Method of screening for genetic predisposition to anticholinesterase therapy

Description

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/non-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 and related narcotics (Kambam et al., 1992) and with their physiological effects (Kambam et al., 1993). This clearly indicates a role for BuChE that must be recognized, especially by those practicing in areas or populations with narcotic usage. Furthermore, exogenous (human) BuChE has been shown to confer protection against cocaine toxicity in rats, both when given prophylactically or therapeutically (Dretchen et al., 1992). Following the elucidation of the pharmacological effects of cocaine, a series of analogs was synthesized, yielding some of the local anesthetics still in use today. As their chemistry is based on that of cocaine, not surprisingly they, too, are subject to hydrolysis and inactivation by BuChE (Baldessarini, 1990).

An aryl acylamidase activity of BuChE, which is strongly inhibited by classical cholinesterase inhibitors, has also been reported. This may have implications for the hydrolysis of analgesics such as paracetamol (Balasubramanian and Bhanumathy, 1993).

Inhibition of Cholinesterases

Inhibition of ChE can be achieved by several different mechanisms. Simple competitive inhibition is caused by such quaternary compounds as edrophonium, which binds selectively to the active site where it is stabilized by interaction of its quaternary nitrogen with the choline-binding pocket, and by hydrogen bonding (Sussman et al., 1992; Harel et al., 1993). In contrast to edrophonium, carbamyl esters serve as hemi-substrates. During catalysis, a carbamoyl enzyme intermediate is formed, which is far more stable than the acetyl-ChE intermediate. The very slow hydrolysis of the intermediate effectively sequesters ChE for several hours. Neostigmine, one of many physostigmine derivatives, has increased stability and potency equal to or greater than physostigmine. Demecarium, two neostigmine molecules linked by a 10-carbon chain, has even greater affinity. As an ACh analog, physostigmine can also block nAChRs (nicotinic acetylcholine receptor) (Shaw et al., 1985; Coleman et al., 1987). In fact, quaternary ammonium anti-ChE compounds have additional direct actions at cholinergic sites, either as agonists or antagonists. For example, neostigmine affects the spinal cord and the neuromuscular junction, both by inhibition of AChE activity and by stimulation of cholinergic receptors.

Over a hundred years ago the Western world became aware of the pharmacological properties of calabar bean extracts (Silver, 1974). These were eventually attributed to the ability of physostigmine to inhibit ChEs. Synthetic versions, neostigmine and pyridostigmine have been made in order to enhance effectiveness or specificity.

Glycoalkaloids and aglycones of the Solanaceae are also inhibitors of ChEs. The Solanaceae include such important foods as the potato, tomato and eggplant. Although both in vitro effects of these substances and cases of poisoning by them have been documented, it is not yet clear whether they exert an evolutionary pressure (Ehrlich et al., 1994a).

Organophosphates (OPs), mainly man-made but also in at least one example, occurring naturally in cyanobacteria (Carmichael, 1994), act as hemi-substrates of ChEs, specifically phosphorylating the active site serine, just as the natural substrate acylates it. Since the rate of hydrolysis of the phosphoryl or phosphonyl enzyme is very much slower than deacylation, OPs are effectively irreversible ChE inhibitors.

OP poisoning has been recently reviewed by Marrs (1993). OPs have also been developed as chemical weapon systems, and these potential battlefield threats have provoked considerable study of their short- and long-term physiological effects. OP anti-ChEs are potent insecticides, due to their inhibition of the insects' flight muscle ChE, with resulting paralysis and death. Because the OPs are environmentally non-persistent—being subject to non-enzymatic hydrolysis—they are increasingly replacing organic chloride compounds which are in disfavor because of their indiscriminate effects (WHO, 1986a,b). As a result of the extensive use of OP pesticides in agriculture, accidental poisoning of humans increased between 1973 and 1984, from half a million to one million cases per year, worldwide (United Nations Security Council, 1984). Particularly affected are locales where their use is poorly regulated.

There are immediate effects of OP poisoning, including respiratory depression, muscular paralysis and convulsions (Foutz et al., 1987), and delayed effects including diarrhea, weight loss, insomnia, myopathy and mental depression (Wecker et al., 1978). Although most modern insecticides are designed to have low vertebrate toxicity, subacute dietary consumption of these poisons (contaminating remnants on vegetables and fruits) may induce chronic cholinergic poisoning of fish and animals (Salte et al., 1987), including humans (Ratner et al., 1983). One very serious effect of exposure to OPs is the increased risk of leukemia (Brown et al., 1990). A molecular description of some secondary effects of OP poisoning on the nervous system has been proposed: the down-regulation of muscarinic receptors following chronic inhibition of AChE (Olianas et al., 1984; Clement, 1991).

Further, in utero exposure to environmental poisons and drugs (i.e. anticholinesterases, cocaine) is frequently associated with spontaneous abortion and placental malfunction. Such poisons include organophosphorus and carbamate anti-cholinesterases (anti-ChEs) used as agricultural insecticides and glycoalkaloid anti-ChEs which occur naturally in edible solanum plants (Soreq and Zakut, 1993). In addition, cocaine abuse during pregnancy is frequently associated with spontaneous abortions (Chasnoff et al., 1985) and fetal growth retardation (MacGregor et al., 1987). In fewer cases, it is correlated with an increased incidence of congenital anomalies (4-fold for mothers of average age of 27 years). (MacGregor et al., 1987). This issue is of growing concern since the current fraction of cocaine-exposed infants in the United States already reaches 6% or more (Volpe, 1992). Genetic polymorphisms involving mutations in genes of drug metabolyzing enzymes are known to cause differences among individuals in drug efficacy and toxicity. This calls for a search for risk groups that are genetically predisposed to placental or fetal intoxication, due to ChE polymorphism.

Neurodegenerative Diseases Related to Cholinergic Malfunction

Defective cholinergic signaling has been found in a number of neurodegenerative disorders in which pathological changes in the levels of AChE and BuChE as well as CHAT (choline-acetyltransferase) and AChR are observed (Rakonczay and Brimijoin, 1988). As these symptoms are organ-specific, rather than global, they may indicate a failure of normal tissue-specific post-transcriptional (alternative splicing) or post-translational modifications. The following is a parital list of these diseases and the type of anticholinesterase therapy that is used as well as the response. More detailed descriptions of the diseases are included herein below as necessary and summarized in Schwartz et al (1995).

1. Alzheimer's Disease (AD) is the most common type of adult-onset dementia. The general malfunction of the cholinergic regions of the brain invariably leads to death. The severity of the disease parallels the reduction in levels of ACHE and CHAT in the frontal and temporal cortices (Perry et al., 1978). A diminished number of cholinergic neurons in basal forebrain nuclei and decreased ACh production in the brain of AD patients, are thought to cause some of the characteristic cognitive impairments. In the affected brain regions, the decrease of AChE is most pronounced in the G4 form (tetrameric globular form; Atack et al., 1983). This loss is accompanied by an increase in BuChE (Atack et al., 1986) and is correlated with selective degeneration of the presynaptic structures (De Kosky and Scheff, 1990). It has been suggested that anti-cholinergic drugs impair the memory of healthy individuals in a manner parallel to that observed early in the development of AD. Therefore, the principal current AD therapeutic approach, and the most promising one in the short term, is the stimulation of the cholinergic system. Precursor loading, with choline or phosphatidyl choline, is ineffective. However, the anti-ChE agent, physostigmine, has been shown to have a small, short-term positive effect on cognitive functions (Davis et al., 1993 and papers therein cited). More recently developed compounds, like SDZ ENA 713 (Sandoz), have greater central selectivity and longer duration of action than physostigmine (Enz et al., 1993) and is thought to bind specifically to the Gl form of AChE, presumed to be the form involved in postsynaptic ACh hydrolysis (Marquis and Fishman, 1985). Its action is concluded to be on the CNS because of its ability to increase the frequency of rapid eye movements during REM sleep (Enz et al. 1991). Nevertheless, higher doses of the drug caused a transient drop in serum BuChE activity, indicating that it inhibits BuchE as well as AChE.

The first anti-ChE drug to be approved for use in AD therapy in the USA is Cognex® (Parke-Davis, 1,2,3,4-tetrahydro-9 aminoacridine, THA, tacrine). Recently, there has been a report of a multi-center, double-blind, placebo-controlled trial of THA therapy, which included 663 patients suffering from mild to moderate AD. It was shown that THA produced statistically significant, dose-related improvements. However, after 30 weeks, significant data were available from only 263 patients. The primary reason for withdrawal of patients from the study was asymptomatic hepatotoxicity, as revealed by elevated serum levels of alanine aminotransferase. The susceptibility to THA was highly variable, the level of the aminotransferase usually being less than three times the upper limit of normal value, but in 2% of the cases it reached 20 times the upper limit of normal, prima facia evidence of hepatocellular necrosis. The adverse effects were rapidly reversed when treatment was terminated, and the majority of patients were able to return to the study with lower dosages of THA (Knapp et al., 1994; Watkins et al., 1994).

The practical benefits of THA therapy have, however, been questioned. It is argued that the improvement of cognitive function in AD patients receiving THA was superficial; the underlying deterioration continued unabated, as became evident when THA therapy was discontinued. Moreover, severe cholinergic side-effects were observed in a significant number (over 10%) of THA treated patients. Finally, only patients suffering from mild to moderate AD respond to THA treatment, while more severe cases do not benefit from this therapy (Winker, 1994). A smaller study (Minthon et al., 1993) has made similar findings.

2. Parkinson's Disease (PD) is a common type of adult-onset chronic degenerative disorder of the CNS. Since PD is associated mainly with the dopaminergic, and not with the cholinergic system, few characterizations of AChE in PD have been performed. However, AChE activity has been observed in dopaminergic brain areas and decreased AChE activity and molecular form changes that parallel those found in AD have been observed in up to 30% of PD. Therefore, cholinergic signaling may be connected with neurodegenerative processes in general, or more specifically with the pathophysiology of PD (Ruberg et al., 1986). PD patients are usually treated with tricyclic anti-depressants. The anti-cholinergic side effect of these drugs may be the basis of some of the benefit shown by this treatment of PD patients. Similarly, there is evidence of worsening of the symptoms in a PD-patient receiving the anti-ChE, THA (Ott and Lannon, 1992).

3. Huntington's Disease (HD) is a dominant inherited autosomal neurodegenerative disorder with symptoms usually evident at the age of 30 to 40, and is associated with genetically programmed cell death in the CNS. The disease, progressing over a 10-20 year period, eventually destroying all motor function. In most HD cases, progressive dementia is a feature when cholinergic neurons of the brain stem are affected. AChE activity is diminished only in selective bundles of the affected area, known for their rich AChE activity and CHAT activity is decreased in these same areas.

4. Amyotrophic lateral sclerosis (ALS), or motor neuron disease, is characterized by motor neuron degeneration and progressive failure of neuromuscular transmission. Both upper and lower motor neurons are affected. In neuromuscular endplates (NMEs) significant decreases of all forms of AChE is observed. The defect is thought to be related to disassembly of the synapses and NMEs due to an excitotoxin, a failure of a trophic factor, or a failure to detoxify a xenobiotic, the consequent decrease in nerve signaling, causing a defect in AChE excretion (Goonetilleke et al., 1994).

5. Myasthenia gravis (MG) an autoimmune neuromuscular disease, characterized by muscle weakness due to autoimmune anti-nAChR antibodies is often treated with myostigmine, a synthetic physostigmine derivative. Edrophonium induces an immediate, but brief, relief of the characteristic symptoms, due to reversible binding to the active site, terminated by rapid excretion of the drug by the kidneys. There has been noted a considerable individual variation in the dosages of anti-ChE agents required to control the disorder.

6. There is ample evidence for perturbations in cholinergic functions being associated with hematological disorders. The increased risk of leukemia following exposure to OP agents has been mentioned. Down's syndrome, like familial Alzheimer's disease, is linked to chromosome 21, (Percy et al., 1993), and is associated with AChE deficiencies, and affected individuals have a high incidence of leukemia. Paroxysmal nocturnal hemoglobinuria, also associated with an elevated risk of leukemia, is characterized by a failure of the post-translational glycosylation of AChE. This prevents transport to and interaction of the enzyme with the erythrocyte membrane (Turner, 1994). Several other hematological disorders associated with the cholinergic system were described by Soreq and Zakut (1993). Recently, antisense inhibition of ACHE gene expression, using phosphorothioated oligodeoxynucleotides, has been shown to induce massive proliferation of meloid cells in bone marrow cultures, an ex vivo mimic of a leukemic syndrome (Soreq et al., 1994). This is a warning that supreme caution must be used in the development and use of anti-ChE drugs or insecticides.

Recent studies provide evidence for the idea of a developmental role for ACh (Soreq et al., 1994; Brown et al., 1990; Schwarz et al, 1995). A recent report (Layer et al., 1993) suggests a cell adhesion role for AChE. The developmental role has also been suggested for BuChE and AChE in ex vivo developing chick motor axons based on the use of selective inhibitors (Layer, 1991; Layer et al., 1988a,b, 1993). Thus, there is accumulating evidence for a developmental role (or roles) for ChEs which is not obviously related to their catalytic activity. Further experiments will be needed to determine whether this function is related to the presumed cell adhesion properties of these enzymes.

The above summary of the actions of ChEs and anti-ChEs show the critical role of cholinesterases and anti-cholinesterases. In particular, several of the reports suggest that there is an at-risk population of people who are exposed to anti-cholinesterase agents, either environmentally or as drugs, some of whom will respond well and others who have side effects as shown in the AD trials.

It would be useful to identify the sub-population who are sensitive to anti-cholinesterase drugs. Conversely, it would be useful to identify people who have a high tolerance to such drugs such that they would be at least risk in environmental exposure.

SUMARY OF THE INVENTION

According to the present invention, a method of screening for a genetic predisposition to anticholinesterase exposure is disclosed. The method includes the steps of obtaining a peripheral blood sample from patients, and then analysing serum from the blood sample for BuChE levels and inhibitor-susceptibilites. The DNA of peripheral white blood cells from the blood sample is also screened for the presence of BuChE alleles thereby identifying patients who have a genetic predisposition to anticholinesterase exposure.

The present invention also includes a kit for screening for a genetic predisposition to anticholinesterase exposure. The kit includes hybridization probes for BuChE alleles, and reagents for determining serum BuChE levels.

The present invention further includes a method for the dissection of sequential enzyme-mediated reactions. The method includes the steps of preparing anti-enzyme antibody-coated wells of microliter plates and then partially purifying the requisite enzymes by adsorption onto the antibody-coated wells of microliter plates. Utilizing the immobilized enzyme the apporpriate activity assay for analysis of the adsobed enzyme can be performed.

BRIEF DESCRIPTION OF THE DRAWINGS

Other advantages of the present invention will be readily appreciated as the same becomes better understood by reference to the following detailed description when considered in connection with the accompanying drawings wherein:

FIGS. 1A-B

is a series of graphs showing the inhibition of BuChE in human sera by carbamate anti-ChEs, pyridostigmine (10 μM), physostigmine (1 μM), N-heptyl physostigmine (0.01 μM) and SDZ ENA713 (10 μM) wherein panels (A) is the percent original acitivity of immobilized ChEs shown as a function of time of exposure to the four carbamates and (B) is the consumption of BuChE activity by the four carbamate inhibitors with the data of (A) used to calculate the activity lost from serum samples during incubation with each inhibitor as a function of incubation time, apparent gains of activity are rendered as zero loss, and wherein serum types are G/G (▪) is a serum sample from a homozygote for the “atypical” BuChE variant, G/D (&Circlesolid;) is a heterozygote for this variant and D/D (▴) is a normal homozygote;

FIGS. 2A-B

is a pair of graphs of the inhibition of serum and recombinant BuChE by tacrine wherein symbols for serum types are as in

FIG. 1

with (A) showing data for serum types inhibited by tacrine and (B) tacrine inhibition is observed on equivalent total amounts of recombinant normal (D/D), “atypical” (G/G) and a 1:1 mixture of the two (D/G);

FIGS. 3A-D

is a series of graphs showing inhibition of serum and recombinant BuChE by cocaine (A, B) and α-solanidine (C, D), in the left panel (A, C), inhibition is shown for serum enzyme from a normal homozygote (D/D, ▪), serum of a heterozygote for normal and the “atypical” BuChE (D/G, &Circlesolid;), and serum of a homozygote of this variant (G/G, ▴), in the right panel (B, D) data are shown for equivalent total amounts of oocyte produced, recombinant normal (D/D) and “atypical” (G/G) BuChE, and a 1:1 mixture of the two (D/G), note that above 5 μM α-solanidine, “atypical” BuChE shows higher activities than the normal, D70 enzyme;

FIG. 4

is a series of graphs showing the time-dependent spontaneous reactivation of recombinant human ChEs after complete inhibition of the immobilized enzymes followed by removal of unreacted inhibitor, inhibitors used were as in FIG.

