A method of treating autoimmune diseases with the use of interferon type i

 

(57) Abstract:

The invention relates to medicine, in particular to immunology and neurology, and for the treatment of autoimmune diseases. This treatment includes a step of oral administration of interferons first type, and each introduction is carried out at a dose of from 50 to 25,000 IU/kg In this regard, I propose to treat multiple sclerosis to reduce the severity or frequency of relapse, and reduce autoimmune inflammation and to inhibit the attack of autoimmune disease. The method, in contrast to the known injecting interferon, it is easy to use, effective and can be used for a long time, as far as is necessary for treatment. 4 C. and 10 C.p. f-crystals, 10 ill.

The present invention relates to the field of neurology, immunology and protein chemistry. More specifically, the present invention relates to new methods of treatment of autoimmune diseases with the use of interferons of the first type.

Acute experimental autoimmune encephalomyelitis /EAE is an inflammatory autoimmune process in the Central nervous system, mediated by T cells, which resembles such a system diseases, which reminds multiple sclerosis /MS/ as in pathological and clinical manifestations, provides a model for evaluation of interventions aimed at changing the course of autoimmune diseases. Myelin basic protein /MBP/ represents an important neuroantigen in the pathogenesis of this disease. CR-EAE in SJL/J mice can be adaptive to transfer after intravenous injection of MBP peptide 89-100 specific T-cell lines. Joint cultivation of MBP peptide 91-103 specific T cells with chemically bound peptide gives T cells tolerance to MBP in CR-EAE in SJL/J mice.

Parenterally, for example, a fourfold introduction natural rat interferon /105units/ partially suppresses acute EAE in male Lewis rats and inhibits passive HiPerStrut localized EAE when injected into the same day as inoculation immunogen. Other parenteral input pitocin, such as TGF-can reduce the clinical picture of the disease and inflammation in the brain and spinal cord during EAE. Parenteral input natural IFN - man can degrade the function of T cells and production of antibody-dependent T cells in humans. Oral introduction of natural IFN - a person can prevent dust for demonstrating the ability of oral input immunoactive substances influence the course of autoimmune diseases IFN - man, is an immunoactive protein, which can be oral to enter in low doses in the treatment of viral diseases in animals. IFN - a man of another interferon first type enhances the function of suppressor cells in vitro with progressive MS and rat IFN - reduces the severity of symptoms in rats with EAE. Given the immunoregulatory and antiviral properties of interferon type 1, their reactions and the production were evaluated in the case of autoimmune diseases. Inactive rheumatoid arthritis are characterized by enhanced /2'-5// oligoadenylates /OAS/, which is easily determined by measuring the activity of interferon type 1 in peripheral leukocytes of the blood, secondary to IFN-/ incentives; active rheumatoid arthritis demonstrate significantly reduced the production of IFN-/ compared to normal donors. Such other autoimmune diseases, such as psoriasis and atopic dermatitis, also show a decrease in the production of interferon. The peripheral blood lymphocytes in patients with MS show similar inefficient production of interferon type 1 in response to viral or mitogenic stimuli, parallel with the severity of the disease. In patients with multiple sclerosis is observed is normalized by treatment with IFN-.

Known abnormality in the production or reactions interferon type 1 autoimmune diseases prompted us to make a few small pilot studies using interferon type 1 as therapeutic agents for MS. In these studies used the systematic introduction of interferon type 1 at doses of 1 million units or more daily without noticeable clinical improvement. Research parenteral IFN - multiple sclerosis with intermittent relapses and remissions suggests that the introduction of the 1.6-8 million units of IFN - subcutaneously three times a week can reduce relapses by 40-50% and reduce inflammation of the brain, as evidenced by the series MBI /images obtained using magnetic resonance.

Known methods of treatment lack of effective means for the treatment of autoimmune diseases by oral administration cytokines such as interferons of the first type. The present invention fills this urgent need.

The aim of the present invention is the demonstration that oral administration of interferon type 1 inhibits proliferation of sensitized antigen suppresses clinical manifestation the present invention is to illustrate the action of interferons of the first type with a dose before and after immunization with GP-MBP in acute EAE.

Another aim of the invention is to suppress the clinical manifestations of asthma autoimmune diseases, reduction of pathological complications and inhibition of inflammatory pitocin when such diseases by oral administration of biological response modifiers such as interferons of the first type.

The aim of the present invention is also a demonstration of the validity of oral administration rat IFN-/ on clinical presentation, histology and IFN - secretion in experimental allergic neuritis in in vivo models of acute inflammatory demieliniziruyushchee radiculoneuropathy or Guillain-Barre syndrome /Guillain - Barre'/.

In one embodiment of the present invention, a method for treatment of autoimmune diseases in animals, which includes a step of oral administration of these animals interferon type 1.

In another embodiment of the invention, a method for decreasing the severity or frequency of relapses of multiple sclerosis in humans, which includes a step of oral administration of interferon type 1 specified patient.

In yet another embodiment of the present invention, a method of reducing inflammation associated with autoimmune diseases is WMD.

Finally, in yet another embodiment of the present invention, a method for inhibiting attacks autoimmune diseases, including the state oral administration to a specified animal interferon of the first type.

Other aspects, features and advantages of the invention will be apparent from the following description presents the preferred variants of the invention, given with the purpose of disclosure.

To clarify the above features, advantages and objectives of the present invention, as well as others that will become clear in the future, and for more detailed explanations, a more particular description of the invention, briefly summarized above, you can perform with links to some versions of the invention, which are illustrated in the accompanying drawings. These drawings are part of the description. However, it should be noted that the accompanying drawings illustrate only preferred variants of the invention and therefore should not be considered as limiting the scope of invention.

Fig. 1 demonstrates that oral administration of murine natural IFN-/ suppresses clinical relapse when mouse CR-EAE. Two groups of six animals in within 7 weeks. Presents one of two representative examples. SEM/average quadratic error/ is less than 10%.

Fig. 2 demonstrates that oral administration of 5000 units of rat IFN-/ inhibits clinical manifestation of acute EAE. Four groups of rats Lewis /6 in each/ enter simulation IFN 1000, 3000 or 5000 units of IFN-/ each rat within 7 days prior to immunization /-7/ and over the next 21 days /+14/. The results are expressed as the mean clinical score for each group on each day of the disease after inoculation SEM. Is one of the representative examples. Data for the cases of the introduction of 1000 and 3000 units not shown,xp less than 0.02 between simulator interferon and 5000 units of IFN-/ introduced animals.

