Why so few drugs for Alzheimer's disease? Are methods failing drugs?

Recent studies of Alzheimer's disease (AD) and other neuropsychiatric drug developments raise questions whether failures of some drugs occur due to flaws in methods. In three case studies of recent AD drug development failures with phenserine, metrifonate, and tarenflurbil we identified methodological lapses able to account for the failures. Errors in complex systems such as drug developments are both almost inescapable due to human mistakes and most frequently hidden at the time of occurrence and thereafter. We propose preemptive error management as a preventive strategy to exclude or control error intrusions into neuropsychiatric drug developments. We illustrate the functions we anticipate for a preemptive error management preventive strategy with a checklist and identify the limitations of this aspect of the proposal with three drug examples. This strategy applies core scientific practices to insure the quality of data within the current context of AD drug development practices.

Effects of time and cholinesterase inhibitor treatment on multiple cerebrospinal fluid parameters in Alzheimer's disease.

Development of neuropathology in Alzheimer's disease (AD) cannot be studied directly in living patients. Therefore, concentrations in cerebrospinal fluid (CSF) of the proteins tau, A beta 42, alpha 1-ACT, apoE and other molecules have been analyzed to elucidate their possible role in degeneration and as biomarkers of the disease. To date, however, studies have not analyzed multiple markers in the same patients over time and as a function of pharmacological interventions. In the present investigation we measured CSF tau, A beta 42, alpha 1-ACT, apoE, total protein and electrophoretic fractions, and leukocytes, as well as MMSE, in 12 AD patients of known APOE phenotype. Two or three CSF examinations were performed during periods of up to 2 1/2 years, while subjects were on and off treatment with the cholinesterase inhibitor (ChEI) metrifonate (MTF). CSF A beta 42 and tau levels were in agreement with clinical diagnosis of AD in all patients. Abnormally high proportions of monocytes were found in CSF at baseline, and these proportions correlated positively with plasma alpha 1-ACT and MMSE scores. A small but significant increase in CSF alpha 1-ACT, which correlated with peripheral alpha 1-ACT, was associated with 6 months' MTF treatment, though alpha 1-ACT levels did not change further when treatment continued for 2 years. Monocyte proportions in CSF declined over time in both treated and untreated patients. Among 5 of 6 patients treated for 2 years or more with MTF, CSF measures remained relatively stable. One patient had changes in CSF parameters apparently associated with a transient ischemic attack. Our findings did not indicate that slowed cognitive decline with MTF treatment is associated with systematic change in any CSF marker analyzed. The results suggest that further investigations of the relationship of tau, A beta 42 and cellular abnormalities in CSF early in the course of AD are warranted.

Employment outcomes for clients with severe mental illness in a PACT model replication. Program for Assertive Community Treatment.

Outcomes of clients with severe mental illness in a psychosocial and vocational rehabilitation program modeled after the Program for Assertive Community Treatment were tracked through record review to determine if clients' employment gains were sustained while they were in the program. A total of 184 clients participated in the program between December 1984 and February 1994, of whom 34 percent remained for one to four years and 33 percent remained for longer than four years. Sixty-four percent of the clients who stayed in the program a year or longer attained employment. The program maintained an average employment rate of 33 percent of all participating clients. More than half of the clients who held jobs worked part time and were employed more than half of the time that they were in the program.

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.

Metrifonate treatment enhances acquisition of eyeblink conditioning in aging rabbits.

The cholinergic system is known to show deterioration during aging and Alzheimer's disease. In response, a therapeutic approach to Alzheimer's disease has been to attempt to compensate for the decrease in central cholinergic function by potentiating the activity of the remaining intact cholinergic cells with cholinesterase inhibitors. In this study treatment with the long-lasting cholinesterase inhibitor metrifonate enhanced acquisition of eyeblink conditioning in aging rabbits without producing interfering side effects. The effects of metrifonate on central and peripheral cholinesterase activity were evaluated, as was the involvement of plasma atropine esterase activity on the central and peripheral response to metrifonate. Results demonstrate that metrifonate can produce predictable, dose-dependent ChE inhibition. Associative learning in the aging rabbit was improved by metrifonate-induced steady state ChE inhibition within a range of 30-80%. Metrifonate was behaviorally effective in the absence of the severe side effects which typically plague cholinesterase inhibitors, suggesting that metrifonate is a possible treatment for the cognitive deficits resulting from normal aging and Alzheimer's disease.

Increased regional cerebral glucose metabolism and semantic memory performance in Alzheimer's disease: a pilot double blind transdermal nicotine positron emission tomography study.

Nicotinic receptor dysfunction and impaired semantic memory occur early in Alzheimer's disease patients (AD). Previous research implied that nicotine's ability to enhance alertness, arousal, and cognition in a number of nonclinical populations was a function of its ability to stimulate CNS nicotinic cholinergic receptors. In this study it was hypothesized that transdermal administration of nicotine would increase both regional cerebral glucose metabolism (rCMRglc) and semantic memory (as assessed by verbal fluency). Two mild AD and two elderly controls underwent positron emission tomography scanning during a double blind nicotinic agonist verbal fluency challenge procedure. rCMRglc increases occurred in both AD patients, but not controls. In the two AD patients, verbal fluency scores increased by an average of 17%. One elderly control's verbal fluency increased, and the other decreased. These findings suggest that nicotine's effect on metabolism and verbal fluency is due to its ability to stimulate the cholinergic system.

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.

