Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/66—Phosphorus compounds
  • A61K31/661—Phosphorus acids or esters thereof not having P—C bonds, e.g. fosfosal, dichlorvos, malathion or mevinphos
  • A61K31/6615—Compounds having two or more esterified phosphorus acid groups, e.g. inositol triphosphate, phytic acid
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/66—Phosphorus compounds
  • A61K31/665—Phosphorus compounds having oxygen as a ring hetero atom, e.g. fosfomycin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
  • A61P25/16—Anti-Parkinson drugs
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/18—Antipsychotics, i.e. neuroleptics; Drugs for mania or schizophrenia
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
  • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
  • A61P35/02—Antineoplastic agents specific for leukemia
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

• the present invention concerns pharmaceutical compositions comprising cyclic glycerophosphates and analogs thereof and treatment of neural-associated conditions and disorders.
• ⁇ GP L- ⁇ -glycerophosphate
• ⁇ GP L- ⁇ -glycerophosphate
• ⁇ GP L- ⁇ -glycerophosphate
• ⁇ GP is a product of enzymatic (Ukita et al, 1955) and alkaline (Clarke and Dawson, 1976) hydrolysis of phospholipids and is formed through the cyclic phosphodiester intermediate 1,2-cyclic glycerophosphate (1,2 cGP) (Ukita et al, 1955; Clarke and Dawson, 1976).
• 1,2 cGP has been detected in algae species (Boyd et al, 1987) as well as in human cancer tissues (Su et al., 1993).
• ⁇ GP can in principle adopt the cyclic form 1,3-cyclic glycerophosphate (1,3 cGP). This compound has been shown to be formed as an intermediate in the phospholipase C hydrolysis of phosphatidyl glycerol (PG) (Shinitzky et al, 1993) and upon further hydrolysis is converted to ⁇ GP.
• PG phosphatidyl glycerol
• cyclic AMP A six-membered cyclic phosphate of foremost biological importance is cyclic AMP.
• the ring of cyclic AMP is actually a derivative of 1,3 cGP backbone.
• Other cyclic phosphates which were detected in biological systems include glucose cyclic phosphodiester (Leloir, 1951), 2',3'-cyclic phosphodiester (Markham and Smith, 1952), ribofiavin-4',5'-cyclic phosphodiester (Forrest and Todd, 1950), myoinositol-l,2-cyclic phosphodiester (Dawson et al, 1971) and cyclic lysophosphatidic acid (Friedman et al, 1996). Except for cyclic AMP and cyclic GMP which have been extensively studied, no specific biological activities have been so far assigned to the other biological cyclic phosphates.
• Parkinson's disease PD
• Parkinson's disease Such diseases often involve degeneration of dopamine-producing neurons.
• Current therapeutic methods are mostly aimed at continuous stimulation of dopamine receptors by drugs which, although initially providing symptomatic relief, gradually lose effectiveness. Furthermore, such drugs do not prevent the progressive degeneration of dopaminergic neurons characteristics of such diseases.
• NGF nerve growth factor
• bFGF basic fibroblast growth factor
• EGF epidermal growth factor
• IGF insulin-like growth factor
• brain derived growth factor and glial derived neurotrophic factor Knusel B., et al, 1990; Knusel et al, 1991; Linn et al, 1993
• TH tyrosine hydroxylase
• CNTF ciliary neurotrophic factor
• Phenyl 1,2, cyclic propanediol phosphate - P-1,2, cPP 15 Cyclic dihydroxyacetone phosphate - cDHAP
• Promoting neural cell differentiation - this term relates to the capability of the CGs used in the invention to cause cells to mature into neural cells after contact therewith. Such activity may be assessed by one of many in vitro and in vivo assays such as those described in the examples below.
• An example for an in vitro assay would be to grow cells capable of differentiating into nerve cells (e.g. PC 12
• In vivo assays may, for example, involve treatment of animals with injured dopaminergic neurons with the tested compounds and testing of motional and limb tremor parameters as well as in situ determination of molecules associated with dopaminergic transmission in the treated animals.
• Target cells any cells which have the potential to mature into neural cells.
• Non-limiting examples of such cells are PC 12 and primary brain cells.
• Analog - relates to any compound which is derived from one of the cyclic glycerophosphates of the invention and which substantially maintains the activity of the cyclic phosphate from which it was derived, including, for example, deoxy analogs and phenyl esters of the cyclic glycerophosphates, preferably, deoxy analogs.
• Substantially maintaining - this term relates to the capability of analogs to promote the activity carried out by the cyclic glycerophosphate from which they were derived to a certain extent.
