L-ALPHA-GLYCEROPHOSPHORYL-D-MYO-INOSITOL FOR THE TREATMENT OF PERIPHERAL NEUROPATHIES AND OF CEREBROPATHIES

Abstract: Pharmaceutical compositions for the treatment of peripheral neuropathies of dysmetabolic or toxic origin and of cerebropathies of organic and functional origin, containing as the active ingredient L- alpha -glycerophosphoryl-D-myo-inosital, as such or as the alkali or alkali-earth metal salt thereof.

Description: - L-ALPHA-GLYCEROPHOSPHORYL-D-MYO-INOSITOL FOR THE TREATMENT OF PERIPHERAL ROPATHIES AND OF CEREBROPATHIES DISCLOSURE The present invention relates to pharmaceutical compositions for the treatment of peripheral neuro¬ pathies of dysmetabolic or toxic origin, and of cere- bropathies of organic and functional origin, containing as the active ingredient L-e(-glycerophosphoryl-D-myo-i- nositol (hereinafter called GFI) of formula 1 or the alkali or alkali-earth metal salts thereof. 15 The present invention also relates to the alkali and alkali-earth metal salts of GFI, particularly the calcium salt of GFI. From a chemical point of view, GFI is structurally 20 similar to phosphatidylinositol (hereinafter called FI); FI being the double-acylated product with fatty acids, mainly unsaturated acids, at the hydroxy groups of the glycerine residue of glycerophosphorylinositol. FI is a molecule of natural origin, almost 25 unsoluble in water, which is generally extracted from bovine brain and/or soy-bean, in admixture with phosphatidylethanolamine ("cephalinic fraction") and subsequently purified. FI turns out to be rather unstable, since the un- saturated fatty acid chains bound to the hydroxy groups of the glycerin residue easily undergo peroxydation re- actions, yielding a number of decomposition products. On the contrary, GFI is the deacylated analogue and therefore it is water-soluble as such or salified, it is stable and the alkali and alkali-earth metal salts thereof, specifically the sodium, potassium, calcium and magnesium salts, are crystalline and particularly suited for use in pharmaceutical formulations. The advantages involved in the use of said salts, particularly the calcium salt, compared with the free acid GFI, consist in a lower hygroscopicity, a higher stability, a better adaptability to the use thereof in pharmaceutical compositions, since the salts themsel¬ ves, being poorly hygroscopic, can be preserved for a long time without appreciable decompositions. GFI, or the salts thereof with alkali and/or alkali-earth metals, particularly the calcium salt, are obtained by controlled saponification of the acylated phospholipid mixture contained in soy-bean, subsequent separation with purification of the obtained free acid GFI and optionally salification with alkali and/or alkali-earth metals. A known method for the preparation of GFI is de¬ scribed in EP-A-0.217.765. From a biochemical point of view, FI catabolism is known to play a very important role in the biochemical events connected with physiological activity, as far as phosphorus turnover is concerned (Ansell G.B. and Dohmen H. ; J. Neurochem. 2^ 1, 1957; Sheltaway A. and Dawson R.M.C. ; Biochem. J. , 111, 157, 1969). When calcium is available as a support for the ac- tivity of phosphatidylinositol phosphodiesterase (phospholipase C) , this enzyme is probably the main re¬ sponsible for FI degradation (Friedel R.O. , Brown J.D. and Durrell J. ; Biochim. biophys. Acta. , 144, 684, 1967; Keough K.M.W. and Thorgpson W. ; Biochim. biophys. Acta. , 270, 324, 1972; Thompson W. ; Can. J. Biochem., 45, 853, 1967; Dawson R.M.C. et al. ; Biochem. J. , 122, 605, 1971; Irvine R.F.; Biochem. J. , 176, 475, 1978), the main metabolits of which are diacylglycerole and inositolphosphate; the whole metabolic cycle (Hawthorne J.N. and Pickard M.R. ; J. Neurochem., _32!, 5, 1979) can be represented as follows: PHOPHATIDYLINOSITOL IACYLGLYCEROL C£>_°-DIACYLGLYCEROL INOSITOLPHOSPHATE Even though a poor acylation of FI can occur under particular physiological conditions (low Ca ++ concen¬ tration) , which acylation being catalysed by the enzyme phospholipase A, and giving raise to lysophosphatidyli- nositol (Hong S.L. and Deykin D. ; J. Biol. Chem. , 256 , 5215, 1981), the specificity of phosphatidylinositol phosphodiesterase, the ubiquitous distribution in animal cells and the extremely high activity thereof, evidenced also in vitro (Friedel R.O. , Brown J.D. and Durrell J. ; Biochim. biophys. Acta., 144, 684, 1967; Keough • * ■ :_.