Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1996-09-17
1999-04-27
Spector, Lorraine
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
435 8, 435 6952, 435 698, 530351, 530413, 536 241, 536 232, 536 234, 536 235, C12N 510, C12N 1511, C12Q 168, G01N 33487
Patent
active
058979905
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND OF THE INVENTION
This invention relates to new assay methods for monitoring the concentration in bodily fluids of immunosuppressants affecting gene expression, e.g., cyclosporins, such as cyclosporin A or cyclosporin G; ascomycins, such as FK-506; and rapamycins, e.g., rapamycin; using a reporter gene assay, e.g., an IL-2 reporter gene assay for immunosuppressive cyclosporins and ascomycins, or a c-jun reporter gene assay for immunosuppressive rapamycins.
Cyclosporins comprise a class of structurally distinct, cyclic, poly-N-methylated undecapeptides, generally possessing immunosuppressive, anti-inflammatory, anti-viral and/or anti-parasitic activity, each to a greater or lesser degree. The first of the cyclosporins to be identified was the fungal metabolite Cyclosporin A, or Ciclosporin, and its structure is given in The Merck Index, 11 th Edition; Merck & Co., Inc.; Rahway, N.J., USA (1989) under listing 2759. Later cyclosporins to be identified are cyclosporins B, C, D and G which are also listed in the Merck Index under listing 2759. A large number of synthetic analogues are also known and representative examples are disclosed in EP 296 123, EP 484 281, and GB 2222770. Cyclosporin A and its structurally similar analogues and derivatives are generally referred to as "cyclosporins" for the purposes of this specification.
Rapamycin is a macrolide immunosuppressant that is produced by Streptomyces hygroscopicus and which has been found to be pharmaceutically useful in a variety of applications, particularly as an immunosuppressant for use in the treatment and prevention of organ transplant rejection and autoimmune diseases. The structure of rapamycin is given in Kesseler, H., et al.; 1993; Helv. Chim. Acta; 76: 117. Large numbers of derivatives of rapamycin have been synthesized, including for example 40-O-alkylated derivatives such as 40-O-(2-hydroxy)ethyl-rapamycin (WO 94/09010), certain acyl and aminoacyl-rapamycins (e.g., U.S. Pat. No. 4,316,885, U.S. Pat. No. 4,650,803, and U.S. Pat. No. 5,151,413), 27-desmethyl-rapamycin (WO 92/14737), 26-dihydro-rapamycin (U.S. Pat. No. 5,138,051), certain pyrazole derivatives (U.S. Pat. No. 5,164,399), certain alkoxyester derivatives (U.S. Pat. No. 5,233,036), and numerous others. Rapamycin and its structurally similar analogues and derivatives are termed collectively as "rapamycins" in this specification.
Ascomycins, of which FK-506 is the best known, are another class of generally immunosuppressive macrolides. FK506 is a macrolide immunosuppressant that is produced by Streptomyces tsukubaensis No 9993. The structure of FK506 is given in the appendix to the Merck Index, as item A5. A large number of related compounds which retain the basic structure and immunological properties of FK506 are also known. These compounds are described in various publications, for example EP 184162, EP 315973, EP 323042, EP 423714, EP 427680, EP 465426, EP 474126, WO 91/13889, WO 91/19495, EP 484936, EP 532088, EP 532089, WO 93/5059 and the like. Ascomycin, FK-506 and their structurally similar analogues and derivatives are termed collectively "ascomycins" in this specification.
Due to their extremely useful pharmaceutical properties, cyclosporins (Cyclosporins A and G in particular), rapamycins (e.g., rapamycin and 40-O-(2-hydroxy)ethyl-rapamycin) and ascomycins (e.g., FK-506) have wide application in, for example the prevention of transplant rejection and in the treatment of auto-immune diseases. However these compounds have side effects at higher doses and therefore their concentration in the blood must be kept within certain therapeutic ranges. Bioavailabilities and metabolic conversion rates tend to be patient specific and hence dosaging is patient specific. It is thus desirable to monitor the blood levels of such drugs and to adjust the dosage to obtain optimum blood levels of the drug thereby maximizing immunosuppression and minimizing adverse side effects.
Up until now, the method of choice for measuring blood levels of immunosuppressants has been using immunoassay
REFERENCES:
patent: 5350574 (1994-09-01), Erlanger et al.
Brombacher et al., IL-2 promoter-driven lacZ expression as a monitoring tool for IL-2 expression in primary T cells of transgenic mice, International Immunol., 6(2): 189-197, Feb. 1994.
Shan et al., The effect of rapamucin on c-jun expression in human lymphocytes, Clin. Immunol. Immunopathol., 69(3): 314-317, Dec. 1993.
Ganong, W.F., Review of Medical Physiology, Appleton & Lange:Norwalk, CT, p. 445, 1989.
Williams et al., "Advantages of Firefly Luciferase as a Reporter Gene: Application to the Interleukin-2-Gene Promoter", Anal. Biochem, vol. 176, pp. 28-32.
Hall et al., "Expression and Regulation of Escherichia Coli LacZ Gene Fusions in Mammalian Cells", J. Mol. Appl. Genetics, vol. 2, No. 1, pp. 101-109.
Dumont et al., "Distinct Mechanisms of Suppression of Murine T-cell Activation by the Related Macrolides FK-506 and Rapamycin", J. Immunology, vol. 144, No. 1, (Jan. 1, 1990).
Derwent Abstract 95-007479 of WPIDS document DE 4317577, Dec. 12, 1994.
Baumann Goetz
Di Padova Franco E.
Wenner Peter
Furman Diane E.
Kaufman Claire M.
Novartis AG
Spector Lorraine
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