Modulation of expression of tn/tn cytokines and ribavirin ribavirin analogues in activated t-lymphocytes

 

(57) Abstract:

The invention relates to immunology and relates to the modulation of expression of TH1/TH2 cytokines and ribavirin ribavirin analogues in activated T-lymphocytes. The invention includes the expression of Th1/Th2 lymphokines in T cells of a patient man ribavirin added to the cells in the dosage, which stimulates the expression of one of lymphokines TNF, IL2 and IFN and suppresses the expression of Th2 lymphokine IL-4. The advantage of the invention is to develop methods of modulating lymphokines, i.e. to increased Th1 response while reversion Th2 phenotin and Vice versa. 7 C.p. f-crystals, 7 ill., table 2.

The scope of the invention

The scope of the invention is immunology.

Background of the invention

Lymphokines are a group of polypeptides belonging to the family of cytokines, i.e. hormone-like molecules that can affect many cell functions and to communicate between different cells. Recent studies have helped to clarify the role of lymphokines in the immune response. The production of lymphokines helper T cells CD4+(and CD8+) often leads to one of the two phenotypes. Th1 and Th2, as in the immune system mytor tumor necrosis (TNF) and interferon gamma (IFN), and they are primarily responsible for cell-mediated immunity such as delayed-type hypersensitivity. Th2 cells produce interleukins, IL-4, IL-5, IL-6, IL-9, IL-10 and IL-13 and primarily involved in providing optimal care humoral immune responses, such as switch isotypes IgE and IgG4 antibodies. (Mosmann, 1989, Annu Rev Immunol, 7:145-173).

Strongly polarized Th1 and Th2 responses not only play different roles in protection, they can also strengthen the various immunopathological reactions. Answers Th1-type involved in organ-specific autoimmunity, such as experimental autoimmune uveoretinitis (Dubey et al., 1991, Eur Cytokine Network, 2:147-152), experimental autoimmune encephalitis (EAE) (Beraud et al, 1991. Cell Immunol, 133:379-389) and insulin-dependent diabetes mellitus (Hahn et al, 1987, Eur J Immunol, 18:2037-2042), contact dermatitis (Kapsenberg et al, Immunol Today 12: 392-395) and some chronic inflammatory diseases. In contrast, responses Th2 dysbalance-type responsible for triggering allergic atopic diseases (against normal environmental allergens), such as allergic asthma (Walker et al, 1992, Am Rev Resp Dis 148:109-115) and atopic dermatitis (van der Heijden et al, 1991, J Invest Derm 97:389-394), they are considered to exacerbate the infection living in the tissues protozoa, Eastern Europe and Caucasus involved in some primary immunodeficiencies, such as Hyper-IgE syndrome (Del Prete et al, 1989. J. Clin Invest 84: 1830-1835) and syndrome OmennOs (Schandene et al, 1993, Eur j Immunol, 23, 56-60), and are associated with reduced Hyper-IgG syndrome (Del prete et al, 1989, J. Clin Invest 84:1830-1835) and syndrome OmennOs (Schandene et al, 1993, Eur j Immunol 23:56-60) and are associated with reduced ability to suppress HIV replication (Barker et al, 1995, Proc Soc Nat Acad Sci USA 92: 11135-11139).

Thus, it is clear that modulation profiles lymphokines the previously mentioned diseases would be therapeutically beneficial. The increased Th1 response is likely to lead to reversion Th2 phenotype and Vice versa. It was shown that monoclonal antibodies to lymphokines, lymphokines and other agents such as thiol antioxidants (Jeannin et al, 1995, J Exp. Med, 182:1785-1792) change the pathogenesis of some diseases by inhibiting the stimulatory disease type cytokines, either Th1 or Th2. For example, intracellular protozoan infection limited to IFN, but increased IL-4, whereas nematode infection are controlled by IL-4 and increased IFN (Heinzel et al, 1989, J Exp Med 162:59-72, Else et al, 1994, J Exp Med 179:347-351). Insulin-dependent diabetes mellitus in NOD mice and EAE in mice and rats can be improved by treatment with IL-4 or anti-IFN monoclonal antibody to the development of a specified disease (Rapoport et al, 1993, J Exp Med 178:87-99, Racke et al, 1994, J Exp Med 180:1961-1966, terisolasi eritematoso syndrome - systemic lupus erythematosus is associated with produced Th2 lymphokines and inhibited by anti-IL-4 antibodies (Umland et al, 1992, Clin Immunol Immunopathol 63:66-73). On the other hand, Th1 cytokines are produced in acute GVHD, in which donor CD8+T cells develop in CTL and destroy the immune system of the host. The treatment with anti-EFN or anti-TNF monoclonal antibodies facilitates disease, and treatment with anti-IL-2 monoclonal antibodies turns acute GVHD in autoimmune GVHD (Via and Finkelman, 1993, Int Immunol 5: 565-572).

