A vaccine for reducing disease progression, and/or protection of motor nerve degeneration, and/or protection from glutamate toxicity in motor neurone disease (MND), particularly amyotrophic lateral sclerosis (ALS), patients, comprising an active agent selected from the group consisting of Cop 1, a Cop 1-related peptide, a Cop 1-related polypeptide, and poly-Glu, Tyr. The active agent is preferably Cop 1 or poly-Glu, Tyr, and can be administered with or without an adjuvant.

Description of the Invention

SUMMARY OF THE INVENTION

It has now been found, in accordance with the present invention, that immunization with Cop 1 can protect transgenic mice overexpressing human SOD1 and mice after facial nerve axotomy, both models for ALS, from motor neuron degeneration. This and the fact that both Cop 1 and PolyYE are effective in protecting retinal ganglion cells from glutamate toxicity, indicates the suitability of these copolymers for the treatment of motor neurone diseases, particularly ALS.

The present invention thus relates, in one aspect, to a method for reducing disease progression, for protection of motor neuron degeneration and/or for protection from glutamate toxicity in a patient suffering from a motor neurone disease (MND), which comprises immunizing said patient with a vaccine comprising an active agent selected from the group consisting of Cop 1, a Cop 1-related peptide, a Cop 1-related polypeptide, and PolyYE.

The motor neurone disease (MND) is any disease affecting the motor neurones in the brain and spinal cord and includes amyotrophic lateral sclerosis (ALS), both familial (FALS) and sporadic (SALS) ALS, primary lateral sclerosis (PLS), progressive muscular atrophy (PMA), progressive bulbar palsy (PBP or bulbar onset), and combined forms thereof such as bulbar onset ALS and bulbar onset PMA.

In one embodiment, the method of the invention includes treatment also with Riluzole or any other drug suitable for treatment of MND, particularly ALS.

In another aspect, the present invention provides a vaccine for reducing disease progression, for protection of motor nerve degeneration and/or for protection from glutamate toxicity in a motor neurone disease (MND), particularly ALS, comprising an active agent selected from the group consisting of Cop 1, a Cop 1-related peptide, a Cop 1-related polypeptide, and poly-Glu, Tyr.

In a further aspect, the present invention relates to the use of an active agent selected from the group consisting of Cop 1, a Cop 1-related peptide, a Cop 1-related polypeptide, and poly-Glu, Tyr, for the manufacture of a vaccine for reducing disease progression, for protection of motor nerve degeneration and/or for protection from glutamate toxicity in motor neurone disease (ND), particularly ALS.

The active agent may be administered without any adjuvant or it may be emulsified in an adjuvant suitable for human clinical use. The adjuvant suitable for human clinical use is selected from aluminum hydroxide, aluminum hydroxide gel, and aluminum hydroxyphosphate. In a preferred embodiment, the vaccine adjuvant is amorphous aluminum hydroxyphosphate having an acidic isoelectric point and an Al:P ratio of 1:1 (herein referred to as Alum-phos).

In one preferred embodiment, the active agent of the vaccine of the invention is Cop 1. In another preferred embodiment, the active agent is poly-Glu, Tyr.

In addition, the vaccine may be administered in a regimen that includes administration of Riluzole or another drug suitable for treatment of ALS.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a vaccine and a method for reducing disease progression, for protection of motor nerve degeneration, for prolonging life span and improving quality of life, and/or for protection from glutamate toxicity in a patient suffering from MND, particularly ALS, which comprises immunizing said patient with a vaccine comprising an active agent selected from the group consisting of Cop 1, a Cop 1-related peptide, a Cop 1-related polypeptide, or PolyYE, either without adjuvant or emulsified in an adjuvant suitable for human clinical use.

As used herein, the terms "motor neurons" and "motor neurons", the terms "PolyYE" and "poly-Glu, Tyr", and the terms "Cop 1" and "Copolymer 1", are each used interchangeably.

For the purpose of the present invention, "Cop 1 or a Cop 1-related peptide or polypeptide" is intended to include any peptide or polypeptide, including a random copolymer, that cross-reacts functionally with myelin basic protein (MBP) and is able to compete with MBP on the MHC class II in the antigen presentation.

The vaccine of the invention may comprise as active agent a random copolymer comprising a suitable quantity of a positively charged amino acid such as lysine or arginine, in combination with a negatively charged amino acid (preferably in a lesser quantity) such as glutamic acid or aspartic acid, optionally in combination with a non-charged neutral amino acid such as alanine or glycine, serving as a filler, and optionally with an amino acid adapted to confer on the copolymer immunogenic properties, such as an aromatic amino acid like tyrosine or tryptophan. Such vaccines may include any of those copolymers disclosed in WO 00/05250, the entire contents of which being hereby incorporated herein by reference.

