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Title:  Method for regulating neuron development and maintenance
United States Patent: 
7,173,001
Issued: 
February 6, 2007

Inventors: 
Bartlett; Perry (North Carlton, AU), Murphy; Mark (Fitzroy, AU)
Assignee: 
Zenyth Operations Pty Ltd. (Richmond, AU)
Appl. No.: 
10/422,552
Filed: 
April 24, 2003


 

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Abstract

The present invention relates to a method for regulating neuron development/maintenance and/or regeneration in a nervous system of a mammal and to pharmaceutical compositions comprising leukaemia inhibitory factor useful for same.

Description of the Invention

The present invention relates to a method for regulating neuron development, maintenance and regeneration in the central and peripheral nervous systems of a mammal and to pharmaceutical compositions comprising leukaemia inhibitory factor useful for same. The present invention is particularly useful in the treatment of developmental and cerebral anomalies and neuropathies in mammals and in particular humans.

Leukaemia Inhibitory Factor (hereinafter referred to as "LIF") is a protein that has been purified, cloned and produced in large quantities in purified recombinant form from both Eschericia coli and yeast cells (International Patent Application PCT/AU88/00093). LIF was originally isolated on the basis its capacity to induce differentiation and suppression of the murine myeloid leukaemic cell line, M1. LIF has no apparent proliferative effect on normal haematopoietic cells although LIF receptors have been detected on cells of the monocyte-macrophage lineage.

The present invention arose in part from an investigation of the effects of LIF on cells of the neural crest. The neural crest is a population of precursor cells which arises from the dorsal lip of the neural tube during embryogenesis and migrates through the embryo along a complex series of pathways. After migration the crest cells give rise to a great variety of cell types including the neurons and Schwann cells of the sensory and autonomic ganglia, the enteric nervous system, adrenal medulla, melanocytes of the skin and facial mesenchyme. When studied at the population level, the crest appears to be a multipotent collection of stem cells. The extensive transplantation experiments of Le Douarin and colleagues, whereby quail neural crest were grafted into chick embryos, showed that the developmental fate of the crest cells was determined by the location of this graft in the chick embryo (1). This not only indicated that the full developmental repertoire of the crest is contained in the different subpopulations of grafted crest cells, but also that environmental factors play a major role in the final differentiated phenotype of the cells.

In the last decade it has become increasingly clear that the neural crest contains subpopulations of cells which are already committed to particular developmental pathways (2,3). However, it is also clear that the differentiation of these cells is determined by environmental factors.

A number of soluble trophic factors have been shown to act as survival agents for neural crest derived neurons, but none of these have been shown to act directly on the neuronal precursor cells within the neural crest. These factors include nerve growth factor (NGF; 4), brain-derived neurotrophic factor (BDNF; 5), ciliary neurotrophic factor (CNTF; 6) and the fibroblast growth factors (FGF's; see 5).

In work leading up to the present invention, experiments were conducted to locate an agent having direct effect on the precursor populations of the neural crest. In accordance with the present invention, it has been surprisingly discovered that neural crest cells differentiate into fully mature neurons in the presence of LIF. This effect is titratable and occurs in the absence of proliferation of neuronal precursor cells. Furthermore, the effect of LIF on the differentiation of neural crest cells into neurons extends to the stimulation of the differentiation of precursor cells in embryonic dorsal root ganglia into mature sensory neurons.

Accordingly, one aspect of the present invention contemplates a method for regulating neuron development and/or maintenance and/or regeneration in a mammal comprising administering to said mammal an effective amount of leukaemia inhibitory factor (LIF) for a time and under conditions sufficient to permit the differentiation and/or maintenance and/or regeneration of neural precursor cells into neurons.

Another aspect of the present invention relates to a method for enhancing and/or stimulating and/or maintaining and/or regenerating the formation and/or survival of neurons in the central nervous system of a mammal which comprises administering to said mammal an effective amount of LIF for a time and under conditions sufficient to effect an increase in and/or to maintain the number of neurons in the central nervous system.

In one embodiment, the LIF enhances, stimulates, maintains (i.e. promotes survival) and/or regenerates immature neurons.

Yet another aspect of the present invention relates to a method for enhancing, stimulating and/or maintaining the formation and/or survival of sensory neurons, for example sensory neuorns, of the peripheral nervous system of a mammal which comprises administering to said mammal an effective amount of LIF for a time and under conditions sufficient to effect an increase in and/or to maintain the number of neurons in the peripheral nervous system.

