Human Plasma Butyrylcholinesterase Research Articles

An engineered cocaine hydrolase blunts and reverses cardiovascular responses to cocaine in rats.

There is increasing evidence that human plasma butyrylcholinesterase can lower the toxicity of cocaine overdose. Recently, with structure-based protein engineering, we converted this enzyme into a more efficient cocaine hydrolase (CocE). When tested in rats, CocE shortened cocaine's plasma half-life and decreased drug accumulation in heart and brain. Here, we have investigated the potential of CocE to antagonize cardiovascular responses to cocaine. Anesthetized rats were instrumented for continuous recording of blood pressure from the femoral artery. Cocaine (7 mg/kg i.v.) caused blood pressure to rise within 30 s by 25 to 37 mmHg, but pressure returned to baseline within 60 s. These transient pressor responses were prolonged up to 5 min when vagal reflexes were blocked with atropine (1 mg/kg). Under such conditions, pretreatment with CocE (3 mg/kg i.v.) reduced cocaine's pressor effect, whereas delayed treatment with CocE rapidly restored normal mean blood pressure. CocE had no hemodynamic effects in control animals not treated with cocaine. The finding that CocE can oppose pre-established physiologic actions of cocaine suggests that similar or improved hydrolases might help rescue patients from the life-threatening toxicity of drug overdose.

Protective effect of polyurethane immobilized human butyrylcholinesterase against parathion inhalation in rat

Organophosphates (OP) are used in large quantities around the world as agricultural insecticides. Exposure to these toxic chemicals is a serious global health problem. Human plasma butyrylcholinesterase is known to be a good scavenger of organophosphorus pesticides and chemical warfare agents. In this study, purified human plasma butyrylcholinesterase (HuBChE) from pooled outdated human plasma, was immobilized onto the polyurethane foam. The immobilized enzyme showed greater stability at and above room temperature (up to 55 °C), compared to the enzyme in solution. Scavenger properties of immobilized enzyme were tested in vitro with parathion and its active metabolite paraoxon. In, in vitro experiments polyurethane foam with immobilized active enzyme removed 40% of parathion and 50% of paraoxon inhibitory effect (based on cholinesterase inhibition). In, in vivo experiments groups of rats inhaled parathion through filters with immobilized active enzyme (Group I), immobilized inactivated enzyme (Group II), and control group (Group III) inhaled solvent only without any parathion or filter. In the Group II animals, activity of plasma and red blood cells cholinesterase was significantly decreased (30 and 28%, respectively) compared to Groups I and III animals. In other tissues such as brain, skeletal muscle and lung, activity of the acetylcholinesterase (AChE) in the Group II animals, was decreased significantly (29, 28, and 22%, respectively). There was no significant differences between Groups I and III animals enzyme activities. In conclusion, immobilized butyrylcholinesterase (BChE) may be useful in scavenging and detoxifying organophosphate compounds both for medical protection and decontamination procedures.

The effect of fluoride on the scavenging of organophosphates by human butyrylcholinesterase in buffer solutions and human plasma

Fluoride ion is a reversible inhibitor of human butyrylcholinesterase (HuBChE) that is a viable drug candidate against organophosphates (OPs) toxicity. Since large numbers of communities in many countries are occasionally exposed to relatively high amount of fluoride, its effect on the kinetics of inhibition of HuBChE by OPs was investigated. In saline phosphate, pH 7.4, fluoride in the lower millimolar range significantly slowed the inhibition of HuBChE by paraoxon, DFP, echothiophate, soman, sarin, and VX. The kinetics of the inhibition was found consistent with the formation of a reversible fluoride–HuBChE complex that is at least 25-fold less active towards phosphorylation or phosphonylation than the free enzyme. Heat inactivation experiments indicate that the binding of fluoride to HuBChE probably involves enhanced cross-domain interaction via hydrogen bonds formation that may decrease enzyme activity. In spite of distinct structural differences among the OP used, the dissociation constants of the fluoride–HuBChE reversible complex varied over a narrow range ( K F, 0.31–0.70 mM); however, K F in human plasma increased to 2.75–3.40 mM. 19F-NMR spectroscopy revealed that fluoride ion is complexed to plasma components, an observation that explains in part the apparent increase in K F. Results suggest that an estimate of the relative decrease in the rate of OPs sequestration in presence of fluoride can be obtained from the fraction of the free HuBChE (1 + [F]/ K F) −1. Considering K F values in human plasma, it is concluded that the scavenging efficacy of OPs by HuBChE is not compromised by the normal concentration range of circulating fluoride ions.

