Abstract:
The present invention is directed to plasmalogens and pharmaceutical compositions thereof which are useful for treating neurodegenerative diseases. Carbons 1 and 2 of the phosphoglyceryl backbone of the plasmaolgens have from 10-24 carbon atoms containing from 1 to 6 double bonds. The phosphoryl group is bonded to choline, ethanolamine, serine or inositol.

Description:
DESCRIPTION OF THE INVENTION 
     The present invention relates to the field of therapeutic chemistry and more particularly to the one of phosphoglycerids. 
     More precisely, it consists in pharmaceutical compositions notably intended for neurodegenerative diseases treatment, based on phosphoglyceroethers and phosphoglyceroesters. 
     STATE OF THE ART 
     Specifically, it consists in pharmaceutical compositions containing phosphoglyceroethers or plasmalogens, combined or not with phospholipids, admixed or diluted into inert, not toxic carriers, suitable for digestive or parenteral administration. 
     Phospholipids are defined as esters constituted of a glycerol molecule esterified in position 1 and 2 with saturated or unsaturated fatty acids chains, and, in position 3 with a phosphoric acid derivative. Most of natural phospholipids contains saturated fatty acids chains such as stearic acid or palmitic acid. Moreover, the phosphoric group is bound to an aminoalkyl chain to form the phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and phosphatidylinositol. These compounds which form the lecithins class are principally of alimentary value. 
     The phospholipids, according to this invention, are characterized in that the glycerol moiety is esterified and/or etherified by non saturated fatty chains. 
     Previous tests have shown the relevance of phospholipids containing polyunsaturated fatty acids as an esterifying group. 
     Their function in nutrition and dietetic has already been the subject to several patent applications depending on whether they origin from cerebral tissues or whether they come from the yolk of hens egg extracts the food of which has been modified. 
     In fact, these phospholipids are carriers of fatty acids chains with a high number of carbons and they have a high unsaturation degree of the n-3 series, especially the acid in C 22  :6. 
     The superiority in nutritional function of the cerebral phospholipids in comparison with other sources of n-3 fatty acids has been more particularly demonstrated (J. M. BOURRE J.Neur.Chem. 60 (1993) 2018-2028). 
     SUMMARY OF THE INVENTION 
     The present invention thus relates to pharmaceutical compositions in which the phosphoglycerids are, partially or predominantly, substituted by plasmalogens the formula of which is: ##STR1## wherein R is an alkyl radical and, preferably, an alkenyl radical having one or several double bonds up to 5 double bonds, and from 10 to 24 carbon atoms R&#39; is a linear or branched, fatty, alkenyl of 10 to 24 carbon atoms and from 1 to 6 double bonds and the nitrogen atom is inserted into a secondary, tertiary or quaternary, linear or cyclic structure. 
     The Merck Index (1989) page 1194, defines plasmalogens as aldehydogenic lipids extracted from the animal kingdom and nothing is known about their therapeutic usefulness. 
     Their nomenclature is not already entirely determined and it is spoken as ethanolamine-phosphatidal or plasmalogen-ethanolamine, or plasmalogen-choline to define more precisely the nitrogenous structure carried by the phosphoric group. 
     Phosphoglycero-ethers of the invention seem to have a high affinity for polyunsaturated fatty acids with a long chain of the n-3 series and it can be deduced therefrom a great physiological significance for eicosanoids. In this case, one characteristic of plasmalogen is that it contains twice more from a polyunsaturated fatty acid (DHA i.e. C 22  : 6 n-3) than other phospholipids. 
     Plasmalogens molecules are essentially present on the membrane and seem endowed with remarkable membrane exchange qualities. So, they would have a greatest metabolic activity at the level of the membrane reconstruction. 
     Moreover, it has been shown that cultured mutant cells which do not synthetize plasmalogen are very sensitive to agents generating free radicals (O. MORAND J.Biol.Chem. 263 (1988) 11.797). This leads to suppose a vitamin E-like protective effect of the plasmalogen with a role of anti-oxidizing in the oxidative stresses (B. ENGELMANN Biochem.Biophys.Res.Commun. 204 (1994) 1235). 
     The relevance of phosphoglycero-ethers appears during various genetic diseases, as well in the animals as in the humans. So, among others, mutant mice breeds, used in laboratory, like &#34;Jimpy&#34; mice and &#34;Quaking&#34; mice have a depressed plasmalogens level, as well in the brain as in the spinal chord (M. H. HACK J.of Chromatogr. 145 (1978) 307), likewise pigs suffering from congenital trembling (D. S. PATTERSON J.Neurochem. 21 (1973) 397). 
     Besides, in the humans, the Zellweger&#39;s cerebrohepatorenal syndrome is a rare genetic disease with a brain degeneracy associated with an etherphospholipids insufficiency due to a lack of peroxisoms which are their biosynthesis sites. 
     In this disease, a great quantity of phospholipids esters with very long fatty acids chains of the n-6 serie is found in affected children&#39;s brain with, simultaneously, a very important decrease in the level of the n-3 series fatty acids, and particularly, of C 22  -6 (DHA) (A. POULOS Biochem.J. 253 (1988) 645). 
     Some other hereditary diseases induce neurological disorders of demyelinisation with a significant decrease in the level of plasmalogen at the cerebrospinal level, it is such the case of the Pelizaeus-Merzbacher&#39;s leukodystrophic disease (J. M. BOURRE Europ.J.Neurol. 17 (1978) 317), as well as in the Refsum infantile disease too, the punctata chondrodysplasia and some others (A. K. HAJRA Plenum Press (1988) 369-380). 
     Some other degenerative diseases are associated with a perturbation of the phospholipids metabolism and, particularly, of plasmalogen at the cerebrospinal level, it is such the case in multiple sclerosis (J. M. BOGGS Neur.Chem.Res. 7 Aout 1982 p.953-954). 
     In several demyelinisation diseases and in ischemia, a rise of the plasmalogen level in cerebral tissues is observed, that is confirming the role of plasmalogens in myelin (L. A. HORROCK Adv.Exp.Med.Biol. 100 (1978) 423-438). 
     The degradation of phospholipids membranes seems to be associated with Alzheimer&#39;s disease and a lowering of cerebral phosphatidylcholine and phosphatidylethanolamine level (R. M. NIBCH Proc.Natl.Acad.Sci.USA 89 (1992) 1671), and also, a lowering of the rate of polyunsaturated fatty acids and, particularly C 22  : 6 (M. SODERBERZ Lipids 26 (1991) p.421-425) are reported. 
     Similar modifications of the same order have been reported in the Down&#39;s syndrome (B. W. BROOKSBANK Mal.Chem.Neuropathol. 11 (1989) p.157-185). 
     Preceeding informations have incited applicants to search phospholipidethers sources and to measure their possible protective efficacy towards oxydizing agents or to degenerative processes. 
     Mammalian brain and spinal chord appear as the best sources of phospholipidethers, in the form of plasmalogens; the used preparation contains about 12% of plasmalogens with a high proportion of polyunsaturated n-3 fatty acids, the composition of which is following: 
     