Fig. 3 demonstrates that oral administration of hrIFN - rats with acute EAE delays the attack, reduces the severity of it, and the rate of return of a clinical attack. Three groups of seven rats Lewis inoculant MBP and CFA at day 0 and orally administered PBS 1000 or 5000 hrIFN daily, starting seven days before immunization /-7/ and within 14 days after immunization /+14/. Values represent experimental clinical parameters in allergic neuritis for each of the groups of 7 animals SEM. Presents one and the th EAE reduces cumulative clinical severity of acute clinical attacks in allergic neuritis. Three groups of seven rats Lewis process, as shown in Fig. 3. The given values correspond to the cumulative clinical indicators in allergic neuritis for each group of 7 animals from 10 to 16 days after inoculation.xp less than 0.001, 5000 units of IFN - a against for 1000 IFN-, and PBS control.

Fig. 5 demonstrates that oral administration of 5000 units hrIFN - reduces the number of inflammatory centres in the spinal cord compared with the control group and the group injected with 1000 units of interferon. After killing the spinal cord of animals, is shown in Fig. 3, is treated in accordance with the description in the methods section. The results are expressed as the number of inflammatory centers for spinal cord SEM. Presents one of the representative examples.xp less than 0.04, 5000 units of IFN against 1000 IFN or PBS control group.

Fig. 6 demonstrates that oral administration of 5000 units hrIFN - without subcutaneous injection reduces the severity of clinical attacks. Three groups of Lewis rats at 6 in the group inoculant MBP and CFA on day 0, and without treatment is administered 5000 or injection subcutaneously 5000 hrIFN daily, starting seven days before immunization /-7/ and over the next 21 days. /+14/. Value represents the s SEM. Presents one of two representative examples.

Fig. 7 demonstrates that oral administration of hrIFN - rats with acute EAE reduces cumulative clinical severity of acute clinical seizures in experimental allergic neuritis. Three groups of six rats Lewis in each process, as shown in Fig. 6. Values represent the cumulative clinical parameters in experimental allergic neuritis for each group of 6 animals from 10 to 16 days after inoculation.xp less than 0,005, for the animals, which were introduced, compared with untreated/ introduced subcutaneously treated animals.

Fig. 8 demonstrates that oral administration of 5000 units hrIFN - reduces the number of inflammatory centres in the spinal cord compared with the control group. After killing the spinal cord of animals, is shown in Fig. 6, process, as described previously, and conduct an independent assessment for inflammation of one observer, who is not a method of processing. The results are expressed as the number of inflammatory centres of the spinal cord SEM. Presents one of two representative examples.xp less than 0.04, 5000 IFN administered orally against 5000, introduced subcutaneously.

Fig. 10 demonstrates that oral administration of rat interferon -/ reduces inflammation and IFN - production in experimental allergic neuritis, Lewis Rats administered orally or 5000 units of IFN-and/ or mock-IFN, starting from 7 days before immunization with bovine myelin of peripheral nerves to the point of killing. Immunocytochemical study lumbosacral nerve roots using ED1 antibody /1:1000/ - and IFN - antibodies /1:10/ 13 days after immunization during an episode of clinical disease shows less ED1 positive macrophages /Fig. 10A, 2B/ IFN-treated rats compared with values in a mock-IFN-treated rats /Fig. 10B, 10E/. Along with the Fig. 100/ in comparison with rats treated with a mock-IFN /Fig. 10F/. Bar = 20 microns.

In the method of the present invention is a modification of the disease and the inhibition of production of inflammatory cytokines, induced due to oral administration of cytokines in EAE, provides a convenient, effective and long term treatment of autoimmune diseases, caused by an unknown antigen in humans, in particular multiple sclerosis.

The present invention is directed to a method of treating autoimmune diseases in animals, which includes a step of oral administration of interferon type 1 specified animals. Usually the interferon that can be used in the method of the present invention, is either interferon alpha or beta interferon. The interferon of the first type can be obtained from any source. It is preferable to choose the interferon from the group consisting of human recombinant interferon, rat murine interferon and interferon.

Usually the interferon of the first type, introduced in accordance with the methods of the present invention, it is necessary to enter in doses that effectively inhibit the occurrence of relapses and so on autoimmune diseases. Cabrette can be used for any animal with autoimmune diseases. It is more preferable to use the method of the present invention to treat people.

The methods of the present invention can treat a wide range of autoimmune diseases. Representative examples of autoimmune diseases include multiple sclerosis, rheumatoid arthritis, diabetes, psoriasis, organ-specific autoimmune diseases, chronic inflammatory demyelinizing polyradiculoneuropathy and Guillain-Barre syndrome.

The present invention is directed also to a method of reducing the severity or frequency of occurrence of relapses of multiple sclerosis in humans, which includes a step of oral administration indicated patient interferon type 1. Moreover, the present invention is directed also to a method of inhibiting attacks autoimmune disease, which involves the step of oral administration indicated patient interferon of the first type.

The following examples are given to illustrate different variants of the present invention, and the present invention is in no way should be limited to experimental allergic neuritis.

Example 1

The induction of experimental autoimmune ence is n and McFarlin, Lab.Invest. /1981/ 45:278-284, modified Miller et al., J. Immunol. /1987/ 138:3776-3784. In short, each mouse subcutaneously injected into the shaved right flank 0.3 ml of an emulsion containing 1 mg syngeneic mouse spinal cord homogenate /MSCH/ in 0.15 ml of phosphate superyoung saline solution and 0.03 mg of Mycobacterium tuberculoses homines H37Ra /MT/ [Difeo Labs. Detroit M1/ 0.15 ml incomplete adjuvant Freund /IFA/. After seven days, mice injected with a similar injection in the left side. The initial clinical signs of disease are observed between 13 and 25 days after immunization. The clinical severity of recurring attacks evaluates the observer /blind/: 0 = no symptoms, 1 = minimal or moderate weakness in the hind limbs; 2 = moderate weakness in the hind limbs or mild ataxia; 3 = moderate to severe weakness in the hind limbs; 4 = severe weakness in the hind limbs to moderate ataxia; 5 = paraplegia with more than moderate weakness of the four limbs; 6 = paraplegia with severe weakness of the four limbs or severe ataxia. Animals evaluated blindly for 6-8 weeks and cumulative weekly rate counting, averaging three indicators per week /Monday, Wednesday, Friday/ for each group of animals.

Priest dose /1-100 units/ mouse of natural IFN-/ /Citymany murine IFN-/ 4,0105IRU /ml, Lee Biomolecular Research, Inc., San Diego, CA/ or simulator murine IFN-/ /Citymany less than 2 IRU/ml, Lee Biomolecular Research, Inc., Diego, CA/ received is identical to IFN -/, except that cultures induce the simulator// using 2.5 cm syringe with 20 gauge needles ball point/ Thomas Seientific, Swedesboro NJ/ three times a week /Monday, Wednesday, Friday/ within 6-9 weeks. Imitation of interferon used as control, as thermodenaturation IFN can save some immunological activity.