1

and serum was Asp

70

BuChE (▴), Gly

70

BuChE (♦), E

6

AChE (▪), E

5

AChE (&Circlesolid;);

FIGS. 5A-B

is a schematic representation of (A) the reactions for catalysis and analogous reactions and (B) the chemical structures of DFP and PAM;

FIG. 6

is a series of schematic representation of active site environment in human BuChE and Torpedo AChE following crystal structure and numbering of residues as in Torpedo.

FIGS. 7A-B

is a series of graphs showing the measurement (A) of inactivation of ChEs by DFP wherein data are presented as percent original activity vs. duration of exposure to DFP times the DFP concentration (k

i

[DFP]) and symbols for BuChE inactivations: ▪ 1 nM, &Circlesolid; 5 nM, ▴10 nM, ▾ 50 nM DFP, for AChE (E6) inactivation: □ 0.1 μM Δ 0.5 μM, ∇ 1 μM DFP, for AChE (E5) 1 μM DFP, for L

286

K BuChE inactivation: ⋄ 50 nM DFP, the BuChE/AChE chimera (χ) ♦ 0.5 μM DFP, is shown in both panels to assist correlation of variants with vastly different inactivation rates and (B) reactivation of DIP-BuChE by PAM wherein a logarithmic function of the regain-in-activity vs. time is presented for BuChE ▪, the L

286

K mutant ⋄ in 1 mM PAM, for AChE (E6) ∇, AChE (E5) , and the chimera ♦, in 0.6 mM (right panel) and 1 mM PAM (left panel), data for PAM is shown in both panels to assist correlation of variants with vastly different inactivation rates;

FIG. 8

is a bar graph showing tests for the rate limiting step with the open bar k

i

, the solid dark bar k

cat

, and the bar with diagonal lines k

r

for the chimera, L

286

K mutant and AChE enzymes;

FIGS. 9A-D

is a series of graphs showing ex-vivo changes in colony counts and cell numbers under AS-BCHE treatment, a reproducible experiment is presented for colony counts (A,C) and cell numbers (B,D) following treatment with partially phosphorothioated oligomers, wherein -&Circlesolid;- AS-BCHE and --∘-- S-BCHE under culture conditions CFU-MK, IL-3 alone, CFU-GEMM, and IL-3+Epo;

FIG. 10

is a photograph of a slab gel electrophoresis showing partially phosphorothioated AS-BCHE does not alter apoptotic DNA fragmentation in ex-vivo cultures of hematopoietic cells, equal quantities (1 μg) of DNA from non-treated control (C) cultures or those treated with S-BCHE (S) or AS-ECHE (AS) or fresh Bone marrow DNA (BM) were electrophoresed, blotted and hybridized, autoradiographic exposure was for 1 day, molecular size markers (Boehringer/Mannheim) were electrophoresed in parallel for size calibration (righthand side) and ladder of nucleosomes (sized on the left side), reflecting apoptosis, appeared in all cultures but not in fresh bone marrow, arbitrary apoptotic index values were calculated by phosphoimage analysis (Fuji, Tokyo, Japan) of exposed plates, determining radioactive labeling of fragmented (<2Kb) over intact DNA;

FIG. 11

is a series of photomicrographs showning AS-BCHE dependent changes in cell composition of bone marrow cultures, representative CFU-MK and CFU-GEMM cytospinned cells grown in the presence of AS-BCHE or S-BCHE are shown wherein Mø: macrophage, E.MK: Early Megakaryocyte, L.MK: Late megakaryocyte, PMN: polymorphonuclear (neutrophil), E.ERB: Early Erythroblast, L.ERB: Late Erythroblast, RBC: erythrocyte;

FIGS. 12A

,

12

B, and

12

C are a series of graphs showing the differential compositions of AS-BCHE treated CFU-GEMM wherein Mø: -&Circlesolid;-, E.Megs: -▪-, L.Megs: -□-, PMN: -∘-, E.ERB: -♦- and L.ERB: -⋄-;

FIGS. 13A-B

shows duration of AS-BCHE effects in vivo in (A) a graph showing labeling decrease through the MK developmental stage wherein PBS: -▪-, AS-BCHE: -∘- and Background: -▴-, and (B) in vivo-ex vivo effect of control (filled bar), AS- (open bar) and S-BCHE (diagonal lines) on CFU-MK colony number, colony counts were scored in three different cultures with average values and standard deviations presented;

FIGS. 14A-B

shows (A) a schematic representation of human ACHE (left) and BCHE (right) genes with exons designated by dark boxes and numbered above and introns light boxes and numbered below, the ACHE promoter (P) and showing that the genes differ in length and exon-intron composition and (B) are photographs of blot-hybridization of placental RNA at gestational ages in weeks subjected to reverse transcription, and PCR amplification with primers used (arrows in schematic representation) were from exons 2 and 3 of the BCHE gene and from exons 3 and 6 of the ACHE gene, RNA from term placentae (T) and cultured syncytiotrophoblast cells isolated from term placentae (C) served for comparison; and

FIG. 15

are bar graphs showing ethnic origin composition of pregnancies at risk study (s) and control groups (c) as percentages, expected allele frequencies for the two groups were calculated, based on these percentages, using values of allele frequencies in distinct Jewish populations from published data (Szeinberg et al., 1972), Inset: PCR-RFLP test showing the DNA fragments obtained from of a heterozygote (lane 1) a D70 homozygote (lane 3), and the uncut PCT product (lane 2).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The present invention provides a method of screening for a subpopulation that has a genetic predisposition to anti-cholinesterase exposure. The method includes the steps of first obtaining a peripheral blood sample, then analysing serum from the blood sample for BuChE levels and inhibitor-susceptibilites, and also screening DNA of peripheral white blood cells from the blood sample for presence of BuChE alleles. From the combination of these assays it is then possible to identify patients who have a genetic predisposition to anticholinesterase exposure.

The invention can be practiced wherein the the anticholinesterase exposure is by anticholinesterase drug therapy for neurodegenerative diseases or other conditions related to cholinergic malfunction. The neurodegenerative diseases or other conditions are selected from the group consisting of adult-onset dementias such as Alzheimer's disease, Parkinson's disease, Huntington's Disease, Amyotrophic lateral sclerosis or motor-neural degenerative diseases like Myasthenia Gravis. The drug therapy giving rise to anticholinesterase exposure can also be related to hematological diseases.

The anticholinesterase exposure can also result from exposure to organophosphates or carbamate insecticides. The method can also include individuals that are exposed to anticholinesterases and who are cocaine addicts. The method can also include pregnant females that are exposed to anticholinesterases or who are cocaine addicts.

In a patient who presents with what may be an adverse cholinergic symptom, the patient's peripheral blood is drawn and serum BuChE is tested against BTCh with the results compared to that of normal BuChE. Further the response to hydrolyzing succinylcholine, will also be evaluated. Responses below normal will indicate an impaired BuChE response. To determine if the impaired response is genetic, PCR amplification, informative SauIIIA restriction and direct sequencing of the corresponding region from the BCHE gene in the peripheral blood DNA by previously established techniques (Ehrlich et al., 1994a) can be undertaken to determine which BCHE alleles are present.

By “normal non-variant BuChE” it is meant that the BuChE encodes butyrylcholinesterase homologous serum esterase known for its ability to hydrolize a wide variety of choline esters and for its capacity for binding selective inhibitors of cholinesterase. Cholinesterase is an acetylcholine (ACh) receptor. (Soreq et al., 1992, Soreq and Zakut, 1993).

Routine screening of serum BuChE can be undertaken prior to administering anti-cholinesterase drugs such as tacrine. For those with impaired BuChE responses, the use of the drug would be contra-indicated. Alternatively, genetic screening of patients can be undertaken as well as those who will be at risk of exposure due to environmental agents such as pesticides. However, since not all mutations may be identified and new ones can arise, the genetic screen is most efficient in combination with screening of serum BuChE.

For the genetic screening in the preferred embodiment is PCR amplification with informative SauIIIA restriction enzyme analysis or with direct sequencing of the corresponding region from the BCHE gene in the peripheral blood DNA by previously established techniques (Ehrlich et al., 1994a). Allele-linked RFLPs may also be useful in a defined population in which there is linkage disequilibrium between specific RFLP haplotypes and variant BuChE. In family studies locus-lined RFLPs can be useful. Utilization of DNA denaturing gradient gel electrophoresis and RNase cleavage techniques taking advantage of mismatches between specific DNA or RNA probes and variant sequences.

In screening the serum BuChE the micro assay as set forth in Example 2 and briefly described herein below is used.

As part of the present invention an assay was developed in order to examine the roles of various regions in human ChEs on specific steps in the catalytic process. This analysis requires the dissection of the various steps in the process, a sequential analysis. The assay employes successive DFP phosphorylation and oxime-induced dephosphorylation as steps analogous to the acylation and deacylation reactions of substrate hydrolysis by these enzymes. Adsorption of the recombinant ChEs onto immobilised selective monoclonal antibodies enriches the enzymes, separates the catalytic steps and prevents oxime-dependent acceleration of substrate hydrolysis. The rate constants thus derived are very close to those calculated by others for the corresponding purified proteins in solution.

The assay, as set forth in the Examples herein below, which employs partial purification by adsorption (Seidman, 1994) onto antibody-coated wells of microtiter plates followed by an activity assay is suitable for screening large numbers of mutant proteins, often the rate-limiting step in a study. Also, it is adaptable to the dissection of other enzyme-mediated sequential reactions, for example, protein phosphorylation and dephosphorylation by kinases and phosphatases, or other reversible covalent modifications that modulate protein properties.

In brief this assay consists of the following steps: coating appropriate anti-ChE monoclonal antibodies in multi-well plates and then immobilizing ChEs from the serum of the patient on the antibody. By appropriate selection of monoclonal antibodies that are not directed against the active site of the enzyme, the enzymes activity can them be measured on the plate by the addition of substrate and spectrophotometric measurement of the substrate conversion.

The present invention further provides a method of screening for a genetic predisposition to anticholinesterase exposure in woman leading to placental malfunction. The method includes obtaining a DNA sample from a woman, generally from peripheral blood white cells. The DNA sample is screened for the homozygous presence of “atypical” BuChE alleles thereby identifying women who on anticholinesterase exposure have a genetic predisposition leading to placental malfunction.

The present invention also provides a kit for screening for a genetic predisposition to anticholinesterase exposure. The kit includes hybridization probes for BuChE alleles (Loewenstein, et al., 1993a, and reagents for determining serum BuChE levels.

The invention is based on the following observations that have lead the applicants to hypothesize and with the data from the Examples herein below determine that there are subpopulations that are genetically predisposed to respond either positively or negatively to anti-cholinesterase compounds or drugs.

1. There is a common natural mutant of BuChE, the “atypical” variant, has been identified that is unable to hydrolyze succinyl choline.

2. There are individuals who carry a variant BCHE allele, one of those that code for catalytically inactive BuChEs, and these people have an increased sensitivity to OP-poisoning (Prody et al., 1989).

3. In a case study, an individual with exceedingly low BuChE activity and a history of exposure to agricultural OPs had a 100-fold amplification of the “atypical” variant BCHE gene, which was not seen in his parents, but was passed on to the next generation (Prody et al., 1989). The clinical consequences of this are not yet apparent, but any potential effect of OPs on the genome is of great concern.

4. There has been noted a considerable individual variation in the dosages of anti-ChE agents required to control MG. This may be due to individual levels of the autoantibodies, or may be due to genetic differences in the BuChEs.

5. In AD, the susceptibility to THA was highly variable, the level of the aminotransferase usually being less than three times the upper limit of normal value, but in 2% of the cases it reached 20 times the upper limit of normal, prima facia evidence of hepatocellular necrosis. The adverse effects were rapidly reversed when treatment was terminated, and the majority of patients were able to return to the study with lower dosages of THA (Knapp et al., 1994; Watkins et al., 1994).

6. In AD, anti-ChE agents are in trial, as discussed herein above. One such agent, Tacrine, was approved in 1994. Higher doses of the drug caused a transient drop in serum BuChE activity, indicating that it inhibits BuChE as well as AChE.

These observations as well as the material following have led the applicants to determine that there are variant BuChE alleles and affect an individual's response to anti-cholinesterase compounds.

Over several decades, large-scale population surveys of BuChE phenotypes have been carried out. Tens of thousands of individuals have been screened from different continents and ethnic origins (Ehrlich et al., 1994a). Since the BCHE gene was cloned in 1986, more than twenty different naturally occurring mutations have been documented (see Table 1 in Schwarz, et al, 1995), with the great majority of the variant phenotype individuals carrying the D70G substitution. The variation was identified as a point mutation of aspartate 70, which was replaced by a glyicne residue (D

70

G) (McTiernan et al., 1987; Lockridge, 1990; Neville et al., 1990b), and the variant was demonstrated to display decreased interactions with inhibitors (Gnatt et al., 1990; Neville et al., 1990a; McGuire et al., 1989) and a 4-fold lower specific activity than the wild-type BuChE (Neville et al., 1992).

The various mutated proteins result in a variety of phenotypes, including the complete absence of any BuChE protein due to premature termination of protein synthesis (“silent” mutation). In at least one area of the brain, BuChE has been demonstrated in cells other than those that have AChE, suggesting a unique function (Graybiel et al., 1981, 1982). The mere existence of a “silent” phenotype, where individuals do well in spite of having no BuChE activity, has been used to argue that BuChE has no important function. However natural selection operates on the level of species, not individuals; what may be tolerated in an isolated individual may, over time and numbers, be disadvantageous to a community. Also, “knock-out” experiments have sometimes found no phenotypes for damaged genes; it would be reckless to conclude therefore, that all such genes have no important biological role. With the exception of two polymorphisms at the 5′ and 3′ non-translated sequence, no mutation in BChE cDNA has yet been found that does not cause alteration in the protein sequence. Together, the catalytically silent mutations comprise 0.001% of homozygotes, which is far less than the catalytically active variants (Ehrlich et al., 1994a). Even in the absence of a well-understood physiological role for BuChE, this in itself suggests a selection advantage for carriers of various genes coding for active proteins, as compared with “silent” gene carriers.

Interestingly, the largest and main coding exon, E2, has 15 of the known mutations found on the BCHE gene. Thus, the average incidence of mutability in the coding domain (approximately 1:100 nucleotides) is exceedingly high. The different BCHE variants were in most cases identified by the analysis of sequences originating from individuals expressing a variant phenotype and not by a random screening of the population. Several of the variants (e.g. D70G) were simultaneously discovered in two continents, while many others were detected only once, an “orphan” allele.

