Application of carbon nanotubes for in-capillary incubations with cytochrome P-450 enzymes

Korte inhoud:Metabolism assessment of drug candidates is an essential part in their development. In recent years, the pharmaceutical industry has implemented metabolism investigation and predictions into the lead-generation phase in order to improve the rate of success in drug development. At this stage, high-throughput metabolic test systems are required to screen the multitude of drug candidates available. Capillary electrophoresis (CE) is an interesting analytical tool for high-throughput in vitro metabolism studies owing to its minute sample and reagent consumption, highly efficient and rapid analyses, and the possibility to integrate incubation and analysis in-capillary, providing additional miniaturisation and automation. The most important obstacle encountered during CE analysis of in vitro metabolic model systems including microsomes and recombinantly expressed cytochrome P450 enzymes (P450s) is adsorption of sample constituents to the bare fused-silica wall, leading to inconsistent results and eventually to capillary blockage. In this work, the possible contribution of carbon nanotubes to in vitro metabolism studies by CE was examined. Taking into account the high affinity of carbon nanotubes towards proteins, nanotube addition to the sample vial was evaluated as an alternative approach to reducing protein adsorption during in-capillary incubations with P450s. Manipulation of carbon nanotubes in incubation buffer demanded the addition of surfactant as dispersion-aiding agent. Initial experiments pointed out that soft dispersion techniques lack sufficient power to disrupt the highly entangled carbon nanotube aggregates. Stable dispersion of carbon nanotubes could only be achieved upon application of high-power probe sonication. In order to curtail potential nanotube damage, a discontinuous sonication procedure was developed, consisting of four sonication stages with sonication-free intermezzi after each stage. Of the three surfactants most commonly utilised for carbon nanotube dispersion, Triton X-100 was selected as a suitable surfactant for combination with P450, since it is generally described as a non-denaturing compound. Stable dispersions of single-walled carbon nanotubes (SWNTs) and three different types of multi-walled carbon nanotubes (MWNTs) in a solution of Triton X-100 in incubation buffer were prepared. A previously reported off-line CE method analysing CYP3A4 activity using verapamil as a probe substrate, was the starting point for carbon nanotube-P450 investigations. The described organic solvent used for quenching of the enzymatic reaction had a detrimental effect on substrate and metabolite peak shape, prompting additional method optimisation. The quenching and filtration step were omitted and replaced by centrifugation of the sample. A rinsing procedure was developed to ensure good migration time repeatability. Unfortunately, the partial co-migration of metabolite with two small P450-related peaks could not be circumvented: several strategies including removal of the interfering compounds by precipitation and modification of CE parameters such as temperature, voltage, and pH, concentration and nature of the background electrolyte, were attempted to no effect. Next, the influence of carbon nanotubes and Triton X-100 on CYP3A4-catalysis was investigated off-line using the optimised CE method. Nanotube dispersions were added to incubation mixtures and the amount of metabolite formed was compared to a standard incubation. Mostly, considerable reductions in metabolite formation were noted upon nanotube addition, although only statistically significant in case of SWNTs. In addition, the rather poor performance of the off-line assay in terms of precision was unveiled. The feasibility of direct injection of incubation mixtures was investigated: the stability of migration times and peak shape was excellent when the rinsing procedure was applied, and a small supplementary benefit of surfactant and nanotube was observed. Subsequently, development of an in-capillary method was tried. Mixing by diffusion and electrophoresis was evaluated and a plethora of parameters was varied. Irrespective of the mixing scheme deployed, in-capillary metabolite formation was always several orders of magnitude lower, compared to the off-line method. An explanation for this phenomenon was found in the increase of the Triton X-100 concentration during in-capillary tests in order to avert nanotube precipitation during prolonged storage of the enzyme vial. In the end, Triton X-100 was abandoned and a more apposite surfactant was sought.A variety of surfactants was screened with respect to nanotube dispersing capability and interference with the enzymatic reaction. Most surfactants were able to achieve stable dispersion of non-functionalised MWNTs. No clear links between surfactant structure and nanotube dispersing capacity were noticed. Even though the surfactant concentration was minimised, the majority of surfactants still negatively affected the enzymatic reaction. The combination of Brij 35 and nanotube was proven the only P450-friendly platform. During direct injection of incubation mixtures, however, the small beneficial effect of nanotubes on migration time stability perceived previously using Triton X-100, could not be confirmed with Brij 35. Finally, an in-capillary CYP3A4 assay was developed. Due to technical shortcomings of the CE instrument, an in-line method based on electrophoretic mixing of zones in the thermostated part of the capillary was conceived. Full separation of metabolite necessitated an increase in capillary length as well as the addition of a viscosity increasing agent to the running buffer. The final method was intricate, incorporating partial filling of the capillary with incubation buffer, zero-potential amplification and a cooling step prior to analysis. Upon repetition of the developed in-capillary method, the favourable effect of MWNTs on capillary life span was unambiguously shown. In the absence of nanotubes, adsorption of P450-components to the capillary wall manifested itself in a gradual increase of migration times and deterioration of peak shape, even when extensive between-run rinsing was applied. These phenomena were not discerned when nanotubes were added to the enzyme vial. The major contributor to run-to-run stability, though, seemed to be the rinsing procedure, but an indispensable benefit of MWNTs became clear in the long term. The suitability of the in-capillary method for a kinetic study was assessed. Poor quantitative results were obtained and attributed to the lack of metabolite reference substance, injection variability and the complexity of the verapamil-CYP3A4 interaction. In conclusion, the outcome of this study justifies further exploration of the use of carbon nanotubes as an expedient for prevention of protein adsorption to the capillary wall in CE. The particular application envisaged in this project, i.e. the development of an in-capillary P450 assay, was proven hard to realise using the aforementioned strategy.

Jaar van publicatie:2011

Trefwoorden:Analytische, anorganische en nucleaire scheikunde, Biochemie & -fysica en Moleculaire biologie

BOF-keylabel:ja

IOF-keylabel:ja

BOF-publication weight:1

CSS-citation score:1

Authors from:Higher Education

Toegankelijkheid:Closed

Reviewstatus:Peer-reviewed