• the analog's activity will be considered to be substantially maintained wherein the activity is 30% or above, preferably 50% or above, more preferably 70% or above, and most preferably 90% or above the level of the activity of the cyclic glycerophosphate.
• Effective amount wherein the method of the invention is intended for prevention of a non-desired condition, the term "effective amount” should then be understood as meaning an amount of the active compound which, when administered, results in the prevention of the appearance of the said condition. Prevention of such a condition, e.g. a neurodegenerative condition, may be required prior to the appearance of any symptoms of a disease, e.g. in individuals having a high disposition of developing the disease, or when the compositions are used for the treatment of nerve rescue which is expected after nerve injury. Wherein the compositions or methods are intended for treatment of an ongoing non-desired condition, the term “effective amount” should then be understood as meaning an amount of the active compound which is effective in ameliorating or preventing the enhancement of the treated condition and related symptoms.
• Neural promoting activity encompasses a variety of neural related activities which may be promoted in target cells upon their contact with the CGs used in the invention. Such activities include but are not limited to promotion of nerve growth, provision of dopaminotrophic supporting environment in a diseased brain, prevention of nerve degeneration, and nerve rescue.
• Prevention or treatment - the term prevention of disorders and diseases is to be understood in accordance with the invention as a reduction in the probability of the appearance of such disorders and diseases in an individual having a high predisposition of developing such disorders and diseases, reducing the extent of the symptoms associated with such disorders and diseases when they occur or completely preventing their appearance.
• Treatment of such disorders or diseases in accordance with the invention means ameliorating the symptoms associated with the disorders or diseases, reducing the extent of such symptoms or completely eliminating them.
• 1,2 cGP, 1.3 cGP and some of their analogs are capable of promoting neuronal outgrowth of PC 12 adrenal tumorigenic cells in culture after a short incubation period.
• the present invention thus provides, by a first of its aspects, a pharmaceutical composition for promoting neural cell differentiation in target cells comprising a pharmaceutically acceptable carrier and, as an active ingredient, a compound of the general formula I:
• acyl refers to an aliphatic saturated or unsaturated Ci - C 24 acyl group, preferably an acyl group having an even number of carbon atoms, most preferably an acyl group derived from a natural fatty acid such as a saturated aliphatic acyl group selected from acetyl, butyryl, caproyl, octanoyl, decanoyl, lauroyl, myristyl, palmitoyl and stearoyl, or an unsaturated aliphatic acyl group selected from palmitoleyl, oleyl, linoleyl, and ricinoleyl; and the term “aryl " refers to a mono- or poly-carbocyclic aryl group, most preferably phenyl, optionally substituted by Ci - C alkyl,
• the composition comprises cyclic dihydroxyacetone phosphate (cDHAP) or phenyl cyclic dihydroxyacetone phosphate (P-cDHAP).
• the composition comprises 1,2 cyclic glycerophosphate (1,2 cGP) or phenyl 1,2 cyclic glycerophosphate (P-1,2 cGP).
• the present invention also provides a pharmaceutical composition for promoting neural activity comprising a pharmaceutical acceptable carrier and, as an active ingredient, a compound of the general formula I above.
• compositions of the invention may also be used for the treatment of neurodegenerative conditions involving damage to dopaminergic neural cells.
• neurodegenerative conditions involving damage to dopaminergic neural cells. Examples of such conditions are Alzheimer's disease (AD) or Parkinson's disease (PD).
• AD Alzheimer's disease
• PD Parkinson's disease
• the present invention provides a method for inducing promotion of neural cell differentiation of target cells comprising contacting said target cells for a suitable period of time with an effective amount of a compound of the general formula I above.
• a suitable period of time is such a period which enables the compositions of the invention to exert their activity.
• This period of time may easily be determined by a person skilled in the art for each kind of composition and target cells using any of the methods described herewith.
• the period of time required for the CGs used in the invention to be in contact with the target cells in order to exert their effect is very short (several minutes).
• a method for promoting neural activity in an individual comprising administering to the individual in need an effective amount of a compound of the general Formula I above.
• a method for the prevention or treatment of disorders and diseases which can be prevented or treated by promoting neural cell differentiation and/or neural activity comprising administering to a person in need a therapeutically effective amount of a compound of Formula I above.
• Fig. 1 shows photographs of PC 12 cells following their incubation for 48 hours with growth medium containing linear ⁇ glycerophosphate as control (Fig.
