Effects of metrifonate on cognitive decline in Alzheimer disease: a double-blind, placebo-controlled, 6-month study.

Forty-seven patients with probable Alzheimer disease (AD) completed a 6-month double-blind study to compare metrifonate with placebo. The Alzheimer Disease Assessment Scale cognitive subscale score of the metrifonate group treated to a 50-70% inhibition of red blood cell acetylcholinesterase activity differed significantly from the placebo group score by 1.8 points (p < 0.03) due to a deterioration in cognitive performance in the placebo group (p < 0.01). Statistically significant deterioration also occurred in the Mini-Mental State Examination scores (p < 0.01) in the placebo-treated group. Adverse effects were uncommon and did not require adjustment of the dose of metrifonate or discontinuation of treatment. These findings extend our previous report of a favorable effect of metrifonate on cognitive symptoms in AD by showing clinical, not only statistical, significance.

Double-blind, placebo-controlled study of metrifonate, an acetylcholinesterase inhibitor, for Alzheimer disease.

Fifty patients with probable Alzheimer disease (AD) completed a 3-month double-blind study to compare metrifonate to placebo. We dosed metrifonate to achieve a 40-60% inhibition of red blood cell acetylcholinesterase activity. The Alzheimer Disease Assessment Scale cognitive subscale score (ADAS-C) served as the primary outcome measure. At the completion of 3 months of treatment, the metrifonate group ADAS-C score differed significantly from the placebo group score by 2.6 points (p < 0.01). A 0.75-point trend toward improvement occurred during treatment in the ADAS cognitive performance of the metrifonate group (p = 0.15), and a 1.10-point deterioration in cognitive performance was found in the placebo group (p < 0.02). On the Global Improvement Scale (GIS), the two groups differed significantly on their changes from baseline to treatment phase (p < 0.02). Significant deterioration occurred in GIS scores (p < 0.01) and in Mini Mental State Examination (MMSE) scores (p < 0.03) in the placebo-treated group. Adverse effects were uncommon and did not require adjustment of the dose of metrifonate or discontinuation of treatment. We achieved a mean of 52.3% decrease in red blood cell acetylcholinesterase activity. During up to 18 months of subsequent open metrifonate treatment of patients, we found a deterioration of 1.68 points per year in MMSE performance. These findings support further study of the effects of metrifonate on deterioration rate in AD.

Oral administration of heptylphysostigmine in healthy volunteers: a preliminary study.

Heptylphysostigmine (HP) is a reversible cholinesterase (ChE) inhibitor with greater lipophilicity and longer inhibitory action than the parent compound, physostigmine (Phy). Single (0.1-0.6 mg/kg) and multiple 5-day (0.1-0.3 mg/kg) doses of HP were administered to 21 young normal volunteers. The relationship between logarithmic dose (mg/kg) and peak inhibition of red blood cell (RBC) ChE was linear with dose. In one subject given 0.6 mg/kg of HP, concentration in plasma was 0.68 ng/ml at 2 h and gradually declined to below the detection limit by 4 h. Peak plasma and RBC ChE inhibitions of 31.2% and 55.8% were achieved at 2 h for both with a 0.6 mg/kg dose. Chronic studies did not result in any accumulation of ChE inhibition up to 0.2 mg/kg b.i.d., whereas at 0.3 mg/kg b.i.d. 10-15% RBC ChE inhibition was maintained. Higher levels of ChE inhibition can be achieved with HP than with its parent compound, Phy. Red blood cell ChE inhibition recovered more slowly than plasma even though the maximum inhibition was similar for both enzymes.

Pharmacokinetics and pharmacodynamics of metrifonate in humans.

We studied the pharmacokinetics and pharmacodynamics [red blood cell (RBC) cholinesterase (ChE) inhibition] of metrifonate (MTF) and its active anti-ChE metabolite, dichlorvos (DDVP) in Alzheimer's disease (AD) patients and normal controls after oral MTF. In Study I conducted for 6 h, 3 patients with prior MTF exposure received oral MTF (7.5 mg/kg). Plasma ChE inhibition peaked to 78.5 +/- 12.3% at 15 min, while maximum RBC ChE inhibition seen at 1 h was 61.0 +/- 11.0%. Plasma ChE inhibition was unchanged at 6 h, whereas RBC ChE recovered with a t1/2 of 7.0 +/- 3.5 h. In Study II, 6 patients and 6 controls with no prior MTF exposure were given oral MTF. Mean plasma t1/2 of MTF was 2.3 +/- 0.3 h with ChE recovery t1/2 of 9.0 +/- 3.3 (plasma) and 26.6 +/- 15.2 days (RBC) after 7.5 mg/kg MTF. The short drug t1/2, long ChE recovery t1/2 and the achievement of high ChE inhibition levels with minimal side effects suggest the potential use of this drug for Alzheimer therapy.

