Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1998-02-20
2001-03-27
Venkat, Jyothsna (Department: 1627)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007900, C436S544000, C530S387100, C530S403000, C546S061000, C546S070000, C546S077000
Reexamination Certificate
active
06207396
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to novel carboxyalkyl 1-position derivatives of lysergic acid diethylamide (LSD) and methods for preparation of these derivatives. The derivatives include immunogens used to stimulate antibody production and polypeptide conjugates useful in immunoassays for detecting LSD. Also provided are hapten intermediates used in the synthesis of the immunogens and polypeptide conjugates and a non-isotopic immunoassay for the determination of LSD.
Although there is widespread public perception that use of LSD is no longer a societal problem, there is considerable evidence that this illicit drug continues to be used, and in some segments of the population, its use is increasing. See Bonner,
Drug Detection Report.
1:5 (1992). LSD was one of the 20 controlled substances most commonly encountered in emergency rooms across the nation in 1985, reflecting continuing abuse and trafficking of this illicit drug. In the United States, seizures of LSD by the Drug Enforcement Agency doubled in 1990 over the previous year, and in England, seizures of LSD have steadily increased since mid-1988. See
Microgram
23:228 (1990). Further cause for concern are reports that LSD is particularly popular among adolescents, and in some areas, it exceeds cocaine in popularity. See Seligmann,
Newsweek,
February 3rd, p. 66, (1992). Factors that have contributed to the continued use of LSD are its wide availability, low cost, and the difficulty of detecting LSD use by analysis of body fluids.
Despite the long history of abuse associated with LSD, little is known concerning the disposition of LSD in humans. The lack of pharmacokinetic data on LSD is partly due to the technical difficulty of detecting and measuring the drug in physiological specimens. LSD is not considered highly toxic, although at least two cases where death was apparently a result of LSD toxicity have been reported. However, the major reason many consider LSD to be highly dangerous is that it can have serious psychological and psychotic effects which sometimes cause users to commit irrational acts resulting in injury or death. LSD is an extremely potent psychedelic drug that acts primarily on the central nervous system; only the d-isomer of the drug is pharmacologically active. Oral doses as low as 25 &mgr;g can cause central nervous system disturbances such as hallucinations, distortions in sensory perception, mood changes and dream-like thought processes, as well as psychotic reactions in apparently predisposed individuals. Therefore, concentrations of LSD and LSD metabolites in blood and urine are likely to be very low. The detection of LSD in body fluids of users is especially difficult because the quantities typically ingested are very small and because the drug is rapidly and extensively converted to metabolic products. Furthermore, the drug's low volatility, its thermal instability, and its tendency to undergo adsorptive losses during gas chromatographic analysis all contribute to the difficulty of developing a method for confirmation of LSD in body fluids.
LSD is a natural product of the rye fungus Claviceps and was first prepared synthetically in 1938. Its psychological effects were discovered following accidental ingestion. Chemically, LSD is an ergot alkaloid and, like other compounds of this class, contains lysergic acid as the basis of its structure. Structurally similar to serotonin (5-hydroxytryptamine), LSD is thought to exert its psychotomimetic effects through antagonism of serotonin activity in the brain stem. Little is known about the tissue distribution, metabolism and excretion of LSD in humans. LSD is absorbed fairly rapidly by the gastrointestinal tract, and its plasma half-life has been calculated to be about 3 hours in man. Animal studies indicate that LSD is inactivated via hepatic oxidation. It is extensively metabolized with only negligible amounts of unchanged drug appearing in the urine and feces, with most of the metabolites being excreted in the urine. Possible metabolic transformations may be hydrolysis to lysergic acid, N-demethylation to nor-LSD and oxidation to 2-oxo-LSD. Studies with urine samples from human volunteers receiving LSD demonstrate that the drug or its closely related metabolites can be detected in the urine by radioimmunoassay (RIA) for several days following administration.
Although continued illicit use of LSD has stimulated efforts to develop effective analytical methods for the detection of the drug and its metabolites in body fluids from suspected LSD users, the methods currently available are complicated, time-consuming, expensive to perform and plagued by other problems. These methods include high performance liquid chromatography (HPLC), gas chromatography/mass spectrometry (GC/MS) and radioimmunoassay. One problem faced by laboratories involved in the determination of LSD is the strong tendency for LSD and derivatized LSD to undergo adsorptive losses when subjected to gas chromatography. This behavior often prevents detection of the drug at the sub-nanogram/milliliter concentrations normally encountered in body fluids from LSD users.
Commercial RIAs for LSD are available from several sources, including ABUSCREEN LSD assay (® Roche Diagnostics Systems, Nutley, N.J.) and COAT-A-COUNT LSD assay (® Diagnostic Products Corp., Los Angeles, Calif.), and these products serve as a useful and relatively inexpensive method of screening for the presence of the drug. However, RIAs are not totally specific for LSD, so that an RIA-positive specimen still has to be confirmed by a second and more specific assay if the results of the analysis could have punitive consequences. The manufacturers' recommended cut-off concentration for considering a sample positive for LSD is 0.5 ng/ml, although lower cut-offs have been used in investigations where legal consequences were not a concern. The actual concentration of LSD in RIA-positive urine specimens is generally lower than that indicated by the RIA, and often considerably lower. Presumably the higher concentrations indicated by RIA are due to the cross-reactivity of LSD metabolites to the RIA antisera, but this conclusion cannot be substantiated until the major LSD metabolites in urine have been identified and their cross-reactivities determined.
In testing for other drugs of abuse, immunoassays, particularly competitive binding immunoassays, have proven to be especially advantageous. In competitive binding immunoassays, an analyte in a biological sample competes with a labeled reagent, or analyte analog, or tracer, for a limited number of receptor binding sites on antibodies specific for the analyte and analyte analog. Enzymes such as &bgr;-galactosidase and peroxidase, fluorescent molecules such as fluorescent compounds, and adioactive compounds such as
125
I are common labeling substances used as tracers. The concentration of analyte in the sample determines the amount of analyte analog which will bind to the antibody. The amount of analyte analog that will bind is inversely proportional to the concentration of analyte in the sample, because the analyte and the analyte analog each bind to the antibody in proportion to their respective concentrations. The amount of free or bound analyte analog can then be determined by methods appropriate to the particular label being used.
One type of competitive binding immunoassay is based upon the reassociation of enzyymatically inactive polypeptide fragments to form active enzyme as a step of generating a detectable signal utilized to determine the amount of analyte present in a sample. This type of assay, known as cloned enzyme donor immunoassay (CEDIA), is described in U.S. Pat. No. 4,708,929. In particular, a &bgr;-galactosidase enzyme donor polypeptide combines with a &bgr;-galactosidase enzyme acceptor polypeptide to form active &bgr;-galactosidase enzyme. Conjugating a hapten, or a small analyte or an analyte analog, to the enzyme donor polypeptide at certain sites does not affect the ability to form active &bgr;-galactosidase by a complementat
Coty William A.
Davoudzadeh David
Morrill Paul R.
Rouhani Riaz
Shindelman Jeffrey E.
Microgenics Corporation
Ponnaluri P
Venkat Jyothsna
Wood Herron & Evans L.L.P.
LandOfFree
Immunoassay reagents and methods for determination of LSD... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Immunoassay reagents and methods for determination of LSD..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Immunoassay reagents and methods for determination of LSD... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2466014