MOLEKULAR BASIS OF THE PATHOGENESIS OF NERVOUS SYSTEM DISEASES.
POSSIBILITIES OF METABOLIC THERAPY
A.P Khokhlov, M.D., Professor
Progress in clinical neurology that loomed in the 1970s did non develop in the next decades, however, and this field of medicine is currently stagnant.
One could easily enumerate the diseases that have "satisfactory" pathogenic therapy.
There is as yet no definite pathogenesis concept at the molecular level for the absolute majority of neurologic deficiencies, this is why treatment is frequentty symptomatic except for hereditary pathology of the nervous system. The primary product of the mutant gene was identified in 105 nosologic forms; in these instances the pathogenesis is clearly determined, therefore effective preventive (for example, in phenylketonuric oligophrenia) or therapeutic (Wilson's disease) measures were worked out.
But even in this field there are numerous unresolved problems. The therapy of chromosomal anomalies is practically inexistent even though the pathogenesis of some of these has been studied in sufficient detail (Down's disease).
Meanwhile, advances in neurochemistry and neurobiology forming the foundation of clinical neurology are immense and, based on progress made, it would not be difficult to revise the pathogenesis concept of the majority of nervous diseases.
On the other hand, we gained clinical experience of using metabolic therapy preparations (in the form of food additives) which should facilitate a rapid
introduction of new pathogenic drugs.
Until recently, present-day pathogenesis hypotheses were of universal nature and explained the decay of nerve cells in most neurologic disorders irrespective of the localization and, frequently, of etiologic factors.
Thus, the "glutamate" hypothesis considered neurocyte impairment as a result of hyperactivation of N-methyl-D-aspartate (NMDA) receptors which
causes accumulation of excess calcium in the cell cytosol. In this case, an increase in cation level occurs in two ways:
1) through the calcium channel system of NMDA i- receptors;
2) by activating the phosphoinositide cycle with c calcium recovery from the endoplasmic reticulum.
Under physiologic conditions, the activation of NMDA receptors and triggering of a cascade of phosphoinositide cycle reactions increase cell adaptation possibilities, initiate a lasting modification of ion conductivity, expression of key genes, etc. Yet, significant excess of calcium paralyzes thenerve cell function. Accumulating in mitochondria,the ion contributes to a dissociation of conjugate tissue breath processes and oxidative phosphorylation by restricting thereby the energetic neurocyte potentialities and stimulating lipid peroxidatic oxidation processes (LPO).
Concurrently, the activation of Ca++-dependent proteinases and hydrolases rapidly produces macromolecule degradation which eventually makes the cell unviable.
Can the cell decay be prevented under these conditions?
According to the glutamate hypothesis, this is theoretically possible. To achieve this, it is required to:
1) eliminate excessive amino acid in the cell and extracellular space of the brain;
2) decrease the affinity of glutamic receptors with the ligand;
3) use new therapeutic drugs, amino acid antagonists;
4) effect a calcium metabolism correction. However, despite the obvious advisability of swift testing and introduction of drugs and treatment techniques in clinical neurology, there is growing skepticism with regard to the effectiveness of new methods of treatment.
Thus, drugs of the first group, glutamatdehydrogenase activators that decrease the glutamic acid level were tested without any result on 40 patients with amyotrophic lateral sclerosis (ALS) where the role of glutamate in central and peripheral motoneuron was considered to be proven .
The testing of rilosol , antagonist of dicarboxilic acid, was more successful. However, due to its insignificant therapeutic effect, this preparation cannot be considered basic in treating the abovementioned severe disease.
Glycine and treonine, 3rd group drugs, showed negative results in some tests .
Accordingly, the value of the glutamate hypothesis in degenerative disorders is questionable now. On the other hand, it was reported about the expediency of the use of glutamatergic drugs in subcortical degenerations and parkinsonism .
Our studies have not confirmed the glutamate hypothesis either. The application of amino acids with a ramified chain in the majority of degenerative diseases did not produce reliable positive results.
In experiments on animals it was clarified, however, that the activity of glutamatdehydrogenase increased after Aminocomposit introduction in subcortical structure nuclei, but not in the cerebral hemispheres, cerebellum or spinal cord neurons.
