Development of luminescent techniques for the enhanced detection of immunosuppressants

Resumen

Immunosuppressants (ISs) are drugs used after an organ transplantation to prevent its rejection by inhibiting the body´s inmunological response. In transplanted patients, one of the most critical aspects is the correct dosage of ISs, which are characterized by narrow therapeutic window. At present, therapeutic drug monitoring (TDM) in post-transplanted patients is a standard practice and it consists in the measurement of the total drug plasma level measured just before the next dose by use of analytical techniques such as HPLC or LC-MS. However, recent clinical studies indicate that the free fraction (unbound to proteins) of ISs, which represents only 1-3% of the administered dose, reflects better the toxic or insufficient drug levels. Its clinical determination requires very low limits of detection. Alternative analytical methods such as fluorescence-based immunoassays (FIAs) are being developed for TDM. Some advantages of FIAs are their rapidity, low instrumental cost and high sensitivity. The main objective of this Thesis is providing tools for developing novel sensitive FIAs for detection of three of the most administered ISs: mycophenolic acid (MPA), cyclosporine A (CyA) and tacrolimus (FK506). Several aspects have been addressed here: 1) Chemical derivatization of IS drugs with –COOH groups for allowing their covalent immobilization to surfaces, fluorescent labels or proteins. Three different carboxy-derivatives of CyA were successfully synthesized by different synthetic routes: i) allylic bromination on the terminal carbon C8′of CyA and further SN2 reaction with methyl 3-mercaptopropanoate to yield the CyA-S-COOH derivative; ii) epoxidation of the CyA C=C bond, followed by a SN2 reaction with an -aminoester, yielding CyA-N-COOH; iii) modification of CyA by its secondary alcohol group providing CyA-O-COOH. In the case of FK506, FK506-COOH was obtained by using carboxymethoxylamine. Derivatization of MPA was not necessary since it already contains a –COOH group. 2) Development of sensitive luminescent markers for ISs detection: 2.i) For fluorescent labelling of the three IS–COOH derivatives an amino-substituted analogue of the Nile Blue fluorophore (ANB) was synthetized. The three ANB-IS fluorescent probes were obtained by amide bond formation, showing good photostability and a strong emission in aqueous buffer at > 650 nm. Fluorescent polarization binding assays performed with the ANB-ISs in the presence of human serum albumin, proved that the technique can be used for the determination of the free fraction of ISs. 2.ii) Innovative fluorescent magnetic nanoparticles (FMNPs) were developed in which intraparticle FRET (Förster resonance energy transfer) processes allow improving ISs Abstract II detection limits. FMNPs were doped with strongly emitting boron-dipyrromethene (BODIPY) dyes. Co-encapsulation of two different types of BODIPY dyes yielded brilliant beads with a very large virtual Stokes shift (> 150 nm, emission max. > 650 nm) due to an intraparticle FRET process. The FRET-FMNPs have been applied as labels in a FIA for tacrolimus detection, yielding a LOD of 0.08 ng mL-1, compared to a LOD of 2.7 ng mL-1 for the immunoassay carried out with direct excitation of the FRET acceptor dye. 2.iii) Polystyrene NPs doped with photosensitizers for 1O2 production have been prepared as alternative to the 1O2 donor beads marketed by Perkin-Elmer (AlphaScreen™ assay). Two types of dyes were chosen: a) a Ru(II) complex (RD3) for doping NPs of 390 and 26 nm. The amount of dye used for doping was optimized by fluorescence spectroscopy; also a method to determine the Δ of a NPs suspension with single photon counting laser kinetic spectrometer system was developed. It was found that a larger amount of 1O2 is produced when the photosensitizer is encapsulated in NPs than when it is free in solution (NPs = 0.50 > RD3 = 0.42); b) Iodinated BODIPY dyes with high Δ were also synthesized as alternative to Ru(II) complexes.