• NGF nerve growth factor
• Fig. IB nerve growth factor
• 1,3 cyclic glycerophosphate (1,3 GP) 1,3 cyclic glycerophosphate
• Fig. 2 shows photographs of PC 12 cells grown in culture medium (control)
• FIG. 2A pulsed for three hours with linear ⁇ and ⁇ glycerophosphates (Fig. 2B and 2C, respectively) with the cyclic glycerophosphates and analogs 1,3 cGP, phenyl- 1.3 cGP, 1,2 cGP, 1,3 cPP, and phenyl- 1,3 cPP (Fig. 2D - Fig. 2H respectively) or with NGF (Fig. 21).
• the cells were washed and grown in growth medium and the photographs show the cells on day 7 after treatment with the various factors. Neural outgrowth is seen only in PC 12 cells treated with the above cyclic glycerophosphates and analogs (Figs. D-H).
• Fig. D-H the above cyclic glycerophosphates and analogs
• FIG. 3 shows photographs showing PC 12 cells treated for a period of 7 days control medium (Fig. 3 A), ⁇ GP (Fig. 3B), ⁇ GP (Fig. 3C), the CGs: 1,3 cGP, phenyl- 1,3 cGP, 1,2 cGP, 1,3 cPP, and phenyl- 1,3 cPP (Fig. 2D - Fig. 2H respectively) at a concentration of 0.5 ⁇ M and with NGF (Fig. 31).
• No neural outgrowth was observed after incubation with either ⁇ or ⁇ GP (Fig. 3B and C respectively) while neural outgrowth is observed to different extents in cells incubated with the various CGs (Fig. 3D-H). Under these conditions, extensive neural outgrowth is seen in cells grown with NGF (Fig. 31).
• Fig. 4 shows photographs showing PC 12 cells treated for 14 days with control medium (4 A), with 50 ng/ml NGF (4B), treated for 7 days with 50 ng/ml NGF and then washed and treated for another 7 days either with a growth medium without NGF (4C), or with 2 ⁇ M, 4 ⁇ M or 6 ⁇ M 1,3 cPP (4D, 4E, and 4F, respectively).
• Cyclic glycerophosphates can be formed by enzymatic degradation of phospholipids which in most cases yields five or six membered ring cyclic glycerophosphates.
• the present invention encompasses within its scope compositions comprising both such cyclic glycerophosphates formed by enzymatic degradation of phospholipids as well as synthetically formed ones.
• CGs having rings of less than five or more than six carbon atoms are also included within its scope.
• cyclic glycerophosphates and analogs thereof used in the invention may generally be synthesized using any one of the methods known in the art for synthesis of phosphate esters. Specific methods which may typically be used for preparing the cyclic phosphates of the invention are described specifically below (see Examples).
• Analogs of these cyclic glycerophosphates of the invention are also within the scope of the invention being typically deoxy analogs as well as phenyl esters of 5 the 1,3 cyclic phosphates. These analogs may also be prepared by enzymatic methods or synthetically by any of the methods known in the art.
• the pharmaceutical compositions may also contain a carrier selected from any one of the carriers known in the art.
• the nature of the carrier will depend on the intended form of administration and l o indication for which the composition is used.
• the compositions may also comprise a number of additional ingredients such as diluents, lubricants, binders, preservatives, etc.
• compositions of the invention will comprise about 1 mg to about 10 mg of the active material per kg body weight of the treated individual.
• compositions of the invention will typically contain a single CG, 20 it is possible at times to include in the composition or to co-administer two or more CGs which may then act together in a synergistic or additive manner to prevent or treat the neurogenerative disorder.
• the CGs used in the invention may be used in any of their isomer forms, (see for example, the four stereoisomers which constitute the synthetic 1,3 cGP 25 depicted in Appendix A). For various purposes, one of the isomers may be preferred over the remaining ones.
• hydroxy groups in the starting compound namely Y is - CH(OH) - and/or X is - CH 2 OH -
• these hydroxy groups have to be protected, e.g. by benzylation, and the benzyl group is then removed after cyclization by conventional catalytic hydrogenation in the presence of a suitable catalyst such as Pt or Pd.
• 1,3 cGP was also produced by the cleavage of phosphatidyl glycerol (PG) with phospholipase C as described (Shinitzky et al., 1993). The product had a trace of approx. 10-20% ⁇ -GP as indicated by paper chromatography.
• Example 2 Synthesis of 1,2 cyclic glycerophosphate (1,2 cGP) This compound was prepared as described (Kugel, L. and Halmann, M., J. Am. Chem. Soc, 89:4125-4128 (1967).
• the disodium salt of ⁇ -glycerophosphate (Sigma) was first converted to the acid form and then cyclized with dicyclohexylcarbodiimide (Aldrich). The product, isolated as the Ba salt, was pure on paper chromatography.
