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Title:  Use of osteopontin for the treatment and/or prevention of neurologic diseases
United States Patent: 
7,217,687
Issued: 
May 15, 2007

Inventors: 
Boschert; Ursula (Troinex, CH), Feger; Georg (Thoiry, FR), Selvaraju; Raghuram (Vandoeuvres, CH), Bernasconi; Lilia (Perly, CH), Papoian; Ruben (Cincinnati, OH)
Assignee: 
Applied Research Systems ARS Holding N.V. (Curacao, AN)
Appl. No.:  
10/477,876
Filed: 
May 8, 2002
PCT Filed: 
May 08, 2002
PCT No.: 
PCT/EP02/05081
371(c)(1),(2),(4) Date: 
April 09, 2004
PCT Pub. No.: 
WO02/092122
PCT Pub. Date: 
November 21, 2002


 

Training Courses --Pharm/Biotech/etc.


Abstract

The invention relates to the use of osteopontin, or of an agonist of osteopontin activity, for treatment or prevention of a neurologic diseases.

SUMMARY OF THE INVENTION

It is the object of the present invention to provide a novel means for the treatment and/or prevention of a neurologic disease.

The invention is based on the finding that the protein osteopontin promotes glial cell proliferation and differentiation, thus promoting myelination and regeneration of nerves. In accordance with the present invention, it has further been found that osteopontin has a beneficial effect in animal models of multiple sclerosis and peripheral neuropathies.

Therefore, the present invention relates to the use of osteopontin, or of an agonist of osteopontin activity, in a neurologic disease, such as traumatic nerve injury, stroke, demyelinating diseases of the CNS or PNS, neuropathies and neurodegenerative diseases.

In accordance with the present invention, osteopontin may also be used in combination with an interferon for treatment and/or prevention of neurologic diseases. The use of nucleic acid molecules, and expression vectors comprising osteopontin, and of cells expressing osteopontin, for treatment and/or prevention of a neurologic disease is also within the present invention. The invention further provides pharmaceutical compositions comprising osteopontin and an interferon, optionally together with one or more pharmaceutically acceptable excipients.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the finding that osteopontin is differentially expressed during oligodendrocyte differentiation and during the development of the cerebellum. It has further been found that expression of osteopontin cDNA in oligodendrocytes leads to a differentiated phenotype of these cells in vitro. Upon expression of osteopontin, oligodendrocytes display a phenotype similar to the phenotype of a differentiating, myelinating cell. In addition to these in vitro findings, it has been shown that osteopontin, and in particular the combination of osteopontin and an interferon, have a beneficial effect in an established model of multiple sclerosis. In an experimental model of peripheral neuropathy, osteopontin had a pronounced beneficial effect on nerve activity, and significantly reduced the percentage of degeneration and enhanced the extent of myelination.

The experimental evidence presented herein therefore provides for a new possibility of treating neurologic diseases, in particular those linked to nervous and glial cell function. These findings are particularly surprising because WO 00/63241 teaches to inhibit osteopontin in order to treat multiple sclerosis.

The invention therefore relates to the use of osteopontin, or of an agonist of osteopontin activity, for the manufacture of a medicament for treatment and/or prevention of neurologic diseases.

Neurologic diseases comprise disorders linked to dysfunction of the CNS or PNS, such as diseases related to neurotransmission, headache, trauma of the head, CNS infections, neuro-ophthalmologic and cranial nerve disorders, function and dysfunction of the cerebral lobes disorders of movement, stupor and coma, demyelinating diseases, delirium and dementia, craniocervical junction abnormalities, seizure disorders, spinal cord disorders, sleep disorders, disorders of the peripheral nervous system, cerebrovascular disease, or muscular disorders. For definitions of these disorders, see e.g. www.merck.com/pubs/manual/section14/sec14.htm.

Preferably, the neurologic diseases of the invention are selected from the group consisting of traumatic nerve injury, stroke, demyelinating diseases of the CNS or PNS and neurodegenerative diseases.

Traumatic nerve injury may concern the PNS or the CNS, it may be brain or spinal cord trauma, including paraplegia, as described in the "background of the invention" above.

Stroke may be caused by hypoxia or by ischemia of the brain. It is also called cerebrovascular disease or accident. Stroke may involve loss of brain functions (neurologic deficits) caused by a loss of blood circulation to areas of the brain. Loss of blood circulation may be due to blood clots that form in the brain (thrombus), or pieces of atherosclerotic plaque or other material that travel to the brain from another location (emboli). Bleeding (hemorrhage) within the brain may cause symptoms that mimic stroke. The most common cause of a stroke is stroke secondary to atherosclerosis (cerebral thrombosis), and therefore the invention also relates to the treatment of atherosclerosis.