ϊ-M.W. and Thompson W. ; Biochim. biophys. Acta., 270, 324, 1972; Thompson W. ; Can. J. Biochem., £5, 853, 1967; Dawson R.M.C. et al. ; Biochem. J. , 122, 605, 1971; Irvine R.F. ; Biochem. J. , 176, 475, 1978), make this enzyme the main responsible for phosphoinositidic metabolism. Now, it has been found that GFI has a marked acti¬ vity in peripheral neuropathies of dysmetabolic or toxic origin and in cerebropathies of organic and func¬ tional origin. The results from pharmacological experimentation are reported hereinbelow, by way of examples. EXAMPLE 1 Activity of GFI in the enteric autonomic neuropathy of diabetic origin The neurological correlates of diabetes have been clearly described as functional and morphological alte¬ rations of both sensory and motor peripheral nerves. These abnormalities are underlined by significant changes of axonal transport with a concomitant axonal atrophy that is followed by degeneration. The autonomic diabetic neuropathy is associated with gastrointestinal problems such as diabetic diar¬ rhea, constipation, reduced small intestinal transit and megacolon, and with an altered innervation of the gut. Alloxan-induced experimental diabetes shows a re- markable correlation between neuronal and gastrointe¬ stinal alterations. Substance P, et-enkephalin and VIP are contained in enteric neurons with different projections and dif- ferent activities. Substance P and VIP axons project orally, while enkephalin neurons project anally. Sub¬ stance P produces direct excytation of enteric neurons and smooth muscle, while enkephalin produces inhibition of enteric neurons and muscle contraction by a direct action, although an indirect effect through acetylcho- line stimulation cannot be excluded. VIP has smooth mu¬ scle relaxant activity. The gastrointestinal functions are likely regulated by the fine interplay among these neuronal components. In the diabetic autonomic neuro- pathy of the gut such coordinated activity is altered thus compromising the gastrointestinal functions. The test was carried out on male Sprague Dowley rats (weighing about 250 g) divided in 3 groups: a con¬ trol group (C) , a diabetic group (D) and a treated dia- betic group (DDTRp). Diabetes was induced by " single subcutaneous injection of 100 g/kg alloxan (glycemia >400 mg/dl) and, starting from the seventh day after diabetic induction, treatment was carried out subcuta¬ neously with 10 mg/kg of (GFI) ? Ca for three months. The following parameters were measured: body wei¬ ght, blood glucose, met-enkephalin, substance P and VIP. All of these parameters, being substantially af¬ fected in the diabetic group, were restored in the tre- ated diabetic group and turned out to be similar to those of the respective control group, with statisti- cally significant variations. Tables 1 and 2 show the met-enkephalin and sub¬ stance P values measured during the test. Besides all rats of the diabetic group D show a typical cataract: the crystalline was very fragile and crumbled during dissection. In the contrary crystalline of diabetic group DDTRp was clear and normally- resi¬ stant to dissection procedure. TABLE 1. MET-ENKEPHALIN (pg/mg prot. ) SMALL INTESTINE C = Control group * p<0.05 D = Diabetic group *** p<0.001 DDTRp=D post-treated with (GFI)_Ca n.s.=not significant TABLE 1. - cont'd MET-ENKEPHALIN (pg/mg prot.) LARGE INTESTINE Rectum pg/mg prot. C 102.4 ± 11.8- (n = 5) D 78.7 ± 5.1 (n = 7)** DDTRp 120.0 ± 1.6 (n = 8)n.s, Colon pg/mg prot. C 101.0 + 10.2 (ι_*= . _.7) D 46.2 ± 3.8 (n = 8)*** DDTRp 90.5 ± 5.6 (n = 8)n.s. C = Control group * p<0.01 D = Diabetic group *** p<0.001 DDTRp=D post-treated with (GFI)_Ca n.s.=not significant TABLE 2. SUBSTANCE P (ng/mg prot.) SMALL INTESTINE Duodenum • C D DDTRp Jejunum C 1.43 ± 0.1 D 0.78 ± 0.05*** DDTRp 1.31 ± 0.04 n.s. Ileum ng/mg prot. C 1.22 ± 0.06 D 0.39 ± 0.03 *** DDTRp 1.13 ± 0.09 n.s. C = Control group * p<0.001 D = Diabetic group n.s.=non significant DDTRp=D post-treated with (GFD-Ca TABLE 2. - cont'd SUBSTANCE P (ng/mg prot.) LARGE INTESTINE Caecum ng/mg prot. C 0.76 ± 0.05 D 0.37 + 0.02*** DDTRp 0.50 ± 0.02** Colon ng/mg prot, C 0.58 ± 0.02 D 0.35 ± 0.02*** DDTRp 0.56 ± 0.03 n.s. Rectum ng/mg prot, C 0.54 ± 0.03 D 0.34 ± 0.02*** DDTRp 0.55 ± 0.02 n.s. C = Control group * p<0.01 D = Diabetic group *** p<0.001 DDTRp=D post-treated with (GFI)_Ca n.s.