WO1998056403A1

WO1998056403A1 - Improvement of the regeneration of myelin sheaths

Info

Publication number: WO1998056403A1
Application number: PCT/EP1998/003232
Authority: WO
Inventors: Ilse Bartke; Jürgen Unger
Original assignee: Roche Diagnostics GmbH; Boehringer Mannheim GmbH
Current assignee: Roche Diagnostics GmbH
Priority date: 1997-06-13
Filing date: 1998-05-29
Publication date: 1998-12-17
Anticipated expiration: 1999-12-13
Also published as: AU8021098A

Abstract

NT-3 is a useful therapeutic agent for the treatment of demyelination diseases, especially for the treatment of multiple sclerosis. It is administered preferably in doses between 0,01 νg and 5νg/kg body weight in 1 to 10 injections at an interval of 1-21 days.

Description

Improvement of the regeneration of myelin sheaths

The present invention concerns a process for improving the regeneration of myelin sheaths, in particular by human oligodendrocytes. In addition 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.

It is known that 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). It is further known that 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).

It is known that neurotrophin-3 (NT-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). It is further known that 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.

Summary of the Invention

It was found that 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).

It was further found that for the regeneration of myelin sheaths, i.e., in MS, an interval application of NT-3 leads to an improved and accelerated remyelination of damaged nerve fibers as compared to continuous application of the therapeutic agent.

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 μg and 5 μg/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 μg and 1 μg/kg.

According to the invention, NT-3 is administered preferably in a dose between 3.5 and 350 μg per injection, preferably 3.5 to 210 μg per injection, at an interval of 1 to 21 days.

It is preferred to administer NT-3 intrathecally, into the cerebrospinal fluid space of the ventricle or spinal cord, preferably of the lateral ventricle or the lumbar spinal cord. Detailed description of the invention

The term "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).

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.

The action of 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. In addition, the composition can contain one or several pharmaceutically tolerated protease inhibitors, for example, aprotinin.

Furthermore, 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. In order to produce pharmaceutical preparations, 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.

In addition 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. Examples of such diseases are, for instance, multiple sclerosis, slow virus encephalitis, various forms of myelitis, or heavy metal poisoning.

The composition according to the present invention is preferably administered systemically. The administration can be carried out by methods familiar to a person skilled in the art, for example, intracisternally, intravenously, or peripherally. For the intracisternal or intravenous administration, NT-3 can be suspended, for example, in physiological saline. However, infusion or bolus injections at regular time intervals are particularly preferred.

The pharmaceutical 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. The amount of NT-3 protein which will be effective in the treatment of MS is in a dose between 0.01 μg and 5 μg/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 μg/injection.

Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, oral, intrapulmonal and intranasal administration. In addition, it may be desirable to introduce the pharmaceutical composition of the invention into the central nervous system by any suitable route, including intrathecal, e.g. intraventricular injection. 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).

Furthermore, it may be desirable to administer the pharmaceutical 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.

The following example and figure are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It is understood that modifications can be made in the procedures set forth without departing from the spirit of the invention.

Description of the Figure

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 μg/h NT-3 with an Alzet minipump for 7 days (B) (remaining demyelination in the center of the lesion). Magnification 200x.

Example

Demyelination in "Gottingen Mini Pigs"

Lesion:

An experimental model of demyelination was established in the female, adult "Gδttingen Mini Pig" (age: 10-14 months, weight: 25-30 kg) by stereotaxic injection of lysolecthin (LL) at multiple sites into periventricular subcortical white matter of the brain (usually 2-3 injection sites located in one hemisphere; 5 μl of 1% LL in 0.9 % saline per lesion infused over a period of 15 min). This infusion causes a rapid reduction of myelin sheaths within the diffusion area of the detergent (Blakemore, W.F., Neuropathol. Appl. Neurobiol. 4 (1978) 47- 59).

NT-3 application:

Immediately after LL-application, 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 μl 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., Schmerz 3 (1987) 115-125; Krames, E.S., and Lanning, R.M., J. Pain Symptom Manage. 8 (1993) 539- 548).