Peripheral Neuropathy may be related to a syndrome of sensory loss, muscle weakness and atrophy, decreased deep tendon reflexes, and vasomotor symptoms, alone or in any combination. Neuropathy may affect a single nerve (mononeuropathy), two or more nerves in separate areas (multiple mononeuropathy), or many nerves simultaneously (polyneuropathy). The axon may be primarily affected (e.g. in diabetes mellitus, Lyme disease, or uremia or with toxic agents), or the myelin sheath or Schwann cell (e.g. in acute or chronic inflammatory polyneuropathy, leukodystrophies, or Guillain-Barre syndrome). Further neuropathies, which may be treated in accordance with the present invention, may e.g. be due to lead toxicity, dapsone use, tick bite, porphyria, or Guillain-Barre syndrome, and they may primarily affect motor fibers. Others, such as those due to dorsal root ganglionitis of cancer, leprosy, AIDS, diabetes mellitus, or chronic pyridoxine intoxication, may primarily affect the dorsal root ganglia or sensory fibers, producing sensory symptoms. Cranial nerves may also be involved, such as e.g. in Guillain-Barre syndrome, Lyme disease, diabetes mellitus, and diphtheria.

Alzheimer's disease is a disorder involving deterioration in mental functions resulting from changes in brain tissue. This may include shrinking of brain tissues, primary degenerative dementia and diffuse brain atrophy. Alzheimer's disease is also called senile dementia/Alzheimer's type (SDAT).

Parkinsons's disease is a disorder of the brain including shaking and difficulty with walking, movement, and coordination. The disease is associated with damage to a part of the brain that controls muscle movement, and it is also called paralysis agitans or shaking palsy.

Huntington's Disease is an inherited, autosomal dominant neurologic disease.

Amyptrophic Lateral Sclerosis, ALS, is a disorder causing progressive loss of nervous control of voluntary muscles, including of destruction of nerve cells in the brain and spinal cord. Amyotrophic Lateral Sclerosis, also called Lou Gehrig's disease, is a disorder involving loss of the use and control of muscles.

Multiple Sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS) that takes a relapsing-remitting or a progressive course. MS is not the only demyelinating disease. Its counterpart in the peripheral nervous system (PNS) is chronic inflammatory demyelinating polyradiculoneuropathy (CIDP). In addition, there are acute, monophasic disorders, such as the inflammatory demyelinating polyradiculoneuropathy termed Guillain-Barre syndrome (GBS) in the PNS, and acute disseminated encephalomyelitis (ADEM) in the CNS.

Further neurologic disorders comprise neuropathies with abnormal myelination, such as the ones listed in the "Background of the invention" above, as well as carpal tunnel syndrome. Traumatic nerve injury may be accompanied by spinal column orthopedic complications, and those are also within the diseases in accordance with the present invention.

Neurologic disorders may further be due to congenital metabolic disorders. In a preferred embodiment of the invention, the neurologic disease is therefore due to a congenital metabolic deficit.

The congenital metabolic disorders encompassed by the present invention may be e.g. phenylketonuria and other aminoacidurias, Tay-Sachs, Niemann-Pick, and Gaucher's diseases, Hurler's syndrome; Krabbe's disease and other leukodystrophies. They may affect the developing myelin sheath, mainly in the CNS.

Neurologic diseases caused by congenital metabolic disorders have also been discussed in detail in the "Background of the invention".

Less well known neurologic diseases are also within the scope of the present invention, such as neurofibromatosis, or Multiple System Atrophy (MSA). Further disorders that may be treated in accordance with the present invention, have been described in detail in the "Background of the invention" above.

In a further preferred embodiment, the neurologic disease is a peripheral neuropathy, most preferably diabetic neuropathy. Chemotherapy associated neuropathies are also preferred in accordance with the present invention.

The term "diabetic neuropathy" relates to any form of diabetic neuropathy, or to one or more symptom(s) or disorder(s) accompanying or caused by diabetic neuropathy, or complications of diabetes affecting nerves as described in detail in the "Background of the invention2 above. Diabetic neuropathy may be a polyneuropathy. In diabetic polyneuropathy, many nerves are simultaneously affected. The diabetic neuropathy may also be a mononeuropathy. In focal mononeuropathy, for instance, the disease affects a single nerve, such as the oculomotor or abducens cranial nerve. It may also be multiple mononeuropathy when two or more nerves are affected in separate areas.

