Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1994-07-07
2001-05-01
Ceperley, Mary E. (Department: 1641)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S188000, C436S815000, C530S403000, C530S404000, C530S405000, C530S406000, C530S388900, C530S389800
Reexamination Certificate
active
06225073
ABSTRACT:
BACKGROUND OF THE INVENTION
FIELD OF THE INVENTION
Mycophenolic acid (“MPA”) is produced by the fermentation of several penicillium species.
It has a broad spectrum of activities, specific mode of action, and is tolerable in large doses with minimal side effects, Epinette, et al.,
Journal of the American Academy of Dermatology
17(6):962-71 (1987). MPA has been shown to have antitumor, antiviral, antipsoriatic, immunosuppressive, anti-inflammatory activities, Lee, et al.,
Pharmaceutical Research
7(2):161-166 (1990), along with antibacterial and antifungal activities, Nelson, et al.,
Journal of Medicinal Chemistry
33(2):833-838 (1990). It inhibits inosine monophosphate dehydrogenase, an enzyme in the de novo synthesis of purine nucleotides. Since T and B lymphocytes depend largely upon this de novo synthesis, MPA is able to inhibit lymphocyte proliferation, which is a major factor of the immune response.
The morpholinoethyl ester of MPA, morpholinoethyl E-6-(1,3-dihydro-4-hydroxy-6-methoxy-7-methyl-3-oxo-5-isobenzofuranyl)-4-methyl-4-hexenoate (“MPA-M”) is rapidly hydrolyzed in vivo to MPA. Administration of MPA in the form of this ester, greatly improves MPA's bioavailability.
MPA-M has a number of other favorable pharmaceutical characteristics, including its stability at pH 2-5 and its good solubility at low pH indicating rapid dissolution in the upper GI tract, Lee, et al., supra.
When used in combination therapy with cyclosporin A (“CsA”), MPA-M and CsA may have a synergistic mode of action. CsA has a selective effect on T cells, but does not suppress B cell antibody production activity, while MPA has an anti-proliferative effect on both T and B cells. Combined CsA/MPA-M therapy may increase survival time and allow for use of lower doses of CsA, which would reduce the side effects associated with CsA, primarily nephrotoxicity.
Because MPA is a potent biologically active material, an effective immunoassay would be useful in monitoring its bioavailability. In addition, it may be important to monitor therapeutic drug levels, i.e., optimal drug levels necessary for adequate immunosuppression. Since MPA-M is rapidly hydrolyzed to MPA, an assay for MPA would allow monitoring of MPA-M dosages.
Such assays, however, are limited by the difficulty of preparing antibodies which bind specifically to MPA and not to any MPA-M or metabolites such as the inactive metabolite mycophenolic acid glucuronide (“MPA-G”), that may be present.
The present invention addresses this need. The present invention provides antibodies that bind MPA and are able to modulate the activity of a label that is bound to another antibody or to an MPA analog and, further, that are capable of distinguishing between MPA and cross-reactive materials such as mycophenolate esters and/or MPA metabolites.
DESCRIPTION OF THE RELATED ART
Jones, et al.,
J. Chem. Soc.
(C) 1725-1737 (1970) discloses numerous transformations that mycophenolic acid undergoes when incubated with select microorganisms.
Nelson, et al., U.S. Pat. No. 4,753,935, pertains to MPA-M, its pharmaceutical uses, and post-dosage monitoring by HPLC of the recipient's plasma concentration of MPA.
SUMMARY OF THE INVENTION
The present invention relates to a method for the determination of mycophenolic acid (“MPA”) in a sample suspected of containing MPA comprising the steps of: (a) contacting the sample with an antibody capable of distinguishing between MPA and mycophenolate esters; and (b) detecting the binding of the antibody to MPA. This method can be homogeneous or heterogeneous. Alternatively, this method uses an antibody capable of distinguishing between MPA and MPA metabolites.
Another aspect of the present invention relates to a method for measuring the amount of MPA in a sample suspected of containing MPA which comprises the steps of: (a) combining in an aqueous medium: the sample, MPA conjugated to a detectable label, and an antibody capable of distinguishing between MPA and a compound selected from the group consisting of mycophenolate esters and mycophenolic acid metabolites; and (b) determining the effect of the sample on the activity of the label.
Another aspect of the present invention relates to a method for homogeneous immunoassay of MPA in a sample suspected of containing this analyte which comprises: (a) combining in a liquid medium: the sample, a conjugate of an MPA analog and an enzyme, an antibody capable of distinguishing between MPA and mycophenolate esters, and substrates for the enzyme; (b) determining the enzymatic activity of the enzyme in the medium; and (c) comparing the activity to the enzymatic activity observed with a sample containing a known amount of the analyte. Alternatively, this method uses an antibody capable of distinguishing between MPA and MPA metabolites.
Another aspect of the present invention relates to a compound comprising MPA bound to a protein by replacement of one or more hydrogen atoms. This invention also relates to an antibody raised in response to this compound, which is capable of distinguishing between MPA and a compound selected from the group consisting of mycophenolate esters and MPA metabolites. The antibody can be bound to a detectable label.
Yet another aspect of the invention relates to a kit for conducting an assay for the determination of MPA, comprising in packaged combination: an antibody capable of distinguishing between MPA and mycophenolate esters, and a compound comprising MPA bound to a detectable label. Alternatively, this kit uses an antibody capable of distinguishing between MPA and MPA metabolites.
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
Before proceeding with the description of the specific embodiments of the invention, a number of terms will be defined.
Sample suspected of containing the analyte: any sample which is reasonably suspected of containing the analyte, i.e., MPA, can be analyzed by the method of the present invention. The sample is typically an aqueous solution such as a body fluid from a host, for example, urine, whole blood, plasma, serum, saliva, semen, stool, sputum, cerebral spinal fluid, tears, mucus, or the like, but preferably is plasma or serum. The sample can be pretreated as described below and can be prepared in any convenient medium which does not interfere with the assay. An aqueous medium is preferred.
Interfering cross-reactive material: material other than MPA that may be recognized by antibodies that bind MPA is an interfering cross-reactive material. These include compounds related to MPA, such as mycophenolate esters and MPA metabolites. The term “mycophenolate ester” includes, but is not limited to, esters of MPA at the carboxylic acid group of the side chain attached at the 1′ position of the MPA isobenzofuranyl ring system such as MPA-M. The term “MPA metabolite” refers to the products of the metabolism of MPA, preferably products containing the isobenzofuranyl ring system, more preferably products also containing a portion of the side chain attached at position 1′, such as MPA-G.
Measuring the amount of MPA: quantitative, semiquantitative, and qualitative methods as well as all other methods for determining MPA are considered to be methods of measuring the amount of MPA. For example, a method which merely detects the presence or absence of MPA in a sample suspected of containing MPA is considered to be included within the scope of the present invention. The terms “detecting” and “determining”, as well as other common synonyms for measuring, are contemplated within the scope of the present invention.
Capable of distinguishing between: the ability of a receptor or antibody to bind preferentially to a first ligand relative to a second ligand. In the present invention, the first ligand is MPA and the second ligand is a mycophenolate ester or MPA metabolite. Usually at least 5-fold more of the first ligand than the second ligand will be bound when the antibody is combined with a sample containing the ligands. Preferably, at least 10-fold more and, more preferably, at least 20-fold more of the first ligand will be bound. A
Alexander Svetlana
Davalian Dariush
Ceperley Mary E.
Dade Behring Marburg GmbH
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