Patent Application
20100144642
The present invention is related with peptides of human heat shock protein of 60 kDa (abbreviated hHsp60), and Altered Peptide Ligands (abbreviated APL) derived from them. Also, the invention is related with the pharmaceutical compositions comprising such peptides for the treatment of Rheumatoid Arthritis (RA).
The RA is an autoimmune disease of unknown etiology that affects approximately 1% of world's population. It is a syndrome characterized by chronic inflammation of the joints, although systemic manifestations can be also observed. This illness begins with the inflammation of the synovial membrane and frequently causes the erosive destruction of the adjacent cartilage and the bone, which result in moderate physical inability of 80% of the patients and an early mortality (Moctezuma, J. F. (2002) Manifestaciones articulares de la Artritis Reumatide. Revista Mexicana de Reumatologia 17:211-219). RA can be presented at any age, without distinctions of races or ethnic groups, but the maximum incidence of its beginning happens between 25 and 55 years old. Among people with RA, the women overcome the men in a proportion from three to one (Emery, P. (2002) Early referral recommendation for newly diagnosed rheumatoid arthritis: evidence based development of to clinical guide. Ann Rheum Dis. 61:290-297).
The cause of RA is unknown. It is an illness that involves the presence of genetic, environmental, immunological and hormonal factors. Certain genes have a role in the immune system, associated with a tendency to develop RA. At the same time, some people with RA don't have these genes and other people having them never develop the illness. Therefore, it has been suggested that the genetic background is important but it is not decisive.
In models of autoimmune disease, microbial antigens with similar structure to own antigens can release a crossed response to autoantigens, producing an alteration in the mechanisms of tolerance and perpetuating an autoimmune response. In general sense, the damage and local necrosis in a tissue produced by an infectious agent could discover the autoantigen cryptic epitope, being able to activate autoreactivate T cells (Albert, L. J. (1999) Mechanisms of Disease: Molecular Mimicry and autoimmunity. N Engl J Med 341:2068-2074)
The phase of transition of T lymphocyte between tolerance and immunity/autoimmunity is regulated at different levels. Two important parameters in this transition are the state of maturation of the Antigen Presenting Cells (abbreviated APC) and the levels of autoantigens that are detected by the immune system (Ohashi, P. S. (2002) Making and breaking tolerance. Current Opinion in Immunology 14:744-759).
One of the current hypotheses that tries to explain the development of autoimmune diseases, outlines that APC in absence of signs of the innate immune system or of signs of danger, remain relatively immature and induce tolerance in autoreactive T cells, when own peptides are presented to them (Steiman, R. M. (2000) The induction of tolerance by dendritic cells that have captured apoptotic cells. J Exp Med. 191:411-416). The induction of peripheral tolerance is also correlated with the concentration of own antigen (Kurts, C. (1999) CD8 T cell ignorance or tolerance to islet antigens depends on antigen dose. PNAS 96:12703-12707). An increment in the presentation of own antigens due to the increment of their expression levels, allows their detection by autoreactive ignorant T cells. If levels of own antigens are increased in absence of events that promote the maturation of APC, the tolerance to these antigens is maintained, otherwise, it happens in presence of proinflammatory signs or other events that promote the maturation of the APC, the tolerance is breaks by activation of the ignorant T cells and autoimmune diseases are developed (Janeway, C. A. (2002) Innate immune recognition. Annu Rev Immunol. 20:197-216). The infectious agents that have been object of study like cause of RA are: Epstein Bar virus, the retrovirus, virus of the hepatitis C, the Mycobacterium tuberculosis (abbreviated MO and the Helicobacter pylori, among others.
The pathogenesis of RA is characterized by the concerted action of different types of cells that cause the progressive destruction of the cartilage and the bone.
In normal situations a balance exists among the inflammatory cytokine as: TNFα, IL-1, IL-6, IL-15, IL-16, IL-17, IL-18 and the IFN-γ, and the anti-inflammatory ones as IL-4, IL-11, IL-13 and antagonistic of IL-1 or TNFα. In the RA this balance moves however in favor of the inflammatory cytokines (Arend, W. P. (2001) Cytokine imbalance in the pathogenesis of rheumatoid arthritis: The role of interleukin-1 receiving antagonist. Semin Arthritis Rheum 30(2):1-6)
The recognition of an exogenous antigen or autoantigen is probably the reason of a series of events that cause destruction of the joints in patients with RA. This phenomenon cause the activation of T CD4+ lymphocytes that in cooperation with the stimulation of different cytokines, induces their differentiation to cells Th1 with the consequent liberation of proinflammatory cytokines (IL-2 and INFγ) (Simón, A. J. (2001) Biological therapy in Rheumatoid Arthritis. Magazine of Clinical Investigation 53(5):452-459). Many investigators coincide that the chronic inflammation of the joints is induced by these activated T cells that infiltrate the synovial membrane. The action of these cytokines on macrophages causes the production of high amount of TNFα and IL-1. These cause a series of local and systemic actions as regulating the expression of the molecules of adhesion in the endothelial cells (LFA1, ICAM-1), which recruit other cells to the inflammation sites. They also stimulate macrophages, fibroblasts, condrocytes and osteoclasts to the liberation of others mediators of the inflammation, as IL-15 and IL-8. TNFα and IL-1 stimulate the proliferation of the synovial membrane that causes formation of pannus, they can also induce the differentiation of B lymphocytes to cells producing antibodies that potentially participate in the destruction of the joint. They also inhibit the production of anti-inflammatory cytokines (IL-4 and IL-14) produced by Th2 cells and stimulate hepatocytes to liberate IL-6. IL-6 favors the production of the proteins of the acute phase, which participate strengthening the immune response (Forre, O. (2000) New possibilities of treatment in AR. Scand J Rheumatol 29(2):73-84).
