Peptide specificity of antibodies to mbp (myelin basic protein), containing pharmaceutical composition and method for the treatment of multiple sclerosis

 

(57) Abstract:

Describes a new peptide or its salt, can neutralize or modulate the formation of antibodies to myelin basic protein, having the formula I R1-Val-His-Phe-Phe-Lys-Asn-Ile-R2where R1selected from the group comprising Asn-Pro-Val-; Pro-Val-; Val-; Lys-Ser-His-Gly-Arg-Thr-Gln-Asp-Glu-Asn-Pro-Val-; Thr-Gln-Asp-Glu-Asn-Pro-Val-; hydrogen and hydroxy; R2selected from the group comprising-Val; -Val-Thr; -Val-Thr-Pro-Val-Thr-Pro-Arg-, hydrogen and hydroxy, provided that R1and R2at the same time are not hydrogen or hydroxyl, with this peptide contains substitutions, additions or deletions. Also described pharmaceutical composition based on compounds of General formula (I) and a method for the treatment of multiple sclerosis. The technical result is increased efficiency in the treatment of multiple sclerosis. 3 s and 5 C.p. f-crystals, 10 ill., table 2.

THE SCOPE OF THE INVENTION

This invention relates to certain polypeptides and their use for the immunoregulation of antibodies to myelin basic protein man. The invention also relates to new pharmaceutical compositions containing these polypeptides, and to method of using these peptides for the treatment of p is dominant disease of the Central nervous system (CNS) of a person, associated with inflammation. Increased synthesis of lgG within the blood-brain barrier (intra-BBB) is a symptom of multiple sclerosis (Tourtelotte, W. W. J. Neurol Sci. 10: 279-304, 1970; Link, H. And Tibbling G., Scand. J. Clin. Lab. Invest. 37: 397-401, 1977; Tourtelotte, W. W. and Ma. B., Neurology 28: 76-83, 1978; Walsh, J. M. And Tourtelotte, W. W. In; Hallpike, J. F., Adams, C. W. M. And Tourtelotte, W. W. eds. Multiple Sclerosis. Baltimore. Williams & Wilkins, 1982: 275-358; and Warren. K. G. and Catz, 1. Ann Neurol 17: 475-480, 1985).

Development lgG within BBB mainly increased in patients with clinically defined disease multiple sclerosis (Schumacher, G. A., Beebe, G., R. E. Kibler et al. Ann NY Acad. Sci. 15: 266-272, 1965), as in the case of active and latent forms of the disease. The specificity of the majority of lgG CNS is unknown. While a small part of them has antiviral activity or acts in interaction with brain antigens, nucleic acids, red blood cells, or antigens of smooth muscles, other nonspecific lg may be involved in polyclonal activation of b-cells (Tourtelotte, W. W., and Ma, W., Neurology 28: 76-83, 1978). In the last decade there has been a considerable interest in the study of antibodies specific for myelin proteins.

Following the detection of circulating immune complexes containing myelin basic protein (MBP) in itry antibodies to MBP (anti-MBP) in spenomegaly fluid (CSF) of patients with the active form of multiple sclerosis (MS) (Warren, K. G. and Catz, I., Ann Neurol 209: 20-25, 1986). The clinical picture of multiple sclerosis is characterized by the active phases of the disease, such as acute recurrent or chronic course, and phases of clinical remission.

For active forms of multiple sclerosis characterized by elevated levels vnutriobolochechnoe anti-MBP (Warren, K. G. and Catz, I., Ann Neurol 209: 20-25, 1986, and Catz, I., and Warren, K. G., Can j Neurol Sci. 13: 21-24, 1986). These antibodies are detected predominantly in the free (F) form during acute relapses and mainly in the coherent (In) form when the disease progresses unnoticed (Warren, K. G. and Catz, I., Ann Neurol 209: 20-25, 1986). During acute relapses titers of anti-MBP spenomegaly fluid (CSF) correspond to the active development of the disease (Warren, K. G. and Catz, I. , Ann Neurol 21: 183-187, 1987). The levels of anti-MBP also increased in patients with first episodes of optic neuritis and in most patients with initial stages of multiple sclerosis (MS) (Warren, K. G., Catz, I. and Bauer, S. , Ann Neurol 23: 297-299, 1988; Warren, K. G. and Catz, I., J Neurol Sci. 91: 143-151, 1989).

Prolonged study of the kinetics of the levels of antibodies to MBP in CSF in patients who are at the stage of recovery after an acute relapse, showed a gradual decline of titers of anti-MBP free (F) form sootvetstvuu; Warren, K. G. and Catz, I., J. Neurol Sci. 88: 185-194, 1988). In remission anti-MBP in CSF may not be detected, suggesting that neutralizing anti-MBP is associated with inactive stage of the disease (MS) (Warren, K. G. and Catz, I. , J. Neurol Sci. 88: 185-194, 1988). On the contrary, chronic progressive multiple sclerosis (MS), characterized by a constant increase in the production of anti-MBP over an extended period of time, associated with inhibition of neutralizing anti-MBP (Warren, K. G. and Catz, I., J. Neurol Sci. 88: 185-194, 1988). Recently, the IgG-fraction obtained from the CSF of patients with MS were identified antibodies to myelin basic protein, including anti-MBP antibodies, neutralizing anti-MBP, and antibodies that inhibit neutralizing anti-MBP (Warren, K. G. and Catz, I., J. Neurol Sci. 96: 19-27, 1990).

Previous studies of the authors of the invention with regard to information about B-cell autoantibodies indicates that there are at least two different from other forms of multiple sclerosis (MS): the first is when most patients develop autoantibodies to myelin basic protein (anti-MBP), and second, when a smaller number of patients developed antibodies to the protein proteolipid nature (anti-PLP) (Warren, K. G., et al., Ann Neurol 35: 280-289, 1994). When the form is affected by free (F) and bound (B) anti-MBP, while the chronic progressive phase is characterized by a lower ratio F/B anti-MBP, and patients in remission rarely have slightly elevated titers of anti-MBP (Warren, K. G. and Catz, I., J. Neurol Sci. 88: 185-194, 1989).

It was shown that part of proliferating T cells in patients with multiple sclerosis directed to MBP (Allegretta et al., Science, 718-721, 1990) and that T-cells can recognize multiple epitopes on the molecule (Richert et al. , J. Neuroimmun 23, 55-66, 1989). MBP, apparently, also the way to the activation of certain T-cells without the involvement of antigen-presenting cells (Altman et al, Eur. J. Immunol. 17, 1635-1640, 1987). It seems that a small MBP peptides can be recognized by T-cells without intracellular processing simply by their ability to bind antigens of the main histocompatibility complex class II on the surface of the presenting cell.