Simplified procedures for purification and stabilization of human plasma butyrylcholinesterase

Human butyrylcholinesterase (HuBChE) is of particular interest because it hydrolyzes a wide range of toxic compounds such as cocaine, succinylcholine and carbamate pesticides. In this study, HuBChE was purified from human plasma using polyethylene glycol (PEG), differential precipitation and ion exchange chromatographic procedures. Purification resulted in a homogeneous enzyme preparation with a 5575-fold purification, and specific activity of 485 U/mg proteins with a reasonable final yield (36%). The final purified HuBChE showed a single electrophoresis band (85 kDa) on SDS-PAGE. Purified enzyme was stabilized with glycerol, PEG, and ammonium sulphate. Kinetic studies of the enzyme showed high affinity for butyrylthiocholine ( k m=18±5 μM) and low affinity for acetylthiocholine ( k m=83±12 μM), paraoxon, parathion, and tetra monoisopropyl pyrophosphomide all inhibited the enzyme with IC 50 of 2.8±0.8, 22±2 and 55±4 μM, respectively. The procedure introduced is inexpensive, simple and comparable to procainamide affinity column chromatography. This procedure can provide large quantities of purified enzyme with acceptable stability.

Pharmacokinetics and pharmacodynamics of a new local anesthetic agent.

We compared the effects of local anesthetics procaine, amethocaine (dicaine), bupivacaine, and a new agent RU-1148 on hydrolytic activity of human plasma butyrylcholinesterase. The butyrylcholinesterase-blocking activity of the test substances decreased in the following order: bupivacaine>amethocaine>procaine>RU-1148. The study of the capacity of these agents to form complexes with human plasma proteins and serum albumin showed that RU-1148 in therapeutic concentrations was transported by human serum albumin,beta-globulin, and acid glycoproteins. Study of the mechanisms of pharmacodynamic interactions between clonidine and RU-1148 demonstrated good prospects of their combined use.

Specificity of ethephon as a butyrylcholinesterase inhibitor and phosphorylating agent.

Butyrylcholinesterase (BChE) is inhibited by the plant growth regulator (2-chloroethyl)phosphonic acid (ethephon) as observed 25 years ago both in vitro and in vivo in rats and mice and more recently in subchronic studies at low doses with human subjects. The proposed mechanism is phosphorylation of the BChE active site at S198 by ethephon dianion. The present study tests this hypothesis directly using [(33)P]ethephon and recombinant BChE (rBChE) with single amino acid substitutions and further evaluates if BChE is the most sensitive esterase target in vitro and with mice in vivo. [(33)P]Ethephon labels purified rBChE but not enzymatically inactive diethylphosphoryl-rBChE (derivatized at S198 by preincubation with chlorpyrifos oxon) or several other esterases and proteins. Amino acid substitutions that greatly reduce rBChE sensitivity to ethephon are G117H and G117K in the oxyanion hole (which may interfere with hydrogen bonding between glycine-N-H and ethephon dianion) and A328F, A328W, and A328Y (perhaps by impeding access to the active site gorge). Other substitutions that do not affect sensitivity are D70N, D70K, D70G, and E197Q which are not directly involved in the catalytic triad. The effect of pH and buffer composition on inhibition supports the hypothesis that ethephon dianion is the actual phosphorylating agent without activation by divalent cations. Human plasma BChE in vitro and mouse plasma BChE in vitro and in vivo are more sensitive to ethephon than any other esterases detected by butyrylthiocholine or 1-naphthyl acetate hydrolysis in native-PAGE. All mouse liver esterases observed are less sensitive than plasma BChE to ethephon in vitro and in vivo. More than a dozen other esterases examined are 10-100-fold less sensitive than BChE to ethephon. Thus, BChE inhibition continues to be the most sensitive marker of ethephon exposure.

