MXPA99006457A - Treatment of multiple sclerosisthrough ingestion or inhalation of copolymer-1 - Google Patents

Abstract

The present invention relates to the treatment of multiple sclerosis by ingestion or inhalation of copolymer-1 and pharmaceutical compositions useful for such treatment.

Description

PHARMACEUTICAL COMPOSITION FOR THE TREATMENT OF MULTIPLE SCLEROSIS COMPRISING COPOLYMER -1 FIELD OF THE INVENTION The present invention relates to the treatment of multiple sclerosis by ingestion or inhalation of the compolymer-1 (as defined below). The present invention also relates to a pharmaceutical composition comprising the copolymer-1 used for the treatment of multiple sclerosis, wherein the pharmaceutical composition is formulated for administration by ingestion or inhalation.

BACKGROUND OF THE INVENTION Copolymer-1, also known as glatiramer acetate and marketed under the trademark Copaxone®, comprises the acetate salts of polypeptides containing L-glutamic acid, L-alanine, L-tyrosine and L-lysine. The average molar fraction of the amino acids are 0.141, 0.427, 0.095 and 0.338, respectively, and the Ref: 30781 average molecular weight of copolymer-1 is between 4,700 and 11,000 daltons. It is not an autoantigen which has been shown to suppress experimental allergic encephalomyelitis (EAE) induced by several encephalogens including ho ogenato of the mouse spinal cord (MSCH) which includes all myelin antigens, such as basic protein of myelin (MBP) (Sela M et al., Bull Inst Pasteur (1990) 88 303-314), proteolipid protein (PLP) (Teitelbaum D et al., J Neuroimmunol (1996) 64 209-217) and oligodendrocyte glycoprotein myelin (MOG) (Ben-Nun A et al., J Neurol (1996) 243 (Suppl 1) S14-S22) in a variety of species. EAE is an accepted model for multiple sclerosis. Copolymer 1 has been shown to be active when injected subcutaneously, intraperitoneally, intravenously or intramuscularly (D. Teitelbaum et al., Eur. J. I munol. (1971) 1: 242-248; D. Teitelbaum et al., Eur. J. Immuno1 (1973) 3: 273-279). In phase III clinical trials, daily subcutaneous injections of copolymer-1 were found to delay the progression of disability and reduce the rate of relapse or reoccurrence in multiple sclerosis of remission-exacerbation (KP Johnson, Neurology (1995) 1: 65-70). The copolymer-1 therapy is currently limited to its daily subcutaneous administration. Commonly, all specifically approved multiple sclerosis treatments 5 involve self-injection of the active substance. Injection site problems frequently observed include irritation, hypersensitivity, inflammation, pain and even necrosis (in the case of at least 1-B treatment with interferon-β) and a low level of condescension of the patient. Therefore, an alternative method is desired "l" * of administration EP 359,783 describes the treatment of autoimmune diseases by oral administration of autoantigens, which describes oral administration of MBP for the treatment of multiple sclerosis. Oral administration of an autoantigen has been called "oral tolerance". PCT International Application Publications, Nos. WO 91/12816, WO 91/08760, and WO 92/06704 describe the treatment of other autoimmune diseases using the "oral tolerance" method with a variety of autoantigens. However, none of these references describe the treatment of multiple sclerosis by oral administration of the no-1 copolymer. autoantigen. The contents of all these patents and all the literary references referred to above are hereby incorporated by reference in their entirety.

DESCRIPTION OF THE INVENTION fi. Therefore, it is an object of the present invention to provide a method for the treatment of multiple sclerosis by oral administration of the copolymer-1 through ingestion or inhalation.

