Pharmacological interventions against aging through the cell plasma membrane: a review of the experimental results obtained in animals and humans.

As was shown in a recent review by this author (Ann. N.Y. Acad. Sci., 928: 187-199, 2001), oxyradicals cannot be considered only as harmful by-products of the oxidative metabolism, but living cells and organisms implicitly require their production. This idea is supported by numerous facts and arguments, the most important of which is that the complete inhibition of the oxyradical production by KCN (or by any block of respiration) kills the living organisms long before the energy reserves would be exhausted. This new theoretical approach not only helps our understanding of the normal functions of the living organisms, such as the basic memory mechanisms in the brain cells, but also helps in identifying the site-specific, radical-induced damaging mechanisms that represent the undesirable side effects of oxygen free radicals. First of all, these effects make the cell plasma membrane vulnerable and cause a series of intracellular functional disorders, as described by the membrane hypothesis of aging (MHA). The logical way for any antiaging intervention therefore should be to increase the available number of loosely bound electrons inside the plasma membrane that are easily accessible for OH(*) free radical scavenging. The present review summarizes the available knowledge regarding the theory of the use of membrane-related antiaging pharmaca, like centrophenoxine (CPH), tested in both animal experiments and human clinical trials. A modified, developed version of CPH coded as BCE-001 is also reported.

In vitro and in vivo effect of the nootropic agent adafenoxate on the 5-HT1 sites in different rat brain structures.

1. The effect of the nootropic agent adafenoxate (a structural analogue of meclofenoxate) on the binding parameters of 5-HT1 receptors in vitro and in vivo in rat cerebral cortex, striatum, hippocampus and hypothalamus was studied. 2. The chronic (100 mg/kg per os for 7 days) adafenoxate treatment produced a significant (24.6%) decrease in the density of 5-HT1 sites in the hippocampus. 3. In vitro adafenoxate inhibited specific [3H]5-HT binding with equal potency in all the regions studied with IC50s in the microM range. 4. It is suggested that the decrease in the density of the 5-HT1 sites in rat hippocampus might contribute to the nootropic action of adafenoxate.

Biogenic monoamine uptake by rat brain synaptosomes during aging. Effects of nootropic drugs.

1. In experiments on young (3-5-month-old), adult (10-11-month-old) and old (21-22-month-old) rats, it was found that significant age-related changes occurred in the high-affinity uptake of dopamine (DA), noradrenaline (NA) and serotonin (5-HT) by cortical and striatal synaptosomes. 2. Changes in DA, NA and 5-HT uptake during aging are suggested to be neurochemical correlates of cognition and memory deficits that develops in senescence. 3. The in vitro effects of the nootropic drugs piracetam, aniracetam, meclofenoxate and adafenoxate on the DA, NA and 5-HT uptake by cortical and striatal synaptosomes from young rats were studied. Administered in increasing concentrations (1 x 10(-4) to 5 x 10(-3) M) these drugs inhibited monoamine uptake. 4. Adafenoxate proved to be a more potent monoamine uptake inhibitor than the other three drugs; it inhibited the uptake in the frontal cortex and striatum without selectivity for either monoaminergic system. It is suggested that adafenoxate affects cognition through the involvement of central neurotransmission and particularly through the inhibition of monoamine uptake systems.

Adafenoxate abolishes the amnesia induced by neonatal 6-hydroxydopamine treatment in rats.

The effect of neonatal 6-hydroxydopamine (6-OHDA) treatment on learning and memory and on the levels of biogenic monoamines in some brain structures, as well as the influence of the nootropic drug adafenoxate on 6-OHDA effect was studied in shuttle box and step down trained rats. In mature rats injected with 6-OHDA postnatal, learning and retention were impaired and the noradrenaline (NA) level in the frontal cortex and hippocampus was decreased. Adafenoxate abolished the amnestic effect of 6-OHDA and restored the NA level to normal in the above-mentioned brain structures. This finding suggests the important role of the noradrenergic neurotransmitter system in 6-OHDA-induced amnesia and the favorable effect of adafenoxate on learning and memory impaired by 6-OHDA.

