Detection and quantification of cyclosporine in body fluids using an interleukin-2 reporter-gene assay

Area of Science:

• Biotechnology
• Immunology
• Pharmacology

Background:

• Current methods for monitoring immunosuppressive drugs lack precision regarding actual patient immunosuppression levels.
• Existing assays do not provide direct information on the biological impact of these drugs.

Purpose of the Study:

• To introduce and validate an interleukin-2 (IL-2) reporter-gene assay (IL-2 RGA) for monitoring immunosuppressant concentrations in biological fluids.
• To assess the sensitivity, selectivity, and reproducibility of the IL-2 RGA for specific immunosuppressive drugs and their metabolites.

Main Methods:

• Development of a chimeric gene construct where the human IL-2 promoter drives reporter gene expression.
• Quantification of reporter gene expression upon mitogenic stimulation to assess drug effects.
• Evaluation of assay performance with immunosuppressive compounds like cyclosporine (CsA) and FK506, including their metabolites and derivatives.

Main Results:

• The IL-2 RGA demonstrated high sensitivity and selectivity for immunosuppressants inhibiting IL-2 gene expression, such as CsA and FK506.
• The assay showed high reproducibility, fast performance, and high capacity, making it suitable for various applications.
• The IL-2 RGA effectively monitored CsA or ascomycin/FK506 analogues and metabolites, even in the absence of specific monoclonal antibodies.

Conclusions:

• The IL-2 reporter-gene assay provides a precise and direct measure of immunosuppression, overcoming limitations of existing drug concentration monitoring methods.
• This assay is a valuable tool for monitoring immunosuppressive drug levels in clinical and preclinical settings, particularly for drugs lacking specific antibody detection methods.
• The IL-2 RGA offers a more suitable alternative to traditional in vitro systems like MLR or mitogen-stimulated PBMC for studying drug immunosuppressive activity in body fluids.