BRIEF DESCRIPTION OF THE FIGURES Figures 1 and 2 show the effect of copolymer-1 on the immunity response to guinea pig myelin basic protein (GPBP) in rats (Figure 1) and mice (Figure 2) as assessed by the spleen cell proliferation. Figures 3 and 4 show the effect of copolymer-1 on cytokine release. Figure 5 shows the deletion of EAE in mice by copolymer-1 administered orally. 25 Mice (SJL / JxBALB / c) Fi were fed with PBS (U). 0. 1 mg of copolymer-1 (D). 0.25 mg of copolymer-1 (A), or 0.5 mg of copolymer-1 (D). Each dose was fed 7 times on days -7; -5; -3; 0; 2; 4 and 6. EAE was induced on day 0 by injection of MSCH. Figure 6 shows the proliferation and secretion of cytokine by the T cell line derived from spleens of rats fed copolymer-1. The cells were cultured with copolymer-1 (50 μg / ml) or concavalin A (With A (5 μg / ml) of the medium.The cytokine proliferation and secretion responses for these antigens were measured.Figure 7 shows the inhibition of EAE per cell line T derived from spleens of rats fed copolymer-1 cells (20xl0d / rat) were injected intraperitoneally 3 days after stimulation with copolymer-1, followed by induction of EAE, Figure 8 shows the EAE inhibition by T-cell line derived from spleens of mice fed copolymer-1 Cells (15x106 / mouse) were injected intravenously 3 days after stimulation with copolymer-1, followed by induction of EAE. shows the Clinical Records against Days of Induction subsequent to the EAE Disease, of the Copolymer-1 in three Rhesus Monkeys.

Figure 10 shows the Clinical Registers against Days of Induction subsequent to the EAE Disease of Copolymer-1 in six Rhesus Monkeys, in an assay comparing the pharmaceutical dosage form uncoated versus enteric coated. The values of zero (0) have been separated on the y-axis, to better show the results.

DETAILED DESCRIPTION OF THE INVENTION Accordingly, the present invention relates to the use of copolymer-1 in the preparation of a medicament for the treatment of multiple sclerosis. The medication is added through either ingestion or inhalation. The present invention is also directed to a method for the treatment of multiple sclerosis comprising the administration of a therapeutically effective amount of copolymer-1, wherein the administration is through either ingestion or inhalation. The present invention is further directed to a pharmaceutical composition administered through ingestion or inhalation, comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of copolymer-1, wherein the pharmaceutical composition is used to treat multiple sclerosis. As stated above, copolymer-1 comprises the acetate salts of polypeptides containing L-glutamic acid, L-alanine, L-tyrosine and L-lysine. The average molar fraction of the amino acids are 0.141, 0.427, 0.095 and 0.338, respectively, and the average molecular weight of copolymer-1 is between 4,700 and 11,000 daltons. The present invention is based on the observation that, for example, oral administration of copolymer-1 is effective in the suppression of EAE, and, therefore, has a therapeutic value for the treatment of multiple sclerosis. As contemplated, copolymer-1 is contacted with those lymphoid tissues in the mucosal lining, which are believed to be a primary source of immune system sensitization. These mucosal coatings can be found (though not necessarily exclusively) in the bronchial passages, trachea, sinusis (where they are known as BALTs or broncho-associated lymphoid tissues) and gastrointestinal coatings (known as GALT or lymphoid tissues associated with the throat). Thus, the administration of the copolymer-1 is understood to include methods wherein the copolymer-1 is introduced into the body by ingestion or inhalation. For example, the copolymer-1 can be administered through the mouth through the feeding, through a stomach tube, through inhalation into the bronchial passages or through nasal inhalation. In an exemplary embodiment of the present invention, copolymer-1 is introduced orally in an amount of 0.1 to 1000 mg per day, which can be administered as a single dose or in multiple dosages. As understood by one skilled in the art, the therapeutically effective dosage is generally a function of a patient's physical condition, age and sex, as well as a function of other common treatments that are administered. The determination of the therapeutically effective optimal dosage is well within the purview of one skilled in the art. When copolymer-1 is introduced orally, it can be mixed with other forms of food and consumed in solid, semi-solid, suspension, or emulsion forms; and can be mixed with pharmaceutically acceptable carriers, including water, suspending agents, emulsifying agents, < £ - flavor improvers, and the like. In one embodiment, the oral composition is enterically coated. The use of enteric coatings is well known in the art. For example, K. Lehman, Acrylic Coatings in Controlled 5 Relay Tablet Manufacturer, Manufacturing Chemist and Aerosol News, p. 39 (June 1973), and K. Lehman, Programmed Drug Relay From Oral Program Forms; Pharma. Int., Vol. ISS 3 1971, p. 34-41, teaches enteric coatings such as Eudragit S and Eudragit L. Handbook of Pharmaceutical Excipients, 2nd ed., Also taught by the applications of Eudragit S and Eudragit L. An Eudragit which can be used in the present invention is L30D55. Copolymer-1 can also be administered nasally in some of the ways mentioned previously by inhalation or nasal drops. In addition, oral inhalation can be used to deliver the copolymer-1 to mucosal coatings of the trachea or bronchial passages. Copolymer-1 can also be prepared by methods known in the art, for example, as described in U.S. Patent No. 3,849,550, wherein the N-carboxyanhydrides of tyrosine, alanine, β-benzyl glutamate and eN-trifluoroacetyl-lysine are polymerized at room temperature in dioxane anhydrous with diethylamine as an initiator. The unblocking of the? -carboxyl group of glutamic acid is effected by hydrogen bromide in glacial acetic acid and is continued by the removal of the trifluoroacetyl groups from the lysine residues by 1M piperidine. As described in PCT / WO95 / 31990, the copolymer-1 having a desired average molecular weight of about 7 + 2 kilodaltons can be preferably prepared by a method comprising copolymer-1 protected by reaction with hydrobromic acid to form the trifluoroacetyl copolymer-1, treating the trifluoroacetyl copolymer-1 with an aqueous piperidine solution to form the copolymer-1, and purifying the copolymer-1 so as to result in the copolymer-1 having the desired average molecular weight. The present invention will be further described in the following examples. However, the present invention should be constructed as being limited thereby. Unless stated otherwise, all parts, percentages, and the like, are by weight.

