Methodological needs in the quality and safety characterisation of nanotechnology-based health products: Priorities for method development and standardisation.

Nanotechnology-based health products are providing innovative solutions in health technologies and the pharmaceutical field, responding to unmet clinical needs. However, suitable standardised methods need to be available for quality and safety assessments of these innovative products prior to their translation into the clinic and for monitoring their performance when manufacturing processes are changed. The question arises which technological solutions are currently available within the scientific community to support the requested characterisation of nanotechnology-based products, and which methodological developments should be prioritized to support product developers in their regulatory assessment. To this end, the work presented here explored the state-of-the-art methods to identify methodological gaps associated with the preclinical characterisation of nanotechnology-based medicinal products and medical devices. The regulatory information needs, as expressed by regulatory authorities, were extracted from the guidance documents released so far for nanotechnology-based health products and mapped against available methods, thus allowing an analysis of methodological gaps and needs. In the first step, only standardised methods were considered, leading to the identification of methodological needs in five areas of characterisation, including: (i) surface properties, (ii) drug loading and release, (iii) kinetic properties in complex biological media, (iv) ADME (absorption, distribution, metabolism and excretion) parameters and (v) interaction with blood and the immune system. In the second step, a detailed gap analysis included analytical approaches in earlier stages of development, and standardised test methods from outside of the nanotechnology field that could address the identified areas of gaps. Based on this analysis, three categories of methodological needs were identified, including (i) method optimisation/adaptation to nanotechnological platforms, (ii) method validation/standardisation and (iii) method development for those areas where no technological solutions currently exist. The results of the analysis presented in this work should raise awareness within the scientific community on existing and emerging methodological needs, setting priorities for the development and standardisation of relevant analytical and toxicological methods allowing the development of a robust testing strategy for nanotechnology-based health products.

Interactions between tenofovir and nevirapine in CD4+ T cells and monocyte-derived macrophages restrict their intracellular accumulation.

OBJECTIVES: There is no pharmacokinetic interaction between tenofovir and nevirapine, but a higher emergence rate of resistance mutations has been reported when these drugs are coadministered. We sought to examine if there is a potential intracellular interaction that may account for the emergence of resistant virus. METHODS: Primary CD4+ and CD14+ cells were isolated from healthy volunteer blood. Monocyte-derived macrophages were differentiated from CD14+ cells. Accumulation of radiolabelled tenofovir and nevirapine was then assessed in these cells. RESULTS: We show here that tenofovir and nevirapine immune cell intracellular concentrations are lower when coincubated in CD4+ cells and monocyte-derived macrophages, but not in CD14+ cells. CONCLUSIONS: These data indicate a potential intracellular drug-drug interaction between these drugs that warrants further investigation.

The impact of cytokines on the expression of drug transporters, cytochrome P450 enzymes and chemokine receptors in human PBMC.

BACKGROUND AND PURPOSE: The function of transporters in peripheral blood mononuclear cells (PBMC) has been characterized, but less is known about cytochrome P450 (CYP) enzyme function in these cells. Given that cytokines are dysregulated in many diseases, the purpose of this work was to assess the impact of cytokines on the expression of CYPs, transporters and chemokine receptors in PBMC. EXPERIMENTAL APPROACH: Human PBMC were incubated with cytokines for 48 h. ATP-binding cassette (ABC)B1, ABCC1, ABCC2, CYP2B6, CYP3A4, CXCR4 and CCR5 expression were measured by quantitative polymerase chain reaction and flow cytometry at 0, 4, 8, 24 and 48 h. Enzyme activity was assessed using fluorescent probes. KEY RESULTS: We show here functional activity of CYP3A4 and CYP2B6 in PBMC. Furthermore, cytokines had a significant impact on the mRNA and protein expression of all proteins. For example, interleukin-2 (IL-2) had a marked impact on ABCB1 mRNA (% control 4745 +/- 11961) and protein (% control 200 +/- 57). Increases in drug efflux transporter expression, in response to cytokines, resulted in reduced cellular accumulation of digoxin [decrease of 17% and 26% for IL-2 and interferon-gamma (IFNgamma) respectively] and saquinavir (decrease of 28% and 30% for IL-2 and IFNgamma respectively). The degree to which drug transporter and chemokine receptor expression changed in response to cytokines was positively correlated (e.g. ABCB1 and CXCR4, r(2) = 0.545). CONCLUSIONS AND IMPLICATIONS: These data have important implications for diseases in which cytokines are dysregulated and for which pharmacological intervention targets immune cells.

Detection of ABCC2, CYP2B6 and CYP3A4 in human peripheral blood mononuclear cells using flow cytometry.

ABCC2 has a wide tissue distribution and can mediate the efflux of a number of therapeutic compounds from cells and contribute to potential treatment failure. Its diverse expression and ability to efflux a number of substrates imply a number of physiological and pharmacological roles. CYP2B6 and CYP3A4 are responsible for the metabolism of a number of therapeutic compounds. Reports on the expression of these proteins in various cells and tissues have been contradictory mainly due to differences in experimental approach and cell type studied. With the advances in commercially available antibodies we describe here a simplified technique for the detection of ABCC2, CYP2B6 and CYP3A4 in human peripheral blood mononuclear cells (PBMC) by flow cytometry. Results are expressed as mean increase in fluorescence compared to isotypically matched controls. Using these assays we confirmed the expression of these proteins in human PBMC. These methods are rapid and reproducible and have potential use for both in vitro and clinical applications.