Effects of T-82, a New Quinoline Derivative, on Cholinesterase Activity and Extracellular Acetylcholine Concentration in Rat Brain

The effects of T-82 (2-[2-(1-benzylpiperidin-4-yl)ethyl]-2,3-dihydro-9-methoxy-1H-pyrrolo [3,4-b]quinolin-1-one hemifumarate), a new quinoline derivative, on acetylcholinesterase (AChE) activity and acetylcholine (ACh) release were compared with those of the well-known cholinesterase inhibitors tacrine and E2020. T-82, tacrine and E2020 all concentration-dependently inhibited AChE in rat brain homogenate (IC50 = 109.4, 84.2 and 11.8 nM, respectively). In addition, although tacrine strongly inhibited butyrylcholinesterase (BuChE), T-82 and E2020 showed only weak activity on BuChE in human plasma. In ex vivo experiments, intraperitoneal administration of T-82 at a dose of 30 mg/kg inhibited AChE activity in the hippocampus, frontal cortex and parietal cortex of rats. The effect of T-82 on the extracellular ACh concentration in rat brain was measured using in vivo microdialysis. T-82 at doses of 10 and 30 mg/kg, i.p. increased the extracellular ACh concentration in the hippocampus and striatum in a dose-dependent manner. These findings suggest that T-82 activates the central cholinergic system by selectively inhibiting AChE activity, while weakly affecting peripheral BuChE activity, and that T-82 increases the extracellular ACh concentration in the brain, which is followed by inhibited AChE activity.

Amitriptyline and procainamide inhibition of cocaine and cocaethylene degradation in human serum in vitro.

Amitriptyline (AMI) and procainamide (PA) have been reported to inhibit the activity of human plasma butyrylcholinesterase, an enzyme important in the metabolic degradation of cocaine (COC) and its ethyl analogue cocaethylene (CE). Because both AMI and PA may be used in the treatment of COC intoxication and abuse, the effect of high pharmacological concentrations of these compounds on the degradation of COC and CE in pooled human serum was studied. AMI (1.8 micromol/L) modestly inhibited the degradation of COC by 4.2% and of CE by 4.0%. PA (42.5 micromol/L) profoundly inhibited degradation of COC by 42.7% and of CE by 47.2%. In contrast, lithium carbonate (1 mmol/L, control) showed no inhibition of degradation of either COC or CE. These results suggest that AMI and PA may prolong the half-life of COC and CE in human serum.

The effect of some iodinated contrast media on human plasma butyrylcholinesterase activity.

The effect of three iodinated contrast media (CM) on human plasma butyrylcholinesterase (BuChE) activity was studied during the intravenous pyelography (i.v.P). BuChE is an enzyme that participates in the metabolism of acetylcholine and hydrolyzes food esters and drugs such as nondepolarizing muscle relaxants and local anesthetics. The three CM were: diatrizoate (an ionic substance) and iohexol and iopentol (two nonionic substances). Five minutes after the intravenous injection, iohexol reduces BuChE plasma activity by 8.5% but this effect disappears within 15 minutes. Diatrizoate and iopentol decrease BuChE plasma activity by 8% and 15.2%, respectively, 5 minutes after the injection and this effect remains stable for the next 15 minutes. The underlying mechanism is unknown. Plasma BuChE activity remains within the normal range in any case and no association with side effects can be made. Thus, according to the above findings, it is not possible to connect or exclude any relationship between BuChE plasma activity and CM-derived adverse reactions during the i.v.P.