Example 3

Receive basic myelin protein Guinea pigs /PD-MBP/

20 g of the spinal cord of Guinea pigs add to the 4.7 ml of methanol at -20oC and homogenized in a tissue homogenizer. Add to 9.4 ml of chloroform at -20oC, homogenize for 2 minutes and stirred for 60 minutes at room temperature; then another 60 minutes at 4oC. the resulting material was filtered through a double folded sterile gauze, through chilled Buchner funnel and filter paper No. 1 Whatman paper. The tissue pellet was washed with 20 ml of acetone at -20oC, and again suspended in 40 ml of cold ddH2O; then stirred for 30 minutes at 4oC, filtered a second time and again suspended in 10 ml of cold; H suspension fabric/H2O adjusted to pH 3 due to 1H. HCl, and stirred for overnight at 4oC. After stirring overnight the pH adjusted to 3 and rotate at 10,000 g for 15 minutes at 4oC. the Supernatant is filtered through paper No. 1 Whatman paper and the liquid filtrate is stored. the pH of the resulting filtrate was adjusted to 9 due to 10 N. NaOH and stirred for 60 minutes at 4oC; then rotate at 10,000 g for 15 minutes at 4oC. Determine the volume of the supernatant liquid and add ammonium sulfate to a final concentration of 27.7 g/100 ml, the supernatant is stirred in the cold for 30 minutes, then spin at 10,000 g for 15 minutes at 4oC and again suspended in 1 ml of ddH2O. Re-suspended sediment cialiswhat in low-molecular /6-8000 MB/ tube for dialysis against ddH2O. the Concentration of protein was determined on A280using UV spectroscopy. To check the purity of the drug myelin basic protein prepared 7-20% SOS-PACE gradient gel and stained with 0.3 M CuCl2. The purity of the drug myelin basic protein Guinea pig /GP-MBP/ demonstrated strip 18 KD.

Example 4

The preparation of cells of the lymph node and spleen

Animal the spleen, and suspension of individual cells receive, rubbing through a sieve made of stainless steel with a cell diameter of 90 mesh. Perform lysis of red blood cells in a cell suspension of the spleen with 2 ml of ASC solution, added to the precipitate and the reaction allow to proceed for 5 minutes at room temperature.

Example 5

The proliferation of T cells in vitro

Seven days after clinical relapse, kill mice, draining inguinal lymph nodes removed and cultured in vitro to determine antigen-specific T cell proliferative responses. Stimulation by antigen carried out, using as antigen either 0 or 100 μg/ml /GP-MBP or MT/ and mitogenic stimulation of Con A at 2.5 µg/ml by incubating cells lymph node 210 cells/cell in RPM1 /Gibco, Grand Island, NY/, supplemented with 10% serum, fetal calf /FCS/ Whittaker Bioproducts Walkersville MD/, 1% sodium pyruvate /Gibco, Crand, Island, NY /, 1% glutamine "Gibcol/, 1% penicillin/streptomycin and 50 ál of 2-mercaptoethanol). Plates are incubated at 5% content of CO2and moisture at 37oC for 4 days. At this point, cells treated with 2 µci titiraupenga [3H] dTd and harvested after 18 hours using an automated collection. Sahwa three parallel experiments, and the results expressed as CPM.

Example 6

Histology

After killing extract the spinal cord and immersion fixed in 10% neutral bateriafina formalin for at least two weeks. After fixation, the brain is cut in the horizontal plane at intervals of about 3 mm and treated with paraffin. Paraffin blocks cut for 6-8 microns and graded sections stained with hematoxylin and Sosina and Luxol-fast blue /PAS/ hematoxylin, and examined under an optical microscope. Sections of brain evaluated independently to determine the centers of inflammation /blind/, without information about the nature of the treatment of the animal prior to killing. Samples of the spinal cord of each animal prepared in an identical manner, and determine the number of centers of inflammation in a section of more than 20 of inflammatory cells in the parenchyma.

Example 7

Analysis of cytokines

Cells from the spleen of animals treated with imitation or 100 units of murine IFN-/ cultured with Con A /2.5 µg/ml/ 1 of 106cells/ml in 75 cm2flasks for tissue culture for 48 hours in a humid incubator with 5% CO2/95% air at 37oC. the Supernatant is harvested after 24 and 48 hours after Con A activation, and the hydrated ELISA analysis. Anti-IL-2, anti-IL-10 or IFN-/Phar-Mingen, San Diego, CA), incubated on polyvinyl microtiter plates with 96 cells in 0.01 M carbonate buffer /pH 9,6/ overnight at 4oC. the Plates are blocked with 3% BSA in phosphate bateriafina saline solution for 3 hours, 100 µl of the supernatant liquids added in different dilutions, which is described for the linear portion of the curve absorption/concentration with a threefold repetition and incubated for 1 hour at room temperature. After the plate is washed five times with phosphate buferizovannyiy saline Tween /0,05%/, within 60 minutes add 100 ál of horseradish peroxidase interlacing monoclonal antibody /epitope determinants, different from the first antibody, which was used for coating polyvinyl plate/ in a concentration of 1:1000. Then add About phenylenedimethylene, and the absorption is determined at 450 nm. Get standard curves for various amounts of different interleukin. Statistical analysis carried out using t-test t-test.

Example 8

Oral administration of murine interferon feather type suppresses clinical signs of illness the PA has immunological effects, but is inadequate when the pressure of clinical relapses. Therefore, two groups of six immunogenic SJL/J mice were treated with simulator IFN or 100 units of murine natural IFN-/ three times a week starting from 30 days after recovery from the first attack. Group clinical parameters after the first attack did not differ significantly for different groups. Clinical recurrence was observed in approximately 40 days after inoculation. Clinical indicators demonstrate significant differences in their occurrence for a group to which oral introduced simulator IFN compared with the group that was injected 100 units of murine natural IFN-a/ less than 0.03, see Fig. 1/. Two major relapse in the group with imitation IFN occur within seven weeks, and observed enhanced neurological deficit. The group, which is administered orally natural murine IFN-/ observed slow single attack without residual neurological deficit. Oral administration of murine natural IFN-/ reduces the severity and reduces group index between the initial attack and relapse.

Con A activation of cells in the draining inguinal lymph nodes is inhibited in animals that were injected peroria with 38095 CPM 3160/. Cells of lymph nodes of animals, which were injected imitation IFN tested with the MBP, produce intense proliferative reaction, but this reaction is significantly inhibited in animals that were injected murine natural IFN-/ /14052 CPM against 842 448 50 CPM/.