One major physiological role of BuChE is thought to be as a scavenger of anti-ChE agents, thus protecting from inactivation the AChE of neuromuscular junctions and other cholinergic sites (Neville et al., 1990a,b). This is deduced from the fact that BuChE interacts with a wider range of anti-ChE agents (Soreq et al., 1992) and in certain cases (e.g. DFP and many carbamates) the rate of inactivation is considerably faster than that of AChE (Loewenstein et al., 1994; Schwarz et al., 1994). Accordingly, there must be an evolutionary pressure that accounts for the need for a scavenger of anti-ChE agents. There are many natural ChE inhibitors in the environment, including glycoalkaloids present in solanaceous plants (Gnatt et al., 1994), fungal antibiotics like puromycin (Hersh, 1981) and its analogs, cocaine derivatives (Gatley, 1991), poisons from several species, like oysters (Abramson et al., 1989), OPs from cyanobacteria (Carmichael, 1994), and polypeptides from snakes (fasciculin, Karlsson et al., 1985) that are offensive or defensive weapon systems, metals (aluminum, scandium and yttrium, Marquis and Lerrick, 1982), and the carbamate of calabar beans, physostigmine (Taylor, 1990). Some of these above ChE inhibitors are extremely poisonous. Several snake venoms contain peptides of 51-59 amino acid residues (e.g. fasciculin) that bind to AChE with Kd values as low as 10

−10

M (Cervenansky et al., 1990; Marchot et al., 1993). However, it is perhaps significant that it is only the glycoalkaloids of the solanaceous food plants (tomato, potato, eggplant) that are inhibitors of both AChE and BuChE. Also, the uneven natural geographic distribution of these food plants must be seen alongside the large series of naturally occurring BuChE variants, also unevenly distributed among different populations—the “atypical” BuChE mutation, D

70

G (heterozygote frequency <5% among Europeans and Americans and up to 11% of other groups; (Ehrlich et al., 1994a)—with variable affinities for them. Of all the classes of natural inhibitors of the ChEs, it seems that only for the glycoalkaloids may BuChE be imagined to have adapted as a scavenger.

The “atypical” mutation also confers resistance to inhibitors of pharmacological interest. It is clinically characterized by the inability of the affected enzyme to hydrolyze succinyl choline and dibucaine, and, compared to the wild-type BuChE, displays a specific activity of 25% of the wild-type enzyme, and at least 10-fold higher IC

50

and K

i

values for bambuterol, physostigmine and echothiophate. The affinity toward ACh is drastically reduced, although the K

m

for BTCh (butyrylthiocholine) is unchanged (Neville et al., 1990a,b). If the mutant BuChE cannot scavenge anti-ChEs and reduce their serum levels, it will not protect synaptic AChE from their effects. The genetic variability of BuChE may well be the basis of the observed variability in the extent and intensity of responses to anti-ChE drugs.

BuChE is reported to hydrolyze heroin, which has a 4-fold higher K

m

for the “atypical” variant than for the usual enzyme (Lockridge et al., 1980). Clearly this has the potential for explaining variations in responses to this narcotic. BuChE hydrolyzes the methyl ester bond of cocaine and its derivatives. The local anesthetic, procaine is hydrolyzed by BuChE, but it has a 15-fold higher K

m

with the atypical variant than with the usual enzyme. Carriers of the atypical allele may not react substantially differently from carriers of the usual enzyme when receiving procaine i.m. as it would be exposed only minimally to BuChE. However, aspirin has a nearly 4-fold higher K

m

with “atypical” BuChE (Valentino et al., 1981). It acts after entering the blood stream where it is exposed to BuChE. This illustrates a potential for significant variations in response to pharmacological agents, arising from natural variations in this drug-processing enzyme.

The above also suggests that variant BuChEs will function in detoxifying cocaine and its derivatives. Cocaine addicts or those who overdoes easily on cocaine have susceptible BuChE variants while those people who do not seem to become addicted, or easily addicted, have resistant variants. Identification of the specific variant can determine the type of treatment and therapy needed for cocaine addiction.

The presence of allelic BuChEs can have an effect on the treatment of OP poisoning. Treatment of OP poisoning includes prophylactic and therapeutic approaches such as protection against the OP agent with reversible inhibitors (Wills, 1970), e.g. pyridostigmine, which protects some AChE molecules from inactivation by the OP agent, allowing time for spontaneously regenerating free active enzyme. Muscarinic symptoms, e.g. increased tracheobronchial and salivary secretion, can be effectively antagonized by a sufficient dosage of atropine, an antagonist of the muscarinic receptor (mAChR), while it has virtually no effect on peripheral neuromuscular activation and subsequent paralysis. The catalytically inactive phosphoryl-enzyme can be reactivated by a cationic oxime through nucleophilic displacement of the phosphoryl moiety from the active site serine (Aldridge and Reiner, 1972). Since reactivation by oximes is most marked at the skeletal neuromuscular junctions, it is an important complement to atropine therapy. These beneficial effects are less evident at autosomal effector sites and insignificant in the CNS. 2-PAM is such an oxime, with features of ACh that permit it to bind to the active site of ChEs.

An experimental approach for treatment has been to test the use of isolated human BuChE, the most prevalent soluble circulating ChE (Ashani et al., 1991; Raveh et al., 1993), as a protective agent for mice and rats. In primates, BuChE from bovine fetal or equine serum (Doctor et al. 1991; Wolfe et al., 1994; Reveh et al., 1993) has been used as a protective agent. These protocols were successful in protecting against subsequent injections of soman (an OP), preventing both acute effects, and long-term (6 weeks) behavioral effects. This approach is claimed (Wolfe et al., 1994) to be much more successful than the established alternative therapy of 2-PAM and atropine, combined with diazepam (to deal with the problem of seizures). However, the combined use of 2-PAM and human BuChE together is potentially more efficacious than either one alone, since phosphoryl-BuChE is rapidly reactivated by 2-PAM, effectively allowing BuChE to catalytically turnover OPs (Schwarz et al., 1994).

It is known that horse serum BuChE, like “atypical” human BuChE, will not hydrolyze succinyl choline (Ehrlich et al., 1994a). The oxime, 2-PAM, reactivates DFP-inactivated D

70

G BuChE at a 5-fold lower rate than does wild type BuChE. Variants having this mutation in tandem with one or two additional natural mutations (Yll4H and S425P) display a rate of reactivation as much as 40-fold lower. Thus, the well established therapy of treating OP-intoxicated patients with 2-PAM (2-pyridine aldoxime methiodide), intended to regenerate active ChE, is less efficient in the case of carriers of variant BuChE as shown in Example 2 herein below. In support of this finding, it has been reported that the response of OP-poisoned patients to 2-PAM therapy varies widely (Willems et al., 1993).

The most frequent variant, “atypical” BuChE, was compared to the common human BuChE and ACHE in its inhibition rate with several anti-ChEs of pharmacological interest (Loewenstein et al., 1994) in Example 1, herein below. With common BuChE, the carbamates physostigmine, heptyl-physostigmine (Modulanum®) and SDZ ENA 713 had inactivation rates higher than or equal to ACHE, but “atypical” BuChE had considerably lower rates. This suggests that BuChE usually reacts with the drugs in preference to AChE. However, heterozygous, and especially homozygous individuals carrying the atypical gene may well show increased sensitivity to the drugs. Moreover, the reversible inhibitor, THA, had a 300-fold higher IC

50

value with “atypical” than with common BuChE. These findings may help explain the variations in response to anti-cholinesterase therapy that have been noted, particularly in AD.

The plethora of mutations in the BCHE gene may be taken as further support for the idea that the major role of BuChE is to function as a scavenger, as the selection of proteins with modified properties will confer better resistance to specific cholinergic poisons. It may also be that the role of scavenger can conflict with another role for BuChE, for instance as a cell membrane element involved in development. In that case, a decreased affinity for an inhibitor in the environment may confer a selection advantage (Ehrlich et al., 1994a).

The recent administration of pyridostigmine bromide to over 400,000 Gulf War soldiers was probably the largest scale ever use of an experimental drug, approved only because of the anticipated exposure of those soldiers to nerve agents (Gavageran, 1994). Previous clinical studies with this drug on healthy volunteers had been limited to small numbers of healthy males and to only several days exposure. Moreover, volunteers who suffered side effects were immediately withdrawn from those studies (see, for example, Glickson et al., 1991). Thus, the Gulf War presents the first real-life experience with this anti-ChE. The war conditions included concurrent exposure to insecticides, chiefly organophosphorus anti-ChEs (Ember, 1994), which increased the level of ChE inhibition in these soldiers to an unknown extent. Adding to the exposure was the insect repellent, DEET, which has been shown to amplify the toxicity of anti-ChEs (Gavageran, 1994). It is thus probable that the total ChE inhibition levels in these soldiers exceeded those intended even for normal individuals. In heterozygote and homozygous carriers of the “atypical” BCHE allele, the capacity of blood BuChE to interact with and detoxify part of the drug would be correspondingly lower. While heterozygotes still possess about 50% of this normal protective detoxifier, “atypical” homozygotes do not. These would hence become most vulnerable to excessive ChE inhibition under treatment by any anti-ChE drug.

Pyridostigmine is not the only anti-ChE that may provoke an adverse response, as is demonstrated by applicants'analyses of several potential drugs for treating Alzheimer's disease as shown in Example 1 herein below. These all emerged as much faster inactivators of BuChE than of AChE, suggesting that when orally administered to patients they will interact primarily with plasma BuChE, so that the clinically effective dose reaching the central nervous system depends heavily on the BCHE genotype. Even without the complication of BCHE polymorphism, BuChE levels can vary with the general state of health (Ehrlich et al., 1994a). Since Alzheimer's patients are far from being as healthy as the pyridostigmine-treated soldiers, they may present yet more drastic symptoms in response to inappropriate dosage of anti-ChEs. Moreover, these patients are usually unable to communicate their difficulties, so that discrimination between effects of the drug and symptoms of the disease depends solely on the subjective observations of a caretaker, which calls for the development of reliable blind tests as set forth in the present invention to detect such responses. With the information from the present invention, appropriate dosages of these drugs can be adjusted to match specific genotypes, for the purpose of avoiding adverse responses.

As shown in Example 2 herein below, the much faster inactivation and reactivation of BuChE than AChE suggests that in vivo, BuChE reacts faster with certain OP and oxime drugs and poisons than AChE. This is relevant to the intravenous PAM administration that is used, in conjunction with other therapies, to treat OP-intoxication (Taylor, 1990). Serum PAM levels that are achieved during therapy for OP poisoning do not exceed 40 μM (Willems et al., 1993), well below its Ka. Because of the accessibility of BuChE as a serum enzyme, this raises the possibility that PAM acts by regenerating BuChE and allowing it to react with more of the OP before the OP has a chance to inactivate neuromuscular AChE, in effect turning serum BuChE into an OPase. This function appears impaired in “atypical” (D

70

G) carriers, about 5% to 10% of the population (Ehrlich et al., 1994a), and in carriers of the double and triple mutations, who would therefore be expected to be poor subjects for oxime therapy for OP poisoning due to the lowered reactivation rates of their diisopropylphosphoryl-(DIP-)enzymes. As BuChE serves as a scavenger of naturally occurring ChE inhibitors (Soreq et al., 1992), this explains the recently reported variable efficacy of PAM treatment (Willems et al., 1993) and the cholinergic side-effects in Alzheimer's disease patients subjected to anti-cholinesterase therapy (Winker, 1994). In addition, Example 2 shows that, the single-step enrichment on immobilized antibody is sufficient for in-depth biochemical analysis and does not alter characteristic kinetic constants.

Cholinesterase inhibitors are primarily designed to inhibit the catalytic activity of acetylcholinesterase, which displays substrate specificity for acetylcholine. However, they frequently interact at least as effectively also with the homologous butyrylcholinesterase, which is less substrate-selective and more susceptible for inhibition by multiple inhibitors than AChE. In addition to brain, BuChE is known to be expressed in liver (Prody et al., 1987) and in the hematopoietic system (Patinkin et al, 1990). Moreover, the BCHE gene encoding BuChE is subject to incomplete somatic amplification (Lapidot-Lifson et al., 1989) and frequent mutability (Zakut et al., 1992) in several blood cell disorders. Furthermore, the BCHE gene maps to the 3q26 chromosomal location, which is often broken in leukemias (Ehrlich et al., 1994b). In addition, the BuChE protein is believed to act as a scavenger of various poisons targeted at acetylcholine binding proteins (Soreq et al., 1992; Loewenstein et al., 1993a). Interference with BuChE activity, an expected outcome of interaction with cholinesterase inhibitors, may hence imply adverse hematopoietic consequences.

To examine if this is the case, and if BuChE inhibition causes distinct effects from those anticipated under AChE inhibition, primary murine bone marrow cultures as an ex-vivo system in which BuChE is expressed were examined and antisense (AS) oligonucleotide inhibition was used to block such expression. These experiments as set forth in Example 3 herein below demonstrate the need in hematopoietic-associated diseases or conditions treatment that patients with varient BuChEs be identified so that they are not receiving anti-cholinesterase drugs. Further, these data indicate that patients with deficient BuChE expression and exposure through treatment or environmentally to anti-ChEs will cause hematopoietic differences in these patients.

The long-term in vivo—ex vivo stability of AS-BCHE inhibition effects in these experiments is of special interest. It indicates that AS-BCHE induced destruction of BCHE mRNA in young promegakaryocytes reduced development of these cells for at least 2 weeks and demonstrates that no feedback responses have occurred to compensate for BCHE suppression and retrieve normal production of megakaryocytes. This, in turn, suggests that individuals with the relatively abundant allelic variants of BCHE, in particular the “atypical” Asp7OGly (D

70

G) substitution (Gnatt et al., 1994; Ehrlich et al., 1994a) may be particularly vulnerable to anticholinesterase therapy employed in neurodegenerative diseases. DNA tests detecting such carriers are therefore useful in predicting the genetic predisposition for hematopoietic damage that may result from anticholinesterase therapy.

Like AS-BCHE, the parallel AS-oligo blocking acetylcholinesterase expression (AS-ACHE) also suppresses megakaryocyte formation (Lev-Lehman et al., 1994, Soreq et al., 1994). However, unlike AS-BCHE, it also suppresses erythropoiesis ex-vivo and in vivo (Lev-Lehman et al., 1994; Soreq et al., 1994), suggesting that acetylcholinesterase participates in the erythropoietic process as well. Moreover, AS-ACHE induces a dramatic ex-vivo expansion of CFU-GEMM colony production and cell proliferation and reduces apoptosis in CFU-GEMM primary bone marrow cultures (Soreq et al., 1994). These differences reveal distinctions between the role(s) played by the two cholinesterases in mammalian hematopoiesis. Development of novel anticholinesterases should therefore take into consideration the hematopoietic involvement of the target proteins of these drugs as well as their distinct role in the hematopoietic process.

The role of BuChE as a potential scavenger and/or degrader of fetotoxic drugs and poisons has recently been the subject of ample research, especially because of its documented involvement in metabolizing cocaine (see, for example, Hahn et al., 1993; Simone et al., 1994 and discussion herein above). Two types of activity can be envisaged for placental BuChE. Being an esterase of wide substrate specificity, its hydrolytic activity may contribute to placental functioning, by controlling the levels of acetylcholine produced by placental choline acetyl transferase (Lu and Hersh, 1994). In addition, or alternatively, it may operate as a scavenger of a wide range of natural and synthetic anti-ChEs: the enzyme may bind or degrade such poisons, thus protecting the cholinergic system of the fetus from them. For both functions, the fully active normal BuChE should be advantageous over the “atypical” enzyme, as both the mutant enzymes' hydrolytic and scavenging capacities are considerably lower. However, in cases of environmental exposure to high concentrations of anti-ChE agents, the “atypical” BuChE might be advantageous: since its ability to interact with many ligands is defective, it should maintain a certain level of activity under inhibitor concentrations that would suppress normal BuChE activity. In particular, heterozygous BuChE would be advantageous, as it combines close to normal activity with effective scavenging capacities under normal circumstances, and provides residual hydrolytic abilities under high anti-ChE concentrations.

Taking into account the possibility that placental BuChE can be a target to drugs and act as a drug hydrolyzing agent, applicants as shown in Example 4 hereinbelow employed several different techniques to demonstrate that the BCHE gene is transcribed in early and late placenta; that its protein product, BuChE, is present and active in differentiated, multinucleated syncytiotrophoblast cells in the early, but not in term placenta; that the enzyme is present along the extracellular surface of the elongated chorionic villi and also in the cytoplasm and rough endoplasmic reticulum of the cells bearing these villi; and that considerable individual variation among specific placenta samples may reflect different individual capacities for drug metabolism. That such differential capacities may have serious clinical consequences was clearly depicted by comparing glycoalkaloid interactions of genotypically different BuChE variants of native and recombinant origins. Genotype/phenotype association analysis indicated that the “atypical” BuChE heterozygotes are not at a disadvantage as compared to normal BuChE homozygotes in their scavenging capacities. This may explain why the incidence of “atypical” BuChE heterozygotes was not higher in pregnant patients with various indications of placental malfunctions than in a control group; in fact, that this incidence was even lower than in the control group, could indicate an advantage to “atypical” heterozygotes under exposure to anti-ChEs. However, placental malfunctions were increased in the “atypical” BuChE homozygotes population.