Among autoantigens involved in the pathogenesis of RA is Hsp60, a protein that belongs to the family of the Hsp, which are immunogenic proteins with exceptionally evolutionary conservation. The immune response against the strange Hsp is an important mechanism of defense against bacterial infections. The antibodies against these proteins can be abundant in healthy people and in patient with autoimmune illnesses and they can cross react with the own antigens (Chen, W. (1999) Human 60-kDa Heat-Shock Protein: To Danger Signal to the Innate Immune System. J Immunol. 162:3212-3219).
The Hsp65 of Mt is homologous to the Hsp60 of the mammals. This suggests that the Hsp60 can be recognized as autoantigen in patient with RA. Comparing patients with osteoarthritis and patients with RA, these last ones have an increase of prolipherative response of B lymphocytes in the synovial liquid to the Hsp65 of mycobacterium. The intensity of the response correlates with the synovial inflammation. This response is not specific for RA compared with other inflammatory illnesses (Life, P. (1993) Responses to Gram negative enteric bacterial antigens by synovial T cells from patiens with juvenile chronic arthritis: recognition of heat shock protein hsp60. J Rheumatol. 20:1388-1396).
The concentration of Hsp is a possible sign of danger for the immune system, which are released from dead cells, and can induce an inflammatory response and to begin the maturation of the APC. The Hsp, are intracellular proteins, which are not expressed in the cellular membrane, neither are secreted, so that the Hsp are attractive candidates for molecules that constitute signs of danger (Van den Berg, W B. (1998): Joint inflammation and cartilage destruction may occur uncoupled. Springer Semin Immunopathol. 20:149-164).
Several preparations have been proposed using the Hsp60 or its derived peptides for the treatment of some autoimmune pathologies. For example in the patent EPO262710, the use of several peptides of the Hsp60 of Mt is proposed for the treatment and diagnosis of autoimmune illnesses, especially of arthritic conditions. This invention is based on the fact that previous infections with several bacteria can trigger the development of autoimmune illnesses, in genetically susceptible people, for example: patients with RA can show a high reactivity to microbial antigens.
These same inventors in the patent EPO322990 propose the use of others peptides of the Hsp60 of Mt with the same purpose of the previous patent.
In the patent application WO9610039 they intend the use of a peptide of the Hsp60 of Mt for the diagnosis and treatment of autoimmune arthritis.
In the patent application WO9711966, the authors intend the use of peptides of non conserved regions of the hHsp60, that don't coincide with the Hsp60 of bacteria that could induce tolerance in T cells of patients with RA.
The patent application WO0143691 proposes the use of pharmaceutical preparations composed specifically by peptides of the hHsp60 and their variants for the prevention of inflammatory illnesses such as RA.
In the U.S. Pat. No. 6,180,103 the authors intend the use of a peptide of the Hsp60 called p277 and its analogous for the diagnosis and prevention of the diabetes type I.
The U.S. Pat. No. 5,993,803 protects the use of the Hsp60, the peptide p277 and a group of derived peptides of this protein to reduce the severity of the immune response during the transplant of organs.
Recently it has been considered that the atherosclerosis presents a series of similar characteristics to autoimmune processes. In the patent application WO0072023 the authors purpose a method for the treatment and diagnosis of the atherosclerosis and coronary illnesses using a preparation that contains the protein Hsp60.
In the patent application WO02057795, a new method is protected for the diagnosis and treatment of the osteoporosis using proteins of the family of Hsp and proteins from pathogens such as: viruses and bacteria.
At the present time, cure doesn't exist for the RA. The current methods of treatment are centered in alleviating the pain, to reduce the inflammation, to retard the damages of the joints and to improve the functions and the well-being of the patients. Recently immunomodulating medications have been elaborated, that blocks cytokines that participate in the beginning and maintenance of the inflammatory response in RA, with the purpose to stop or slow the progression of the illness. For this kind of therapy two types of medications exist: blocking the action of the Tumor Necrosis Factor (abbreviated TNFα) and those that inhibit the action of the interleukin 1 (IL-1).
Although the results are promissory with the therapies anti-TNFα and anti-IL-1, the percentage of infections is high. Many of treated patients with these drugs develop serious infections that are fatal in some cases; including other autoimmune illnesses, neoplasias, etc. Besides, they are very expensive medications (Breshinan, B. (1998) Treatment of rheumatoid arthritis with recombinant human anti-interleukin-1 antagonist. Arthritis Reum. 41:2196-2204)
Oral tolerance has been proposed as a method of creating peripheral tolerance in front of certain antigens. This can be induced by mechanisms of active suppression, anergy or clonal deletion, depending on the doses and the frequency of the administration of the antigen. The method can induce regulatory T cells that are activated in a specific way by the antigen, but exert its action independently (active suppression). For the regulatory action takes place, it is not necessary to administer the supposedly pathogenic antigen, but any other able to induce the active suppression in the inflammatory focus, inhibiting the activity of the pathogenic effector cells. Collagen type II (abbreviated CII) is the autoantigen that has been used more frequently in this sense. The results of the studies carried out in patients with RA, using chicken and bovine CII have given contradictory results (Trentham, D. E. (1993) Effects of oral administration of type II collagen in rheumatoid arthritis. Science 261:1727-1730; Sieper, J. (1996) Oral type II collagen treatment in early rheumatoid arthritis: to double-blind, placebo-controlled, randomize trial. Arthritis Rheum. 39:41-51).