Clinical trials of native and recombinant IL-2 in HIV-infected patients conducted since 1983 (Volberding et al, 1987, AIDS Res Hum Retroviruses, 3:115-124). In this case the cause was the fact that it has been shown that the development of AIDS (AIDS) is associated with a switch of the type produced lymphokines (Clerici and Shearer, 1994, Immunol Today 15: 575-581). Over time, the infected individual with the disease is detected reduced expression of Th1 lymphokines, such as IL-2 (Maggi et al, 1987, Eur j Immunol, 17:1685-1690, Grutes et al, 1990, Eur j Immunol, 20: 1039-1044, Clerici et al, 1993, J Clin Invest, 91:759-765), which is accompanied by increased production of Th2 lymphokines, such as IL-4 and IL-10 (Clerici et al, 1994, J Clin Invest 93:768-775, Hoffman et al, 1985, Virology. 147:326-335). T cells from asymptomatic or long ivesia their ability to suppress HIV replication and to produce IL-2 (Barker et al, 1995. Proc Soc Nat Acad Sci USA, 92: 11135-11139).

These modern immunomodulatory therapies (monoclonal antibodies and recombinant cytokines), however, have limitations. For example, with continuous treatment with monoclonal antibodies animal-host produces antibodies against monoclonal antibodies, thereby limiting their useful effect. Received "humanized" monoclonal antibodies that have significantly reduced the risk of induced immune response to these monoclonal antibodies. However, it is still under development, and in addition, these new monoclonal antibodies remain large proteins and therefore may have difficulty in achieving site-targets. Therapeutic agents based on cytokines, also have limitations. For example, the treatment of autoimmune GVHD using IL-2 leads to the development of acute GVHD in mice.

Ribavirin (1-b-D-ribofuranosyl-1,2,4-triazole-3-carboxamide) is a synthetic nucleoside that is able to inhibit the replication of RNA and DNA virus (Huffman et al, 1973, Antimicrob Agents Chemother, 3:235, Sidwell et al, 1972, Science, 177:705). The authors confirmed the observations of those who suggested that ribavirin in addition to its antiviral activity affects some and the fact ribavirin effect on the proliferation of mitogen - or antigen-activated T and b lymphocytes (Tam et al, 1995 (data not shown), Peavy et al, 1980, Infection and Immunity, 29:583-589), and then, when combined with cyclosporine, ribavirin has shown efficacy in long-term allograft survival (Jolley et al, 1988, Transplantation Proc, 20:703-706).

In addition, the authors greatly promoted the original research showing that ribavirin modulates the type of cytokine in the immune response, at least in part by stimulating Th1 response and suppressing Th2 response. When compared with previously obtained data, this discovery is not incompatible with the previous studies. First, it is known that ribavirin inhibits as a functional humoral immune response (Peavy et al, 1981, J Immunol, 126: 861-864, Powers et al, 1982, Antimicrob Agents Chemother 22:p.108-114), and IgE-mediated modulation of secretion of stem cells (Marquardt et al, 1987, J Pharmacol Exp Therapeutics 240:145-149, (both Th2 mediated by lymphokines)). Secondly, ribavirin antagonisitic antiviral effect of azidothymidine (AZT) in peripheral blood lymphocytes of HIV patients (Vogt et al, 1987, Science 235:1376-1379). This is a significant discovery, because AZT reduces the expression of IL-2 receptor (IL-2R), but not the expression of IL-2 (Viora and Camponeschi, 1995, Cell Immunol 163:289-295). Thus, it is possible that ribavirin is an antagonist of AZT, modulating the expression of D (Th-2-mediated disease) has led to a sharp termination of the disease, the outcome, which does not occur with conventional immunosuppressive therapy, for example in the treatment of cyclosporine and glucocorticoids (Cassano, 1991, Bone Marrow Transplantation, 7:247-248). Finally, treatment with ribavirin (one year) patients with hepatitis C (HCV) showed less aggregation of lymphocytes and less liver damage than with placebo control (Dusheiko et al, 1994, Hepatology, 20:206A). This observation may reflect the fact that although the dominant immune response to hepatitis C is mediated by the Th1 lymphokines, T cell phenotype Th0/Th2 can be infected with HCV (Zignego et al, 1994, unpublished data) and this infection can lead to further mediated by antibodies destruction of hepatocytes.