Previous studies addressed the issue placental of ChEs by measuring catalytic activities in placental homogenates in the presence of selective inhibitors or by employing cytochemical staining followed by light microscopy (Simone et al., 1994; Hahn et al., 1993). While these observations demonstrated the presence of ChEs in placental preparations, they could not unequivocally distinguish between endogenous production or importation of these proteins or pinpoint the subcellular sites where they may operate. The combination of RT-PCR analyses with cytochemical electron microscopy solves both these difficulties. In addition, no population diversity study has ever performed on patients in whom the “atypical” enzyme might exert a clinically observable effect, such as the patients with placental malfunction that were probably exposed to anti-ChEs, either from solanum vegetables intake or by exposure to insecticides. Applicants present study determined that heterozygous carriers of the “atypical” allele do not have an increased risk of placental malfunction on exposure to anti-ChEs and indicates that risk is limited to fetuses of mothers that are homozygous with regard to variant alleles of the BCHE gene.

The above discussion provides a factual basis for the method of screening for a genetic based adverse predisposition to anticholinesterase therapy. The methods used with, and the utility of, the present invention can be shown by the following examples.

EXAMPLES

General Methods and Reagents

Numbering of human BuChE residues is according to its published sequence (Prody et al, 1987). Residues can be indicated by the single letter amino acid abbreviation followed by the position generally as a superscript. Point mutations are indicated by the wild-type residue, the position in the sequence, and the mutant residue, thus, L

286

K is the replacement of leucine 286 by arginine.

Varient enzymes: Serum BuChE activity against ETCh was measured spectrophotometrically as detailed by Neville et al, (1990a,b; 1992). Recombinant normal and “atypical” BuChE were produced in Xenopus oocytes microinjected with in vitro transcribed BuChEmRNAs prepared from the corresponding cDNA types (Neville et al, 1990a). Alternative recombinant AChEs were produced in ACHEDNA-injected oocytes under control of the cytomegalovirus CMV promoter, using either the brain-characterixtic 3′ exon 6 (Seidman et al, 1994) or the hematopoietically-expressed domain composed of the fourth pseudo-intron and the 3′-exon 5 (Karpel et al, 1994b).

Inhibitors: Tacrine and physostigmine were purchased from Sigma Chemical Co. (St. Louis, Mo.). SDZ ENA-713 and N-heptyl physostigmine were gifts of Sandoz (Bern, Switzerland) and Merck Sharp & Dohme (Harlow, U.K.), respectively. Pyridostigmine was from Research Biochemicals International (Natik, Mass.).

Antibody immobilizations: Monoclonal mouse anti-human serum BuChE (No. 53-4; Gift from Dr. Nordward Petersen, Copenhegen, Denmark) or anti-human AChE (No. 101-1, 0.2 mg/ml) were absorbed to multiwell plates overnight at 4° C. in carbonate buffer (Seidman et al., 1994). Free binding sites were blocked with PBS-T buffer (144 mM NaCl, 20 mM Na phosphate pH 7.4, 0.05% Tween 20 and 0.01% Tymerosal) for 60 minutes at 37° C. (as described generally in Harlow and Lane,

Antibodies: A Laboratory Manual

, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1988.). Homogenates of microinjected oocytes or serum samples were diluted 1:20 to 1:40 in PBS-T to achieve similar activity levels and were incubated in the antibody-coated wells for four hours at room temperature with agitation, and overnight at 4° C. Plates were washed three times with PBS-T prior to use.

Inactivation and reactivation measurements: Antibody-immobilized enzymes were exposed to the tested anti-ChEs in PBS-T buffer for varying times (0.5 to 80 minutes) following an initial determination of catalytic activities. At the noted time points, plates were washed three times with PBS-T and remaining substrate hydrolysis rates were determined (Loewenstein et al., 1993a). Spontaneous reactivation was measured for immobilized recombinant ChEs following complete inhibition, three washes with PBS-T, and subsequent incubation for the noted time and activity determination.

Specific activity: To determine enzyme quantities, immobilized BuChE was incubated with a rabbit anti-human polyclonal antiserum (Dako, Glostrup, Denmark) at 1:4,000 dilution in PBS-T for 70 to 80 min at 37° C. After washing with PBS-T, horseradish peroxidase-conjugated goat-anti rabbit antibody (HRP, Jackson Laboratory, Bar Harbor, Me.) was added at 1:10,000 dilution in PBS-T. Peroxidase activity was thereafter assayed using o-phenylenediamine dihydrochloride at 1 mg/ml in phosphate/citrate buffer, pH 9.6 and Na perborate as substrates. Purified human BuChE was used for calibration, and absorbance at 45 nm was recorded on a Molecular Devices microliter plate reader.

Single-step enrichment for AChE or BuChE on immobilized antibody: Monoclonal mouse anti-human serum BuChE (53-4) or anti-human ACHE (101-1) antibodies (Liao et al., 1993), were adsorbed to microliter plates (Nunc, Roskilde, Denmark) at 0.5 pg/ml in 0.1 M carbonate buffer, pH 9.6, for at least four hours at room temperature. Plates were then washed three times in PBS-T buffer (144 mM NaCl, 20 mM Na phosphate, pH 7.4, 0.05% Tween-20). Free binding sites on the well surface of the microliter plate were blocked with PBS-T for one hour at 37° C. (as described generally in Harlow and Lane,

Antibodies: A Laboratory Manual

, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1988.). Microinjected oocyte homogenates containing the enzyme were then added at a dilution of 1:20 in PBS-T or at a concentration of 100 mIU/ml in PBS-T for at least 3 hours at room temperature with agitation. Plates were washed three times with PBS-T before use. Inactivation was performed at pH 7.4 in order to facilitate comparison with similar studies in the literature.

Measurement of inactivation of ChEs by DFP: Immobilised enzymes utilizing the single-step method were exposed to DFP in PBS-T buffer for varying times. Hydrolysis rates were determined in 96-well microtiter plates. To each well were added 200 μl of 30 mM butyrylthiocholine (BTCh) in 0.5 mM 3,3′-dithiobis (6-nitrobenzoic acid) (DTNB), 100 mm Na phosphate, pH 7.4. This achieved a substrate concentration at least 10 times the K

m

for BuChE and the natural mutants and 1.5- to 5-times that of the site-directed mutants (Gnatt et al., 1994). In the case of AChE, 2 mM acetylthiocholine was used. Absorbance at 405 nm was automatically recorded on a Molecular Devices microliter plate reader (Menlo Park, Calf., USA).

Rate of reactivation of DIP-BuChE by PAM: In order to minimise the extent of aging—the progressive, refractoriness of OP-inhibited enzyme to reactivation due to hydrolysis of one of the two alkyl groups on the phosphate (Taylor, 1990)—inactivations were performed at a sufficiently high DFP concentration to bring residual activities to below 2% of the uninhibited level within 10 minutes, and reactivations were begun as soon as possible, usually within 5 minutes. The wells containing the DIP-ChE (prepared utilizing the single-step method) were exposed to 1 mM PAM in PBS-T, 22° C. for various times, then washed several times with PBS-T and assayed for enzyme activity.

Example 1

Genetic and Serum Analysis

The most frequent phenotypically effective mutation of BuChE and, therefore, the most likely to be seen affecting inhibitor interactions is the substitution of aspartate at position 70 with glycine, known as the “atypical” BuChE variant (McGuire et al, 1989, Neville et al, 1990a,b, 1992). Homozygous carriers of this mutation average 1:2,500 among Caucasians (Ehrlich et al, 1994a), reaching an incidence of 1:1,000 in some sub-populations originating in the middle-East (Ehrlich et al, 1994a), which implies a frequency of approximately 3 to 7.5% heterozygotes. The frequency of this variant among Blacks is very much lower (Ehrlich et al, 1994a). The atypical variant, in contrast to the normal enzyme, was shown to be unable to hydrolyze succinylcholine, and to be much less sensitive to several inhibitors: physostigmine (Kalow & Davis) and several organophosphates, including diisopropylfluorophosphate (DFP), iso-OMPA and echothiophate (McGuire et al., 1989; Gnatt et al, 1994, Schwarz et al., 1994). Homozygous carriers of this variant allele were reported by several groups, including the applicants, to be particularly vulnerable to parathion exposure and to the use of succinylcholine at surgery, which causes in them post-anesthesia apnea (see, for example, Prody et al., 1989; reviewed by Soreq and Zakut, 1990). These would hence be logical candidates for genetic predisposition to adverse effects of anti-ChE therapies as well. Applicants learned of a family with a member (proband) who had experienced both succinylcholine-induced apnea and, during the Gulf War and under treatment with pyridostigmine, symptoms of cholinergic deficits, see following case study. The succinylcholine incident signaled a genetic variation, and applicants suspected that the adverse cholinergic symptoms reflected the patient's response to anti-ChE therapy and might well have the same genetic basis. Therefore, applicants initiated a study of the inhibitor interactions of serum ChEs from members of this family, and compared them with the enzyme from normal serum and with recombinantly produced variant ChEs.

Case Study

H.K., born in 1970, was first referred to applicants in 1989 following an incident of two hour post-anesthesia apnea caused by succinylcholine administration in the course of knee surgery, which was treated by respiration. At the time, applicants assayed his serum BuChE against BTCh and found it to be approximately 30% of normal level and totally incapable of hydrolyzing succinylcholine, suggesting that it was the “atypical” enzyme (Neville et al., 1990a). Following both PCR amplification, informative SauIIIA restriction and direct sequencing of the corresponding region from the BCHE gene in his peripheral blood DNA by previously established techniques (Ehrlich et al., 1994a), H.K. was indeed diagnosed as being homozygous for the “atypical” allele, but not a carrier of other frequent point mutations of BuChE. The same methods revealed that both his parents and his sister are heterozygous carriers of the “atypical” BuChE allele. The patient was advised to avoid anti-ChE insecticides or drugs. H.K. served in the Israel Defense Forces in 1991, during the period of the Gulf War and, with others, received 90 mg prophylactic daily doses of pyridostigmine. He developed insomnia, weight loss and general fatigue, which worsened consistently, and a deep depression. Following discontinuation of pyridostigmine, his condition improved gradually over the following 10 weeks and H.K. is currently without symptoms.

Analysis: To analyze on a micro scale the interactions of irreversible inhibitors with ChEs, and to enrich each of the examined CHES, applicants immobilized native human BuChEs through monoclonal antibodies to multiwell microliter plates. BuChEs from sera of individuals identified as homozygous or heterozygous carriers of the “atypical” BuChE allele were compared to those homozygous for the normal BuChE allele. For reference, applicants also immobilized recombinant Xenopus oocyte-produced variant ChEs, including normal and atypical BuChEs (Neville et al., 1992) and 3′-alternative AChEs (Karpel et al., 1994a). The enzymes immobilized in multiwell plates were subjected to successive inactivation by an anti-ChE and allowed to spontaneously reactivate. By this procedure inhibition rates could be conveniently determined, irreversible inhibitors could be removed prior to activity measurements, and amounts of the enzyme could be determined for each sample.

The specific activity of “atypical” BuChE, which cannot hydrolyze succinylcholine, was found in H.K.'s serum to be about 3-fold lower than that of the normal enzyme, which hydrolyzed 80 nmol butyrylthiocholine/min/μl serum (an average from 15 individuals), in agreement with values obtained previously for other patients homozygous for the “atypical” allele (Lockridge, 1990). Heterozygotes presented intermediate specific activities, 60-70% of normal homozygotes (average of three genetically confirmed individuals). Recombinant “atypical” and normal BuChEs confirmed his difference in specific activities (Neville et al., 1990a). Thus carriers of the “atypical” allele have a less active BuChE, although they carry amounts of serum BuChE protein similar to individuals with the normal enzyme.

To test the interactions of “atypical” BuChE with various anti-ChEs, in 1994 applicants prepared fresh serum samples from H.K.'s peripheral blood. Serum from his heterozygous father and from genomically diagnosed normal homozygotes served for comparison. Irreversible inactivation measurements demonstrated that the “atypical” BuChE reacts with four carbamates, pyridostigmine, physostigmine, N-heptyl physostigmine and SDZ-ENA 713, much slower than its normal counterpart (FIG.

1

A). Moreover, differences between the serum enzyme of the heterozygous father of the proband and homogzygous normal sera could also be discerned in the inactivation rates. Both these effects varied with the particular inhibitor being tested. For example, “atypical” BuChE displayed a decrease of about 40% in its activity when incubated with SDZ ENA 713 for 30 minutes, as compared with an 95% loss of activity in the normal enzyme at this time and with 80% in the enzyme from heterozygous serum (FIG.

1

A). The loss and rate of loss of BuChE activity thus depends quite significantly on the individual's genotype. Calculations of inactivated enzyme levels reveal that the differences observed in inactivation rates reflect drastically variable capacities for scavenging each of the tested drugs in sera of patients with the normal and the atypical alleles, as displayed in FIG.

1

B.

To examine whether such genetic predisposition to weakened drug interactions can also be expected for the reversible Alzheimer's disease drug, tacrine, applicants determined the IC

50

values for this drug (FIG.

2

A and Table 1). To test whether the observed differences were indeed due to the examined point mutation and to mimic the heterozygote state, similar dose-dependence curves were prepared also for recombinant, Xenopus-oocyte produced normal and “atypical” monomeric BuChE and for 1:1 mixtures of the normal and the “atypical” enzyme (equal volumes of oocyte homogenates, which was close to equal amounts of the ChEs) (FIG.

2

B).

In both cases, applicants observed a drastic reduction in the capacity of “atypical” BuChE as compared with the normal enzyme to interact with tacrine. Mixtures of normal and “atypical” enzyme, whether from heterozygous sera or prepared by genetic engineering, yielded the expected intermediary inhibition curves (FIG.

2

B), demonstrating that these differences were not affected by multisubunit assembly and/or competition between the two types of enzyme subunits. Thus, for tacrine as well, one might expect drastically different scavenging capacities of serum BuChE depending on the genotype of the individual. For example, in the presence of 1 μm of this drug, normal BuChE would be totally inhibited whereas the “atypical” enzyme shows virtually no interaction, and sera from heterozygotes show intermediate levels of activity.

To understand the origin of the differences between tacrine interactions with the normal and the “atypical” enzymes, applicants employed the crystal structure of tacrine-soaked AChE (Sussman et al., 1993), and compared it with the corresponding computer-modelled region of human BuChE (Harel et al., 1992). The expanded active site domain in these proteins indeed revealed conspicuous differences in the rim of the gorge area. This includes Asp7O, approximately 3.7 Å from the drug, which explains why substitution of this residue may prevent the enzyme from interacting with it. The dynamic equilibrium of drug concentrations in a patient's serum depends, in addition to inactivation and removal rates, and to general hemodynamic parameters, on the rate of reactivation of drug-enzyme complexes.

To get a more complete picture of the expected outcome of treating genetically-distinct individuals with anti-ChEs, applicants further used the antibody-immobilized recombinant enzymes to follow spontaneous rates of reactivation for each of the examined drugs (FIG.

4

). These measurements revealed considerable differences between drugs and for each drug between AChE and BuChE. However, there was no dramatic difference between the C-terminal alternative forms of AChE or between normal and “atypical” BuChE in these experiments (FIG.

4

). Of the examined drugs, applicants noted rapid and efficient reactivation rates for complexes of AChE with physostigmine. While ACHE reactivated up to 90% in 45 minutes, in that time normal and atypical BuChE regained only 10 to 20% of its original activity. In contrast with these fast recovery rates from physostigmine inhibition, an effect reflected in the short in vivo half life of this drug, applicants observed quite limited capacities of the N-heptyl physostigmine inhibited enzymes to regain activity. With this particular drug, normal and “atypical” BuChE were faster to reactivate, yet it could only reach 10% of its original activity by 20 minutes. N-Heptyl physostigmine inactivation of both AChE forms was yet more severe, and it could only reactivate by 5%. SDZ-ENA 713 was yet more stable in its ChE interactions, with reactivation levels for all of the examined enzymes never exceeding 8%. Finally, pyridostigmine reactivation reached 50 and 10% for AChE and BuChE, respectively (FIG.

