Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1999-08-11
2001-03-27
Ceperley, Mary E. (Department: 1641)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C436S526000, C436S527000, C436S529000, C436S530000, C436S531000, C530S389800, C530S404000, C530S405000
Reexamination Certificate
active
06207398
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to novel cyclosporine derivatives that have improved protein and solid surface conjugatibility and hydrolytic stability. The cyclosporine derivatives of the present invention are useful in assays measurement of cyclosporin A levels, as well as in the production of cyclosporine immunogens and capture conjugates.
2. Background
Cyclosporine A (cyclosporine) is a potent immuno-suppressant that has been widely used in the United States and other countries to prevent the rejection of transplanted organs such as kidney, heart, bone marrow and liver, in humans.
To prevent allograft rejections, minimum level cyclosporine A in the blood is required throughout the lifetime of the patient. Chronic high doses can result in kidney and liver damage. Distribution and metabolism of the drug varies greatly between individuals, as well as in a single individual during the course of therapy. Accordingly, monitoring cyclosporine A levels in the blood or serum of allograph recipients is considered essential.
Laboratory methods for detection of cyclosporine have been developed. These techniques typically involve high performance liquid chromatography (HPLC), radioimmunoassay (RIA) and non-radioimmunoassay.
It has been reported that CsA, itself, is non-immunogenic (Donatsch, p. et al., J. Immuno Assay 1981; 2:19). To obtain antibodies, therefore, it is necessary to link CsA to a protein carrier. The side chain of CsA, however, consists most of alliphatic groups. Few of the functional groups customarily used to link a hapten to a carrier. Previous workers have made immunogenic cyclosporine C (CsC) protein conjugates because the CsC has a threonine residue in position 2. Linkage to a protein was via a hemisuccinate linker through an ester group (U.S. Pat. No. 5,169,773). In addition, hemisuccinate coupling chemistry has been used to immobilize CsC to a solid support such as stabilized chromium dioxide particles (U.S. Pat. No. 5,151,348). Due to a number of factors including, for example, short chain length of the hemisuccinate linker, hydrophobicity of the cyclosporine-C hemisuccinate molecule and hydrolytic instability of the hemisuccinate ester linkage, the CsC hemisuccinate derivatives conjugate poorly to protein and solid surface. Furthermore, CsC protein conjugates and CsC immobilized on a solid support by hemisuccinate coupling, are hydrolyticly unstable. Thus, immunoassays developed by using such hemisuccinate CsC derivatives suffer from low sensitivity and poor reagent stability. There is a strong desire to replace the widely used radioimmunoassays and HPLC methods with a more robust and sensitive immunoassay for CsA. Accordingly, there is a need in the art for cyclosporine derivatives that are capable of being conjugated to solid supports and carriers more efficiently and stably.
SUMMARY OF THE INVENTION
The present invention provides novel cyclosporine C (CsC) derivatives having improved protein conjugatibility and hydrolytic stability. The present invention further provides a CsC derivative conjugated to a carrier, e.g., a solid support. Preferably, the solid support is a latex or magnetic particle.
The invention also provides improvements in assays for the determination of cyclosporin levels in a sample, e.g., whole blood, suspected of containing cyclosporin.
Furthermore, the invention includes kits for conducting an assay for the determination of cyclosporin. The present invention also provides for the production of cyclosporine immunogens and capture conjugates comprising the CsC derivatives of the present invention.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to cyclosporine C (CsC) derivatives having the structure:
X is selected from the group consisting of:
Y is selected from the group consisting of:
wherein Z is selected from the group consisting of:
wherein R
1
and R
2
are each a C1-C8 alkyl group;
wherein R
3
is a C
0
-C
8
alkyl group; and
wherein m is 1-200
A particular embodiment of the present invention includes CsC derivatives having the following structures:
The CsC derivatives of the present invention were prepared by activation of the CsC position 2 hydroxy group using disuccinimidyl carbonate followed by coupling with linkers such as diamine linkers, e.g., ethylene glycol bis(2-aminoethyl)ether (DA-10). The following scheme illustrates the application of this procedure to the synthesis of the CsC derivatives of this invention:
Starting materials used in the above-described scheme are either known or commercially available.
The CsC derivatives of the present invention may be used in immunoassay for the measurement of cyclosporin A levels in whole blood samples. An example of such an assay comprises the steps of:
(a) lysing red blood cells in a sample of whole blood containing cyclosporin A;
(b) contacting the lysed whole blood sample with excess labeled anti-cyclosporin antibody, e.g., beta-D-galactosidase labeled, to form a labeled antibody-cyclosporin A complex;
(c) separating unbound antibody from the complex by contacting the mixture formed in step (b) with a solid phase comprising a cyclosporin derivative of the present invention immobilized on a solid support; and
(d) determining the amount of the label in the complex as a measure of cyclosporin A, using, for example, a beta-D-galactosidase substrate selected from the group consisting of chlorophenol red-beta-D-galactopyranoside (CPRG) and resorufin-beta-D-galactopyranoside (ReG) if a beta-D-galactosidase label is used.
The enzyme-linked immunoassay of this invention is useful for measuring cyclosporin A levels in whole blood samples of patients receiving cyclosporin A. Monitoring of cyclosporin A blood levels and subsequent cyclosporin A dosage adjustment are necessary to prevent toxic effects caused by high cyclosporin A blood levels and to prevent organ rejection caused by low cyclosporin A blood levels.
The immunoassay of the present invention is performed by contacting a lysed whole blood sample containing cyclosporin A with excess labeled anti-cyclosporin antibody, e.g., beta-D-galactosidase-labeled, to form a reaction mixture containing a complex of cyclosporin A with labeled antibody and free labeled antibody, separating free antibody from the reaction mixture by contacting the reaction mixture with a solid phase comprising an immobilized CsC derivative of the present invention on a solid support, e.g. magnetic particles, separating the solid phase from the liquid phase, and measuring the amount of the bound label in the liquid phase by adding, for example, to the liquid phase CPRG or ReG as a beta-D-galactosidase substrate if a beta-D-galactosidase label is used.
Specifically, the red blood cells of a whole blood sample containing cyclosporin A must be lysed to release cyclosporin A. Red blood cell lysis can be accomplished by many methods, such as sonication, detergent lysis and distilled water lysis. The lytic agent chosen should be compatible with the labeled anti-cyclosporin antibody. Although some detergents can denature-beta-D-galactosidase, it has been found that by using CPRG and ReG as beta-D-galactosidase substrates, the sample volume can be made to be sufficiently small to minimize the denaturing effect of the detergent. The preferred lysis method uses detergent.
After lysis, a reaction mixture is formed by contacting the lysed whole blood sample with excess labeled anti-cyclosporin antibody and incubating the reaction mixture for a time and at a temperature sufficient to permit the labeled antibody to form a complex with all of the cyclosporin A in the sample. This usually takes 1-5 minutes at room temperature. Anti-cyclosporin antibody can be obtained commercially, prepared by known methods, or prepared using the derivatives of the present invention. The anti-cyclosporin antibody can be polyclonal or monoclonal. A monoclonal anti-cyclosporin antibody specific for cyclosporin A is preferred. The anti-cyclosporin antibody can be labeled using standard techniques with any molecule tha
Ceperley Mary E.
Dade Behring Inc.
Gattari Patrick G
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