The U.S. Pat. No. 6,153,200 intends the use of a peptide of the Hsp70, protein belonging to the family of Hsp, to induce tolerance for oral route in patients with RA.
Another variant that has been suggested to induce tolerance is through APL peptides, based on the fact that T cells are activated if the specific T CD4+ lymphocytes for a certain antigenic peptide recognize the antigen presented by competent APC. Nevertheless, if the same T cell is first activated with a different form from the antigen, in which one of the contact sites with the TcR are lightly altered, it can result in a partial activation or even inactivation of the T cells. These antigens are named APLs. The APLs are similar to immunogenic peptides with one or several substitutions in the essential positions of contact with the TcR or with MHC that interfere the cascade of necessary events for the complete activation of the T cells.
Conceptually, APLs can be designed with similar properties to the immunogenic peptide (agonist) among other effects for increasing the response of T cells toward specific antigens. This effect is advantageous under pathological conditions as infectious illnesses. Peptides can also be designed with antagonistic properties to the immunogenic peptide that could be beneficial in the control of autoimmune illnesses since they can block the response of T cells acting as antagonistic of the TcR (M. De Magistris (1992) Antigen analog-major complex histocompatibility complexes act as antagonist of the T cell receptor. Cell 68:625-634), partial agonist or inducing a population of regulatory T cells that mediate the active suppression (Evavold, B. D. (1991) Separation of IL-4 production from Th cell proliferation by an altered T cell ligand. Science 252:1308-1310). The capacity to experimentally manipulate the intrinsic properties of peptide ligands allows altering the nature, the course and the power of the immune cellular response appropriately.
Up to this moment, two clinical trials in humans using type APL peptides have been performed for the treatment of autoimmune diseases. In both assays, the peptides are derived from an epitope in position 83 to 93 of the myelin basic protein. One of these trials included 142 patients with multiple sclerosis, and it was suspended because 9% of the patients developed hypersensitivity (Ludwig Kapposi (2000)
Induction of non-encephalitogenic type 2 T helper-cell autoimmune response in multiple sclerosis after administration of an altered peptide ligand in to placebo controlled, randomized phase II trial. Nature Medicine 10:1176-1182).
The other trial included 25 patients and was also interrupted, because in three patients was observed an exacerbation of the illness (Bibiana Bielekova (2000) Encephalitogenic potential of the myelin basic protein peptide (amino acids 83-99) in multiple sclerosis: Results of to phase II clinical trial with an altered peptide ligand. Nature Medicine 6:1167-1175). The main author of this work analyzes the possible factors that determined these negative results, and outlines that the changes carried out in the APL's sites may originate new motifs of union to the HLA (like it happened in the case of the APL binding to DRB1*0404 present in patient I), the complex APL-HLA can also stimulate T cells not eliminated in the negative selection that can be cross-activated by the native autoantigen. In this trial high concentrations of the APL were used in the pharmaceutical preparations, which can stimulate T cells with high similarity for the autoantigen and to induce a heteroclitic T cells response. (Bibiana Bielekova and Roland Martin (2001) Antigen-specific immunomodulation via altered peptide tying. J Mol Med. 79:552-556).
For carrying out these two clinical trials, the authors didn't previously analyze the in vitro response of the T cells from the patients to the APL. They also did not consider the types of HLA molecules expressed by the treated patients
The main challenge in the treatment of the autoimmune diseases is the development of therapeutic strategies that can eliminate pathogenic T cells with specificity, without affecting other non related T cells. Therefore, therapeutic search should find a secure, specific and effective way of turning off an advanced autoimmune process.
The present invention, in contrast to the state of the previous technique, proposes the use of peptides of the hHsp60 and its derived type APL, which induce inhibitory molecular mechanisms, in a specific way, of the course of RA.
The present invention solves the problem previously mentioned, providing peptides of the human protein of heat shock of 60 kDa that constitute epitopes for T cells, to induce mechanisms of tolerance, in particular anergy induction mechanisms or mediated by clones of regulatory T cells in patients with RA and that present the sequences:
The patent application WO9711966, intends the use of peptides of the hHsp60 and of Mt based on the fact that they could induce tolerance in T cells and to prevent the RA. These authors corroborate certain improvement of the illness by administering these peptides to mice (Arthritis was previously induced with pristine). Contrary to this application, we have demonstrated that our peptides induce mechanisms of active suppression which induce peripheral tolerance in a very efficient way, corroborating a significant increase of IL-10 in two animal models of Arthritis, as well as in “ex vivo” assays using mononuclear cells of patients.
It is well known that fundamental response of T cells in the model of adjuvant-induced arthritis (AIA) is directed against the Hsp60. We checked in this model that our derived peptides of the hHsp60 exert a very marked therapeutic protection, producing an increase of the IL-10 and a decrease of TNFα levels. The same therapeutic effect exerted by these peptides, we have also checked in the animal model of collagen-induced arthritis (CIA), where the major response is against the type II collagen.
