Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1997-10-06
2001-12-04
Marschel, Ardin H. (Department: 1631)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S011000, C435S015000, C435S018000, C435S019000, C435S020000, C435S183000, C435S196000, C435S197000, C435S069100, C514S002600, C514S014800
Reexamination Certificate
active
06326139
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Technical Field
The present invention relates to a method for screening for a genetic predisposition for adverse responses to anticholinesterase therapy and exposure.
2. Background Art
The clinical uses of anticholinesterases (anti-ChEs) have recently been extended in two major developments, involving many new subjects. First, during the 1991 Gulf War, the carbamate, pyridostigmine was administered prophylactically to over 400,000 soldiers, with the intention of transiently blocking (and thus protecting) a fraction of their nervous system acetylcholinesterase (AChE, EC 3.1.1.7), in anticipation of nerve agent attacks (Gavageran, 1994; Ember, 1994). Yet more recently, the reversible cholinesterase (ChE) inhibitor, tetrahydroamino acridine (THA, tacrine, Cognex®) was approved for use in patients with Alzheimer's disease, for the purpose of enhancing the availability of acetylcholine at synapses and improving residual cholinergic neurotransmission in patients suffering from massive degeneration of cholinergic neurons (Knapp et al., 1994).
Adverse symptoms were reported in both groups (Ember, 1994; Gavageran, 1994; Winker, 1994), including responses characteristic of cholinergic deficits, such as depression, general fatigue, insomnia and weight loss. However, these were only a few out of many symptoms in a complex and diverse list, the interpretation of which was complicated by incomplete medical records and the stressful situation experienced by the first group and the generally bad condition of the aging patients from the second group.
To identify the molecular basis of these adverse responses to anti-ChEs, applicants have focused on the protein targets of these agents. Most anti-ChEs were designed as selective AChE inhibitors; however, many, if not all of these drugs also interact quite efficiently with the closely related serum butyrylcholinesterase (BuChE). In fact, some consider one of BuChE's biological roles to be a scavenger of natural anti-ChEs. No allelic variant with modified biochemical properties is known for AChE, perhaps because the fully active enzyme is absolutely essential to ensure good quality cholinergic neurotransmission. In contrast, there are over 20 different allelic variants of BuChE, some of which display altered interactions with certain inhibitors (Neville et al., 1990a; Gnatt et al. 1994). This raised the possibility of a genetic basis, rooted in the polymorphism of BuChE, of the adverse symptoms experienced by some patients undergoing anti-ChE therapy.
Acetylcholinesterase (EC 3.1.1.7; AChE) and butyrylcholinesterase (EC 3.1.1.8; BuChE) are two closely homologous proteins. Both are present in all vertebrates, and both are capable of hydrolyzing the neurotransmitter, acetylcholine (ACh). Reviews, by Taylor (1991), Massoulie et al. (1993), Soreq and Zakut (1993), and Taylor and Radic (1994) contain specialized information on sub-topics, especially on cholinesterases (ChEs) of non-human species and on the cell biology aspects of these enzymes.
The most obvious and best studied function of AChE is the hydrolysis of ACh to terminate neurotransmission at the neuromuscular junction and nicotinic or muscarinic brain synapses and secretary organs of various sorts. AChE is characterized by a narrow specificity for ACh and certain inhibitors and by substrate inhibition. In humans, AChE is produced in muscle and nerve, in hemopoietic cells (Patinkin et al., 1990; Lev-Lehman et al., 1994; Soreq et al., 1994), embryonic tissues (Zakut et al., 1985; Zakut et al., 1990), several tumors (Lapidot-Lifson et al., 1989) and germ cells (Malinger et al., 1989).
The role of BuChE, beyond hydrolyzing ACh at concentrations that would cause inhibition of AChE (Augustinsson, 1948), has not been identified with certainty, but as it has a wider substrate specificity and interacts with a broader range of inhibitors, it has been proposed that it scavenges anti-ChE agents, protecting synaptic AChE from inhibition and the multitude of ACh receptors from blockade (Soreq et al., 1992).