         ______________________________________sphingomyeline       5 to 10%phosphatidylcholine       20 to 30% of which 1/20 is in plasmalogens formphosphatidylserine       15 to 20%phosphatidylinositol       3 to 5%phosphatidylethanol-       30 to 40% of which 2/3 are in plasmalogensamine       formphosphatidic acid       3 to 5%lysophospholipids       2 to 10% of which total plasmalogens are 10 to       15%fatty acids n-3 total =       11%ratio n-6/n-3 =       15%ration C.sub.22 : 6 (n-3)/       10%fatty acids total) =______________________________________ 
    
     By comparison, hens eggs fed with a rich feeding of n-3 series fatty acids contain a lower proportion but however significative of plasmalogens as well as a lesser quantity of n-3 serie long chain fatty acids as it is shown in Table I below: 
     
                       TABLE 1______________________________________HEN EGGS PHOSPHOLIPIDS     ETHER LIPIDS______________________________________Cholinephosphatidyl             70%      0Phosphatidyl ethanolamine             16%      2.8 to 4%Other phospholipids             12%      0Total amount of lipids ethers                      0.5 to 1%n-3 fatty acids total             6 to 7%Ratio n-6/n-3     5 to 6%Ratio C.sub.22 : 6 (n-3)/             5%Fatty acids total______________________________________ 
    