In yet a further preferred embodiment, the neurologic disorder is a demyelinating disease. Demyelinating diseases preferably comprise demyelinating conditions of the CNS, like acute disseminated encephalomyelitis (ADEM) and multiple sclerosis (MS), as well as demyelinating diseases of the peripheral nervous system (PNS). The latter comprise diseases such as chronic inflammatory demyelinating polyradiculoneuropathy. (CIDP and acute, monophasic disorders, such as the inflammatory demyelinating: polyradiculoneuropathy termed Guillain-Barre syndrome (GBS).

A further preferred embodiment of the invention relates to the treatment and/or prevention of a neurodegenerative disease. The neurodegenerative disease is selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease and ALS.

Preferably, the osteopontin is selected from a peptide, a polypeptide or a protein selected from the group consisting of:

(a) A polypeptide comprising SEQ ID NO: 1;

(b) A polypeptide comprising amino acids 1 to 168 or 170 of SEQ ID NO: 1;

(c) A polypeptide comprising amino acids 1 to 16 and 170 to 314 of SEQ ID NO: 1;

(d) A polypeptide comprising amino acids 170 to 314 of SEQ ID NO: 1;

(e) A polypeptide comprising SEQ ID NO: 2;

(f) A polypeptide comprising SEQ ID NO: 3;

(g) A mutein of any of (a) to (f), wherein the amino acid sequence has at least 40% or 50% or 60% or 70% or 80% or 90% identity to at least one of the sequences in (a) to (f);

(h) A mutein of any of (a) to (f) which is encoded by a DNA sequence which hybridizes to the complement of the native DNA sequence encoding any of (a) to (f) under moderately stringent conditions or under highly stringent conditions;

(i) A mutein of any of (a) to (f) wherein any changes in the amino acid sequence are conservative amino acid substitutions to the amino acid sequences in (a) to (f);

(j) a salt or an isoform, fused protein, functional derivative, active fraction or circularly permutated derivative of any of (a) to (f).

Active fractions or fragments may comprise any portion or domain of any of the osteopontin isoforms, such as an N-terminal portion or a C-terminal portion, or any of OPN-a, -b, or -c, as shown in FIG. 2. The GRGDS motif may be present, or absent, or mutated. The heparin binding site may be mutated so as to render osteopontin devoid of heparin-binding. Full length osteopontin, or any active fragment thereof, may be phosphorylated at one or more of the following serine residues, such as the serine residues at the following positions: 8, 10, 11, 33, 46, 47, 60, 62, 65, 83, 86, 89, 92, 101, 104, 107, 110, 113, 153, 155, 175, 179, 199, 203, 208, 212, 218, 223, 227, 238, 242, 247, 251, 254, 259, 264, 275, 287, 292, 294, 295. Additionally, the serine phosphorylation sites may be mutated from serine to glutamate residues, in order to mimic phosphorylation.

The person skilled in the art will appreciate that even smaller portions of osteopontin may be enough to exert its function, such as an active peptide comprising the essential amino acid residues required for osteopontin function.

The person skilled in the art will further appreciate that muteins, salts, isoforms, fused proteins, functional derivatives of osteopontin, active fractions or circularly permutated derivatives of osteopontin, will retain a similar, or even better, biological activity of osteopontin. The biological activity of osteopontin and muteins, isoforms, fused proteins or functional derivatives, active fractions or fragments, circularly permutated derivatives, or salts thereof, may be measured in a co-culturing assay, such as the one described below in Example 8. Mixed cortical cultures contain oligodendrocytes, as well as other CNS derived cells (such as neurons, astrocytes, microglia), and induce or up-regulate the typical genes involved in myelination, like P0, MBP or MAG, upon incubation with OPN or the mutein, isoform, fragment, active fraction, functional derivative or salt. Expression of these genes can be measured by quantitative real time RT-PCR (TaqMan.RTM. RT-PCR) analysis, which is explained in detail in the examples below. A further simple assay to measure OPN activity is an oligodendrocyte proliferation assay, comprising incubating an adequate oligodendrocyte cell line, such as oli-neu or CG4 cells, with OPN or the mutein, isoform, fragment, active fraction, functional derivative or salt, as described in Example 7 below, for example.