By "LIF" as used herein is meant to include naturally occurring, recombinant and synthetic LIF comprising the naturally occurring amino acid sequence or any single or multiple amino acid substitutions, deletions and/or additions therein including single or multiple substitutions, deletions and/or additions to any molecules associated with LIF such as carbohydrate, lipid and/or peptide moieties. Accordingly, the term "LIF" as used herein contemplates naturally occurring LIF and LIF-like polypeptides which include mutants, derivatives, homologues and analogues of LIF. Regardless of the LIF molecule used, however, the only requirement is that it can assist in regulating neuron development and/or maintenance and/or regeneration in a mammal. In a preferred embodiment the mammal is human and the LIF is of human origin or from a different mammal but which still has activity in a human. Hence, the source of LIF and the mammal to be treated may be homologous, i.e. from the same mammal or may be heterologous, i.e. from a different mammal. In some circumstances, the mammal to be treated may itself be used to isolate the LIF for use in the method of the present invention.

By "regulating neuron development, maintenance and regeneration" as used herein is meant to include stimulating, enhancing and/or maintaining the formation and/or survival of neurons in the central and peripheral nervous systems of a mammal. It also includes the ability of said factor to assist the regeneration of properties associated with neuronal function following damage caused by disease or trauma. It is also includes stimulating, enhancing, maintaining and/or regenerating those properties associated with neurons such as, but not limited to, neurotronsmitter type, receptor type and other features associated with this phenotype. In particular, LIF has been shown herein to induce, stimulate, enhance, maintain and/or regenerate the differentiation of neural crest cells into fully mature neurons. This effect is titratable and occurs in the absence of proliferation of neuronal precursor cells. The effect of LIF also extends to the stimulation of the differentiation of precursor cells in embryonic dorsal root ganglia (DRG) into mature sensory neurons. The sensory neurons of the peripheral nervous system are derived from precursor cells in the embryonic neural crest. After crest migration, these precursor cells aggregate into the DRG and then differentiate into mature sensory neurons. The survival of sensory neurons has been shown to be dependent on two characterised growth factors, nerve growth factor (NGF) and brain derived neurotrophic factor (BDNF) and other undefined factors at critical stages during development. However, nothing is known about the identity of factors which might stimulate the differentiation of the sensory precursor cells. It was, therefore, surprisingly found in accordance with the present invention that LIF stimulated the differentiation of precursor cells in the embryonic DRG into mature sensory neurons and that LIF acted as a survival factor for these neurons throughout embryogenesis and into postnatal life.

LIF also affects the central nervous system. The early steps in the development of the central nervous system from the embryonic precursor cells of the neural tube involves expansion of the precursor population and differentiation of these cells into mature neurons and glia. This phase is followed by a selective survival of neurons which have appropriately innervated the correct targets and is believed to be based on the limited availability of survival factors which are produced by the target cells.

It has been recently shown (9) that the fibroblast growth factors are involved in the expansion and differentiation phases of development of the embryonic brain and in addition it has also been shown that FGF can act as a survival agent for mature neurons. Work from Barde (5) indicates that the survival of a subset of CNS neurons, the retinal ganglion cells, is dependent on BDNF. However, little is known about other factors which are operative in the development of the embryonic brain and spinal cord.

Accordingly, it has now been surprisingly found that LIF acts as a differentiation/survival and/or regenerating agent for spinal cord neurons and enhances, stimulates and/or promotes spinal cord development and promotes neurite extension.

This method is particularly applicable to regulating spinal cord development and in treating a disease, injury and/or an abnormality to a nervous system. For example, the method of the present invention can be used in relation to the central and/or peripheral nervous system to treat one or more of Cerebral Palsy, trauma induced paralysis, vascular ischaemia associated with stroke, neuronal tumours, motorneurone disease, Parkinson's disease, Huntington's disease, Alzheimer's disease, multiple sclerosis, peripheral neuropathies associated with diabetes, heavy metal or alcohol toxicity, renal failure and/or infectious diseases such as herpes, rubella, measles, chicken pox, HIV and/or HTLV-1.

Another aspect of the invention relates to a method for enhancing, stimulating, maintaining and/or regenerating spinal cord development and spinal cord neuron number which comprises administering to said mammal an effective amount of LIF for a time and under conditions sufficient to effect an increase in spinal cord neuron number and spinal cord development.

Yet another aspect relates to a method of enhancing, stimulating, maintaining and/or regenerating neurite extension from spinal cord and other central nervous system neurons and further relates to the central nervous system other than the spinal cord.

Still yet another aspect of the invention contemplates a method of treatment of disease and injury in both the central and peripheral nervous systems in a mammal, said disease or injury including but not limited to one or more of Cerebral Palsy, trauma induced paralysis, vascular ischaemia associated with stroke, neuronal tumours, motorneurone disease, Parkinson's disease, Huntington's disease, Alzheimer's disease, multiple sclerosis and peripheral neuropathies associated with diabetes, heavy metal or alcohol toxicity, renal failure and/or infectious diseases such as herpes, rubella, measles, chicken pox, HIV and/or HTLV-1 which comprises administering to said mammal an effective amount of LIF for a time and under conditions sufficient to ameliorate the disease or injury.