     This is the reason why a cerebral phospholipids preparation rich in plasmalogens (phosphoglycero-ethers) the composition of which and the plasmalogens content seems to be particularly suitable, is preferably used. 
     In a more particular way, phospholipids used in the present invention contain from 5 to 25% of total plasmalogens and preferably from 10 to 15%. 
     The compositions, according to this invention, also find a use to fight against intoxications or nervous degeneracies, diseases in which either a deficiency in the biosynthesis or a phospholipids degradation containing an ester link or an ether link with an hydrocarbonated polyunsaturated fatty acid chain, particularly, the one of the n-3 series, can be shown. It results in a more or less reversible damage of the nervous cell membrane wall. 
     The compositions according to this invention are intended to digestive or parenteral administration. For these aims, there will be added to excipients or diluents suitable for these ways of administration, such as inert mineral products, for example, calcium carbonate, tricalcium phosphate, magnesium phosphate, alumina, colloidal silica, kaolin, clays, aluminium silicate, calcium silicate or iron oxide for the digestive way of administration, of aqueous liquids for the parenteral way of administration. 
     The compositions according to this invention, can also contain organic inert supports of organic nature such as starches, dextrins, lactose, cellulose, cellulose synthetic derivatives, alginates, carrhagenates, fatty acids, waxes or resins. 
     The compositions according to this invention can also contain other active agents with complementary action such as the group B vitamins (vitamin B1, vitamin B2, vitamin B6, folic acid, pantothenic acid, pangamic acid, vitamin PP) or mineral salts or trace elements such as selenium, lithium or rubidium. 
     The compositions according to this invention thus appear in the form of drinkable vials, flasks, soft gelatine capsules, uncoated or coated tablets, lozenges, granules or flavoured or not, sweetened or not powders. 
     The compositions according to this invention can also appear in the liquid form such as, for example, a gelified preparation or a drinkable suspension or even more an oil in water emulsion. 
    
    
     The present invention is also described with more details in the following examples: 
     EXAMPLE I 
     A preparation based on phospholipids rich in ether lipids (plasmalogen) and preferably extracted from mammalians or fishes brains or spinal chord and optionally from hens eggs. 
     Cerebral phospholipids 10 to 300 mg of which 10 to 15% of ether lipids: 
     
         ______________________________________• Vitamin B1  0.30 to 4.2 mg• Vitamin B2  0.30 to 4.8 mg• Vitamin B6  0.35 to 6 mg• Folic acid  30 to 600 μg• Vitamin B12 0.15 to 3 μg______________________________________ For one capsule 
    
     EXAMPLE II 
     Capsules based on cerebral phospholipids rich in ether lipids (plasmalogen) 
     
         ______________________________________• Preparation according to example I                   70.0   g• Lactose         175.5  g• Vitamin E       20.0   g• Microcrystalline cellulose                   30.0   g• Calcium stearate                   4.5    g______________________________________ For 1000 capsules 
    
     EXAMPLE III 
     Capsules based on cerebral phospholipids rich in ether lipids (plasmalogen) 
     
         ______________________________________• Preparation according to example I                   65.0   g• Magnesium oxide 75.0   g• Zinc gluconate  100.0  g• Maltodextrin    59.25  g• Colloidal silica                   0.75   g______________________________________ for 1000 capsules 
    
     EXAMPLE IV 
     Capsules based on cerebral phospholipids rich in ether lipids (plasmalogen) 
     
         ______________________________________• Preparation according to example I                   90.5   g• Magnesium oxide 100.0  g• Yeast autolysate titrated in vitamins                   55.0   gof the B group• Carboxymethyl cellulose                   53.6   g• Colloidal silica                   0.9    g______________________________________ for 1000 capsules 
    
     EXAMPLE V 
     Plasmalogen-based capsules 
     
         ______________________________________• Preparation according to example I                   100.0  g• Lactose         770.0  g• Magnesium stearate                   20.0   g• Colloidal silica                   3.0    g______________________________________ for 1000 capsules 
    
     EXAMPLE VI 
     Tablets based on cerebral phospholipids rich in ether lipids (plasmalogen) 
     
         ______________________________________• Preparation according to example I                   95.5   g• Magnesium oxide 250.0  g• Sorbitol        637.5  g• Microcrystalline cellulose                   15.0   g• Colloidal silica                   2.5    g______________________________________ for 1000 capsules 
    
     EXAMPLE VII 
     Ampuls to be drunk based on cerebral phospholipids rich in ether lipids (plasmalogen) 
     
         ______________________________________• Preparation according to example I                   45.0   g• Ammonium glycyrrhizinate                   12.5   g• Purified water  10     l______________________________________ for 1000 ampuls of 10 ml 
    
