WO2002043654A2

WO2002043654A2 - Pharmaceutical composition and method for treatment of brain injury, spinal cord injury, stroke, neurodegenerative disease and other conditions

Info

Publication number: WO2002043654A2
Application number: PCT/SE2001/002656
Authority: WO
Inventors: Milos Pekny
Original assignee: Milos Pekny
Priority date: 2000-12-01
Filing date: 2001-11-30
Publication date: 2002-06-06
Also published as: WO2002043654A3; AU2002223170A1; SE0004455D0; WO2002043654A8

Abstract

Disclosed is a pharmaceutical composition comprising a substance that upon administration to a patient leads to an inhibition of extension of cellular processes of astrocytes and/or a retraction of said cellular processes. Disclosed is also use of said substance for the production of a pharmaceutical composition for treatment of a condition selected from the group consisting of brain injury, spinal cord injury, stroke, neurodegenerative diseases, neuronal and/or synaptic loss associated with ageing, disorders of the brain associated with ageing and diabetic retinopathy, and also a method for treatment of said conditions wherein said substance is administered to a patient. Examples of such substances are quercetin, endothelin, hyaluronectin, L2 antibody, L2 glycolipid, L2 tetrasaccharide, TGF-alpha, or a tyrosine phosphatase inhibitor such as orthovanadate or phenylarsine oxide.

Description

PHARMACEUTICAL COMPOSITION AND METHOD FOR TREATMENT OF

BRAIN INJURY. SPINAL CORD INJURY. STROKE, NEURODEGENERAT1NE

DISEASE AND OTHER CONDITIONS

Technical field of the invention The present invention relates to a method for neuron regeneration in the central nervous system, CNS, as well as means for carrying out said method.

Background of the invention

Glial fibrillary acidic protein, GFAP, is a major component of the intermediate filaments present in cells of astroglial lineages, including astrocytes in the CNS, non-myelin forming Schwann cells and enteric glia (Pekny et al, EMBO, vol. 14, pp. 1590-1598 (1995)). In said reference it was reported that mice lacking GFAP developed normally, reached adulthood, and reproduced. Loss of GFAP intermediate filaments was not compensated for by up- regulating other intermediate filament proteins, such as vimentin, and nestin, (as shown in Pekny, M., et al., Exp. Cell Res., vol. 239 ρp.332-343 (1998) and Eliasson, C. et al., JBC vol 274, pp. 23996-24006, (1999)). Intermediate filaments are general constituents of the cytoskeleton, and are present in astroglial cells. The astroglial cells express three different intermediate filament proteins, viz. glial fibrillary acidic protein, vimentin, and nestin. Nestin can not form filaments on its own, vimentin can form filaments together with nestin or GFAP, and GFAP can form filaments on its own. (Eli- asson, C. et al, JBC, vol 274, pp. 23996-24006, (1999)). It was shown that the ability of reactive astrocytes to up-regulate intermediate filaments following an CNS injury is crucial for CNS wound healing, in particular the formation of glial scar (Pekny, M. et al. Jour. Cell Biol. Vol. 145, no. 3, pp. 503-514 (1999)). The results shown suggest requisite intermediate filament protein partnerships for intermediate filament formation in normal and reactive astrocytes.

Xu et al (Glia, 1999) showed that GFAP null astrocytes are neither better, nor worse than control astrocytes in supporting neural outgrowth in vitro. Such astrocytes still contain intermediate filaments which are composed of vimentin and nestin (Pekny et al., Exp. Cell Res., supra). The finding of Xu et al., was contradicted by a report by Menet, V. et al, GLIA, 31:267-272 (2000)) claiming that GFAP null astrocytes are favourable substrate for neuronal survival and neurite growth and showing that inhibition of GFAP synthesis leads to increased neurite outgrowth. However, this paper is based on comparison of mice of one genetic background with mice of another genetic background, and thus no reliable conclusions can be drawn from this experimental set-up.

The statements by Xu et al compared to the statements by Menet et al go in opposite direction, and the statement by Menet et al is impossible to in- terpret, viz. different genetic backgrounds. Thus, the Xu et al statements seem to be more true, also because these authors provide more robust evaluation paradigm and statistical analysis.