Chromatographic analysis of 1,3-bis(dimethylamino)isopropyl 4-chlorophenoxyacetate dihydrochloride, a new CNS stimulant.

A reversed-phase liquid chromatographic method is developed for the assay of the new CNS stimulant, 1,3-bis(dimethylamino)isopropyl 4-chlorophenoxyacetate dihydrochloride (BCE-001), and its main contaminant, 4-chlorophenoxyacetic acid (PCPA). In the first place the possible reactions of BCE-001 with mobile phase components are investigated and it is found that BCE-001 is readily hydrolysed in water and undergoes transesterification in methanol or ethanol. The methyl ester is formed rapidly and quantitatively so that BCE-001 can be assayed as methyl 4-chlorophenoxyacetate. PCPA is formed as a result of the hydrolysis of BCE-001 during the sample pretreatment and chromatographic separation and this causes an overestimate of the PCPA impurity in the bulk drug. An effective method is developed to prevent the hydrolysis of BCE-001 during sample pretreatment.

Effects of serotoninergic receptor antagonists and their combination with scopolamine on memory.

Using the method for passive avoidance with punishment reinforcement (step-down), it was found that the serotonin (5-HT)-receptor blockers methergoline, methysergide and ritanserin at a dose of 5 mg/kg b.w. administered i.p. 30 min before the training session as well as the M-cholinergic-receptor blocker scopolamine administered at a dose of 2 mg/kg b.w. 90 min before training impaired acquisition and retention of memory traces. The memory impairment was also manifested by the poor habituation of methergoline- and scopolamine-treated rats placed in an unfamiliar environment. Memory deficit was increased by the combination of methergoline or ritanserin with scopolamine. On multiple administration before training the nootropic drug adafenoxate completely prevented the amnestic effect of the combination methergoline plus scopolamine.

Memory impairment induced by combined disturbance of noradrenergic and dopaminergic neurotransmissions: effects of nootropic drugs.

The effect of the combined application of the alpha 2-adrenoreceptor agonist clonidine and of the dopaminergic blocker haloperidol on the memory processes was tested on albino rats. The changes in the memory were studied using the following methods: two-way active avoidance with negative reinforcement (shuttle-box) and passive avoidance (step-through). Both clonidine (0.05 mg/kg) and haloperidol (0.5 mg/kg), injected intraperitoneally immediately after the end of the training session, slightly impaired retention in the memory tests used with both training methods. Their combined application, however, caused a marked amnesia. This amnesia model was used to study the effects of the nootropic drugs: adafenoxate and the newly-synthesized compound benzoyl-1, 4-dipyrolydinone (p-P). Administered orally in a dose of 100 mg/kg for 5 days prior to the training session, both adafenoxate and p-P fully eliminate the amnesia caused by the combined application of clonidine and haloperidol. The paper discusses the role of the noradrenergic and dopaminergic neurotransmitter systems for the amnestic effect of the clonidine + haloperidol combination, as well as for the favourable effect on the cognitive functions of the tested nootropic drugs adafenoxate and p-p.

Age-related changes of cyclic AMP phosphodiesterase activity in rat brain regions and a new phosphodiesterase inhibitor--nootropic agent adafenoxate.

1. The low- and high-KM cyclic AMP phosphodiesterase (cAMP PDE) activity in cerebral cortex, striatum, hypothalamus and hippocampus of young (4-5-month-old) and aged (22-month-old) rats has been studied. 2. A significant rise in the high-KM cAMP PDE activity in the cerebral cortex, hypothalamus and hippocampus in aged rats has been found. 3. The activity of the low-KM cAMP PDE does not change during senescence in all the brain structures studied. 4. In a series of increased concentrations (from 5 x 10(-4) to 1 x 10(-5) M) adafenoxate inhibits low- and high-KM cAMP PDE in most of the brain structures studied in both age groups. 5. The present results provide evidence for realization of the CNS effects of adafenoxate through inhibition of cAMP PDE activity and regulation of the intracellular level of cAMP.

