The theoretical approach to the problems of cellular aging, elaborated and followed by this author during the last 30 years, called the membrane hypothesis of aging (MHA), helped to understand the normal functions of the living organisms, and also to identify the site-specific, radical-induced molecular damaging mechanisms, which represent the non-desired side-effects of the oxygen free radicals, the constant formation of which is an attribute of the living state. These effects make vulnerable first of all the cell plasma membrane and cause a series of intracellular functional disorders, first of all slow down the replacement of the damaged components. The logical way of any anti-aging intervention should be, therefore, to increase the available number of loosely bound electrons inside the plasma membrane, which are easily accessible for OH· free radical scavenging process. The present paper gives a brief survey of the available knowledge regarding the theory and practice of the use of a membrane-related anti-aging pharmacon, called centrophenoxine (CPH). It was tested in both animal experiments and human clinical trials. The results strongly suggest that CPH may be efficient also in prevention of the neuronal damages involved in the development of Alzheimer disease.
The central dogma of the free radical theory of aging FRTA1,2,3.4 is that oxygen free radicals are harmful byproducts of the aerobic life and as such, are responsible for aging and numerous diseases. A deeper analysis, however, revealed various contradictions, and paradoxical situations. This author has analyzed the contradictory items of the FRTA5,6,7,8,9,10, outlining a new, comprehensive interpretation of the possible biological role of oxygen free radicals in the living state, cell differentiation and aging, called the membrane hypothesis of aging (MHA)7,8,11,12,13.
Although the MHA implicitly contradicts the central dogma of the FRTA, it does not deny the possibility of damaging side effects of these radicals. The MHA attributes an implicite role to the constant flux of oxygen free radicals in the living state, i.e., offers a much wider basis for the interpretation of the free radical function12,13,14. In terms of the MHA, the FRTA may remain valid with a series of modifications14.
This chapter summarizes the available knowledge regarding the effects of a nootropic drug, centrophenoxine (CPH), the neurostimulating effect of which may be useful in the prevention of the age-dependent deterioration of brain cell functions.
The MHA attributes a leading role in differentiation and aging processes to the plasma membrane, undergoing inevitable, continuous alterations during the life5,6,7,10,12,15,16. The alterations are due in part to free radical induced molecular damage, and also to the "residual heat" formed during each depolarization of the resting potential. Due to these membrane alterations, an accumulation of dry mass (i.e., a decrease of the intracellular water content) takes place continuously in the intracellular space. This is a necessary process for the development and maturation, but becomes a rate-limiting factor above a certain physical density of the cell colloids. This latter statement is supported by the fact that the in situ enzyme activities in the cells are all strongly dependent on the density of their microenvironment. MHA is valid mainly for the postmitotic cells, like neurons, muscle cells, etc.. It is particularly evident in the erythrocytes where no de novo protein synthesis takes place. The MHA has been strongly supported by the recent developments of molecular genetics10,12.
CPH is an ester of p-chlorophenoxyacetic acid (PCPA) and dimethylaminoethanol (DMAE)17,18, called also meclophenoxate. The relevant early literature considering its in vitro and in vivo effects have been reviewed11. It is important to note that this drug originally classified as ‘neuro-energeticum’ became nowadays one of the most frequently used representative of nootropica in various countries11. Numerous beneficial effects of CPH have been observed in cases like cerebral atrophy, brain injury, postapoplectic disorders, chronic alcoholism, barbiturate intoxications, etc.19,20,21,22.
Prolonged administration of CPH to healthy old animals reduced significantly the accumulation of lipofuscin in the brain cells23 and myocardium. The medium life span of the CPH-treated old animals increased significantly, and the learning ability of the old, treated mice improved as compared to their age-matched controls24,25,26,27. On the basis of these results, CPH was considered as a potential anti-aging drug11.
It was suggested that the DMAE moiety enters the choline synthesis cycle (and improves the acetylcholine supply of the brain)28,29. This explanation had been contradicted by others30,31. It is evident that an increase of choline availability by transforming a fraction of DMAE into choline cannot be the only explanation of the effect of this drug, since in this case a choline-rich diet alone should have the same effect as CPH has, which is not the case30,31. Further relevant data have also been discussed32.
