Bertrand Blankert

Biosensors in Drug Discovery and Drug Analysis

Abstract Biosensors are, by definition, sensing devices comprising a biological component (enzyme, antibody, animal or plant cell, oligonucleotide, lipid, microorganisms, etc.) intimately connected to a physical transducer (electrode, optical fiber, vibrating quartz, etc.). This dual configuration permits a quantitative study of the interaction between a drug compound and an immobilized biocomponent. Ideally, biosensors should be readily implemented and allow for low reagent and energy consumption. Enzyme‐based biosensors can be applied in the pharmaceutical industry for monitoring chemical parameters in the production process (in bioreactors). Affinity biosensors are suitable for high‐throughput screening of bioprocess‐produced antibodies and for candidate drug screening. They are suitable for selective and sensitive immunoassays in clinical laboratories and for decentralized detection of drug residues. Enzyme‐based biosensors may be used in hospitals for bedside drug testing, emergency control, in patient treatment control (anticancer therapy) etc. Current research efforts are focused on proteins, tissues, or living cells immobilized in microfabricated configurations for high‐throughput drug screening and discovery. Such devices can comprise several different microelectronic sensors and biosensors sensitive, for example, to pH, temperature, impedance, dissolved oxygen, etc. for a multiparametric monitoring. Of equal new interest are the oligonucleotide‐immobilized biosensors for interactions studies between a surface linked DNA and the target drug or for hybridisation studies. This short review summarizes several recent trends dedicated to the development and application of biosensors in the pharmaceutical arena.

Organocatalysis Paradigm Revisited: Are Metal-Free Catalysts Really Harmless?

Catalysts are commonly used in polymer synthesis. Traditionally, catalysts used to be metallic compounds but some studies have pointed out their toxicity for human health and environment, and the removal of metal impurities from synthetic polymer is quite expensive. Organocatalysts have been intensively synthesized and are now widely used in ring-opening polymerization (ROP) reactions to address these issues. However, for most of them, there is not any evidence of their safety. The present study attempts to assess whether well-established organo-based ROP catalysts used for the preparation of FDA-approved polyesters may present a certain level of cytotoxicity. In vitro toxicity is evaluated using a methyl-thiazol-tetrazolium cytotoxicity assay on two cell models (FHs74Int and HepaRG). Among the investigated organocatalysts, only functionalized thiourea shows an important cytotoxicity on both cell models. 4-Dimethylaminopyridine (DMAP), 1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD), and meta-(trimethylammonio)phenolate betaine (m-BE) show cytotoxicity against HepaRG cell line only at a high concentration.

Horseradish peroxidase electrode for the analysis of clozapine

Abstract A horseradish peroxidase (HRP) immobilized electrode was developed for the assay of the antipsychotic compound clozapine (CLZ). The biosensor was made of HRP crosslinked with glutaraldehyde and bovine serum albumin (BSA) and blended in the electrode matrix. The latter was a carbon paste based on solid paraffin and graphite particles. A dialysis membrane was secured at the tip of the enzyme based electrode. Cyclic voltamperometry at the solid carbon paste electrode (sCPE) permitted to point out a reversible pattern for CLZ electrooxidation attributed to a relatively stable nitrenium ion. The formation of the latter and of a newly generated species, was inferred at the biosensor. The electroreduction of these generated species was performed at the biosensor at an applied potential of 0.0 V vs. Ag/AgCL 3 M KCl. Several experimental parameters influencing the biosensor response were studied such as pH, buffer composition, and detection potential. The resulting biosensor offered, at pH 4.5 in Britton–Robinson buffer (BRb) in the presence of 0.1 mM H2O2 and at 0.0 V vs. Ag/AgCl, a linear response in the concentration range comprized between 1.0 × 10−6 M and 1 × 10−5 M with a detection limit of 1.7 × 10−7 M and a quantification limit of 5.6 × 10−7 M. In addition to the mechanistic information provided, the biosensor was found useful for the determination of CLZ in tablets. The accuracy of the assay was checked by capillary electrophoresis (CZE).

