Organic compounds -- part of the class 532-570 series – Organic compounds – Heterocyclic carbon compounds containing a hetero ring...
Reexamination Certificate
1999-06-18
2001-07-17
Chin, Christopher L. (Department: 1641)
Organic compounds -- part of the class 532-570 series
Organic compounds
Heterocyclic carbon compounds containing a hetero ring...
C546S061000, C546S069000, C546S070000, C546S074000, C546S077000, C546S106000, C435S004000, C435S006120, C435S007800, C435S007210, C435S007600, C435S007900, C436S544000, C436S545000, C436S546000, C530S300000, C530S350000, C530S387100, C530S403000
Reexamination Certificate
active
06262265
ABSTRACT:
TECHNICAL FIELD
This invention relates generally to the field of determining drug metabolites in biological samples. More specifically, it provides a system of analogs, conjugates and specific antibodies that can be used in assay systems for specific detection or quantitation of heroin abuse.
BACKGROUND
Testing for heroin abuse is complicated by the fact that heroin undergoes rapid metabolism to 6-O-acetyl-morphine (also known as 6-monoacetylmorphine, 6MAM). After an intramuscular administration of heroin, 6MAM appears in urine almost immediately. Levels of 6MAM remain positive in urine for about 8 hours, as detected by standard techniques such as GC/MS (Cone et al., Anal. Toxicol. 15:1, 1991). Heroin is then broken down into morphine, which is a metabolite of other opiates such as codeine.
A number of tests have been developed for measuring opiates in biological samples. Generally, immunoassays have been unsuccessful at discriminating between 6MAM and related compounds. Other opiate metabolites, such as morphine-3-glucuronide and morphine-6-glucuronide, may be present at levels approximately four to five orders of magnitude greater than 6MAM. Investigators have had to resort to more cumbersome and expensive techniques to determine the identity of an opiate in an unknown sample.
Solans et al. (J. Pharmaceut. Biomed. Anal. 8:905, 1990) determined various opiates in urine, including 6MAM, using GC-MS. Jenkens et al. (J. Anal. Toxicol 22:173, 1998) used GC-MS to detect 6MAM in postmortem cerebrospinal fluid. Bogusz et al. used chemical ionization-mass spectrometry-liquid chromatography to determine 6MAM and other morphine metabolites in samples collected from overdose victims on autopsy. Zuccaro et al. (J. Anal. Toxicol. 21:268, 1997) describe simultaneous determination of 6MAM, morphine glucuronides, and other opiates by liquid chromatography-atmospheric pressure ionspray-mass spectrometry.
Holt et al. (Anal. Chem. 68:1877, 1996) report a bioluminescent assay for heroin and its metabolites. The assay is based on heroin esterase, and morphine dehydrogenase, using bacterial luciferase from
Vibrio harveyi.
Heroin, morphine, and codeine are all detected.
International patent publication WO 93/20079 (Buechler et al.) report opiate derivatives and protein conjugates. In their synthetic compounds, the group at the 6-O-position is typically H—, CH
3
(CO)—, or CH
3
CH
2
—. Some of the conjugates are proposed as immunogens, but the ability of the antibodies obtained to discriminate between opiates is not reported.
European patent application EP 0363041-A1 (Uda et al.) report monoclonal antibodies to morphine, made using a N-(4-bromobutyl) derivative. An immunoassay is reported using the antibody and a BSA normorphine conjugate. Cross-reactivity between morphine and each of cocaine, codeine, dihydrocodeine, ethylmorphine, fentanyl or methadone is reported as <1.0% or better, depending on the antibody tested.
U.S. Pat. No. 4,064,228 (Gross et al.) report antigens and immunoassays for morphine and related compounds. 3-Oxybenzomorphan derivatives are conjugated to immunogenic carriers using a linking site on an aryl group, and used to produce antibodies. The assays are reported as being primarily reactive with morphine, with an 11% cross-reactivity to codeine and a 7% cross-reactivity to monoacetylmorphine.
Fogerson et al. (J. Anal. Toxicol. 21:451, 1997) describe the qualitative detection of opiates such as heroin, 6MAM and morphine, in sweat. Immunoassay was conducted using an enzyme immunoassay kit from STC Technologies. Standardized to morphine, the assay also reacted with 6MAM (30% cross-reactivity), hydrocodone (143%), and codeine (588%). Discrimination between opiates was only possible using GC-MS. Similarly, the sweat testing by Kintz et al. (Clin Chem. 43:736, 1997) is done strictly by GC-MS.