Based on the results obtained, the amino acid compound (Aminocomposit) was administered in patients with extrapyramidal disorders, the dosage being 1-2 g daily.
A manifest therapeutic effect was recorded in parkinsonism patients. Diminished extrapyramidal tonus and an increased amount of movements were observed already on the 3rd-4th day in 87% of patients with akinetic-rigid forms and in 68% of patients with rigid-trembling forms.
A decrease (by 30-60%) of the parkinsonian tremor rhythm amplitude occurred on the 10th-12th day and practically in all cases it was possible to cut (by 30-50%) the L-DOPA dosage.
The therapeutic effect lasted for several months after which a course of treatment was repeated.
Besides, an important favorable effect was achieved in treating degenerative subcortical diseases, for instance, in olivopontocerebellar degeneration. In 1.5 months, neurologic deficiency could be diminished by 70-85%. The drug proved effective in eliminating extrapyramidal symptoms in ICP patients.
The positive results obtained only indirectly confirm the adequacy of the glutamate hypothesis, however. The elimination of the direct toxic action of excessive glutamate on the mitochondrial and genetic cell apparatus was not accompanied, at least in the experiment. by a change in the calcium level and principal indicator of the activity of NMDA receptors.
On the contrary. studies conducted in the recent years point to a diminished  function of glutamatergic neurons in some subcortical diseases, including in parkinsonism patients. The administration of glutamatergic medications favorably influenced the pathologic process in the disease concerned.
To correct this deficiency, we developed the amino acid compound Neoprim that contains a minimum amount (less than 8%) of L-glutamic acid and possesses a powerful glutamatergic effect.
The application point of the drug was assessed by three methods:
1) biochemical: measurement of the affinity degree of hippocampus NMDA receptors with the ligand;
2) electrophysiologic studies of the phenomenon of prolonged potentiation (hippocampus sections); 3) change in EEG range and rhythm in animals with implanted electrodes.
An increase (by 1.2 times) of the affinity of receptors with glutamate was accompanied by a clear-cut change of the characteristics-parameters of glutamatergic synaptic transmission.
The drug produced a manifest exciting effect. Upon introduction (10 minutes later) an increase of responses amounted to over 300%. As the dose increased, responses trustworthily grew in number.
The increase of not only post-, but also presynaptic pop-spikes points to increased excitability of membranes, decreased excitability threshold of axons and neuron bodies.
Similar data were obtained following EEG investigations of the brain of animals.
Neoprim became a basic drug in treating a series of nervous system disorders, in particular, peripheral motoneuron, subcortical diseases, oligophrenia, etc. The course of treatment lasts 15-30 days in combination with other amino acid compounds. In a limited amount, the drug proved useful in treating patients with consequences of severe cerebral circulation impairment.
The treatment stretches over 10-15 days, the dosages being 5-10 mg/kg daily. The results as manifested in motor activity recovery are impressive.
The literature reports that a successful use of glycine amino acid in the therapy of patients with severe cerebral circulation impairment brought a first serious corroboration of the suggested hypothesis .
It is supposed that the mechanism of the antiischemic effect of glycine consists in inactivating NMDA receptors as a result of interaction between the amino acid and the glycine loci. Thereby, excessive calcium accumulation in the cell is prevented.
According to Ye. Gusev, glycine intake at a dosage of 1-2 g daily resulted in a rapid withdrawal of neurologic deficiency .
In all fairness it should be stated that 3 years before this report was published, Prof. Khokhlov's team successfully used this amino acid in treating acute cerebral circulation impairment (ACCI) patients. However, because of the opposition of Prof. FI. Gorbacheva, head of the clinic where the testing was carried out, the study had not won acceptance and the publication resulted only in the application for an inventor's certificate No. 4120032 from June 25th, 1986.
Another aspect is the therapeutic effect of the said amino acid in cerebeltar involvement. Reports on this problem were widely published and confirmed by a number of leading Russian clinics.
The mechanism of the action of the drug in question consists in regulating calcium metabolism by an increased release of active phosphoinositides. The amino acid was used as a solution at a dosage of 3-6 g a day. The treatment lasted 30-50 days. In this context, a decrease in ataxia and other clinical cerebellar impairment manifestations was reported already at the end of the first week following the beginning of treatment. Concurrently, a rise in the number of metabolites of the phosphoinositide cycle  was recorded.