Because of experimental allergic encephalomyelitis (EAE), a recognized model of multiple sclerosis (MS) in animals can be induced by the introduction of sensitive rodents as MBP and PLP in combination with complete adjuvant's adjuvant, the process of demyelination in multiple sclerosis may have an autoimmune mechanism (Fritz, R. C. et al., J. Immunol., 130, 1024-1024, 1983; Trotter, which targeted disease, localized near Tripropylene sequence (residues - 99-100-101-) in the area between 80-m and 100-m residue (Warren, K. G., et al, Ann Neurol 35: 280-289, 1994). This B-cell epitope overlaps with the immunodominant epitope T-cell activity against MBP, which are found in parts of the brain affected with multiple sclerosis (MS) (Oksenberg, g. R. et aL, Nature, 362, 68-70,1993).

Previous studies showed that anti-MBP neutralized MBP. However, prior attempts to treat multiple sclerosis (MS) intramuscular or subcutaneous injection of heterologous MBP did not lead to success (Campbell, B. , Vogel, R. J., Fisher, E. and Lorenz, R., Arch Neurol 29: 10-15, 1973; Gonsette, R. E., Delmotte, P. and Demonty, L., J Neurol 216: 27 - 31, 1977; and Romine, J. S. and Salk, J., In: Hallpike, J. F., Adams, C. W. M. and Tourtelotte, W. W., eds. Multiple sclerosis. Baltimore, Williams & Wilkins, 1982: 621-630). The problem of using native MBP has two sides. Protein obtained from samples of the human brain and therefore there is a potential danger that it contains latent nairovirus.

In addition, despite the fact that, as a rule, the ISI is not immunogenic when administered to persons with compromised immune systems ISI can manifest itself as the antigen and cause the production of antibodies to MBP.

Accordingly, the present invention sclerosis (MS) in the stage of acute relapse neutralized through certain peptides MBP person (h-MBP). For this purpose, synthetic peptides corresponding to the full length h-MBP was used to determine possible epitope region on the h-MBP, which will neutralize anti-MBP obtained from these patients. Therefore, the selected peptides, which neutralize the anti-MBP can be used for the treatment of multiple sclerosis (MS) is more efficient than full length MBP. These peptides obtained by artificial means and therefore do not carry the potential threats associated with nairoviruses. In addition, due to the small size of these peptides do not manifest themselves as immunogens. Therefore, the use of these peptides as a means of treatment of multiple sclerosis will help to solve problems arising from the use of a native protein.

In addition, peptides, obtained according to the present invention, are examined to determine the effectiveness of their binding or modulation of the production of anti-MBP multiple sclerosis (MS) in vivo.

THE INVENTION

In accordance with the invention claimed peptides, the sequence of which is largely homologous to part of the amino acid sequence of myelin basic protein man. These peptides are able to neutralize or modulate e-Phe-Lys-Asn-Ile-R2< / BR>
and their salts, where Val-His-Phe-Phe-Lys-Asn-Ile - amino acid residues 87-93 SEQ ID NO:1 and R1and R2independently selected from the group comprising hydrogen, hydroxy, the residue of the amino acid and the residue of the polypeptide, and R1and R2are not hydrogen or hydroxyl at the same time. The peptide may contain substitutions, deletions or additions, and its function neutralize or modulate the generation of anti-MBP saved.

Examples of these peptides:

MBP 75-95 (amino acid residues 75-95 of SEQ ID NO: 1)

Lys Ser His Gly Arg Thr Gln Asp Glu Asn Pro Val Val His Phe Phe Lys Asn Ile Val Thr

MBP 64-78 (amino acid residues 64-78 of SEQ ID NO: 1)

Ala Arg Thr Ala His Tight Gly Ser Leu Pro Gln Lys Ser His Gly

MBP 61-75 (amino acid residues 61-75 of SEQ ID NO: 1)

His His Pro Ala Arg Thr Ala His Tyr Gly Ser Leu Pro Gln Lys

MBP 69-83 (amino acid residues 69-83 of SEQ ID NO: 1)

Tyr Gly Ser Leu Pro Gln Lys Ser His Gly Arg Thr Gln Asp Glu

MBP 80-97 (amino acid residues 80-97 of SEQ ID NO: 1)

Thr Gln Asp Glu Asn Pro Val Val His Phe Phe Lys Asn Ile Val Thr Pro Arg

MBP91-106 (amino acid residues 91-106 of SEQ ID NO: 1)

Lys Asn Ile Val Thr Pro Arg Thr Pro Pro Pro Ser Gln Gly Lys Gly

MBP 84-93 (amino acid residues 84-93 of SEQ ID NO: 1)

Asn-Pro-Val-Val-His-Phe-Phe-Lys-Asn-Ile

MBP 85-94 (amino acid residues 84-94 of SEQ ID NO: 1)

Pro-Val-Val-His-Phe-Phe-Lys-Asn-Ile-Val

MBP 86-95 (amino acid residues e-Val-Thr-Pro

Further, according to the present invention, the claimed pharmaceutical composition containing as active ingredient a peptide as described above, individually or in the composition of the mixture with a pharmaceutically acceptable carrier.

In addition, in accordance with the present invention claims a method of treating multiple sclerosis, involving the administration of an effective amount of a peptide as described above, both individually and in combination to effectively neutralize or modulate the production of antibodies to MBP person.

BRIEF DESCRIPTION OF DRAWINGS

Fig. 1 shows the localization of eighteen synthetic peptides relative to the intact molecule MBP person. The peptides presented in the form of vertical strips and placed after the corresponding area on the MBP molecule. The scale shows the amino acid residues of the MBP molecule man.

Fig. 2 shows curves of inhibition of antibodies to MBP, purified and obtained from 10 different patients with multiple sclerosis, with the introduction of the ISI man and MBP-peptide.

Fig. 3 shows the neutralization of anti-MBP obtained from the same patient multiple (MS) ISI man and MBP peptides 80-97, MBP is chronic multiple sclerosis (MS):

levels F (free) and b (related) anti-MBP were consistently elevated in the study of 26 samples during the 11-year period from 1983 to 1993

cpm: count per minute

< / BR>
- o - linked (In) anti-MBP, determined after acid hydrolysis of immune complexes in the cerebrospinal fluid (CSF).

-- free (F) anti-MBP.

Fig. 5 control patients: levels of anti-MBP CSF in 2 time controls (1F56, Fig. 5A and B, Fig. 5B) and 2 two time controls when using saline solution. All four patients level (F) and (C) anti-MBP remains stubbornly high in relation to the base level when CSF was taken for analysis every 30 min during the first two hours, as well as 24 hours later. Designations are the same as in Fig. 4.

