Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1998-08-06
2001-02-13
Housel, James C. (Department: 1641)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S004000, C435S007900, C436S501000, C436S536000, C514S885000, C530S317000, C530S321000, C530S350000
Reexamination Certificate
active
06187547
ABSTRACT:
This invention relates to an assay procedure and kit for use in determining the levels of drug substances in unextracted blood in the presence of specific binding proteins. The assay is particularly suitable for determining the blood levels of immunophilin-binding drugs, e.g., cyclosporins, rapamycins, or FK506 compounds.
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, 11th Edition; Merck & Co., Inc.; Rahway, New Jersey, 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 296 122, EP 484 281, and GB 2222770.
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 rapainycin 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 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 40-O-alkylated derivatives (WO 94/09010). Rapamycin and its structurally similar analogues and derivatives are termed collectively as “rapamycins” in this specification.
FK506 is a macrolide immunosuppressant that is produced by
Streptomvces tsukubaensis
No 9993. The structure of FK506 is given in the appendix to the Merck Index, as item A5. Also a large number of related compounds which retrain the basic structure and immunological properties of FK506 are also known. These compounds are described in a large number of 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. These compounds are termed collectively “FK506 compounds” in this specification.
Due to their extremely useful pharmaceutical properties, cyclosporins (and Cyclosporins A and G in particular), rapamycins and FK506 compounds 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 therefore necessary to monitor the concentration of these immunosuppressants in the blood at regular intervals.
Certain assay procedures based upon high pressure liquid chromatography (HPLC) have been developed but are either cumbersome to use or are not specific enough. For cyclosporin A and FK-506, specific monoclonal antibodies have been developed and assay procedures based on the antibodies provided. However all the assay procedures provided to date require the blood or plasma sample to be first extracted with a solvent (such as methanol) which is then removed by evaporation or dilution. The antibody is then added to the sample and a radioimmunoassay (RIA) analysis performed. The assay procedure based on the specific monoclonal antibody works well but the need for the extraction step and the subsequent removal of the solvent can result in the assay becoming less sensitive and less precise if care is not taken. Therefore the assay must be carried out by skilled technicians and is a time consuming procedure.
Hence, given the importance of cyclosporins, rapamycins and FK506 compounds as pharmaceuticals, there is a need for simple, sensitive assays to determine their concentrations in blood.
Accordingly this invention provides an assay procedure for determining the concentration of a immunophilin-binding pharmaceutical in blood; the procedure comprising adding a binding competitor that displaces the pharmaceutical from immunosuppressant-immunophilin complexes in the blood; adding a receptor that binds to the pharmaceutical but not significantly to the binding competitor, separating the receptor-pharmaceutical complex from the sample; and determining the amount of the pharmaceutical.
It has been found that a portion of a cyclosporin, rapamycin or FK506 compound present in blood exists in the form of a pharmaceutical-immunophilin complex. If the pharmaceutical is displaced from the complex using a binding competitor, it is then not necessary to extract the blood sample using methanol and hence the disadvantages associated with methanol extraction and removal are removed. The resultant assay procedure gives accurate results, is simple and is a surprising break-through in the assaying of cyclosporins, rapamycins or FK506 compounds. For example, the assay procedure is able to detect concentrations as low as 0.7 ng (cyclosporin A)/ml (of whole blood) with a coefficient of variation of less than 30%. This is much better than the cyclosporin A assay that is commercially available.
Immunophilins are a family of intracellular binding proteins which bind cyclosporins, rapamycins or FK506 compounds. Two distinct families of immunophilins are presently known; cyclophilins which bind to cyclosporins, and macrophilins which bind to rapamycins and FK506 compounds. The structures of certain immunophilins are described in Walkinshaw et al; 1992
; Transplantation Proceedings
, 24, 4(2), 8-13. Specific examples are cyclophilin A and macrophilin-12 (often known as FKBP-12).
The amount of the binding competitor to be used to displace the pharmaceutical from the immunophilin-pharmaceutical complex is likely to vary from pharmaceutical to pharmaceutical and from binding competitor to binding competitor. However, in each case, an optimum range may be readily determined carrying out the assay procedure at several concentrations (including a blank) of the pharmaceutical and at several concentrations of the binding competitor. The samples are then diluted two or three times and the procedures carried out again on each dilution. The sensitivities of the tests are then compared and those concentrations of binding competitor that give decreased sensitivity are discarded.
The receptor which binds to the pharmaceutical may be any specific binding compound as is used in conventional assays, e.g., polyclonal, monoclonal, or recombinant antibodies, antibody fragments, or molecular imprinted polymers (e.g., as described by Vlatakis, et al., (1993) Nature, 361:645), preferably a monoclonal antibody.
Once the pharmaceutical is released from the pharmaceutical-immunophilin complex, the amount of the pharmaceutical bound to the receptor may be determined using any assay method, preferably a monoclonal antibody based assay, e.g., a competitive assay measuring the ability of the pharmaceutical to compete for binding to the antibody or receptor, or a noncompetitive assay. A competitive assay preferably uses, e.g., a labeled pharmaceutical (tracer) as competitor for the antibody, in the presence and absence of the test sample. The tracer may be labeled with a label capable of providing a suitable readout, e.g., radioactive, fluorescent, luminescent or colorimetric readout as is conventional in the art. Alternatively, the competitor for the receptor may be unlabeled pharmaceutical (optionally the pharmaceutical-protein immunogenic conjugate used to raise the antib
Legay Francois
Wenger Roland
Furman Diane E.
Housel James C.
Novartis AG
Ryan V.
LandOfFree
Assay kit does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Assay kit, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Assay kit will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2598671