These facts indicate that therapeutic capacities of our peptides are independent of the induction agent and can mediate mechanisms of active suppression in the site of inflammation that could be extended to other autoantigens present in joints, which progressively contribute to the immunopathogenic process that takes place during the course of RA. These results reinforce the therapeutic possibilities of the use of our preparations for the treatment of RA.
These peptides are in particular useful, according to the present invention, for the design of derived type APL variants of the peptides E18-12 (SEQ. ID. NO: 1) and E18-3 (SEQ. ID. NO: 2), modified in the contact sites with the human MHC molecule and a derived type APL of the peptide F19-6 (SEQ. ID. NO: 3), modified in the contact sites with the rat MHC molecule. The respective sequences of amino acids are:
it is substituted in the position 1 for: A, F, I, L, M, V, W, or Y
position 2 for: A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y
position 3 for: A, K, V, R, T, I, P, L, N, S, G, Y, or M
position 4 for: A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y
position 5 for: A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, or Y
position 6 for: A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y
position 7 for: A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, or Y
it is substituted in the position 1 for: L, I, V, M, Y, W, F, or A
position 2 for: A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y
position 3 for: A, F, I, K, L, M, P, R, S, T, V, W, or Y
position 4 for: L, I, V, M, Y, W, F, or A
position 5 for: A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y
position 6 for: A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y
position 7 for: A, F, I, K, L, M, P, R, S, T, V, W, or Y
position 8 for: A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y
position 1 is substituted by H.
position 2 is substituted by E.
In a particular carrying out, derived type APL peptides have amino acid sequence selected from the group that consists of SEQ. ID. NO: 4, SEQ. ID. NO: 5, SEQ. ID. NO: 6, SEQ. ID. NO: 7, SEQ. ID. NO: 8, SEQ. ID. NO: 9, SEQ. ID. NO: 10, SEQ. ID. NO: 11, SEQ. ID. NO: 12, SEQ. ID. NO: 13, SEQ. ID. NO: 14, SEQ. ID. NO: 15, SEQ. ID. NO: 16, SEQ. ID. NO: 17, SEQ. ID. NO: 18, SEQ. ID. NO: 19, SEQ. ID. NO: 20 y SEQ. ID. NO: 21.
The peptides of the present invention can be produced by routine methods of peptide synthesis. The specific composition of each peptide can be assayed by the level and the quality of the immune response that it induces in experiments as those are described in the examples that will be presented later on.
All the sequences described above and in the examples are useful and can also be used as a base for the design and synthesis of derived peptides with improved properties.
The invention also includes pharmaceutical compositions that include one or more of previously enumerated peptides and optionally a pharmaceutically acceptable vehicle.
The administration of the pharmaceutical preparations to the patients will be in correspondence with the HLA class II molecules that are expressed by the specific patient.
The way of administration of the pharmaceutical compositions will depend on the cytokines pattern that these preparations induce in the “ex vivo” tests. The pharmaceutical preparations containing APL peptides inducing a regulatory response will be administered by intradermal or subcutaneous route. The pharmaceutical preparations containing the original peptides inducing a TH1 response will be able to be administered by oral route.
The amounts of peptides in the pharmaceutical compositions of this invention are those that produce an effective immune response in the host. The effective amount is the quantity that when is administered induces molecular mechanisms that significantly diminish the inflammatory signs characteristic of the RA and stop the damages in the joints characteristic of the course of this illness. The amount of the pharmaceutical composition administered to the host can also vary depending on of several factors that include: the used peptides, ACR score (American College of Rheumatology, abbreviated ACR), HLA type II, time of having diagnosed the illness, age, sex, general health, as well as the level of immunological response in general.
The present invention also relates a method of treatment of the RA, which includes the administration to a patient of effective amounts of peptides or the pharmaceutical compositions referred previously.
In part, results are shown graphically in the following figures:
The selected peptides of the hHsp60 were those reported as epitopes of human T cells by the used programs: SYFPEITHI (Hans-Georg Rammensee, Jutta Bachmann, Niels Nikolaus Emmerich, Oskar Alexander Bachor and Stefan Stevanovic. 1999. Immunogenetics 50:213-219. SYFPEITHI: database for MHC ligands and peptide motifs. (access via: http://www.uni-tuebingen.de/uni/kxi/)) and ProPred (Singh, H. and Raghava, G. P. S. 2001. ProPred: Prediction of HLA-DR binding sites. Bioinformatics, 17(12):1236-37).
The sequences corresponding to these peptides are shown in Table 1.
Starting from the sequence of hHsp60 we select a peptide that binds to the molecule of MHC-II of rat RT1.BI according to the sequence motif described in the database SYFPEITHI (Hans-Georg Rammensee, Jutta Bachmann, Niels Nikolaus Emmerich, Oskar Alexander Bachor, Stefan Stevanovic. 1999. Immunogenetics 50:213-219. SYFPEITHI: database for MHC ligands and peptide motifs. (access via: http://www.uni-tuebingen.de/uni/kxi/)).
A program that analyzes all peptides of 9 amino acids was designed to search those that fulfill with the motif. We also included variants, which in one of the positions were not included the described residuals as anchorage or preferentials.
The sequence corresponding to one of these peptides (F19-6) it is shown in the Table 2.
Starting from the peptides selected in examples 1 and 2, we have modified the positions involved in the binding to the specific MHC (positions 1, 3, 4, 6, 8 or 9) trying to increase the affinity of the peptide for the HLA molecule. It was searched for the optimal residuals in each one of these positions that could be anchorage residuals if the peptide was selected for methods that use binding motifs (SYFPEITHI) or residuals able to increase the score of the peptide as MHC ligand if the selection of the peptide was made using another algorithm type (ProPred).
