Inhibitor Report for: Heptylphysostigmine

Cholinergic replacement therapy for Alzheimer's disease using existing cholinesterase inhibitors is compromised by short duration, meagre benefits restricted to subgroups of patients, and peripheral toxicity. Heptyl physostigmine is a lipophilic carbamate derivative of physostigmine. In rhesus monkeys, heptyl physostigmine (0.2-0.9 mg/kg i.m.) fully reversed a scopolamine-induced cognitive impairment. Following oral administration in squirrel monkeys, heptyl physostigmine (8 mg/kg) induced long-lasting hypothermia (greater than or equal to 4 h), a centrally-mediated cholinergic effect. Erythrocyte acetylcholinesterase activity was inhibited by 86% at the time of peak hypothermia (180 min). Clinical trials with heptyl physostigmine will enable a more rigorous evaluation of cholinomimetic therapy for dementia.

Heptastigmine is a new long acting cholinesterase inhibitor that affects behaviour in a number of cognitive tests in animals. We have studied its pharmacokinetics in rats: plasma kinetics were evaluated after single intravenous dose (2 mg/kg), intramuscular (4 mg/kg) and oral (4 and 8 mg/kg) administration. Tissue distribution (heart, liver, kidney and brain) was studied after single intramuscular (4 mg/kg) and oral (8 mg/kg) administration. Plasma and tissue kinetics were also investigated after repeated oral doses (8 mg/kg b.i.d. for 7 days). Heptastigmine levels in plasma and tissues were determined using an HPLC method with an electrochemical detector. After a single dose, heptastigmine remained for a long time in plasma (the terminal half-life was about 12 h), distributed widely in tissues (the volume of distribution was about 61) and brain concentrations were very high (4-22 times those found in plasma). After repeated oral doses, the drug levels increased in plasma and, to a lesser extent, in liver and kidney.

The aim of the study was to evaluate the effects of food on the rate and extent of eptastigmine absorption in healthy volunteers. METHODS: The study was carried out according to a double-blind, randomized, placebo-controlled, three-way cross-over design. On three separate occasions, six young subjects received 30 mg eptastigmine after a 12-h overnight fast (reference treatment), 30 mg eptastigmine 15 min after a standard breakfast (test treatment) and placebo 15 min after a standard breakfast (control treatment). Acetylcholinesterase activity in red blood cells was assayed 24 h after drug administration as a biological marker of eptastigmine plasma concentrations. RESULTS: Mean maximum acetylcholinesterase inhibition (Imax) was 39.9% after eptastigmine without food and 33.1% after eptastigmine with food. Maximum inhibitions occurred at 4.75 h and 4.88 h after eptastigmine without and with food, respectively. Areas under the curve of acetylcholinesterase per cent inhibition from 0 to 8 h after drug administration (AUC0-8) were 198% h after eptastigmine without food and 124% h after eptastigmine with food. Ninety per cent confidence intervals of test/reference ratios for AUC0-8 and Imax exceeded the 0.80 to 1.20 limits, thus indicating that the two eptastigmine treatments cannot be considered bioequivalent. Mild and transient adverse events were recorded in three subjects receiving eptastigmine without food, one subject receiving eptastigmine with food and one subject receiving placebo. CONCLUSIONS: The ingestion of food significantly reduces the bioavailability of eptastigmine estimated by the assay of red blood cell acetylcholinesterase activity.

The potency of a series of anticholinesterase (anti-ChE) agents and serotonin-related amines as inhibitors of the aryl acylamidase (AAA) activity associated with electric eel acetylcholinesterase (AChE) (EC 3.1.1.7) and horse serum butyrylcholinesterase (BCHE) (EC 3.1.1.8) was examined and compared with the potency of the same compounds as ChE inhibitors. Neostigmine, physostigmine, BW 284C51, (+/-)-huperzine A, E2020, tacrine, edrophonium and heptyl-physostigmine were, in that order, the most potent in inhibiting eel AChE-associated AAA activity, their inhibitor constant (Ki) values being in the range 0.02-0.37 microM. The rank order of the same compounds as AChE inhibitors basically paralleled that of AAA, although they were in general stronger on AChE (Ki = 0.001-0.05). The peripheral anionic site inhibitors propidium and gallamine were inactive on AChE-associated AAA. Serotonin and its derivatives were slightly stronger on AAA (Ki = 7.5-30 microM) than on AChE (Ki = 20-140 microM). Tacrine (IC50 = 0.03 microM), diisopropylfluorophosphate (IC50 = 0.04 microM), heptyl-physostigmine (IC50 = 0.11 microM), physostigmine (IC50 = 0.15 microM) and tetra-iso-propylpyrophosphoramide (iso-OMPA) (IC50 = 0.75 microM) were the most potent in inhibiting horse serum BCHE-associated AAA activity. Serotonin and related amines were very weak on BCHE-associated AAA activity. These results indicate that the inhibitory potencies of the active site anti-ChE agents on the AAA activity associated with eel AChE and horse serum BCHE are closely correlated with their action on the respective ChE. In addition, the efficacy of tacrine, E2020, heptyl-physostigmine and (+/-)-huperzine A in the treatment of Alzheimer's disease is unlikely to be related to the action of these drugs on ChE-associated AAA.