EXAMPLE 1 Antigens - Copolymer-1, which is prepared according to the method described in PCT / WO95 / 31990, was obtained from Teva Pharmaceutical Industries Ltd., Israel. GPBP was prepared from the spinal cord of the guinea pig by acid extraction and precipitation of ammonium sulfate as described in Hirshfeld H et al., Febs Lett (1970) 7 317-320.

Animals - Female mice (PL / JxSJL / J) Fl (8-10 weeks of age) were obtained from Jackson Laboratories (Bar Harbor, ME). Female Lewis rats (8-12 weeks of age) were obtained from Harlan-Olac (Bicester, G.B.).

Induction and Evaluation of EAE - Mice were injected with 200 μg of GPBP emulsified in an equal volume of complete Freund's adjuvant (CFA) containing 4 mg / ml of tuberculosis mycobacterium (H37Ra) (Difco Lab, Detroit, Mich.). The emulsion at a total volume of 0.1 ml was injected into the four soles of the legs. Immediately after and 24 hours later, pertussis toxin (250 ng / mouse) was injected intravenously (Sigma). The rats were immunized with 25 μg of GPBP emulsified 1: 1 in CFA containing 4 mg / ml H37Ra. The emulsion at a total volume of 0.1 ml was injected into the two plants of the hind legs after induction of EAE, as described above. Cells from 3 animals in each group were pooled and cultured in triplicate (5xl05 mouse cells and 2xl05 rat cells) in microtiter plates with various concentrations of antigen (GPBP) in a final volume of 0.2 ml. Microtiter plates containing RPMI 1640 culture medium (available from Sigma Biochemicals, St. Louis, Missouri) supplemented with 1% autologous serum or derivative it. After 72 hours of incubation, the cells were pulsed with 1 μCi. { 3H} -thymidine for 18 hours and then collected on filter papers and counted by radioactivity.

Cytokine Secretion Assay - Spleens were removed 10-11 days after the induction of EAE and the cells of 3 mice from each group were concentrated or pooled. Cells (5xl06 / ml) were grown in duplicates in 24-well plates in RMPI 1640 supplemented with 10% FCS (Fetal Calf Serum) in the presence or absence of antigen.

(GPBP '100 Fg / ml). The supernatants were harvested after 24-40 hr of culture. Quantitative ELISA for IL-2, IFN- ?, IL-4, IL-6 and IL-10 were performed using mAbs in specific pairs for the corresponding cytokines (Pharmmgen, La Jolla, CA) according to the manufacturer's instructions.

Results: The efficiency of copolymer-1 administered «. orally to prevent the clinical manifestations of EAE in Lewis rats was compared to that in GPBP, when tested under previously reported conditions to induce oral suppression by GPBP (PJ Higgins &HL Weiner, J Immunol (1988) 140 440-445). The results set forth in Table 1 demonstrate that copolymer-1 was more effective than GPBP and significantly decreased both the incidence (54% inhibition) and the severity (57% inhibition) of EAE, which were compared with the rats fed PBS which served as control.

Table 1: Suppression of EAE in Rats by Oral Administration of Copolymer-1.