And yet, notwithstanding theoretical prerequisites, glycine proved to be an inefficient anti-convulsive drug. In spite of the popularity of the glutamate hypothesis in the disease concerned, the literature does not carry reports on a favorable effect of the amino acid .
All this calls into question not only the "glutamate" hypothesis but the above interpretation of the glycine action mechanism as well.
The following natural anti-convulsive drugs are used in clinical practice: glutamic acid, tryptophan, cerebrolysine, aminolone (y-aminobutyric acid).
Therapeutic effectiveness of the enumerated drugs is widely discussed in the literature . We have tested and introduced 4 amino acid drugs possessing an anti-convulsive effect.
The action mechanism is different. Aminovil, the most widely used drug, blocks absorption of yaminobutyric acid by astrocytes and simultaneously enhances the capture of dicarboxilic amino acids. This results in a changed ratio of the inhibition and excitement mediators in neurocytes and glial cells.
The testing revealed that the drug had not only an anti-depressive but also neotropic effect, this is why it was employed in the treatment of not only epilep- sy. but also in the therapy of cerebrovascular illnesses, including chronic cerebrovascular insufficiency Daily dosages were 1-2 g. The therapeutic effect occurred on the 2nd-4th day. The drug was frequently used in combination with Glucaprim.
In epileptology, the action of this drug is universal The therapeutic effect was recorded in all forms o of the disease, including Jackson's seizures.
A manifest anti-diuretic effect is a unique feature of the drug. This is the reason why Aminovil was largely applied in hydrocephaly patients; in terms of therapeutic efficiency. it outperforms other diuretic drugs such as Diacarb.
Trevit acts in much the same way. Its diuretic effect is less pronounced, however, and, in case o - prolonged administration, it may cause side effect: and complications.
Evit, an amino acid compound, was used only to stop nocturnal attacks. It was applied for long peri- ods in two-week courses of treatment with 10-day intervals. The action mechanism: serotonin exchan- ge correction and suppression of LPO processes.
Concluding a review of the drugs that indirectly confirm the leading role of the "glutamate hypothesis" in the mechanism of the decay of nerve cells one more group of drugs, glutamic acid antagonist; (of the rilosol type), should be singled out the testing of which is performed in a number of clinics in the world. As yet, positive results are identical and little conclusive.
Thus, the administration of a daily dosage of 10C g rilosol in ALS patients could prolong their life time by 12 months .
The "arginine" hypothesis advanced in the earl) 90s has not won acceptance either. A lot of thera- peutic drugs are still being clinically tested. And while in cardiology and gastroenterology the role o arginine metabolites in the pathogenesis of a num- ber of diseases is considered proven, clinical neu- rology is still making its first steps in this direction.
NO (or relaxation factor) is called a substance o the century. It is a proven fact that the absolute majority of vascular drugs produce a therapeutic effect by changing the NO level in the smooth muscle cells .
Arginine amino acid is a metabolite source in the tissues whereas NO is formed in two stages .
The first reaction catalyzed by NO-synthetase produces hydroxyarginine, a rather stable com-pound that, under the influence of an unknown enzyme (the second reaction), is disintegrated into nitric oxide and citrulline.
The NO-synthetase activity and, hence, the speed of nitric oxide formation in the tissues are controlled by the calcium level and determined by the function. al state of NMDA receptors. In its turn, nitric oxide that also activates the cGMP-synthetase triggers of a cascade of reactions that lead to a decrease in the cation level in the cells by suppressing the inositide cycle .
It should be pointed out that most NO effects in neurocytes are due to a changed cGMP-synthetasE activity. The highest enzyme activity was recorded in the cerebellum, the region of the reticular cerebra trunk formation, the subcortical structures as well a: the occipital lobule of the cerebral hemispheres There is clear evidence that in some cerebral struc- tures NO act as a kind of neuromediator .
It should not be overlooked, however, that a large amount of NO represents a toxic agent. The duration of the radical is several seconds. But if superoxide radicals in the cell are in excess, a combined longliving toxic peroxinitrate anion, a powerful initiator of LPO processes, is formed .