• Example 3 Synthesis of phenyl 1,3 cyclic glycerophosphate (P-1,3 cGP) The method described in Example 1 for 1,3 cGP was followed by reacting
• Example 5 Synthesis of 1,2 cyclic propanediol phosphate (1,2 cPP) 1,2 cPP was prepared by the same procedure as in Example 4 but using
• Example 9 Synthesis of cyclic dihydroxyacetone phosphate (cDHAP) This novel compound was prepared by reaction of POCl 3 with dihydroxyacetone .
• PC 12 cell line is one of the most investigated systems in neuronal differentiation. In the presence of nerve growth factor these cells differentiate to neuronal cells.
• PC 12 cells originated from rat pheochromocytoma were grown as monolayers in Eagle's medium (EM) supplemented with 10% fetal calf serum, 50 ⁇ g/ml gentamicin and 5 mM glutamine, in a humidified incubator buffered with 5% C0 2 , at 37°C. The culture medium is changed every four days and the cells are passaged every eight days and performed confluent monolayers (1.5 x 10 ' in a 10 cm plate or 10 cells per well in 24 wells plate).
• PC 12 cells are originally round cells which, following several days in the presence of nerve growth factor (NGF) process nerves. Upon withdrawal of the NGF, the nerves retract and a process of apoptosis is initiated in the cells.
• NGF nerve growth factor
• Sprague-Dawley (SD) rats (weighing 230-250 g) are anesthetized with ketamine plus xylazine administered i.p. and their head secured in a stereotaxic frame.
• 60H-DA (8 mg/4ml) is then injected into the median-forebrain-bundle to destroy the dopaminergic terminals unilaterally (Fitoussi, N., et al. Neuroscience, 85(2):405-413) (1998)). Manifestations of the disease are evident within 2-3 weeks.
• Dopaminergic ablation is assessed behaviorally using a rotometer test, which is based on upregulation of dopamine receptors on the lesioned side.
• Systemic administration of a DA agonist induces rotation in rats with unilateral dopaminergic ablation, with rotation occurring in the direction contralateral to the side of the lesion.
• Cyclic phosphates are administered into the brain using ALZET osmotic pumps (ALZET Co ⁇ oration, Palo Alto, CA).
• ALZET Co ⁇ oration Palo Alto, CA.
• a canulla (30 gauge) is implanted
• Cyclic phosphates are microperfused at a rate of 1 ⁇ l/h for 3 or 14 days.
• Rats are decapitated and their brains rapidly removed.
• the brains are then placed in a rat brain mold on ice and 0.5 mm serial sections are cut and placed on chilled microscope slides.
• Tissue punches are rapidly taken using a stainless steel cannula with an inner diameter of 1.1 mm, according to the following coordinates: Al .5-2.0 mm for the striatum; P5.5-5.0 mm for the SN, and include most of the desired regions.
• the tissue samples are immediately frozen in liquid nitrogen and stored at -70°C until extraction. Extraction is achieved by thawing the punches and subjecting them to probe sonication (80 watts for 5 sec.
• cyclic glycerophosphates The effect of cyclic glycerophosphates on the release and production of GDNF from the SVG-cells and brain tissue extracts is determined as follows. Cells are incubated for 12, 24 and 48 hours with or without cyclic phosphates. Supernatants are taken after centrifugation and analyzed for GDNF using an ELISA kit (ENDOGEN, MA, USA and PROMEGA, Madison, USA, respectively).
• RNA is isolated from cultured cells or tissue extracts, using Tri
• RNA is estimated spectrophotometrically at 260 nm and 280 nm and stored at -80°C until use.
• First strand cDNA synthesis is carried out in a reaction volume of 20 ⁇ l containing 3 ⁇ g of total RNA, 10 mM primer dT (Boehringer Mannheim, Germany) and 1 mM dNTP mix (Boehringer Mannheim, Germany). After heating for 2 min. at 65°C and cooling back to 4°C, the reaction is initiated by the addition of 50 units M-MuLV reverse transcriptase and 20 units RNAse inhibitor (Boehringer Mannheim, Germany). The mixture is then brought to 37°C for 60 mins.
• PCR on the cDNA was carried out in a reaction volume of 50 ⁇ l.
• First strand cDNA (2 ⁇ l) is added to the PCR mixture containing the following components: 0.2 mM dNTP mix (Boehringer Mannheim, Germany), 1 mM each oligonucleotide primer (primers were designed according to the published GDNF cDNA sequence. 5'-TCACCAGATAAACAAATGGC-3' ⁇ 5' ⁇ and 5'-TACATCCACACCTTTTAGCG -3' ⁇ 3' ⁇ corresponding to bases 81-101 and 460-480 respectively) (Biosource, CA, USA), and 2.5 U Taq DNA polymerase (Boehringer Mannheim).