If 14C-labelled DMAE or CPH was administered intravenously, much higher levels of DMAE were encountered in the brain after CPH treatment, than with DMAE alone, since the esterified form of DMAE with PCPA penetrates much better the blood-brain barrier33. CPH is hydrolyzed on both sides of the barrier in its two component parts in vivo. However, the DMAE moiety becomes phosphorylated in the brain to yield phosphoryl-DMAE, which was converted to phosphatidyl-DMAE, seemingly the end-metabolite of DMAE in the brain33. Phosphatidyl-DMAE is incorporated in the nerve cell membranes, and remains in this form for a relatively long time on the place of choline33, i.e., it forms a special class of phospholipids in the brain cell membranes. The PCPA moiety is excreted in the urine, apparently without any metabolic change33. Although some fraction of DMAE administered either alone or in form of CPH, was found in acid soluble and lipid cholines in the brain33, evidence is available that trimethylation of DMAE takes place only in the liver, but not in the brain33. Therefore, the presence of DMAE in the nerve cell membrane had to be considered as the starting point of any approach to the mechanism of action of CPH.
Experiments have revealed the effects of DMAE on the brain functions in terms of local oxyradical protecting effect on the cell membrane components, such as (i) the protein cross-linking model34,35, (ii) electron spin resonance spectroscopy (ESR) in spin trapping experiments36.
Obviously, neither the presence of DMAE in the brain nerve cell membranes33, nor the in vitro OH· radical scavenging ability of this compound36 prove directly that it acts as a OH· radical scavenger also in vivo. However, numerous animal experimental results demonstrate indirectly that the presence of DMAE in the cell membrane is of physiological significance, which can be attributed to their local OH· radical scavenger properties.
Aging causes an increase of microsviscosity of the membrane lipids in the synapto-somal membranes. Measured by fluorescence polarization techniques after diphenyl-hexa-triene (DPH) labeling of brain cortical synaptosomal membranes37, the membrane microviscosity decreased again significantly after treatment of old rats with CPH for 60 days37. Synaptosomal membranes were protected considerably by CPH pretreatment against the toxic effect of in vivo acute Fe2+ overload in the cerebrospinal fluid of young rats38.
The molecular weight distribution of the proteins is considerably shifted toward the higher values in the synaptosomes of the rat brain cortex during aging. A treatment of 60 days with CPH39 reversed this tendency to a significant extent in old rats.
The fluorescence recovery after photobleaching (FRAP) technique40 measures the lateral mobility of membrane proteins and lipids. The lateral diffusion constant of hepato-cyte membrane proteins (Dp) displayed a characteristic, negative, linear age correlation in Fischer 34440,41, Wistar rats42, in C57BL black mice43, in wild mouse strains of considerably different longevity (Peromyscus leucopus, Mus musculus)44, BN/Bi rats45, as well as SAM mice46. The same finding has been described for the Con-A-receptor proteins in the skeletal muscle cells of mice47, and in the large brain cortical cells of rats48. Although membrane lipid lateral mobility (Dl) is almost by an order of magnitude faster than Dp, it showed a very similar age-dependence in hepatocyte membranes49. The age-dependent decays of Dp and Dl of various tissues are inversely proportional to the life span of the given strain50. FRAP experiments on 2-year-old Fischer 344 rats pretreated per os through gastric tube with aqueous solutions of 80 mg/kg CPH for 5 weeks revealed a significantly higher value of Dp than in the controls. Fischer 344 male rats above the age of 2 years display a linear body weight loss until death. This loss was slightly inhibited by CPH44. This fact indicates that the increase in Dp under the effect of CPH indicates an overall improvement of the status of the animals. It is also noteworthy that caloric restriction experiments on mice also increased the Dp values to such an extent which might correspond the life prolonging effect if this intervention51.
As explained by the MHA, the age-dependent decline of the passive potassium permeability in the nerve cells is an important, key issue in brain aging. CPH treatment of 60 days (80-100 mg/kg) in old rats re-increased the passive potassium permeability of the neuronal cell membrane, i.e., it decreased the intracellular potassium content and rehydrated the cytoplasm of brain neurons significantly52,53. These findings indicate that CPH may be useful in the prevention or therapy of the age-dependent brain disorders.