Brief description of drawings

Table 1 represents the level of resting and PMA/ionomycin-activated level lymphokines, IL-2, IL-4, TNF, and IFN (PG/ml) after 48 and 72 hours, measured in the extracellular supernatant, and the expression on cell surface IL-2 (IL-2R) and IL-4 (IL-4R) receptors (medium intensity channel fluorescence) in T cells.

Fig.1 is a graphical representation of the effect of ribavirin and interferon alpha on the extracellular expression of IL-2, IL-4, TNF and IFN in lymphocytes, activated with PMA/ionomycin. Results BS="ptx2">

Fig. 2 is a graphical representation of the influence of 2, 10 or 50 μm ribavirin in the presence of 2000 U/ml interferon alpha (left panel) and the effect of 500, 1000 or 2000 U/ml interferon alpha (right panel) in the presence of 10 μm ribavirin on the extracellular expression of IL-2 (a and C) and IL-4 (b and D) T lymphocytes, activated by PMA/ionomycin.

Fig.3 is a graphical representation of the effect of ribavirin and interferon alpha on the expression of mRNA of IL-2, IL-4 and IFN T lymphocytes, activated RM/ionomycin.

Fig.4 is a graphical representation of the effect of ribavirin and interferon on the expression of cell surface IL-2 and IL-4 receptors on T lymphocytes, activated by PMA/ionomycin. The results are presented as the percentage increase in the expression of the receptor of lymphokine obtained after processing only PMA/ionomycin.

Fig.5 is a graphical representation of the expression of intracellular expression of IL-2 in resting (a and E) or activated CD4+(upper panel) or CD8+(lower panel) T cells treated only PMA/ionomycin (b and F) or in the presence of 10 μm ribavirin (C and G) or 5000 U/ml interferon alpha (D and H). Data shown is one experiment and are presented as% of the geographical representation of the considered analogues of ribavirin.

Fig. 7 is a set of graphs showing the effects of different concentrations of ribavirin analogs on IL-2, TNF-, IFN-, IL-4 and IL-5.

Summary

In accordance with one aspect of the present invention specified nucleoside ribavirin is administered to the patient in the interval dosage that is effective to modulate the expression of lymphokines by activated T cells. In particular, ribavirin is used to suppress Th-2 mediated responses of T cells and stimulation of Th-1 mediated response of T cells.

Thus, instead of introducing ribavirin as well recognized antiviral agent ribavirin here is used to treat imbalances in the expression of lymphokines. Such imbalances can be companions allergic atopic disorders such as allergic asthma and atopic dermatitis, helminth infection and leishmaniasis, and various primary and secondary immunodeficiencies that may be associated and not associated with viral infection.

In accordance with other aspects of the present invention, one or more analogues of ribavirin is administered to the patient in the range of dosages, which is effective for modulating the expression of lymphokines 2-mediated responses of T cells.

Detailed description of specific embodiments of the

In the preferred embodiment of ribavirin is introduced orally to humans in a dosage that achieves levels of serum on average 0.25 to 12.5 μm, and most preferably 2.5 μm. In typical individuals, this optimal level in serum is expressed approximately 4.5 mg/kg/day of body weight that can be put in doses of 200-1200 mg Preferably the dosage is divided into a number of smaller doses, which are then entered in the day.