High-performance liquid chromatographic method using ultraviolet detection for measuring metrifonate and dichlorvos levels in human plasma.

We report high-performance liquid chromatographic methods using ultraviolet detection, developed for the first time in our laboratory with sensitivity to detect clinically significant concentrations of metrifonate (MTF), an experimental drug for Alzheimer disease, and its active anticholinesterase metabolite, dichlorvos (DDVP). The determination limit of the method for MTF and DDVP was 1 microgram/ml and 40 ng/ml, respectively. Stability of MTF and DDVP at various temperatures in water, buffered solutions and in human plasma were also studied.

Determination of heptylphysostigmine in plasma by high-performance liquid chromatography with electrochemical detection.

An analytical method was developed with sensitivity to detect clinically significant concentrations of heptylphysostigmine (HP), a new physostigmine derivative with potent and long-lasting inhibitory activity on cholinesterase. HP, an experimental drug for Alzheimer disease, was measured in human plasma by high-performance liquid chromatography with electrochemical detection with use of a normal-phase column and acetonitrile buffer containing tetrahydrofuran and sodium acetate, pH 4.6. The limit of detection of the method was 0.125 ng/ml using a 2-ml sample of plasma. Analytical recovery of HP was 53.04 +/- 7.75% for plasma in the range 0.25-2.5 ng/ml. Stability studies conducted at 37 degrees C indicated that the drug was stable in 1 M hydrochloric acid, 1 M hydrogen peroxide and sodium acetate-buffered solution at pH 4 for at least 6 h but at pH 7 it degraded slightly to 79% at 6 h and was unstable in 1 M sodium hydroxide with only 9% of the parent compound remaining at 30 s. HP was stable when exposed to ultraviolet light at 22 degrees C or 100% relative humidity at 37 degrees C, with almost 80 and 75% of the parent compound remaining after 4 and 28 days, respectively. HP was stable in plasma at 4 degrees C for 0.25 h, and it slowly degraded to 56 and 28% of the original concentration by 1 and 2 h, respectively. HP was highly unstable in plasma at higher temperatures; at 22 and 37 degrees C it degraded immediately to 48 and 36% of the original concentration and was not detected after 0.5 and 0.25 h, respectively.

Physiological pharmacokinetic and pharmacodynamic model of physostigmine in the rat.

A physiological model for physostigmine disposition was developed in the rat which incorporated anatomical, physiological, and biochemical parameters, i.e. tissue volume, plasma flow rates, drug metabolism, and tissue-to-plasma partition coefficients. Predicted concentrations of physostigmine in different tissue compartments were consistent with the experimental observations in the rat following an iv dose. Part of this study also compared the time course changes in measured effect, as percentage change in cholinesterase activity in brain, and related these changes to the plasma or brain drug level in either a combined pharmacokinetic-pharmacodynamic (plasma physostigmine-effect relationship) or a dynamic model (brain physostigmine-effect relationship). Fitting the time course of the effect in a pharmacokinetic-pharmacodynamic model required an effect compartment with the equilibration rate constant between it and the plasma compartment. Both models help to understand whether the cholinesterase activity is homogeneous or heterogenous in the brain.

Determination of physostigmine in plasma and cerebrospinal fluid by liquid chromatography with electrochemical detection.

Physostigmine (Phy) was determined in plasma and cerebrospinal fluid (CSF) by HPLC with electrochemical detection, with use of a normal-phase column and methanolic sodium acetate buffer, pH 4.6. The detection limit of the method was 0.5 micrograms/L for a 2-mL sample of plasma or 0.5 mL of CSF. Analytical recovery of Phy in the range from 0.5 to 40 micrograms/L was 60% (SD 5%) for plasma and 78% (SD 8%) for CSF. Excellent chromatographic separation of Phy without column deterioration during extended usage and constant recovery for a wide range of Phy concentrations makes the routine monitoring of plasma from patients with Alzheimer's disease economically feasible. Using our method, we measured Phy in 13 such patients' plasmas at 105 and 135 min after a 135-min intravenous infusion of 300, 600, and 900 micrograms of Phy per square meter of body surface. Mean values significantly increased with dose (P = 0.001), but differences between 105 and 135 min (P = 0.229) or between dose and time (P = 0.949) were not significant.