Bush, T. G. et al, Neuron, vol. 23, 297-308 (1999) disclose the outcome of physical elimination of all dividing astrocytes, i.e., a sub-population of reac- tive astrocytes. Such elimination triggers activation of the immune system.

Bush et al draw the conclusion that astrocyte loss or dysfunction represents a potentially significant cause of neuronal degeneration. They also show increased regrowth of nerve fibres in the injured area with loss of dividing reactive astrocytes and massive accumulation of immunocompetent cells. The experimental paradigm used in this study does not allow to discriminate between the effect of ablation of a subpopulation of reactive astrocytes and the effect of massive invasion of the lesion area by immunocompetent cells producing many cytokines and other growth promoting molecules.

WO 98/29547 - Hatten, M. et al - relates to modulators of radial glia- astrocyte differentiation and transformation, and diagnostic and therapeutic uses thereof. The modulators hereby act as inhibitors of the astroglial cells.

WO 97/26901 - Eisenbach-Schwartz, M. et al - relates to regeneration of nerves in the central nervous system, whereby astrocytes in vitro are subjected to inflammation-associated cytokines and are then transplanted, in a therapeu- tically effective amount, into the site of an injury of the CNS.

US-A-5,202,120 - Silver, J., et al - relates to method for promoting central nervous system axon regeneration by providing activated immature astrocytes, and administering an effective amount of the activated immature astrocytes to damaged axons to promote axon regeneration. Brief description of the drawings In the summary, description and examples below reference is made to the accompanying drawings on which:

Figure 1 shows the effect of an injury or other damage on astroglial cell activity. The top two drawings illustrate the normal extension of astrocytic processes for untreated astroglial cells, and the bottom two drawings show how it, according to the invention, is possible to even retract those astrocytic processes.

Figure 2 shows high-resolution laser-scanning confocal microscopy im- ages of the inhibition of the extension of astrocytic processes as seen in reactive astrocytes which activity has been modulated as compared to normal reactive astrocytes.

Figure 3 shows the change in the length of the cellular processes of reactive astrocytes for modulated astrocytes (B and C: modulation to a lower de- gree, D: modulation to a higher degree) compared to normal reactive astrocytes (A) after an injury. 100% is the length of astrocytes prior to the injury.

Figure 4 shows the change in the volume of the cellular processes of reactive astrocytes for modulated astrocytes (B and C: modulation to a lower degree, D: modulation to a higher degree) compared to normal reactive astrocytes (A) after an injury. 100% is the volume of astrocytes prior to the injury.

Summary of the invention The inventor has found that it is possible to improve recovery/regeneration after brain or spinal cord injury, stroke, and neurodegenerative dis- eases by modulating astroglial cell activation by means of the present invention, which is characterized in that the astroglial cell activation is modulated preferably within a certain time after an injury, or stroke event.

The inventor has found that astrocyte activation has a positive effect in early stage of a CNS injury, contributing to wound healing, glial scar forma- tion, and blood-brain barrier reconstruction (Pekny, M., et al, J Cell Biol, supra; Pekny, M. et al, GLIA, vol 22, pp 390-400, (1998)). Additionally, it has now been found that astrocyte activation has also a positive neuroprotective effect in early stage of a CNS injury. However, it has also been found that reactive astrocytes have a negative influence on wound healing in the CNS at a late stage of the healing process i.e., in synaptic remodelling. The object of the present invention is to obtain a possibility of regenerating damaged neurons in the central nervous system, neurons that might have been damaged due to injury, stroke or degenerative processes or lost due to ageing in the CNS. Normally, when astrocytes become reactive, e.g. in response to an injury, stroke or a neurodegenerative disease, they show signs of hypertrophy, in particular extension of astrocytic processes. The inventor has now found that it is possible to revert or prevent this, and even to achieve retraction of astrocytic processes compared to the situation prior to the injury, stroke or neurodegen- erative disease, which leads to neuroregeneration. This is further illustrated in Figures 1 and 2, further explained below.