To determine whether inhibition on the other sensitized with an antigen, investigate antigen-specific proliferation of draining inguinal lymph nodes on the second sensitized with an antigen of Mycobacterium tuberculoses homines /MT/ component MSCH of inoculum. Cells of lymph nodes of animals, which were injected imitation IFN produce intense proliferative response to MT, but this response is significantly reduced in animals that were injected murine natural IFN-/ /52401 CPM 857 against 5214 CPM 808/.

Animals were also examined histologically after 65 days after immunization. It turned out that there is considerably less inflammatory centres the group, which was administered IFN /0,5 0,1/ compared with the control group, which was introduced imitation IFN /1,8 1,1/p less than 0.05/. Thus, murine natural interferon of the first type when oral administration can suppress the clinical manifestations of the disease, reduce inflammation and ingerii IFN-/ correlated with a decrease in IFN - secretion

The intensity of the disease EAE is associated with IFN - a secretion after Con A stimulation of spleen cells. Therefore, the United spleen cells stimulate both for animals treated with oral simulator IFN /n=5/, and for oral processed 100% /n= 5/ mouse IFN-a/ Con A /2.5 µg/ml/ 1106cells/ml for two days, and the supernatant analyzed in solid-phase ELISA/IL-2, IL-10, IFN- . Oral administration of IFN-/ consistently reduces IFN - a mediator of inflammation /exp. 1, a simulator, spleen 2500 ng/ml 300 vs. 100%, spleenx1100 ng/ml 200; exp. N 2 simulator, spleen 2180 ng/ml 100 units, spleen against 247x143 ng/ml 13,xp less than 0.001 compared with control-imitation IFN/. There is also a decrease of IL-2, growth factor T cells and increased production of IL-10, although these changes did not reach statistical significance.

The present invention illustrates that oral administration of interferons first type can modify the biological response CP-EAE in the introduction after the animal is recovering from the first attack. With the introduction in adequate doses, oral administration of interferon type 1 suppresses clinical relapses, reduces inflammation and inhibits Strait is astate activated T cells by subcutaneous injection of antigen. Thus, the type 1 interferons are active in oral method of administration with significant immunological effects in exposed to the immune sites. Oral administration of IFN consistently reduces Con A-induced IFN - secretion in the cells of the spleen. The suppression of cell proliferation to MBP can occur even after a period of time of 6 weeks after inoculation and 4 weeks after the initial clinical attack. Thus, oral administration of cytokines without a protective matrix to prevent the digestion of protein in the oropharynx and the rest of the alimentary canal is able to inhibit proliferation to previously sensitized antigen.

The weakening of established or new immune response is an important therapeutic moment. Oral administration of myelin antigens animals can improve the clinical course of CR-EAE in rats and Guinea pigs and reduce pathological changes on milenova antigens in CR-EAE. Oral administration of myelin proteins may reduce the severity of attacks in men DR2-MS patients and the frequency of MBP specific t-cell lines treated with myelin individuals. However, in the observation of patients who then suggests, that oral administration of other immunoactive substances may exceed myelin antigens.

Immunomodulatory mechanism of oral administration of interferon first type is unknown. Modulatory effects Co A activated lymphocytes to mitogenic response of cells normal responders can be cancelled by adding anti-human leukocyte IFN serum in vitro; this can prevent the production of inhibitory factors, induced IFN-, for example, soluble suppressor of immune responses /SIRS/ and derived from macrophage suppressor factor . After such mutagenic stimulation may be required peripheralization T cells to produce interferon. However, oral administration of low doses of IFN - mice does not lead to detectionin levels of IFN - in the blood, in contrast with intraperitoneal introduction, his action cannot be blocked by circulating anti-IFN antibodies. Neutropenics effect of oral introduced IFN can be replaced by the injection of blood cells, but not serum, animal-recipients. Analysis of mRNA of cytokines CNS during EAE suggests that IL-2, IL-6 and IFN - increased in acute disease, but in the process of stabilization of symptoms number of these Chitipa 1 can be immunomodulatory molecules, which induce suppressor factors, such as IL-10, inhibiting the reaction to immunizable antigens such as MBP and MT.

Thus, oral administration of such biological response modifiers, as interferons first type representing a potentially non-toxic, convenient and continuous means of inhibiting immune reactivity. The biological response modifiers, injected oral, provide additional funds for the treatment of autoimmune diseases.

Example 10

Induction of acute experimental autoimmune encephalomyelitis. EAE induce 8-10 weeks in female rats, Lewis /Harlan Sprague Dawley, Indianapolis, IN/ using the emulsion containing equal parts of the myelin basic protein in PBS and full adjuvant Freund /CFA/; Mycobacterium tubereuloses homines H37Pa /MT/ /Difco Labs, Detroit, MI/. Rats Lewis immunizat subcutaneous injections in the sole of the hind legs of 0.1 ml of an emulsion containing 50 µg and 500 µg MT in adiuvante Freund. The initial clinical signs of disease are observed between 9 and 11 days after immunization. The clinical severity of the initial attack is subdivided as follows /assessment blind/: 0 = no disease, 1 = drop tail; 2 = slight weakness in the hind konechnoraznostnoi clinical indicator count, determining the average for each group on each day of the attack. Cumulative clinical indicator of experimental allergic neuritis was calculated, and it included a complete clinical disease severity, incidence and duration of clinical signs in a single comparative measure by summing the daily indicators for all rats in the treated group between the beginning and the end of the attack and dividing by the total number of rats in the treated group. Statistical analysis carried out for the combined data from at least two experiments.

Example 11

Introduction

Seven days before immunization /-7/ and within 21 days after the start /+14/ to rats orally, injected or imitation IFN, or 1000, 3000 or 5000 rat natural IFN-/ /citymany rat IFN-+ 4,0105IRU /ml Lee Biomolecular Research, Inc. San Diego, CA/ or recombinant human IFN- /hrIFN- / / interferon-113,0106EgH/ml, Schering Corp., Kenilworth, NJ/ using a syringe with gauge boll point needle /Thomas Seientifie, Swedesboro, NJ/ placed in the back of the oropharynx or injectively subcutaneously with PBS placebo or 5000 hr IFN - daily. Lyophilized IFN preparations recreate with DPBS and diluted 1/600 to 1/3000. Imitation of natural preparationwork main protein of Guinea pigs /GP-MBP/, i.e., homogenization and dilapidation, acid elution MBP, preparations of cells of lymph nodes and spleen, analyses of in vitro T cell proliferation and cytokine carried out as described previously.