The effect of subchronic administration of the acetylcholinesterase (AChE) inhibitor heptastigmine (HEP 0.6 mg/kg s.c. daily for 15 days) was investigated on cortical extracellular acetylcholine (ACh) levels and on memory function in aged male rats (26 months old at the beginning of the experiments) using microdialysis and behavioural techniques. Twenty-four hours after the last treatment, cortical ACh levels were significantly higher in rats subchronically treated with HEP than in rats treated with saline and AChE activity was still inhibited in cortex, hippocampus and striatum. The injection of a challenge dose of HEP (0.6 mg/kg s.c.) 24 h after the last treatment produced a faster and a more sustained increase of ACh in the cortex of subchronically treated rats compared to those repeatedly injected with saline. However, the maximum increase of ACh levels after injection of the challenge was comparable in both groups. In an object recognition test in which the pretest and test phase were spaced by 45 days, HEP prevented the deterioration of spatial memory occurring during this period, but had no effect on non-spatial memory. The present results suggest that moderate inhibition of brain AChE is able to maintain high levels of cortical extracellular ACh in aged rats and that this increase matches facilitatory effect of HEP on spatial memory.

This review starts with an historical background of the pharmacological development of tacrine almost fifty years ago (1949). Tacrine is the first drug to be tested, clinically, on a large scale and to be registered (1993) for treatment of Alzheimer's disease. For the first time, clinical results of four second generation cholinesterase inhibitors (ChEI) (donepezil, ENA 713, eptastigmine and metrifonate) are reviewed and compared with other ChEI such as tacrine, physostigmine and galanthamine. Data based on more than 6000 patients show that second generation drugs are well tolerated and show evidence of clinical efficacy. Differences are mainly due to frequency of side effects, number of drop outs and percentage of improved patients. These results also demonstrate the presence of clinical efficacy for all ChEI tested so far. Clinical mechanism of action, levels of efficacy and differences among various ChEI are discussed. Future potential indications are suggested. The present data indicate that optimization of effects prolongation and maintenance of clinical gains will depend on further knowledge of the compounds pharmacodynamic properties.

Eptastigmine is a new acetylcholinesterase (AChE) inhibitor currently under development for the symptomatic treatment of Alzheimer disease. This study was conducted to establish the maximum tolerated dose and the pharmacodynamics of eptastigmine in nine healthy elderly volunteers. Subjects received single oral doses of 8 mg, 20 mg, 32 mg, and 40 mg eptastigmine and placebo according to a double-blind, randomized, rising-dose, five-way crossover design. Adverse events, blood pressure, heart rate, body temperature, forced expiratory volume, salivary flow, and pupilar activity were closely monitored during treatment. Pharmacodynamic activity of eptastigmine was evaluated with an assay of AChE activity in red blood cells. Eptastigmine doses of 8 mg, 20 mg, and 32 mg were well tolerated. Two of four subjects receiving the 40-mg dose developed profound AChE inhibition (58-59%) and reported severe adverse events (nausea, vomiting, syncope, and bradycardia), precluding further administration in the remaining subjects. Eptastigmine administration produced a weak effect on supine heart rate, body temperature, and pupil diameter. There were no effects on blood pressure, forced expiratory volume, salivary flow, and near point of focus. Acetylcholinesterase activity was inhibited in a dose-related fashion according to a sigmoidal (logistic) function. The mean (+/- SEM) maximum inhibition of AChE activity (Imax) was 14.5+/-3.3%, 20.4+/-2.3%, 28.7+/-2.9%, 45.2+/-1.3% and 53.6+/-2.9% after placebo, 8 mg, 20 mg, 32 mg, and 40 mg of eptastigmine, respectively. The theoretical maximum response (Emax) was 72.9%, and the dose that produced half of the maximum response (ED50) was 29.5 mg. At 24 hours, residual AChE inhibition ranged from 9% to 15%, with a half-life of recovery of the enzyme of approximately 10 hours. The maximum tolerated dose of eptastigmine after single-dose oral administration in healthy elderly subjects is 32 mg. Single oral doses of eptastigmine produce sustained, dose-related inhibition of AChE activity. Adverse events are related to the degree of AChE inhibition.