Description of the present invention The invention is based on neuron regeneration in the central nervous system, CNS, in particular recovery/regeneration after brain or spinal cord injury, stroke, and neurodegenerative diseases by modulation of the astroglial cell activation, preferably within a certain time after an injury, or stroke event, or at the discovery of a neurodegenerative disease. By this modulation it is possible to suppress the expression of intermediate filaments in the astroglial cells of the brain or spinal cord. It is also possible to suppress the expression of intermediate filaments in astrocytes present in the brain or spinal cord. In order to achieve this a therapeutically effective amount of a compound modulating the astroglial cells is administered. The modulation is preferably performed at the transition into chronic phase of recovery after injury or stroke event. How- ever, it may also be performed after the initial acute recovery phase by the astroglial cells. Moreover, it was found that after injury, reactive astrocytes extend their processes and create an environment that prevents/does not support neuroregeneration. When this extension of astrocytic processes was inhibited or the processes were made even shorter, neuroregeneration of the damaged brain tissue and restoration of neuronal synapses was achieved. This is further illustrated in figures 1 and 2. Figure 1 shows how modulation of astroglial cell activity results in the inhibition of the extension of astrocytic processes. Moreover, astrocytic processes retract, i.e. become shorter (see at the bottom as compared to normal extension of astrocytic processes on the top). This leads to neuroregeneration in the CNS which was determined at the ultrastructural level as the increase in the number of synaptic complexes, i.e. the functional connec- tions between neuronal cells. According to the invention it is thus possible to affect astrocytes in such a way that normal astrocytes (upper left) that have been exposed to an injury or trauma resulting in extension of their cellular processes (upper right) can be made to retract their cellular processes to a nor- mal state or even to a shortened state (lower right), resulting in neuron regeneration. This can also be shown as in figure 2, in high-resolution laser-scanning confocal microscopy images of the inhibition of the extension of astrocytic processes as seen in reactive astrocytes which activity has been modulated. Figure 2 corresponds to the schematic presentation shown in Fig. 1. Astroglial cells were visualized by an immunohistochemical method which uses antibodies against glutamine synthase, an enzyme produced by astrocytes. The cell nuclei are also visualized and can be seen in this picture.

The present invention thus relates to a pharmaceutical composition comprising a substance that upon administration to a patient leads to an inhibi- tion of extension of cellular processes of astrocytes and/or a retraction of said cellular processes.

The invention also relates to the use of a substance that upon administration to a patient leads to an inhibition of extension of cellular processes of astrocytes and/or a retraction of said cellular processes for the production of a pharmaceutical composition for treatment of a condition selected from the group consisting of brain injury, spinal cord injury, stroke, neurodegenerative diseases and disorders of the brain associated with ageing.

Furthermore, the invention relates to a method for neuron regeneration in the central nervous system, CNS, for treatment of a condition selected from the group consisting of brain injury, spinal cord injury, stroke, neurodegenerative diseases and disorders of the brain associated with ageing, wherein a therapeutically effective amount of a substance that upon administration to a patient leads to an inhibition of extension of cellular processes of astrocytes and/or a retraction of said cellular processes is administered to a patient. Moreover, the invention relates to an assay system for screening of substances that can be used for neuroregeneration based on their ability to inhibit extension of cellular processes of astrocytes and/or their ability to cause retraction of said cellular processes, said assay system comprising co-cultures of astrocytes and neurons. The invention also relates to pharmaceutical compositions comprising a substance screened with the above assay. In the present description and the enclosed claims the expression astrocytes is used. This expression includes astrocytes as well as astroglial cells.

With the substances used according to the invention, or with the pharmaceutical composition according to the invention or with the method accord- ing to the invention it is possible to treat all conditions that benefits from a reduction of the process extension of reactive astrocytes. Examples of such conditions are conditions selected from the group consisting of brain injury, spinal cord injury, stroke, neurodegenerative diseases and disorders of the brain associated with ageing. It is also possible to slow down the ageing of the brain, which is known to be accompanied by gradual activation of astroglial cells. More specific examples of such conditions are Parkinson's disease, Alzheimer's disease, multiple sclerosis, and diabetic retinopathy, the latter being a common pathology affecting the eye retina which is a part of the CNS, and all these conditions are connected with substantial activation of astroglial cells. For the purpose of this disclosure, the terms "illness", "disease", "medical condition", "abnormal condition", "ageing of the CNS" and the like may be used interchangeably with "condition". The term "treatment" used herein relates to both treatment in order to cure or alleviate a disease or a condition, and to treatment in order to prevent the development of a disease or a condition. The treatment may either be performed in an acute or in a chronic way.