Fig. 6 - comparative studies of peptides from different patients: levels of anti-MBP in cerebrospinal fluid in a group of four patients (10F38, Fig. 6A; 13F43, Fig. 6C; M, Fig. 6D; and M, Fig. 6G), which increased the number (1, 2.5, 5 and 10 mg, respectively) unbound control synthetic peptide MBP-35-58 and in a group of four other patients (6F53, Fig. 6B; M, Fig. 6D; and M, Fig. 6F; and 1F56, Fig. 6N), which received increasing amounts (1, 2.5, 5 is isimon the ratio of peptide MBP 75-95, and they did not engage in interaction with MBP peptide 35-58. Related anti-MBP remained virtually directionspanel.

Fig. 7 - comparative studies of peptides from different patients: when MS patients were subjected to temporary control (1F56, Fig. 7C and M, Fig. 7D) or temporary control using physiological solution (M, Fig. 7A and B, Fig. 7B), and that they were unrelated control peptide MBP 35-58 (M and M), levels (F)-anti-MBP and anti-MBP remained constantly unchanged. On the contrary, when the same patients M, 1F56 and M later received 5-10 mg anti-MBP-related peptide MBP 75-95, (F) anti - MBP was no longer detected in the period up to 7 days and returned to baseline between 10 and 21 day.

Fig. 8 is repeated intradermal injections of synthetic peptide: the patient with chronic progressive MS received 10 weekly injections of 10 mg MBP 75-95 introduced directly into the cerebrospinal fluid; captions (F) and (C) anti-MBP was measured before (indicated by circles) and after 30 min after injection (indicated by squares). Free (F)-anti-MBP (shaded squares and circles) became undetectable within 10 weeks, although the level is, 9. Intravenous synthetic peptide: the levels of anti-MBP in cerebrospinal fluid correspond to a single intravenous injection of 500 mg MBP 75-95; levels connected () and free (F)- anti-MBP significantly reduced when studies after 10, 16 and 30 days after injection. Designations are the same as in Fig. 4.

Fig. 10. Further study of the MBP epitope for anti-MBP MS using a group of 41 Decapeptide that overlap areas between 61 and 110 remains.

Description:

the bands indicate the percentage of inhibition =100 - units of radioactivity;

MBP and MBP peptide 75-95 were used as a positive control and provided 100% inhibition of free (F) and bound (B) antibodies;

peptides MBP 51-60 and MBP 111-120 were used as negative control and resulted in a slight inhibition (0-10%) related () and free (F)-anti-MBP;

the Decapeptide MBP 84-93, MBP 85-94, MBP 86-95 and MBP 87-96, which ensured maximum inhibition (90-100%) as free (F) and free ()-antibodies are closely associated with the MBP epitope;

dotted line: 95% credible interval studies on inhibition.

A DETAILED DESCRIPTION OF THE INVENTION

N. the part of the amino acid sequence of myelin basic protein man. The term "virtually homologous" means that small differences between the amino acid sequence of myelin basic protein of human rights and peptides can be provided that the peptides with small differences in amino acid sequence capable of functioning in accordance with an inherent purpose, i.e. is able to neutralize or modulate the production of antibodies to myelin basic protein human (anti-MBP). Conducted in the present invention explanations make it obvious for specialists in this area the fact that empirically small variations in amino acid sequences of these peptides can be produced without compromising their function.

In accordance with the present invention on the basis of research by competitive inhibition using a set of 41 Decapeptide it was found that MBP epitope for anti-MBP MS localized at the site between amino acids 82 and amino acid 98 (more than 40% inhibition associated with anti-MBP and more than 60% ingibirovaniya free anti-MBP).

Given the high level of inhibition of MBP epitope anti-MBP probably is between 84 amino acid and amino acid 96.


R1-Val-His-Phe-Phe-Lys-Asn-Ile-R2,

including their salts, where Val-His-Phe-Phe-Lys-Asn-Ile - amino acid residues 87-93 SEQ ID NO 1 and where R1and R2- independently from each other selected from the group comprising hydrogen, hydroxy, the residue of the amino acid and the residue of the polypeptide; provided that R1and R2are not hydrogen or hydroxyl at the same time. Plot amino acid sequence corresponding to the seven amino acid occupying position 87-93, probably not too large to efficiently bind to anti-MBP. Therefore, R1and R2cannot be hydrogens or hydroxyl.

When R1or R2- amino acid, it may be selected from natural amino acids. R1or R2not limited by the amino acid located in position "upstream" and "downstream" relative to Val 187 and Ile 93 in myelin mostly human protein as shown in SEQ ID NO:1. Can be used with various modifications, including substitutions, additions and deletions in the sequences located in the "upstream" and "downstream" with respect to R1and R2. In addition, the same modification, VK is ovii, what is thus obtained peptides retain the intended them to function, i.e., neutralize or modulate the production of antibodies to myelin basic protein man.

The term "residue of a peptide or polypeptide residue" refers to a polypeptide consisting of two, three or more amino acid residues, including proteins and fragments thereof. As mentioned above, when R1or R2- polypeptide residue, R1and R2not limited to peptides, located in the "upstream" and "downstream" relative to Val 187 and Ile 93 myelin basic protein man. Can be used any residue of the polypeptide.

R1and/or R2may be repetitions of the sequence-Val-His-Phe-Phe-Lys-Asn-Ile or its modifications, including substitutions, additions or deletions. Thus, the peptide may contain multiple repetitions of the binding site of anti-MBP (epitope).

The compounds claimed according to the present invention, can be obtained in accordance with conventional and well known methods of synthesis of polypeptides. The term "peptide" refers to peptides derived from natural myelin basic protein as a result of the controlled hydrolysis, the implementation of the IDA, obtained using recombinant DNA technology. Based on the known sequence of the claimed peptides in the scope of the present invention also includes the establishment of appropriate DNA sequences encoding the claimed amino acid sequence. The appropriate DNA sequence may be obtained by conventional well-known methods of synthesis of DNA sequences. Thus obtained DNA sequence can be cloned in an appropriate cloning vector and used to transform a suitable host cell to obtain a recombinant peptide. All the above approaches are conventional and well known to specialists in this field.

The peptides claimed in accordance with the present invention, mainly homologous in their amino-acid sequences of part of the amino acid sequence of myelin basic protein man. The concept of "part of the amino acid sequence" means that the sequence can be of any length provided that it is sufficiently long in order to neutralize or modulate the production of antibodies to MBP, but is not so long, which is one example of the present invention described peptides, ranging from ~ 10 to ~ 25 amino acid residues. If the claimed peptides are used as part of fusion proteins, the total amount of peptide can be much more.