New Method for Retrospective Detection of Exposure to Organophosphorus Anticholinesterases: Application to Alleged Sarin Victims of Japanese Terrorists

With regard to detection of exposure to anticholinesterase, the presently used methods have the disadvantage that they cannot detect either low-level exposures with certainty or the structure of the agent and the extent of poisoning. In principle, organophosphate-inhibited butyrylcholinesterase in human plasma is the most persistent and abundant source for biomonitoring of exposure to organophosphate anticholinesterases. Fluoride ions reactivate the inhibited enzyme readily at pH 4, converting the organophosphate moiety into the corresponding phosphofluoridate. Subsequent quantitation of the latter product provides a reliable, highly sensitive and retrospective method for detection of exposure to, or handling of, organophosphates such as nerve agents and organophosphorus pesticides. We applied the new procedure to serum samples from victims of the Tokyo subway attack by the AUM Shinriyko sect and from an earlier incident at Matsumoto. In serum of 10 of 11 victims from the Tokyo incident and of 2 of the 7 samples from the Matsumoto incident, reactivation with fluoride ions yielded sarin concentrations in the range of 0.2–4.1 ng/ml serum. Evidently, these victims had been exposed to an organophosphate with the structure PriO(CH3)P(O)X, presumably with X = F (sarin). Several applications of the new procedure to establish nerve agent and/or organophosphate (OP) pesticide exposure can be envisaged, e.g., (i) in biomonitoring of exposure for health surveillance of those handling organophosphates, (ii) in cases of alleged exposure to nerve agents and/or OP pesticides in armed conflict situations or terrorist attacks, (iii) in medical treatment of intoxication, and (iv) in forensic cases against suspected terrorists that may have handled anticholinesterases.

Cocaine Detoxification by Human Plasma Butyrylcholinesterase

The ability of human plasma butyrylcholinesterase (BChE) to detoxify cocainein vivowas evaluated. Intravenous administration of BChE, at doses sufficient to increase the plasma levels of the enzyme as much as 800-fold, produced no adverse effects on the cardiovascular, autonomic, or central nervous systems of rats. Most of the enzyme could be recovered in the plasma immediately after administration and remained active with a β-t12 of 21.6 ± 2.4 hr. Pretreatment of chloralose–urethane anesthetized rats with BChE, 0.1–7.8 mg/kg, decreased the hypertensive and arrhythmogenic effects produced by cocaine and increased the lethal dose of cocaine by three- to fourfold. Treatment of conscious rats with 1 and 10 mg/kg BChE decreased the incidence of seizures and deaths produced by a prior dose of cocaine (80 mg/kg, ip). These results suggest that BChE would provide a safe and highly efficacious treatment for cocaine intoxication.

Inhibition of human plasma and serum butyrylcholinesterase (EC 3.1.1.8) by alpha-chaconine and alpha-solanine.

The purpose of these experiments was to determine the reversibility of alpha-chaconine and alpha-solanine inhibition of human plasma butyrylcholinesterase (BuChE). For the substrate alpha-naphthylacetate, optimal assay conditions were 0.50 M sodium phosphate buffer and a substrate concentration of 3-5 x 10(-4) M. Dibucaine (1 x 10(-5) M) indicated the usual phenotype for all subjects; alpha-chaconine and alpha-solanine at 2.88 x 10(-6) M inhibited BuChE about 70 and 50%, respectively. One- and 24-hr incubations at 1 x 10(-5) M with alpha-chaconine, alpha-solanine, paraoxon, eserine, and ethanol yielded reversible inhibition with dilution except for paraoxon. Twenty-four-hour dialyses of incubations showed no inhibition except for paraoxon. PAGE enzyme activity gels of 1- and 24-hr incubations also showed no inhibition except for paraoxon. alpha-Chaconine and alpha-solanine are reversible inhibitors of human butyrylcholinesterase. At estimated tissue levels, alpha-chaconine, alpha-solanine, and solanidine inhibited BuChE 10-86%. In assays which combined alpha-chaconine, alpha-solanine, and solanidine, inhibition of BuChE was less than additive. No inhibition of albumin alpha-naphthylacetate esterase (an arylesterase) was noted with any inhibitor. The importance of these data to adverse toxicological effects of potato alkaloids is discussed.