=not significant EXAMPLE 2 Activity of GFI in early enteric autonomic neuropathy of diabetic origin Experimentally induced diabetic rats will be trea- ted according to the above said modalities (see EXAMPLE 1) to evaluate the gastroenteric protection in the same moment in which the neuropathic damage is established. Treatment was carried out i.p. with 10 mg/ g of (GFI)-Ca for 35 days, starting from the seventh day af- ter diabetic induction. The observed parameters (body weight, blood glucose and met-enkephalin) were reesta¬ blished in the treated diabetic group DDTRp in a simi¬ lar way as described in EXAMPLE 1. EXAMPLE 3 Activity on receptor and second messenger in peripheral organs in experimental diabetic neuropathy The animals, treatment, groups and dosage are si¬ milar to those reported in EXAMPLE 1, while days of treatment are 35 like in EXAMPLE 2. The measured parameter was insulin receptor in li- ver, by binding of 125I-ιnsulιn, analized by the method of Scatchard (G. Scatchard; Ann. N.Y. Acad. Sci. ; 51, 660, 1970), using purified hepatic plasma membranes, prepared by the method of Ray (T.K. Ray; Biochem. Biophys. Acta; 196, 1, 1970). The number of high affinity receptor binding sites 125 for I-insulin ( B maχ1 ) n liver is increased in dia¬ betic rats. The treatment with (GFI) 2 Ca prevents par¬ tially this increase in diabetic rats. EXAMPLE 4 Anti-amnesic activity of GFI using a one-trial passive avoidance test with electro-convulsive shock as the amnesic agent. This passive avoidance test was carried out using a conventional apparatus consisting of a chamber with a grid floor and an elevated runway protruded from the front wall of the chamber. The runway is illuminated while the chamber is dark. When placed on the runway, a rat can enter the dark chamber through an opening. A scramble footshock can be delivered through the grid floor of the- dark compartment. The test was subdivided during 3 days : on the first day, each rat was subjected to the usual learning training; on the second day, the treated group was administered orally with (GFI) Ca at three dose levels: 3-30-100 mg/kg, and after 60 rain. , training was carried out by means of both the footshock, as a further learning agent, and electro-shock, as the amnesic agent. On the third day, training was repeated measuring the latency time necessary for the rat to enter the room. The test was carried out in comparison with Pira- cetam, administered at doses of 300-1000 mg/kg per os, and significance of the results in favour of GFI was evidenced comparing the doses of 30-100 mg/kg of GFI with those of 300-1000 mg/kg of Piracetam. EXAMPLE 5 Activity of GFI in the nerve conduction velocity Male Fischer F-344 rats with an initial weight of 271 g were used. The diabetes was induced with 70 mg/Kg of streptozocin and, in order to prevent an uncontrolled deterioration of the general physiological state of the diabetic animals, they were given daily s.c. injections of 0.75 U insulin. The animals were assigned to the three groups C, D and DDTRp (see EXAMPLES 1, 2 , 3) . The treatment was performed with (GFI)_Ca 10 mg/Kg i.p. for 8 weeks, starting from the first day of diabe- tes. The measured parameter was the tail nerve conduc¬ tion velocity (CV) as reported by Spϋler et al. (M._ Spϋler, W. Dimpfel, H.-U. Tϋllner; Arch. Int. Phar a- codyn., 287, 211, 1987). In particular it was evalua- ted: 1. The CV of proximal section of motor nerve 2. The CV of proximal section of sensory nerve 3. The CV of distal section of sensory nerve The CV was measured before induction of diabetes (3 month old rats) and- after 8 weeks• of treatment. It decreased in diabetic group D which confirmed the deve¬ lopment of a peripheral diabetic neuropathy, while, as reported in table 3, in DDTRp group it was statisti¬ cally increased, suggesting the therapeutic effect of (GFI)2Ca in diabetic neuropathy. TABLE 3 H•t- p < 0.001 D vs. C and DDTRp vs, It is known (D.A. Green, P.V. de Jesus and A.I. Winegrad; J. Clin. Invest., 5J5, 1326, 1975) that this kind of test was positively performed in the past trea¬ ting streptozotocin-induced diabetic rats with 650 mg/Kg of myo-inositol, but this result was not confir¬ med later by other tests, although the doses of myo- inositol were always very high (J.G. Salway, J.A. Fin-- negan, D. Barnett, L. Whitehead, A. Karunanayaka and R.B. Payne; 2_, 1282, 19781. For this reason the possi- ble therapeutic use of myo-inositol failed. As it is evident from the above results, GFI can conveniently be used as the active ingredient in phar¬ maceutical compositions for the treatment of peripheral neuropathies and of cerebropathies of organic or functional origin, such as vasculopathies, Alzheimer disease, involutive syndromes in the elderly and similar pathologies. Examples of pharmaceutical compositions suited to the oral administration comprise capsules, soft cap- sules, tablets, granulates, powders, solutions, sa¬ chets, sustained-release forms, containing 10 to 500 mg of GFI (as such or as an alkali or alkali-earth metal salt, preferably as the calcium salt), per unitary dose, to be administered 2-3 times a day, according to the diagnosis and the patient's conditions. For the parenteral administration, both intrave¬ nous and intramuscular, suited forms are lyophilized vials or sterile solutions containing 2 to 250 mg of GFI (as such or as an alkali or alkali-earth metal salt, preferably as the calcium salt), per unitary dose, to be administered 1 to 3 times a day. The compositions of the invention can possibly contain other active ingredients having a complementary or anyhow useful activity. The physico-chemical characteristics of GFI and of the calcium salt thereof used in the above described pharmacological tests and prepared according to the above reported conventional procedures are shown he- reinbel GFI free acid MW = 334 g/mole m.p. = 140°C Elemental analysis : C (theoretical: 32.33%) = 32.25% H (theoretical: 5.69%) = 5.73% GFI calcium salt (GFI) 2 Ca ; MW = 706 g/mole [όU D = -15.5° ± 1 (c=2.07; H 2 0) 1 H-NMR (300 MHz; D 2 0) : ppm 3.37 (dd; CH-5; J 5 _ g =J 5 _ 4 =9.27 Hz) ppm 3.58 (dd; CH-3; 3 _ 2 = 2 - 78 Hz J J 3_ = 9 * 27 Hz) ppm 3.65-3.75 (m; CH-4 + CH 2 ~9) ppm 3.79 (dd; CH-6; J 6 _ 5 =J g _ 1 =9.27 Hz) ppm 3.90-4.10 (m; CH-1 + CH-8 + CH 2 -7) ppm 4.31 (dd; CH-2 ; J 2 _3 =J 2_1 = 2 ' 78 Hz) ppm 4.80 (s; DHO) 13 C-NMR (D 2 0) (fully decoupled) ppm 69.03 (s; C-9)

Claims: CLAIMS 1. Pharmaceutical compositions for the treatment of pe¬ ripheral neuropathies of dysmetabolic or toxic origin and of cerebropathies of organic and functional origin, containing as the active ingredient L-ol-glyceropho- sphoryl-D-myo-inositol, as such or as the alkali or- alkali-earth metal salt thereof. 2. Pharmaceutical compositions as claimed in claim 1, containing as the active ingredient L-o^-glyceropho- sphoryl-D-myo-inositol as the calcium salt. 3. Pharmaceutical compositions as claimed in claims 1 or 2, for the oral administration. 4. Pharmaceutical compositions as claimed in claims 1 or 2, for the parenteral administration. 5. Pharmaceutical compositions as claimed in any one of the preceding claims, containing 5 to 500 mg of L-o6- glycerophosphoryl-D-myo-inositol per unitary dose, as such or as the alkali or alkali-earth metal salt. 6. Pharmaceutical compositions as claimed in claim 5, ~ containing L-o^-glycerophosphoryl-D-myo-inositol as the calcium salt thereof. 7. The use of L-ol-glycerophosphoryl-D-myo-inositol and of the alkali or alkali-earth metal salts thereof for the preparation of pharmaceutical compositions for the treatment of peripheral neuropathies of dysmetabolic or toxic origin and of cerebropathies of organic and func¬ tional origin. 8. The use of L-tf-glycerophosphoryl-D-myo-inositol cal- cium salt for the preparation of pharmaceutical compo¬ sitions for the treatment of peripheral neuropathies of dysmetabolic or toxic origin and of cerebropathies of organic and functional origin. 9. L-o-glycerophosphoryl-D-myo-inositol salts with alkali or alkali-earth metals. 10. L-o^-glycerophosphoryl-D-myo-inositol calcium salt.

Inventors: PALAZZI CAMILLO MARIA FRANCESC, PROCIDA CARLA, SCOLASTICO CARLO, PALAZZI, CAMILLO, MARIA, FRANCESCO, GIULIO, PROCIDA, CARLA, SCOLASTICO, CARLO
Applicants: DEPHA TEAM SRL, DEPHA TEAM S.R.L.
Application year: 1990
Main classification: C07F9
IPC codes: A61K31, A61P25, C07F9