The invention relates to the use of IL6RIL6 chimera in the treatment and prevention of neurological diseases and disorders.

SUMMARY OF THE INVENTION

It is the object of the present invention to provide means for the treatment and/or prevention of neurological diseases or disorders. In particular, it is an object of the present invention to provide a means to stimulate or enhance the differentiation of progenitors or differentiated glial cells into myelinating cells. The basis of the invention is in the use of the recombinant IL6RIL6 chimera protein, which has a markedly higher affinity for gp130 than does the mixture of IL-6 and sIL-6R.

It is further an object of the present invention to provide a means to stimulate or enhance the myelination or remyelination of injured fibers as illustrated by the induced remyelination of axons following sciatic nerve axotomy in vivo further to the myelinating effects of the IL-6 chimera on the induction of myelin genes in vitro.

It is also an object of the present invention to use the IL6RIL6 chimera for increasing the number of Schwann cells developing in dorsal root ganglia (DRG) cultures. Furthermore, it is an object of the present invention to use the IL6RIL6 for induction of the differentiation of these cells to the point where they wrap around axons and produce myelin basic protein as illustrated in cocultures of Schwann cell lines with primary DRG.

It is another object of the present invention to use the IL6RIL6 chimera for induction the transcription of the myelin basic protein (MBP) genes in a system of transdifferentiation in which cells with melanocytic phenotype are converted into a Schwann, myelinating phenotype, as illustrated by the effect of IL6RIL6 on a murine melanoma.

It is another object of the present invention to use the IL6RIL6 chimera as a neuroprotective agent and for prevention of neuronal cell death in the hippocampus, a region involved in memory encoding and one that exhibits early degeneration in Alzheimer's disease and ischemia.

It is another object of the present invention to use the IL6RIL6 chimera as a protective agent against the neurotoxicity process triggered by excitatory amino acids as illustrated by the protection from glutamate-induced neurotoxicity provided by the IL-6 chimera in primary cultures of new born rat cerebella neurons.

A molecular mechanism is proposed by which the IL6RIL6 induces and represses specific transcription factors that bring about the induction of the MBP gene differentiation into myelinating phenotype.

Thus, the present invention provides the use of IL6RIL6 chimera for the manufacture of a medicament for the treatment and/or prevention of neurological diseases and disorders. In particular, the present invention provides the use of IL6RIL6 chimera for the manufacture of a medicament for treating traumatic nerve degeneration, demyelinating diseases of the CNS or PNS and/or neurodegenerative diseases.

More particularly, the invention provides for the use of IL6RIL6 chimeras in the treatment of multiple sclerosis (MS), Alzheimer's disease, Parkinson's disease or ALS.

The invention also provides pharmaceutical compositions comprising IL6RIL6 chimera, optionally together with one or more pharmaceutically acceptable excipients, for the treatment and/or prevention of neurological disease and disorders. In particular the pharmaceutical composition is for treating traumatic nerve degeneration, demyelinating diseases of the CNS or PNS and/or neurodegenerative diseases.

A preferred use for the pharmaceutical compositions of the present invention is the treatment of MS, Alzheimer's disease, Parkinson's disease or ALS.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the invention it has been found that addition of IL6IL6R recombinant protein to cultures of dorsal root ganglia cells or of melanoma cells stimulates the differentiation of those cells into myelinating cells. It was also found that the addition of the IL6IL6R recombinant protein to cocultures of neurons and Schwann cells induces the latter to form a regular sheath around the axons. The invention therefore relates to the use of the IL6RIL6 chimera for the manufacture of a medicament to generate myelinating cells or to stimulate, enhance or accelerate the generation of myelinating cells.

The IL-6 chimera further induces in vivo remyelination of transected fibers following sciatic nerve axotomy in rats. The invention therefore further relates to the use of IL6IL6 chimera for manufacture of a medicament to induce, enhance or accelerate remyelination, in particular after nerve damage or trauma or axonal injury.

The addition of IL6IL6R recombinant protein to organotypic cultures was further found to induce a prolonged protective effect from neurotoxic agents. The invention therefore also relates to the use of IL6RIL6 chimera for manufacture of a medicament to induce, enhance, prolong or accelerate neuroprotection, in particular from neurotoxic agents, and to inhibit, reduce or decelerate neuronal death, which may be due to apoptosis, for example.

The present invention concerns the use of the "IL6RIL6 chimera" (also called "IL6RIL6" or IL-6 chimera), which is a recombinant glycoprotein obtained fusing the entire coding sequence of the naturally-occurring soluble IL-6 Receptor 6-Val to the entire coding sequence of mature naturally-occurring IL-6, both from human origin. The IL6RIL6 chimera may be produced in any adequate eukaryotic cells, such as yeast cells, insect cells, and the like. It is preferably produced in mammalian cells, most preferably in genetically engineered CHO cells as described in WO 99/02552. Whilst the protein from human origin is preferred, it will be appreciated by the person skilled in the art that a similar fusion protein of any other origin may be used according to the invention, as long as it retains the biological activity described herein.