Moeller et al. (Forensic Sci. Int. 70:125, 1995) report the detection of 6MAM in biological samples by GC/MS and radioimmunoassay. The radioimmunoassay used was obtained in kit form from Diagnostic Products Corporation.
FIG. 4
of that article shows the reactivity of potential interfering substances in comparison with 6MAM. Based on 50% B/Bo points, cross-reactivity with heroin is ~50%, morphine ~30%, morphine-6-glucuronide ~20%. Reactivities with codeine are not titrated to the 50% point, but codeine and other interfering substances are clearly detectable.
Preferable to separation immunoassays (in which antibodies are used to capture the analyte) are homogeneous type assays, in which analyte-antibody complexes are detected in situ. A particularly powerful homogeneous assay system is the cloned enzyme donor immunoassay (CEDIA®), described in U.S. Pat. No. 4,708,929, and in Henderson, Clin. Chem. 32:1637, 1986. In a preferred form of the CEDIA® assay, two subunits of the enzyme beta-galactosidase associate to provide the detectable signal, which is quantitatively affected by analyte-specific antibody except in the presence of a sample containing free analyte.
Specific immunoassays of either type require the availability of an antibody that binds the analyte of interest but not potential interfering substances. An immunoassay for 6MAM capable of differentiating samples containing codeine or morphine requires an antibody with a very high relative affinity for 6MAM.
The limited specificity in current art 6MAM assays is attributable to the limited specificity in the antibody in the assay. This in turn is attributable to the unsuitability of current art immunogens for generating antibodies with a better ability to discriminate between the analyte and potential interfering substances.
DISCLOSURE OF THE INVENTION
The present invention provides a system for the improved detection of heroin metabolites in biological samples. New chemical analogs are provided for the metabolite 6-O-acetyl-morphine (also known as 6-monoacetylmorphine, 6MAM). These analogs are more stable than 6MAM, but have been found to mimic 6MAM in several important functional parameters. The hapten analogs can be used to construct immunogens, enzyme or enzyme donor conjugates, and other hapten conjugates.
The immunogens reproducibly generate antibodies with an exquisite ability to distinguish 6MAM in biological samples from morphine, codeine, and other potential interfering substances. The specific antibodies and the hapten conjugates can be used to distinguish and measure 6MAM in biological samples, such as those obtained from an individual suspected of substance abuse. Additional embodiments relate to certain reagents embodied in this invention, reagent combinations, and kits for performing an assay method for 6MAM in a biological sample.
In one embodiment, the invention encompasses 6MAM analog compounds having the following structure:
and its salts,
wherein X is —O—, —S—, —NH— or —CH
2
— and
wherein R is selected from the group consisting of:
wherein K is —CH
3
, —CF
3
, —CHF
2
, or —CH
2
F; with the proviso that when R is
and K is —CH
3
, X is not —O—; and
Q is —L
1
—Z, where L
1
is a linker containing at least one carbon atom;
wherein Z is selected from the group consisting of the moieties
—NH
2
,
—COOH,
—SH,
—NH—C(═O)—L
2
—M,
a moiety which reacts with a protein to form a covalent bond, or any combination or repetition of the aformentioned moieties;
where L
2
is a linker containing at least one carbon atom; where M is halide or maleimide; and wherein J is —O—, —S—, —NH— or —CH
2
—. A preferred X group is —O—. Preferred R groups include
L
1
and L
2
are preferably independently selected from the group consisting of C
1
-C
20
hydrocarbon chains, containing zero to ten heteroatoms selected from the group consisting of N, O, and S, and which contain at least as many carbon atoms as heteroatoms.
In certain embodiments, the compound is covalently attached to a label, either directly or through a linking group, to form a conjugate. The attachment can be anywhere on the molecule, including the normorphine nitrogen, and is preferably attached to the Z group of the compound. The label is preferably selected
Rouhani Riaz
Sigler Gerald F.
Chin Christopher L.
Cook Lisa V.
Microgenics Corporation
Wood Herron & Evans LLP
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