The ability of central cholinesterase inhibition to improve cerebral blood flow in the ischemic brain was tested in Sprague-Dawley rats with tandem occlusion of left middle cerebral and common carotid arteries. Cerebral blood flow was measured with lodo- 14C-antipyrine autoradiography in 170 regions of cerebral cortex. The regional distribution of blood flow was characterized in normal animals by cerebral blood flow maxima in the temporal regions. After 2 h ischemia, minimum cerebral blood flow values were found in the lateral frontal and parietal areas on the left hemisphere, and a new maximum was found in the right hemisphere in an area approximately symmetrical to the ischemic focus. Heptyl-physostigmine (eptastigmine), a carbamate cholinesterase inhibitor with prolonged time of action improved cerebral blood flow in most regions, with the exception of the ischemic core. The drug also enhanced the ischemia-induced rostral shift of cerebral blood flow maxima in the right hemisphere. The effects of eptastigmine were more marked 24 h after ischemia. Discriminant analysis showed that data from only 22 regions was sufficient to achieve 100% accuracy in classifying all cases into the various experimental conditions. The redistribution of cerebral blood flow to the sensorimotor area of the right hemisphere of animals with cerebral ischemia, a phenomenon possibly related to recovery of function, was also enhanced by eptastigmine.

The potential of heptylphysostigmine tartrate (pyrrolo [2,3b] indol-5-ol, 3,3a,8,8a-hexahydro-1,3a,8-trimethylheptylcarbamate [ester, (3aS-cis)]) (MF201), a new second-generation cholinesterase inhibitor, to antagonize scopolamine-induced amnesia in rats was assessed in an 8-arm radial maze. Upon completing the training session, the rats were orally administered increasing doses of MF201 (2, 3, 4, 6 and 8 mg/kg) 60 min prior to a s.c. injection of scopolamine (0.25 mg/kg). 9-Amino-1,2,3,4-tetrahydroamino-acridine hydrochloride hydrate (tacrine) (0.25, 0.37, 0.5, 1 and 2 mg/kg), 1-benzil-4-[(5,6-dimethoxy-1-indanon)-2-yl]-methyl piperidine (E2020) (0.125, 0.18, 0.25 and 0.5 mg/kg) and physostigmine (0.15, 0.25, 0.5 and 1 mg/kg) were orally administered and rats were tested in the same task. As previously described, scopolamine induced an impairment in radial maze performance, measured in terms of total number of errors, total time taken to complete the task and the percentage of amnesic animals. The reversal of scopolamine-induced impairment was characterized by the presence of an inverted U-shaped dose-response curve. A significant antagonistic effect was achieved with a dose (mg/kg) of 0.25 for E2020, 0.5 for tacrine and physostigmine and 3, 4 and 6 for MF201, the latter manifesting a broader spectrum of activity (3-6 mg/kg). While the maximal active doses restored the scopolamine-induced modified pattern of arm entry, they were ineffective in reducing hypermotility, suggesting the drugs have a specific effect on cognitive function.

Enhancing the availability of endogenous acetylcholine by inhibition of cholinesterase with physostigmine, eptastigmine or soman at sub-toxic doses increases cerebral blood flow (CBF) and the response of this variable to changes in PaCO2. These effects are not correlated with metabolic activation, suggesting that the function of the cholinergic vasodilation is not merely to supply metabolic substrates. Since choline (Ch) can exchange between blood and the brain extracellular milieu the stage is set for possible feedback interactions between ACh synthesis and CBF. A negative feedback of CBF on ACh synthesis under conditions of a negative arteriovenous (A-V) difference for Ch across cerebral capillaries may contribute to stabilize GBF in ischemia. Eptastigmine and physostigmine significantly improve perfusion in experimental models of focal cerebral ischemia and traumatic brain injury respectively. During the short periods of time in which the A-V difference for Ch across the brain is positive, a positive feedback between cerebral free Ch and CBF may enhance the ability of the brain to recover Ch from the circulation for synthesis of membrane phospholipids. A loss of cholinergic cerebrovascular control may thus impair the survival of all cells within the CNS and contribute to the pathophysiology of dementia. Perhaps the view that the loss of cholinergic cells is the end point of Alzheimer's dementia could be modified to state that a cholinergic deficit may be the starting point of a decline in cerebral phospholipid turnover and cell membrane renewal that could lead to a generalized deterioration of cerebral function.