Unlike nitric oxide, peroxynitrate anion stimulates the capture of calcium by mitochondria dissociating tissue breath and phosphorylation processes which eventually produces a drop in the energetic potential of the nerve cells with ensuing consequences.
According to the literature, genetically determined superoxide dismutase insufficiently occurs in some degenerative diseases, in particular, in ALS patients .
In an experiment, a restriction of NO-synthetase activity by injecting inhibitors (N-nitro-L-arginine) produced a favorable effect. Thus, 2-3 injections of the drug (in subcortical degenerations) resulted in a decreased progression of the pathologic disorder. In another group of animals with experimental cerebral ischemia a significant reduction of the size of foci was reported when an NO-synthetase inhibitor was previously injected [11, 6].
There is still another risk of producing enzyme hyperactivation consequences: instead of the end product, N0, the cell may accumulate an excessive amount of an intermediate product of the reaction, Nw-hydroxy-L-arginine which is a potent cytostatic agent that inhibits the key DNA synthesis enzymes .
Thus, the literature corroborates the important role played by nitrogen oxide in the pathogenesis of nervous system diseases. Unfortunately, laboratory research does not make it possible to determine the metabolite content in bioliquids, this is why we have to confirm only indirectly our assumptions regarding disturbed metabolism of this compound. In particular, we often determine the content of nitrites and nitrates, end products of the nitric oxide metabolism. For this purpose, patients were put on a diet without any nitric products after which the content of the nitrate and nitrite level was determined in the blood and liquor of the patients; in this case a set of reagents of the German Merck company was used.
While in healthy persons the nitrate content in 1 ml of cerebrospinal liquid was in the 3-8 mkg range, in ALS patients, irrespective of their age, form and duration of the disease, invariably zero results were recorded. A proved metabolite decrease was also found in parkinsonism, multiple sclerosis patients and in other diseases.
On the basis of the results arrived at it was assumed that an accumulation of intermediate NOsynthetase reaction products occurred in some neurologic illnesses. The process was especially active in ALS patients. Neurovit that represents an amino acid compound was employed to regulate the level of nitrocompound; its intake during 2-3 days at a dosage of 0.5-2.0 g produced not only an essential increase of nitrates in blood and liquor but had a manifest therapeutic effect in many nervous disorders.
Thus, the bulbar syndrome of whatever origin (ALS, consequences of severe cerebral circulation impairment, neuroinfection, etc.) was dealt with by administrating the pharmaceutical preparation during 10-14 days.
And yet the produced therapeutic action dwindled to nothing in a matter of 14-22 days which called for continued drug injection with short 7-12 day intervals. As a result (supervision lasted for 2 years), we could manage to stabilize the pathologic process in patients with central motoneuron involvement.
Recovery of minor movements in ICP and ACCI patients is another Neurovit application. In the concluding phase the course of treatment lasts 14-18 days.
Hence, as far as the effectiveness and the manifestation speed of the therapeutic effect are concerned, the drug has no likes in world practice. Its area of application as well as that of other pathogenic preparations is limited. however.
On the contrary, the hypothesis on genetic determination of the decay of nerve cells (the apoptosis phenomenon) still remains universal and wins an ever growing number of supporters. In 1988, a gene was identified whose derepression leads to the generation of a specific protein that destroys DNA cells and triggers apoptosis .
It is hard to identify in vivo the apoptosis characteristics: DNA fragmentation and a change of neuron membrane permeability. All the more so that, depending on the amount of the protein produced, the process may be prolonged and occasionally go on for many years. Injuries, hypoxia, frequent convulsive seizures, neuroinfectious diseases and other factors can significantly accelerate the cell decay program implementation which modern medicine is unable to prevent despite the impressive armamentarium of therapeutic drugs.
Postmortal studies of the brain of patients who died of traumas or somatic diseases point to the possibility of a similar mechanism of the death of nerve cells. Yet it should not be ignored that apoptosis is a physiological process. In this fashion, 3-5% of nerve cells with metabolic defects are rejected in the prenatal (and, partially, in the postnatal) period. However, the process of the decay of nerve cells risks (under the influence of certain etiologic factors) to degenerate into a large-scale process and then large cell groups may lose their viability .