     EXAMPLE VIII 
     Sachets of drinkable powder based on cerebral phospholipids rich in ether lipids (plasmalogen) 
     
         ______________________________________• Preparation according to example I                   100.0  g• Magnesium oxide 500.0  g• Zinc gluconate  125.0  g• Vitamin E       30.0   g• Maltodextrin with a high absorption                   2.5    gcapacity• Diluent q.s.______________________________________ for 1000 sachets 5 g 
    
     EXAMPLE IX 
     Injectible suspension based on cerebral phospholipids rich in ether lipids (plasmalogen) 
     
         ______________________________________• Cerebral phospholiplds             10 mg• Sodium chloride             15 mg• PPI water QS______________________________________ for 1 ampuls of 5 ml 
    
     EXAMPLE X 
     Ether lipids dosage in mammalians brains extract has been performed by two methods: 
     A) Bidimensionnal chromatographic method 
     Firstly, total lipid extraction by the Folch method as it is described by B. Entressangles, in &#34;Manuel des corps gras&#34; (editions Lavoisier-Paris) 1992-P. 1414-1418. Ether lipids are separated from total phospholipids by bidimensionnal chromatography CIH vapors intermediate hydrolysis. The thus hydrolysed ether lipids into corresponding lyoderivatives, separate from diacylated phospholipids during the second dimension course. 
     B) By NMR spectrometry 
     Phospholipids are quantified by  31P! NMR spectroscopy using triphenyl phosphate as an internal control. The samples are treated in a special way to obtain lines and a good resolution. The resulting values are % of phospholipids moles in relation to internal standard. 
     In order to convert % mole into % per weight, the values are multiplied by the corresponding phospholipid molecular weight. 
     Cholesterol and cerebrosides are quantitatively determined by  1H!NMR spectroscopy while by adding internal corresponding standards. Ethers lipids data are obtained from signals included in the three spectra. Free fatty acids data are calculated from the  13C! NMR spectrum. Additionally to  31P! NMR measurements,  1H! NMR spectra and and a  13C! NMR spectrum have been carried out. Cholesterol and cerebrosides are added as a standard in order to obtain quantitative values for these two lipids types through the  1H! NMR spectrum. By using the  13C! NMR spectrum, free acids and alkenyl ethers content, can be calculated. Table 2 shows the values obtained on cerebral phospholipids. 
     
         ______________________________________Total phospholipids             53.6%PC-Ether                  0.7%PE-Ether                  12.0%Cholesterol       7.0%Cerebrosides      9.0%Free fatty acids  5.0%Total             74.6%______________________________________ PC-Ether = phosphatidylcholine ether PEEther = phosphatidylethanolamine ether 
    
     EXAMPLE XI 
     Lipids ethers preparations from pig cerebral tissues 
     Cerebral phospholipids according to this invention are obtained by extracting pig brains removed from freshly slaughtered animals according to the following methods: 
     pig brains are removed from freshly slaughtered animals stemming from breedings without any infectious diseases and rigorously followed by veterinary services for the sanitary conditions. 
     brains are immediately frozen at -20° C. and kept at this temperature 
     brains are then reduced to a temperature comprised between -5° to 0° C. before getting through an industrial mincer and grinders, in order to obtain a liquid paste the content of water of which, is about 80%. 
     the brain paste is transferred to the top of an atomizing room wherein water is immediately evaporated through a hot air flow at 190°/195° C. 
     the resulting powder is fed into a reactor which contains a mixture of aliphatic hydrocarbons based on hexane and kept under stirring. 
     after filtration, the liquid phase is vacuum-concentrated and give rise to a crude extract 
     the crude extract is then run into acetone in the presence of an alimentary antioxidising agent 
     the obtained precipitate is filtered under nitrogen pressure 
     the collected product is vacuum-dried and contains the purified cerebral phospholipids. 
     EXAMPLE XII 
     Pharmacological effect demonstration of the preparations according to this invention. 
     A plasmalogen-rich (phosphoglycero ethers) cerebral phospholipids preparation has been used as a protective agent in a certain number of animal tests. 
     a) On pyrithiamin-induced experimental neuritis 
     Pyrithiamin is an antimetabolite which at a 5 mg/Kg IP dose, induces in the mice a tetanic hyperpolarization. 
     Phospholipids protective activity was tested, on the one hand as a preventive treatment i.e. administered at the same time than pyrithiamin and, on the other hand, as a curative agent, that is when the first neurological symptoms appear. 
     Results are reported Table 3 below. 
     It is stated that the protective activity during preventive treatment is total until doses as low as 2.5 mg of phospholipids per 10 g of food. Knowing that a 20 g mouse eats about 3 g of food a day, the effective dose is about 0.35 mg of phospholipids rich in plasmalogens according to the invention, per animal. 
     