In accordance with one embodiment of the invention, it was found that the claimed peptides corresponding to substantially the amino acid sequence of human MBP, effective in neutralizing and modulation generate anti-MBP. These peptides correspond to amino acid sequences of human MBP in the range from 61 to 106 amino acid residue. In one example, these peptides correspond to the section 75 - 106 amino acid sequence of human MBP, when the peptides are used to neutralize free anti-MBP. In the following example, these peptides correspond to section 82 - 99 amino acid sequence of human MBP, when the peptides are used to neutralize and modulation generate related anti-MBP. Therefore, the selected peptides consisting of 10 to 25 amino acid residues, of a continuous amino acid sequence within the sequence shown below (amino acid residues of SEQ ID NO:1), provided that the sequence/P> Amino acid residues 61-106 SEQ ID NO:1

61 His His Pro Ala Arg Thr Ala His Tight Gly Ser Leu Pro Gln Lys Ser His

Gly Arg Thr Gln Asp Glu Asn Pro Val Val His Phe Phe Lys Asn Ile Val Thr Pro

Arg Thr Pro Pro Pro Ser Gln Gly Lys Gly 106

Below are examples of peptides selected from the group including:

MBP 61-75 (amino acid residues 61-75 SEQ ID NO: 1)

His His Pro Ala Arg Thr Ala His Tight Gly Ser Leu Pro Gln Lys

MBP 64-78 (amino acid residues 64-78 SEQ ID NO: 1)

Ala Arg Thr Ala His Tyr Gly Ser Leu Pro Gin Lys Ser His Gly

MBP 69-83 (amino acid residues 69-83 SEQ ID NO: 1)

Tyr Gly Ser Leu Pro Gln Lys Ser His Gly Arg Thr Gln Asp Glu

MBP 75-95 (amino acid residues 75-95 SEQ ID NO: 1)

Lys Ser His Gly Arg Thr Gln Asp Glu Asn Pro Val Val His Phe Phe Lys Asn Ile Val Thr

MBP 80-97 (amino acid residues 80-97 SEQ ID NO: 1)

Thr Gln Asp Glu Asn Pro Val Val His Phe Phe Lys Asn Ile Val Thr Pro Arg

MBP 91-106 (amino acid residues 91-106 SEQ ID NO: 1)

Lys Asn Ile Val Thr Pro Arg Thr Pro Pro Pro Ser Gln Gly Lys Gly

One aspect of the present invention relates to peptides represented by the formula:

R1- Val-His-Phe-Phe-Lys-Asn-Ile-R2< / BR>
and their salts, where R1and R2independently from each other selected from the group comprising hydrogen, hydrocity, the remainder of the amino acid and the residue of the polypeptide; provided that R1R2are not both hydrogen or hydroxyl. The peptide may contain substitutions, additions or deletions p. the century.

Below are examples of peptides selected from the group including:

MBP 84-93 (amino acid residues 84-93 SEQ ID NO: 1)

Asn-Pro-Val-Val-His-Phe-Phe-Lys-Asn-Ile

MBP 85-94 (amino acid residues 84-93 SEQ ID NO: 1)

Pro-Val-Val-His-Phe-Phe-Lys-Asn-Ile-Val

MBP 86-95 (amino acid residues 84-93 SEQ ID NO: 1)

Val-Val-His-Phe-Phe-Lys-Asn-Ile-Val-Thr

MBP 87-96 (amino acid residues 84-93 SEQ ID NO: 1)

Val-His-Phe-Phe-Lys-Asn-Ile-Val-Thr-Pro

The possible role of anti-MBP in the pathogenesis of multiple sclerosis continues to be explored. Higher titers of anti-MBP in patients with active form of multiple sclerosis was first reported Panich et al. (Panitch, H. S., Hooper, S. S., and Johnson, K. P., Arch Neurol 37: 206-209, 1980), who used a solid-phase radioimmunoassay analysis with MBP Guinea pigs. Patients with acute relapse of the disease, as a rule, had a high content of anti-MBP, mainly in the free form, whereas some patients in clinical remission could have undetectable levels of anti-MBP. During the transition from acute doses to remission credits of free anti-MBP progressively decreased over a period of several weeks to several months, while the level of related antibodies was comparable to the original. Other patients in remission was the place, what antibodies (antibody), neutralizing anti-MBP, are associated with anti-MBP. Sometimes patients that match the criteria clinically defined multiple sclerosis, or patients with neuropathologically identified multiple sclerosis had undetectable levels of anti-MBP during the active stage of the disease. It is possible that such patients have antibodies to other myelin proteins. The absence of a schema production of specific antibodies does not diminish the potential of the possible role of anti-MBP in the mechanism of demyelination in the majority of patients with multiple sclerosis.

Recently, various antibodies to MBP were detected in fractions lqG isolated from the cerebrospinal fluid of patients with multiple sclerosis (Warren, K. G. and Catz, I., J Neurol Sci 96: 19-27, 1990). Primary antibodies to MBP in free and bound form was associated with the active phase of the disease: F/B > 1 in patients with acute and < 1 in patients with chronic progressive form of multiple sclerosis (Warren, K. G. and Catz, I. , Ann Neurol 209: 20-25, 1986; Catz, I., and Warren, K. G. Can J Neurol Sci 13: 21-24, 1986; and Warren, K. G. and Catz, I., Ann Neurol 21: 183-187, 1987). Secondary antibodies that neutralize anti-MBP, appear when the disease becomes inactive form. Tertiary antioppression and goes into an inactive form. The fact that the cascade of antibodies to MBP associated with phases (MS), suggests that the formation of such antibodies may play an important role in the development of this disease. Despite the fact that anti-MBP can be determined in the CSF of patients with the active form of multiple sclerosis (MS), their main role in the pathogenesis of demyelination remains unconfirmed. Part of the anti-ICBM in the mechanism of development of multiple sclerosis (MS) may best be established by their neutralization in vivo, possibly with the introduction of the claimed peptides and monitoring the clinical course of the disease. If anti-MBP are only primary antibodies associated with demyelination in multiple sclerosis (MS) might block demyelinization vnutriobolochechnoe, and/or intravenous and/or oral introduction stated MBP peptides that would neutralise the anti-MBP and would provide tolerance to MBP in situ. Other myelin proteins person may also participate in demyelinization in multiple sclerosis (MS) and, accordingly, the scope of the present invention includes the use of peptides substantially homologous amino acid sequence of part of the amino acid follower, that previous attempts at treatment of multiple sclerosis (MS) intramuscular or subcutaneous injection of heterologous MBP was not fully successful (Campbel, C., Vogel, R. J., Fisher, E. and Lorenz, R., Arch. Neurol 29: 10-15, 1973; Gousette, R. E., Delmotte, P. and Demonty, L. J Neurol 216: 27-31, 1977; and Romine, J. S. and Salk; J. , In: Halpike, J. F., Adams, C. W. M. and Tourtelotte, W. W., eds. Multiple Sclerosis. Baltimore, Williams & Wilkins, 1982: 621-630), vnutriobolochechnoe and/or intravenous administration of MBP peptides that neutralize or modulate the production of anti-MBP, in accordance with the invention showed better results.