The rates of synthesis of total- as well as mRNA, measured in vivo by means of radioisotope methods in rats, display an age-dependent decrease in the brain cortex of rats between 1 and 2 years of age (to 40-50 % of the young-adult value)54. In vivo treatments of 60 days with CPH re-increased these rates to 80-90%55.
CPH treatment prolonged the medium life span of laboratory animals, especially if the treatment started at relatively young ages. It improved also the learning ability of several animal species53,56. In some cases an increase of the medium life span up to 30 % was described in mice, but only if the CPH treatment started at the age of 6 months.
Several persons around the age of 40 years, including this author, have started to take CPH (500 mg/day) in 1976, and maintained this experimental treatment up to now. Due to the relatively low number of these subjects, this cannot be considered as a scientific experiment, nevertheless, all the participants agree that they maintained their better physical and mental performance than the untreated persons of similar age in their family. Apart from this observation, it is important to note that the treated persons had no side effects even after 31 years. Fifty persons of 76 years of age with male/female ratio of 1/1 were recruited in a double blind, randomized, human clinical trial. CPH treatment of 8 weeks improved the psychometric and behavioral performance in about 50 % of patients with medium level dementia. The placebo group displayed improvement only in 27%57. A considerable rehydration of the intracellular mass was observed in the verum group at the expense of the extracellular liquid, meanwhile the body weight remained unchanged57. These data are mentioned here to demonstrate that nootropica like CPH behave also in humans in the same way as predictable on the basis of MHA.
i. The synthetic interpretation of the biological aging phenomena offered by the MHA represents an interdisciplinary approach describing a cellular mechanism explaining the effects of the oxygen free radicals with general validity and offers good chances for an experimental testing.
ii. The assumption, according to which only site-specific OH· free radical scavengers may be considered seriously as potential preventive anti-aging drugs, seems to be well supported by the experimental facts. The most important theoretical message of the reviewed results is that a proper improvement of the defense against OH· free radical attacks in the brain cell membrane is beneficial.
iii. It should be emphasized that even the future life span-prolonging strategy should be based on the FRTA. Due to the properties of the OH· radical reactions, only a site-specific, non-toxic and physicochemically feasible radical protection offers the hope of any success. It should also be stressed that in highly developed living systems where the natural radical protection is much more efficient than in the rodents, most probably only a multifactorial radical protection can be successful in improving the natural defense system.
iv. Since CPH is able to retard or even avoid the age-dependent deterioration of the brain functions, one can assume that it may act also as a useful drug in retarding (or even avoiding?) the structural and functional declines of typical brain diseases like Alzheimer’s disease (AD). Unfortunately, systemic investigations, to best of our knowledge, have not been performed so far in this respect, although they would be necessary to see the applicability of CPH for such purposes.
1. Harman, D. (1956): Aging, a theory based on free radical and radiation chemistry. J. Ger-ontol., 11, 298-300.
2. Harman, D. (1981): The aging process. Proc. Natl. Acad. Sci. USA, 78, 7124-7128.
3. Harrnan, D. (1988): Role of free radicals in aging and disease. Annal. N.Y. Acad. Sci., 673, 126-141.
4. Harman, D. (1994): Free radical theory of aging. Increasing the functional life span. Annal. N.Y. Acad. Sci., 717, 1-15.
5. Zs.-Nagy, I. (1986): Common mechanisms of cellular aging in brain and liver in the light of the mewbrane hypothesis of aging. In: K. Kitani (Ed): Liver and Aging - 1986, Liver and Brain, pp. 373-387, Elsevier Science Publishers, Amsterdam, New York, Oxford.
6. Zs.-Nagy, I. (1987): An attempt to answer the questions of theoretical gerontology on the basis of the membrane hypothesis of aging. Adv. Biosci., 64, 393-413.
7. Zs.-Nagy, I. (1992): A proposal for reconsideration of the role of oxygen free radicals in cell differentiation and aging. Annal. N.Y . Acad. Sci., 673, 142-148.