In these conditions, moderate hypoxia, birth traumas, neuroinfection, etc. produce irreversible changes precisely in these regions.
In this case, the cell decay process may last for a long time and gradually form a clinical picture of infantile cerebral palsy. Many years ago, Prof. K.A. Semyonova proved such a possibility although the development and prolongation mechanism of the pathologic process remained unclear.
One could think: should the hypothesis be confirmed, the treatment tactics for neurologic diseases would have to be radically revised. Yet, this is unfeasible in the present stage; it is advisable to perform the therapy along the following three lines:
1) suppression of the apoptosis process;
2) correction of the disturbed metabolism processes (pathogenic therapy);
3) elimination of factors stimulating apoptosis (prevention).
Examples and significance of the pathogenic therapy were mentioned above. Apoptosis pharmacology is still in its infancy and most of pharmaceutical preparations are being experimentally tested.
As our experiments and two-year clinical studies have shown, the application of the drug Provit permits not only to arrest apoptosis but to simultaneously correct the disturbed metabolic processes as well.
It is recommended to administer Provit in children of the "risk group" aged 5-60 days when the nonformed hematoencephalitic barrier does not limit penetration of amino acids through nerve cells. The course of treatment lasts 20 days. The treatment results are presented below.
Another problem. Is it possible to prevent apoptosis development in adults? To do so, it is necessary to identify in every specific case factors stimulating this process, that is, an in-depth study of the pathogenesis of disease at the molecular level is required.
Take, for instance, multiple sclerosis, a severe demyelinating autoimmune disease. Etiology is unknown. Specificity, as compared with other autoimmune diseases, was not revealed. The pathogenesis at the molecular level, though, was established in sufficient detail.
Furthermore, genetic predisposition of individuals to multiple sclerosis has been established in the recent years. This applies not only to the immune component.
Apoptosis in oligodendrocytes triggered at a certain developmental stage severely inhibits remyelination processes preparing the ground for myelin loss .
Under these conditions, myelin loses the set of antiinflammatory means: antioxidants, proteolytic enzyme inhibitors, unique lipids; the flammatory reaction produced by autoimmune mechanisms can therefore be indefinitely long.
Yet, the process is not of total nature. For some unclear reasons, single cell groups are affected which contributes to the formation of separate foci. Apoptosis is stimulated by the following factors: g-interferon produced by T-lymphocytes; tumor necrosis factor (TNF) produced by astrocytes and macrophages; interleukin-I and others.
The cited biologically active compounds stimulate apoptosis in lesioned oligodendrocytes closing the vicious circle. This is why the disease is continually progressing. In these conditions, drugs of the (3-interferon group, y-interferon and TNF antagonists, may substantially inhibit the pathologic process activity, yet they are unable of suppressing apoptosis.
The triggered cell death mechanism in oligodendricytes is going on and there are noguarantees against repeated exacerbation.
For this purpose, we suggest the following preparations.
"Halovit" that inhibits the activity of macrophages, T-lymphocytes and astrocytes and restores the myelin's antioxidant background. Until recently, the drug was used to suppress the activity of macrophages in patients with intestinal infections. The use of the drug in the treatment of demyelinating diseases will allow cell death suppression and will vigorously strengthen the remyelinating process. The application areas of other drugs, cholamine and dechol, are T-lymphocytes. The drugs stimulate the triggering of apoptosis in these immunocompetent cells by restricting their activity.
Primavit, being an activator of ATP activity, was used as a pathogenic drug in treating multiple sclerosis patients.
The drug produced a pronounced therapeutic effect 3-4 days following the beginning of its intake. Its main destination is elimination of pyramidal symptoms and pelvic disorders. In this respect, composit surpassed other preparations (especially, hormones) used to this end.
Combined application of the enumerated drugs made it possible to arrest the pathologic process in 83% of multiple sclerosis patients. Over a 4-year period of supervision of the group of patients con cerned, not more than 2 exacerbations were report ed while prior to treatment most of the patients were considered as those with progredient disease form.
As of today, the determined cell death hypothesis prevails both in terms of experiment and practice The fight against apoptosis is complicated and requires a detailed knowledge of fine pathogenesis mechanisms and adequate metabolic therapy.
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