More specifically, the vaccine for use in the present invention comprises at least one copolymer selected from the group consisting of random copolymers comprising one amino acid selected from each of at least three of the following groups: (a) lysine and arginine; (b) glutamic acid and aspartic acid; (c) alanine and glycine; and (d) tyrosine and tryptophan.

The copolymers for use in the present invention can be composed of L- or D-amino acids or mixtures thereof. As is known by those of skill in the art, L-amino acids occur in most natural proteins. However, D-amino acids are commercially available and can be substituted for some or all of the amino acids used to make the terpolymers and other copolymers used in the present invention. The present invention contemplates the use of copolymers containing both D- and L-amino acids, as well as copolymers consisting essentially of either L- or D-amino acids.

In one embodiment of the invention, the copolymer contains four different amino acids, each from a different one of the groups (a) to (d). A preferred copolymer according to this embodiment comprises in combination alanine, glutamic acid, lysine, and tyrosine, of net overall positive electrical charge and of a molecular weights of about 2,000-40,000 Da, preferably of about 2,000-13,000 Da, and is most preferably Copolymer 1 of average molecular weight of about 4,700-13,000 Da. Preferred molecular weight ranges and processes for making a preferred form of Cop 1 are described in U.S. Pat. No. 5,800,808, the entire contents of which being hereby incorporated in the entirety. It is clear that this is given by way of example only, and that the vaccine can be varied both with respect to the constituents and relative proportions of the constituents if the above general criteria are adhered to. Thus, the copolymer may be a polypeptide from about 15 to about 100, preferably from about 40 to about 80, amino acids in length, and is preferably the copolymer having the generic name glatiramer acetate.

In another embodiment, the copolymer contains three different amino acids each from a different one of three groups of the groups (a) to (d). These copolymers are herein referred to as terpolymers.

In one embodiment, the terpolymers for use in the present invention contain tyrosine, alanine, and lysine, hereinafter designated YAK. The average molar fraction of the amino acids in these terpolymers can vary. For example, tyrosine can be present in a mole fraction of about 0.005-0.250; alanine can be present in a mole fraction of about 0.3-0.6; and lysine can be present in a mole fraction of about 0.1-0.5. The average molecular weight is between 2,000-40,000 Da, and preferably between about 3,000-35,000 Da. In a more preferred embodiment, the average molecular weight is about 5,000-25,000 Da. It is possible to substitute arginine for lysine, glycine for alanine, and/or tryptophan for tyrosine.

In another embodiment, the terpolymers for use in the present invention contain tyrosine, glutamic acid, and lysine, hereinafter designated YEK. The average molar fraction of the amino acids in these terpolymers can vary: glutamic acid can be present in a mole fraction of about 0.005-0.300, tyrosine can be present in a mole fraction of about 0.005-0.250, and lysine can be present in a mole fraction of about 0.3-0.7. The average molecular weight is between 2,000-40,000 Da, and preferably between about 3,000-35,000 Da. In a more preferred embodiment, the average molecular weight is about 5,000-25,000 Da. It is possible to substitute aspartic acid for glutamic acid, arginine for lysine, and/or tryptophan for tyrosine.

In another embodiment the terpolymers for use in the present invention contain lysine, glutamic acid, and alanine, hereinafter designated KEA. The average molar fraction of the amino acids in these polypeptides can also vary. For example, glutamic acid can be present in a mole fraction of about 0.005-0.300, alanine can be present in a mole fraction of about 0.005-0.600, lysine can be present in a mole fraction of about 0.2-0.7. The average molecular weight is between 2,000-40,000 Da, and preferably between about 3,000-35,000 Da. In a more preferred embodiment, the average molecular weight is about 5,000-25,000 Da. It is possible to substitute aspartic acid for glutamic acid, glycine for alanine, and/or arginine for lysine.

In another embodiment, the terpolymers for use in the present invention contain tyrosine, glutamic acid, and alanine, hereinafter designated YEA. The average molar fraction of the amino acids in these polypeptides can vary. For example, tyrosine can be present in a mole fraction of about 0.005-0.250, glutamic acid can be present in a mole fraction of about 0.005-0.300, and alanine can be present in a mole fraction of about 0.005-0.800. The average molecular weight is between 2,000-40,000 Da, and preferably between about 3,000-35,000 Da. In a more preferred embodiment, the average molecular weight is about 5,000-25,000 Da. It is possible to substitute tryptophan for tyrosine, aspartic acid for glutamic acid, and/or glycine for alamine.

In a more preferred embodiment, the mole fraction of amino acids of the terpolymers is about what is preferred for Copolymer 1. The mole fraction of amino acids in Copolymer 1 is glutamic acid about 0.14, alanine about 0.43, tyrosine about 0.10, and lysine about 0.34. The most preferred average molecular weight for Copolymer 1 is between about 5,000-9,000 Da. The activity of Copolymer 1 for the vaccine disclosed herein is expected to remain if one or more of the following substitutions is made: aspartic acid for glutamic acid, glycine for alanine, arginine for lysine, and tryptophan for tyrosine.

