Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/18—Growth factors; Growth regulators
  • A61K38/185—Nerve growth factor [NGF]; Brain derived neurotrophic factor [BDNF]; Ciliary neurotrophic factor [CNTF]; Glial derived neurotrophic factor [GDNF]; Neurotrophins, e.g. NT-3

Definitions

• the present invention concerns a process for improving the regeneration of myelin sheaths, in particular by human oligodendrocytes.
• the invention concerns a pharmaceutical composition for treating diseases in which a demyelination of nerve fibers occurs, as well as its use and a process for its production.
• the covering of nerve fibers in the central nervous system (CNS) with myelin is essential for the function of neuronal signal transmission.
• the myelin sheath is formed by oligodendrocytes which wrap myelin sheaths around the axon of a nerve cell.
• Diseases such as multiple sclerosis in which the myelin sheath of the axon is damaged or destroyed also lead to impairments of the oligodendrocytes.
• the identification and characterization of factors which are responsible for the regeneration of myelin sheaths is very important for the treatment of demyelination diseases.
• NGF has potent and beneficial effects on cholinergic neurons after axotomy (see, for example, Hoffman et al., Exp. Neurol. 110 (1990) 39-44).
• NGF infusions stimulate the regeneration of cholinergic neurons in a transsection model (Gage, F.H., J. Comp. Neurol. 269 (1988) 147-155, Tuszynski, M.H., et al, Neuroscience 36 (1990) 33-44).
• infusions of NGF stimulate the expression of choline acetyltransferase (ChAT) (Hefti, F., et al., Brain Res. 293 (1984) 305-311) and p75NGF receptor mRNA (Gage, F.H., et al., Neuron 2 (1989) 1177-1184).
• ChAT choline acetyltransferase
• p75NGF receptor mRNA Gage, F.H., et
• NT-3 neurotrophin-3 regulates the proliferation and survival of developing rat brain oligodendrocytes (Cohen, R.I., J. Neuro. Sci. 16 (1996) 6433-6442). It is further known that NT-3 prevents axotomy-induced death of rat corticospinal neurons in vivo (Giehl, K.M., et al., Euro. J. Neurosci. 8 (1996) 1167-1175). It is further known that NT-3 promotes the survival and development of neurons in the cerebellum of hypothyroid rats in vivo (Neveu, I., et al, J. Cell. Biol. 133 (1996) 631-646).
• NT-3 has an effect on the survival of adult rat retinal ganglion cells (Peinado-Ramon, P., et al., Invest. Ophthalmol. Vis. Sci. 37 (1996) 489-500). Further described is an effect of NT-3 on the promotion of supraspinal axonal regeneration into Schwann cell grafts (Xu, X.M., Ex. Neurol. 134 (1995) 261-272). NT-3 enhances sprouting of corticospinal tract during development and after adult spinal cord lesion (Schnell, L., et al., Nature 367 (1994) 170-173). However, an influence of NT-3 on the myelin sheaths is not known from the state of the art.
• the object of the present invention was therefore to find a substance which acts to improve the regeneration of myelin sheaths and at the same time is not toxic.
• NT-3 improves the regeneration of myelin sheaths in mammals, preferably in humans.
• NT-3 is a protein belonging to the group of neurotrophins, and plays a pivotal role in the neuronal development.
• NT-3 is known to support and regulate the survival and regulation of the phenotypic expression of a multiplicity of neurons of the central and peripheral nervous system.
• NT-3 thus has an influence on neurological diseases such as Alzheimer's disease and Parkinson's disease (H.S. Phillips, Neuron 7 (1991) 695-702, and R.M. Lindsay et al, Expl. Neurol. 124 (1993) 103-118).
• the invention concerns a method of preparing a therapeutic agent for the treatment of multiple sclerosis, the method being characterized in that NT-3 is brought into a pharmaceutically acceptable formulation for administering preferably in a dose between 0.01 ⁇ g and 5 ⁇ g/kg body weight in preferably 1 to 10 bolus injections at an interval of 1 to 21 days, most preferably 1 to 12 days. In a preferred embodiment of the invention, it is preferred to use a dose between 0.1 ⁇ g and 1 ⁇ g/kg.
• NT-3 is administered preferably in a dose between 3.5 and 350 ⁇ g per injection, preferably 3.5 to 210 ⁇ g per injection, at an interval of 1 to 21 days.
• NT-3 intrathecally into the cerebrospinal fluid space of the ventricle or spinal cord, preferably of the lateral ventricle or the lumbar spinal cord.