Each figure represents the cumulative results of 3-5 independent experiments. The p-values represent the statistical meaning of difference of the control group (Fisher's exact test). The average maximum response was calculated for the entire group.

Effect of Antigen Feeding on the Immune Response to GPBP - The effect for oral administration of copolymer-1 and GPBP on the immunity response to the disease that induces the GPBP-antigen was tested in mice and rats. The results are shown in Figure 1 which shows the reduction in cell proliferation for each of the orally administered compounds (copolymer-1 or GPBP) in a suspension of rat spleen cells stimulated with GPBP (FIG. 2 shows the similar results of the mouse). As can be seen, oral administration of copolymer-1 resulted in almost complete inhibition of proliferative responses to GPBP in these two species. In both species, copolymer-1 was more effective than GPBP in inhibiting the response to GPBP. Cytokine levels were measured in the supernatants of cultures of spleen cells derived from mice (Figures 3 and 4). The control mice were fed PBS secreted from IL-2; IFN-? and IL-6 (not shown) in response to GPBP. In mice fed with copolymer-1 or GPBP the amounts of the proinflammatory cytokines of Thl, IL-2 and IFN-α produced in response to stimulation by GPBP was lower than in the control groups, with copolymer-1 being a more effective supernatant. IL-4 and IL-10 were not detected with any treatment of the group. The results demonstrate that copolymer-1 is effective in suppressing EAE when given orally. The clinically protective effect of orally administered copolymer-1 is associated with down-regulation of immune responses of the T cell to GPBP such as proliferation and release of proinflammatory cytokines (IL-2 and IFN-γ).

EXAMPLE 2 Additional studies of EAE suppression by oral administration of copolymer-1 were performed in rats and mice. These studies establish the optimal dose for the treatment in each species. To understand the fundamental mechanism of oral deletion of EAE by copolymer-1, a T-cell line specific for copolymer-1 was isolated from spleens of animals fed copolymer-1. The in vitro reactivity of the lines and their effect in vivo in the induction of the disease were studied.

Materials and methods: Isolation of Copolymer-1 specific T cell lines - Lewis rats were fed 5 times with 1 mg copolymer-1 and mice (SJL / J x BALB / c) Fi 7 times with 250 μg of copolymer-1, at intervals of 2-3 days. Four to eleven days after the last feeding the animals were sacrificed and their spleens were removed.

Spleen cells from 3 animals were collected and incubated (50 x 10d / plate) with copolymer-1 (500 μg) in medium containing 1% autologous serum or derived from itself, for 4 days. Every 14-21 days the cells (4-6 x 106 / plate) were restimulated for 3 days of exposure to copolymer-1 (500 μg) presented in rat thymocytes (100 x 106 / plate) irradiated syngeneic (3000 rad) or mouse splenocytes (50 x 106 / plate). Stimulation was followed by propagation in 10% supernatant of normal mouse spleen cells activated with Con A as the T cell growth factor (TCGF).

Proliferation assay - T cell lines (1 x 104 cells) were cultured with irradiated thymocytes (3000R) (rat-1 x 106) or splenocytes (mouse-5 x 105) and with the indicated antigens (10 μg of copolymer-1) 1 μg of Con A) in a final volume of 0.2 ml in microtitre plates. At the end of 48 hours the incubation cultures were pushed with 3H-thymidine and harvested 6-12 hours later.

Assay with Cytokine - T cells from the rat line (0.5 x 10 6 / ml) were incubated with irradiated thymocytes (10 x 10 6) with or without the indicated antigen (50 μg of copolymer-1, 5 μg With A). Cells were cultured 5 > for 24 hours in RPMI 1640 supplemented with 10% FCS for measurements of IL-2, TNFa, IL-4 and IL-10 and in serum-free medium - DCCM-1 (Biological Industries, Kibbutz Beit Haemek, Israel) for 72 hours pair of TGFß. Cytokine levels in supernatants are "F measured in a quantitative ELISA using pairs of monoclonal antibodies specific for the corresponding cytokines.

Induction of EAE - Mice (SJL / xBALB / c) Fi were injected into the four leg plants with 2 mg spinal cord from the homogenized mouse (MSCH) emulsified in the 1: 1 ratio in CFA containing 1 mg / ml of H37Ra (Difco Lab. Detroit, Mich.), La Toxoplasma toxin (250 ng / mouse, Sigma) was injected twice intravenously, once immediately after and again 48 hours later.