As a result of these analyses, the peptide E18-12 has been substituted in the positions 1 to 7 by each one of the amino acids that are specified as follow:
it is substituted in the position 1 for: A, F, I, L, M, V, W, or Y
position 2 for: A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y
position 3 for: A, K, V, R, T, I, P, L, N, S, G, Y, or M
position 4 for: A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y
position 5 for: A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, or Y
position 6 for: A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y
position 7 for: A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, or Y
it is substituted in the position 1 for L, I, V, M, Y, W, F, or A
position 2 for A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y
position 3 for A, F, I, K, L, M, P, R, S, T, V, W, or Y
position 4 for L, I, V, M, Y, W, F, or A
position 5 for A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y
position 6 for A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y
position 7 for A, F, I, K, L, M, P, R, S, T, V, W, or Y
position 8 for A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y
Peptide F19-6 has been substituted in the positions 1 and 2 by the amino acids that are specified as follow:
position 1 is substituted by H.
position 2 is substituted by E.
Female inbred Lewis rats (RT1.BI MHC) were used in the experiment supplied by the National Center for the Production of Laboratory Animals (CENPALAB, Cuba). The rats were randomly selected, with an approximate weight of 101-120 g and 5-8 weeks of age.
One of the inductor agents of the illness used was the Mt (H37Ra, Difco, England) in Incomplete Freund's Adjuvant (IFA) (Sigma, USA). The other inductor agent of the Arthritis was the bovine type II collagen in IFA.
In this example, two different induction agents were used for developing arthritis in rats by an immune response against two different autoantigens. It is very well known that in the animal model for Arthritis using Mt as inductor of the disease, the fundamental response of the T-cells is against the Hsp60 (Anderton, S M (1995) Activation of T cells recognizing self 60-kD heat shock protein can protect against experimental arthritis. J. Exp. Med. 181: 943-952). In the model using collagen as inductor, noticeable antibody response as well as T-cell clones are developed against this antigen (M Griffiths (1988) Immunogenetics of collagen-induced arthritis in rats. Inten. Rev. Immunology 4: 1-15).
Our fundamental objective for developing these two Animal Models was to evaluate our peptides in both models, with the purpose of demonstrating if the protective effect of these peptides was independent or not to the autoantigen used as induction agent of the Arthritis in rats.
The inflammation grade associated with the development of the arthritis that presented each rat was evaluated according with the following average prepared for this purpose:
0 point: normal paw.
1 point: Slight, but definitive redness of the ankle or doll, apparent redness and inflammation limited to an individual finger, independently of the number of affected fingers.
2 points: Moderated redness and inflammation of the ankle and the doll.
3 points: Severe redness and inflammation of the whole paw including the fingers.
4 points: Maximum inflammation and deformity of the paw involving multiple articulations.
The four paws were evaluated separately according to the established score, each paw could receive up to 4 points. Therefore, each rat could receive a maximum of 16 points.
The visual evaluation was carried out after the induction of the illness every five days.
The clinical evaluation of the signs of the Arthritis demonstrated that there were significant statistical differences among animals corresponding to the healthy control group (group IV), where the illness was not induced, and animals corresponding to the sick control group (group III), where illness was induced but they were not treated with peptides, like it is shown in the
The peptides were synthesized by the strategy Fmoc/tBu in syringes. The resin used was Fmoc-AM-MBHA (0.54 mmol/g) and the synthesis protocol was continued with mechanical agitation. After the treatment with TFA, the peptide was lyophilized and it was characterized by HPLC and mass spectrometry.
The animals were separated in four groups of 12 rats each one. The illness was induced in three groups of animals (Group I-Group III), and two groups were treated with peptides and one was the control of the induction of the illness. The two groups of animals that were treated with peptides received 200 μg of peptide per each rat in a volume of 100 μL of PBS, the days 12, 17 and 22 after the induction of the disease with Mt.
The animals separated in four groups were treated in the following way:
Days 15, 21, 28 and 35 after the induction, three animals were sacrificed by group, spleens were extracted and sample was taken from joints for histopathological analysis
As it is shown in the
In this figure it is evident that the administration of peptide F19-6, corresponding to the original epitope of the hHsp60, and their variant APL F19-7, induced a significant reduction of the clinical signs of the Arthritis in the animals treated with both peptides. It was obtained statistically significant differences among the groups treated with the peptides F19-6 and F19-7 and the group taken as positive control of the illness (P<0.05).
The results obtained in this assay corroborate that peptides F19-6 and F19-7 exert a protective effect in treated animals, since it was possible to observe an almost complete remission of the illness in the rats.
The mononuclear cells isolated from spleens of rats, were incubated approximately during 20 hours. Later, supernatants were discarded and 1 mL of the reagent: TRI REAGENT™ (SIGMA, USA) was added to each well and it was left during 5 minutes at room temperature. Later on, the isolation of the mRNA was carrying out following the protocol recommended by the suppliers.
For obtaining the complementary DNA the system Gene Amp RNA PCR Kit (Perkin-Elmmer, USA) was used. 1 mg of mRNA corresponding to each sample and the protocol was used and the protocol was continued as recommended the suppliers.
The reaction was incubated in a thermocycler (Minicycler, MJ Research, USA), according to the following outline: a first cycle of 1 hour at 42° C., subsequently 5 minutes at 95° C. and 5 minutes at 4° C.
