Epidermal growth factor as a medicament for the restoration of neurons of the substantia nigra in the treatment of neurodegenerative disorders

Abstract: Pharmaceutical compositions containing Epidermal Growth Factor (EGF) as active ingredient and their use in the treatment of neurodegenerative disorders are described.

Description: The present invention relates to the use of Epidermal Growth Factor for the manufacture of a medicament for the treatment of neurodegenerative disorders caused by a neuronal depauperation of the substantia nigra such as, for example, Parkinson's disease or disorders of traumatic origin. Epidermal Growth Factor (EGF) is a natural protein existing both in human and in mammals which has a stimulating activity on the cell proliferation of epithelial tissue and so accelerates the healing of ulcers of various origin. At first EGF was identified and isolated from the mouse submaxillary glands [J. Biol. Chem., 237 , 1555, (1962)]. The analysis of the protein showed the presence of 53 aminoacids with three disulfide bonds. Generally, this protein is indicated as mouse-EGF or more shortly "m-EGF". The different methods for separation and purification of m-EGF or some suitable enzymatic treatments can bring to obtain a protein in which 2 or 5 terminal aminoacids are lost [J. Biol. Chem., 247 , 7609, (1972)]. These derivatives, which are called "EGF-2" and "EGF-5" respectively, show properties analogous to EGF with 53 aminoacids. EGF is also present in different human biological liquids such as plasma, saliva, urine, amniotic liquid and milk. Human EGF or more shortly "h-EGF" is a protein having substantially the same physico-chemical characteristics and biological activity of m-EGF. For these reasons, from now on, the term EGF includes m-EGF as well as EGF-2, EGF-5 and h-EGF or mixtures thereof. The stimulating activity on the proliferation of epithelial tissue and, more generally, the mitogenic activity has suggested the use in human therapy to enhance epidermal and corneal healing for instance in case of ulcers and ophthalmological surgery. Parkinson's disease is a neurodegenerative disease of the central nervous system which affects about 1-2% of people aged over 60 and which is characterized by a constant depigmentation of the substantia nigra due to a neuronal depauperation of this mesencephalic nucleus. Other neurodegererative disorders are diseases related to cerebral traumas such as traumas due to car crashes or similar accidents. The world incidence of nervous parenchyma effects due to cranial penetrating traumas is extremely high and related to the rate of industrialization and motorization of the considered country. In Europe and in the U.S.A., head traumas represent the main cause of severe motor deficiencies in young people. These motor deficiencies have substantially the symptoms generally known as parkinsonism. At present, only symptomatic therapies such as the therapies with dopaminergic agonists are available for the treatment of Parkinson's disease or of parkinsonism. Levodopa (Merck Index XI Ed., No. 5344, page 860), the most used drug, is able to partially restore the mesencephalic-striatal level of dopamine. Nevertheless, the therapy with levodopa has several limitations which can be summarized in a choreic-akinetic syndrome appearing after about five years from the beginning of the use of the drug. In fact, the patient, who has lost substantially all the neurons of the substantia nigra , which neurons transform levodopa into dopamine, less and less responds to the drug. As a consequence, the patient has a feeling of well-being for a progressively shorter time and shows fluctuations in the daily motory performance, that is hyperkinesia during the phase of efficacy of the drug (chorea) and akinesia (motory block) when the plasmatic levels of the drug are low. The use of slow-release pharmaceutical compositions containing levodopa did not substantially change patient conditions with respect to the chronic treatment. The use of drugs able to directly stimulate the receptor has not even shown to be better than the therapy with levodopa. During the last years some growth factors, and particularly NGF (Nerve Growth Factor), have shown to be able to act as trophic factors towards some neuronal types, promoting neuronal cell survival as well as neurite outgrowth in rat brain cells. We have now surprisingly found that EGF is able to restore, at least partially, in vivo the functionality of the dopaminergic system and therefore it is useful in the therapeutic treatment of neurodegenerative disorders of the central nervous system and, in particular, of Parkinson's disease and of parkinsonism. Therefore, object of the present invention is the use of EGF for the manufacture of a medicament for the treatment of neurodegenerative disorders, caused by a neuronal depauperation of the substantia nigra , by restoration of the neurons of the substantia nigra . Preferably, the medicament containing EGF is in the form of injectable solutions or of freeze-dried powders from which injectable solutions are obtained by dilution at the moment of use. The injectable solutions, optionally prepared at the moment of use by dilution of a freeze-dried powder are particularly suitable for intracerebroventricular administration using osmotic minipumps. Examples of such minipumps are those commercialized as Alzet 2002 ® (trademark of Alza Corp.). Furthermore, when EGF is administered by a route different from the intracerebroventricular route, the use of suitable administration systems containing EGF such as liposomes or nano-particles may be preferred, in order to allow a higher amount of EGF to cross the blood-brain barrier. The in vivo efficacy of EGF in the treatment of neurodegenerative disorders has been evaluated in rodents by studying, before and after EGF treatment, the change in the rotational behaviour of the animal to which a lesion of the nigrostriatal pathway was surgically effected [Acta Physiol. Scand., Suppl. 