The molar ratios of the monomers of the more preferred terpolymer of glutamic acid, alamine, and tyrosine, or YEA, is about 0.21 to about 0.65 to about 0.14.

The molar ratios of the monomers of the more preferred terpolymer of glutamic acid, alanine and lysine, or KEA, is about 0.15 to about 0.48 to about 0.36.

The molar ratios of the monomers of the more preferred terpolymer of glutamic acid, tyrosine, and lysine, or YEK, is about 0.26 to about 0.16 to about 0.58.

The molar ratios of the monomers of the more preferred terpolymer of tyrosine, alanine and lysine, or YAK, is about 0.10 to about 0.54 to about 0.35.

The terpolymers can be made by any procedure available to one of skill in the art. For example, the terpolymers can be made under condensation conditions using the desired molar ratio of amino acids in solution, or by solid phase synthetic procedures. Condensation conditions include the proper temperature, pH, and solvent conditions for condensing the carboxyl group of one amino acid with the amino group of another amino acid to form a peptide bond. Condensing agents, for example dicyclohexyl-carbodiimide, can be used to facilitate the formation of the peptide bond. Blocking groups can be used to protect functional groups, such as the side chain moieties and some of the amino or carboxyl groups against undesired side reactions.

For example, the process disclosed in U.S. Pat. No. 3,849,650, can be used wherein the N-carboxyanhydrides of tyrosine, alanine, .gamma.-benzyl glutamate and N .epsilon.-trifluoroacetyl-lysine are polymerized at ambient temperatures in anhydrous dioxane with diethylamine as an initiator. The .gamma.-carboxyl group of the glutamic acid can be deblocked by hydrogen bromide in glacial acetic acid. The trifluoroacetyl groups are removed from lysine by 1 molar piperidine. One of skill in the art readily understands that the process can be adjusted to make peptides and polypeptides containing the desired amino acids, that is, three of the four amino acids in Copolymer 1, by selectively eliminating the reactions that relate to any one of glutamic acid, alanine, tyrosine, or lysine. For purposes of this application, the terms "ambient temperature" and "room temperature" mean a temperature ranging from about 20 to about 26.degree. C.

The molecular weight of the terpolymers can be adjusted during polypeptide synthesis or after the terpolymers have been made. To adjust the molecular weight during polypeptide synthesis, the synthetic conditions or the amounts of amino acids are adjusted so that synthesis stops when the polypeptide reaches the approximate length which is desired. After synthesis, polypeptides with the desired molecular weight can be obtained by any available size selection procedure, such as chromatography of the polypeptides on a molecular weight sizing column or gel, and collection of the molecular weight ranges desired. The present polypeptides can also be partially hydrolyzed to remove high molecular weight species, for example, by acid or enzymatic hydrolysis, and then purified to remove the acid or enzymes.

In one embodiment, the terpolymers with a desired molecular weight may be prepared by a process which includes reacting a protected polypeptide with hydrobromic acid to form a trifluoroacetyl-polypeptide having the desired molecular weight profile. The reaction is performed for a time and at a temperature which is predetermined by one or more test reactions. During the test reaction, the time and temperature are varied and the molecular weight range of a given batch of test polypeptides is determined. The test conditions which provide the optimal molecular weight range for that batch of polypeptides are used for the batch. Thus, a trifluoroacetyl-polypeptide having the desired molecular weight profile can be produced by a process which includes reacting the protected polypeptide with hydrobromic acid for a time and at a temperature predetermined by test reaction. The trifluoroacetyl-polypeptide with the desired molecular weight profile is then further treated with an aqueous piperidine solution to form a low toxicity polypeptide having the desired molecular weight.

In a preferred embodiment, a test sample of protected polypeptide from a given batch is reacted with hydrobromic acid for about 10-50 hours at a temperature of about 20-28.degree. C. The best conditions for that batch are determined by running several test reactions. For example, in one embodiment, the protected polypeptide is reacted with hydrobromic acid for about 17 hours at a temperature of about 26.degree. C.

As binding motifs of Cop 1 to MS-associated HLA-DR molecules are known (Fridkis-Hareli et al, 1999), polypeptides of fixed sequence can readily be prepared and tested for binding to the peptide binding groove of the HLA-DR molecules as described in the Fridkis-Hareli et al (1999) publication. Examples of such peptides are those disclosed in WO 005249, the entire contents of which being hereby incorporated herein by reference. Thirty-two of the peptides specifically disclosed in said application are reproduced in Table 1 (see Original Patent), hereinbelow. Such peptides and other similar peptides would be expected to have similar activity as Cop 1. Such peptides, and other similar peptides, are also considered to be within the definition of Cop 1-related peptides or polypeptides and their use is considered to be part of the present invention.