Because ribavirin exists in the sale of several years, there are many dosage forms and routes of administration, and all acceptable dosage forms and routes of administration can be used. For example, in addition to the oral introduction of ribavirin can be given intravenously, intramuscularly, intraperitoneally, topically, and in this way, all of these methods are known. Pharmaceutical compositions comprising ribavirin, may also contain one or more pharmaceutically acceptable carriers, which may include excipients such as stabilizers (for long-term storage), emulsifiers, binding agents, thickening agents, salts, preservatives, solvents, dispersion media, covering the use of such media and agents for pharmaceutically active compounds is well known in the world. Except when any conventional medium or agent is incompatible with ribavirin, we can assume his/her use in therapeutic compositions and preparations. Supplementary active ingredients can also be introduced into the compositions and preparations.

In addition to therapeutic uses of ribavirin discussed here, ribavirin may also be used as a laboratory tool for the study of absorption, distribution, cellular uptake and effectiveness.

It was also revealed that some analogs of Virazole (previously known and unknown) that were previously rejected as having minimal activity, also have significant cytokine activity. Our research has shown that there are several classes of analogs virazole with such activity, and examples are given below under the heading "ribavirin Analogues". These examples are intended to characterize the positions in which Mirasol can be modified to obtain active compounds, and this description, therefore, should not be limited to the illustrated specific modifications. Next, it is assumed that these modifications can be applied to standard the cells

Mononuclear cells from peripheral blood (PBMCs) were isolated from leucocytes film by centrifugation in density gradient Ficoll-Hypaque 60 ml of blood from healthy donors. T cells were then isolated from PBMCs using Lymphokwik reagent isolation of lymphocytes that are specific for T cells (LK-25T, One Lambda, Canoga Park CA). 40-60106T cells, which is the average output were then incubated overnight at 37oWith 20-30 ml of RPMI-AP5 (RPMI-1640 medium (ICN, Costa Mesa, CA) containing 20 mm HEPES buffer, pH 7.4, 5% of the original plasma, 1% L-glutamine, 1% penicillin/streptomycin and 0.05% 2-mercaptoethanol) to remove all contaminating adherent cells. In all experiments, T cells were washed RPMI-AP5 and then was applied to 96-well microtiter tablets at a cell concentration 1106cells/ml.

Activation of T cells and treatment with ribavirin

T cells were activated by the addition of 500 ng ionomycin and 10 ng of phorbol 12-myristate 13-acetate (PMA) (Calbiochem, La Jolla, CA) and were incubated for 48-72 hours at 37oC. PMA/ionomycin-activated T cells were treated with 0.5-5.0 µm ribavirin or 250-10000 IU/ml control antiviral interferon-alpha (Accurate, Westbury, NY) immediately after activation and processing poweriscsi.sles for measurement of extracellular cytokines. After activation of 900 μl of cell supernatant from each blade was transferred to another microplate for analysis on the production of cytokines from the cells. Cells then were used for immunofluorescence analysis of intracellular levels of cytokines and the expression of receptors of cytokines.

Analysis of extracellular cytokines

The concentration of cytokines from human cells was determined in cell supernatant for each blade. Caused by activation changes in the levels of interleukin-2 (IL-2) were determined using commercially available ELISA kit (R& D systems Quantikine kit, Minneapolis, MN) or Biotest using IL-2-dependent cell line CTLL-2 (ADS, Rockville, MD). Caused by activation changes in the levels of interleukin-4 (IL-4), tumor necrosis factor (TNF), interleukin-8 (IL-8) (R & D systems (Quantikine kit, Minneapolis, MN) and interferon-gamma (IFN) (Endogen (Cambrige, MA) were determined using ELISA kits. All ELISA results are expressed in PG/ml, and the results of CTLL-2 Biotest as counting pulses per minute, representing IL-2 - dependent cellular inclusion of 3H-thymidine (ICN, Costa Mesa, CA) cells CTLL-2.

The study of direct immunofluorescence (receptors cytokines)

For direct staining with antibodies to ant is olivem solution pH 7.4 (Becton Dickinson, Mansfield, MA) and was resuspendable in 50 ml of isotonic saline and divided into two samples. One aliquot of the sample was Saakashivili or PE-anti D25/FI-anti-CD4 or PE-rat anti-mouse IgG + anti-CDw124/FI-anti-CD4 monoclonal antibodies and nonspecific fluorescence was determined by staining the second aliquot RE/FI-labeled appropriate isotype control monoclonal antibody. All labeled with fluorescent-labeled monoclonal antibodies were obtained from Becton Dickinson (San Jose, CA), in addition to anti-CDwl24, which were obtained from Pharmingen, San Diego, CA. Incubation was carried out at 4oC in the dark for 45 minutes at saturating concentrations of monoclonal antibodies. Without the label was removed by washing PBS before analysis on a FACScan flow cytometer (Becton Dickinson).