                       TABLE 3______________________________________Dosis of phospholipids in          Preventive effect                       Curative effect10 g food in the groups under          Con-             Con-experiment     trols   Experim. trols Experim.______________________________________10 mg          +++     0        +++   +5 mg           +++     0        +++   ++2,5 mg         +++     0        +++   ++2 mg           +++     +        +++   ++1 mg           +++     +++      +++   +++______________________________________ +++Acute polyneuritis: al animals die in few days ++Attenuated symptoms: survival of more than 50% of animals +light symptoms: all animals survive 0 no symptom 
    
     b) Protective effect of phospholipids rich in plasmalogen on oxotremorine-induced neurological symptoms 
     Oxotremorine induces in the mice very characteristic static tremblings, of the Parkinson&#39;s disease type, as well as peripheral symptoms such as lachrymation and salivation. 
     
                       TABLE 4______________________________________Dosis of phospholipids       Duration of  Intensity of the symptomsin 10 g food       the treatment                    as a function of time afterin the groups under       with phospholipids                    arrest of the treatmentexperiment  before tremorine                    1 j    2 j  3 j  4 j______________________________________ 0 mg                    +++    +++50 mg       3 days       0      0    ++30 mg       3 days       0      0    ++20 mg       3 days       0      ++   +++10 mg       3 days       0      +++ 0 mg                    +++    +++50 mg       1 days       0      0    +++30 mg       1 days       0      0    +++20 mg       1 days       0      +++10 mg       1 days       0      +++______________________________________ 
    
     Table 4 clearly shows that 50 mg of phospholipids per 10 g of food administered during 3 days before oxotremorine injection, completely inhibit symptoms appearance (tremblings), this inhibition to the oxotremorine action lasts 3 days. 
     c) Effects of phospholipids rich in plasmalogens on experimental allergic encephalomyelitis 
     An encephalomyelitis is induced among 20 rats by injecting a spinal chord suspension (Freund&#39;s adjuvant). Disease grows up from the 10th to the 14th day. These animals divided into 3 groups have been treated every day with increasing dosis of phospholipids. 
     
                       TABLE 5______________________________________Dosis of brain      Lack of symptomsphospholipids I.M      after 7 j               after 14 j                        after 21 j                                Not cured______________________________________0,01 mg    1        2        1       160,02 mg    8        17               3 0,5 mg    6        18               2______________________________________ 
    
     The curative effect is thus very clear. 
     EXAMPLE XIII 
     Clinical effect demonstration of preparations according to this invention 
     A clinical trial have been undertaken with a group of children suffering from severe mental handicaps. One treated group of 104 children has been compared to an untreated group of 85 children aged from 1 to 14. Treatment consists in a 30 mg phospholipids supply a day per os for 6 to 12 months periods as the case may be. Psychometric tests, I.Q. measurements or electroencephalographic recording have been achieved according to the age or the symptomatology. 
     It will be noted in Table 6 below, that more of six children out of ten, have gained an important benefit from this treatment. 
     
                                           TABLE 6__________________________________________________________________________  RESULTS TREATED CHILDREN                        RESULTS UNTREATED CHILDREN  Number      None         Weak             Significant                  Very marked                        Number                            None                                Weak                                    Significant                                         Very marked__________________________________________________________________________DOWN&#39;S  40 0  13  21    6    24   6  15  3    0syndromLITTLE&#39;S   22 6   6   8    2    11   9   2  0    0diseasePost meningo-encephalitic   7  0   3   4    0     7   5   2  0    0conditionPost    9  0   3   4    2     5   4   1  0    0ictero-nuclearconditionEndogenous   13 2   2   6    3    18  11   7  0    0debilityPetit mal   4  0   0   3    1     6   3   2  1    0Grand mal   9  1   2   4    2    10   6   3  1    0TOTAL  104 9  29  50   16    81  44  32  5    0      8,6%         27,8%             48%  15,3%     42,3%                                30;7%                                    4,8%             63,3%__________________________________________________________________________