367, 69, (1971)] (see Example 1). This experimental model is an art-recognized model and it is considered to be predictive in studying the efficacy of drugs in the treatment of parkinsonisms. It is worth noting that, in this experimental model, other neurotrophic factors resulted to be inactive. For example, NGF did not show, in practice, any efficacy in changing the rotational behaviour of the animal [Brain Research, 459 , 398, (1988)]. The preparation of the compositions containing EGF is carried out according to traditional techniques bearing in mind, of course, that EGF is a protein. The amount of EGF to be administered to the patient depends on several factors such as the seriousness of the neurodegenerative disease, the individual response of the patient, associated therapies, the selected pharmaceutical formulation and the selected administration route. In case of intracerebroventricular administration, solutions containing an EGF concentration between 0.1 and 10 mg/l will be administered. In order to better illustrate the present invention, the following examples are now given. Example 1 To a group of Sprague Dowley rats weighing about 200 grams a lesion of the nigrostriatal pathway was surgically effected. After about two weeks, the animals were selected on the basis of the number of rotations in one direction after dopaminergic stimulation, indicative of a lesion of the nigrostriatal pathway. For this purpose, the rats were treated with amphetamine (0.5 mg/kg i.p.) and put into apparatus able to monitor the number of rotations homolateral to the lesion. About 40 days after the lesion, the rats were randomized into two groups showing the same number of rotations. Osmotic minipumps were subcutaneously inserted at intra-scapular level. Shortly before the use, the minipumps "Alzet 2002 R " were filled with a saline solution (200 µl) containing bovine serum albumin fraction V (BSA) 0.1% (Fluka) and EGF (0.6 µg) for each group of treated animals, whereas a saline solution containing BSA only was administered to the control animals. The minipumps were inserted according to the technique described by Jankovic J. in South Med. J., 81 (8), August 1988, 1021-7. In order to realize an infusion for 28 days, it was necessary to substitute the minipumps with other minipumps containing a fresh EGF solution after 14 days. At the end of the infusion, the rats were tested by subcutaneous administration of amphetamine (0.5 mg/kg) every 10 days in order to evaluate the rotational behaviour in comparison with the rotational behaviour, under the same conditions, of not treated lesioned rats. The not treated lesioned rats showed, in behavioural tests carried out after about 2 months from the end of the infusion, an average rotation number of 98±20 (14 animals) while the EGF-treated lesioned rats showed an average rotation number of 45±11 (11 animals) (p≥0.05 student t). These results show that the lesioned animals, which have received EGF, progressively reduced their rotational activity in response to amphetamine; this is indicative of a progressive restoration of the functionality of the previously lesioned nigrostriatal pathway [Nature, 292 , 351, (1981); Science, 204 , 643, (1979)]. Example 2 After the rotational behaviour studies as described in example 1, the animals were sacrificed after about 3 months from the infusion and the brains were studied by an immunohystochemical method in order to evaluate the amount of tyrosine hydroxylase enzyme (TH) present in the nigrostriatal pathways [Istochemie, 33 , 231, (1973); J. Histochem., 25 , 1222, (1977)]. The presence of TH is a recognized sign of the functionality of dopaminergic nigrostriatal neurons. Assuming equal to 100% the TH value present in the not lesioned nigrostriatal pathways, the TH amount in the lesioned nigrostriatal pathways of the not EGF-treated animals was 22%. On the contrary, in the lesioned nigrostriatal pathways of the EGF-treated animals, the TH amount was 78%. In addition, counts of TH-positive neurons in the substantia nigra , starting point of the nigrostriatal pathway, were carried out on each brain. In the EGF-treated lesioned animals, the number of neurons resulted to be about three times higher than that of the not treated lesioned animals.

Claims: Use of Epidermal Growth Factor for the manufacture of a medicament for restoration of the neurons of the substantia nigra in the the treatment of neurodegenerative disorders, caused by a neuronal depauperation of the substantia nigra . Use according to claim 1 wherein Epidermal Growth Factor is m-EGF, EGF-2, EGF-5, h-EGF or mixtures thereof. Use according to claim 1 or 2 for the treatment of Parkinson's disease or of parkinsonism.

Inventors: CARENZI ANGELO, PEZZOLI GIANNI, RICCIARDI SANTE, CARENZI, ANGELO, PEZZOLI, GIANNI, RICCIARDI, SANTE
Applicants: ZAMBON SPA, ZAMBON GROUP S.P.A.
Application year: 1991
Main classification: A61K38
IPC codes: A61K38