The density of the antigen was determined by an indirect method in noise live CD4+T cells and was expressed as the mean channel fluorescence (MCF). Surface expression of specific antigen (Cdwl24, CD25) was expressed as the average shift of the channel (MCS) obtained by subtracting MCF cells stained control FITC - or PE-labeled appropriate isotype (IgG1) monoclonal antibody from MCF cells, ukrasheniya CD4+-subgroups of cells stained CD28 monoclonal antibodies was determined by subtracting MCF cells D28+CD4+of MCF cells CD28-CD4-.

The viability of the control not treated and treated with ribavirin and interferon - cells was determined in each batch of all oligonucleotides many donors by intravital staining dye, propidium the iodide (5 mg/ml final concentration). The percentage of living cells that did not include propidium iodide was determined by flow cytometry and was > 90% (90-99%) after treatment with all concentrations used.

Immunofluorescence analysis of intracellular expression of cytokines

For analysis of intracellular expression of IL-2 in CD4+and CD8+subgroups of T cells T cells were first treated in the last 4 48-72 hours from the time of activation 10 mg Brefeldin A (Gibco BRL, Gaithersburg, MD) to minimize the secretion of newly synthesized IL-2 in the extracellular space. After activation with 900 ml of cell supernatant from each blade were transferred to another microplate for analysis of cytokine production from cells. Before direct staining (30 min, 4oC, in darkness) associated with the or, pH 7.4, and resuspendable in 100-150 ml of staining buffer (phosphate saline buffer, pH 7.4, containing 1% fetal calf serum (FCS) (Hyclone, Logan, UT) and 0.1% of sodium azide) and divided into two samples. Stained cells were washed in 1 ml of staining buffer and the cell sediment resuspendable in 100 ml of fixation buffer (4% paraformaldehyde in PBS), followed by removal of supernatant. Fixed cells kept at 4oC for 20 minutes, then washed in 1 ml of staining buffer and the cell sediment resuspendable by stirring in 50 ml permeabilizing buffer (0.1% saponin (ICN, Costa Mesa, CA) in PBS). Permeabilization cells were stained PE-labeled antibodies to IL-2 for 30 minutes at 4oIn the dark and then were washed in 1 ml permeabilizing buffer, resuspendable in 250 ml of staining buffer before FACS analysis.

Analysis of mRNA of cytokines

Total RNA was extracted from resting T cells and treated with ribavirin and interferon - and untreated activated T cells using commercial varieties of guanidyl thiocyanate/phenol extracting method (Trizol reagent (GIBCO/BRL). RNA was washed with 70% ethanol and finally resuspendable 10 ál Madion, WI). Briefly, 1 µg of total RNA was heated at 65oWith 10 minutes and cooled on ice before mixing with 2 μl of 10 x reverse transcription buffer (100 mm Tris model HC1 (pH 8.8), 500 mm KS1, 1% Triton X-100), 5 mm MgCl, 2 μl 10 mm dNTPs (1 mm each dNTP), and 0.5 μl of RNase inhibitor, 1 ál oligo (dT)15primer (0.5 μg/μg RNA) and 0.65 µl AMV reverse transcriptase (N. With.) The reaction was incubated at 42oC for 1 hour, and then 10 minutes at 95oWith 5 min on ice.