Example 12

Histology

After killing the spinal cord extract and imersion fixed in 10% neutral bateriafina formalin for at least two weeks. After fixing the chord cut across the horizontal plane of from about 3 mm intervals and treated with paraffin. Paraffin blocks, cut into sections 6-8 MK and stages section /n=5/ stained with hematoxylin, Sosina and Luxol - fast blue /PAS/ hematoxylin, and examined under an optical microscope. Section chords evaluated independently to determine centers of inflammation /blind/ without information about the nature of the treatment of the animal prior to killing. Tissue samples of the spinal cord are selected in an identical manner for each animal, and count the number of inflammatory centres on a section of more than 20 of inflammatory cells in the parenchyma. Statistical analysis carried out using t-test t-test.

Example 13

Natural rat interferon Alfa-beta modifies the clinical nature of the disease, in cinedelic rats Lewis immunizat equal parts MBP and CFA, the attacks then begin to 9 days and last up to 16 days. Seven days before immunization /-7/ s through the 21st day after that /+14/, each group of animals orally administered either the simulator or 1000, 3000 or 5000 rat IFN-/ the first type daily in 0.1 ml PBS. All animals evaluated for clinical symptoms of the disease until 16 days after immunization. Research results /blind/ daily group clinical indicators demonstrate significant differences in clinical manifestations in animals that were injected imitation of interferon, and animals, which were injected 5000 IFN-/, especially on 13-16 day, but not for the animals, which were introduced 1000 or 3000 units. In rats, which were injected 5000 rat natural IFN-/ the peak of the disease was less severe than the group which was administered simulator interferon, and this group recovered more quickly and returned to baseline faster than the group treated simulator /Fig. 2, the data for 1000 and 3000 units not represented. Clinical cumulative performance of experimental allergic rewrite for animals treated with 5000 rat natural IFN- , were significantly lower than the control, which was introduced in the simulator IFN /0,8 0,2 at 5000 IU; 1.2 Itachi. There are fewer inflammatory lesions in rats treated with IFN-/ /266/compared with the group that was injected imitation interferon /502/, although this has no statistical significance because of the small number of samples of the spinal cord in group /n=3/ p < 0.06/.

The proliferation of Con A in the draining popliteal lymph nodes inhibited with 162091234 CPM in animals with simulator interferon to 8120765 CPM in animals that were injected 5000 IFN-/. Cells from the draining popliteal lymph nodes of animals processed by the simulator and IFN 5000 IFN were stimulated with ionomycin + PMA and show a decrease in proliferation from simulator /20505 SRM/ up to 5000 units /6111 SRM/ for treated animals. In the draining popliteal lymph nodes of animals processed by the simulator and interferon, there is no significant difference in MBP or MT proliferation. No was also observed inhibition in the spleen or negreiros lymph nodes briggate Con A or ionomycin /PMA in IFN-treated animals.

Thus, the specific samples of type 1 interferons inhibit the severity of acute clinical disease when introduced into the respective doses. The inhibition of proliferation at the expense of peroral secnik or directly. In vitro treatment with interferon type 1 cells in the draining popliteal lymph node and spleen cells taken from immunogenic, but imitation treated animals, demonstrates effects similar to the processing in vivo. Not observed a clear effect on Con A proliferation of cells in the draining popliteal lymph nodes or spleen cells exposed IFN /1-100 units/ in vitro, in contrast to the in vivo oral introduction of IFN, in which the proliferation of draining popliteal lymph nodes was reduced. Thus, the indirect effect of IFN of the first type may be at the expense of lymphoid tissue associated with the gut.

Example 14

Modification of the acute rat EAE due to oral administration of human recombinant interferon alpha.

As human interferon of the first type may exhibit cross-specific activity in mice, Guinea pigs, gnotobiotic calves, horses and pigs, and cats, was used recombinant human interferon hrIFN- / homogeneous material, which can provide a more effective immunosuppression per unit activity than natural drugs. For behold the 00 unit IFN - daily. Three groups of seven or eight to ten weeks of Lewis rats was immunizable, the result has been an attack since 10 days, which lasted 16 days. All animals were monitored for the evaluation of the clinical nature of the disease up to 16 days after immunization. As can be seen in Fig. 3, the animals, which were injected PBS and 1000 hrIFN - showed serious acute episodes, starting with 10 days, and reached maximum values on day 14. The animals, which were injected 5000 IFN-, showed a delayed onset of seizures, low weight at the peak and earlier recovery from attack. Cumulative clinical characteristics of experimental allergic neuritis in animals treated with 5000 hrIFN - were significantly lower than in animals that were administered PBS /control/ or 1000 units /Fig. 4/. Thus, oral administration of hrIFN - suppresses the occurrence of clinical seizures EAE in Lewis rats with the introduction to inoculation. After killing on day 16 after immunization were also carried out by histological examination of animals. It turned out that much smaller foci of inflammation accounts for the group, which was introduced by 5000 units of IFN - compared with the PBS control group or the group, which enter the human recombinant interferon-alpha modifies the clinical nature of the disease

Oral administration hrIFN - modifies the clinical nature of the disease and reduces inflammation of the spinal cord. Investigated the relative effectiveness of equivalent quantities of oral and parenteral input hrIFN-. Three groups of six rats Lewis were immunitary and either left untreated or were injected with 500 units hrIFN-or they were injectively subcutaneously 5000 hrIFN - within seven days prior to immunization /-7/, and over the next 21 days /+14/. All animals were evaluated for clinical signs of disease for up to 18 days after immunization. In rats that orally was administered at 5000 hrIFN - was less severe course of the disease in the peak and a faster recovery compared with the untreated group /Fig. 6/. Really raw and subcutaneously treated groups had very similar rates of clinical curves, which suggests that subcutaneous administration hrIFN - has little or no effect on the clinical nature of the disease. Cumulative clinical parameters in experimental allergic neuritis demonstrate significant differences in clinical manifestations in raw/subcutaneously injected animals compared with odnosno injectionem animals. 18 days after immunization, animals were slaughtered and performed histological examination. Acatalog that much smaller foci of inflammation observed in the case of 5000 units of interferon compared to 5000 units subcutaneously introduced /Fig. 8/.

Similar results were obtained for oral input and subcutaneously injected PBS. In this case, four groups of six rats Lewis was administered either orally PBS, or PBS subcutaneously, or orally 5000 hrIFN or subcutaneously 5000 hrIFN - within seven days before immunization /-7/ and for 21 successive days /+14/. All animals immunized on day 0. Animals appreciate the nature of the clinical symptoms up to 16 days after immunization and then kill. Cumulative clinical characteristics of experimental allergic neuritis are significantly less in animals that were injected hrIFN- /1,00,2/ compared to those who were administered PBS /2,50,4/ /p of less than 0,005/. No significant difference between those animals, which were injected subcutaneously imitation /1,70,4/ and those that were injected subcutaneously 5000 hrIFN-even though treated with subcutaneous injection the animals were supposed to have a higher cumulative clinical parameters in experimental allergic evritania with the introduction of clinically preventive oral dose.