RESULTS: 20 1. Study of the dose response in rats and mice The rats were fed 5 times with 0.5, 25 1 or 2 mg of the copolymer-1 according to the established protocol (see Materials and Methods, above), and then stimulated for the induction of EAE. The results are shown in Table 2, and it is indicated that the most effective dose was 1 mg of copolymer-1 - 0.5 mg or 2 mg of copolymer-1, they were less efficient in the suppression of EAE. The mice (SJL / JxBALB / c) Fi were fed 7 times with 0.1, 0.25 or 0.5 mg of copolymer-1 at days -7; -5; -3; 0; 2; 4 and 6 by gastric intubation. EAE was induced on day 0 by injection of MSCH. The results shown in Figure 5 demonstrate that oral administration of copolymer-1 could suppress the disease in mice and the most effective dose was 0.1 mg of copolymer-1. 0.25 mg of copolymer was less effective and a dose of 0.5 mg was completely inactive. Accordingly, the results in both rats and mice demonstrate that the oral copolymer-1 had an optimal dose response curve, and exceeded the effective oral dose that resulted in the inefficient suppression of EAE. 2. Studies with specific Ts lines of copolymer-1 established from animals fed with copolymer-1.

The T-specific suppressor cell lines of copolymer-1 were isolated from spleens of rats and mice that became insensitive to EAE by feeding with copolymer-1. The response to cytokine proliferation and secretion from such a line isolated from rats is demonstrated in Figure 6. This line proliferated in response to copolymer-1 and secreted IL-2, some IL-10 and TGFβ but no TNFa or IL -4. This cytokine profile is compatible with Th3 cells which were shown to be induced by oral MBP (Chen et al., Science 265, 1237, 1994). The ability of the specific lines of copolymer-1 to prevent EAE in vivo was studied. Cell lines were injected 3 days after stimulation in vivo with copolymer-1 (20 x 106 cells / i.p. injected rats and 15 x 106 cells / i.v. injected mouse). The animals were stimulated for the induction of EAE immediately followed by cell transfer. The results illustrated in Figures 7 and 8 show that the disease was significantly inhibited in the recipient animals. Accordingly, both the rat and the T cell lines of murine copolymer-1 adoptively transferred the EAE insensitivity induced by oral administration of copolymer-1. These cells actively regulate the pathological immune response in vivo in a decreasing manner.

Table 2: Study of the Oral Dosage Response of Copolymer-1 in Rats * calculated by average brand.

Each incidence figure represents the cumulative results of 2 individual experiments. The average maximum clinical record or record was calculated for the entire group.

Example 3 The effect of oral administration of copolymer-1 on the induction of EAE in Rhesus monkeys was studied. materials Copolymer-1 was provided by Teva Pharmaceutical Industries Ltd. in an enteric coated hard gelatin capsule comprising two dosage levels: 1 mg of copolymer-1 and 20 mg of copolymer-1. Each dosage level was formulated using mannitol, and coated with Eudragit L30D55. The control and placebo capsules comprise capsules containing 5 mg of sugar. Bovine MBP was purchased from Life Technologies, Grand Island, New York. This material represents a highly purified preparation that provides a unique band at 18.5 Kd followed by Silver staining and SDS-PAGE.

Power Protocol Three rhesus monkeys were treated as follows: One monkey served as control and was fed with placebo capsules (containing 5 mg of glucose only). The second and third monkeys were fed with capsules containing copolymer-1 at a dose of 1 mg / feed and 20 mg / feed, respectively. The animals were fed each day for a total of 10 feeds: 5 times before induction of the disease (immunization on day 0) and then 5 times after immunization.

Induction of Disease The disease was induced on day 0 by an intradermal injection of 8 mg of bovine MBP and 3 mg of H37Ra M. tuberculosis in FCA in the plants of the hind legs, the total volume injected between 0.1 and 0.15 ml per plant of the paw. Animals were followed for clinical manifestations of EAE, a variety of immunological CSF and serum markers, and spinal cord and cranial MRIs.

Clinical Marks or Records Symptom marks were given as follows: 0, normal neurological examination; 1, weight loss, anorexia, yawning, slow responses to stimulation, irritability or drowsiness; 2, mild or mild neurogenic signs, indifference, drooling or delay, disability using limbs, tremors, altered cry and disturbed gaze; 3, moderate neurogenic signs, blindness (the pupils do not react to light), akinesia, weakness in the legs, or paralysis; 4, severe neurogenic signs, semi-coma, coma, quadriplegia; 5, death.