8. Zs.-Nagy, I. (1994): The Membrane Hypothesis of Aging. CRC Press, Boca Raton, USA.
9. Zs.-Nagy, I. (1995): Semiconduction of proteins as an attribute of the living state, The ideas of Albert Szent-Györgyi revisited in the light of the recent knowledge regarding oxygen free radicals. Exp. Gerontol., 30, 327-335.
10. Zs.-Nagy, I. (1997): The membrane hypothesis of aging, its relevance to recent progress in genetic research. J. Mol. Med., 75, 703-714.
11. Zs.-Nagy, I. (1994): A survey of the available data on a new nootropic drug, BCE-001. Ann. N.Y. Acad. Sci., 717, 102-114.
12. Zs.-Nagy, I. (2001): Membranes, aging and genome. In: Bertoni-Freddari, C. and Nieder-müller, H. (Eds.): Current Concepts in Experimental Gerontology. Vienna Aging Series, Vol. 6. 3-14. Facultas, Vienna.
13. Zs.-Nagy, I. (2001): On the true role of oxygen free radicals in the living state, aging and degenerative disorders. Annal. N.Y. Acad. Sci., 928, 187-199.
14. Zs.-Nagy, I. (2002): Pharmacological interventions against aging through the cell plasma membrane. A review of the experimental results obtained in animals and humans. Annal. N.Y. Acad. Sci., 959, 308-320.
15. Zs.-Nagy, I. (1978): A membrane hypothesis of aging. J, theor. Biol., 75, 189-195.
16. Zs.-Nagy, I. (1979): The role of membrane structure and function in cellular aging, a review. Mech. Ageing Dev., 9, 237-246.
17. Pfeiffer, C., Jenney, E.H., Gallagher, W., Blaciunore, W., Smith, R.P., W. Bevan, W. jr., Killam, K.F. and Killam. E.K. (1957): Stimulant effect of 2-dimethylaminoethanol, possible precursor of brain acetylcholine. Science, 126, 610-611.
18. Thuiller, J., Rumpf, P. and Thuiller, G. (1959): Derivés des acides regulateurs de crois-sance des vegetaux. I. Proprietés pharmacologiques de l'ester dimethylamino-ethylique de l'acide p-chlorophenoxyacetique (235 ANP). C. R. Seanc. Soc. Biol., 153, 1914-1918 (in French).
19. Schmidt, H. and Broicher, H. (1970): Klinische Erfahrungen bei der Behandlung von Zustanden zerebraler Insuffizienz mit centrophenoxin (Helfergin). Medsche Welt, 33, 1432-1436 (in German).
20. Vojtechovsky, M., Soukupova, B. Safratova, V. and Votava, Z. (1970): The influence of cen-trophenoxine (Lucidril) on learning and memory in alcoholics. Int. J. Psychobiol., 1, 49-56.
21. Herrschaft, H., Gleim, F. and Duus, P. (1974): Die Wirkung von Centrophenoxin auf die regionale Gehirndurchblutung bei Patienten mit zerebrovascularer Insuffizienz. Dtsch. Med. Wochenschr., 99, 1707-1714 (in German).
22. Kugler, J. (1977): Hirnstoffwechsel and Hirndurchblutung. Berichstband Centrophen-oxin-Arbeitstagung, Timmendorfer Strand, 1976, Schnetztor Verlag, Konstanz (in German).
23. Nandy, K. and Bourne, G. (1966): Effects of centrophenoxine on the lipofuscin pigment in the neurons of senile guinea pigs. Nature, 210, 313-314.
24. Nandy, K. (1978): Centrophenoxine, effects on aging mammalian brain J. Am. Geriatr Soc., 26, 74-81.
25. Hochschild, R. (1971): Lysosomes, membranes and aging. Exp. Gerontol., 6, 153-166.
26. Hochschild, R. (1973): Effect of dimethylaminoethanol on the life span of senile male A/J mice. Exp. Gerontol., 8, 185-192.
27. Hochschild, R. (1973): Effect of dimethylaminoethyl-p-chlorophenoxy acetate on the life span of male Swiss Webster albino mice. Exp. Gerontol., 8, 177-183.
28. Haubrich, D.R., Wang, P.F., Clody, D.E. and Wedecking, P.W. (1975): Increase in rat brain acetylcholine induced by choline or Deanol. Life Sci., 17, 975-980.