More particularly, the present invention concerns the use of IL6RIL6 chimera to stimulate the differentiation of progenitor or differentiated glial cells into myelinating cells.

As demonstrated herein the IL6RIL6-induced myelinating cells differentiation process involves both, activation of genes required for the formation of the myelin sheath around neuronal axons, as well as repression of a gene required for the maintenance of non myelinating phenotypes. In accordance with the present invention it has been observed that the addition of IL6RIL6 chimera to cultures of embryonic dorsal root ganglia (eDRG) cells, isolated from mice embryos at days 14 15 of gestation, has a profound effect on the development of the Schwann cell precursors present in the DRG. After 2 5 days in culture, there is a marked increase in the number of the embryonic Schwann cells, a marked phenotypic change in these cells, which start to wrap their membrane around the DRG axons, and an induction of MBP. In accordance with the present invention it has been further observed that in a co-culture of Schwann cells with neurons the IL-6 chimera can induce a significant increase in the binding of Schwann cells along the non-myelinated axons within 5 hours. The Schwann cells, which were labeled with fluorogold, elongate and after a few days their fluorescent cytoplasm could be seen to form a regular sheath around the axons. Without the chimera, or with NGF, the Schwann cells bind much less and do not form a sheath.

Therefore, the invention further relates to the use of IL6RIL6 chimera for the manufacture of a medicament to induce Schwann cell proliferation and/or differentiation, as well as to the myelination by Schwann cells in the peripheral neural system. In accordance with the present invention it has further been shown that the IL-6 chimera can induce remyelination of peripheral nerves in vivo. Following sciatic nerve axotomy in rats and juxtaposition of proximal and distal stumps the IL-6 chimera could induce the regeneration of the peripheral nerves and the remyelination of the transected fibers in vivo. In the presence of the IL-6 chimera a 4 fold increase in the number and thickness of myelinated fibers was found and an increase in the remyelination of the more distant fibers. Therefore, the invention further relates to the use of IL6RIL6 chimera for the manufacture of a medicament to induce remyelination in the peripheral nervous system.

Furthermore it has also been found according to the present invention that the IL-6 chimera can induce the expression of the genes encoding myelin protein components such as MBP, PLP and P0 genes in myelinating cells of the peripheral nervous system such as Schwann cells, and in cells of the central nervous system such as oligodendrocytes. Therefore, the invention further relates to the use of IL6RIL6 chimera for the manufacture of a medicament to induce myelination and/or remyelination by oligodendrocytes in the central nervous system.

It has also been found according to the present invention that the addition of IL6RIL6 chimera to cultures of the B16/F10.9 murine melanoma cell line, induces the expression of the MBP gene within 6 12 hours. Other genes, which encode proteins of the myelin, such as the CNPase gene, are induced whereas expression of genes which are involved in melanogenesis (formation of melanin pigments) such as tyrosinase, are strongly repressed. The F10.9 cells treated by IL6RIL6 also undergo a marked morphological change, and acquire a Schwann-like phenotype. The phenotypic changes and the induction of specific myelin genes support the hypothesis that IL6RIL6 causes a transdifferentiation of the cells from a melanocytic to myelinating state. Since in the embryo, cells migrating from the neural crest can give rise to either melanocytes or myelinating Schwann cells and oligodendrocytes, it is suggested that IL6RIL6 can influence the fate of the cells and promote the formation of myelinating cells. Therefore, the invention also relates to the use of IL6RIL6 chimera for the manufacture of a medicament to induce, promote, enhance or accelerate the formation of myelinating cells in the peripheral and in the central nervous system and/or to induce transdifferentiation of melanocytic cells into myelinating cells.

Moreover, it is shown in accordance with the present invention that IL6RIL6 acts by down-regulating the homeobox gene Pax-3, a gene expressed in embryonic neural crest cells before they differentiate into myelinating Schwann cells (Kioussi and Gruss, 1996). Pax-3 is known to repress MBP gene. Therefore, Pax-3 repression appears to be a key event in the final maturation of the myelinating cells. Hence IL6RIL6 acts on a key differentiation switch (i.e. pax-3 repression).