Changes in brain biogenic monoamines induced by the nootropic drugs adafenoxate and meclofenoxate and by citicholine (experiments on rats).

1. The effects of Adafenoxate (Adf), meclofenoxate (Mf) and citicholine (CCh) administered at a daily dose of 100 mg/kg for 7 days on the levels of noradrenaline (NA), dopamine (DA) and serotonin (5-HT) in the frontal cerebral cortex, striatum, hippocampus and hypothalamus of rats were studied. 2. Adafenoxate increased the NA level in the striatum and decreased it in the hypothalamus; it increased the DA level in the cerebral cortex and hypothalamus and decreased it in the striatum; it increased the 5-HT level in the cerebral cortex and decreased it in the hippocampus. 3. Meclofenoxate decreased the NA level in the cerebral cortex and hypothalamus; it increased the DA level in the hippocampus and hypothalamus and the 5-HT level in the cerebral cortex, striatum, hippocampus and hypothalamus. 4. Citicholine increased the NA level in the cerebral cortex and hypothalamus; it increased the DA level in the striatum and the 5-HT level in the cerebral cortex, striatum and hippocampus. 5. An attempt is made to explain some similarities and differences in the behavioral effects of the drugs tested (and those observed in other studies) by the changes they induce in brain biogenic monoamines.

Memory effects of the new derivative of the p-chlorophenoxyacetic acid adafenoxate compared to the effects of some cognition-enhancing drugs in rats.

In experiments on male rats the effects of adafenoxate (Adf), meclofenoxate (Mf), piracetam (Pc) and citicholine (CCh) on learning and memory were studied using the maze active avoidance method with punishment reinforcement. The drugs tested were administered twice daily for seven days at doses of 10 and 50 mg/kg body weight for Adf, Mf and CCh and only at a dose of 50 mg/kg body weight for Pc. The effects of these drugs on scopolamine-treated and scopolamine-untreated rats were also studied using the step-through method. Retention tests were given 24 h and 7 days after the end of the training session in the punishment-reinforcement active avoidance and 3 and 24 h after training in the passive avoidance situation. With the maze method statistically significant results about the favourable effects of the four drugs were obtained by most of the indices for learning and memory. However, the effects of the drugs tested were differently pronounced depending on the dose utilized. With the step-through method all four drugs prevented the scopolamine-induced amnesia. Comparing the present results with other data previously obtained about the effects of the drugs tested and of other nootropic drugs on brain biogenic monoamines, it is suggested that induced changes in biogenic monoamines are responsible for the similarities and the differences in the effects of nootropic drugs on learning and memory.

Effect of adafenoxate on different rat brain structures monoamine oxidase activity in vitro.

1. The effect of the nootropic drug adafenoxate on monoamine oxidase (MAO) activity in rat brain cortex, striatum, hypothalamus and hippocampus has been studied using the following substrates: tyramine (total MAO), serotonin (MAO A) and beta-phenylethylamine (MAO B). 2. In a series of increased concentrations (from 5 x 10(-4) up to 1 x 10(-5) M) adafenoxate inhibits total MAO, MAO A and MAO B in the brain structures studied. 3. The adafenoxate IC50 values obtained illustrate its inhibitory properties and its lack of selectivity toward MAO in the brain structures isolated. 4. The results of our research prove the participation of MAO in the mechanisms through which adafenoxate affects the brain monoaminergic systems and realises its central effects.

Effects of the nootropic agents adafenoxate and meclofenoxate on brain biogenic monoamines in aged rats.