The term "patient", as it is used herein, relates to any human or non- human mammal in need of treatment according to the invention.

The pharmaceutical composition according to the invention, used according to the invention, or produced according to the invention may also comprise other substances, such as an inert vehicle, or pharmaceutically acceptable adjuvants, carriers, preservatives etc. which are well known to persons skilled in the art.

The substances and/or pharmaceutical compositions according to the invention can be administered in a number of ways, including oral, rectal, in- travenous, intramuscular, intraperitoneal, intracerebral, subcutaneous and according to other administration protocols.

The term "therapeutically effective amount" relates to an amount that will lead to the therapeutical effect desired according to the invention. The substances used according to the invention are compounds modulating regeneration in the CNS following brain or spinal cord injury, stroke, or neurodegenerative diseases which pathologies exhibit distinct similarities at a cellular level, by modulating astroglial cell activation , w a i — — v ■ , _w ^ y

7 cellular level, by modulating astroglial cell activation (including astrocyte activation) and reactive gliosis, as well as compounds increasing the efficiency of gene therapy and stem cell therapy protocols in the CNS by rendering the CNS environment more permissible to survival, expansion, spread-out and function of the transplanted cells. The invention also relates to the use of the substances, the pharmaceutical compositions and the methods according to the invention in connection with transplantation of neurons and/or stem cells into a human brain or another part of the CNS, such as the retina.

Non-limiting examples of substances suitable for use according to the present invention are quercetin (further described in e.g. J Neurosci Res 2000 Dec 1; 62(5): 730-6), endothelins such as endothelin-1 (ET-1) (further described in e.g. Neurochem Res 1998 Mar;23(3):377-84) and endothelin-3 (further described in e.g. Neuroscience 1994, Aug; 61(4): 1007- 16), hyaluronectin (HN) (further described in e.g. Neuroreport 1995 Oct 23;6(15):2037-40), L2 antibod- ies, L2 glycolipid, and L2 tetrasaccharide (further described in e.g. J Cell Biol 1988 Jan;106(l):213-23), TGF-alpha (further described in e.g. Neurochem Res 1998 Mar;23(3):377-84)Exp Cell Res 2000 Feb l;254(2):269-78), and tyrosine phosphatase inhibitors, orthovanadate and phenylarsine oxide (further described in e.g. Neurochem Res 1998 Mar;23(3):377-84)Glia 2000 Oct;32(l):60-70).

Injury to the adult CNS is devastating because of the inability of CNS neurons to regenerate correct axonal and dendritic connections and the inability of the stem cells and neuronal progenitors present in the CNS to develop into cells of the CNS and thus replace cells lost due to a pathological condition or ageing. The consequences of injury are not just a break in communication between healthy neurons, but a cascade of events that often leads to neuronal degeneration and cell death. The failure of CNS neurons to regenerate is not an intrinsic deficit of neurons, but rather a characteristic feature of normal or damaged CNS environment that either does not support or prevents regenera- tion. Strategies rendering the CNS environment permissible to regeneration will be applicable to many CNS disorders such as spinal cord and brain injury, genetic disorders that result in aberrant axonal pathfinding or neuronal cell loss, degenerative diseases (such as Parkinson's disease, Alzheimer's disease, dementia, or amyotrophic lateral sclerosis and multiple sclerosis), stroke, either of ischemic or hemorrhagic origin, tumours, and infections of the CNS. The present invention is based on the modulation, preferably suppression, of the function of astroglial cells as a means to create a neuroregenera- tion-supporting environment.

The impact of astrocyte activation on different pathological conditions affecting the brain has not been clear due to the absence of suitable experimental models. It was recently shown that by manipulating astrocyte cytoskeleton, specifically intermediate filaments, formation of glial scar following brain or spinal cord trauma was impaired (Pekny et al, J. Cell Biol. Vol. 145, no. 3, pp503-514 (1999)). The present invention is based on an experimental system which combines genetically modified mice lacking astrocytic intermediate filaments and quantitative assessment of the repair/regenerative capacity of the CNS following injury. As a great surprise, this system identified two distinct effects of astrocyte activation. At an early stage, activated astrocytes (in this model, normal mice whose astrocytes can produce intermediate filaments compared to genetically modified mice whose astrocytes can not produce intermediate filaments) clearly play a positive role, limiting the number of synaptic complexes (connections between neuronal cells) which are lost as a consequence of the injury. However, at a later stage, suppression of astrocyte activation (in this model, genetically modified mice whose astrocytes can not pro- duce intermediate filaments compared to normal mice whose astrocytes can produce intermediate filaments ) results in substantially better recovery/regeneration. Regenerative capacity between day 4 and day 14 after the lesions was 10.6% in mice with normal astrocyte activity and 76.9% in mice in which astrocyte activity was suppressed (measured as restored synapses and expressed as a percentage of synapses in undamaged brain).