Antigen-induced lymphocyte proliferation Heparinized blood samples were diluted 1: 1 with Hanks balanced salt solution (BSS) containing 5% hot inactivated calf fetal serum (FCS), stratified in a hypaca-ficol gradient. The centrifugation (2000 rpm for 20 minutes at room temperature) allowed the recovery of diluted plasma and the separation of lymphocytes at the interface. The recovered lymphocytes were washed three times in BSS of Hanks-5% FCS and turned into suspension in RPMI 1640 complete medium containing RPMI 1640 medium, 10% FCS, and 1% of the following reagents: non-essential amino acids , sodium pyruvate, L-glutamine, 2-mercaptoethanol, and penicillin / streptomycin. The recovered lymphocytes were counted and resuspended in a final concentration of 2 x 106 / ml. The cultures containing 100 microliters of the cell suspension and 100 microliters of MBP (20 micrograms / well). 100 microliters of copolymer-1 (10 micrograms / well) or 100 microliters of Con A (1 microgram / well) were fitted into 96-well round bottom microtitre plates. The cultures were maintained at 37 ° C in 5% C02 for 6 days. On day 5 of the culture, each well was pulsed with 1 μCi of 3H-thymidine for 16 to 18 hours. On day 6, cultures were harvested by an automatic cell harvester, and counted by liquid scintillation methods. The stimulation indices were determined as follows: stimulation index = cpm expepmental-cpm background cpm background Clinical Results The animals do not exhibit clinical symptoms for 24 days. Animal 18374 treated with placebo developed the disease in 25 days, and due to severe manifestations of EAE (mark or record = 4+ on a scale of 5) had to be sacrificed on day 28. Animal 18498 treated with copolymer-1 at high dose, treated with 20 mg of copolymer-1, did not show any significant clinical symptoms during the 60-day observation period. Animal 18497 treated with low-dose copolymer-1, showed minimal symptoms on day 25, however, in contrast to animal 18374 treated with placebo,? 497 showed a much slower increase in clinical symptoms and was equaled in 2 to 3"1" from day 28 to day 33. At this time point animal 97 was fed 20 mg capsules 5 times on alternating days for a total of 10 days. As can be seen in Figure 9, within 3 days the clinical signs of the animal fell to zero and remained here until the two animals 97 and 98 fed with the copolymer-1 were sacrificed on day 60 by histology.

Flow Cytometry The Epics C flow cytometer was used to analyze both peripheral blood lymphocytes (PBL) and cerebral spinal fluid (CFS) collected from each monkey. The red blood cells were treated with lysis and the remaining white blood cells (WBCs) were washed and stained using standard methods with appropriate reagents. The Tables 3 and 4 below show the data of the WBCs coloration of PBL and CSF of the three animals in this Example.

The number of cells that were colored CD4 + was slightly higher in animals 18497 and 18498 fed with copolymer-1 than in control animal 18374. The number of CD4 + cells that are also CD45RA- seems to be increased in both control animal 18374 and animal 18497 fed low-dose copolymer-1, around the time when both animals exhibited clinical symptoms (days +27 to +34), but remains clearly constant in the animal 18498 fed high-dose copolymer-1. The number of CD8 + CD45RA + staining cells decreases permanently in control animal 18374 and animal 18497 fed with low dose copolymer-1, but is ^ increases slightly in animal 18498 fed with X, 15 high-dose copolymer-1 (indicating the production of new CD4 + cells). The number of cells found in the CSF of the i _t > Control animal 18374 was permanently increased from day 20+ to day 28+ when the animal died. i 20 The analysis shows that more of the CD4 + cells were CD45RA-. The number of cells collected from the CSF of animal 18497 was too small to count or color. The number of cells collected from animal 18498 was so few to count or color except on day +27. The "CD4 + cells were collected then predominantly CD45RA-.

Table 3: Analysis of PBL obtained from Rhesus monkeys immunized with MBP.