4

). Based on these cumulative experiments, applicants conclude that the different k

1

, and IC

50

values, determined in vitro (Tables 1 and 2) should influence the fate of the drugs in patients sera in vivo.

These findings show that AChE is a relatively vulnerable target for anti-ChEs in carriers of “atypical” BuChE (up to 7.5% of some populations). These observations explain at least some of the symptoms reported recently among pyridostigmine-treated soldiers and tacrine-treated Alzheimer patients, and indicate that particular DNA and serum tests will identify individuals at risk for such responses.

Applicants' study was conducted with antibody-immobilized ChEs, which enabled them to stop the inactivation or reactivation processes at any given time, remove the inhibitors and measure remaining ChE activities for obtaining correct rates of these processes. While this approach does not take into consideration pharmacodynamics, it does provide accurate values for the target molecule, i.e. the human ChEs themselves. In previous studies, inhibition levels, but not rates were determined, and measurements were performed in the presence of the inhibitors which had changed these levels. A second major feature distinguishing this study from previous ones is the comparison to human recombinant ChEs. Inhibition observed in immobilized serum enzymes confirmed with the recombinant enzyme may with confidence be taken as a true interaction of inhibitor and enzyme.

The strength of interaction of tacrine with ChEs can be understood by reference to the crystallographic model of the enzymes (FIG.

3

). Tacrine is held in the active site gorge of ACHE by interaction of its aromatic rings with the aromatic rings of Trp84. At the mouth of the active site gorge Phe330 (ACHE) or Ala328 (BuChE) encloses tacrine. The alanine residue of BuChE provides a less crowded space for tacrine than the more bulky phenylalanine of AChE, reflected tacrine's slightly smaller IC

50

, 0.05 for BuChE vs. 0.15 mM for AChE. In normal BuChE, the distance from the Asp7O carboxyl group to the tacrine anilinic nitrogen is only 3.7 Å, indicating the possibilty of a salt bridge. This interaction is removed in “atypical” BuChE, reflected in a 100-fold increase in tacrine's IC

50

.

Applicants' analyses predict different effects of the “atypical” genotype on individual responses to the several Alzheimer's drugs. With all of the tested carbamates, significant differences are to be expected in homozygous “atypicals”, and less so in heterozygotes, due to the over 10-fold differences in the inactivation rates of normal and “atypical” BuChE by these drugs. In addition to the 0.03-0.10% homozygotes among the treated population difficulties perhaps may be predicted also for those heterozygotes suffering from liver malfunction and reduced BuChE levels from other causes. The situation would be more severe under treatment with tacrine. This drug reacts with “atypical” BuChE so much more weakly than with its normal counterpart, that the “atypical” enzyme becomes a negligible factor in its interaction. Under these circumstances, even heterozygotes might show adverse responses, as their AChE levels would be reduced because of lack of scavenger under doses that cause no reduction in homozygous normals. The reported high percentage of cholinergic deficits under tacrine treatment (up to 15%, 1994) may perhaps reflect such heterozygotes and, in addition, patients with liver malfunctions and, consequently, with low serum BuChE levels.

Example 2

Evaluation of Pam Therapy for OP Poisoning

The effect of PAM therapy on OP poisoning in BuChE variants was examined. An established therapy for OP poisoning is a combination of 2-PAM and atropine, combined with diazepam (to deal with the problem of seizures). To study the effects of variant BuChE requires an investigation of the dynamics of catalysis. This includes attraction of the ligand into the deep gorge, formation of an acyl-enzyme intermediate by displacement of the choline alkoxy group by the enzyme's active site serine hydroxyl oxygen, and hydrolysis of the acyl-enzyme (Taylor, 1990; FIG.

5

). Detailed dissection of catalysis by any ChE variant would discriminate between effects on one or the other of these stages of catalysis.

The inactivation of ChEs by an organophosphate agent (OP) is analogous to the acylation step (Wilson, 1954; Taylor, 1990). Phosphorylation of the active site serine (Ordentlich et al., 1993b) is just as specific for S

198

as is the acylation stage of catalysis. Furthermore, it has the same dependence on the integrity of the catalytic triad (Ordentlich et al., 1993a). Certain steps included in the action of an oxime that displaces the phosphoryl-serine bond (Hackley et al., 1955) are similarly analogous to hydrolysis (Taylor, 1990; FIG.

5

). Cleavage of the phosphoryl-ChE bond is extremely slow, making OPs hemi-substrates. Their reactivation rate can be enhanced by nucleophiles (B:) such as choline, which acts from its customary binding site. More effective nucleophiles than choline accelerate the reactivation reaction much more.

PAM is Pyridine-2-aldoxime methiodide, a most successful nucleophile (Wilson, 1954), and is a rigid zwitterionic molecule that extends from the choline-binding site and juxtaposes its nucleophilic group precisely against the phosphoryl bond, which it displaces. PAM is a competitive inhibitor of catalysis (Rosenberry, 1975), and natural substrates compete with PAM in the reactivation reaction (Liu et al., 1985). The order of effectiveness of non-assisted hydrolysis of the variety of dialkylphosphoryl-ChEs formed by a spectrum of OP agents (e.g. dimethyl>diethyl>diisopropyl) is maintained in the PAM-assisted reactivations (Taylor, 1990). This suggests that reactivation shares mechanistic characteristics with the deacylation step of catalysis.

To analyse on a micro-scale and to avoid extensive purification of each of the studied proteins, applicants immobilised recombinant Xenopus oocyte-produced variant ChEs on selective monoclonal antibodies in multi-well plates and subjected the bound enzymes to successive OP inactivation and oxime-promoted reactivation. This procedure also provided undisturbed examination of each reaction. Applicants exploited this approach by measuring major changes in rates of the reaction with an OP agent and an oxime (FIG.

5

), for a large series of human ChE variants differing within the gorge lining, the acyl-binding site or the C-terminus, always by comparison to the wild-type enzyme.

Spatiotemporal dissociation of catalytic steps: To address involvements of several regions of ChEs in the catalytic process, applicants focused on the gorge lining and the acyl-binding site of human BuChE, since residues in these regions differ from those in ACHE (FIG.

6

). Applicants collected rate constants for each of the two recombinant human enzymes as produced in microinjected Xenopus oocytes from the corresponding cloned, in vitro transcribed mRNA (Neville et al., 1992) or cDNA (Seidman et al., 1994). Because applicants expected that variations in the C-terminus would not affect catalytic properties of the enzyme (Massoulie et al., 1993; Soreq & Zakut, 1993), applicants used their approach also to test this expectation and compare variations in different regions of these proteins. To this end applicants included products of ACHEDNA vectors that encode the major form of human AChE expressed in brain and muscle (Soreq et al., 1990), and designated E6 (exons 2, 3, 4, and 6) and the alternative, that encodes a different C-terminus and represents a variant ACHEmRNA species expressed in hematopoietic and tumor cells (E5; exons 2, 3, 4, intron 4 and exon 5) (Karpel et al., 1994a). Applicants also examined a chimera of human AChE and BuChE, in which the gorge rim, the gorge-lining, the conserved oxyanion hole and the choline-binding site of BuChE (residues 58 to 133) were substituted with the homologous peptide of AChE (Loewenstein et al., 1993a). The major difference in the chimeric enzyme is that its gorge lining is more aromatic than BuChE. Several natural mutants of recombinant human BuChE (Neville et al., 1992) and site-directed point mutations its acyl-binding site (Gnatt et al., 1994) completed this series. Table 3 lists these alterations, and

FIG. 6

positions these changes on the ribbon model of human BuChE.

To examine the kinetic consequences of changing these defined regions in the enzyme for the different steps of catalysis, each of the variants was reacted with diisopropylfluorophosphonate (DFP). This was followed by regeneration of activity by PAM (FIG.

5

). DFP and PAM concentrations were chosen to yield phosphorylation and reactivation half-times measured in minutes. The Xenopus oocyte-produced enzymes were enriched by adsorption onto an immobilised antibody (Seidman et al., 1994; Liao et al., 1993). ELISA measurements provided determinations of the amounts of recombinant proteins for evaluation of k

cat

values and confirmed that all of the examined proteins were produced in quantities of the same order of magnitude.

The gorge lining contributes to enzyme phosphorylation: Inactivation by DFP is shown for five representative ChEs (FIG.

7

A). The DFP-inactivation second order rate constant for AChE, 7×10

4

min

−1

M

−1

(Table 3), was found to be 160-fold lower than that of BuChE. This value is in good agreement with the value of 3×10

4

min

−1

M

−1

determined for native, purified human AChE (Main & Iverson, 1966) and is almost identical to the 9.1×10

4

min

−1

M

−1

value determined more recently for purified recombinant human AChE (Ordentlich et al, 1993a). Introduction of a positive charge into the acyl-binding site in the L

286

K mutant slowed the inactivation rate of BuChE approximately 8-fold (FIG.

7

A). In contrast, in-the chimera, 15 conservative and 12 non-conservative changes were sufficient to reduce the inactivation rate of BuChE 60-fold, down to the level displayed by AChE (FIG.

7

B).

In general, inactivation rates of natural BuChE mutants were not severely affected. There was no effect of D

70

G or S

425

P alone, but the D

70

G/S

425

P double mutant displayed a lowered inactivation rate that was paradoxically (and inexplicably) restored in the D

70

G/S

425

P/Y

114

H triple mutation. Most acyl-site mutants of L

286

and F

329

also displayed inactivation rates moderately (2- to 7-fold) lower than native BuChE, with the exception of F

329

Q, which remained unchanged. Finally, both the alternative AChE forms displayed similar inactivation rates (FIG.

7

A and Table 3), demonstrating, as expected, that differences in the acyl-binding site and gorge lining of ChEs contribute to the acylation rate far more than the natural variability of their C-termini.

Active site charles hamper deacylation: To study the deacylation step of catalysis, the same series of enzymes was inhibited by DFP and then assessed for rates of reactivation by PAM. The Kd for diisopropylphosphoryl-(DIP-)BuChE, 0.3 mM, is close to the value of 0.2 mM found for purified diethylphosphoryl-BuChE (Wang & Braid, 1967). Thus for this enzyme as well, the single-step enrichment on immobilized antibody is sufficient for in-depth biochemical analysis and did not alter characteristic kinetic constants.

The pseudo-first order rate constant for reactivation of DFP-inhibited BuChE (Table 3) is 25-fold higher than for either form of AChE (FIG.

7

B). In contrast, the chimera displayed a reactivation rate constant only moderately (5-fold) lower than that of native BuChE. The lower rate for AChE and the chimera is attributable to a lower true first-order rate constant (k

r

) its affinity for PAM, K

a

, remaining unchanged (Table 4). In contrast, the BuChE L

286

K mutant displayed a 40-fold reduction in the pseudo-first order rate constant for reactivation, reflecting a reduction in both reactivation rate and affinity for PAM. Applicants further substituted an acidic, a basic, a sulphydryl and a polar group for F

329

. Of these, only the sulphydryl and acidic groups disrupted the reactivation (Table 3) by 10- to 20-fold, emphasizing the importance of the acyl-binding site in the reactivation process. However, altering the C-terminus of AChE affected neither the affinity for PAM nor the true first order rate constant (Table 3).

For the natural BuChE variants, effects of D

70

G and Y

114

H on reactivation are cumulative. The natural substitution of D

70

by glycine makes the resulting variant reactivate at a rate 5-fold lower than that of the wild-type enzyme. Addition of the Y

114

H or S

425

P mutation to the D

70

G mutation results in an even slower reactivating enzyme, and a combination of all three mutations in one variant causes the most severe decrease, 40-fold, in the rate of reactivation (Table 3). Thus, reactivation rates, as compared to the parent enzyme, are seriously impaired, in certain natural and site-directed mutants of BuChE, more than in the chimera. Such impairment in reactivation underlies the response to anti-cholinesterase drugs in carriers of these mutations and the need to identify them in the population.

Effects of variations on kcat: To compare effects on the analogous reactions with effects on catalysis, the consequences of each variation were further evaluated by determining turnover numbers. The turnover number for human BuChE as determined by applicnats, 96,000 min

−1

, is in good agreement with that reported for recombinant human and mouse BuChE (Ordentlich et al., 1993a; Vellom et al., 1993). Effects on k

cat

in the various mutants are not cumulations of effects on k

i

, and k′

r

, nor are they expected to be, as only the slowest step is reflected in the catalytic rate. Thus, substitution of L

286

with a basic residue which affects reactivation, led to a 5-fold reduction in the turnover number (Table 3). Other replacements at this position, and substitution of the gorge lining in the chimera, had considerably smaller effects on this value.

Search for the rate-limiting step of catalysis: To see if conclusions could be drawn about the effect of mutations on the rate limiting step for a sampling of variants, the changes in the approach of DFP to the active site and its reaction with the active site serine (normalised k

i

), and reaction of bound PAM with the DIP group bound to that serine (normalised k

r

) , to the overall catalytic cycle (normalised k

cat

) (

FIG. 8

) were compaared. An OP does not interact with a ChE exactly as does a substrate, and PAM may distort the active site in ways different from binding of an acyl group. Nevertheless, the application of this approach could add another level to the conclusions that are inferred solely from consideration of the rate constants alone and shows the value of the assay for dissecting the steps of sequential reactions.

Normalised kinetic constants were used (FIG.

8

): normalised k

i

, k

i

(variant)/k

i

(BuChE), normalised k

r

, k

r

(mutant)/k

r

(BuChE) and normalised k

cat

, k

cat

(mutant)/k

cat

(BuChE). At one extreme, one of the events leading to acylation by a variant is rate-limiting for catalysis, and the decrease from the value for BuChE in k

cat

will be in the same proportion as in k

i

. At the other extreme, deacylation is rate-limiting and the decrease in k

cat

is in the same proportion as in k

r

. Thus, normalised k

r

in one case, or normalised k

r

, in the other, will be equal to normalised k

cat

. If acylation is not rate-limiting for a variant, there is no coupling of the normalised k

i

with normalised k

cat

; the rate-limiting step of catalysis may be seriously impaired while inactivation rate remains high (normalised k

i

normalised k

cat

) , or catalysis may remain unaffected while inactivation is impaired (normalised k

i

<<normalised k

cat

). Similarly, if deacylation is not rate-limiting, normalised k

r

, has no relation to normalised k

cat

. However, for each variant either inactivation or reactivation should track catalysis.

The test for the rate-limiting step is the near equivalency of normalised k

cat

with the normalised constant for inhibition (acylation) or for reactivation (deacylation). Inspection of

FIG. 8

suggests that for the chimera and AChE using ETCh as a substrate, deacylation is rate-limiting, whereas for the L

286

K variant both acylation and deacylation may be rate-limiting. The strikingly lower inactivation rate seen in AChE and the chimera (Table 3) can be accommodated without a great decrease in the catalytic rate, precisely because it is deacylation (reactivation), not acylation (inactivation) that is rate-limiting in these enzymes. The hindered access of ETCh to the AChE active site, due to the aromatic groups of the gorge lining of these enzymes, seems not to overshadow the decrease in deacylation rate, which is apparently rate-limiting. Rather, there has presumably been a minor realignment of the reactive groups at the active site that cleave the butyryl- and DIP-enzymes and regenerate the free enzyme. In contrast, when a basic group is substituted for L

286

at the acyl-binding site, proper orientation of the acyl group of the substrate is apparently disrupted, affecting both acylation and deacylation. Altogether, applicants' findings highlight precise alignments at the gorge lining and the active site as determining the catalytic distinctions between AChE and BuChE and exclude the C-terminus region from such involvement.

Example 3

Antisense Inhibition Of BuChE Gene Expression Predicts Adverse Hematopoietic Consequences of Cholinesterase Inhibitors

Antisense (AS) oligodeoxynucleotide inhibition was used to explore the hematopoietic effects of interference with butyrylcholinesterase expression. These experiments demonstrate the need in hematopoietic-associated diseases or conditions treatment that patients with varient BuChEs be identified so that they are not receiving anti-cholinesterase drugs. Further, these data indicate that patients with deficient BuChE expression and exposure through treatment or environmentally to anti-ChEs will cause hematopoietic differences in these patients.