Drug interaction for plasma protein binding: physostigmine and other drugs.

Physostigmine (Phy) has gained prominence recently because of its use in the treatment of Alzheimer's disease and also as a prophylactic agent in the treatment of organophosphate intoxication. Since Alzheimer's disease is usually confined to the older population, the patients generally receive more than one medication. The present study examined whether Phy is displaced from the plasma protein binding site in vitro by a number of commonly prescribed drugs used in this patient population, using therapeutic range of blood levels. The binding of Phy to plasma proteins decreased in the presence of quinidine (3-15 micrograms/ml), from 45.5-5.3%, furosemide (1-100 micrograms/ml) from 45.5-28.4%, acetaminophen (10-160 micrograms/ml) 48.8-37.9%, theophylline (6.67-80 micrograms/ml) 52.2-43.7%, verapamil (0.1-0.5 micrograms/ml) 44.8-34.9%. However, imipramine (0.05-0.2 micrograms/ml), chlorpromazine (0.1-2 micrograms/ml) and propranolol (0.02-0.04 micrograms/ml) did not affect the binding of Phy irrespective of whether the plasma samples were preincubated with Phy or not. The binding of Phy to alpha 1-acid glycoprotein (50 mg/dl), gamma-globulin (1.3 mg/dl) and low density lipoprotein (0.83 mg/ml) was very small (less than 15%), contributing to about one-third of the total plasma protein binding. It appears that the binding of Phy to human plasma proteins was altered to a significant degree in the presence of quinidine, furosemide, acetaminophen, theophylline and verapamil. Free fraction of Phy in plasma has increased which could lead to greater toxicity of Phy.

Hepatic and muscle clearance of physostigmine in the rat.

Physostigmine (Phy) was rapidly metabolized by rat liver after iv (100 micrograms/kg) or im (650 micrograms/kg) administration. The half-life of Phy in liver after iv and im administration was found to be 24 and 26 min, respectively, indicating that Phy does not show dose-dependent kinetics. The half-life of Phy in muscle after iv administration was 20 min. The extraction ratio and the clearance from the liver and muscle were calculated. Hepatic clearance ws found to be 23.08 ml/min/kg (34.90% of systemic clearance) and the intrinsic clearance from the liver was 83.73 ml/min/kg. The percentage dose was found to be maximal in the muscle and the clearance was about 5.2 ml/min/kg. It appears that 30-40% of radioactivity was bound to the liver precipitate and could not be washed off by 10% trichloroacetic acid or organic solvents. Phy and the metabolites, eseroline (Es), M1, M2, and M3 were separated by HPLC, collecting the radioactive fractions. The time course of radioactivity per gram of liver after iv dosing (100 micrograms/kg), showed the major metabolite to be M1, followed by M2 and Es, whereas after im dosing (650 micrograms/kg), Es was present in larger amounts than were other metabolites. Phy appeared to be slowly metabolized in the muscle after iv administration. Liver played a major role in the metabolism of Phy.

Binding of physostigmine to rat and human plasma and crystalline serum albumins.

The binding of 3H-physostigmine (3H-Ph) to human and rat plasma proteins and crystalline serum albumin was studied by ultrafiltration technique. This study showed that the percentage of 3H-Ph bound to rat plasma slightly decreased from 49% to 41% whereas human plasma showed an increase in binding from 29% to 43% over a 50-fold increase in drug concentration. Human plasma samples which were collected in a bag coated with citrate phosphate dextrose adenine-1 solution bound 50% less 3H-Ph than samples collected with EDTA indicating a drug-drug interaction between 3H-Ph and anticoagulants. No significant change in binding was observed if the samples were frozen prior to use. Scatchard plots for binding of 3H-Ph resulted in a positive slope for human plasma and a negative slope for rat plasma; whereas curvilinear Scatchard plots with negative slopes were obtained for binding to human and rat crystalline serum albumin.