The protein chemistry, enzymology, non-CNS
on-catalytic role(s) and genetics of AChE and BuChE has been extensively reviewed by Schwarz, et al (1995) incorporated herein by reference. In addition, see Loewenstein-Lichtenstein, Y. (1995) “Structural and Molecular Dissection of Biologically Active Domains in Human Cholinesterases”, Ph.D. Thesis, Hebrew University of Jerusalem, incorporated herein by reference, for a review of this material. Of particular interest for the present application is the following.
In man, the two functionally distinct ChEs, AChE and BuChE, which share a high degree of amino acid sequence homology (>50%), are encoded by two separate genes, ACHE and BCHE, respectively (Soreq et al., 1990). The two genes have similar exon-intron organization but radically different nucleotide composition, ACHE being G,C-rich while BCHE is A,T-rich. The presence of two distinct ChE genes in all vertebrates studied to date, indicates that both protein products are biologically required in these species, and presumably that they have distinct roles.
The human ACHE gene spans about 7 kb and includes 6 characterized exons and 4 introns. It can, through alternative splicing, give rise to several different mRNA transcripts (Sikorav et al., 1988; Maulet et al., 1990). The BCHE gene is much larger than ACHE, spanning 70 kb, and consists of 4 exons, the first of which is non-translatable but contains two potential translation initiation sites, and the second of which contains 83% of the coding sequence (Arpagaus et al., 1990; Gnatt et al., 1991). The use of fluorescent in situ hybridization with biotinylated ACHE DNA, mapped the refined position of the ACHE gene to chromosome 7q22 (Ehrlich et al., 1994a; Getman et al., 1992).
Mapping of the human BCHE gene to its defined chromosomal location, 3q26-ter, was first performed by in situ hybridization to lymphocyte chromosomes and by blot hybridization to DNA of hybrid somatic cells (Gnatt et al., 1990). Direct PCR amplification of human BCHE-specific DNA fragments from somatic cell hybrids and chromosome sorted libraries later confirmed this mapping of the BCHE gene to chromosome 3q26-ter (Gnatt et al., 1991). When using ACHE specific primers, a prominent PCR product was observed with DNA from two different cell-lines and from one chromosome sorted library, all containing DNA from human chromosome 7.
These findings confirmed predictions that the two closely related CHE genes are not genetically linked in the human genome (Gnatt et al., 1991). They further revealed that these two apparently unrelated genes are both located at chromosomal sites subject to frequent breakage in leukemias (Ehrlich et al., 1994a).
Drugs that are Hydrolyzed or Scavenged by Cholinesterases
When a drug enters the body through the blood stream, its first encounters with a ChE are with AChE of the erythrocyte membrane outer surface and with circulating BuChE. However, since BuChE is capable of interacting with a wider range of ligands than AChE, and in some cases, at much higher rates (Schwarz et al., 1994), it appears to be the major scavenger of anti-ChE agents. Support for this can be found in the absence of a correlation between IC
50
values of AChE for a range of carbamate anti-ChE's, and their LD
50
values, while a positive correlation between these parameters exists for BuChE (Loewenstein et al., 1993b).
Succinyl choline, an inhibitor of AChE, is a commonly used muscle relaxant. BuChE recognizes succinyl choline as a substrate, and the slow hydrolysis of the agent limits the duration of its action in vivo. BuChE has been shown in vitro to hydrolyze the methyl ester bond of cocaine and its derivatives (Isenschmid et al., 1989; Gatley, 1991). In vivo, in contrast to cytochrome P-450-catalyzed destruction of cocaine, which produces hepatotoxic norcocaine nitroxide, BuChE-catalyzed hydrolysis of cocaine generates innocuous products. The serum levels of BuChE, too, as modulated by ChE inhibitors, correlate with serum levels of cocaine
Soreq Hermona
Zakut Haim
Kohn & Associates
Marschel Ardin H.
Yissum Research Development Company of Hebrew University of Jeru
LandOfFree
Method of screening for genetic predisposition to... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Method of screening for genetic predisposition to..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Method of screening for genetic predisposition to... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2596602