The effect of the nootropic agents meclofenoxate (Mf) and adafenoxate (Adf) on the content of biogenic monoamines in the frontal cerebral cortex, striatum, hypothalamus, and hippocampus of 22-month-old rats was studied. Mf and Adf were administered orally for seven days twice daily in a dose of 50 mg/kg weight. Both agents tested increased the content of serotonin (5-HT) in the cortex and striatum. Adf also raised the noradrenaline (NA) content in the cortex and hippocampus, lowering the dopamine (DA) level in the striatum. Comparison of the results obtained in the present study with the finding of earlier experiments of ours on 4-5-month-old rats revealed the following: 1. Biogenic monoamines (BMA) in the brain tend to decrease with ageing. 2. In addition to the unidirectional effects of Mf and Adf on the BMA content in the brain of both young and old rats, but in some cases there were also differences. 3. The comparison of the effects of Mf and Adf on the BMA content in the different brain regions with the changes in this content, characteristic of certain diseases, reveals prospects for selectivity in the application of these nootropic agents, which are generally rather similar in pharmacological characteristics.

Comparative studies on the effects of the nootropic drugs adafenoxate, meclofenoxate and piracetam, and of citicholine on scopolamine-impaired memory, exploratory behavior and physical capabilities (experiments on rats and mice).

The effects of adafenoxate (Adf), meclofenoxate (Mf), piracetam (Pc), and citicholine (CCh) on scopolamine (Scop)--impaired memory and exploratory behavior (experiments on rats) and on physical capabilities (experiments on mice) were studied. In the experiments with scopolamine (2 mg/kg i.p.) we used the step-through passive avoidance method to determine the memory changes. In the case of single treatment with the drugs tested scopolamine was injected immediately after training and Adf, Mf, and CCh at doses of 20 and 100 mg/kg and Pc at a dose of 100 mg/kg were administered immediately after scopolamine. In the case of multiple administration the drugs were applied at the same doses for 7 days before training. Scopolamine was injected immediately after training. Retention tests were given 3 and 24 hours later. All the four drugs tested prevented to a large extent or completely the scopolamine-induced retrograde amnesia. However, significant quantitative differences in the antiamnestic effects of the drugs tested were observed. The effects of the four drugs on exploratory behavior were tested in the Opto Varimex apparatus. After 7-day treatment with the drugs at the doses utilized, the behavior of experimental animals was observed for 10 min, checking out the changes in the frequency of rearing, ambulation, and rotation. Only Adf at a dose of 50 mg/kg significantly decreased rearing and ambulation frequencies; this effect was considered to be an expression of accelerated habituation. The physical capabilities of mice were studied, using the method of treadmill (revolving drum activity cage) training. Before the experiment the mice received orally Adf, Mf, and Pc at a dose of 100 mg/kg or were injected intraperitoneally with CCh at doses of 50 and 100 mg/kg once daily for 7 days. The number of revolutions of the drum cages was counted for 4 hours. Only Pc significantly increased the physical capabilities of mice and much delayed the occurrence of fatigue.

Comparative studies on the effects of the nootropic drug adafenoxate and of the cerebral vasodilator flunarizine on arterial smooth muscles.

The effects of the nootropic drug adafenoxate and of the cerebral vasodilator flunarizine were studied on smooth-muscle preparations isolated from rabbits (perfused central ear artery and segments of the thoracic aorta). Both drugs tested did not change the smooth muscle tone. When applied extralumenally adafenoxate decreased the noradrenaline (NA)-evoked arterial contractions. Adafenoxate (especially in high concentrations) markedly relaxed the NA-contracted arterial preparations. The arterial contractions in response to low-frequency electrical stimulation (ES) were moderately potentiated by high concentrations of extralumenal adafenoxate, but were decreased by intralumenal administration of the drug. Administered prior to or after NA adafenoxate antagonized its contractile effects on the aortic segments (this effect was much pronounced when the smooth-muscle preparations were contracted by NA). Flunarizine exerted similar but less pronounced inhibitory effects on the NA- or ES-evoked contractions of the smooth-muscle preparations. The only essential difference between the two drugs tested was that extralumenal flunarizine inhibited the arterial contractions in response to ES. It is suggested that if adafenoxate exerts an antivasoconstrictory effect not only on the peripheral arteries but also on the cerebral blood vessels, this effect might play an important role for the realization of the nootropic action of this compound.