The Polymerase Chain Reaction (PCR) was carried out for the cytokines: TNFα and IL-10 and the constituent gene that codes for the enzyme GAP-DH (glyceraldehyde-3-phosphate dehydrogenase, abbreviated GAP-DH).
For each reaction 10 pmol of each primer was used, 5 mL of the DNA mold, 0.25 mL of the DNA Thermostable Polymerase (Taq-Pol) (Perkin-Elmmer, USA) in the buffer recommended by the suppliers (KCl 50 Mm, Tris-HCl 10 mM, pH 9.0, Triton X-100 to 0.1% and MgCl2 1.5 mm) in a final volume of 25 μL.
The reaction for the GAP-DH was incubated in a thermocycler (Minicycler, MJ Research, U.S.) according to the following program: a first cycle of 3 minutes at 94° C. (denaturating temperature), 1 minute at 94° C., 1 minute at 58° C. (hybridization temperature) and 1 minute at 72° C. (elongation temperature), subsequently 35 cycles of: 1 minute at 94° C., 1 minute at 58° C. and 1 minute at 72° C. in each one. Lastly an extension step of 3 minutes at 72° C.
For the cytokines, the previous outline was continued, varying only the hybridization temperature. For the IL-10, 54° C. and for the TNFα 64° C.
The result of the PCR was analyzed by electrophoresis in agarose gel. The images of the electrophoretical running were captured and analyzed with the Molecular Program Analyst (BioRad). The results were expressed as relative levels of mRNA for each cytokine.
To determine the differences in the pattern of cytokines (according to the relative levels of mRNA for each one) among groups of treated animals, the tests of Kruskal-Wallis and Student-Newman-Keuls were used. (Statistical package Sigma Stat v 2 1997, GraphPad Software, Inc).
The statistical analyses demonstrated that in the day 15 after the induction of the disease, the peptides significantly diminish the levels of TNFα in comparison with the control group of the illness.
These results are shown in the
With the purpose of determining if the peptides used in this study, could stimulate the production of anti-inflammatory cytokines as the IL-10, the relative levels of mRNA corresponding to this cytokine were evaluated.
The IL-10 is involved in the induction of peripheral tolerance through a T cell mediated response. The IL-10 regulates directly the T cells to inhibit the production of IL-2, IL-5 and TNFα.
The
The treatment of the animals with these peptides induces an increment in the relative expression levels of the IL-10. We did not obtain statistically significant differences between groups treated with both peptides, but differences exist between these groups and animals that constitute positive and negative controls. Starting from these results we can infer that the peptides act inducing a change in the functional phenotype of the T cells involved in the pathogenesis of the illness, increasing the levels of production of anti-inflammatory cytokines like the case of IL-10 and diminishing the levels of production of the pro-inflammatory cytokines like TNFα.
With the objective to corroborate the clinical signs observed in the animals of this animal model and to analyze the capacity of both peptides to revert or to diminish the destructive process generated by the Mt, we carried out the pathological analysis of the animal's joints of each group.
The sheets corresponding to the joints were analyzed by the group of Pathology of CIGB.
The joints were fixed in a neutral buffer of formalin at 10% and decalcified. The tissues were dehydrated in an alcohol gradient, absorbed in paraffin and cut in sections of 5 μm. Later on, the samples were mounted on glass slides and stained with a hematoxiline-eosine mixture and observed in an optical microscope.
The results of the histological analysis are shown in the table 3.
In Table 3 P: represents the pannus; EC: erosion of the cartilage; IM: invasion of the bone marrow; HO: hyperplasia of the osteoclasts; G: granulematose reaction; PMN: polymorphonuclears.
In the animals of the group III including those sacrificed at day 15, was evident the presence of the pannus that cause the erosion of the cartilage and the invasion of the bone marrow. Also, the typical granulomatose reaction of the induction with Mt is observed and frequently the hyperplasia of the osteoclasts that are giant cells that have a great destructive power of the bony substance. These histological results reproduce the characteristic signs of the modifications in the joint that occurring in patients with AR and demonstrate that we have developed an appropriate animal model to evaluate the pharmacological candidates.
The
In the treatment with F19-7 peptide by intradermal route (group II) as is shown in
As it is shown in Table 3, the groups of treated animals with the F19-6 and F19-7 peptides didn't show practically histological affectations during the course of the first assay. A bigger protection against to the development of the Arthritis was evidenced in the animals treated with F19-7 peptide. Half of the rats involved in this group of treatment (6 rats) didn't present any histological signs, in correspondence with the group of animals treated with F19-6, where only two rats didn't present histopathological damage. In the groups treated with F19-6 and F19-7 peptides the presence of pannus was not observed at days 15 and 21 after induction of the Arthritis, which indicates that the administration of these peptides caused a retard in the appearance of the cell events characteristic of the inflammatory reaction during the development of the Arthritis. It was significant that in treated animals with F19-7 peptide that were sacrificed at 35 day after induction of the Arthritis, only one rat evidenced the histological affectations characteristic of the development of the illness. These results indicate that the treatment of the animals with this peptide induced, besides a retard in the clinical manifestation of the illness, practically a total remission of the histo-pathological signs of the Arthritis, showing a therapeutic superiority of the F19-7 APL with regard to the peptide containing the original epitope F19-6.