Preferred active fractions have an activity which is equal or better than the activity of full-length osteopontin, or which have further advantages, such as a better stability or a lower toxicity or immunogenicity, or they are easier to produce in large quantities, or easier to purify. The person skilled in the art will appreciate that muteins, active fragments and functional derivatives can be generated by cloning the corresponding cDNA in appropriate plasmids and testing them in the co-culturing assay, as mentioned above.

The proteins according to the present invention may be glycosylated or non-glycosylated, they may be derived from natural sources, such as body fluids, or they may preferably be produced recombinantly. Recombinant expression may be carried out in prokaryotic expression systems such as E. coli, or in eukaryotic, such as insect cells, and preferably in mammalian expression systems, such as CHO-cells or HEK-cells.

As used herein the term "muteins" refers to analogs of osteopontin, in which one or more of the amino acid residues of a natural osteopontin are replaced by different amino acid residues, or are deleted, or one or more amino acid residues are added to the natural sequence of osteopontin, without changing considerably the activity of the resulting products as compared with the wild-type osteopontin. These muteins are prepared by known synthesis and/or by site-directed mutagenesis techniques, or any other known technique suitable therefor.

Muteins of osteopontin, which can be used in accordance with the present invention, or nucleic acid coding thereof, include a finite set of substantially corresponding sequences as substitution peptides or polynucleotides which can be routinely obtained by one of ordinary skill in the art, without undue experimentation, based on the teachings and guidance presented herein.

Muteins in accordance with the present invention include proteins encoded by a nucleic acid, such as DNA or RNA, which hybridizes to DNA or RNA, which encodes OPN, in accordance with the present invention, under moderately or highly stringent conditions. The term "stringent conditions" refers to hybridization and subsequent washing conditions, which those of ordinary skill in the art conventionally refer to as "stringent". See Ausubel et al., Current Protocols in Molecular Biology, supra, Interscience, N.Y., .sctn..sctn.6.3 and 6.4 (1987, 1992), and Sambrook et al.(Sambrook, J. C., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).

Without limitation, examples of stringent conditions include washing conditions 12 20.degree. C. below the calculated Tm of the hybrid under study in, e.g., 2.times.SSC and 0.5% SDS for 5 minutes, 2.times.SSC and 0.1% SDS for 15 minutes; 0.1.times.SSC and 0.5% SDS at 37.degree. C. for 30 60 minutes and then, a 0.1.times.SSC and 0.5% SDS at 68.degree. C. for 30 60 minutes. Those of ordinary skill in this art understand that stringency conditions also depend on the length of the DNA sequences, oligonucleotide probes (such as 10 40 bases) or mixed oligonucleotide probes. If mixed probes are used, it is preferable to use tetramethyl ammonium chloride (TMAC) instead of SSC. See Ausubel, supra.

In a preferred embodiment, any such mutein has at least 40% identity or homology with the sequence of SEQ ID NO: 1, 2 or 3 of the annexed sequence listing. More preferably, it has at least 50%, at least 60%, at least 70%, at least 80% or, most preferably, at least 90% identity or homology thereto.

Identity reflects a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, determined by comparing the sequences. In general, identity refers to an exact nucleotide to nucleotide or amino acid to amino acid correspondence of the two polynucleotides or two polypeptide sequences, respectively, over the length of the sequences being compared.

For sequences where there is not an exact correspondence, a "% identity" may be determined. In general, the two sequences to be compared are aligned to give a maximum correlation between the sequences. This may include inserting "gaps" in either one or both sequences, to enhance the degree of alignment. A % identity may be determined over the whole length of each of the sequences being compared (so-called global, alignment), that is particularly suitable for sequences of the same or very similar length, or over shorter, defined lengths (so-called local alignment), that is more suitable for sequences of unequal length.

Methods for comparing the identity and homology of two or more sequences are well known in the art. Thus for instance, programs available in the Wisconsin Sequence Analysis Package, version 9.1 (Devereux J et al 1984), for example the programs BESTFIT and GAP, may be used to determine the % identity between two polynucleotides and the % identity and the % homology between two polypeptide sequences. BESTFIT uses the "local homology" algorithm of Smith and Waterman (1981) and finds the best single region of similarity between two sequences. Other programs for determining identity and/or similarity between sequences are also known in the art, for instance the BLAST family of programs (Altschul S F et al, 1990, Altschul S F et al, 1997, accessible through the home page of the NCBI at www.ncbi.nlm.nih.gov) and FASTA (Pearson W R, 1990; Pearson 1988).