Model MS animal is experimental allergic encephalomyelitis (EAE), which is mediated by T-cells demyelinizing disease. Symptoms of EAE can be skorrigirovanna the introduction of mice with synthetic peptides MBP (Gaur, A. et al, Science, 258, 1491-1494, 1992). Moreover, the introduction of high doses of MBP delegated self-reactive T cells and abolished the clinical and pathological symptoms of EAE in mice (Gritchfield, J. M. et al., Science, 263, 1139-1143, 1994). Even oral introduction MBP modulated during EAE induction of peripheral tolerance (Chen, W. et aL, Science, 265, 1237-1240, 1394). Recently on the basis of dual pilot experiments it has been suggested, ctse 259, 1321-1324, 1993). The combination of myelin antigens and synthetic peptides of these antigens introduced oral and/or intravenous, and/or vnutriobolochechnoe way, it may be necessary to modulate T cells, b cells and macrophages involved in the destruction of myelin in patients with multiple sclerosis (MS).

Accordingly, the invention also relates to pharmaceutical compositions containing as active ingredient a peptide described above, alone or in composition, in a mixture with a pharmaceutically acceptable carrier. Examples of pharmaceutically suitable carriers are well known and include, for example, saline.

The peptides claimed in the present invention, can be administered to humans for the treatment or modulation MS. Therapeutic dose for intravenous and/or oral administration in the treatment of MS can range from about 1.0 mg per 1 kg body weight to about 10.0 mg per 1 kg of body weight. If the introduction is vnutriobolochechnoe, the dose may be from about 1 to about 10 mg. In one of the examples of the invention the peptide is applied either intravenously or vnutriobolochechnoe or a combination of these methods. If required, the peptides may be administered as single doses, the center set out the preferred options for its implementation, the following examples illustrate but do not limit the invention.

Example 1.

Neutralizing antibodies to MBP human in vitro.

Fig. 1 shows the localization of the 18 peptides of human MBP used in this experiment, with respect to the molecule intact MBP. Native MBP was isolated from the brain tissue that is not affected with multiple sclerosis (MS) (Diebler, O. E., Martenson, R. E., Kies, W. W. Prep Biochem 2: 139-165, 1972) and further purified by gel-filtration. The final antigenic preparations were tested for purity SDS-polyacrylamide gel electrophoresis. Further studies have used only those drugs that are moved, respectively, the molecular weight of 18.5 KD. Purified MBP was used in antigen-specific affinity chromatography, in experiments on neutralization and solid-phase radioimmunoassay using anti-MBP.

Eighteen peptides, overlapping MBP person and containing from 8 to 25 amino acid residues, were synthesized Fmocmethod, as described previously (Groome, N. P., Dawkes, A, Barry, R. et al. J. Neurommun 19: 305-315, 1988). The purity of the peptides was checked obraniakowi liquid chromatography high-pressure column 18 and the gradient of water/acetonitrile (0.1% T isawanya in this experience, contained a balance of non-natural cysteine, because they were synthesized to function as immunogens. This is unlikely to have influenced the findings.

Cerebrospinal fluid was obtained within one week from the moment of appearance of initial symptoms in 35 patients with acute relapses (MS), and the levels of IgG was determined values. This experience was selected samples of CSF with original brand high levels of IgG (80 g/l) and elevated titers of anti-MBP (F/B ratio > 1.0). All patients had clinically definite disease.

IgG was isolated from the concentrated cerebrospinal fluid of patients with acute multiple sclerosis on protein a-sepharose (PharmaciaTM) affinity chromatography as described previously (Warren, K. G. and Catz, I. J. Neurol Sci 96: 19-27, 1990). The purity of each preparation IgG was checked by polyacrylamide gel electrophoresis and isoelectric focusing. When elevated levels of anti-MBP purified from the IgG was absorbed by the ISI to zero, obtained after absorption supernatant contained residual amounts of IgG.

Purified MBP was associated with activated Enrichment by separate 4B (Pharmacia) according to the manufacturer's instructions. Acidaemia unbound anti-MBP using affinity chromatography on MBP-sepharose (Warren, K. G. and Catz, I. J. Neurbl/ Scil03: 90-96, 1991). Purified samples of anti-MBP were compared with the original source of IgG using polyacrylamide gel electrophoresis. When purified anti-MBP were absorbed to zero using the ISI obtained after absorption supernatant did not contain residual IgG.

Constant amount of anti-MBP (with the aim of expanding the scale of 15 units of binding of radioactivity taken for 100%) incubated with elevated amounts of human MBP (0-10000 ng) or individual peptides MBP (0-10000 ng) in the liquid phase of the study and after 1.5 hours of incubation, the content (level) free anti-MBP was detected in all mixtures. Anti-MBP derived separately from the 7 patients with MS, or combined fraction of anti-MBP from 10 different patients were used in experiments on neutralization. Histone calf thymus and albumin human serum was used as negative antigen controls (range: 10-1000 ng).

One monoclonal antibody to the peptide MBP 64-78 (clone 26) and the polyclonal anticavity rabbit to the MBP peptide 1-8 (R 155) was used as a positive control antibody (Groome, N., Harland, J. and Dawkes, A, Neurochem Int. 7: 309-317, 1985; Barry, R., Payton, M. And Groome N., Neurochem Int 2: 291-300, 1991). Another monoclonal anti is determined by a solid phase radioimmunoassay when using MBP man (Warren, K. G. And Catz, I, Ann. Neurol 209: 20-25, 1986; Warren, K. G. And Catz I, Ann. Neurol 21: 183-187, 1987; Warren, K. G. And Catz, I, J Neurol Sci 91: 143-151, 1989). The content of free anti-MBP was measured in all fractions after affinity chromatography, and in all the reaction mixtures used for neutralization. All individual samples were examined four times using the same itinerating material in order to minimize the variability of results between experiments.