• Reactions are overlaid with mineral oil, and initially denatured at 94°C for 2 min.
• PCR is performed using a MJ Research thermal cycler programmed for 40 cycles consisting of denaturation at 94°C for 1 min. followed by primer annealing at 55°C for 1 min. and primer extension at 72°C for 1 min. At the end of the 40 cycles, the reaction mixture is kept at 72°C for 10 min.
• the PCR product is electrophoretically analyzed on a 2% agarose gel containing ethidium bromide).
• the animals are anesthetized with ketamine and xylazine i.p. and then perfused via cardiac puncture with PBS followed by 4% paraformaldehyde.
• the brains are then removed and post-fixed in 4% paraformaldehyde for 24 hrs and then transferred into 20% sucrose for 48 hours.
• Tissue sections of 35 ⁇ m are obtained using a cryostat and placed in 24 wells plate in PBS. The sections are incubated overnight in 4°C with a primary rabbit polyclonal antibody to Tyrosine hydroxylase (TH) (Chemicon, CA, USA) or a primary mouse monoclonal antibody to glial fibrillary acidic protein (GFAP) (Chemicon, CA, USA).
• TH Tyrosine hydroxylase
• GFAP glial fibrillary acidic protein
• the sections are then washed with PBS, incubated (1 hr) with a HRP conjugated secondary antibody (sheep anti-rabbit or anti-mouse) (Chemicon, CA, USA) and washed with PBS. Then, the sections are incubated with the chromagen diaminobenzidine (DAB), counter-stained with hematoxylin, and screened by light microscopy. Positive staining for TH indicates the amount of dopaminergic- cells in the striatum and substantia nigra, i.e. dopaminergic- cells survival. Positive staining for the GFAP in the injection tract indicates gliorna processes.
• DAB chromagen diaminobenzidine
• PC 12 cells were grown in culture as explained above, the cells were divided into three groups and different factors were added to the growth medium of each group for 48 hours as follows:
• Group B - nerve growth factor at a concentration of 50 ng/ml.
• A growth medium
• B ⁇ GP
• C ⁇ GP
• D 1,3 cGP
• E phenyl 1,3 cGP
• F 1,2 cGP
• Example 15 PC 12 cells were grown as described above and divided into the same groups as described in Example 14 above. The cells were incubated with the various factors for a consecutive period of seven days. Neural outgrowth was monitored and microscopic photographs were taken following the seven day incubation with the above factors. As seen in Fig. 3, neural outgrowth was seen in cells incubated with the various above CGs(Fig. 3B-3H) as well as in cells grown in the presence of NGF (Fig. 31) but not in the control cells grown with the linear glycerophosphates (Fig. 3A).
• PC 12 cells are grown in culture in the presence of NGF under conditions allowing neuronal outgrowth of the cells.
• the NGF is then withdrawn by washing the cells and replacing their growth medium with a medium comprising no NGF.
• the cells then retract and the nerves disintegrate (analogous to the delayed neurodegeneration observed in the vicinity of injured nerves).
• the tested CG is added to the culture either for a short period of time after which it is washed or for a longer period of time and the CGs capability of "rescuing" the nerves is assessed by evaluating the re-differentiation of the cells into neuronal cells.
• Parkinson's disease is induced in rats as described in the Materials and Methods part above by injection of 60H-DA into their brains.
• the rats are then treated either with ⁇ and ⁇ linear GPs or with CG by administration of the either topically, per os, or directly into the brain using osmotic pumps as described above.
• the effect of the linear GPs and of the CGs is assessed by evaluating the in situ production of L-DOPA, dopamine (DA), the dopamine metabolites DOPAC and HNA and the growth factor GD ⁇ F by using microdialysis techniques and by the methods described above. Motional and limb tremor parameters are also quantitatively evaluated in the rats treated with each of the above factors.
• Rats having injured optical nerves are treated with ⁇ and ⁇ linear glycerophosphates or with a CG as explained above and the effect of the above CG on the visual response and nerve generation of the treated rats is monitored.
• Fig. 4A wherein the PC 12 cells were grown in growth medium with no additives added, no neuronal spreading was observed (control). Growth of the cells in the presence of NGF (50 ng/ml) for the full period of 14 days resulted in full neuronal differentiation as seen in Fig. 4B. As seen in Fig. 4C, when the cells were grown for the first 7 days in the presence of NGF (50 ng/ml) and then cultured without NGF for an additional period of 7 days, complete nerve retraction was observed and the level of differentiation of the cells returned to control level.
• NGF 50 ng/ml

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