The PCR reaction was performed using the GeneAmp PCR kit (Perkin-Elmer Cetus, Foster City, CA). In a new test tube RT reaction mixture (3 μl) was mixed with 5 μl of 10 fold PCR buffer (500 mm KS1, 100 mm Tris-HCl, pH 8.3, 15 mm MgCl2and 0.01% (wt./about.) gelatin), 1 μl 10 mm dNTPs and 1 U Taq DNA polymerase. We used the following primers: primers interleukin-2, interleukin-4, interferon- (man) (Stratagene, La Jolla, CA) and RNA ribosomal gene. The amplification conditions were 45 s at 94oC, 1 min at 57oC and 2 min at 72oS for 35 cycles, followed by 8 min at 72oC. the PCR Products were analyzed on 2% agarose gel containing ethidium bromide. After electrophoresis the PCR products were transferred to Hybond N+ membrane (Amersham, Arlington Heights, GL) 20 times SSC during the night and immobilizovana using 0.4 M NaOH. The blots is. is each primers mix of cytokines was used as a probe with a radioactive label (instructions). The equivalent load was determined after hybridization with a sample formed RNA sense primer. The washed blots were then analyzed using a Phosphorlmager.

Effect of ribavirin on the extracellular levels of cytokines in activated T cells

Processing PMA/ionomycin (48-72 hours) T cells significantly increases the levels of all analyzed cytokines i.e. IL-2, IL-4, TNF, IFN (table 1) the First number in each cell indicates the arithmetic value, and the numbers in brackets refer to the corresponding ranges. N=4. In the demonstration experiment, shown in Fig. 1, the addition of ribavirin dose in the range of 0.5-5.0 µm activated increases the levels of Th1 cytokines, IL-2 and TNF maximum at 5 μm (30%) and 20 μm (36%), respectively. In contrast, interferon - inhibits the expression of IL-2 and TNF depending on the dose (interval 250-10000 U/ml, maximum inhibition of 33% and 38%, respectively) when compared with levels in untreated activated T cells. In addition, ribavirin mediates the concomitant decline in levels of activated Th2 cytokine, IL-4 (peak inhibition of 74% at 2 microns), while in the interferon Alfa, as shown in Fig.2, a constant concentration of 2000 U/ml interferon alpha inhibits dose-dependent ribavirin increased levels of activated IL-2 (a) and reverses the inhibition levels of activated IL-4 (C). Similarly, the constant value of 10 μm ribavirin draws mediated interferon Alfa dose-dependent inhibition of the levels of activated IL-2 (b) and suppresses the increase in levels of activated IL-4 (D).

Effect of ribavirin on the mRNA levels of cytokines in activated T cells.

These opposite effects of ribavirin and interferon - levels of activated extracellular cytokines was also observed at the level of transcription. In Fig. 3 shows that treatment of PMA/ionomycin T cells significantly increases the mRNA levels of IL-2, IL-4 and IFN. Treatment with ribavirin (2, 5 and 10 μm), following the activation of the T cells, enhances IL-2, reduces IL-4 and does not affect IFN mRNA. In contrast, interferon-, at 1000, 2000 and 5000 U/ml reduces IL-2, increases IL-4 and reduces IFN mRNA. Thus, the corresponding dose-dependent effect of ribavirin and interferon on the expression of mRNA of IL-2, TNF, and IL-4 were consistent with the ELISA assays. These data suggest that ribavirin enhances the synthesis of Th1 cytokines, IL-2 and levels of receptors for IL-2 and IL-4 in activated T cells.

Using FACS analysis, the authors compared the effects of ribavirin and interferon on the expression of IL-2 (CD25) and the expression of IL-4 (CDwl24) receptor in activated T cells. Processing PMA/ionomycin increases the expression of CD25 and CDwl24 from the level of the rest 50,160,45 and 62,311,46 activated to levels 162,482,89 and 87,533,98 respectively (n=4). In the present three experiments in Fig.4 it is shown that ribavirin (1-50 μm) has little effect on the activated levels of IL-2 and IL-4 receptors, whereas interferon-, dose interval 250-10000 U/ml, reduces the expression of IL-2 receptor and increases the expression of GL-4 receptor, depending on the dose when compared with the levels of the receptor in the control of activated T cells. Thus, these data show that the effect of ribavirin on the synthesis of the cytokine exists independently of the expression of the receptor of the cytokine. On the contrary, the treatment effect of interferon - a on IL-2 and IL-4 receptor correlates with that observed in its effect on the expression of activated IL-2 and IL-4.

Effect of ribavirin on the intracellular levels of IL-2 in CD4 and CD8+subgroups of activated T cells.