The definition of "Cop 1 related-polypeptide" according to the invention is meant to encompass other synthetic amino acid copolymers such as the random four-amino acid copolymers described by Fridkis-Hareli et al., 2002, as candidates for treatment of multiple sclerosis, namely copolymers (14-, 35- and 50-mers) containing the amino acids phenylalanine, glutamic acid, alanine and lysine (poly FEAK), or tyrosine, phenylanine, alanine and lysine (poly YFAK), and any other similar copolymer to be discovered that can be considered a universal antigen similar to Cop 1 and polyYE.

According to the present invention, the preferred copolymer for use in the vaccine of the invention is Copolymer 1, herein referred to also as Cop 1, most preferably in the form of its acetate salt known under the generic name Glatiramer acetate. Glatiramer acetate has been approved in several countries for the treatment of multiple sclerosis (MS) under the trade name, COPAXONE.RTM. (a trademark of Teva Pharmaceuticals Ltd., Petah Tikva, Israel). Several clinical trials demonstrated that Cop 1 is well tolerated with only minor side reactions which were mostly mild reactions at the injection site (Johnson et al, 1995).

As mentioned before, mutations in the SOD1 gene are one genetic cause for familial ALS (Rosen et al., 1993; Brown, 1995). Several mouse models that express the mutated SOD1 genes develop motor neuron degeneration similar to that in humans (Gurney et al., 1994; Ripps et al., 1995; Kong and Xu, 1998). The initial characterization of these mouse lines has proven that a dominant gain of an adverse property by the mutated enzymes causes motor neuron degeneration (for review, see Bruijn and Cleveland, 1996). In addition, these analyses confirmed numerous pathological features that have been observed in humans (Hirano, 1991; Chou, 1992). Understanding this mutation, called SOD1 alteration, yielded an accepted animal model (ALS mice) for testing therapies for familial ALS. Since SOD1-related familial ALS and sporadic ALS (which accounts for 90% of all ALS cases) have similar symptoms and pathological features, the transgenic mouse carrying a mutated SOD1 gene is an accepted animal model for testing therapies for both familial and sporadic ALS forms, and is the model used by the ALS Therapy Development Foundation (ALS-TDF). ALS mice develop a motor disease that closely resembles ALS. The motor dysfunction eventually causes their death.

According to the present invention, ALS mice which were immunized with a vaccine of Cop 1 emulsified in CFA or in an adjuvant suitable for human use were shown to be protected from motor nerve degeneration, in spite of the oxidative stress conditions created by the overexpression of SOD. Thus, vaccination with the "universal" weak self-reactive antigen Cop 1 in CFA prolonged by 52 days the life span of ALS mice (mean.+-.SD, 263.+-.8 days, n=14) compared to untreated matched controls (211.+-.7 days; n=15; P<0.0001). The vaccination significantly improved motor activity in the clinical and pre-clinical stages. In addition, vaccination with Cop 1 also prevented acute motor neuron degeneration after facial nerve axotomy: almost 200% more motor neurons survived in vaccinated mice than in axotomized controls (P<0.05). These results suggest that the concept of autoimmunity as protective can be extended to include motor neuron diseases. They also have potentially dramatic clinical implications.

The adjuvants used for the immunization according to the invention are aluminum-based adjuvants. More commonly used in vaccines containing virus-derived antigens such as hepatitis B surface antigen or Haemophilus influenza type b capsular polysaccharide, these adjuvants are for the first time used together with synthetic copolymers, particularly with Cop 1.

The dosage of Cop 1 or PolyYE to be administered will be determined by the physician according to the age of the patient and stage of the disease and may be chosen from a range of 10-80 mg, although any other suitable dosage is encompassed by the invention. The administration may be made at least once a month or at least once every 2 or 3 months, or less frequently, but any other suitable interval between the immunizations is envisaged by the invention according to the condition of the patient.

The vaccine of the invention may be administered by any suitable mode of administration, including orally, intramuscularly, subcutaneously and intradermally, with or without adjuvant.

When administered together with Riluzole or any other drug suitable for treatment of MND, particularly ALS, the additional drug is administered at the same day of vaccination, and daily thereafter, according to the manufacturer's instructions, with no association to the vaccine regimen. For example, the daily dose of Riluzole is 100 mg.
 

Claim 1 of 11 Claims

1. A method for protection against motor nerve degeneration, and/or protection from glutamate toxicity in a patient suffering from amyotrophic lateral sclerosis (ALS), which comprises immunizing said patient with a vaccine comprising a therapeutically effective amount of an active agent selected from the group consisting of Copolymer 1, a Copolymer 1-related peptide, and a Copolymer 1-related polypeptide.

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