Molecularly Imprinted Polymers: Compromise between Flexibility and Rigidity for Improving Capture of Template Analogues

New synthetic strategies for molecularly imprinted polymers (MIPs) were developed to mimic the flexibility and mobility exhibited by receptor/enzyme binding pockets. The MIPs were prepared by bulk polymerization with quercetin as template molecule, acrylamide as functional monomer, ethylene glycol dimethacrylate as cross-linker, and THF as porogen. The innovative grafting of specific oligoethylene glycol units onto the imprinted cavities allowed MIPs to be obtained that exhibit extended selectivity towards template analogues. This synthetic strategy gives promising perspectives for the design of molecular recognition of molecules based on a congruent pharmacophore, which should be of interest for drug development.

Targeted extraction of active compounds from natural products by molecularly imprinted polymers

One of the most promising separation techniques that have emerged during the last decade is based on the use of molecularly imprinted polymers (MIPs). MIPs are stable polymers that possess specific cavities designed for a template molecule, endowed with excellent selectivity compared to regular solid phase extraction techniques. Molecularly imprinted solid-phase extraction (MISPE) has already shown a high efficiency for the sample preparation from complex matrices. Natural products received huge attention in recent years. Indeed, the application of MISPE for the screening of natural products appears extremely interesting not only for the selective extraction of a target compound but also for the concomitant discovery of new drug candidates, promising sources of therapeutic benefits. In the present review, examples of recognition and separation of active components from natural extracts are emphasized. MIPs are very promising materials to mimic the recognition characteristics exhibited by enzymes or receptors although further developments are necessary to fully exploit their wide potential.

Quercetin-imprinted chromatographic sorbents revisited: optimization of synthesis and rebinding protocols for application to natural resources.

Molecularly imprinted polymers (MIPs) based on quercetin and synthesized by either bulk, precipitation or suspension polymerization were characterized in terms of size and shape by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). After a study of rebinding protocols, the optimal materials were evaluated as sorbents for solid-phase extraction (SPE) and high-performance liquid chromatography (HPLC) to confirm the presence of imprinted cavities and to assess their selectivity. Besides quercetin, other structurally related natural compounds, naringenin, daidzein and curcumin, were employed for selectivity tests of MIPs. Although rebinding protocols previously described for such MIPs are typically based on binding, washing and eluting methanol-based solutions, we show that this highly polar solvent leads to weak specific interactions (imprinting factor<1) and poor sorbent properties, most probably because of hydrogen-bonding interferences between the MIP and MeOH. Similar experiments performed in tetrahydrofuran yield to much more improved properties (imprinting factor>2.4). This calls for reviewing most of previously published data on quercetin-MIPs; in proper binding conditions, published MIPs may prove more performing than initially assessed. As expected, tested MIPs exhibited the highest selective rebinding towards quercetin template (imprinting effect, quercetin, 3.41; naringenin, 1.54; daidzein, 1.38; curcumin, 1.67); the differences in selectivity between quercetin analogues were explained by the ligand geometries and H-bonding patterns obtained from quantum-chemical calculations. The evaluation of MIPs under identical analytical conditions allowed investigating the effect of the production method on chromatographic performances. The MIPs in bead materials (for quercetin, peak width, 0.69; number of theoretical plates, 143; symmetry factor, 2.22) provided a significant improvement in chromatographic efficiency over the bulk materials (for quercetin, peak width, 1.25; number of theoretical plates, 115; symmetry factor, 2.92). Using the quercetin-beaded MIP as SPE sorbent, quercetin was selectively extracted from Allium cepa L. extract. The MIP developed in this work therefore appears highly promising for the enrichment and determination of quercetin in natural products.

Enzyme immobilized magnetic nanoparticles for in-line capillary electrophoresis and drug biotransformation studies: application to paracetamol.

Enzyme Immobilized Magnetic Nanoparticles (EMNPs) were injected and magnetically retained, as a microreactor, in the capillary of a capillary electrophoresis (CE) setup with UV detection. The enzyme horseradish peroxidase (HRP) was chemically immobilized onto commercially available magnetic 300 nm diameter nanoparticles. Paracetamol (acetaminophen: N-acetyl-p-aminophenol), a common analgesic drug, was used as model drug compound. The enzymatic reaction was studied in-line by CE in 12.5 mM phosphate buffer pH 7.4 containing 20 mg/ml sulfated- beta -cyclodextrin and 0.1 mM hydrogen peroxide. By means of the developed setup, the apparent Michaelis Menten constant between HRP and acetaminophen (APAP) was determined as K(m)(app) = 53+/-5 microM. This approach was found to be of interest for enzyme kinetics studies with short time resolution condition. Based on our results and from literature data, it was possible to infer that the in-line generated product was an APAP dimer. Higher enzyme immobilized beads loading in the CE setup generated the APAP dimer with two additional minor peaks likely attributed to APAP trimer and tetramer. N-acetyl-p-benzoquinone imine (NAPQI) was not generated during APAP short time migration through the in-line microreactor.