Thus, modulation of astrocyte activity following brain or spinal cord trauma (also other conditions, such as stroke and degenerative diseases) could be used in patients as a part of therapeutic protocols. This is achieved either by interfering directly with the production of intermediate filaments (up- regulation of intermediate filaments is the hallmark of astrocyte activation) or by suppressing or modulating astrocyte activation by interfering with other biochemical pathways.

Specifically, by inhibiting production of astrocytic intermediate filaments, a part of the cytoskeleton, it was possible to achieve complete restora- tion of the number of synapses (functional connections between nerve cells) following surgical lesions in the brain. This opens up the possibility to screen - - i - ^■- w <J fc

9 for pharmacological substances which inhibit astrocyte activation, and to validate these in the animal models with the prospect of using them in patients suffering from brain or spinal cord trauma, stroke or e.g., Alzheimer's disease. More specifically, complete restoration of the number of synapses was achieved by inhibition of cellular processes of astrocytes and/or retraction of said cellular processes as this creates a situation where the astroglial cells cannot access sufficiently large volume of the brain tissue to exert their regeneration inhibitory functions.

As stated above, the invention also relates to a an assay system for screening of substances that can be used for neuroregeneration based on their ability to inhibit extension of cellular processes of astrocytes and/or their ability to cause retraction of said cellular processes. This assay system is based on co-cultures of astrocytes and neurons made in a microtiter plate format, or in a similar way. The effect of the substance(s) to be tested, which e.g. may be ob- tained from a compound library, is recorded and evaluated, e.g. visually.

The scope of the invention also encompass substances identified with this assay systems as well as pharmaceutical compositions comprising such substances.

Experiment

Entorhinal cortex lesions were carried out in wild-type and GFAP-/- vim-/- mice (Eliasson, C. et al, supra), whereby lesions were made on one hand at one side of the brain, and on the other hand allowing the other side to form a control side, in each type of mice. The synaptic complexes m 1000 μm in the outer part of the molecular layer in the fascia dentata of the hippocampus were determined after 4 days, and 14 days, respectively. GFAP -/-vim-/- mice do not produce any intermediate filaments in reactive astrocytes. The results obtained are given in Table 1 below as mean+SEM. Table 1

As evident from the table, the recovery in wild-type mice, producing intermediate filaments in reactive astrocytes, is only 10.6% between days 4 and 14, while it is 76.9% in GFAP -/-vim-/- mice having no production of intermediate filaments in the reactive astrocytes. From a biological point of view this can be regarded as an almost complete recovery, particularly considering the variation in SEM for control side versus injured side. The data indicate that a suppression, in this case complete abolition of the formation of intermediate filaments, will support the recovery/regeneration/healing.

The extension (or retraction) of cellular processes of reactive astrocytes after injury were quantified, and this is illustrated in figures 3 and 4. A in the figures shows a normal situation. B and C show a situation wherein the activation of astrocytes was modulated to a lower degree. D shows a situation wherein the activation of astrocytes was modulated to a higher degree. Figure 3 shows the change in the length of the cellular processes of reactive astrocytes, and figure 4 shows change in the average volume a reactive astrocyte can access via its processes. This volume is about 9 times decreased in astrocytes which activation has been modulated compared to the situation without any intervention.

Thus, by inhibiting the extension of cellular processes of reactive astrocytes or by causing retraction of said processes, it is possible to induce substantial regeneration in the CNS.

Claims

1. A pharmaceutical composition comprising a substance that upon administration to a patient leads to an inhibition of extension of cellular proc- esses of astrocytes and/or a retraction of said cellular processes.