A novel therapy against organophosphate exposure, the combination of a carbamate eptastigmine and an organophosphate hydrolase (phosphotriesterase) was studied in mice against diisopropylfluorophosphate (DFP) (1.75 mg/kg) exposure. Mice received eptastigmine (0.9 mg/kg; iv) 10 min prior to the ip injection of DFP. Phosphotriesterase (83 U/g body weight) was injected iv 10 min after DFP. Eptastigmine (1.5 mg/kg; iv) inhibited the acetylcholinesterase activities in brain and erythrocytes for a longer time than physostigmine. Eptastigmine caused only minor changes in the behavior and activity of the animals, whereas physostigmine clearly reduced their activity for about 30 min. The eptastigmine pretreatment clearly supplemented the protective effect of phosphotriesterase against DFP: the plasma butyrylcholinesterase activity was doubled and the activity recovered faster than in animals treated with phosphotriesterase alone. In lung, butyrylcholinesterase activity was initially lower after eptastigmine-phosphotriesterase than phosphotriesterase treatment alone. However, the activity returned 24 hr later to normal in eptastigmine-phosphotriesterase-treated groups. With phosphotriesterase only, it recovered only to 75% of the control level. Presumably eptastigmine, by preventing the binding of DFP to cholinesterases, caused an elevation of free DFP levels in body fluids and promoted phosphotriesterase hydrolysis of DFP.

The protective action of i.v. administered eptastigmine, an organophosphate hydrolase (phosphotriesterase), or pralidoxime-2-chloride (2-PAM) and their combination in acute diisopropylfluorophosphate (DFP) intoxication were evaluated in mice. The mice received the physostigmine derivative, eptastigmine (0.9 mg/kg body wt, i.v.), 10 min prior to the i.p. injection of DFP (1.8 mg/kg body wt). Phosphotriesterase (66 micromol/min x ml/g and 6 microg/g body wt) or 2-PAM (30 mg/kg body wt) were given i.v. 30 min after DFP. The animals also received atropine sc (37.5 mg/kg body wt) immediately after DFP. The cholinesterase (ChE) activities were not protected or reactivated by 2-PAM alone. The ChE activities in brain and plasma were protected by phosphotriesterase. Eptastigmine alone assisted the recovery of the brain ChE activities. Also the combination of eptastigmine-phosphotriesterase protected the brain enzymes. It did not, however, provide any additional protection compared with phosphotriesterase-treatment on its own. In brain, the combination of eptastigmine with 2-PAM resulted in partly restored enzyme activities 24 hr after DFP exposure. In plasma, eptastigmine did not prevent the inhibition of ChE by DFP. However, when it was combined with phosphotriesterase, it significantly promoted the recovery of plasma ChE activity. In lung and in erythrocytes, the various combinations of antidotes caused only minor changes in the ChE activities.

Eptastigmine is a long-lasting acetyl-cholinesterase inhibitor, currently being developed for the symptomatic treatment of Alzheimer's disease. In the present study, we investigated the relationship between pharmacokinetics and pharmacodynamics of eptastigmine in young healthy volunteers. Eight male subjects received single oral doses of 10, 20, and 30 mg of eptastigmine and placebo according to a double-blind, randomized, crossover design. Blood was collected before and 0.5, 1, 1.5, 2, 3, 4, 6, and 24 hours after drug administration. Cholinesterase activity was measured using a potentiometric method in both plasma (butyryl-cholinesterase) and in red blood cells (acetyl-cholinesterase). Eptastigmine plasma levels were measured by a very sensitive high-performance liquid chromatography method (limit of quantitation 0.2 ng/mL). Eptastigmine plasma concentrations increased proportionally with the dose (mean +/- SEM AUC0-24 was 0.74 +/- 0.58, 3.61 +/- 1.15, and 6.25 +/- 1.51 ng.h/mL with 10, 20, and 30 mg, respectively) and were undetectable at 24 hours. The inhibition of acetyl-cholinesterase was dose-dependent (peak inhibition was 15 +/- 2%, 30 +/- 4%, and 36 +/- 6% with 10, 20, and 30 mg, respectively) and long-lasting, with a residual inhibition of 8 to 11% at 24 hours. Acetyl-cholinesterase inhibition and drug plasma levels were related over time with a counterclockwise hysteresis curve, suggesting the formation of active metabolites and/or a slow association to and dissociation from the enzyme in red blood cells. Butyryl-cholinesterase inhibition was weak and not dose-dependent (peak inhibition was 12 +/- 4%, 13 +/- 3%, and 12 +/- 2% with 10, 20, and 30 mg, respectively). The drug was well tolerated by all subjects.

Rapid and reliable measurement of acetylcholinesterase (AChE) activity is of crucial importance to the pharmacodynamic monitoring of anticholinesterase drugs. A new assay has been developed to measure AChE from 10 microliter samples of capillary blood. AChE activity was calculated from the change in pH of the reaction medium caused by the hydrolysis of acetylcholine and measured with a highly sensitive differential pH apparatus (CL-10, Eurochem, Rome, Italy). Interference by butyrylcholinesterase was eliminated by a specific inhibitor, quinidine sulfate. The assay lasts 1 min. The coefficient of variation (CV) for replicated measurements was 2.8% (3267 U/L, n = 33). Linearity ranged from 0 to 10,000 U/L. The correlation coefficient between the new technique and Ellman's colorimetric method on washed erythrocytes was r = 0.987 (y = 1.299x - 63, n = 29). The correlation coefficient between assays on capillary and venous samples was r = 0.979 (y = 0.974x + 174, n = 47). A cross-laboratory validation study was performed in 10 centers using glycerol-stabilized hemolysates with normal and reduced AChE activity. Samples were assayed in triplicate. The within- and between-laboratory CVs for samples with normal AChE activity (6,018 U/L) were 2.2 and 8.1%, respectively. The new method was applied to a double-blind, placebo-controlled multicenter study of eptastigmine in Alzheimer patients and proved to be a simple, noninvasive, rapid, and reliable method for pharmacodynamic monitoring of this drug.