Transdermal patch delivery of acetylcholinesterase inhibitors.

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.

Effect of monoamine oxidase A and B inhibition on the uptake and metabolism of serotonin within serotonergic neurons of rat brain.

The effects of inhibiting monoamine oxidase (MAO) A and B on metabolism and uptake of serotonin (5-HT) in serotonergic synaptosomes were studied. To avoid contamination by extrasynaptosomal MAO, synaptosomes were separated from other components of rat brains by discontinuous sucrose density gradient centrifugation. Kinetic analysis of 5-HT uptake demonstrated that 5-HT was selectively transported into serotonergic synaptosomes through the high affinity 5-HT uptake process. Selectivity of the uptake and subsequent deamination of 5-HT within serotonergic synaptosomes were confirmed using selective and non-selective 5-HT and norepinephrine (NE) uptake inhibitors. MAO inhibitor analysis of 5-HT deamination occurring within serotonergic synaptosomes indicated that, at physiologically relevant concentrations of 5-HT, MAO A deaminates 5-HT, maintaining a low cytoplasmic concentration of 5-HT. When the cytoplasmic concentration of 5-HT is increased above physiologically relevant levels by the inhibition of MAO A, MAO B becomes active. [( 14)C] 5-HT uptake into synaptosomes was reduced by decreasing the V( max) of [(14)C] 5-HT uptake. One mechanism for a decrease in the V(max) could be the increase in the cytoplasmic concentration of 5-HT.

Inhibition of human brain and RBC acetylcholinesterase (AChE) by heptylphysostigmine (HPTL).

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.

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.

Parallel distributed processing and neural networks: origins, methodology and cognitive functions.

Parallel Distributed Processing (PDP), a computational methodology with origins in Associationism, is used to provide empirical information regarding neurobiological systems. Recently, supercomputers have enabled neuroscientists to model brain behavior-relationships. An overview of supercomputer architecture demonstrates the advantages of parallel over serial processing. Histological data provide physical evidence of the parallel distributed nature of certain aspects of the human brain, as do corresponding computer simulations. Whereas sensory networks follow more sequential neural network pathways, in vivo brain imaging studies of attention and rudimentary language tasks appear to involve multiple cortical and subcortical areas. Controversy remains as to whether associative models or Artificial Intelligence symbolic models better reflect neural networks of cognitive functions; however, considerable interest has shifted towards associative models.

Stability of peripheral hematological parameters after chronic acetylcholinesterase inhibition in man.

Acetylcholinesterase (AChE; EC 3.1.1.7) is present in both primitive and mature erythroid cells, and a role has been suggested for the enzyme in regulation of differentiation in the human erythron. AChE is also a major enzyme in the central nervous system; alteration of its activity has been proposed as a therapeutic strategy in Alzheimer disease. We recently treated 18 Alzheimer disease patients with metrifonate, a long-acting AChE inhibitor, over periods up to 7 months, with resulting erythrocyte AChE inhibition as high as 82 per cent of baseline values. Despite chronic reduction of enzyme activity, no significant alterations were noted in erythrocyte, leukocyte or platelet characteristics or numbers that would suggest a deleterious effect of AChE inhibition on normal differentiation. Thus, any modification of developmental pathways appears to be compensated by other regulatory mechanisms in the intact organism.

Regulation of low molecular weight insulin-like growth factor binding proteins in experimental diabetes mellitus.

Circulating insulin-like growth factor (IGF) bioactivity is reduced in animals and patients with diabetes mellitus. We sought to determine whether the availability and levels of specific IGF binding proteins (BPs) are altered in animals with experimental diabetes, and might contribute to changes in circulating IGF bioactivity in experimental diabetes. Female Sprague-Dawley rats were administered streptozotocin or citrate buffer iv, and then killed either 3 days later, or else after 4-day insulin treatment (7.5 U/kg human NPH twice daily), or 2 days after insulin was discontinued. Serum [125I]IGF-I binding activity was markedly increased in diabetic animals compared to controls when analyzed by Sephacryl S-200 chromatography, dot blot, and affinity labeling techniques, due to increased binding to low mol wt BPs (81 +/- 4% of ligand eluting with low mol wt BPs in diabetic serum vs. 22 +/- 3% in control, P less than 0.001). In contrast, activated charcoal removed ligand from these BPs and underestimated the availability of BPs in diabetes. Serum binding activity fell toward control levels during insulin therapy, then rose again after insulin was withdrawn, corresponding to changes in metabolic status. To distinguish changes in specific BPs, serum proteins were separated by 13% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, then transferred to nitrocellulose. Ligand blotting with [125I]IGF-I demonstrated that serum levels of a 32 K mol wt IGF BP are markedly increased in diabetic rats and decline during insulin therapy. Levels of this 32 K IGF BP rose again after insulin was discontinued, demonstrating regulation in accordance with changes in insulin and metabolic status. Western analysis and affinity labeling with immunoprecipitation revealed that this 32 K protein is distinct from the 34 K fetal rat BP, and is immunologically related to the type 1 human IGF BP. We conclude that circulating [125I]IGF-I binding activity is markedly increased in animals with acute streptozotocin-induced diabetes, due to changes in low mol wt proteins, including a 32 K type 1 IGF BP that is regulated by changes in insulin and/or metabolic status. Regulation of low mol wt IGF BPs by insulin, and perhaps other factors, may play an important role in the modulation of tissue growth factor bioactivity in metabolic disease.