In this assay sick rats were used where the Arthritis was induced using bovine type II collagen in IFA. The spleen of each analyzed rat was extracted and the mononuclear cells were isolated.
The mononuclear cells isolated from the spleen were cultured in 1.5 mL of RPMI 1640 in 24 well plates (Costar, USA). The assay was carried out by triplicate, and a total of 3×106 cell/well was cultured during 24 hours with F19-6 and F19-7 peptides at a concentration of 5 mg/mL and incubated at 37° C., in humid atmosphere of CO2 at 5%. The cultivated cells without the peptides were used as negative control.
Later on the mRNA was isolated and the reactions of reverse transcription of the nucleic acid and the PCR were carried out in the same way as described in the previous examples. The analyses of the reaction products were performed by electrophoresis in agarose gels as described in the example 12. To determine the differences in the cytokine pattern according to the relative levels of mRNA for each sample, statistical tests were used as described in example 13.
In
As it is observed in these two figures, F19-6 and F19-7 peptides diminish the levels of TNFα in vitro, but in the case of the levels of IL-10, only the F19-7 peptide increases it significantly.
These results indicate that F19-7 peptide is able to modulate the cytokines pattern to a regulating phenotype in the animal model where the Arthritis is induced with collagen. Therefore we can affirm that the immunomodulating action of this peptide derived from the hHsp60 in vitro can be extended to animal models of Arthritis, where the induction agent that participates in the development of the illness is not Mt or related autoantigens.
These results suggest that the F19-7 peptide can induce tolerance for structurally related antigens, being able to mediate in vivo active suppression in the site of the inflammation.
With these results we can suggest that F19-7 peptide induces a protection against arthritis induced by Mt or type II collagen in the animals, which is mediated by regulatory T cells that produce IL-10. This effect could extend to other autoantigens present in the joints that progressively contribute to the immunophatogenic process that takes place in the course of RA.
Using blood of patients with RA was carried out assays of measurement of cytokines: TNFα, IFNδ and IL-10. The potentiality of the peptides of the hHsp60 to increase or to diminish the different cytokines involved in the pathogenesis of the illness was evaluated in this assay.
Starting from 10 mL of blood of each patient that was diluted ½ in PBS 1×, 5 mL from this dilution was added to 3 mL of Ficoll-Paque (Amershan) in centrifugation tubes of 15 mL, which were centrifuged during 30 min. to 1200 rpm. The ring corresponding to mononuclear cells was extracted. Later on, the cells were washed twice with 15 mL of PBS 1× and after each washing, they were centrifuged at 900 rpm. Finally, the pellet of the cell was resuspended in RPMI 1640 containing 10% of bovine fetal serum and supplemented with penicillin (100 U/mL), streptomycin (100 μg/mL), HEPES 25 mM/L and L-glutamine 2 mM (all acquired from Gibco BRL).
The mononuclear cells obtained were seeded in number of 106 cells/well in plates of 24 wells (Costar) in a volume of 800 μL. Later on, peptides of the hHsp60 were added at a concentration range from 0.5 μg/mL up to 100 μg/mL, in order to know the optimimal concentration of each peptide to modulate the cytokines levels. Phytohemagglutinin (PHA) 1% was used as positive control of cellular stimulation, while the RPMI 1640 alone was used as control of the basal cell growth.
The cells were cultured during 24 hours and later on the supernatant of each well was taken, diluted ½, and cytokine concentration was determined by specific kits (Quantikine®, R&D Systems) according to recommendations of suppliers.
The levels of TNFα modulated by F19-6 and F19-7 peptides in mononuclear cells of patients with RA are represented in the
In the patients represented as P19 and P21, the levels of TNFα diminished significantly by the F19-6 and F19-7 peptides in comparison with the non stimulated cells as it is observed in
In the patient represented as P13, the levels of TNFα were increased by the F19-6 peptide, however the levels of this cytokine diminished significantly, when this patient's mononuclear cells are stimulated with the F19-7 peptide. These results evidence that F19-7 peptide, although it was designed to be presented by the MHC from rat, they can diminish the levels of this cytokine, which is largely responsible of the inflammatory process characteristic of RA, thus this peptide is a potential therapeutic candidate for the treatment of this disease.
Assays for cytokines measurement using blood of patients with RA were carried out: TNFα, IFNδ and IL-10. The potentiality of the peptides of the hHsp60 to increase or to diminish the different cytokines involved in the pathogenesis of the illness was evaluated in these assays.
Ten mL of blood were extracted from each patient, which were diluted ½ in PBS 1×. It was added 5 mL of this dilution to 3 mL of Ficoll-Paque (Amershan) in centrifugation tubes of 15 mL, and centrifuged during 30 min at 1200 rpm. The ring corresponding to mononuclear cells was extracted. Later, the cells were washed twice with 15 mL of PBS 1× and after each washing they were centrifuged at 900 rpm. Finally, the precipitate was resuspended in RPMI 1640 medium containing 10% of bovine fetal serum supplemented with penicillin (100 U/mL), streptomycin (100 ìg/mL), 25 HEPES mM/L and L-glutamine 2 mM (all acquired of Gibco BRL).
The mononuclear cells obtained were seeded in a number of 106 cells/well in plates of 24 wells (Costar) in a volume of 800 μL. Afterwards, the peptides of the hHsp60 were added in a concentration range from 0.5 μg/ml up to 100 μg/ml, in order to know the optimal concentration of each peptide to modulate the cytokine levels. The Phytohemagglutinin (PHA) to 1% it was used as positive control of cell stimulation, while the RPMI 1640 alone was used as control of the basal cell growth.