Pharmacological treatment of patients with Alzheimer's disease is becoming more important, as evidenced by the number of drugs being developed in different countries. It has been shown in the majority of clinical trials that cholinesterase inhibitors, such as tacrine (tetrahydroaminoacridine), are able to induce beneficial effects in cognition and memory. Tacrine, like most of the other oral antidementia agents, is rapidly absorbed from the gastrointestinal tract. It is excreted mainly through the kidney, with a terminal elimination half-life of about 3 hours. Tacrine has nonlinear pharmacokinetics and there are large interindividual differences in pharmacokinetic parameters after oral, intravenous and rectal administration. A positive relationship between cognitive changes and plasma tacrine concentrations has been recently described. Similarly, velnacrine exhibits evidence of nonlinearity in some pharmacokinetic parameters, but renal excretion is a minor route of elimination for this drug. Pharmacokinetic data pertaining to eptastigmine, a third cholinesterase inhibitor, is more limited. However, the drug is rapidly distributed to the tissues after oral administration and readily enters the central nervous system, where it can be expected to effectively inhibit acetylcholinesterase in the brain for a prolonged period. Pharmacokinetic data for the nootropic agents are more limited. However, of the 3 agents reviewed only pramiracetam penetrates the central nervous system (CNS) poorly. Indeed, oxiracetam crosses the blood-brain barrier and persists for longer in the CNS than in the serum. Selegiline (deprenyl), a neuroprotective agent, is readily absorbed from gastrointestinal tract. It is metabolised mainly in the liver, and to a minimal extent in the lung or kidneys. The steady-state concentrations of metabolites in the cerebrospinal fluid (CSF) and serum are very similar, reflecting their easy penetration into the CNS. Idebenone, another neuroprotective agent, likewise is rapidly absorbed and achieves peak concentrations in the brain comparable to those in plasma. Similarly, CSF concentrations of metabolites of ST 200 (acetyl-L-carnitine) parallel those in plasma, suggesting that they easily cross the blood-brain-barrier. Gangliosides (GM1) can be given intramuscularly or subcutaneously, but the latter route of administration provides a concentration 50% higher both in the serum and the ganglioside fraction. However, because of its longer elimination, the intramuscular route is the best form of administration when the brain is the target organ for the treatment. Absorption of nimodipine is quite rapid. The pharmacokinetics of nimodipine during multiple-dose treatment have not been studied extensively; however, the drug does not appear to accumulate during repeated administration of standard doses.

A placebo-controlled multiple dose study was conducted to evaluate the safety, tolerability, and pharmacodynamics of multiple dose levels of eptastigmine in 25 patients with probable Alzheimer's disease (AD). Twenty patients (12 M, 8 F; mean age 74, range 57-84) were randomized to receive 12mg (N = 3), 20mg (N = 6), 28mg (N = 6) or placebo (N = 5) tid on a double-blind basis for 14 days, followed by seven days of single blind placebo, in successively rising dose groups. All patients completed the study without intolerable or severe adverse events. All doses significantly (p < 0.001) reduced peak and trough RBC cholinesterase (AChE) activity as compared to baseline. Percent inhibition for Day 14 peak and trough RBC AChE peak and trough values, respectively, appeared proportional to dose: 18% and 21% (12mg); 36% and 35% (20mg); 40% and 44% (28mg). In order to determine the maximum tolerated dose of eptastigmine, an additional single-blind study was performed in five patients (2 M, 3 F; mean age 78, range 72-80) utilizing a rising dose schedule of eptastigmine (N = 4) or placebo (N = 1), starting with the previously tolerated 28mg tid dose and increasing by 4mg tid up to a potential maximum of 56mg tid. Dose-limiting adverse events occurred requiring discontinuation of medication in one patient at 48mg tid and two patients at 52mg tid; RBC AChE inhibition was proportional to dose, with peak values up to 70% inhibition at 48mg tid. The maximum tolerated dose of 48mg tid was identified as a basis for potential Phase II multicenter efficacy trials.