Preferred changes for muteins in accordance with the present invention are what are known as "conservative" substitutions. Conservative amino acid substitutions of osteopontin polypeptides, may include synonymous amino acids within a group which have sufficiently similar physicochemical properties that substitution between members of the group will preserve the biological function of the molecule (Grantham, 1974). It is clear that insertions and deletions of amino acids may also be made in the above-defined sequences without altering their function, particularly if the insertions or deletions only involve a few amino acids, e.g. under thirty, and preferably under ten, and do not remove or displace amino acids which are critical to a functional conformation, e.g. cysteine residues. Proteins and muteins produced by such deletions and/or insertions come within the purview of the present invention.

Preferably, the synonymous amino acid groups are those defined in Table I -- see Original Patent. More preferably, the synonymous amino acid groups are those defined in Table II -- see Original Patent; and most preferably the synonymous amino acid groups are those defined in Table III -- see Original Patent.

Examples of production of amino acid substitutions in proteins which can be used for obtaining muteins of osteopontin, polypeptides or proteins, for use in the present invention include any known method steps, such as presented in U.S. Pat. Nos. 4,959,314, 4,588,585 and 4,737,462, to Mark et al; U.S. Pat. No. 5,116,943 to Koths et al., U.S. Pat. No. 4,965,195 to Namen et al; U.S. Pat. No. 4,879,111 to Chong et al; and U.S. Pat. No. 5,017,691 to Lee et al; and lysine substituted proteins presented in U.S. Pat. No. 4,904,584 (Shaw et al).

The term "fused protein" refers to a polypeptide comprising osteopontin, or a mutein or fragment thereof, fused with another protein, which, e.g. has an extended residence time in body fluids. An osteopontin may thus be fused to another protein, polypeptide or the like, e.g. an immunoglobulin or a fragment thereof.

"Functional derivatives" as used herein, cover derivatives of osteopontin, and their muteins and fused proteins, which may be prepared from the functional groups which occur as side chains on the residues or the N- or C-terminal groups, by means known in the art, and are included in the invention as long as they remain pharmaceutically acceptable, i.e. they do not destroy the activity of the protein which is substantially similar to the activity of osteopontin, and do not confer toxic properties on compositions containing it.

These derivatives may, for example, include polyethylene glycol side-chains, which may mask antigenic sites and extend the residence of an osteopontin in body fluids. Other derivatives include aliphatic esters of the carboxyl groups, amides of the carboxyl groups by reaction with ammonia or with primary or secondary amines, N-acyl derivatives of free amino groups of the amino acid residues formed with acyl moieties (e.g alkanoyl or carbocyclic aroyl groups) or O-acyl derivatives of free hydroxyl groups (for example that of seryl or threonyl residues) formed with acyl moieties.

As "active fractions" of osteopontin, muteins and fused proteins, the present invention covers any fragment or precursors of the polypeptide chain of the protein molecule alone or together with associated molecules or residues linked thereto, e.g. sugar or phosphate residues, or aggregates of the protein molecule or the sugar residues by themselves, provided said fraction has substantially similar activity to osteopontin.

The term "salts" herein refers to both salts of carboxyl groups and to acid addition salts of amino groups of OPN molecule or analogs thereof. Salts of a carboxyl group may be formed by means known in the art and include inorganic salts, for example, sodium, calcium, ammonium, ferric or zinc salts, and the like, and salts with organic bases as those formed, for example, with amines, such as triethanolamine, arginine or lysine, piperidine, procaine and the like. Acid addition salts include, for example, salts with mineral acids, such as, for example, hydrochloric acid or sulfuric acid, and salts with organic acids, such as, for example, acetic acid or oxalic acid. Of course, any such salts must retain the biological activity of OPN relevant to the present invention, i.e., exert a proliferative effect on oligodendrocytes.

In a preferred embodiment of the invention, osteopontin is fused to a carrier molecule, a peptide or a protein that promotes the crossing of the blood brain barrier ("BBB"). This serves for proper targeting of the molecule to the site of action in those cases, in which the CNS is involved in the disease. Modalities for drug delivery through the BBB entail disruption of the BBB, either by osmotic means or biochemically by the use of vasoactive substances such as bradykinin. Other strategies to go through the BBB may entail the use of endogenous transport systems, including carrier-mediated transporters such as glucose and amino acid carriers; receptor-mediated transcytosis for insulin or transferrin; and active efflux transporters such as p-glycoprotein. Strategies for drug delivery behind the BBB further include intracerebral implantation.