Synthesis and Characterization of Photolabile Choline Precursors as Reversible Inhibitors of Cholinesterases: Release of Choline in the Microsecond Time Range

Three o-nitrobenzyl ether derivatives of choline (compounds A−C) were synthesized as photolabile cholinesterase inhibitors in order to study the mechanism of choline release from the enzyme active site. The key step of the synthesis was a simple and efficient Lewis acid-catalyzed opening of dioxolane rings derived from o-nitrobenzaldehyde and o-nitroacetophenone. Laser flash photolyses of compounds A−C were analyzed by UV spectroscopy, HPLC, and an enzymatic assay for choline. The quantum yields of photoconversion were determined, and the kinetics of choline release were analyzed by studying the decay of the transient aci-nitro intermediate at 405 nm. The observed rates varied considerably in function of both pH and the substituent at the α-benzylic position. Furthermore, we demonstrated that all three compounds possessed reversible inhibitory properties on both purified Torpedo acetylcholinesterase and purified human plasma butyrylcholinesterase. Compound A, O-[1-(2-nitrophenyl)ethyl]choline iodide, which displayed the highest quantum yield (0.27) and the most rapid photolysis rate (6.8 × 104 s-1 at pH 6.5), represents therefore an interesting tool for the study of the fast process of choline release from cholinesterases by time-resolved Laue crystallography.

Inhibition of Human Plasma and Serum Butyrylcholinesterase (EC 3.1.1.8) by α-Chaconine and α-Solanine

The purpose of these experiments was to determine the reversibility of alpha-chaconine and alpha-solanine inhibition of human plasma butyrylcholinesterase (BuChE). For the substrate alpha-naphthylacetate, optimal assay conditions were 0.50 M sodium phosphate buffer and a substrate concentration of 3-5 x 10(-4) M. Dibucaine (1 x 10(-5) M) indicated the usual phenotype for all subjects; alpha-chaconine and alpha-solanine at 2.88 x 10(-6) M inhibited BuChE about 70 and 50%, respectively. One- and 24-hr incubations at 1 x 10(-5) M with alpha-chaconine, alpha-solanine, paraoxon, eserine, and ethanol yielded reversible inhibition with dilution except for paraoxon. Twenty-four-hour dialyses of incubations showed no inhibition except for paraoxon. PAGE enzyme activity gels of 1- and 24-hr incubations also showed no inhibition except for paraoxon. alpha-Chaconine and alpha-solanine are reversible inhibitors of human butyrylcholinesterase. At estimated tissue levels, alpha-chaconine, alpha-solanine, and solanidine inhibited BuChE 10-86%. In assays which combined alpha-chaconine, alpha-solanine, and solanidine, inhibition of BuChE was less than additive. No inhibition of albumin alpha-naphthylacetate esterase (an arylesterase) was noted with any inhibitor. The importance of these data to adverse toxicological effects of potato alkaloids is discussed.

Amino Acid Residues Controlling Reactivation of Organophosphonyl Conjugates of Acetylcholinesterase by Mono- and Bisquaternary Oximes