The time course and dose dependence of acetylcholinesterase inhibition in three regions of brain were measured for the long-acting physostigmine derivative, heptylphysostigmine (HP). Behavioral studies were performed on rats lesioned with ibotenic acid in the nucleus basalis magnocellularis (nBM) using doses of HP that inhibit cholinesterase activity 20%, 40%, and 60% 2 h after injection. Spatially-cued learning and memory were tested in the water maze. Lesioned animals that received higher doses of HP showed a trend towards improvement in the acquisition of this task, but this was not statistically significant. Swimming speed was reduced in the group receiving the highest dose of HP in comparison with a lower dose. The acoustic startle response was diminished in all groups given HP in comparison with both lesioned and sham-lesioned saline-injected controls. Open field activity was slightly enhanced by the presence of the nBM lesion. HP reduced the hyperactivity in a dose-dependent manner. Deficiencies in limb strength or coordination were not detected. These results suggest that HP may decrease spontaneous, stressed, or reflexive activity, although an effect on the spatial learning deficit produced by an ibotenic acid lesion of the nucleus basalis was not detected.

The synthesis of 2'-heptylcarbamoyloxy-2-methyl-6,7-benzomorphan is described. The compound is structurally related to the cholinesterase inhibitor heptylphysostigmine (MF 201) because the angular methyl group of the esoroline nucleus has been changed into a bridging carbon and the anilinic nitrogen has been replaced by a methylene group. This compound proved to be a potent cholinesterase in vitro inhibitor.

A microdialysis technique was used to investigate the effect of physostigmine (PHY) and heptylphysostigmine (HEP), administered systemically or locally, on the extracellular levels of acetyl-choline (ACh), norepinephrine, dopamine and 5-hydroxytryptamine in the cerebral cortex of the rat. Levels of these neurotransmitters in dialysates were assayed simultaneously with two different high pressure liquid chromatography systems. No cholinesterase inhibitor was added into the probe to increase detection of ACh after systemic administration. Cholinesterase inhibition and its relation to ACh levels were also studied. Systemic administration of two doses of cholinesterase inhibitor [PHY (30 and 300 micrograms/kg) and HEP (2 and 5 mg/kg)] produced a dose-dependent increase in ACh levels. Local perfusion of these drugs through the probe elicited a strong increase in extracellular ACh. HEP produced a longer lasting inhibition of cholinesterase and a more prolonged elevation of ACh in cerebral cortex than PHY. After systemic administration of PHY (both doses), we observed a significant increase of norepinephrine levels. This effect was weaker after HEP. Local administration through the probe did not modify norepinephrine concentration. Dopamine levels were also increased after systemic administration. ONly HEP perfused into the probe elicited a significant increase in extracellular dopamine. Systemic or local administration did not modify 5-hydroxytryptamine levels. These observations suggest a more favorable pharmacological profile for HEP as a potential drug for Alzheimer disease, as compared to PHY.

In recent years muscarinic receptor agonists and cholinesterase inhibitors have been developed for the treatment of Alzheimer's disease. We have evaluated examples from both classes of compounds in rodent tests of reference and working memory, as well as tests that are sensitive to the side-effects of these compounds. Thus, three selective muscarinic receptor partial agonists L-689,660, (M1/M3), AF102B (M1/M3) and L-687,306 (M1) and two cholinesterase inhibitors, E2020 and eptastigmine, were compared in a mouse tail-flick (TF) test, a rat response sensitivity (RS) test, in rat tests of reference memory, passive avoidance (PA) or conditioned suppression of drinking (CSD), and working memory (delayed-matching-to-position, DMTP). In the TF test, all of the compounds tested, with the exception of L-687,306, (1.0-30.0 mg/kg) dose-dependently induced antinociception of which L-689,660 was the most potent (minimum effective dose (MED) = 0.03 mg/kg). In the RS test, all of the compounds, but again with the exception of L-687,306, (1.0-30.0 mg/kg), dose-dependently reduced response rates, of which L-689,660 was again the most potent (MED = 0.1 mg/kg). In the reference memory test, all the compounds reversed the effects of a scopolamine-induced deficit with L-687,306 being the most potent (MED = 0.01 mg/kg). By contrast, in the DMTP test, although both the cholinesterase inhibitors and L-687,306 reversed the effects of scopolamine-induced deficit, L-689,660 and AF102B were without effects. These results suggest that cholinesterase inhibitors and low efficacy M1 selective muscarinic receptor agonists can reverse the effects of a scopolamine-induced deficit in animal tests of reference and working memory at doses that do not induce the side-effects usually associated with cholinomimetics.