Ways of application:

1. Chronic infusion of NT-3 via Alzet pump directly intracerebral, beyond the blood- brain-barrier:

— application interval: 7-20 days

— concentration of NT-3: 0.4 mg/ml , dissolved in PBS, pH 7.2-7.4

— dosages: 0.5 μg/h corresponding to 1.2-4 μg/kg/interval

2. Pulsative injections of NT-3:

After fixation of the catheter with Ionocem® bone cement (IONOS, Seefeld, Germany) 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) injected every 2nd day through the skin into the port, starting 2 days after initial LL lesions, by single bolus injections into the port at a dose of 0.05-5 μg/kg body weight (3-350 μg per injection). 3-5 injections were carried out into the cerebrospinal fluid within a period of 6-12 days after LL-induced demyelination. The effect of single bolus injections of NT-3 described above can be compared with the continuous infusion of NT-3 into the lateral ventricle via osmotic minipumps (Alzet for 6-12 days at a dose of 0.5 μg/h) .

3. Bolus NT-3 injection every second day:

Significant regeneration was also noted under NT-3 treatment in this system, with no apparent differences to animals receiving chronic infusions of NT-3. Furthermore, NT-3 injected in the CSF space in the area of the spinal cord showed the same positive effect on remyelination as found for intraventricular injections.

Controls :

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.

Evaluation of NT-3 effects:

At the end of the NT-3 (control, respectively) application period, 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.

Findings:

— Injection of LL causes a widespread demyelination throughout the area of diffusion.

— Chronic infusion of NT-3 via Alzet pumps:

An improved and accelerated remyelination of the lesioned myelin sheaths of nerve fibers was detected within the application interval as compared to controls. Significant regeneration was observed as early as 7 days after initial lesion whereas severe demyelination was still present in the white matter of sham treated. Effect of intracerebroventricular infusion of Neurotrophin-3 for the stimulation of remyelination in the lysolecithin model of demyelination in minipigs

Method:

Animals received an icv. cannula connected to an Alzet osmotic minipump. NT-3 (concentration: 0.4 mg/ml) was infused into the left ventricle at a rate of 0.2 μg/h immediately after a demyelinating lesion was carried out in the subcortical white matter of the right brain hemisphere by stereotaxic infusion of 25 μl 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.

Results:

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 μg/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.

List of References

Blakemore, W.F., Neuropathol. Appl. Neurobiol. 4 (1978) 47-59

Cohen, R.I., J. Neuro. Sci. 16 (1996) 6433-6442

Gage, F.H., et al., Neuron 2 (1989) 1177-1184

Gage, F.H., J. Comp. Neurol. 269 (1988) 147-155

Giehl, K.M., et al, Euro. J. Neurosci. 8 (1996) 1167-1175

Hefti, F., et al., Brain Res. 293 (1984) 305-311

Hoffman et al., Exp. Neurol. 110 (1990) 39-44

Krames, E.S., and Lanning, R.M., J. Pain Symptom Manage. 8 (1993) 539-548

Lindsay, R.M., et al., Expl. Neurol. 124 (1993) 103-118

Motsch, J., and Robert, B., Schmerz 3 (1987) 115-125

Neveu, I., et al, J. Cell. Biol. 133 (1996) 631-646

Peinado-Ramon, P., et al., Invest. Ophthalmol. Vis. Sci. 37 (1996) 489-500 Phillips, H.S., Neuron 7 (1991) 695-702 Schnell, L., et al, Nature 367 (1994) 170-173 Tuszynski, M.H., et al, Neuroscience 36 (1990) 33-44 Windisch, J.M., et al., Biochemistry 34 (1995) 11256-11263 Xu, X.M., Ex. Neurol. 134 (1995) 261-272

Claims

Patent Claims

1. A process for improving the regeneration of myelin sheaths at lesioned nerve fibers of a mammal, comprising administering human neurotrophin-3 (NT-3) or an active fragment of NT-3 to a mammal in need of such treatment.

2. The process as claimed in claim 1, wherein said mammal is suffering from a demyelinating disease.

3. The process as claimed in one of the preceding claims, wherein said mammal is a human.

4. The process as claimed in one of the preceding claims, wherein said demyelinating disease is multiple sclerosis.

5. The process as claimed in one of the preceding claims, wherein said NT-3 is administered in a dose between 0.01 and 5 μg/kg body weight.

6. The process as claimed in one of the preceding claims, wherein said NT-3 is administered at a treatment interval of between 1 and 21 days.

7. A pharmaceutical composition for use in the treatment of diseases in which demyelination of nerve fibers occurs, comprising human neurotrophin-3 (NT-3) or an active fragment thereof in combination with a pharmaceutically acceptable carrier.

8. A method for the production of a pharmaceutical composition for use in the treatment of diseases in which demyelination of nerve fibers occurs, characterized in that human neurotrophin-3 (NT-3) or an active fragment thereof is used as an essential component of the composition.