Functional derivatives of osteopontin may be conjugated to polymers in order to improve the properties of the protein, such as the stability, half-life, bioavailability, tolerance by the human body, or immunogenicity. To achieve this goal, osteopontin may be linked e.g. to Polyethlyenglycol (PEG). PEGylation may be carried out by known methods, described in WO 92/13095, for example.

Therefore, in a preferred embodiment of the present invention, osteopontin is PEGylated.

In a further preferred embodiment of the invention, the fused protein comprises an immunoglobulin (Ig) fusion. The fusion may be direct, or via a short linker peptide which can be as short as 1 to 3 amino acid residues in length or longer, for example, 13 amino acid residues in length. Said linker may be a tripeptide of the sequence E-F-M (Glu-Phe-Met), for example, or a 13-amino acid linker sequence comprising Glu-Phe-Gly-Ala-Gly-Leu-Val-Leu-Gly-Gly-Gln-Phe-Met (SEQ ID NO: 6) introduced between osteopontin sequence and the immunoglobulin sequence, for instance. The resulting fusion protein has improved properties, such as an extended residence time in body fluids (half-life), or an increased specific activity, increased expression level. The Ig fusion may also facilitate purification of the fused protein.

In a yet another preferred embodiment, osteopontin is fused to the constant region of an Ig molecule. Preferably, it is fused to heavy chain regions, like the CH2 and CH3 domains of human IgG1, for example. Other isoforms of Ig molecules are also suitable for the generation of fusion proteins according to the present invention, such as isoforms IgG.sub.2 or IgG.sub.4, or other Ig classes, like IgM, for example. Fusion proteins may be monomeric or multimeric, hetero- or homomultimeric. The immunoglobulin portion of the fused protein may be further modified in a way as to not activate complement binding or the complement cascade or bind to Fc-receptors.

The invention further relates to the use of a combination of osteopontin and an immunosuppressive agent for the manufacture of a medicament for treatment and/or prevention of neurologic disorders, for simultaneous, sequential or separate use. Immunosuppressive agents may be steroids, methotrexate, cyclophosphamide, anti-leukocyte antibodies (such as CAMPATH-1), and the like.

The invention further relates to the use of a combination of osteopontin and an interferon for the manufacture of a medicament for treatment and/or prevention of neurologic disorders, for simultaneous, sequential, or separate use.

The term "interferon", as used in the present patent application, is intended to include any molecule defined as such in the literature, comprising for example any kinds of IFNs mentioned in the above section "Background of the Invention". The interferon may preferably be human, but also derived from other species, as long as the biological activity is similar to human interferons, and the molecule is not immunogenic in man.

In particular, any kinds of IFN-.alpha., IFN-.beta. and IFN-.gamma. are included in the above definition. IFN-.beta. is the preferred IFN according to the present invention.

The term "interferon-beta (IFN-.beta.)", as used in the present invention, is intended to include human fibroblast interferon, as obtained by isolation from biological fluids or as obtained by DNA recombinant techniques from prokaryotic or eukaryotic host cells as well as its salts, functional derivatives, variants, analogs and fragments.

"Functional derivatives", as used herein, covers derivatives which may be prepared from the functional groups which occur as side chains on the residues or the N- or C-terminal groups, by means known in the art, and are included in the invention as long as they remain pharmaceutically acceptable, i.e., they do not destroy the biological activity of the proteins as described above, such as the ability to bind the corresponding receptor and initiate receptor signaling, and do, not confer toxic properties on compositions containing it. Derivatives may have chemical moieties, such as carbohydrate or phosphate residues, provided such a derivative retains the biological activity of the protein and remains pharmaceutically acceptable.

For example, derivatives may include aliphatic esters of the carboxyl groups, amides of the carboxyl groups by reaction with ammonia or with primary or secondary amines, N-acyl derivatives or free amino groups of the amino acid residues formed with acyl moieties (e.g. alkanoyl or carbocyclic aroyl groups) or O-acyl derivatives of free hydroxyl group (e.g. that of seryl or threonyl residues) formed with acyl moieties. Such derivatives may also include for example, polyethylene glycol side-chains which may mask antigenic sites and extend the residence of the molecule in body fluids.