ND = Not determined Table 4: Analysis of CSF obtained from Rhesus monkeys immunized with MBP. ND not determined Analysis For the antigen-specific T cell suppressor factor (Tcsfi) inducer The analysis for specific Tsfi of MBP in the plasma of the three monkeys in this Example is shown in Tables 5 and 6 below. None of the animals produced specific Tsfi-MBP until day +13 followed by the induction of EAE with MBP. Control animal 18374 does not produce any Tsfi until day +20 and the level produced after this was adjusted to the previous context. Animals 18497 and 18498 fed with copolymer-1 consistently produce significant levels of Tsfi from day +13 to day +41 shortly before termination. Table 6 shows that plasma samples which do not contain Tsfi do not react with the anti-TGF-beta antibody. The plasma which exhibits the binding of MBP with the 3C9 antibody (anti-Tsfi) also reacts with the anti-TGF-beta antibody. Recombinant human TGF-beta reacts with the anti-TGF-beta, but not with the 3C9 antibody.

Table 5: Assay for the inducer factor3 of the T-specific suppressor MBP a = The data represent OD at 405nm, from plasma samples at a dilution of 1:20 ND = Not Determined Table 6: The association between specific Tsfi of MBP and TGF-beta in plasma of rhesus monkeys treated with copolymer-1. to. It represents OD405 nm of material bound by MBP (2.5 μg of protein) and reacts with the anti-human antibody Tsfi (3C9). b. It represents OD405nm of material bound to MBP and reacts with the anti-human TGF-beta antibody. c. Represents cavity coated with 100 ng of recombinant human TGF-beta.

Example 4 Six monkeys were treated and subsequent analyzes were performed in substantial manner according to the protocol described by Example 3 described above. Control animal 18746 fed with placebo was fed with capsules containing glucose. Animal 18586 was fed with 1 mg of copolymer-1 in capsules with cracked enteric coating. Animal 18639 was fed 20 mg of copolymer-1 in capsules with cracked enteric coating. Animal 18724 was fed with 1 mg of copolymer-1 in intact, enteric coated capsules. Animal 18810 was fed with 10 mg of copolymer-1 in intact enteric coated capsules.

Animal 18962 was fed 20 mg of copolymer-1 in intact enteric coated capsules. The feeding program, the induction of the disease and the following were substantially the same as those of Example 3 described above.

Results As can be seen with reference to Figure 10, the control monkey 18746 develops the acute disease starting on day +21 and dies on day +23 of manifestations of the disease. The animals 18586 and 18639 treated with the "cracked" enteric coating, which opens in the stomach (in both doses), were not protected from the disease and died within 2-3 days of the manifestations of the disease. All monkeys fed with copolymer-1 in enteric coated capsules, were totally protected from the disease, and did not develop signs of EAE until day 60, when they were sacrificed for histology.

It is noted that in relation to this date, the best method known by the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention. Having described the invention as above, the content of the following is claimed as property

Claims (15)

1 . A method for treating multiple sclerosis characterized in that it comprises an ingestion or inhibition of a therapeutically effective amount of copolymer-d

2. A method according to claim 1, characterized in that the therapeutically effective amount of copolymer-1 is from 0.1 to 1000 mg / day.

3. A method according to claim 1, characterized in that the copolymer-1 is administered orally.

4. A method according to claim 1, characterized in that the copolymer-1 is administered through the nose.

5. A method according to claim 3 or 4, characterized in that the copolymer-1 is inhaled.

6. A method according to claim 3, characterized in that the copolymer-1 is administered orally in an enteric coated form.

7. The use of copolymer-1 for the manufacture of a medicament for the treatment of multiple sclerosis, wherein the medicament is administered through ingestion or inhalation.

8. The use according to claim 7, wherein the medicament comprises from 0.1 to 1000 mg / day of copolymer-1.

9. The use according to claims 7 or 8, wherein the medicament is formulated for oral or nasal administration.

10. The use according to claim 9, wherein the administration is by inhalation.

11. The use in accordance with the claim 9, where the medication is encapsulated enterically.

12. A pharmaceutical composition for the treatment of multiple sclerosis comprising a therapeutically effective amount of the copolymer-1 and a pharmaceutically acceptable carrier, characterized in that the pharmaceutical composition is formulated for administration by either ingestion or inhalation.

13. The pharmaceutical composition according to claim 12, characterized in that the pharmaceutical composition is in the solid form, liquid form, aerosol or powder for inhalation.

14. The pharmaceutical composition according to claim 13, characterized in that the solid form is enterically coated.

15. A method for treating multiple sclerosis in a subject, characterized in that it comprises the administration of a therapeutically effective amount to the subject of the claimed pharmaceutical composition in any of claims 12 to 14.