• NT-3 or "active fragment of NT-3" within the sense of the present invention refers to natural NT-3, in particular, to natural human or murine NT-3, and all fragments or derivatives of NT-3 which have its biological activity, i.e., cause an improvement of the regeneration of myelin sheaths in vivo.
• the process according to the present invention is preferably carried out with a human NT-3, particularly preferably, with human recombinant NT-3.
• NT-3 binds to a single leucin which is a motif of TrkC (Windisch, J.M., et al., Biochemistry 34 (1995) 11256-11263).
• NT-3 Increasing active concentrations of NT-3 did not result in a more rapid regeneration of the myelin sheaths; it was, however, found that the extent of fiber elongation and branching was dependent on the concentration. The fiber production in all oligodendrocytes which survived at least the examination period (2 weeks) could be improved by NT-3.
• NT-3 can also be improved when the myelin sheaths are treated with a combination of NT-3 and one or several protease inhibitors.
• An example of a suitable and preferred protease inhibitor is aprotinin, which is, for example, sold by Bayer (Leverkusen, DE) under the trade name Trasylol®.
• the present invention also concerns a pharmaceutical composition for the treatment of diseases in which a demyelination of nerve fibers occurs and which contains NT-3 or an active fragment thereof, as the active substance and as an essential component of the composition, if desired, together with the usual pharmaceutical vehicles, auxiliary substances, fillers, and diluents.
• the pharmaceutical composition preferably contains human NT-3, especially human recombinant NT-3.
• the composition can contain one or several pharmaceutically tolerated protease inhibitors, for example, aprotinin.
• the invention concerns a process for the production of a pharmaceutical composition for the treatment of diseases in which a demyelination of nerve fibers occurs which contains NT-3 or an active fragment thereof as the active substance and as an essential component of the composition, if desired, together with the usual pharmaceutical vehicles, auxiliary substances, fillers, and diluents, in which one preferably uses human NT-3, particularly preferably, human recombinant NT-3, as the active substance.
• the composition according to the present invention can be processed with therapeutically acceptable vehicles. Suitable vehicles for the production of such solutions are water, polyols, sucrose, invert sugar, and glucose.
• Suitable vehicles for injection solutions are water, alcohols, polyols, glycerol, and vegetable oil.
• the pharmaceutical preparations can contain preservatives, solvents, stabilizing agents, wetting agents, emulsifiers, salts for changing the osmotic pressure, buffers, and, if desired, other therapeutic drugs.
• Diseases in which a demyelination of nerve fibers occurs and which can be treated with the aid of the pharmaceutical composition according to the present invention can, for example, be caused by inflammations, autoimmune processes, enzymes, or toxins.
• diseases are, for instance, multiple sclerosis, slow virus encephalitis, various forms of myelitis, or heavy metal poisoning.
• composition according to the present invention is preferably administered systemically.
• administration can be carried out by methods familiar to a person skilled in the art, for example, intracisternally, intravenously, or peripherally.
• NT-3 can be suspended, for example, in physiological saline.
• infusion or bolus injections at regular time intervals are particularly preferred.
• compositions which are used according to the invention and contain NT- 3 may be administered in any sterile biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline or dextrose solution, preferably in an acidic solution having a pH of about 4 to 5, preferably in an acetate buffer.
• saline buffered saline or dextrose solution
• the amount of NT-3 protein which will be effective in the treatment of MS is in a dose between 0.01 ⁇ g and 5 ⁇ g/kg body weight at an interval of 1 to 21 days, preferably in 1 to 10 injections. It is further preferred to use 3.5 to 350 ⁇ g/injection.
• Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, oral, intrapulmonal and intranasal administration.
• Intraventricular injection may be facilitated by an intraventricular catheter, for example attached to a reservoir, such as an implantable port catheter system, such as the Periplant ® filtrosafe (B. Braun GmbH, Spangenberg, DE).