Example 16

Oral administration of IFN - inhibits induced mitogen IFN - and in the draining popliteal lymph nodes.

The intensity of the disease EAE was associated with IFN - a secretion after Con A stimulation. Therefore, cells of the spleen and draining popliteal lymph nodes /on day 18 after immunization from animals that oral was administered simulator interferon or 5000 oral hrIFN - stimulated with Con A /2.5 µg/ml for two days and the supernatant was analyzed using ELISA. Presented in table 1, the results demonstrate that oral administration of hrIFN - reduces IFN - a mediator of inflammation in the draining popliteal lymph nodes, but not in the spleen.

Table 1

Inhibition of clinical manifestations of the disease due to oral administration of interferon correlates with a decrease in the secretion of IFN - in the draining popliteal lymph nodes. LN imitation LN 5000 spleen, imitation spleen, 5000 IFN- /ng/ml

46160 9632*36075 31062

Cells of the spleen and draining popliteal lymph nodes /18 day/ from rats that were administered oral simulator IFN and 5000 units of IFN immunogenic rats were cultured with Con A /2.5 µg/ml at a concentration of 1106cells/ml in t is after centrifugation. The content of IFN - determine, using solid-phase ELISA analysis. The results are expressed in ng/ml. Presents the combined data from the two experiments. LN 5000 compared to LN imitationxp less than 0.05.

Oral introduced interferons of the first type, as opposed to identical doses given subcutaneously, disclosed in the present invention as biological response modifiers on MBP in cases of acute EAE in Lewis rats, if they are administered before sensitization and clinical attack. Oral ingested interferon type 1 partially modify clinical attacks, reduce the number of inflammatory centres in the spinal cord, reduce nonspecific proliferation at the expense of Con A and ionomycin/PMA, and reduce the production of IFN - in the draining popliteal lymph nodes. This suggests that IFN - more active in oral method of administration, and have defined immunological effect, and confirms that specific cytokines can inhibit clinical disease when introduced through the gastrointestinal tract.

It was unexpectedly found that in the present invention are as human recombinant IFN-, and species-specific crycry may be only a small component of natural type. Human IFN shows some cross-species activity, however, the exclusive use of interferon-11subtype may lead to more due to the inhibitory activity/total activity of the most important components for immunosuppression in rats.

Antiproliferative effects of oral IFN input is greater than in the draining popliteal lymph nodes than in Negreira popliteal lymph nodes. Oral administration of IFN - a also inhibits the production of IFN - in the draining popliteal lymph nodes. Draining popliteal lymph nodes present natural drainage areas for subcutaneous injection of antigens and therefore are presumably tank high frequency sensitized MBP-specific T cells. Oral administration of cytokines may predominantly affect the proliferation and production of cytokines in places the immune Activati compared with systemic administration. Inhibition of IFN - secretion in activated regional immune Department by IFN - can reduce the inflammatory effect by MBP-specific cells in the CNS.

Oral administration of IFN - Oka did not inhibit the proliferation of Con A in the population LN in contrast with in vivo by oral administration. Thus, the method of administration is critical and can determine the immunological mechanism for IFN. Proteins that can not survive the transit through the alimentary canal, can still demonstrate immunoinhibitory activity for the account associated with the intestinal lymphoid tissues /GALT/ in the oropharynx. Therefore, oral administration provides an alternative system of drug delivery for therapeutic cytokines in the treatment of autoimmune diseases due to generation of immunoregulatory cells through the intestinal immune system.

Immunomodulatory mechanism of oral ingested interferon first type is unknown. Modulatory effect of Con A-activated lymphocytes on michanie reaction cells normal responders " can be canceled by adding anti-human leukocyte IFN serum in vitro, which may modify the production of inhibitory factors, induced IFN- , for example, soluble immune response suppressor /STRS/ and suppressor factor, derived from the macrophage . Peripheralization T cells may be required for the production of interferon after mitogenic stimulation. However, oral administration of low doses of IFN mice does not lead to detektivami UB is stimulating anti-IFN antibodies. Neutropenics effect of oral IFN input can be transferred by injecting blood cells but not animal serum-recipients. Similarly, cells can be induced in Meyerovich plaques after oral administration of antigen to mice, which reduces the humoral response in vitro. Therefore, IFN of the first type may be immunomodulatory molecule produced by activated T and other immune cells, which induce suppressor factors such as M-SF inhibiting reactions to immunizable antigens such as MBP.

The present invention demonstrates that oral IFN - a therapy is suitable for relapsing-imitiruemogo multiple sclerosis and other chronic neurological autoimmune diseases, as oral ingested IFN - appears to be more effective than equivalent doses of parenteral introduced IFN-. Thus, oral administration of modifiers biologic response cytokines, such as IFN - provides an effective means for modification of clinical attacks in response to antigens with the introduction of prior sensitization. Oral route of administration is a convenient delivery system of drugs, which allows the use of significantly Bo>Immunization

Female rats of the Lewis /Harlan/ 150-170 g immunizat 20 mg wet weight/ peripheral nerve myelin, emulsified in an equal volume of complete adjuvant Freund /CFA/, which contains 10 mg. M. tuberculoses per ml adjuvant. 200 µl immunogen injection in the bottom of the right hind paws. The myelin of peripheral nerves isolated from bovine spinal roots /Pel-Freeze, TX/ by way of Norton. Methods Enzymol., 31 /par A/, 435, 1975.

Example 18

Interferon

Starting seven days prior to immunization and to killing 20 days after immunization, rats administered either imitation IFN or 5000 rat natural IFN-/. Citymany rat IFN-/ 1,5106IU/ml Lee Biomolecular Researeh Inc., San Diego, CA/. Lyophilized IFN restore at the expense of sterile water, diluted in phosphate bateriafina saline /PBS/, and is administered in a daily dose of 0.1 ml using a syringe with 20 gauge needle ball point/Thomas Seientific, Swedesboro, NJ/ placed in the back of the oropharynx.

Example 19

Clinical assessment

Rats weighed daily starting 7 days after immunization, and conducting clinical assessment, using the following scheme: 0, normal; 1 = limp tail; 3 moderate prepares; 4=serious prepares; 5 = paraplegia; 6 = paraplegia, including art in accordance with the definition of Hahn et al. Acta Neuropathol. 82 : 60 - 65 /1991/. Weight loss calculated for each animal as the difference between the weight at the time of the killing and maximum weight and expressed as a percentage of the maximum weight.

