1. Field of the Invention
The body relies upon a complex immune response system to distinguish self from non-self. The proper functioning of the immune system is vital for the long term health of the body.
Deficient immune response can lead to the body's inability to protect itself from non-self matter. Excessive immune response can lead to the body's over reaction to what would otherwise be innocuous matter.
At times, the body's immune system must be controlled in order to either augment a deficient response or suppress an excessive response. For example, when organs such as kidney, heart, heart-lung, bone marrow, and liver are transplanted in humans, the body will sometimes reject the transplanted tissue by a process referred to as allograft rejection.
In treating allograft rejection, the immune system is frequently suppressed in a controlled manner through drug therapy. Immunosuppressant drugs are carefully administered to transplant recipients in order to help prevent allograft rejection of non-self tissue.
One drug which finds use as an immunosuppressant in the United States and other countries is cyclosporin A (CsA). CsA is a cyclic undecapeptide of the general structure: ##STR1##
wherein all of the .alpha.-amino acid residues that form the cyclic backbone of cyclosporin A are of the L-configuration except .alpha.-amino acid 8 which is of the D-configuration. Amino acid residue 1 is derived from an unusual 9 carbon amino acid [2S,3R,4R,6E]-3-hydroxy-4-methyl-2-methylamino-6-octenoic acid. Amino acid residues 1, 3, 4, 6, 9, 10 and 11 are N-methylated on the amide nitrogen atoms of the cyclic backbone of cyclosporin A. Cyclosporin A is described in U.S. Pat. Nos. 4,117,118 (1978) and 4,396,542 (1983).
CsA may have other useful properties such as antibiotic, anti-arthritic and anti-inflammatory activities and may find use in the treatment of other conditions such as diabetes, malaria and autoimmune diseases.
A large number of CsA metabolites that retain the undecapeptide ring have been identified and reported (see Maurer, G.; Loosli, H. R.; Schreier, E.; Keller, B. Drug Metabolism and Disposition 1984, 12(1), 120-126, the structures, nomenclature and analytical data of the metabolites are incorporated herein by reference). It is not known what role, if any, these metabolites play in either the desired immunosuppressant activity or any unwanted adverse reactions when CsA is used for the prevention of allograft rejection.
Even though CsA is a highly effective immunosuppressant drug, its use must be carefully managed because the effective dose range is narrow and excessive dosage can result in serious side effects. Renal dysfunction, hypertension, cardiovascular cramps, hirsutism, acne, tremor, convulsions, headache, gum hyperplasia, diarrhea, nausea, vomiting, hepatotoxicity, abdominal discomfort, paresthesia, flushing, leukopenia, lymphoma, sinusitis and gynecomastia have been observed in kidney, heart or liver transplant patients undergoing CsA treatment. Too little CsA can lead to graft rejection.
Management of CsA dosage involves careful control of the level of the drug present in the patient. Because the distribution and metabolism of CsA varies greatly between patients, and because of the wide range and severity of adverse reactions, accurate monitoring of drug level is considered essential.
Laboratory methods for detection of cyclosporin have been developed. These techniques typically involve high performance liquid chromatography (HPLC), radioimmunoassay (RIA) or florescence polarization immunoassay (FPIA). See for example Wolf, B. A.; et al. Clinical Chem. 1989, 35(1), 120-124; Vernillet, L.; et al. Clinical Chem. 1989, 35(4), 608-611; Ball, P. E.; et al. Clinical Chem. 1988, 34(2), 257-260; Schran, H. F.; et al. Clinical Chem. 1987, 33(12), 2225-2229; Sanghvi, A.; et al. Clinical Chem. 1988, 34(9), 1904-1906; McBride, J. H.; et al. Clinical Chem. 1989, 35(8), 1726-1730; Quesniaux, V.; et al. Clinical Chem. 1987, 33(1), 32-37.
Each of these techniques has certain limitations with regard to safety and complexity of the procedure and level of specificity for cyclosporins of interest. For example, HPLC requires long sample preparation and/or run times using high cost labor-intensive procedures; RIA presents the well-known hazards of handling radioactive materials; and FPIA, when based on non-specific mono- or polyclonal-antibodies, often fails to distinguish between CsA and its metabolites.
A simple analytical method specific to selected cyclosporins is needed for use in cyclosporin treatment management.
Immunoassay is a technique suited to the requirements of a simple method for measuring the amount of cyclosporin in a sample suspected of containing cyclosporin. However, most available antibodies capable of recognizing cyclosporins of interest also recognize and cross-react with closely related compounds such as cyclosporin metabolites. Because of this cross-reactivity, immunoassays dependent on these antibodies are less specific to cyclosporins of interest than might be desired. Cyclosporin immunoassays dependent on antibodies which cross-react with compounds other than cyclosporins of interest can be improved if the cross-reactive compounds are substantially inactivated toward recognition by the antibodies which recognize the cyclosporins of interest.
The methods and compositions of the present invention relate to a simple, specific immunoassay method for cyclosporins. The methods and compositions of the present invention also related to a method for inactivating cross-reactive compounds in immunoassay methods for cyclosporin.
2. Brief Description of the Related Art
World Patent Application No. 8,602,080 (1986), describes monoclonal antibodies selective for certain cyclosporins. These antibodies are prepared in response to cyclosporins bound to antigenic carriers through a modified amino acid residue such as a (D)-Lysine at position 8 or an (L)-threonine at position 2.
European patent application No. 283,801 (1988), describes a fluorescence immunoassay method and cyclosporin A derivatives used to raise antibodies. Cyclosporin A is linked to an immunogenic carrier via the amino acid side-chain at amino acid residue no. 1.
Cacalano, N. A.; Cleveland, W. L.; Erlanger, B. F. J. Immunol. Meth. 1989, 118, 257-263, describes the, presumably but not certainly, random photochemical grafting of cyclosporin A alkyl side-chains onto 4-benzoylbenzoic acid. These grafts are used to couple to immunogenic carriers for antibody preparation.
U.S. Pat. No. 4,727,035 (1988), describes a method of immunoassay for cyclosporins. The method involves either radioiodine or fluorescence immunoassay.
Quesniaux, V. F. J.; Tees, R.; Schreier, M. H.; Wenger, R. M.; Van Regenmortel, M. H. V. Molecular Immunology 1987, 24(11), 1159-1168, describes antibodies grown in response to cyclosporin-immunogen conjugates. The conjugates are attached to cyclosporin through a modified side chain on amino acids 2 or 8.
U.S. Pat. Nos. 4,764,503 (1988), 4,639,434 (1987), and 4,384,996 (1983), describe cyclosporins modified at amino acid residue no. 8. These cyclosporins are hydroxylated on the amino acid side-chain and are useful as immunosuppressive drugs.