Interference with BuChE activity, an expected outcome of interaction with cholinesterase inhibitors, suggests adverse hematopoietic consequences. To examine if this is the case, and if BuChE inhibition causes distinct effects from those anticipated under ACht inhibition, primary murine bone marrow cultures as an ex-vivo system in which BuChE is expressed were examined and antisense oligonucleotide inhibition was used to block such expression.

In primary bone marrow cell cultures interleukin 3 (IL-3) enables expansion of a small fraction of the existing pluripotent stem cells into multipotent progenitors. These can differentiate within four days into megakaryocyte colony forming units (CFU-MK) composed of megakaryocytes, granulocytes and macrophages (Metcalf, 1992). Addition of erythropoietin and transferrin to IL-3 leads, within 8-9 days, to the development of CFU-GEMM colonies composed of granulocytes, erythroid cells, megakaryocytes and macrophages (Koury and Bondurant, 1990). Therefore, CFU-MK and CFU-GEMM colony counts reflect the capacity of these cultures for expansion and survival of progenitors, whereas total cell numbers indicate proliferation rates. Finally, differential cell compositions of surviving colonies demonstrate which cell lineages developed under the experimental conditions employed and in what fractions. To investigate whether any of these parameters is affected by interfering with BCHE gene expression, applicant employed phosphorothioated antisense oligodeoxynucleotides (AS-oligos, Eckstein, 1985) targeted towards the BCHE gene. Recent reports demonstrate that AS-oligos toward several key proteins interfere with hematopoiesis ex-vivo and in vivo (Stein and Cheng, 1993; Gewirtz, 1993; Ratajczak and Gewirtz, 1994). More specifically, certain AS-oligos were shown to selectively block megakaryopoiesis. These include AS-oligos to the proto-oncogenes c-mpf (Methia et al., 1993), c-myb (Szczylik et al., 1993), bcr-abl and fos/jun (Lord et al., 1993) as well as to metabolically important enzymes such as cdc kinases (Lapidot-Lifson et al., 1992) or 5-lipoxygenase (Anderson et al., 1993). In vivo studies further demonstrated inhibition of erythropoiesis with AS-oligo to the c-kit ligand and to mixed colony stimulating factors (Pech et al., 1993).

In addressing the hematopoietic function of cholinesterases by this approach applicnats have previously shown that AS-oligos toward BCHE impair CFU-MK formation (Patinkin et al., 1990) and that the related AS-ACHE causes hematopoietic changes in vivo (Lev-Lehman et al., 1994) and ex-vivo (Soreq et al., 1994). To examine if the involvement of BCHE gene expression in megakaryopoiesis depends on erythropoietin, and to evaluate the duration of such interference, applicants extended their AS-BCHE analysis in CFU-MK to CFU-GEMM cultures and to the in vivo administration of AS-BCHE. In addition, the capacity of bone marrow-cells subjected in vivo to AS-BCHE treatment to develop ex-vivo into CFU-MK colonies was examined. The experimental findings demonstrate considerable erythropoietin-independent impairment induced by AS-BCHE over megakaryopoiesis ex-vivo and in vivo and predict hematopoietic abnormalities in individuals with suppressed BuChE or in those patients subjected to cholinesterase inhibition.

Specific Materials and Methods

Primary bone marrow cell cultures: Primary murine bone marrow of 8-12 week-old C3H/Hei mice was grown as described by Patinkin et al. (1990). For CFU-MK, 1×10

5

cells were seeded in LPM (Low protein medium, Biological Industries, Bet Haemek, Israel) containing 1% bovine serum albumin (BSA), 1×10

−4

M thioglycerol, 10% conditioned medium from WEHI cells and 1% methyl cellulose and were incubated for four days. For CFU-GEMM, an additional 2.8×10

−4

M human transferrin and 2 units/ml erythropoietin (Epo) (Terry Fox Laboratories, Vancouver, Canada) were added to cultures incubated for eight days at 5% CO

2

and 37° C. In liquid cultures of CFU-MK, the methyl cellulose was deleted and medium increased accordingly. Oligonucleotides were added at day 0, either in totally phosphorothioated forms (Lapidot-Lifson et al., 1992) or phosphorothioated at the three 3′-terminal internucleotidic bonds to reduce cytotoxicity (Ehrlich et al., 1994b).

Differential Cell Analysis: Colonies of either CFU-MK or CFU-GEMM were cytospinned as described by Ehrlich et al. (1994b) and stained with May-Grunwald-Giemsa. Cells were characterized as megakaryocytes, macrophages, granulocytes or erythrocytes as detailed by Ehrlich et al. (1994b). From 500-1,500 cells were counted for each experimental section.

Evaluation of Apoptotic DNA index: For apoptosis experiments, cells were grown at 1.5-2.5×10

5

cells/ml and peak concentrations of 5 μM AS- or S-BCHE for CFU-MK and 10 μM AS- or S-BCHE for CFU-GEMM. CFU-MK were seeded in liquid culture while CFU-GEMM were grown in methyl cellulose serum-free cultures. For DNA analysis, 1 μg of total DNA extracted from control cells or those treated with sense or antisense oligos was electrophoresed at 60 V for 1.5 hours on 1.5% agarose gels, then DNA was blotted on a nylon membrane (Zeta probe, Bio-Rad, Hercules, Calif.) and hybridized with [

32

P] random primed labeled genomic mouse DNA. Exposure was for one day. Molecular size markers were electrophoresed in parallel. A ladder of oligonucleosome-sized DNA fragments reflecting apoptosis, appeared in all culture lanes and its intensity served to evaluate the apoptotic index of the corresponding cultures.

In vivo administration of AS-BCHE: Totally phosphorothioated oligodeoxynucleotide, AS-BCHE (5′-GACTTTGCTATGCAT-3′) (SEQ ID NO. 1) was intraperitoneally injected into a group of four three week old female Sabra mice to reach a final concentration of 5 μg/gr weight. The injected volume did not exceed 10 μl/gr weight. Phosphate buffered saline (PBS) was injected to control mice.

In Situ Hybridization: Bone marrow smears were prepared from adult female mice 20 days post-injection (once, with 5 μg/gr weight totally phosphorothioated AS-BCHE or with up to 10 μl/gr weight PBS). In situ hybridization on fresh bone marrow smears was performed using [

35

S]-labeled in vitro transcribed RNA probes from the sense and antisense directions of BCHEcDNA essentially as described by Lev-Lehman et al. (1994). In situ hybridization results were analysed using a Nikon Microphot microscope connected through an interface to a Magiscan Image Analysis microscope controller (Applied Imaging Int. Ltd., U.K.) as detailed by Lev-Lehman et al. (1994). BCHEmRNA levels in megakaryocytes (MK) were determined as average numbers of silver grains per cell as detailed in Patinkin et al (1990).

Results

The effects of AS-BCHE treatment on primary bone marrow cell cultures containing erythropoietin were first evaluated by counting CFU-GEMM colonies. A rise of about 60% in CFU-GEMM number above control values was observed in a representative experiment shown in

FIG. 9

upon application of 5 μM AS-BCHE. This colony increase was retained with rising concentration until at least 30 μM of AS-oligo (FIG.

9

A and data not shown). Sense-BCHE-treated cultures exhibited a similar rise in CFU-GEMM until 5 μM concentration. However, addition of higher S-BCHE concentrations reduced colony counts down to ⅕ of control values at 20 μM oligo (FIG.

9

A), demonstrating cytotoxicity. Cell counts peaked at 5-15 μM AS-BCHE with 2-fold over controls and then slowly declined, an effect which was not observed with S-BCHE which caused a significant decrease in cell counts above 10 μM (FIG.

9

B). Thus, AS-BCHE did not change the number of surviving CFU-GEMM progenitors yet improved their capacity to expand in culture.

In the absence of Epo, CFU-MK numbers dropped sharply by concentrations higher than 5 μM AS-BCHE (Patinkin et al., 1990). This significant decrease in colony numbers was reproduced in this present work, using a partially phosphorothioated oligo with reduced cytotoxicity (Ehrlich et al., 1994b and FIG.

9

C). Colony counts of corresponding S-BCHE treated cultures were essentially similar to those of control cultures (FIG.

9

C), demonstrating the specificity of CFU-MK impairment by AS-BCHE. No significant change was observed in CFU-MK cell counts with either oligo (FIG.

9

D). The AS-BCHE oligo hence suppressed CFU-MK but not CFU-GEMM colonies. In contrast, the S-BCHE oligo exerted cytotoxic effects on CFU-GEMM, but not on CFU-MK colonies, demonstrating that culture conditions affect the vulnerability of stem cells to such cytotoxicity.

The reproducibility of these oligonucleotide effects was examined by performing repetitious experiments using primary bone marrow cells from different mice (Table 5). This analysis revealed, in addition to the reduced CFU-MK counts in the presence of AS-BCHE, similar variability between experiments, from ±86.7 colonies in control cultures to ±96.7 in the presence of 15 μM AS-BCHE, in spite of the drastic reduction in mean colony counts. A significantly higher variability between CFU-MK counts occurred upon S-BCHE addition (up to ±255.0 at 15 μM oligo, P<0.0092). Since S-BCHE has no counterpart sequence in the cell, this change in variability probably reflects structure-related cytotoxicity particular to the S-BCHE sequence.

In contrast, the variability, but not absolute number of CFU-GEMM colony counts was reduced with addition of AS-BCHE (from ±12 in control cultures to ±6 with 30 μM AS-BCHE). However, S-BCHE did not alter the variability in CFU-GEMM counts, which remained ±10.8 in the presence of 15 μM S-BCHE (Table 5). Thus, in addition to the sequence-dependent capacity of AS-BCHE to reduce expansion of progenitor cells into CFU-MK colonies, AS-BCHE also lowered the inter-experimental variability in CFU-GEMM experiments in a sequence-dependent manner, without changing the mean colony counts.

To determine whether the administration of AS-BCHE caused non-specific changes in programmed cell death within the various cultures, yields and integrity of DNA were evaluated as detailed by Shi et al. (1992). To this end equal quantities of DNA preparations from control, AS-BCHE and S-BCHE treated cell cultures were electrophoresed, blotted and hybridized with a [

32

P]-labeled probe from mouse genomic DNA. DNA from all of these cultures, either CFU-MK or CFU-GEMM, all exhibited extensive fragmentation, typical of the apoptosis expected to occur in these primary cell cultures (Shi et al., 1992; Okumura et. al., 1992). The CFU-GEMM cultures exhibited a 25% higher degree of apoptosis than did the CFU-MK cultures, possibly due to their longer incubation period (FIG.

10

). However, there was no discernible difference between control lanes and AS-BCHE or S-BCHE lanes in either type of colony. Thus AS-BCHE effects in both culture types were apparently due to changes in cellular differentiation rather than associated with induction of apoptosis.

Colonies grown in the presence of S-BCHE included the cell types expected under the growth conditions employed. In the presence of IL-3 alone, these were primarily megakaryocytes and macrophages (

FIG. 11

, top panel), whereas the addition of Epo and transferrin and longer incubation times permitted erythropoiesis to occur as well (

FIG. 11

, bottom panel). In contrast, cultures grown with AS-BCHE included considerably less megakaryocytes and correspondingly more macrophages and neutrophils. This change was observed both in the presence of IL-3 (

FIG. 11

, top panel) and in the presence of IL-3 together with Epo and transferrin, (

FIG. 11

, bottom panel), where megakaryocytes but not erythroblasts appeared to be depleted by AS-BCHE treatment.

The arrest of megakaryopoiesis in CFU-MK by AS-BCHE was in line with applicants'previous findings (Patinkin et al., 1990, Lapidot-Lifson et al., 1992, Ehrlich et al., 1994b), yet the finding that it is Epo-independent was novel. To examine if this AS-BCHE-dependent interference with megakaryopoiesis reflected a significant change also in the presence of Epo, differential analysis was performed on CFU-GEMM colonies following treatment with AS-BCHE or S-BCHE. This revealed a 2-fold increase in the fractions of macrophages and neutrophils (

FIG. 12A

) and a corresponding decline in early, but not late MKs (

FIG. 12B

) with rising concentration of AS-BCHE up to 15 μM. Early and late erythroid cells exhibited little or no change in percent values (FIG.

12

C), demonstrating that the erythropoietin-independent effect of AS-BCHE was limited to megakaryopoiesis and did not affect erythropoiesis in these cultures.

The duration of AS-BCHE effects in vivo was first evaluated by in situ hybridization. Twenty days following a single intraperitoneal injection of AS-BCHE, almost no BCHEmRNA could be detected over MK in bone marrow smears from treated mice (data not shown). ACHEmRNA labeling of these MK was also reduced, yet to a more limited extent of 40%. Labeling decreased in both intermediary (14-20 pm diameter) and mature (>20 μm) megakaryocytes, demonstrating that cells created at or after the time of injection survived in these mice in spite of BCHEmRNA destruction in them (FIG.

13

A). CFU-MK colony numbers produced ex vivo from bone marrow derived from mice injected with AS-BCHE were about 60% of those of controls, while marrow from S-BCHE-injected mice showed values slightly higher than controls (FIG.

13

B). Both these results mirror exactly applicants' previously demonstrated in vitro studies (Patinkin et al., 1990, Lapidot-Lifson et al., 1992) yet extend them to reveal lowered capacity of CFU-MK progenitors subjected in vivo to AS-BCHE treatment to give rise to colonies in culture.

Discussion

The ex-vivo findings demonstrated certain enhancement in myeloid cell fractions and corresponding suppression of the megakaryocyte fractions in both CFU-MK and CFU-GEMM cultures. This erythropoietin-independent effect was sequence-dependent and not associated with general apoptotic changes. Complementary in vivo studies revealed continuation of the antisense-induced destruction of BCHEmRNA for over two weeks, no effect on megakaryocytes survival and ex-vivo suppression of CFU-MK expansion capacity following the in vivo treatment. In view of the parallel increase in cell counts in CFU-GEMM colonies, this study predicts increased myeloid cell fraction as a long-term effect of cholinesterase inhibitors blocking BuChE activity irreversibly.

To avoid non-specific cytotoxicity of the ex-vivo oligonucleotides, partial phosphorothioate protection of the relevant oligos, replacing only the three 3′-terminal internucleotidic bonds with phosphorothioate groups (Ehrlich et al., 1994b) was employed. Demonstration of a non-disturbed apoptotic index in experimental cell cultures, evidenced in unchanged ladders of fragmented DNA, indicated that the studied effects did not result from non-specific induction of programmed cell death. This, in turn, suggests that these effects were primarily due to selective destruction of the target BCHEmRNA.

In both CFU-MK and CFU-GEMM cultures, partially protected AS-BCHE but not S-BCHE enhanced myeloid and granulocyte counts while reducing the fraction of early megakaryocytes. In CFU-MK cultures, sequence-independent effects of the employed S-BCHE oligo increased the variability in colony counts; in contrast, the variability in CFU-GEMM colony counts was reduced under AS-BCHE treatment, together with suppression of megakaryocytes. These observations confirm and extend applicnats previous findings (Patinkin et al., 1990, Lapidot et al., 1992, Lev-Lehman et al., 1994, Ehrlich et al., 1994b) while unexpectedly demonstrating that the hematopoietic diversion induced by AS-BCHE from megakaryopoietic toward the myeloidogenic lineages is erythropoietin-independent, involves increases in myeloid proliferation and occurs also under in vivo conditions. The occurrence of myeloid leukemia in farmers exposed to organophosphorous anti-cholinesterase insecticides (Brown et al., 1990) may hence be related with the “ageing” capacity of these insecticides (Soreq and Zakut, 1993), causing long-term inhibition of BuChE.

In addition, these findings demonstrate a variable sensitivity of hemopoietic progenitors of CFU-MK colonies, but not those forming CFU-GEMM to the sequence independent cytotoxicity exerted by the S-BCHE oligo. Also, these findings indicate that CFU-GEMM progenitors respond to AS-BCHE in a less variable manner than CFU-MK progenitors. Individual progenitor cells may therefore be expected to respond to specific oligos with different levels of variability, dependent both on the oligo and on the cell type.