A microdialysis technique was used to sample acetylcholine (ACh) from the cerebral cortex of conscious rats. We thus investigated the effects of systemically administered cholinesterase inhibitors (ChEI) such as physostigmine (300 micrograms/kg), heptylphysostigmine (5 mg/kg) and tetrahydroaminoacridine (tacrine, 5 mg/kg) on extracellular ACh levels. Baseline quantities of extracellular ACh could be detected, even in the absence of ChEI. Acetylcholine levels increased to 1100% over baseline within 30 min of physostigmine administration and returned to control levels after 1.25 hr. Heptylphysostigmine elicited a maximal increase of 1000% within 1.5 hr, and the effect persisted up to 9.5 hr. A 500% increase was observed 1.5 hr after tacrine administration, and ACh returned to control levels after 4 hr. Although the ACh effects observed in this study correlated with previously determined levels of acetylcholinesterase (AChE) inhibition, we conclude that measures of cortical AChE activity alone are not sufficient to predict extracellular ACh levels following systemic ChEI administration.

Transdermal delivery of cholinesterase inhibitors (ChEI) for treatment of dementia would have advantages associated with continuous dosing and enhanced compliance, but feasibility depends on achieving desired levels of central nervous system enzyme inhibition. We developed a patch technique for assessing delivery of ChEI in rats and examined two organophosphate compounds, metrifonate and DDVP, and a carbamate, heptylphysostigmine, for production of peripheral and central nervous system ChE inhibition at target levels. With DDVP, a log-dose/percent brain AChE inhibition was obtained over a range of 10-65% inhibition within a 10-fold concentration of inhibitor in the patch. Brain cholinesterase was inhibited up to seven days after a 24-h patch application. Long-term inhibition was greater than that attained after intramuscular injection, but without the rapid initial inhibition peak seen with the latter route. In contrast to DDVP, sustained high levels of brain enzyme inhibition could not be produced by transdermal delivery of metrifonate or heptylphysostigmine. Apparently DDVP has features, i.e., liquid state in pure form and high inhibitor potency, which make it particularly suitable for patch administration.

The cerebrovascular and metabolic effects of the novel cholinesterase inhibitor eptastigmine were tested in conscious rats. The drug was administered by single intravenous injection, and blood flow or glucose utilization were assessed in 38 brain regions by quantitative autoradiographic techniques. A dose-dependent increase in regional cerebral blood flow (rCBF) was obtained for i.v. doses ranging from 0.5 to 3 mg kg-1. Forty minutes after the dose of 1.5 mg kg-1, average rCBF of the 38 regions studied was (mean +/- SD) 2.62 +/- 0.62 ml g-1 min-1, a value significantly higher than that of saline-injected controls (1.46 +/- 0.26; p < 0.005). In contrast, a similar dose of eptastigmine did not significantly alter regional cerebral glucose utilization (rCGU) (0.90 +/- 0.21 mumol g-1 min-1) when compared with saline-injected controls (0.99 +/- 0.08 mumol g-1 min-1). A linear correlation between rCBF and rCGU was observed both in saline (r = 0.871) and eptastigmine (r = 0.873)-injected animals but the slope of the regression line of rCBF on rCGU was significantly higher (p < 0.01) in the eptastigmine group (2.863 +/- 0.266) than in the controls that received saline (1.00 +/- 0.094). The cerebral vasodilatation induced by eptastigmine peaked at 40 min after drug administration. No toxic signs were observed at the doses used. Mean arterial blood pressure decreased after 0.5 mg kg-1 (control = 109.3 +/- 10.56 mm Hg; eptastigmine = 96.6 +/- 8.10 mm Hg) but did not differ from control at the higher doses. It is concluded that eptastigmine induces a long-lasting increase in rCBF and a significant enhancement of the rCBF:rCGU ratio in most regions. The results suggest an important role of endogenous acetylcholine in the control of cerebral perfusion.

A sensitive (50 pg/ml) method for the determination of heptylphysostigmine in human plasma is described. The procedure is based on liquid-liquid extraction of the drug from buffered plasma, and analysis of the concentrated organic extract using high-performance liquid chromatography on a silica column, under normal-phase chromatographic conditions, with fluorescence detection. Physostigmine was used as an internal standard. The assay has been fully validated in the concentration range 50-2000 pg/ml and utilized for the analysis of clinical samples from subjects dosed with heptylphysostigmine.

This study sought to determine the effect of heptylphysostigmine (H-PHY), a reversible cholinesterase (ChE) inhibitor with greater lipophilicity and longer duration of action than physostigmine, on resting and basal forebrain (BF)-elicited increases in cortical cerebral blood flow (CBF). Laser-doppler flowmetry (LDF) was used to monitor changes in frontal cortical microvascular perfusion in urethane anesthetized rats. Responses were measured before, early after, and 1 hr following H-PHY, 3 mg/kg, i.m. Electrical stimulation (100 microA) of the BF elicited up to 220% increases in CBF at 50 Hz, an effect that was graded with frequency. At 15 min following H-PHY (3 mg/kg) resting cortical CBF was unchanged, whereas BF-elicited increases were potentiated 47% at 50 Hz. At 1 hour, resting cortical CBF remained unchanged, and the BF-elicited responses were remarkably potentiated by 354% at 10 Hz and 67% at 50 Hz. Acetylcholinesterase (AChE) activity measured in the tissue directly beneath the LDF probe was decreased by 84% at a time when these CBF responses were enhanced. These data suggest that H-PHY substantially enhances the regulation of cortical CBF by the BF, an effect that may be linked to inhibition of cortical AChE activity. This enhancement of cortical CBF may contribute to the efficacy of H-PHY as a treatment for Alzheimer's disease.