In all such methods of the present invention, the enhancing, stimulating, maintaining and/or regenerating of neurons is referred to as "regulating neuron development". Furthermore, use of the term "LIF" includes LIF-like polypeptides and derivatives thereof as discussed above.

The effective amount of LIF used in accordance with the present invention will be that required to regulate the neurons and will generally be from about 0.01 to about 10,000 microgram (.mu.g) per kilogram (kg) of body weight and preferably 0.1 to 10,000 .mu.g/kg and most preferably 1 to 1000 .mu.g/kg of body weight. However, depending on such factors as the disease treated, the treatment and the patient, more or less LIF may be used while still being within the scope of the present invention. Furthermore, it may be convenience to determine the effective amount of LIF in Units/ml or Units/kg. The definition of a Unit of LIF activity can be found in PCT/AU88/00093. For example, and not by way of limitation LIF may be used from 10 to 10.sup.8 U/ml. Administration may be per hour, per day, per week or per month or may be a single administration. Administration may also need to be continuous infusion.

In accordance with the present invention, LIF may be administered alone or in combination with one or more other neuron stimulating factors such as, but not limited to, FGF, CNTF and/or BDNF and/or other neurotrophic factors. In "combination" means either the simultaneous addition of LIF and the one or more other factors in the same composition or the sequential addition of the LIF and one or more other factors where a first factor is given followed by a second factor. The exact order of addition and time between additions is best determined by the practicing physician and may depend on the patient and/or the treatment required.

Accordingly, the one or more other neuron stimulating factors may be given by simultaneous or sequential administration with LIF. The effective amount of other neuron stimulating factors will be from about 0.01 to about 10,000 .mu.g/kg body weight, preferably 0.1 to 10,000 .mu.g/kg and most preferably 1 to 1,000 .mu.g/kg body weight. Again, administration may be a single dose or repeated per hour, per day, per week or per month. Administration may also be continuous infusion.

The route of administration is preferably by intramuscular or intravenous injection or using gene therapy although other routes of administration are possible such as by infusion, drip, intracerebral injection and/or implants.

Another aspect of this invention relates to administration of LIF to target tissue, or the precise location of the nerve so as to facilitate uptake by retrograde transport as outlined in Example 5.

The present invention is also directed to a pharmaceutical composition comprising LIF and one or more neuron stimulating factors and one or more pharmaceutically acceptable carriers and/or diluents. Such a composition is useful for regulating neuron development and/or maintenance in a mammal such as in enhancing, stimulating, maintaining and/or regenerating the formation and survival of neurons in the peripheral nervous system and/or enhancing, stimulating, maintaining and/or regenerating the formation and survival of sensory neurons in the central nervous system and/or enhancing, stimulating and/or maintaining the formation and survival of spinal cord neurons and/or spinal cord development.

Preferably, the composition is suitable for administration into a human. In accordance with the present invention, the LIF used in the composition is as previously herein defined and includes, for example, LIF-like polypeptides and mutants, derivatives, homologues and/or analogues of LIF. The LIF and other neuron stimulating molecules and/or neurotrophic factors may be the same or different in terms of their mammalian source and whether they are naturally occurring, recombinant or synthetic. As with the method, the mammalian source of the LIF and other neuron stimulating factor may be homologous or heterologous to the mammal being treated. The compositions of the present invention are also useful in treating the, diseases, injuries and/or abnormalities of a nervous system as previously described.

The preparation of pharmaceutical compositions is well known in the art and reference can conveniently be made to Remington's Pharmaceutical Sciences, 16th ed., 1980, Mach Publishing Co., Edited by Osol et al.

Another aspect of the present invention is directed to the use of LIF including its derivatives for the manufacture of a medicament for enhancing, stimulating, maintaining and/or regenerating the formation and/or survival of neurons in the peripheral nervous system and/or enhancing, stimulating maintaining and/or regenerating the formation and/or survival of neurons in the central nervous system and/or enhancing, stimulating, maintaining and/or regenerating the formation and/or survival of spinal cord neurons and/or spinal cord development in a mammal. Preferably, the mammal is a human and the LIF used is as hereinbefore defined. The use in accordance with the present invention may also include the use of one or more other neuron stimulating factors such as FGF, CNTF and/or BNDF.



Claim 1 of 36 Claims

1. A method for regulating neuron development and/or neuron regeneration and/or neuron maintenance in a mammal comprising administering to said mammal an effective amount of leukaemia inhibitory factor (LIF) for a time and under conditions sufficient to permit the development of neural precursor cells into neurons and/or to promote survival of said neural precursor cells.

 

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If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.

 

 

     
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