Three optimized and validated (using accuracy profiles) LC methods for the determination of pentamidine and new analogs in rat plasma

Three novel LC-UV methods for the determination of pentamidine (PTMD) and two of its new analogs in rat plasma are described. The chromatographic conditions (wavelength, acetonitrile percentage in the mobile phase, internal standard) were optimized to have an efficient selectivity. A pre-step of extraction was simultaneously developed for each compound. For PTMD, a solid phase extraction (SPE) with Oasis(®) HLB cartridges was selected, while for the analogs we used protein precipitation with acetonitrile. SPE for PTMD gave excellent results in terms of extraction yield (99.7 ± 2.8) whereas the recoveries for the analogs were not so high but were reproducible as well (64.6 ± 2.6 and 36.8 ± 1.6 for analog 1 and 2, respectively). By means of a recent strategy based on accuracy profiles (β-expectation tolerance interval), the methods were successfully validated. β was fixed at 95% and the acceptability limits at ± 15% as recommended by the FDA. The method was successfully validated for PTMD (29.6-586.54 ng/mL), analog 1 (74.23-742.3 ng/mL) and analog 2 (178.12-890.6 ng/mL). The first concentration level tested was considered as the LLOQ (lower limit of quantification) for PTMD and analog 1 whereas for analog 2, the LLOQ was not the first level tested and was raised to 178.12 ng/mL.

Study of nucleic acid-ligand interactions by capillary electrophoretic techniques: A review.

The understanding of nucleic acids-ligand (proteins, nucleic acids or various xenobiotics) interactions is of fundamental value, representing the basis of complex mechanisms that govern life. The development of improved therapeutic strategies, as well as the much expected breakthroughs in case of currently untreatable diseases often relies on the elucidation of such biomolecular interactions. Capillary electrophoresis (CE) is becoming an indispensable analytical tool in this field of study due to its high versatility, ease of method development, high separation efficiency, but most importantly due to its low sample and buffer volume requirements. Most often the availability of the compounds of interest is severely limited either by the complexity of the purification procedures or by the cost of their synthesis. Several reviews covering the investigation of protein-protein and protein-xenobiotics interactions by CE have been published in the recent literature; however none of them promotes the use of these techniques in the study of nucleic acid interactions. Therefore, various CE techniques applicable for such interaction studies are discussed in detail in the present review. The paper points out the particular features of these techniques with respect the estimation of the binding parameters, in analytical signal acquisition and data processing, as well as their current shortcomings and limitations.

Ultra High Performance Liquid Chromatography Method for the Determination of Two Recently FDA Approved TKIs in Human Plasma Using Diode Array Detection

Generally, tyrosine kinase inhibitors have narrow therapeutic window and large interpatient variability compared to intrapatient variability. In order to support its therapeutic drug monitoring, two fast and accurate methods were developed for the determination of recently FDA approved anticancer tyrosine kinase inhibitors, afatinib and ibrutinib, in human plasma using ultra high performance liquid chromatography coupled to PDA detection. Diclofenac sodium was used as internal standard. The chromatographic separation was achieved on an Acquity UPLC BEH C18 analytical column using a mobile phase combining ammonium formate buffer and acetonitrile at a constant flow rate of 0.4u2009mL/min using gradient elution mode. A µSPE (solid phase extraction) procedure, using Oasis MCX µElution plates, was processed and it gave satisfying and reproducible results in terms of extraction yields. Additionally, the methods were successfully validated using the accuracy profiles approach (β = 95% and acceptance limits = ±15%) over the ranges 5–250u2009ng/mL for afatinib and from 5 to 400u2009ng/mL for ibrutinib in human plasma.