader and KinExA 3200 immunosensor.

Afatinib (AFT) is a potent and highly selective drug used to treat various solid tumors including non-small cell lung cancer (NSCLC). To ensure safe and effective treatment of cancer patients with AFT, its plasma concentrations should be monitored. Thus, sensitive immunoassays are required for measuring AFT concentrations in plasma samples. In this study, two different formats of heterogeneous fluorescent immunoassays were developed and validated for AFT bioanalysis. These assays were microwell-based fluorescence immunoassay (FIA) using fluorescence plate reader and kinetic exclusion assay (KinExA) using KinExA 3200 immunosensor. Both FIA and KinExA were developed using the same reagents: mouse anti-AFT antibody, solid-phase immobilized AFT conjugated with bovine serum albumin protein (AFT-BSA), and goat anti-mouse IgG labelled with fluorescein isothiocyanate (FITC-IgG) for signal generation. The analytical performances of both assays were comparatively evaluated, and the results revealed that although both assays had comparable accuracies, KinExA was superior to FIA in terms of sensitivity and precisions. Moreover, both FIA and KinExA were better alternatives to the existing chromatographic methods for bioanalysis of AFT. The proposed FIA and KinExA are anticipated to effectively contribute in ensuring safe and effective treatment with AFT in clinical settings.

Development and comparative evaluation of two immunoassay platforms for bioanalysis of crizotinib: A potent drug used for the treatment of non-small cell lung cancer.

This study describes, for the first time, the development of two platforms of competitive fluorescent immunoassays for bioanalysis of crizotinib (CZT), a potent drug used for the treatment of non-small cell lung cancer (NSCLC). These platforms were microwell-based heterogeneous fluoroimmunoassay (FIA) and a kinetic exclusion assay (KinExA) with KinExA™ 3200 immunosensor. Both FIA and KinExA were developed using same reagents; mouse anti-CZT antibody and a capturing reagent of CZT conjugated with bovine serum albumin (CZT-BSA). In the FIA, the CZT-BSA coated onto the microwells of the assay plate was present simultaneously in the assay mixture (CZT and its antibody). In the KinExA, the antibody was allowed to pre-equilibrate with CZT, and then the incubation mixture was rapidly passed through a microcolumn containing CZT-BSA coated onto polymethyl methacrylate (PMMA) beads. The analytical performances of both assays were comparatively evaluated in terms of assay working range, limit of detection, precision profile, and accuracy. The results revealed that KinExA yielded higher sensitivity and better precision than FIA; whereas, both assays had comparable accuracies. Both FIA and KinExA were superior to all the existing chromatographic methods for CZT in terms of the assay sensitivity, convenience, analysis throughputs. The proposed FIA and KinExA are anticipated to effectively contribute to the therapeutic drug monitoring (TDM) of CZT in clinical settings.

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.

Automated flow fluorescent noncompetitive immunoassay for measurement of human plasma levels of monoclonal antibodies used for immunotherapy of cancers with KinExA™ 3200 biosensor.

This study describes, for the first time, the development of an automated sensitive flow fluorescent noncompetitive immunoassay based on kinetic-exclusion analysis (KinExA) for the quantitative determination of human plasma levels of monoclonal antibodies (mAbs) used for cancer immunotherapy. The assay was adapted on KinExA™ 3200 biosensor and optimized and validated for bevacizumab (BEV) and cetuximab (CET), as representative examples of the mAbs, using their specific antigens. These antigens were the human vascular endothelial growth factor (VEGF) and epidermal growth factor receptor (EGFR) for BEV and CET, respectively. The limits of detection were 1.28 and 52.64 ng mL-1 for BEV and CET, respectively. The accuracy of the assay was demonstrated with analytical recovery of analytes from spiked plasma at 96.2-104.3 and 96.8-105.3% for BEV and CET, respectively. The precision of the assay was satisfactory as shown by relative standard deviation (RSD) at 2.2-5.7 and 2.5-6.1% for assay of BEV and CET, respectively. The high sensitivity of the assay allowed the use of very small volumes (~ 1 µL) of plasma sample for analysis. Automated analysis by the proposed KinExA-based assay facilitates the processing of large numbers of mAbs-containing specimens in studies of pharmacokinetics (PK), pharmacodynamics (PD), and therapeutic drug monitoring (TDM) of therapeutic mAbs. The proposed assay can be used to overcome the problems encountered in the existing conventional immunoassays for mAbs.

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.

Development and validation of generic heterogeneous fluoroimmunoassay for bioanalysis of bevacizumab and cetuximab monoclonal antibodies used for cancer immunotherapy.