Of particular importance is a protein that has been derivatized or combined with a complexing agent to be long lasting. For example, PEGylated versions, as mentioned above, or proteins genetically engineered to exhibit long lasting activity in the body, can be used according to the present invention.

The term "derivatives" is intended to include only those derivatives that do not change one amino acid to another of the twenty commonly-occurring natural amino acids.

The term "salts" herein refers to both salts of carboxyl groups and to acid addition salts of amino groups of the proteins described above or analogs thereof. Salts of a carboxyl group may be formed by means known in the art and include inorganic salts, for example, sodium, calcium, ammonium, ferric or zinc salts, and the like, and salts with organic bases as those formed, for example, with amines, such as triethanolamine, arginine or lysine, piperidine, procaine and the like. Acid addition salts include, for example, salts with mineral acids, such as, for example, hydrochloric acid or sulfuric acid, and salts with organic acids, such as, for example, acetic acid or oxalic acid. Of course, any such salts must retain the biological activity of the proteins (osteopontin and IFN-beta, respectively) relevant to the present invention, i.e., the ability to bind to the corresponding receptor and initiate receptor signaling.

Interferons may also be conjugated to polymers in order to improve the stability of the proteins. A conjugate between Interferon .beta. and the polyol Polyethlyenglycol (PEG) has been described in WO99/55377, for instance.

In another preferred embodiment of the invention, the interferon is Interferon-.beta. (IFN-.beta.), and more preferably IFN-.beta.1a.

Osteopontin is preferably used simultaneously, sequentially, or separately with the interferon.

In a preferred embodiment of the present invention, osteopontin is used in an amount of about 0.0001 to 100 mg/kg of body weight, or about 0.01 to 10 mg/kg of body weight or about 1 to 5 mg/kg of body weight or about 2 mg/kg of body weight.

The invention further relates to the use of a nucleic acid molecule for manufacture of a medicament for the treatment and/or prevention of a neurologic disease, wherein the nucleic acid molecule comprises a nucleic acid sequence encoding a polypeptide comprising an amino acid sequence selected from the group consisting of:

(a) A polypeptide comprising SEQ ID NO: 1;

(b) A polypeptide comprising amino acids 1 to 168 or 170 of SEQ ID NO: 1;

(c) A polypeptide comprising amino acids 1 to 16 and 170 to 314 of SEQ ID NO: 1;

(d) A polypeptide comprising amino acids 170 to 314 of SEQ ID NO: 1;

(e) A polypeptide comprising SEQ ID NO: 2;

(f) A polypeptide comprising SEQ ID NO: 3;

(g) A mutein of any of (a) to (f), wherein the amino acid sequence has at least 40% or 50% or 60% or 70% or 80% or 90% identity to at least one of the sequences in (a) to (f);

(h) A mutein of any of (a) to (f) which is encoded by a DNA sequence which hybridizes to the complement of the native DNA sequence encoding any of (a) to (f) under moderately stringent conditions or under highly stringent conditions;

(i) A mutein of any of (a) to (f) wherein any changes in the amino acid sequence are conservative amino acid substitutions to the amino acid sequences in (a) to (f);

(j) an isoform, fused protein, functional derivative, active fraction or circularly permutated derivative of any of (a) to (f).

The nucleic acid may e.g. be administered as a naked nucleic acid molecule, e.g. by intramuscular injection.

It may further comprise vector sequences, such as viral sequence, useful for expression of the gene encoded by the nucleic acid molecule in the human body, preferably in the appropriate cells or tissues.

Therefore, in a preferred embodiment, the nucleic acid molecule further comprises an expression vector sequence. Expression vector sequences are well known in the art, they comprise further elements serving for expression of the gene of interest. They may comprise regulatory sequence, such as promoter and enhancer sequences, selection marker sequences, origins of multiplication, and the like. A gene therapeutic approach is thus used for treating and/or preventing the disease. Advantageously, the expression of osteopontin will then be in situ.

In a preferred embodiment, the expression vector is a lentiviral derived vector. Lentiviral vectors have been shown to be very efficient in the transfer of genes, in particular within the CNS. Other well established viral vectors, such as adenoviral derived vectors, may also be used according to the invention.

A targeted vector may be used in order to enhance the passage of osteopontin across the blood-brain barrier. Such vectors may target for example the transferrin receptor or other endothelial transport mechanisms.

In a preferred embodiment of the invention, the expression vector may be administered by intramuscular injection.