Purified anti-MBP were completely neutralized MBP and MBP peptides 80-97, MBP 91-106 and MBP 75-95 and were partially offset by peptides MBP 64-78, MBP 69-83 and MBP 61-75 (PL. 1 and Fig. 2). The remaining 12 peptides are not neutralized purified anti-MBP, and their kinetic curves show a decline within the shaded area, see Fig. 2. Histone calf thymus and albumin human serum did not react with purified anti-MBP even at concentrations above 1000 ng. Clone 26 inhibited only the MBP peptide 64-78. R 155 inhibited only the MBP peptide 1-8. Clone 16 did not react with either MBP or with other peptides (for clarity, monitoring data not shown). Control samples show the validity of the approach neutralization, since each control anti interaction takes place even at high concentrations of peptides (10 000 ng).

Anti-MBP obtained from 7 different patients, were completely neutralized MBP person and MBP peptides 80-97, MBP 91-106 and MBP 75-95 (see Fig. 3 as an illustration). As obtained from individual patients, the number of antibodies was limited, anti-MBP was not subjected to the reaction with the remaining 15 peptides.

As stated above, anti-MBP were neutralized by peptides corresponding to the interval between 61-106 amino acid residues. Peptides, which are not neutralized by anti-MBP, cover aminobenzene (about 1 to 63 residues) and carboxy-terminal (from about 117 to 162 balance) plots MBP person. Apparently, peptides from different non-overlapping areas MBP neutralize the same antibodies. This can be explained by the fact that the antibodies recognize discontinuous epitope containing amino various sites. This fact was previously observed Hruby et al. (Hruby, S., Alvord, E. C., Groome, N. P. et al., Molec Immun 24: 1359-1364, 1987), which showed that rat monoclonal antibody has the main epitope sequence MBP 112-121, and in that time the strong cross-reactivity with other epitope 39-91. It is more likely than the possibility that antibody cross-reactivity to the two completely different 44: 637-650, 1985). Data on the neutralization can be explained by the ability of the peptides corresponding to different sections of the ISI, partly to occupy the binding region of the antibodies interacting with various amino acid side chains. This explanation is confirmed by the observation that peptides that provides complete inhibition (IMM 80-97, MBP 91-106 and MBP 75-95), approximately 100 times less effective in molar ratio than the intact MBP. Based on the above hypothesis, it could be due to the fact that each peptide clone are not able to achieve the binding energy with the actual epitope MBP.

Example 2.

Neutralization or modulating the formation of antibodies to MBP person, in vivo

Selection of patients and control studies

Patients who were selected for this research project, was observed in the clinic for the care and study of patients with multiple sclerosis University of Alberta, Edmonton, Canada. Patients were diagnosed as patients with multiple sclerosis according to the criterion Schumacher (1965) with confirmation of the diagnosis method of obtaining images of the brain using magnetic resonance and/or immunochemical profiles of cerebrospinal fluid (CSF). To generate chivo elevated for extended periods of time, from months to several years, patients re-took the puncture CSF lumbar control of free and bound anti-MBP. In patients with chronic progressive disease (MS) level of autoantibodies remains steadily increased for 11 years and spontaneous decline in the level of anti-ICBM does not occur (Fig. 14 illustrates an example).

In order to determine what initially elevated levels of anti-MBP in cerebrospinal fluid (CSF) was relatively constant within 24 hours, in two patients (IF56 and M) took samples of CSF every 30 min for 2 hours as well as 24 hours later, to controlling the free and bound forms of anti-MBP (Fig. 5A and 5B, respectively). Patients 1F56 and M served as time controls. Levels of free and bound anti-MBP remained consistently elevated in the CSF studies every 30 min for 2 hours as well as after 24 hours.

In addition, in this way was determined the effect of introducing 5 cm3saline solution into the cerebrospinal fluid the other two patients (M and M) (Fig. 5C and 5D, respectively). These patients served as time controls with saline solution. With the introduction of 5 cm3physiology, when samples of CSF were investigated, as described above, thus confirming that the effect of cultivation on the titers of anti-MBP was insignificant.

The levels of anti-MBP was determined by solid phase radioimmunoassay analysis with MBP person adsorbed in the wells of the panel for micrometrology "Immunlon". The wells were covered with 100 μl of MBP concentration of 10 µg/ml (1 μg/well) and incubated overnight at 37oC. After the application of BSA and three times laundering water panels were stored at room temperature. Samples (100 µl) CSF or tissue extract, dissolved in 0.010 g lgG/l (0.01 M PBS buffer, 0.05% tween-20), incubated in ISI-covered cells for 1-2 hours at room temperature. After five ottavani buffer (0.01 M PBS, 0.05% tween-20) cells were incubated with antibodies goat to IgG-Fc rabbit (0.01 M PBS, 0.05% tween-20, 0.5% BSA) for 1 hour at room temperature and then washed as described above. Finally, we added labeled125I-protein A (or125I-protein G) and incubated for 1 hour at room temperature. When the label used125I-protein G BSA in the buffer for research and treatments exhaustion was replaced with ovalbumin. After three final laundering water wells obsc the odd sample - account control) - (total score of radioactivity account control). All the specimens were examined in 10 repetitions, and the time the account was 10 min in order to register > 10,000 units of radioactivity in each positive sample.

Before the study all samples of CSF and/or tissue samples were diluted to a final concentration of IgG 0.010 g/liter Free (F) anti-MBP was determined directly in CSF or tissue extract, whereas the levels of related antibodies was determined after acid hydrolysis of immune complexes. Nonspecific binding was observed for each sample in the uncovered holes. To localize the epitope synthetic peptides were first subjected to interaction with purified antibodies in liquid-phase competitive examination, after which anti-MBP was determined by radioimmunoassay analysis of all the final mixes. The results of a competitive binding radioimmunoassay was expressed as % inhibition of the synthetic peptide, defined as 100 units of radioactivity. Samples were studied in 10 repetitions and cheated for 10 min at LKB1275 Minigamma-counter. United extracted from tissue anti-MBP was used in 5 pre-prepared dilutions as Olam. The reproducibility of the studies ranged from 3% to 5%, and the scatter of the data is less than 7%.

Define constant elevated levels of anti-MBP in CSF in the control experiments was the next stage of research.