This study describes, for the first time, the development and validation of a highly selective and sensitive heterogeneous fluoroimmunoassay (FIA) for the bioanalysis of two monoclonal antibodies (mAbs) used for cancer immunotherapy: bevacizumab (BEV) and cetuximab (CET). The assay combines reliable non-competitive binding of BEV and CET to their specific cell receptor proteins (human vascular endothelial growth factor (VEGF) and epidermal growth factor receptor (EGFR), respectively) with the highly specific fluorescence activity of the fluorescein isothiocyanate labeled anti-human IgG (FITC-IgG) used as label. The limits of detection were 14.14 and 1.27 × 103 ng mL-1 for BEV and CET, respectively. The accuracy and precision of the assay were demonstrated. The assay is simple, convenient, and requires very small volume (~ 5 µL) of plasma sample for analysis. The assay can offer high throughput analysis in clinical settings when modern microplates of multiplies of 96 (up to 6144-wells) are used and/or integrated as a part of automated robotic system. The proposed assay can be used for routine clinical bioanalysis of mAbs with potential application in pharmacokinetics, pharmacodynamics and therapeutic drug monitoring (TDM).

Spectrophotometric and molecular modelling studies on in vitro interaction of tyrosine kinase inhibitor linifanib with bovine serum albumin.

Linifanib (LNF) possess antitumor activity and acts by inhibiting receptor tyrosine kinase VEGF and PDGF. The interaction of BSA with the drug can provide valuable information regarding the pharmacokinetic and pharmacodynamics behavior of drug. In our study the spectrophotometric methods and molecular docking studies were executed to understand the interaction behavior of BSA and LNF. BSA has an intrinsic fluorescence and that fluorescence was quenched by LNF. This quenching process was studied at three different temperatures of 288, 300and 308 K. The interaction between LNF and BSA was due to static quenching because the Ksv (Stern-Volmer constant) at 288 K was higher than at 300 and 308 K. Kq (quenching rate constant) behaved in a similar fashion as the Ksv. Several other parameters like binding constants, number of binding sites and binding energy in addition to molecular docking studies were also used to evaluate the interaction process. A decrease in the binding constants was observed with increasing temperatures and the binding site number approximated unity. The decreasing binding constant indicates LNF-BSA complex stability. The site mark competition experiment confirmed the binding site for LNF was located on site II of BSA. UV-visible studies along with synchronous fluorescence confirm a small change in the conformation of BSA upon interaction with LNF. The thermodynamic analysis provided the values for free energy ΔG0, ΔH0 and ΔS0. The ΔG0 at the 288, 300 and 308 K ranged in between -21.5 to -23.3 kJ mol-1, whereas the calculated values of ΔH (-55.91 kJ mol-1) and ΔS0 (-111.74 J mol-1·K-1). The experimental and molecular docking results suggest that the interaction between LNF and BSA was spontaneous and they exhibited hydrogen bonding and van der Waals force between them.

Kinetic-exclusion analysis-based immunosensors versus enzyme-linked immunosorbent assays for measurement of cancer markers in biological specimens.

Kinetic exclusion analysis (KinExA)-based immunosensors and enzyme-linked immunosorbent assays (ELISA) have been developed and validated for measurement of five different cancer markers in biological specimens. These markers were: 2'-deoxycytidine (dCyd), 8-hydroxy-2'-deoxyguanosine (8HdG), carbohydrate antigen (CA15-3), α-fetoprotein (AFP), and ß-subunit of human chorionic gonadotropin (ß-HCG). The KinExA-based assays were conducted on the KinExA™ 3200 instrument. The ELISA assays employed the competitive immunoassay format for dCyd and 8HdG, however they employed the direct sandwich-type format for CA15-3, AFP, and ß-HCG. Each assay was validated in terms of its limit of detection, working range, precision profile, and accuracy. The analytical performances of the KinExA-based sensors were found to be superior to the ELISA for the five markers. The data demonstrated that the format of the assay may influence its performance characteristics (sensitivity, precision, etc.), even when exactly the same reagents are employed. The superior performance of the KinExA format is most likely due to: (1) the high surface area of beads containing the immobilized capture in the flow cell of the instrument, (2) the high flow rate of the reagents passing through the beads, which minimizes the diffusion limitations at the reaction surface, and (3) the limited time that the antibody is in contact with the capture reagent. The KinExA-based assays exhibited three noteworthy properties compared with ELISA: (1) avoiding the problems of mass transport limitations, and mobility effects, (2) KinExA analysis with automated sampling increase the assay convenience; and (3) providing high sensitivity with a lower limit of detection and better precision than ELISA. The proposed KinExA-based immunosensors are anticipated to have a great value in measurement of the cancer markers where more confident results are needed.