A study of nootropic drugs for anti-anxiety action.

The effects of adafenoxate (100 mg/kg), aniracetam (50 mg/kg), meclofenoxate (100 mg/kg), piracetam (250 mg/kg), and standardized ginseng extract-G115 (100 mg/kg) applied for five days were studied with Vogel's conflict procedure in which thirsty naive rats were periodically administered shocks for licking water. The results showed a significant anti-anxiety effect (increases in licking) with adafenoxate and meclofenoxate. Piracetam significantly suppressed licking behaviour and this effect was evaluated as anxiogenic (resulting from the non-specific stimulant action of the drug). Adafenoxate was found to increase also the number of entries into and escapes from the dark compartment without punishment responding. This effect of adafenoxate was considered to be an expression of facilitated conditioned-reflex activity. A beneficial role for the observed anti-anxiety effects of adafenoxate and meclofenoxate in the nootropic action of these drugs is suggested.

Effects of the nootropic agents adafenoxate, meclofenoxate and the acetylcholine precursor citicholine on the brain muscarinic receptors (experiments on rats).

The effect of adafenoxate (Af), meclofenoxate (Mf) and citicholine on the brain muscarinic receptors was studied in groups of ten male Wistar rats. The compounds were administered in doses of 50 mg/kg body weight twice daily for 7 days. One hour after the last treatment the animals were killed and the frontal cerebral cortex striatum, the hypothalamus and the hippocampus were removed immediately. Af and Mf were found to diminish significantly and to an analogous extent the density (Bmax) of the muscarinic receptors in the cerebral cortex, striatum and the hippocampus. At the same time, however, the greater decrease of Kd induced by these two nootropic agents, i.e. the increased affinity of the muscarinic binding sites, exceeded considerably the decreased number of the binding sites. These differences in the effects on Bmax and Kd suggest that the functional capacity of the cerebral cholinergic system increases under the action of Mf and Af. The surprising increase in the number of muscarinic receptors in the striatum, observed in citicholine-treated animals, is assumed to be due to the great increase of the dopamine content in this structure, induced by this acetylcholine precursor, observed in other experiments. This increase would result in reduced acetylcholine production by the inhibited cholinergic neurones, with a subsequent increase in the number of the muscarinic receptors.

Individually-determined differences in the effects of psychotropic drugs on memory (experiments on rats).

Using the methods for "staircase-maze" training with positive (alimentary) reinforcement and "steps down" passive avoidance with punishment (electroshock) reinforcement, we divided the experimental animals into three groups: "good", "intermediate", and "poor" learners. In the course of seven days the animals of the three groups were treated with five substances and were then tested for retention with both methods. Amphetamine at single doses of 0.1 and 0.5 mg/kg was injected s. c. 30 min before the retention test given on the 7th day after the end of training. The other four drugs were administered twice daily throughout the period between the end of training and retention testing (seven days). Adafenoxate was applied at a dose of 10 mg/kg, piracetam at doses of 50, 150, and 300 mg/kg, and aniracetam at doses of 50 and 150 mg/kg--all the three drugs were administered orally; citicholine was injected at doses of 10 and 50 mg/kg i. p. The effects of the substances tested significantly differed depending on the animal's belonging to one or another group. Furthermore, depending on the retention test, the drug tested and the dose used, we observed differences in the effects not only in the different groups but also in one and the same group. Not taking into account the individual capabilities of experimental animals to acquire a retention task might lead to an incorrect characterization of such an important property of psychotropic drugs as their effect on memory process.