Example 20

Proliferation analysis

When the killing on the 20th day after immunization remove the draining popliteal lymph nodes and spleen. Suspension of individual cells receive, passing the tissue through a stainless steel mesh with cell diameters of 90 μm. Lysis of red blood cells is carried out in a suspension of spleen cells by adding 2 ml of ACK solution to the precipitate. Cells from spleen and draining lymph nodes collected for each of the treated groups for in vitro culture. Full cell population in the spleen or lymph nodes incubated with 2 105cells/ml with the addition of M. tuberculosis with 10 μg/ml, SP26 at 1 µg/ml /SP26 is neuritogenic peptide of 26 amino acids/ Rostami et al., J. Neuroimmunol. , 30 : 145 - 151 /1990/, synthesized in the Department of Analitical Chemistry at the Uneversity of Texas/, Concanavalin A /Con A/ at 2.5 µg/ml /Sigma Chemical Co., St.Louis, MO/ in RPMI (Gibco, Crand Island, NY), ionomycin at 100 ng/ml Calbiochem, La Jolla, CA/ combined with Piletilevi complex ester of myristic acid /PMA/ when 1 ng/ml /Sigma/, supplemented with 10% fetal serum t is the susceptibility and 50 μm 2-mercaptoethanol. Plates are incubated in 5% CO2in a humid environment at 37oC for 4 days. At this point, cells treated with 2 µci labeled Triticum /3N/ a dTd, and harvested after 18 hours using automatic collection. Capture /3N/ a dTd is determined using a liquid scintillation counter Beckman. Culture are three instances and the results expressed as CMP.

Example 21

Analysis of cytokines

Cells United draining popliteal lymph nodes and the United spleens from animals that were treated with imitation interferon or 5000 rat IFN with experimental allergic neuritis cultured with Con A /2.5 µg/ml of M. Tuberculosis /10 μg/ml or PS26 /1 μg/ml/ 1106cells/ml in 75 cm2the flask for tissue culture for 48 hours in a humid environment incubator containing 5% CO2/ 95% air at 37oC. the Supernatant collected after centrifugation, and frozen at -70oC. the Content of IFN - determine, using solid-phase ELISA analysis. Anti-IFN- /Pharmingen, San Diego, CA), incubated on polyvinyl microtiter plates with 96 cells 0.01 M carbonate buffer /pH 9,6/ overnight at 4oC. the Plates are blocked with 3% albumin bovine savor the s for the linear portion of the curve absorption/concentration/ three experiment and incubated for 1 hour at room temperature. Then the plates are washed 5 times with PBS Tween /0.05% and added dropwise within 60 minutes 100 μl of horseradish peroxidase IFN - a monoclonal antibody /epitope determinants, different from the first antibody used for the coating of polyvinyl plate/ at a concentration of 1: 1000. Then peroxidase substrate add On-phenylenedimethylene, and the absorption was measured at 450 nm. Get standard curves for various amounts of IFN-.

Example 22

Histology

Rats killed by perfusion of saline under anesthesia phenobarbital on the 20th day after immunization. The tail is removed and divided. The distal portion is fixed with 2.5% paraformaldehyde /2.5% of the glutaraldehyde in PBS for 4-6 hours, treated with osmium overnight in 2% osmium tetroxide, dehydration and enter in Epon. From each animal prepared from 4 to 5 units lower lumbosacral roots. Representative 0,5 MK slices of each of the blocks stained with toluidine blue.

Histological studies of demyelination and inflammation in every back exercise, using the following rating scale: /Hahn et al., 1991/: demyelination; 1 + = some demyelinated axons perivenular or Russ is luento; inflammation; 1+ = few scattered managernew inflammatory cells often superintegrable 2+ = perivenular education "cuff" with managername inflammatory cells; 3+ = intense multicenter perivenular education "manzhetok and widespread endoneural inflammation. The indicators are calculated for each animal by dividing the total number of demyelination and inflammation in the number of nerve roots in the slices. The slides are read with the help of the researcher /MP/ who don't know the Protocol of immunization and treatment of prior samples.

The proximal part of the cauda equina / 2 cm/ frozen in methylbutane cooled in liquid nitrogen and stored at -70oC. Womentrannies cross sections get into the cryostat, fixed in ether cooled with ice and used for immunocytochemical studies.

Example 23

Immunocytochemical studies

Immunocytochemical studies carried out using the following antibodies: ED1 1:1000 /macrophages/, W3/13 /lymphocytes; Harlan Bioproduets for Science, IN), CDll b/c 1: 100 /CR3 on macrophages, granulocytes, dendritic cells; Pharmingen, CA, IFN - 1:10/ Genzyme, MA/. Womentrannies frozen slices block in Siva the t PBS, incubated with biotinylated secondary antibody for 1 hour, washed in PBS, incubated with ABC-peroxidase /Vectastain/ for 5 minutes, washed and show diaminobenzidine and hydrogen peroxide. Blocking of 0.01% hydrogen peroxide is preceded by incubation with primary antibodies to IFN - Intensity and distribution of staining nerve roots is estimated as 1+, weak; 2+, moderate, and 3 + strong.

Example 24

Clinical symptoms

Rats begin to lose weight on day 10 after immunization, and the first neurological symptoms appear on day 12, whereas the recovery begins on the 16th day or later. In 11 of the 22 rats /50%/ on to develop severe experimental allergic neuritis with clinical indicator 4 /heavy parport/ or above, and 2 of 11 animals that enter the simulation, die 14 and day 15 after immunization, whereas none of the animals that were injected SHAT-/ not killed. Clinical indicator in the group with imitation IFN /n= 11, figure 4,12,4; experimental allergic neuritis /was significantly higher than in the group of rats which were administered IFN-/ /n=11, figure 3,31,4; p less than 0.03, the test Wilkinson/. A larger number of rats in the group with imitation IFN demonstrated clinically RALO more than 25% of the weight, than the group that was administered IFN-/ /Fig. 9B/.

Example 25

Cell proliferation and cytokines

Proliferation of spleen cells to Con A in animals that were injected IFN-/ was reduced compared to animals that were injected simulator IFN /spleen: the group with the simulator 11811415194 against rats with IFN-/ 83594, mean SEM, p less than 0.006, t-test/.

The production of IFN - a after stimulation through P26 cells of lymph nodes and spleen in rats, which were injected imitation IFN was much higher than the production of IFN - cells in the lymph nodes and spleen in rats, which were injected IFN-/ according to ELISA, but the difference does not reach statistical significance /lymph nodes: 18,92,4 ng/ml against 11,93, ng/ml; spleen: 11,90,4 ng/ml compared with 6.3 1.8 ng/ml, mean SEM, in rats with simulated against rat IFN-a/ p<0,08/. After immunization with Con A or M. tuberculosis was observed no differences in the production of IFN-

Example 26

Histology

Histological studies included in Epo sections, shows that demyelinative much less seriously in rats, which were injected IFN-/ /index 0,880,11 mean +SD/ compared with rats that were injected simulator IFN /index 1,10,26; p less than 0.05, t-test), but the inflammation in these groups Zam is health a greater number of inflammatory cells as perivascular, and diffuse infiltra nerve roots of the cauda equina as in rats, which were injected simulator IFN and rats, which were injected IFN-/, when their slain on the 20th day. The majority of these cells were phagocytic ED1 positive macrophages, and there was no significant difference between groups of animals which were injected simulator IFN or IFN-/ in the degree of inflammation.