Control "blind" experiment phase 1 duplicate peptide-vnutriobolochechnoe injection

Conduct phase 1 of the experiment for determining the action of a synthetic peptide MBP 75-95 on titles F and anti-MBP in cerebrospinal fluid. After approval by the Research ethics Committee of the University of Alberta project was carried out with the participation of patients with clinically diagnosed multiple sclerosis (MS) (Schumacher et al., Ann NY Acad. Sci., 122, 552-568, 1965), seriously ill persons with strongly progressive disease. Fourteen patients voluntarily agreed to participate in this study; 8 patients were selected on the initial titer F anti-MBP in CSF (about 8 units of radioactivity) (Table. 2) for receiving one vnutriobolochechnoe injection or peptides MBP 75-95 that bind anti-MBP in vitro or non-binding control peptide MBP 35-58 (Warren and Catz, 1993 b). The experiment was carried out using a double blind" option when neither the researchers nor the subjects did not know the nature of the input is m Paired peptides, dissolved in 5 cm3saline and injected into the CSF by lumbar puncture, were used in increasing doses (1, 2.5, 5 and 10 mg). CSF was investigated before injection (baseline), with 30 minute intervals for 2 hours after the injection, 24 hours later, and then with utmost interval within 3-4 weeks up until the levels of anti-MBP did not return to baseline (reference). Counting cells, total protein, glucose, levels of IgG and albumin were determined in all samples of CSF. Levels of free and bound anti-MBP was determined by radioimmunoassay, as described above.

Table. 2: Clinical data and the levels of anti-MBP in CSF in 14 patients with chronic progressive multiple sclerosis (MS) who have agreed to participate in the phase 1 research, providing vnutriobolochechnoe single injection of synthetic peptides MBP. Because it was necessary initially high level of free (F) anti-MBP (> 8 units of radioactivity) in order to obtain significant postinjection change, for the study were selected only 8 out of 14 patients.

All peptides used in these studies was synthesized under the code of "good quality product" (GMP) on retinopathy chromatography on a column of 18 with water and the combined acetonitrile gradient, containing 0.1% THU. Mass spectroscopy and amino acid analysis was performed based on standard techniques. Before the introduction of all peptides was tested on progenote (Vancouver General hospital, Vancouver, Canada), sterility (local Public Health laboratory Northern Alberta, Edmonton, Canada) and acute toxicity (Service Laboratory for medical research, University of Alberta, Canada). It has been shown that they are "suitable" for introducing people. The appropriate number of encoded synthetic peptides were dissolved in 5 cm3sterile saline (0.9% NaCl, injection USP, pyrogen-free, Baxter Corp, Toronto, Canada) was filtered 2 times through a sterilizing membrane with a pore size of 0.22 μm (Millex-GX, Millipore Corp., Bedford, M A, USA) and injected into the cerebrospinal fluid (CSF) by lumbar injection.

Comparative (between patients) studies of peptides

Patients 6F33, M, M and 1F56 received synthetic peptide MBP 75-95, capable of binding anti-MBP in vitro, and patients 10F36, 13F43, M and M received "control" nswazwi synthetic peptide MBP 35-58 in increasing doses of 1, 2.5, 5 and 10 mg, respectively (Fig. 6). The patient 6F53 (Fig. 6B), which received 1 mg MBP 75-95, nabludalos is that received 2.5 mg MBP 75-95, there was complete linkage-neutralization of free anti-MBP, and then their levels returned to baseline by 24 hours; the patient M (Fig. 6F), which introduced 5 mg MBP 75-95, sharp and full binding-neutralization of free anti-MBP that lasted for 7 days with a return to the original level was found after 21 days; the patient 1F56 (Fig. 6H), which was introduced 10 mg MBP 75-95, was also full binding-neutralization of free anti-MBP that lasted for 7 days and then the antibody levels returned to baseline values, it was found that in the period from 14 to 28 day. Levels associated anti-MBP insignificant change when a single vnutriobolochechnoe introduction MBP 75-95. Patients 10F38, 13F43, M and M, which was introduced on 1, 2.5, 5 and 10 mg control nesvezhego peptide MBP 35-58, the levels of free and bound antibodies in CSF remained altered in relation to baseline within 24 hours of the experiment (Fig. 6A, 6C, 6E and 6G, respectively). Traditional CSF parameters of inflammation in MS, such as bills of cells, the absolute levels of total protein, IgG and albumin, oligoclonal binding, index, IgG and IgG synthesis in the CNS, remained invariant before and after the introduction of the peptide.

th, to minimize the variability of the results of studies of several patients. The patient M, which was administered either saline (temporary control) or 5 mg nesvezhego peptide MBP 35-58, the levels of free anti-MBP remained elevated relative to baseline during both experiments (Fig. 7A). Patient M initially injected with saline (temporary control), and two months later he introduced 5 mg MBP 75-95. The levels of free patients with anti-MBP remained persistently elevated in all samples collected during the control experiment, and became undetectable after the introduction of the ISI 75-95 (Fig. 7B). Similar results were obtained and the patient 1F56, which had persistently elevated levels of free antibodies during the control experiment, and after administration of 10 mg MBP 75-95 its free anti-MBP became undetectable, (Fig. 7C). The full study was conducted with patients M. He levels of free anti-MBP were consistently elevated during the control experiment or with the introduction of 10 mg MBP 35-58; however, when they were introduced 10 mg MBP 75-95, free anti-MBP were completely neutralized and were not found within 7 days (Fig. 7D).

When patients are chronically progressive form of multiple sclerosis (MS) has been re-vnutriobolochechnoe injection 10 mg MBP 75-95 with a one week interval (for up to 10 weeks), the initially high level of free anti-MBP could become undetectable until introduced peptide; when the peptide ceased to enter, the level of free anti-MBP returned to the source within 1-2 months of the ptx2">

Patients who participated in these studies who received either a single injection of synthetic peptides or repeated weekly injections, had chronic progressive multiple sclerosis with a high degree of neurological damage.

None of these patients reported worsening of neurological symptoms or exacerbations of the disease (MS), followed vnutriobolochechnoe introduction peptides, and cell response in GSF did not develop. Patients who received re-introduction MBP 75-95 was controlled on the development of possible physical complications, including changes in electrolytes, and dysfunction of the heart, liver, kidney, and hematological changes, and any unwanted complications were observed.

Injecting MBP 75-95

After establishing the fact that vnutriobolochechnoe introduction peptide MBP 75-95 leads to the binding of the free anti-MBP without changing the level of related antibodies, it was decided to determine the effect of intravenous injection of the same peptide on the titles in the CSF of free and bound anti-MBP; 500 mg MBP 75-95 was dissolved in 100 cm3saline and injected intravenously after 30 min the patient most. The blood was collected before injection and after 16 and 30 days and analyzed for electrolytes, creatinine, heart and liver enzymes and haematological parameters. In the cerebrospinal fluid were determined by the number of cells, total protein, glucose, IgG and albumin. As shown in Fig. 8, intravenous administration of 500 mg MBP 75-95 makes no changes in the levels of related titles and free anti-MBP in the CSF during the first two hours; 30% drop in the level of free anti-MBP in CSF was observed after 18 hours. When CSF was re-analysed after 10, 16 and 30 days, the levels of free and bound anti-MBP has dropped below the original with 11 units of radioactivity up to 4, 2 and 1 radioactive units, respectively.