Pax-3 is a transactivator of the micropthalmia associated transcription factor MITF, which in turn induces and maintains the expression of the tyrosinase and other genes responsible for the melanocytic phenotype. The discovery of the rapid repression of Pax-3 by IL6RIL6 can therefore explain the molecular events which promote the myelinating activity of the neural crest derived cells. After nerve injury, myelinated axons undergo demyelination as part of the Wallerian degeneration. During that process, Schwann cells turn down the expression of MBP gene and other related myelin protein genes. Concomitantly there is an upregulation of Pax-3 and GFAP that denotes a reversion from myelinating SC to non-myelinating and proliferating SC (Kioussi and Gruss, 1996). In accordance with the present invention, the IL6RIL6 chimera appears to be a potent cytokine to revert the process of Wallerian nerve degeneration by repressing Pax-3 and inducing the SC to resume their myelinating activities. The same considerations would apply to brain demyelinating diseases since, like in traumas, the neurodegeneration in these diseases is spurred by a demyelination process driven by macrophages and other inflammatory cells. The invention therefore also relates to the use of IL6RIL6 chimera for the manufacture of a medicament for treatment of nerve damage and/or traumatic nerve degeneration and/or axonal injury.

IL6RIL6 can be injected to mice in which an autoimmune demyelination has been induced by immunization with MBP as a model system for chronic relapsing multiple sclerosis (Cannella et al, 1998). The capacity of IL6RIL6 to induce myelin protein genes and differentiation of myelinating glial cells can be observed in vivo, using this pharmaceutical paradigm. The invention therefore further relates to the use of IL6RIL6 chimera for the manufacture of a medicament for treatment and/or prevention of autoimmune demyelinating diseases, in particular to the treatment and/or prevention of Multiple Sclerosis.

The IL-6 chimera has been also shown to have neuroprotective effects, preventing loss of neuronal cell viability induced by exitotoxic agents in regions involved in memory encoding and exhibiting early degeneration in Alzheimer's disease and ischemia. The IL-6 chimera was found to protect neurons from glutamate induced neurotoxicity. Therefore, the invention relates to the manufacture of a medicament for the protection of neural cells against cell death, in particular due to the effects of neurotoxic agents, and to the treatment and/or prevention of neurodegenerative diseases like Alzheimer's disease, Parkinson's disease or ALS, for example.

Further neurological diseases that may be treated with the IL6RIL6 chimera are strokes, movement disorders, epilepsy, pain and the like.

The definition of "pharmaceutically acceptable" is meant to encompass any carrier, which does not interfere with effectiveness of the biological activity of the active ingredient and that is not toxic to the host to which it is administered. For example, for parenteral administration, IL6RIL6 chimera may be formulated in a unit dosage form for injection in vehicles such as saline, dextrose solution, serum albumin and Ringer's solution.

The IL6RIL6 chimera can be administered to a patient in need of administration thereof in a variety of ways. The routes of administration include intradermal, transdermal (e.g. in slow release formulations), intramuscular, intraperitoneal, intravenous, subcutaneous, oral, epidural, topical, and intranasal routes. Any other therapeutically efficacious route of administration can be used, for example absorption through epithelial or endothelial tissues or by gene therapy wherein a DNA molecule encoding the IL6RIL6 chimera is administered to the patient (e.g. via a vector) which causes the IL6RIL6 chimera to be expressed and secreted in vivo. In addition the IL6RIL6 chimera can be administered together with other components of biologically active agents such as pharmaceutically acceptable surfactants, excipients, carriers, diluents and vehicles.

For parenteral (e.g. intravenous, subcutaneous, intramuscular) administration, IL6RIL6 chimera can be formulated as a solution, suspension, emulsion or lyophilized powder in association with a pharmaceutically acceptable parenteral vehicle (e.g. water, saline, dextrose solution) and additives that maintain isotonicity (e.g. mannitol) or chemical stability (e.g. preservatives and buffers). The formulation is sterilized by commonly used techniques.

An "effective amount" refers to an amount of the active ingredients that is sufficient to affect the course and the severity of the diseases described above, leading to the reduction or remission of such pathology. The effective amount will depend on the route of administration and the condition of the patient.

The dosage administered, as single or multiple doses, to an individual will vary depending upon a variety of factor, including IL6RIL6 chimera pharmacokinetic properties, the route of administration, patient conditions and characteristics (sex, age, body weight, health, size), extent of symptoms, concurrent treatments, frequency of treatment and the effect desired. Adjustment and manipulation of established dosage ranges are well within the ability of those skilled, as well as in vitro and in vivo methods of determining the remyelination of the nerves.

 

Claim 1 of 4 Claims

1. A method for inducing myelination and remyelination of neurons, comprising administering to a patient in need thereof an effective amount of interleukin-6 receptor interleukin-6 (IL6RIL6) chimera, thereby resulting in the induction of myelination or remyelination of neurons.
 

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