Interestingly, the suppression of megakaryopoiesis by AS-BCHE occurred throughout the dose-response curve of CFU-GEMM and seemed to be dominant over the induction of this differentiation process by Epo (Metcalf, 1992) yet unrelated with erythropoiesis. This implies that the AS-BCHE effects are not related with the differing erythropoietin sensitivities in individual proerythroblasts (Kelly et al., 1993). Since erythroid cells and megakaryocytes are believed to stem from a common progenitor (Okumura et al., 1992), this places the function of BuChE at a later time in the megakaryopoietic pathway, after the separate commitment of these two lineages. Defects in BCHE gene expression, analogous to AS-BCHE inhibition, may therefore interfere with platelet production while enhancing myeloid cell counts.

Overexpression of hematopoietic growth factors such as IL-6 was recently shown to induce age-dependent neurodegenerative disease intransgenic mice (Campbell et al., 1993). Likewise, these current findings predict that suppression of brain enzymes like BuChE may cause adverse hematopoietic effects distinct to this particular suppression. The interrelationship between the brain and the hematopoietic system previously shown for cytokines such as LIF, (Escary et al., 1993) thus extends beyond currently known agents also into the realm of enzymes.

Example 4

Placental Malfunction

In order to investigate fetal protection from anti-ChE poisons that may be associated with pregnancy complications, applicant examined whether catalytically active BuChE is produced in the early placenta, where it could hydrolyze such drugs. To clearly associate each of the ChE activities with distinct cell types during placental development, and to reveal the spatiotemporal pattern of placental ChE expression applicant combined cytochemical staining in the presence of selective inhibitors with electron microscopy. In addition, applicant subjected placenta and cultured placental cell extracts to RT-PCR analyses of gene expression. Since, in general, BuChE binds and/or hydrolyzes anti-ChEs but the “atypical” enzyme is relatively inert to many of these, applicant quantified the capacity of normal and “atypical” BuChE to interact with cocaine and a-solanine. Finally, to examine fetal protection by their mother's serum, applicant determined the incidence of carriers of the “atypical” BCHE allele among pregnant women with various indications of placental malfunction.

Specific Methods

Patients: Patients composing the study group included 23 cases of spontaneous abortions, or a history of such abortions, six pregnancy induced hypertension, five pre-eclamptic toxemia, four intrauterine growth retardation, three gestation diabetes melitus, two premature contractions, one oligohydramnios, one premature rupture of membranes, one secondary infertility and two premature ovarian failure. The control group consisted of 76 women after normal births, or non-pregnant patients with a history of normal pregnancies.

Samples: DNA samples were taken from the blood of the above-mentioned patients and controls. Term placenta samples were taken during normal births; early placenta samples were derived from normal pregnancies terminated in the first trimester for non-physiological reasons. Without exception, participants had given their informed consent to the use of their blood and tissue samples according to the guidelines of the local Institutional Review Board.

DNA extraction and genotype analysis: Peripheral blood cells were separated using UNI-SEP tubes (Eldan Technologies Co., Ltd., Jerusalem, Israel) as previously described (Ehrlich et al., 1994) and were stored at −70° C. until use. For DNA extraction, cells were thawed and lysed overnight by proteinase K (0.2 mg/ml) in 5 ml final volume of 10 mM Tris-HCl, 1 mM EDTA, 100 mM NaCl and 0.5% SDS, at 50° C. Lysates were extracted with phenol-chloroform and DNA was precipitated with 0.1 volume 3 M Na-acetate and 2.5 volumes ethanol. “Atypical” BuChE alleles were genotyped by PCR amplification and SauIIIA restriction analysis as described by Ehrlich et al. (1994).

Native and recombinant enzvme studies: In vitro transcription of plasmid DNAs, Xenopus oocyte microinjection, homogenization and enzyme activity measurements were all performed as detailed previously (Neville et al., 1990; 1992) and as herein above with butyrylthiocholine as substrate and with different concentrations of α-solanine (both from Sigma Chemical Co., St. Louis, Mo.) or cocaine-HCl (from E. Merck, Darmstadt, Germany). Serum BuChE was tested for butyrylthiocholine hydrolysis using previously established procedures (Loewenstein-Lichtenstein et al., 1995) and as detailed herein above. Wherever noted, recombinant AChE activity was followed by hydrolysis of acetylthiocholine.

RT-PCR: RNA extraction and RT-PCR procedures were essentially as described by Karpel et al. (1994), except that annealing temperature for reactions using primers corresponding to the BCHE gene was 55° C. Primers used were 1281+ (5′-AGACTGGGTAGATGATCAGAGACCTGAAAACTACCG-3′) (SEQ ID NO: 2) and 1635− (5′-GACAGGCCAGCTTGTGCTATTGTTCTGAGTCTCAT-3′) (SEQ ID NO: 3) from BCHE and 1522+ (5′CGGGTCTACGCCTACGTCTTTGAACACCGTGCTTC-3′) (SEQ ID NO: 4) and 2003− (5′-CACAGGTCTGAGCAGCGATCCTGCTTGCTG-3′) (SEQ ID NO: 5) for the ACHE gene. Numbers denote nucleotide positions in the corresponding cDNA sequences (Prody et al., 1987 and Soreq et al., 1990 for BCHE and ACHE, respectively). Reaction products (20%) were electrophoresed on a 1.5% agarose gel, with TAE buffer (40 mM Tris-acetate, 2 mM EDTA). To intensify signals, gels were blotted and membranes subjected to hybridization with the relevant oligonucleotide probes end-labeled by polynucleotide kinase with

32

P-α-ATP before autoradiography Lev-Lehman et al., 1994).

Cytochemical AChE staining and electron microscopy:

Tissues were fixed, cytochemically stained and prepared for electron microscopy as previously described (Seidman et al., 1995). To distinguish between butyryl- and acetylcholinesterase activities, cytochemical staining (Karnovsky and Roots, 1964) was carried out in acetate buffer (pH 6.0) overnight at 4° C. in the presence of 10

−5

M tetraisopropylpyrophosphoramide (iso-OMPA), a selective inhibitor of BuChE, or 1,5-bis-(4-allyldimethylammoniumphenyl)-pentane-3-one dibromide (BW 284C51), a selective inhibitor of AChE.

Syncytiotrophoblast cultures: Primary cells from seven to nine gestational weeks placenta were grown in culture and RNA extracted from them as detailed by Rachmilewitz et al. (1995).

ELISA: To determine the BuChE protein content, monoclonal mouse anti-human serum BuChE antibodies (no. 53-4), 4 μg/ml, were adsorbed to multiwell plates (Nunc, Denmark) overnight at 4° C. in carbonate buffer (Seidman et al., 1995). Free binding sites were blocked with PBS-T buffer (144 mM NaCl, 20 mM Na phosphate, pH 7.4, 0.05% Tween 20 and 0.01% thimerosal) for 60 to 80 minutes at 37° C. Homogenates of microinjected oocytes or serum samples were diluted 1:20 to 1:40 in PBS-T and further incubated in the antibody-coated wells for four hours at room temperature with agitation, and overnight at 4° C. Plates were washed three times with PBS-T and incubated with a rabbit anti-human polyclonal antiserum (Dako, Glostrup, Denmark) at 1:4000 dilution in PBS-T for 70 to 80 minutes at 37° C. After washing with PBS-T, horseradish peroxidase-conjugated goat anti-rabbit antibody (Jackson Laboratory, Bar Harbor, Me., USA) was added at 1:10,000 dilution in PBS-T. Peroxidase activity was thereafter assayed using o-phenylenediamine dihydrochloride at 1 mg/ml in 0.05 M phosphate/citrate buffer with Na-perborate, pH 9.6. Purified human BuChE was used for calibration and change of absorbance at 450 nm was recorded on a Molecular Devices (Menlo Park, Calif., USA) microtiter plate reader.

Results: Placental BuChE expression was first pursued by RT-PCR analysis using placental RNA from different developmental stages. While analyses from different samples of similar gestational age revealed considerable variabilities in the intensity of signals obtained, all of the RNA preparations examined yielded the expected PCR product, from the seventh gestational week through term placenta (FIG.

14

). That this BuChEmRNA could be produced in cytotrophoblast cells was confirmed by testing RNA from cultured primary cytotrophoblasts (

FIG. 14

) and two malignant cytotrophoblast cell lines (JAR and JEG, not shown)(Rachmilewitz et al., 1995).

In addition to BuChEmRNA, placental RNA preparations also included AChEmRNA, at levels that were rather variable between specimens, yet appeared throughout placental development (FIG.

14

). Within the same samples, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels were largely similar in all mRNA preparations (data not shown), indicating comparable mRNA quantities and suggesting that the differences in ChE RNA levels reflected genuine variabilities between individuals. Because of the different PCR primers employed, the wide variability between individuals and the distinct nucleotide composition of ACHE and BCHE genes (Soreq and Zakut, 1993), applicants did not compare the absolute concentrations of these two placental transcripts with each other. However, applicants noted that several preparations, including that of syncytiotrophoblast cells, created very faint AChEmRNA signals. In contrast, the same cells showed clear BuChEmRNA-derived bands (FIG.

14

), suggesting that BCHE might be the primary ChE gene being expressed in the early placenta.

To examine in situ the protein products of these placental ChE mRNA transcripts, applicants analyzed by electron microscopy cross sections from early (seven weeks) and term placenta following cytochemical staining for ChE activities. Electron microscopy of cytochemically stained syncytiotrophoblasts from both early and late placenta clearly revealed the crystal reaction products reflecting acetylthiocholine hydrolysis. Some of these products appears on chorionic villi, indicating that at least part of the catalytically active enzyme could have originated from serum. However, reaction products were also observed in the cytoplasm of examined cells and in rough endoplasmic reticulum structures, reflecting endogenous production. Both the distribution of reaction products within cells and staining intensity were essentially similar in early and term placenta. To distinguish between AChE and BuChE, staining was further performed in the presence of selective inhibitors. When stained in the presence of 10

−5

M iso-OMPA, a selective BuChE inhibitor, early placenta sections remained totally unlabeled. However, no reduction in labeling intensity of either chorionic villi or intracellular structures could be observed in iso-OMPA treated term placenta. In contrast, reciprocal inhibition by the AChE inhibitor BW284C51 in the same concentration, largely suppressed staining in term, but not early placenta, leaving only a few crystal reaction products associated with the chorionic villi, perhaps reflecting exogenous activity of the serum enzyme. Applicants, therefore, concluded that early syncytiotrophoblast cells produce mainly BuChE, whereas term placenta cells produce primarily AChE. Interestingly, applicants could not detect in situ ChE activity in cytotrophoblast cells.

To reveal the cellular origin(s) of the two ChEs within the placenta, applicants further analysed blood vessels in the examined sections. Epithelial cells surrounding the inner part of these blood vessels, as well as hematopoietic cells present in them, both stained intensely for ChE activities, either with or without the above inhibitors. This observation explained the previously reported AChE and BuChE activities in both first trimester and term placental homogenates (Hahn et al., 1993; Simone et al., 1994). In addition, these staining results may explain the presence of both mRNA transcripts throughout placental development.

The syncytiotrophoblast BuChE expression in first trimester placenta, makes it the major ChE at this stage, its role, presumably, being protective of AChE. This, in turn, raised the question to what extent less active BuChE variants (e.g. “atypical” BuChE) could serve as scavengers. To test this issue, applicants prepared serum from an individual who had experienced succinylcholine-induced apnea and was genotyped as a homozygous carrier of the “atypical” BCHE allele (Ehrlich et al., 1994). The interactions of BuChE from this individual's serum with cocaine and with the glycoalkaloid α-solanine were compared (

FIG. 3

) with those of serum BuChE from a heterozygote family member of this subject, with enzyme from normal serum and with recombinant normal and “atypical” BuChEs produced by microinjection of mRNAs to Xenopus oocytes (Neville et al., 1992). To mimic the heterozygous state with the recombinant enzymes, applicants tested 1:1 mixtures of oocyte homogenates, expressing the normal and “atypical” enzymes in approximately equal amounts (FIGS.

3

A,C: DG curves).

The “atypical” enzyme, both recombinantly produced and in serum, presented normal protein level yet low specific activity (Table 6). Moreover, it remained relatively inert to cocaine and α-solanine inhibition up to 1 mM and 10 μM concentrations of these drugs, respectively. In addition, recombinant AChE was insensitive to inhibition by cocaine at concentrations up to 0.1 mM (Table 6). Therefore, at this range of exposure, normal, but not “atypical”, BuChE or AChE can bind cocaine. However, at α-solanine concentrations higher than 5 μM the “atypical” enzyme retains higher activity than the normal and the heterozygote enzymes (FIG.

3

). This is especially prominent for sera, but is also noticeable for the recombinant enzyme in oocyte mixtures. Thus, under exposure to high concentrations of anti-ChE agents, the “atypical” enzyme may be advantageous.

The serum and recombinant “atypical” enzymes displayed >10 folder higher IC50 values for cocaine as compared to corresponding normal BuChEs (4.5 and 0.3 mM, respectively, Table 6A). For α-solanine, the IC50 difference was >100 fold (170 and 2.5 (M, Table 6B). Interestingly, while heterozygous mixtures of normal and “atypical” recombinant BuChE yielded the expected intermediate inhibition curve, heterozygous serum displayed curves similar to those of normal serum BuChE (FIG.

3

). Indeed, the IC50 value of heterozygous recombinant BuChE for cocaine was >2 higher than that of the enzyme produced from normal DNA, whereas normal and heterozygous sera yielded similar values (Table 6A). To verify that these differences were not due to different enzyme amounts in different sera, applicants quantified these amounts by ELISA, and found that the heterozygous and homozygous “atypical” sera contained normal concentrations of BuChE (approximately 2 ng/μl, Table 6).

Since the oocyte-produced BuChE remains primarily monomeric (Neville et al., 1992), the different poison-interactions of native heterozygous BuChE, as compared with 1:1 mixture of recombinant BuChE may be attributed, at least in part, to heteromeric assembly. Therefore, this analysis predicted that the scavenging capacities of homozygous BuChE variants in vivo should differ from those of normal BuChE, depending exclusively on the genotype of the individual, and that “atypical” BuChE within syncytiotrophoblast cells should detoxify much less poisons than the normal enzyme. In contrast, heterozygote carriers of “atypical” BuChE were predicted to be similar to normal homozygotes in situations of intoxication by drugs hydrolyzed by or targeted at BuChE.

To test this hypothesis on one possible outcome of such intoxications, placental malfunction, applicants genotyped BCHE alleles in 49 patients with complicated pregnancies of various origins (Table 7). The common element to most of these is that they reflect placental malfunction. Only one of these patients admitted drug abuse, and their sera were not checked for drug metabolites. However, the frequent use of agricultural anti-ChEs and the high dietary intake of solanum vegetables in the central region of Israel predict that these patients were all exposed to at least some ChE inhibitors. Interestingly, the frequency of the “atypical” BCHE allele was 0.01 in this group and 0.03 in a control group of 76 women with histories of normal pregnancies. While “atypical” allele frequency in the complicated pregnancy group was considerably lower than in the control group, the group sizes were too small to discern statistically significant differences between these allele frequencies (p<0.2). Since the “atypical” allele incidence is known to differ between ethnic groups, applicants further tested the possibility that this difference was due to the population diversity within the examined groups. To this end, the expected G70BCHE allele frequencies in the study and control groups were calculated as based on published values of G70 allele frequencies in different Jewish populations (Szeinberg et al., 1972; Ehrlich et al., 1994), and using the ethnic origin distribution within each group (FIG.

15

). This analysis ascertained that the allele frequency differences were not due to different ethnic origin distributions of the two groups, as the expected average allele frequency was found to be 0.03 in both groups. Therefore, “atypical” heterozygotes may be excluded from being at risk for placental malfunction under exposure to anti-ChEs.