29. London, E.D. and Coyle, J.T. (1978): Pharmacological augmentation of acetylcholine levels in kainate lesioned rat striatum . Biochem. Pharmacol., 27, 2962-2965.
30. Zaniser, N.R., Chou, D. and Hanin, I. (1977): Is 2-dimethylamino-ethanol (Deanol) indeed a precursor of brain acetylcholine? A gas chromatographic evaluation. J. Pharmacol. Exp. Ther., 200, 545-559.
31. Jope, R.S. and Jenden, D.J. (1979): Dimethylaminoethanol (Deanol) metabolism in rat brain and its effect on acetylcholine synthesis. J. Pharmacol. Exp. Ther., 211, 472-479.
32. Bertoni-Freddari, C., Giuli, C. and Pieri, C. (1982): The effect of acute and chronic centro-phenoxine treatment on the synaptic plasticity of old rats. Arch. Gerontol. Geriatr., 1, 365-373.
33. Miyazaki, M., Nambu, K., Minaki, A.Y., Hashimoto, M. and Nakamura, K. (1976): Comparative studies on the metabolism of beta-dimethylaminoethanol in the mouse brain and liver following administration of beta-dimethylaminoethanol and its p-chlorophen-oxyacetate, Meclofenoxate. Chem. Pharm. Bull., 24, 763-769.
34. Zs.-Nagy, I. and Nagy, K. (1980): On the role of cross-linking of cellular proteins in aging. Mech. Ageing Dev., 14, 245-251.
35. Nagy, K., Dajkó, G., Uray, I. and Zs.-Nagy, I.. (1994): Comparative studies on the free radical scavenger properties of two nootropic drugs, CPH and BCE-001. Ann. N.Y. Acad. Sci., 717, 115-121.
36. Zs.-Nagy, I. and Floyd, R.A. (1984): Electron spin resonance spectroscopic demon-stration of the hydroxyl free radical scavenger properties of dimethylaminoethanol in spin trapping experiments confirming the molecular basis for the biological effects of centrophenoxine. Arch. Gerontol. Geriatr., 3, 297-310.
37. Nagy, K., Zs.-Nagy, V., Bertoni-Freddari, C. and Zs.-Nagy, I. (1983): Alterations of the synaptosomal membrane microviscosity in the brain cortex of rats during aging and centrophenoxine treatment. Arch. Gerontol. Geriatr., 2, 23-39.
38. Nagy, K., Floyd, R.A., Simon, P. and Zs.-Nagy, I. (1985): Studies on the effect of iron over-load on rat cortex synaptosomal membranes. Biochim. Biophys. Acta, 820, 216-222.
39. Nagy, K. and Zs.-Nagy, I. (1984): Alterations in the molecular weight distribution of proteins in rat brain synaptosomes during aging and centrophenoxine treatment. Mech. Ageing Dev., 28, 171-176.
40. Zs.-Nagy, I., Kitani, K., Ohta, M., Zs.-Nagy, V. and Imahori, K. (1986): Age-dependent decrease of the lateral diffusion constant of proteins in the plasma membrane of hepatocytes as revealed by fluorescence recovery after photobleaching in tissue smears. Arch. Gerontol. Geriatr., 5, 131-146.
41. Zs.-Nagy, I., Kitani, K., Ohta, M., Zs.-Nagy, V. and Imahori, K. (1986): Age-estimations of rats based on the average lateral diffusion constant of hepatocyte membrane proteins as revealed by fluorescence recovery after photobleaching. Exp. Gerontol., 21, 555-563.
42. Kitani, K., Zs.-Nagy, I., Kanai, S., Sato, Y. and Ohta, M. (1988): Correlation between the bili-ary excretion of ouabain and the lateral mobility of hepatocyte plasma membrane prote-ins in the rat. The effects of age and spironolactone pretreatment. Hepatology, 8, 125-131.
43. Zs.-Nagy, I., Kitani, K. and Ohta, M. (1989): Age dependence of the lateral mobility of pro-teins in the plasma membrane of hepatocytes in C57BL/6 mice, FRAP studies on liver smears. J. Gerontol. Biol. Sci., 44, B83-B87.