Heptylphysostigmine (HPTL), a derivative of the AChE inhibitor physostigmine (PHY), is under investigation as a therapeutic agent in Alzheimer's disease. HPTL is active against human RBC AChE both in vitro and in vivo. Activity of HPTL against human brain has not been documented. We have developed an in vitro assay system using particulate membrane fractions which permits comparison of inhibition and recovery kinetics of human RBC (primarily globular dimer) and brain (primarily globular tetramer) membrane-bound forms. Under these conditions the HPTLIC50 is similar for the two forms. RBC AChE inhibition spontaneously reverses in 24 h, as occurs in vivo. In striking contrast, activity of inhibited brain enzyme does not recover on overnight incubation. DDVP-induced inhibition, but not HPTL inhibition, can be reversed by the oxime 2-PAM. Some recovery of HPTL inhibition, but not to the level seen with RBC AChE, occurs on addition of heat-stable fractions from serum or CSF. Brain enzyme recovers rapidly from PHY in this system. Responses of brain and RBC AChE to HPTL indicate that these forms are functionally as well as structurally distinct. Since brain inhibition apparently does not spontaneously reverse like RBC inhibition, peripheral measurements of patient responses should be assessed with caution during treatment with HPTL.

The effect of eight different acetylcholinesterase inhibitors (AChEIs) on the activity of acetylcholinesterase (AChE) molecular forms was investigated. Aqueous-soluble and detergent-soluble AChE molecular forms were separated from rat brain homogenate by sucrose density sedimentation. The bulk of soluble AChE corresponds to globular tetrameric (G4), and monomeric (G1) forms. Heptylphysostigmine (HEP) and diisopropylfluorophosphate were more selective for the G1 than for the G4 form in aqueous-soluble extract. Neostigmine showed slightly more selectivity for the G1 form both in aqueous- and detergent-soluble extracts. Other drugs such as physostigmine, echothiophate, BW284C51, tetrahydroaminoacridine, and metrifonate inhibited both aqueous- and detergent-soluble AChE molecular forms with similar potency. Inhibition of aqueous-soluble AChE by HEP was highly competitive with Triton X-100 in a gradient, indicating that HEP may bind to a detergent-sensitive non-catalytic site of AChE. These results suggest a differential sensitivity among AChE molecular forms to inhibition by drugs through an allosteric mechanism. The application of these properties in developing AChEIs for treatment of Alzheimer disease is considered.

Cholinergic replacement therapy for Alzheimer's disease using existing cholinesterase inhibitors is compromised by short duration, meagre benefits restricted to subgroups of patients, and peripheral toxicity. Heptyl physostigmine is a lipophilic carbamate derivative of physostigmine. In rhesus monkeys, heptyl physostigmine (0.2-0.9 mg/kg i.m.) fully reversed a scopolamine-induced cognitive impairment. Following oral administration in squirrel monkeys, heptyl physostigmine (8 mg/kg) induced long-lasting hypothermia (greater than or equal to 4 h), a centrally-mediated cholinergic effect. Erythrocyte acetylcholinesterase activity was inhibited by 86% at the time of peak hypothermia (180 min). Clinical trials with heptyl physostigmine will enable a more rigorous evaluation of cholinomimetic therapy for dementia.

Heptastigmine is a new long acting cholinesterase inhibitor that affects behaviour in a number of cognitive tests in animals. We have studied its pharmacokinetics in rats: plasma kinetics were evaluated after single intravenous dose (2 mg/kg), intramuscular (4 mg/kg) and oral (4 and 8 mg/kg) administration. Tissue distribution (heart, liver, kidney and brain) was studied after single intramuscular (4 mg/kg) and oral (8 mg/kg) administration. Plasma and tissue kinetics were also investigated after repeated oral doses (8 mg/kg b.i.d. for 7 days). Heptastigmine levels in plasma and tissues were determined using an HPLC method with an electrochemical detector. After a single dose, heptastigmine remained for a long time in plasma (the terminal half-life was about 12 h), distributed widely in tissues (the volume of distribution was about 61) and brain concentrations were very high (4-22 times those found in plasma). After repeated oral doses, the drug levels increased in plasma and, to a lesser extent, in liver and kidney.