The authors investigated whether the effect of ribavirin on the expression of IL-2-specific CD4-or CD8+T cell. Expressly two-color flow fluorimetry using antibodies to CD4 or CD8 and IL-2, labeled with a fluorescent label. In Fig. 5 shows that after treatment with ribavirin at 10 μm, the percentage of CD4+T cells expressing IL-2, increasing from 82 to 91% and the percentage of CD8+expressing IL-2, increases from 81 to 91%. In contrast, the percentage expressing IL-2 CD4+and CD8+cells after treatment with interferon - a (5000 IU/ml) was 81% and 71% respectively. These data suggest that ribavirin effect on the expression of intracellular IL-2, which do not differ between CD4+and CD8+subgroups of T cells. In contrast, treatment with interferon - has little effect on CD4+T cells and even reduces the expression of IL-2 in CD8+the subset of T cells.

Analogues of ribavirin.

There are several classes of ribavirin analogs, which are considered to be clinically effective to modulate Th1 or Th2 dysbalance-mediated responses of T cells. They include molecules conforming to General formula of Fig.6, where X represents O, S, CH2SNON or N-CO-R11; A, b and C are independently N, P, SN,-HE,-CH3, S-alkyl,-alkenyl, C-CH2, -CN,-halogen,-CN,-SOON3C-NH2C-SNH2C-SO2-NH2C-CONH2C-CS-NH2C-C(NH)NH2SRO2-NH2or S-heterocyclic number(CH2)pONH2, (CH2)pNH2CH3CH2SPH or (CH2)-heterocyclic ring; R2represents H, HE, co3, SH, SCH3, halogen, CN, NH2, ONH2, NH3, (CH2HE, (CH2)pNH2CH3, Sooma; R3, R4, R5, R6, R7and R8are independently H, HE, co3, SH, SCH, halogen, CN, NH2, ONH2, NHCH3, (CH2HE, (CH2)pNH2CH3, Sooma or phenyl; R9represents H, halogen, NH2CH3, CONH, CSNH2, Sooma, SNH2, SONH2, RHO2NH2, (CH2)R, (CH2)R-heterocyclic ring or (CH2)p-glucose; R10represents H, halogen, NH2CH3, CONH, CSNH2, Sooma, SNH2, SO2NH2, RHO2NH2, (CH2)R, (CH2)R-heterocyclic ring,

(CH2)p-glucose, O-CH3, O-CH2CH3or amino; Y represents O, S, NHHC1, NOH, NOCH3or NOCH2PH; R10& Y in combination are heterocyclic systems, such as thiazole, imidazole, etc.; R11represents CH3(CH2)pNH2, (CH2)R-heterocycle, (CH2)R-amino acid or (CH2) is Sidi.

Such molecules can be created in accordance with one or more than one of the following exemplary schemes.

1. Synthesis ICN1369 (Parasailin): methods of synthesis for this compound are described in the following references: (a) J. Farkas, Z. Flegelova and F. Sorm, Tetrahedron Letts, 22, 2279 (1972); (b)S. De Bernardo and M. Weigele, J. Org. Chem. , 41, 287 (1976); ((C) J. G. Buchanan, A. Stobie and R. H, Wightrnan, J. Chem. Soc. Chem Commun, 916 (1980); N. Karagiri, K. Takashima, T. Haneda and T. Kato, J. Chem. Soc. Perkin Trans., 553 (1984).

2. Synthesis ICN3438 (1--D-xylofuranosyl-1,2,4-triazole-3-carboxamide): the synthesis of this compound performed by following the procedure described in J. T. Witkowski, M. Fuertes, P. D. Cook and R. K. Robins. J. Carbohydr. Nucleosides, Nucleotides 2(1), 1 (1975).

3. Synthesis ICN3844 (1 (5-O-sulfamoyl--D-ribofuranosyl)-1,2,4-triazole-3-carboxamide): This compound was prepared by following the procedure described in G. D. Kini, B. M. Henry, R. K. Robins, S. B. Larson, J. J Marr, R. L. Berens, C. J. Bacchi, H. C. Nathan and S. Keithly, J. Med. Chem., 33, 44 (1990)/

4. Synthesis ICN4625 (1 (3-deoxy-D-erythropoiesis)-1,2,4-triazole-3-carboxamide): This compound is prepared using synthesis method shown in scheme 1 (see the end of the description).

