Assays for measuring immunosuppressants by reporter gene expression
AssigneeNovartis AG
InventorsGoetz Baumann, Franco E. Di Padova, Peter Wenner
Life of this patent
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Abstract
Quantitative reporter gene assays allowing for highly precise measurements of immunosuppressants in bodily fluids are provided, e.g., an IL-2 reporter gene assay for measuring blood levels of immunosuppressive cyclosporins and ascomycins (e.g., cyclosporin A and FK-506), and a c-jun reporter gene assay for measuring blood levels of immunosuppressive rapamycins.
Claims20
A method for measuring ex vivo the concentration of an immunosuppressant or its pharmacologically active metabolites in a bodily fluid, wherein said immunosuppressant is a cyclosporin or ascomycin, comprising:
- subjecting cells of a T-cell line transfected with a reporter gene under the control of an IL-2 promoter to conditions sufficient to stimulate IL-2 transcription,
- treating at least a portion of said cells with a bodily fluid diluted to a concentration of less than 5%, whereby expression of said reporter gene in a treated cell is inhibited relative to expression of said reporter gene in an untreated cell, and
- comparing the degree of inhibition of reporter gene expression effected by said bodily fluid with the degree of inhibition of reporter gene expression effected by a known concentration of the immunosuppressant, wherein said comparing indicates the concentration of said immunosuppressant or metabolite thereof in said bodily fluid.
A method for measuring ex vivo the concentration of an immunosuppressant or its pharmacologically active metabolites in a bodily fluid, wherein said immunosuppressant is a rapamycin, comprising:
- subjecting cells of a T-cell line transfected with a reporter gene under the control of a c-jun promoter to conditions sufficient to stimulate c-jun transcription,
- treating at least a portion of said cells with a bodily fluid diluted to a concentration of less than 5%, whereby expression of said reporter gene in a treated cell is inhibited relative to expression of said reporter gene in an untreated cell, and
- comparing the degree of inhibition of reporter gene expression effected by said bodily fluid with the degree of inhibition of reporter gene expression effected by a known concentration of the immunosuppressant, wherein said comparing indicates the concentration of said immunosuppressant or metabolite thereof in said bodily fluid.
A method for measuring ex vivo the concentration of an immunosuppressant or its pharmacologically active metabolites in a bodily fluid, wherein said immunosuppressant is a cyclosporin, comprising:
- subjecting cells of a T-cell line transfected with a reporter gene under the control of a promoter selected from the group consisting of promoters for the cytokines IL-2, TGF-.beta., IFN-.gamma. and IL-4, under conditions sufficient to stimulate transcription of the cytokine,
- treating at least a portion of said cells with a bodily fluid diluted to a concentration of less than 5%, whereby expression of said reporter gene in a treated cell is inhibited relative to expression of said reporter gene in an untreated cell, and
- comparing the degree of inhibition of reporter gene expression effected by said bodily fluid with the degree of inhibition of reporter gene expression effected by a known concentration of the immunosuppressant, wherein said comparing indicates the concentration of said immunosuppressant or metabolite thereof in said bodily fluid.
An assay kit for monitoring the concentration of an immunosuppressant in a bodily fluid comprising:
- cells of a T-cell clone transfected with a reporter gene under the control of a promoter, said promoter being a promoter for a cytokine or protein the transcription of which is stimulated or suppressed in said cells by the immunosuppressant to be monitored, and
- one or more concentration standards of the immunosuppressant to be monitored, wherein
- (a) the immunosuppressant is an ascomycin and the promoter is an IL-2 promoter, or
- (b) the immunosuppressant is a cyclosporin and the promoter is selected from the group consisting of IL-2, TGF-.beta., IFN-.gamma., and IL-4 promoters, or
- (c) the immunosuppressant is a rapamycin and the promoter is a c-jun promoter.
Dependencies are inferred from the claim text.