The use of a vector for inducing and/or enhancing the endogenous production of osteopontin in a cell normally silent for expression of osteopontin, or which expresses amounts of osteopontin which are not sufficient, are also contemplated according to the invention. The vector may comprise regulatory sequences functional in the cells desired to express osteopontin. Such regulatory sequences may be promoters or enhancers, for example. The regulatory sequence may then be introduced into the appropriate locus of the genome by homologous recombination, thus operably linking the regulatory sequence with the gene, the expression of which is required to be induced or enhanced. The technology is usually referred to as "endogenous gene activation" (EGA), and it is described e.g. in WO 91/09955.

The invention further relates to the use of a cell that has been genetically modified to produce osteopontin in the manufacture of a medicament for the treatment and/or prevention of neurologic diseases.

The invention further relates to a cell that has been genetically modified to produce osteopontin for manufacture of a medicament for the treatment and/or prevention of neurologic diseases. Thus, a cell therapeutic approach may be used in order to deliver the drug to the appropriate parts of the human body.

The invention further relates to pharmaceutical compositions, particularly useful for prevention and/or treatment of neurologic diseases, which comprise a therapeutically effective amount of osteopontin and a therapeutically effective amount of an interferon, optionally further a therapeutically effective amount of an immunosuppressant.

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, the active protein(s) may be formulated in a unit dosage form for injection in vehicles such as saline, dextrose solution, serum albumin and Ringer's solution.

The active ingredients of the pharmaceutical composition according to the invention can be administered to an individual 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, intrathecal, rectal, 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 active agent is administered to the patient (e.g. via a vector), which causes the active agent to be expressed and secreted in vivo. In addition, the protein(s) according to the invention 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, the active protein(s) can be formulated as a solution, suspension, emulsion or lyophilised 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.

The bioavailability of the active protein(s) according to the invention can also be ameliorated by using conjugation procedures which increase the half-life of the molecule in the human body, for example linking the molecule to polyethylenglycol, as described in the PCT Patent Application WO 92/13095.

The therapeutically effective amounts of the active protein(s) will be a function of many variables, including the type of protein, the affinity of the protein, any residual cytotoxic activity exhibited by the antagonists, the route of administration, the clinical condition of the patient (including the desirability of maintaining a non-toxic level of endogenous osteopontin activity).

A "therapeutically effective amount" is such that when administered, the osteopontin exerts a beneficial effect on the neurologic disease. The dosage administered, as single or multiple doses, to an individual will vary depending upon a variety of factors, including osteopontin 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.

As mentioned above, osteopontin can preferably be used in an amount of about 0.0001 to 10 mg/kg or about 0.01 to 5 mg/kg or body weight, or about 0.01 to 5 mg/kg of body weight or about 0.1 to 3 mg/kg of body weight or about 1 to 2 mg/kg of body weight. Further preferred amounts of osteopontin are amounts of about 0.1 to 1000 .mu.g/kg of body weight or about 1 to 100 .mu.g/kg of body weight or about 10 to 50 .mu.g/kg of body weight

The route of administration, which is preferred according to the invention is administration by subcutaneous route. Intramuscular administration is further preferred according to the invention.

In further preferred embodiments, osteopontin is administered daily or every other day.

The daily doses are usually given in divided doses or in sustained release form effective to obtain the desired results. Second or subsequent administrations can be performed at a dosage which is the same, less than or greater than the initial or previous dose administered to the individual. A second or subsequent administration can be administered during or prior to onset of the disease.

According to the invention, osteopontin can be administered prophylactically or therapeutically to an individual prior to, simultaneously or sequentially with other therapeutic regimens or agents (e.g. multiple drug regimens), in a therapeutically effective amount, in particular with an interferon. Active agents that are administered simultaneously with other therapeutic agents can be administered in the same or different compositions.

The invention further relates to a method for treating a neurologic disease comprising administering to a patient in need thereof an effective amount of osteopontin, or of an agonist of osteopontin activity, optionally together with a pharmaceutically acceptable carrier.

A method for treating a neurologic disease comprising administering to a patient in need thereof an effective amount of osteopontin, or of an agonist of osteopontin activity, and an interferon, optionally together with a pharmaceutically acceptable carrier, is also within the present invention.


Claim 1 of 19 Claims

1. A method of treating a neurologic disease, comprising administering to a patient in need thereof a medicament comprising osteopontin, or an agonist of osteopontin activity, optionally together with a pharmaceutically acceptable carrier.

 

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