• compositions which are used according to the invention, locally to the area in need of treatment.
• This may be achieved by, for example, and not by way of limitation, injection by means of a catheter or by means of an implant, said implant being of a porous, non-porous or gelatinous material, including membranes, such as sialastic membranes or fibers.
• NT-3 is preferably administered during the relapse period. If high doses are used, only a few or even one bolus application is sufficient.
• Figure 1 Representative sections through the area of lysolecithin lesion in the subcortical white matter of the frontal cortex in minipigs, receiving placebo treatment (cytochrome C (A)) or continuous intracerebroventricular infusion of 0.2 ⁇ g/h NT-3 with an Alzet minipump for 7 days (B) (remaining demyelination in the center of the lesion).
• a stainless steel needle was implanted in the left ventricle space and affixed with Ionocem®, a ionomeric bonding bone cement (IONOS Seefeld, Germany) and an Alzet® osmotic pump (200 ⁇ l volume, infusion rate was connected with the needle and implanted subcutaneously, or a catheter system (Periplant Filtrosafe, Braun, Melsungen, Germany is implanted either into the lateral ventricle of the brain or in the subarachinoidal space of the lumbar spinal cord (Motsch, J., and Robert, B., care 3 (1987) 115-125; Krames, E.S., and Lanning, R.M., J. Pain Symptom Manage. 8 (1993) 539- 548).
• the intrathecal catheter is connected with a subcutaneous drug delivery system (periplant Filtrosafe, Braun , Melsungen, Germany) which is implanted subcutaneosly in the upper back region.
• NT-3 dissolved in phosphate-buffered saline
• 3-5 injections were carried out into the cerebrospinal fluid within a period of 6-12 days after LL-induced demyelination.
• Controls were carried out by replacement of NT-3 with an equal dose of Cytochrome C® applied in the same ways and time interval as described.
• the experimental animals were sacrificed, the brain was removed and prepared for histological examination: The areas of interest were studied with routine histological (H.E . staining, Luxol Fast Blue staining), immunocytochemical (myelin basic protein-MBP) and MRI-analysis.
• NT-3 (concentration: 0.4 mg/ml) was infused into the left ventricle at a rate of 0.2 ⁇ g/h immediately after a demyelinating lesion was carried out in the subcortical white matter of the right brain hemisphere by stereotaxic infusion of 25 ⁇ l of a 1% solution of lysolecithin (LL) as previously described.
• Pigs were sacrificed 7 days after operation, the brain was removed and fixed in 10% formalin. Paraffin sections through the lesion area were cut and stained for myelin with Luxol Fast Blue or myelin basic protein immunocytochemistry.
• Fig. 1 depicts representative sections through the area of lysolecithin lesion in the subcortical white matter of the frontal cortex in minipigs, receiving placebo treatment (cytochrome C (A)) or continuous intracerebroventricular infusion of 0.2 ⁇ g/h NT-3 with an Alzet minipump for 7 days (B). Sections were stained by immunochemistry for myelin basic protein (MBP). NT-3 infusion results in a profound increase of MBP-immunoreactivity as compared to cytochrome C-treatment (remaining demyelination in the center of the lesion). Magnification: 200x.

Landscapes

• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Engineering & Computer Science (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Medicinal Chemistry (AREA)
• Psychology (AREA)
• Neurology (AREA)
• Zoology (AREA)
• Gastroenterology & Hepatology (AREA)
• Chemical & Material Sciences (AREA)
• Biomedical Technology (AREA)
• Immunology (AREA)
• Neurosurgery (AREA)
• Pharmacology & Pharmacy (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Epidemiology (AREA)
• Animal Behavior & Ethology (AREA)
• General Health & Medical Sciences (AREA)
• Public Health (AREA)
• Veterinary Medicine (AREA)
• Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Priority Applications (1)

Applications Claiming Priority (2)

Publications (1)

Family

ID=8226913

Family Applications (1)

Country Status (2)

Cited By (2)

Citations (3)

• 1998
  • 1998-05-29 AU AU80210/98A patent/AU8021098A/en not_active Abandoned
  • 1998-05-29 WO PCT/EP1998/003232 patent/WO1998056403A1/de active Application Filing

Patent Citations (3)

Non-Patent Citations (1)

Also Published As

Similar Documents

Legal Events