See: J/Med. Chem., 8, 659 (1965)

5. Synthesis ICN5531 (5-amino-1--D-ribofuranosylthiazole-4-carboxamide): to obtain the specified connection has followed the methodology of synthesis reported in B. K. Bha): the synthesis of this compound was carried out, following the procedure described in J. T. Witkowski, M. Fuefles, P. D. Cook and R. K. Robins. J. Carbohydr. Nucleosides, Nucleotides, 2(1), 1 (1975).

7. Synthesis ICN5839 (1--D-erythropoiesis-1,2,4-3-carboxamide): this compound was prepared using the sequence of the synthesis shown in scheme 2 (see the end of the description) 8. Synthesis ICN6242 (2-bromo-1--D-ribofuranosylthiazole-4 carboxamide): ICN6242 was prepared according to the method shown in scheme 3 (see end of description).

Cm. : R. C. Srivastava, G. A. Ivanovics, R. J. Rousseau and R. K. Robins, J. Org. Chem., 40, 2920 (1975).

9. Synthesis ICN11808 (1--D-ribofuranosyl-pyrazole-3,4-dicarboxamide): synthesis of this compound is described in Y. S. Sanghvi, V. K. Bhattacharaya, G. D. Kini, S. S. Matsumoto, S. B. Larson, W. J. Jolley, R. K. Robins and G. R. Revankar, J. Med. Chem., 33, 336 (1990), and the compound was prepared in accordance with this.

10. Synthesis ICN 12204 (2--D-ribofuranosyl)imidazol-5-carboxamide): the synthesis of this compound was carried out by following the procedure described in J. Igoleo, T. N. Dinh, A. Keib and S. Perreur, Chimie Therapeutique, 207 (1972).

For carbocyclic derivatives of 4'-tishara and 4'-asakhara ribavirin analogues following sugar could be condensed with the appropriate heterocyclic compounds and derivatives obtained as described in the above schemes, and links.

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2. Specified 4'-atasehir was obtained as described in the literature procedure: E. J. Reist, D. E. Gueffroy and L. Goodman, J. Am. Chem. Soc., 87, 677 (1965).

3. Specified 4'-thio-D-ribofuranose was obtained by following the literature procedure: M Hobek and R. L. Whistler in "Methods in Carbohydrate Chemistry", Vol. 1.292 (1962).

Proof of the effectiveness of ribavirin analogs in the modulation of Th1 and Th2 cytokines set forth in Fig. 7 and in table 2. The tests shown here, include a short list of 35 analogues analogues tested ribavirin and all tests were performed three times. In General, it can be seen that the tested compounds are divided into three main categories: (I) compounds such as ICN1369 and ICN3844 that suppress IFN, IL-2, IL-4, IL-S and TNF; (2) compounds, such as ribavirin, which increases IL-2 and TNF and inhibits ILA and IL-5; and (3) compounds such as ICN6242, ICN3839 and ICN3531 that increase IL-2 and IFN inhibit IL-4 and IL-5. Such activity listed above, would be potentially suitable for determining in which modulation of cytokines would be appropriate immune response.

Thus, it was shown that ribavirin and ribavirin analogs effect is osenia and applications were given, it should be obvious for the skilled professionals that many more modifications are possible without separation from the one shown here concepts of the invention. The invention, therefore, should not be limited only by the nature of the attached claims.

1. A method of modulating the expression of Th1/Th2 lymphokines in cells through the use of ribavirin, characterized in that these cells are T cells of the patient man, and ribavirin add to these cells in the dosage, which stimulates the expression of at least one Th1 lymphokines TNF, IL-2 and IFN and suppresses the expression of Th2 lymphokine IL-4.

2. The method according to p. 1, characterized in that the patient has the disease, which includes allergies.

3. The method according to p. 1, characterized in that the patient has the disease, which includes autoimmune disease.

4. The method according to p. 1, characterized in that the patient has the disease, which includes helminth disease.

5. The method according to p. 1, characterized in that the patient has the disease, which includes primary immunodeficiency.

6. The method according to p. 1, wherein the patient-ChELOVEKA fact, the patient has the disease, which includes human immunodeficiency virus.

8. The method according to p. 1, characterized in that the patient has the disease, which includes hepatitis C.

Priority points:

23.01.1996 - PP.1-7;

14.01.1997 - p. 8.

 

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