Single and multiple site mutants of recombinant mouse acetylcholinesterase (rMoAChE) were inhibited with racemic 7-(methylethoxyphosphinyloxy)-1-methylquinolinium iodide (MEPQ) and the resulting mixture of two enantiomers, CH3PR,S(O)(OC2H5)-AChE(EMPR,S-AChE), were subjected to reactivation with 2-(hydroxyiminomethyl)-1-methylpyridinium methanesulfonate (P2S) and 1-(2'-hydroxyiminomethyl-1'-pyridinium)-3-(4"-carbamoyl-1"- pyridinium)-2-oxapropane dichloride (HI-6). Kinetic analysis of the reactivation profiles revealed biphasic behavior with an approximate 1:1 ratio of two presumed reactivatable enantiomeric components. Equilibrium dissociation and kinetic rate constants for reactivation of site-specific mutant enzymes were compared with those obtained for wild-type rMoAChE, tissue-derived Torpedo AChE and human plasma butyrylcholinesterase. Substitution of key amino acid residues at the entrance to the active-site gorge (Trp-286, Tyr-124, Tyr-72, and Asp-74) had a greater influence on the reactivation kinetics of the bisquaternary reactivator HI-6 compared with the monoquaternary reactivator P2S. Replacement of Phe-295 by Leu enhanced reactivation by HI-6 but not by P2S. Of residues forming the choline-binding subsite, the E202Q mutation had a dominant influence where reactivation by both oximes was decreased 16- to 33-fold. Residues Trp-86 and Tyr-337 in this subsite showed little involvement. These kinetic findings, together with energy minimization of the oxime complex with the phosphonylated enzyme, provide a model for differences in the reactivation potencies of P2S and HI-6. The two kinetic components of oxime reactivation of MEPQ-inhibited AChEs arise from the chirality of O-ethyl methylphosphonyl moieties conjugated with Ser-203 and may be attributable to the relative stability of the phosphonyl oxygen of the two enantiomers in the oxyanion hole.

Semi-empirical molecular orbital calculations on bambuterol, an inhibitor of human butyrylcholinesterase

The molecular geometry and conformations of bambuterol, a selective inhibitor of human plasma butyrylcholinesterase, has been determined using the semiempirical quantum chemical program mopac and the AM1 and PM3 methods. The conformational flexibility has also been investigated by CHARMm energy calculations. The electronic and electrostatic properties of bambuterol are discussed, because the molecular electrostatic maps may be useful in understanding the inhibitor-enzyme interaction and activity in molecular terms.

Pressure and Propylene Carbonate Denaturation of Native and "Aged" Phosphorylated Cholinesterase

Organophosphate-inhibited cholinesterases may become progressively refractory to reactivation by nucleophilic compounds due to the dealkylation of an alkoxy group from the covalently bound phosphonate ester. This process is termed "aging". It has been found that "aged" cholinesterases are more resistant to protein unfolding than the non-inhibited ones. The pressure-induced denaturation of the native (non-inhibited) and "aged" tetrameric form of human plasma butyrylcholinesterase was investigated in the presence and absence of a denaturing agent (propylene carbonate). This study was undertaken to determine whether the stability of aged butyrylcholinesterase varies with the structure with the structure of the alkyl/aryl (R2) group remaining attached to the phosphorus atom of the organophosphoryl moiety."Aged" organophosphoryl-cholinesterase conjugates were formed by reacting the enzyme with organophosphates: soman (trimethylpropylmethyl-phosphonofluoridate), sarin (isopropylmethyl-phosphonofluoridate), tabun (ethyl-N-dimethyl-phosphoramido-cyanidate), DFP (diisopropyl phosphorofluoridate) and PBPDC (pyrenebutyl-phosphorodichloridate). The dual effects of hydrostatic pressure up to 3·5 kbar and propylene carbonate up to 1·2 M were investigated in 10 Mm Tris·Hcl (Ph 7·0). Non-inhibited and aged enzymes were subjected to pressure/propylene carbonate for 12 hours at 20°C. The perturbing effects of this treatment upon cholinesterase structure were analyzed after pressure release by non-denaturing electrophoresis.Pressure and propylene carbonate induced progressive inactivation of the native enzyme. The loss in activity was correlated with irreversible denaturation of the tetramer and its subsequent aggregation. Similarly, pressure and propylene carbonate induced the formation of irreversibly denatured forms of aged butyrylcholinesterase. These denatured forms are partially unfolded enzyme conformations. The native enzyme was found to be more susceptible to denaturation than aged enzymes, with the exception of the PBPDC-aged enzyme. Methyl phosphono adducts, i.e. soman or sarin-aged conjugates were found to be the most stable aged species. Phenomenological analysis of the pressure/propylene carbonate denaturation maps at half-way of the denaturation process indicated that denaturation is a multistep process. The lowest stability of tabun-aged and DFP-aged conjugates suggested that the size, the orientation and the hydrophobicity of the remaining alkyl/aryl (R2) of the organophosphoryl moiety play a role in determining the overall stability of aged enzymes.Molecular modelling of aged adducts shed light on steric constraints exerted by the R2 chain on the bridge formed between the negatively charged P-O- of the dealkylated organophosphoryl moiety and protonated His438 N4. This allowed us to interpret the stability differences between aged conjugates in terms local conformation changes and hydration changes: the R2 chain may induce a local rearrangement of the three-dimensional conformation.