44. Zs.-Nagy, I., Ohta, M. and Kitani, K. (1989): Effect of centrophenoxine and BCE-001 treatment on the lateral diffusion constant of proteins in the hepatocyte membrane as revealed by fluorescence recovery after photobleaching in rat liver smears. Exp. Gerontol., 24, 317-330.
45. Kitani, K.,Tanaka, S. and Zs.-Nagy, I. (1998): Age-dependence of the lateral diffusion coefficient of lipids and proteins in the hepatocyte plasma membrane of BN/BiRijHsd rats as revealed by the smear-FRAP technique. Arch. Gerontol. Geriatr., 26, 257-273.
46. Zs.-Nagy, I., Tanaka, S. and Kitani, K. (2001): Comparison of the lateral diffusion coefficient of hepatocyte plasma membrane proteins in 3 strains of senescence accelerated mouse (SAM ). Arch. Gerontol. Geriatr., 32, 119-137.
47. Zs.-Nagy, I., Tanaka, S. and Kitani, K. (1998): Age-dependence of the lateral diffusion coefficient of Con-A-receptor protein in the skeletal muscle membrane of C57BL/6J mice. Mech. Ageing Dev., 101, 257-268.
48. Zs.-Nagy, I., Tanaka, S. and Kitani, K. (1999): Age-dependence of the lateral diffusion coefficient of Concanavalin-A receptors in the plasma membrane of ex vivo prepared brain cortical nerve cells of BN/BiRijHsd rats. Exp. Brain Res., 124, 233-240.
49. Zs.-Nagy, I. and Kitani, K. (1996): Age-dependence of the lateral mobility of lipids in hepato-cyte plasma membrane of male rats and the effect of life-long dietary restriction. Arch . Gerontol . Geriatr., 23, 81-93.
50. Zs.-Nagy, I., Cutler, R.G., Kitani, K. and Ohta, M. (1993): Comparison of the lateral diffu-sion constant of hepatocyte membrane proteins in two wild mouse strains of consi-derably different longevity, FRAP studies on liver smears. J. Gerontol. Biol. Sci., 48, B86-B92.
51. Zs.-Nagy, I., Kitani, K., Ohta, M. and Cutler, R.G. (1993): The effect of caloric restriction on the lateral diffusion constant of hepatocyte membrane proteins in C57BL/6 male mice of various ages, FRAP studies on liver smears. Mech. Ageing Dev., 71, 85-96.
52. Zs.-Nagy, I., Pieri, C., Giuli, C. and Del Moro, M. (1979): Effects of centrophenoxine on the monovalent electrolyte contents of the large brain cortical cells of old rats. Gerontology, 25, 94-102.
53. Lustyik, Gy. And Zs.-Nagy, I. (1985): Alterations of the intracellular water and ion concen-trations in brain and liver cells during aging as revealed by energy dispersive X-ray microanalysis of bulk specimens. Scanning Electron Microscopy 1985/I, 323-337.
54. Semsei, I., Szeszák, F. and Zs.-Nagy, I. (1982): In vivo studies on the age-dependent decrease of the rates of total and mRNA synthesis in the brain cortex of rats. Arch. Gerontol . Geriatr., 1, 29-42.
55. Zs.-Nagy, I. and I. Semsei. (1984): Centrophenoxine increases the rates of total and mRNA synthesis in the brain cortex of old rats, an explanation of its action in terms of the membrane hypothesis of aging. Exp. Gerontol., 19, 171-178.
56. Pék, Gy., Fülöp, T. and Zs.-Nagy, I. (1989): Gerontopsychological studies using NAI ("Nürnberger Alters-Inventar") on patients with organic psychosyndrome (DSM III, Category 1) treated with centrophenoxine in a double blind, comparative, randomized clinical trial. Arch. Gerontol. Geriatr., 9, 17-30.
57. Fülöp, T.jr., Wórum, I., Csongor, J., Leövey, A., Szabó, T., Pék, Gy. and Zs.-Nagy, I. (1990): Effects of centrophenoxine on body composition and some biochemical parameters of demented elderly people as revealed in a double blind clinical trial. Arch. Gerontol. Geriatr., 10, 239-251.