Groups of rats treated with either mouse /h=3, or simulator IFN /n=3/ slew earlier in the course of the disease on day 13 after immunization. Immunocytochemical studies were to evaluate IFN - production in situ, that is visible only up to 14 days for experimental allergic neuritis, and to assess inflammation. Infiltration of ED1 positive macrophages and W 3/13 positive cells was much less severe in rats, which were injected IFN -/ than in rats, which were injected simulator IFN /control/. IFN - coloring attended the dense areas of inflammation on the surface of inflammatory cells. Fig. 10 demonstrates that IFN - staining significantly less in rats, which were injected in parallel with less intense inflammation.

Oral administration species-specific IFN-/ compared to simulator IFN reduces the severity of experimental is less weight loss. Histological data obtained 20 days after immunization, when it had already started healing, show less demyelination in the group treated with IFN -/, without changing the degree of inflammation. On day 13 after immunization, in situ, IFN - production was lowered along with inflammation, according to immunocytokine. Studies of cell proliferation in the draining lymph nodes and spleen gives reduced proliferation to Con A in IFN-treated rats with experimental allergic neuritis compared with rats that were injected simulator IFN. Analysis of cytokines showed reduced production of IFN - a after stimulation SP26, antigens of myelin of peripheral nerves, able to induce experimental allergic neuritis. In General, the present invention demonstrates that oral administration of IFN-/ reduces the severity of experimental allergic neuritis by reducing the production of IFN-. Thus, the present invention demonstrates immunomodulatory effect of oral administration IFN-/ on the immune system of the host.

The mechanism of action of oral administration IFN-/ on the immune system is unknown, but may involve effects on Peyronie plaques in lymphoid tissues, attornye factors derived from CD8 + T cells, which are responsible for the modification of the disease. Reduced production of IFN-associated with orally administered IFN-/ suggests the possible inhibition of the functions of the system T 1-like helper T cells detected during EAE, which produce IFN-. This leads to reduced T cell encephalitogenic actively or passively induced diseases.

As was shown injecting - IFN - intensifies as induced by myelin and mediated by T cells in experimental allergic neuritis with enhanced clinical signs and histological disorders, and increases oxidative germination of macrophages, whereas the opposite effect is observed with parenteral anti-IFN - antibodies. IFN - also induces MHC class 1 and 11 the expression on Schwann cells in vitro. IFN - inflammatory cytokine secreted by Th1 CD4+T cells, may enhance MHC class 11 expression on macrophages and endothelial cells and activate macrophages, which play an important role in the phagocytosis of myelin. Reduced IFN - expression and reduced inflammation observed in asthma clinical disease in animals that were injected IFN-/. Histological OC the e inflammation in animals which was injected IFN-/. The decrease in IFN - and reducing inflammation in the early stages and decrease demyelination in the later stages of the disease suggest a critical role of interferon in the pathogenesis of experimental allergic neuritis. The inability to reduce inflammation in the later stages of the disease may be related to a partial suppression of IFN - production, the influence of other cytokines and mediators of inflammation and induction too heavy experimental neuritis.

In the end, the insertion oral interferon is a biological response modifier, effective for the modification of the disease in different experimental autoimmune disease, experimental allergic neuritis. The results of the present invention in experimental allergic neuritis, demonstrating the attenuation of the disease in rats that orally administered IFN-/ illustrate that oral administration of IFN-/ should be useful in the treatment of immuno-oposredstvovanii neuropathies in humans, such as Guillain-Barre syndrome or chronic inflammatory demyelinizing polyradiculoneuropathy /CIDP/.

All patents and publications referred to in this description are pointers priklucheni as references.

Specialist it is easy to understand that the present invention is well adapted to accomplish the goals and achieving those results and benefits. Examples, and the methods, procedures, treatments, molecules, and specific compounds described herein are representative and preferred variants are examples and in no way limit the scope of the invention. Those changes, and other applications that are obvious to experts, included in the essence of the invention defined by the scope of the claims.

1. A method of treating autoimmune disease in a mammal, including humans, by administration of interferon of the first type, characterized in that it includes a step of oral administration of interferon of the first type, and each introduction produce at a dose of from 50 to 25,000 IU/kg

2. The method according to p. 1, characterized in that the said interferon is selected from interferon-alpha and beta-interferon.

3. The method according to p. 2, characterized in that the said interferon is selected from the group consisting of human recombinant interferon, rat murine interferon and interferon.

4. The method according to p.,1 characterized in that the interferon is up from the group, consisting of multiple sclerosis, rheumatoid arthritis, diabetes, psoriasis, organ-specific autoimmune diseases, chronic inflammatory demyelinizing polyradiculoneuropathy syndrome Hiera-Barre.

6. The method of reducing the severity or frequency of relapses of multiple sclerosis in a mammal, including humans, by administration of interferon of the first type, characterized in that it includes a step of oral administration of interferon of the first type and each injection produced in a dose of from 50 to 25,000 IU/kg

7. The method according to p. 6, characterized in that the said interferon is selected from interferon-alpha and beta-interferon.

8. The method according to p. 6, characterized in that the said interferon is selected from the group consisting of human recombinant interferon, rat murine interferon and interferon.

9. The method according to p. 6, characterized in that the interferon is injected through the day.

10. A method of reducing inflammation associated with an autoimmune disease of a mammal, including humans, by administration of interferon of the first type, characterized in that it includes a step of oral administration of interferon of the first type and each introduction is interferon selected from alpha-interferon, beta-interferon.

12. The method according to p. 10, characterized in that the said interferon is selected from the group consisting of human recombinant interferon, rat murine interferon and interferon.

13. The method according to p. 10, characterized in that the autoimmune disease is chosen from the group consisting of multiple sclerosis, rheumatoid arthritis, diabetes, psoriasis, organ-specific autoimmune diseases, chronic inflammatory autoimmune demyelinative polyradiculoneuropathy syndrome Hiera-Barre.

14. Method of inhibiting attack autoimmune disease, in a mammal, including humans, by administration of interferon first type is characterized in that it includes a step of oral administration of interferon of the first type and each of interferon produced in a dose of from 50 to 25,000 IU/kg

 

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3 ex, 1 tbl

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