Ricinus communis agglutinin I reacting and non-reacting butyrylcholinesterase in human cerebrospinal fluid

Differences in glycosylation of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) in human brain, plasma and cerebrospinal fluid (CSF) have been investigated by means of their interaction with agarose-immobilized lectins. Most of the AChE in brain and CSF was associated to concanavalin A (Con A), Lens culinaris (LCA) and Triticum vulgaris (WGA) agglutinins, but little activity was adsorbed to Ricinus communis agglutinin I (RCAI). Brain, plasma and CSF BuChE was almost fully bound to Con A, LCA and WGA-agarose. Brain BuChE was unable to react with RCA (RCA-BuChE), the plasma enzyme was completely bound to the lectin (RCA+BuChE) and BuChE from CSF of normal children was partially fixed to RCA (RCA +/- BuChE). BuChE in CSF of children with meningitis fully reacts with the lectin. The data suggest that the proportion of RCA+BuChE in CSF of children with meningitis is increased, this enzyme probably coming from plasma.

The long-acting cholinesterase inhibitor heptyl-physostigmine attenuates the scopolamine-induced learning impairment of rats in a 14-unit T-maze

Heptyl-physostigmine (heptyl-Phy), a new carbamate derivative of physostigmine (Phy), has been assessed for potential clinical value by evaluating its in vitro activity against human erythrocyte acetylcholinesterase (AChE) and plasma butyrylcholinesterase (BChE), its duration of in vivo activity against rat plasma AChE, and its effects on attenuating a scopolamine-induced impairment in learning performance of young rats in a 14-unit T-maze. Heptyl-Phy demonstrated potent cholinesterase inhibition, with activity similar to that of Phy against AChE, IC 50 values 21.7±2.0 nM and 27.9±2.4 nM, respectively, and significantly greater than that of Phy against BChE, IC 50 values 5.0±0.1 nM and 16.0±2.9 nM, respectively. Heptyl-Phy achieved maximum AChE inhibition of 92.5% at 60 min and maintained a high and relatively constant inhibition for more than 8 h. For analysis of effects on learning performance, heptyl-Phy at 1.0, 1.5, 2.0 or 3.0 mg/kg, or vehicle was administered i.p. to 52 3-month-old male Fischer-344 rats 60 min prior to maze training. Thirty minutes prior to training, each animal received either 0.9% NaCl or scopolamine hydrochloride (0.75 mg/kg). Only a 2.0 mg/kg dose of heptyl-Phy significantly reduced the number of errors in scopolamine-treated rats. The other doses did not improve any aspect of maze performance. Although the therapeutic window of heptyl-Phy did not appear wide enough for clinical use, the longer duration of action of heptyl-Phy would appear beneficial.

Evidence for a single butyrylcholinesterase gene in individuals carrying the C 5 plasma cholinesterase variant (CHE2)

DNA of 3 unrelated individuals carrying the human plasma butyrylcholinesterase C 5 variant (CHE2) was isolated from white blood cells. Southern blot patterns of DNA restriction fragments probed with each of the 4 butyrylcholinesterase exons provided evidence that the production of C 5 is not directed by a second butyrylcholinesterase gene. This finding supports the suggestion that the C 5 variant is a hybrid enzyme resulting from the association of butyrylcholinesterase subunits with a non-cholinesterase protein.