Discussion: Applicants electron microscopy data demonstrate endogenous BuChE activity in multinucleated terminally differentiated syncytiotrophoblasts as well as in blood vessels, and hemopoietic cells. Interestingly, these latter cells express BuChE at the first trimester but not in term placenta, when they switch to expressing AChE; and their progenitor, less differentiated cytotrophoblast cells do not express either of these enzymes. Nevertheless, RT-PCR analyses on placental homogenates demonstrated BuChE and AChE mRNA expression throughout placental development. In addition, BuChEmRNA, and to a lesser extent, AChEmRNA was also found in cultured cytotrophoblast cells, showing the potential of these cells to express ChEs although they did not stain for ChE activity. The mRNA transcripts in homogenates could probably originate in epithelial blood vessel cells and hemopoietic cells, where applicants noted the corresponding enzyme activities. In addition, these mRNAs could also be expressed in trophoblast cells with the production and activity of their protein products is suppressed by posttranscriptional control mechanisms. The latter possibility is supported by the finding that cultured cytotrophoblast cells produce BuChEmRNA while the same cells in the placental sections do not produce the active BuCuE protein. Furthermore, in an earlier study, plasmid cDNA libraries were generated from chorionic villi of nine weeks gestation separated from blood vessels by dissection. In screening these libraries, BuChEcDNA but not AChEcDNA was found, showing that first trimester trophoblast cells express BuChEmRNA (Zakut et al., 1991). In either case, the pattern of ChE production in placenta resembles that in the nervous and bone marrow systems: while the expression of both enzymes appears to initiate with the onset of the terminal differentiation process, BuChE expression generally precedes that of AChE and ceases thereafter (for reviews, see Rakonczay and Brimijoin, 1988; Soreq and Zakut, 1993; Taylor and Radic, 1994; Massoulie et al., 1994; Layer, 1994).

That “atypical” heterozygous serum displayed inhibition curves similar to those of normal homozygous serum both with cocaine and with (-solanine, might be due either to upregulation of normal BuChE expression in these heterozygotes, to allosteric effects on heteroligomers of normal and “atypical” BuChE which may have different affinities for the drugs than the homomers, or to another unidentified factor(s) in the serum absent in the oocyte homogenate which interacts with the drug. The first possibility is unlikely since the BuChE levels in serum of heterozygous and homozygous “atypical” BuChE carriers was found to be similar to that of normal serum. Furthermore, the activity of “atypical” heterozygous serum compared to those of normal and homozygous ones implies that the level of expression of the two alleles is similar in heterozygotes (data not shown). This makes the allosteric theory the most plausible one.

This study was conducted on DNA of pregnant woman who live in the central region of Israel where use of anti-ChEs as insecticides is widespread. Also, pregnant women from agricultural regions are likely to be concurrently exposed to organophosphorus or carbamate anti-ChE insecticides, which further increases the level of poisonous metabolites in these women. Moreover, the possibility that poisonous alkaloids may cause fetal damage becomes a pertinent issue in view of the recent increase in cocaine exposure during pregnancy. That this damage is dependent upon the mother's BCHE phenotype is due to the orders of magnitude greater metabolic activity of the mother as compared to the fetus. However, applicants present study excludes heterozygous carriers of the “atypical” allele from this risk and indicates that it is limited to fetuses of mothers with homozygous genotype of variant alleles of the BCHE gene.

Throughout this application various publications are referenced by citation or by patent number. Full citations for the cited publications are listed below. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.

The invention has been described in an illustrative manner, and it is to be understood that the terminology which has been used is intended to be in the nature of words of description rather than of limitation.

Obviously, many modifications and variations of the present invention are possible in light of the above teachings. It is, therefore, to be understood that within the scope of the appended claims, the invention may be practiced otherwise than as specifically described.

TABLE 1

Affinity of Tacrine for ChEs: IC

50

values

Recombinant

Serum

AChE

brain-type (E6)

0.15 ± 0.08

—

hematopoietic (E5)

0.15 ± 0.04

—

BuChE

normal

0.054 ± 0.036

0.082 ± 0.009

“atypical”

11.4 ± 1.4

8.94 ± 2.46

IC

50

values for tacrine were measured for recombinant human ChEs and for sera in the presence of 1 mM butyrylthiocholine (BuChE) for 1 mM acetylthiocholine (AChE). The data shown are averages of 2 for serum samples and 3 for the recombinant enzymes.

TABLE 2

Rates of reaction of ChEs with carbamate inhibitors, second order rate constants, k

i

(M

−1

min

−1

),

for inactivation of recombinant ChEs by several carbamate inhibitors.

BuChE

AChE

normal

“atypical”

E6

E5

pyridostigmine

1.9 × 10

3

± 0.2 × 10

3

ND

2.2 × 10

4

± 0.9 × 10

4

2.5 × 10

4

± 0.7 × 10

4

(10

−5

M)

physostigmine

3.8 × 10

5

± 1.6 × 10

5

2.6 × 10

4

± 0.9 × 10

4

ND

ND

(10

−5

M)

heptyl

1.1 × 10

7

± 0.3 × 10

7

7.7 × 10

5

± 4.4 × 10

5

1.6 × 10

6

± 0.7 × 10

6

1.4 × 10

6

± 0.4 × 10

6

physostigmine

(10

−8

M)

SDZ ENA-713

1.4 × 10

4

± 0.3 × 10

4

4.7 × 10

2

± 4.6 × 10

2

4.3 × 10

3

± 1.8 × 10

2

3.3 × 10

3

± 0.6 × 10

3

(10

−5

M)

From plots such as those in

FIG. 1

, pseudo-first order rate constructs were calculated - slope of plot - ln (activity vs. time) then divided by the reagent concentration (in parentheses), to calculate second order rate constants. Data are averages and standard deviation of at least four determinations.

ND - Not Determined

TABLE 3

Kinetic rate constants for DFP-inactivation,

PAM-reactivation and catalysts of ChEs

a

k

i

× 10

−4

k′

r

× 10

3

k

cat

× 10

−3

Variant

(M

−1

min

−1

)

(min

−1

)

(min

−1

)

BuChE

1220 ±

4 (3)

150 ±

30 (11)

96 ±

22 (6)

Chimera

19 ±

8 (2)

40 ±

18 (6)

36 ±

14 (5)

AChE (E6)

7 ±

1 (4)

6 ±

2 (3)

7.5

c

AChE (E5)

5

(1)

8 ±

3 (2)

L

286

D

188 ±

24 (3)

120 ±

30 (3)

38 ±

15 (5)

L

286

Q

166 ±

40 (3)

120 ±

20 (4)

42 ±

24 (5)

L

286

R

268 ±

164 (3)

6 ±

3 (3)

13 ±

7 (5)

L

286

K

138 ±

4 (3)

4 ±

1 (3)

13 ±

5 (4)

F

329

R

43

(1)

F

329

Q

1398 ±

532 (3)

44 ±

15 (5)

F

329

C

552 ±

408 (2)

14 ±

2 (4)

F

329

D

442 ±

190 (3)

8 ±

1 (4)

S

425

P

b

1054 ±

408 (3)

134 ±

8 (4)

D

70

G

b

1008 ±

418 (3)

32 ±

4 (4)

D

70

G + Y

114

H

b

2112 ±

1074 (3)

12 ±

5 (3)

D

70

G + S

425

P

b

260 ±

12 (2)

11

(1)

D

70

G + Y

114

H +

1598 ±

294 (3)

4 ±

1 (3)

S

425

P

b

a

Rate constants for inactivation (k

i

) were calculated by linear regression analysis of ln (A

t

) vs. t, where t is the time of exposure to DFP, and A

t

is the remaining activity at time t (e.g. FIG. 7A). Second order rate constants for inhibition (k

i

), were calculated for each of the noted variants of human ChEs from rates observed between 1 nM and 1 μM DFP. The pseudo-first order rate constant for reactivation, k′

r

, was calculated by linear

# regression analysis of ln (A∞-A

i

)/(A∞-A

t

) vs. t, where t is the time of exposure to 1 mM PAM, and A∞ is the potential activity, A

t

, the activity at time t, and A

i

, the residual activity of the inhibited enzyme (e.g. FIG. 7B). k

cat

values were calculated from the rates of reaction of BuChE and its variants with 30 mM BTCh and the quantity of enzyme evaluated in comparison to human serum BuChE by ELISA assay of the enzyme

b

Natural variant of BuChE. Numbers of experiments, in parentheses, and standard deviations are shown.

c

Value from Ordentlich et al., 1993a.

TABLE 4

Constants for PAM reactivation of

diisopropylphosphoryl-ChEs

a

k

r

× 10

3

·

K

d

Variant

(min

−1

)

(mM)

BuChE

220 ± 60 (4)

0.30 ± 0.08

Chimera

48 ± 20 (3)

0.34 ± 0.13

AChE (E6)

7 ± 3 (3)

0.27 ± 0.04

AChE (E5)

10 ± 5 (2)

0.19 ± 0.07

L

286

K

35 ± 27 (3)

>5

a

True first order rate constants for reactivation (k

r

) and dissociation constants for the DIP-enzyme/PAM complex (Ka) were evaluated from a plot of the reciprocals of the pseudo-first order rate constants vs. the reciprocals of PAM concentrations between 0.1 and 0.6 mM. Numbers of experiments, in parentheses, and standard deviations are shown.

TABLE 5

AS-BCHE reduces the variability in CFU-GEMM colony counts

CFU-GEMM

CFU-MK

Oligo [μM]

N

Colony Counts (Mean ± STD)

Oligo [μM]

N

Colony counts

None

0

9

26.2 ± 12.6

0

7

299.3 ± 86.5

AS-BCHE

2.5

—

—

2.5

8

274.4 ± 111.0

5.0

9

33.9 ± 9.8

5.0

9

173.4 ± 86.5

7.5

—

—

7.5

3

80.7 ± 4.5

10.0

5

32.6 ± 7.0

10.0

9

142.4 ± 102.2

15.0

9

38.0 ± 16.4

15.0

5

130.2 ± 96.7

20.0

9

31.0 ± 8.5

—

—

—

30.0

3

25.7 ± 6.4

—

—

—

S-BCHE

2.5

—

—

2.5

6

313.5 ± 121.8

5.0

2

38.0 ± 2.8

5.0

5

312.2 ± 143.2

7.5

—

—

7.5

3

165.7 ± 12.7

10.0

5

35.4 ± 10.7

10.0

7

231.3 ± 155.3

15.0

6

21.5 ± 10.8

15.0

5

264.2 ± 255.4

20.0

2

6.0 ± 0.0

—

—

—

Cell culture conditions were as detailed under Methods. The noted oligonucleotides were added at the given concentrations at day 0 and colony counts scored at the noted (N) Number of experiments on day 4 or 8 for CFU-MK and CFU-GEMM colonies, respectively. Averge cell number per colony was 50 for CFU-MK and 10,000 for CFU-GEMM. Mean colony counts ± standard deviation (STD) is presented for each set of conditions.

TABLE 6

D/D

D/G

G/G

Rec. BuChE

Protein, ng/ml

IC

50

Cocaine, mM

0.28 ± 0.02

0.67 ± 0.07

4.05 ± 0.05

IC

50

α-solanine, μM

1.95 ± 1.04

3.54 ± 2.41

171

Serum BuChE

Protein, ng/ml

IC

50

Cocaine, mM

0.32 ± 0.08

0.29 ± 0.005

5.2 ± 0.3

IC

50

α-solanine, μM

2.88 ± 0.33

3.61 ± 0.81

165

Rec. AChE

Protein, ng/ml

IC

50

Cocaine, mM

3.45 ± 2.15

TABLE 7

Ethnic origins in

Age

D70G

Diagnosis

No.

numbers

range

het.

spontaneous

22

A.-4; A./T.-1; B. &

22-42

1

abortion(s)

T.-4; In.-2; Iq.-3; N.

A.-6; N. A./Y.-1 S.-

1.

spontaneous

1

A.

70

abortions history

pregnancy induced

6

A.-2; A./N. A.-1;

24-30

hypertension

In.-2; S.-1

pre-eclamtic toxemia

5

A.-1; A./N. A.-1;

25-37

B./T.-1; Iq.-2

intrauterine growth

4

A.-1; In./N. A.-1;

28-35

retardation

Iq.-1; Y.-1

gestational diabetes

3

B.-1; Iq.-1; N. A.-1;

28-32

melitus

premature

2

A.-1; N. A.-1

19, 23

contractions

oligohydramnios

1

N. A.

28

premature rupture of

1

In.

21

membranes

hyperhermesis

1

In.

25

gravidarum

secondary infertility

1

T.

27

premature ovarian

2

In.-1; Iq.-1

24, 27

failure

control

76

A.-12; A./In.-1; A./N.

18-77

5

A.-1; B. & T.-9; In.-

8; Iq.-14; Iq./T.-1; L.

& S.-4; N. A.-24;

Y.-2

A.-Ashkenazi; B.-Balkan; In.-Iranian; Iq.-Iraqi; L.-Lebanese; N. A.-North African; S-Syrian; T-Turkish; Y-Yemenite.

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5

1

15

DNA

Homo sapiens

1
gactttgcta tgact 15

2

36

DNA

Homo sapiens

2
agactgggta gatgatcaga gacctgaaaa ctaccg 36

3

35

DNA

Homo sapiens

3
gacaggccag cttgtgctat tgttctgagt ctcat 35

4

35

DNA

Homo sapiens

4
cgggtctacg cctacgtctt tgaacaccgt gcttc 35

5

30

DNA

Homo sapiens

5
cacaggtctg agcagcgatc ctgcttgctg 30

Claims

1. A method of screening for a genetic predisposition to anticholinesterase exposure due to variant BuChE alleles including the steps ofobtaining a peripheral blood sample from a patient, analysing serum from the blood sample for BuChE levels and BuChE inhibitor-susceptibilites assays for response to an anticholinesterase differing from normal, and screening DNA of peripheral white blood cells from the blood sample for presence of variant BuChE alleles and from the results of the analyses and of the DNA screening thereby identifying patients who have a genetic predisposition to anticholinesterase exposure differing from normal BuChE due to variant BuChE alleles.
2. The method as set forth in claim 1 wherein the anticholinesterase exposure is by anticholinesterase drug therapy for neurodegenerative diseases or other conditions related to cholinergic malfunction.
3. The method as set forth in claim 2 wherein the neurodegenerative diseases or other conditions are selected from the group consisting of adult-onset dementias Alzheimer's disease, Parkinson's disease, Huntington's Disease, Amyotrophic lateral sclerosis, and Myasthenia Gravis.
4. The method as set forth in claim 1 wherein the anticholinesterase exposure is exposure to organophosphates or carbamate insecticides.
5. The method as set forth in claim 1 wherein the anticholinesterase exposure is related to hematological diseases.
6. The method as set forth in claim 1 wherein the anticholinesterase exposure occurs in drug addicts.
7. The method as set forth in claim 1 wherein the anticholinesterase exposure occurs in pregnant females.
8. The method as set forth in claim 1 wherein the genetic predisposition is identified as able to stand exposure to above normal levels of anticholinesterses.
9. The method as set forth in claim 1 wherein the genetic predisposition is identified as unable to stand exposure to normal levels of anticholinesterses.
10. A method of screening for a genetic predisposition to anticholinesterase exposure in a woman leading to placental malfunction due to the homozygous presence of “atypical” BuChE alleles including the steps ofobtaining a deoxyribonucleic acid (DNA) sample from a woman, and screening the DNA sample for a homozygous presence of “atypical” BuChE alleles thereby identifying the woman who has a genetic predisposition to anticholinesterase exposure leading to placental malfunction, if the presence of said homozygous “atypical BuChE alleles is found in said screening.
11. A kit for screening for a genetic predisposition to anticholinesterase exposure includinghybridization probes for BuChE alleles, and reagents for determining serum BuChE levels.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a National Phase of PCT/US96/00322, filed Jan. 11, 1996.

PCT Information

Filing Document	Filing Date	Country	Kind	102e Date	371c Date
PCT/US96/00322		WO	00	10/6/1997	10/6/1997

Publishing Document	Publishing Date	Country	Kind
WO96/21744	7/18/1996	WO	A

US Referenced Citations (3)

Number	Name	Date
4391904	Litman et al.	Jul 1983
5595903	Soreq et al.	Jan 1997
5807671	Soreq et al.	Sep 1998

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Method of screening for genetic predisposition to anticholinesterase therapy

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