With repeated observations in patients with vnutriobolochechnoe injection of MBP were often observed resistance to elevated levels of related antibodies, whereas free anti-MBP were undetectable after vnutriobolochechnoe injection MBP 75-95. This suggests that the inflammatory process caused by autoantibodies to MBP, remains active during and after vnutriobolochechnoe introduction MBP 75-95. To confirm this observation MBP 75-95 was injected, Pacini and free and bound antibodies in CSF decreased significantly during the observation month. The decrease in both free and associated antibodies CSF after intravenous MBP 75-95 means that reducing regulation of the autoimmune inflammatory process responsible for the synthesis of anti-MBP.

MBP ezetop for anti-MBP in MS

To determine the localization of MBP epitope for anti-MBP in MS free and bound anti-MBP, purified by affinity chromatography from CSF and brain tissue of patients with multiple sclerosis (MS) (Warren, K. G., et al., Ann. Neurol. 35, 280-283, 1994), were subjected to interaction in research on competitive inhibition with 41 consecutive synthetic peptide MBP equal length (each peptide consists of 10 residues and blocks adjacent to 9 residues), which occupies the area between 61 and 110 residues of MBP person. It was found that the peptide(s), providing maximum inhibition, the most strongly bound to the antigen-binding region of the antibody.

Maximum inhibition (80%) treated free and bound anti-MBP from the brain tissue of patients with multiple sclerosis (Fig. 10) was provided by four Decapeptide labeled MBP 84-93, MBP 85-94, MBP 86-95 and MBP 87-96, suggesting that the MBP epitope for anti-MBP is localized between 84 and 96 remains. Minimum a more limited focus, than available antibodies.

The role of antibodies to anti-MBP in the pathogenesis of demyelination in multiple sclerosis is not yet established and can only be determined by modulating the anti-MBP in vivo and sequential monitoring of clinical and pathological manifestations. For example, during the acute stages of MS, when the ratio of free and bound antibodies above 1, a peptide which is known as the binding free anti-MBP, can be entered vnutriobolochechnoe to associate freely circulating antibodies and korrigirovat clinical symptoms of acute relapse; weekly introduction may be necessary before the onset of remission. For patients with chronic progressive disease intravenous peptide, as well as vnutriobolochechnoe doing, it may be necessary to regulate the mechanisms of inflammation, which is caused by anti-MBP.

Various modifications of the preferred embodiments of the invention may be made without limitation of the scope of the invention, which is covered by the following claims.

1. The peptide or its salt, can neutralize or modulate the formation of antibodies to myelin basic white is Pro-Val-; Pro-Val-; Val-; Lys-Ser-His-Gly-Arg-Thr-Gln-Asp-Glu-Asn-Pro-Val-; Thr-Gln-Asp-Glu-Asn-Pro-Val-; hydrogen; hydroxy;

R2selected from the group including Val; Val-Thr; Val-Thr-Pro-Val-Thr-Pro-Arg-; hydrogen; hydroxy,

provided that R1and R2at the same time are not hydrogen or hydroxyl, with this peptide contains substitutions, additions or deletions.

2. The peptide under item 1, characterized in that when R1Asn-Pro-Val-, R2is hydrogen or hydroxy; when R1Pro-Val-, R2- Val; when R1Val-, R2- Val-Thr; when R1hydrogen or hydroxy, R2- Val-Thr-Pro; when R1Lys-Ser-His-Gly-Arg-Thr-Gln-Asp-Glu-Asn-Pro-Val-, R2- Val-Thr; when R1Thr-Gln-Asp-Glu-Asn-Pro-Val-, R2- Val-Thr-Pro-Arg.

3. The pharmaceutical composition active against multiple sclerosis, comprising the peptide or its salt, can neutralize or modulate the formation of antibodies to milenova basic protein, alone or in combination, in a mixture with a pharmaceutically acceptable carrier, in which the said peptide has the formula

R1-Val-His-Phe-Phe-Lys-Asn-Ile-R2,

where R1selected from the group comprising Asn-Pro-Val-; Pro-Val-; Val-; Lys-Ser-His-Gly-Arg-Thr-Gln-Asp-Glu-Asn-Pro-Val-; Thr-Gln-Asp-Glu-Asn-Pro-Val-; hydrogen; hydroxy;

R2selected from the group including Val; Val-Thr; Val-Thr-Pro-Val-Droxicam, this peptide contains substitutions, additions or deletions.

4. The pharmaceutical composition according to p. 3, characterized in that when R1Asn-Pro-Val-, R2is hydrogen or hydroxy; when R1Pro-Val-, R2- Val; when R1Val-, R2- Val-Thr; when R1hydrogen or hydroxy, R2- Val-Thr-Pro; when R1Lys-Ser-His-Gly-Arg-Thr-Gln-Asp-Glu-Asn-Pro-Val-, R2- Val-Thr; and when R1Thr-Gln-Asp-Glu-Asn-Pro-Val-, R2- Val-Thr-Pro-Arg.

5. A method of treating multiple sclerosis by assigning the patient a peptide or its salts, characterized in that the peptide has the formula

R1-Val-His-Phe-Phe-Lys-Asn-Ile-R2,

where R1selected from the group comprising Asn-Pro-Val-; Pro-Val-; Val-; Lys-Ser-His-Gly-Arg-Thr-Gln-Asp-Glu-Asn-Pro-Val-; Thr-Gln-Asp-Glu-Asn-Pro-Val-; hydrogen; hydroxy;

R2selected from the group including Val; Val-Thr; Val-Thr-Pro-Val-Thr-Pro-Arg-; hydrogen; hydroxy,

provided that R1and R2at the same time are not hydrogen or hydroxyl, with this peptide contains substitutions, additions or deletions.

6. The method according to p. 5, characterized in that when R1Asn-Pro-Val-, R2is hydrogen or hydroxy; when R1Pro-Val-, R2- Val; when R1Val-, R2- Val-Thr; when R1hydrogen or hydroxy, R2-Thr-Pro-Arg.

7. The method according to p. 5, characterized in that the peptide is injected via lumbar puncture, oral or a combination of these methods.

8. The method according to p. 5, characterized in that the peptide is administered intravenously at a dose ranging from 1 mg/kg body weight to 10 mg/kg body weight, once or several times, if necessary.

 

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