2. A pharmaceutical composition according to claiml, for treatment of a condition selected from the group consisting of brain injury, spinal cord injury, stroke, neurodegenerative diseases, neuronal and/or synaptic loss associated with ageing, disorders of the brain associated with ageing and diabetic retinopathy.

3. A pharmaceutical composition according to claiml or 2, for treatment of Parkinson's disease.

4. A pharmaceutical composition according to claiml or 2, for treatment of Alzheimer's disease.

5. A pharmaceutical composition according to claiml or 2, for treatment of multiple sclerosis.

6. A pharmaceutical composition according to any one of the claims 1 - 5, wherein said substance is quercetin.

7. A pharmaceutical composition according to any one of the claims 1 - 5, wherein said substance is an endothelin.

8. A pharmaceutical composition according to any one of the claims 1 - 5, wherein said substance is endothelin- 1 or endothelin-3.

9. A pharmaceutical composition according to any one of the claims 1 - 5, wherein said substance is hyaluronectin.

10. A pharmaceutical composition according to any one of the claims

1 - 5, wherein said substance is a L2 antibody, a L2 glycolipid, and/or a L2 tetrasaccharide.

11. A pharmaceutical composition according to any one of the claims

1 - 5, wherein said substance is TGF-alpha.

12. A pharmaceutical composition according to any one of the claims

1 - 5, wherein said substance is a tyrosine phosphatase inhibitor, orthova- nadate and/or phenylarsine oxide.

13. Use of a substance that upon administration to a patient leads to an inhibition of extension of cellular processes of astrocytes and/or retraction of said cellular processes for the production of a pharmaceutical composition for treatment of a condition selected from the group consisting of brain injury, spi- nal cord injury, stroke, neurodegenerative diseases, neuronal and/or synaptic loss associated with ageing, disorders of the brain associated with ageing, and diabetic retinopathy.

14. Use according to claim 13, wherein said condition is Parkinson's disease.

15. Use according to claim 13, wherein said condition is Alzheimer's disease.

16. Use according to claim 13, wherein said condition is multiple sclerosis.

17. Use according to any one of the claims 13 - 16, wherein said substance is quercetin.

18. Use according to any one of the claims 13 - 16, wherein said substance is an endothelin.

19. Use according to claim 18, wherein said substance is endothelin-1 or endothelin-3.

20. Use according to any one of the claims 13 - 16, wherein said substance is hyaluronectin.

21. Use according to any one of the claims 13 - 16, wherein said substance is a L2 antibody, a L2 glycolipid, and/or a L2 tetrasaccharide.

22. Use according to any one of the claims 13 - 16, wherein said substance is TGF-alpha.

23. Use according to any one of the claims 13 - 16, wherein said substance is a tyrosine phosphatase inhibitor, orthovanadate and/or phenylarsine oxide.

24. A method for neuron regeneration in the central nervous system,

CNS, for treatment of a condition selected from the group consisting of brain injury, spinal cord injury, stroke, neurodegenerative diseases, neuronal and/or synaptic loss associated with ageing, disorders of the brain associated with ageing and diabetic retinopathy, wherein a therapeutically effective amount of a substance that upon administration to a patient leads to an inhibition of extension of cellular processes of astrocytes and/or retraction of said cellular processes is administered to a patient.

25. The method of claim 24, wherein said condition is Parkinson's disease.

26. The method of claim 24, wherein said condition is Alzheimer's disease.

27. The method of claim 24, wherein said condition is multiple sclerosis.

28. The method of claim 24, wherein said substance is selected from the group consisting of quercetin, endothelins including endothelin- 1, endothelin- 3, hyaluronectin, L2 antibodies, L2 glycolipids, L2 tetrasaccharides, TGF- alpha, tyrosine phosphatase inhibitors, orthovanadate and/or phenylarsine oxide.

29. Use of a pharmaceutical composition according to any one of the claims 1 - 12, use according to any one of the claims 13 - 23 or use of a method according to any one of the claims 24 - 28, in connection with trans- plantation of neurons and/or stem cells into a human brain or another part of the CNS.

30. An assay system for screening of substances that can be used for neuroregeneration based on their ability to inhibit extension of cellular processes of astrocytes and/or their ability to cause retraction of said cellular proc- esses, said assay system comprising co-cultures of astrocytes and neurons.

31. A pharmaceutical composition comprising a substance screened with an assay system according to claim 30.