The cells were cultured during 24 hours and later on, the supernatant of each well was taken, diluted ½, and cytokine concentration was determined by specific kits (Quantikine®, R&D Systems) according to the recommendations of the suppliers.
The levels of TNFα modulated by the E18-3 and E18-12 peptides in mononuclear cells of patients with RA are represented in
In the figure when exists statistically significant difference among the levels of the cytokines secreted by the cells stimulated with the peptides with respect to non stimulated cells, a different letter is assigned in the bars for levels of the corresponding cytokine.
As it is observed in
This same effect takes place in the patient P9 of genotype DR 0506 and the patient P10 DR 0204.
In patients P7, P8 and P12 presenting a genotype DR 0303, any difference in levels of TNFα was not observed, when mononuclear cells are stimulated with our peptides.
The modulation of IL-10 levels for E18-3 and E18-12 peptides are represented in
In the case of IL-10, as it is observed in
From these observations we can infer that E18-12 and E18-3 peptides induce a TH1 phenotype in peripheral mononuclear cells.
A therapeutic variant of original peptides: E18-12 or E18-3 could be their administration by oral route, so they will induce mechanisms of tolerance that contributed significantly to decrease the inflammation.
From previous results, we decided to modify these original peptides corresponding to epitopes of T cells, at the sites of contact with HLA molecule, using Bioinformatic tools, so that the modified type APL peptides would be able to change the pattern of cytokines to a regulating phenotype. In addition, modifications made in each of these peptides fulfill with peptide binding motifs reported for most of the HLA molecules expressed by the patients that theoretically will favor the interaction of each of them with different HLA molecules.
Assays for measurement of the cytokines: TNFα and IL-10, were carry out using blood of patients with RA. The potentiality of derived type APL peptides from the hHsp60 to increase or to diminish the different cytokines involved in the pathogenesis of the disease was evaluated in these tests. In addition, the original peptides were included in this evaluation (E18-3, E18-12) as experimental controls.
The APL peptides evaluated in these tests were the following:
Ten mL of blood were extracted from each patient and diluted ½ in PBS 1×. It was added 5 mL of this dilution to 3 mL of Ficoll-Paque (Amershan) in centrifugation tubes of 15 mL, and centrifuged during 30 min at 1200 rpm. The ring corresponding to mononuclear cells was extracted. Later, the cells were washed twice with 15 mL of PBS 1× and after each washing they were centrifuged at 900 rpm. Finally, the precipitate was resuspended in RPMI 1640 medium containing 10% of bovine fetal serum supplemented with penicillin (100 U/mL), streptomycin (100 μg/mL), 25 HEPES mM/L and L-glutamine 2 mM (all acquired of Gibco BRL).
Obtained mononuclear cells were cultured in number of 106 cells/well in plates of 24 wells (Costar) in a volume of 800 μL. Cells were stimulated with peptides [E18-3, E18-12, E18-3APL1 (SEQ. ID. NO: 18), E18-3APL2 (SEQ. ID. NO: 21), E18-12APL1 (SEQ. ID. NO: 5), E18-12APL3 (SEQ. ID. NO: 12)] at a concentration of 40 μg/mL and by triplicate. RPMI 1640 was used as control of the basal cellular growth.
Cells were cultured during 24 hours and later supernatants of each well were taken, diluted ½ and the concentration of cytokines were determined by specific kits (Quantikine®, R&D Systems) according to the recommendations of the suppliers.
In
In
These results could be contradictory, since such APLs that increase the production of IL-10 promote an increase in the levels of TNFα. In
The results of the study are extremely promissory, because it suggested that these two APLs could be administered to patients and could induce a subpopulation of regulatory T cells secretory of IL-10 that mediate an active suppression at systemic level. Another partial conclusion that we can arrive by means of the analysis of these results, is that these APLs practically display a homogenous behavior in all the studied patients, independently of the genotype that they have. These results are extremely encouraging, because it would allow that the administration of these APL had the same effect in all the patients, in spite of the existing differences with respect to the type of HLA-II molecules that they have.
From our experimental results, the fundamental advantage of the present invention is the use of pharmaceutical preparations containing immunomodulating peptides of the human stress protein Hsp60, corresponding to T cell epitopes of or variants type APL of these peptides, that can be effectively used in the treatment of patients with RA, inducing a specific immune response directed to silence the clones of involved autorreactive T cells in the immunopathogenic mechanisms of this disease.
The treatment we propose in this invention is reasonable and can be extended to a large number of patients and also it can be used in combination with current therapies for this disease.
The animals were separated in twelve groups of eight rats each one. The disease was induced in eleven animal groups (Grupo I-Grupo XI), ten of them were treated with peptides and one stayed as control of the induction of the disease. Animal groups that were treated with peptides, received 50 μg of peptide by rat in a volume of 100 μL of PBS, days 12, 14, 16, 18, 20, 22, 24, 26 after induction of the disease with Mt.
The separated animals in twelve groups were treated with the following form:
Days 21 and 35 after the induction, several animals from each group were sacrificed, spleens were extracted and sample was taken from joints for histopathological analysis.
In the groups of animals treated with peptides type APL, the clinical improvement was significant in comparison with the non treated animal group and with the groups treated with E18-12 and E18-3. These results were corroborated by histopathological analysis and by measurement of cytokines, demonstrating in all cases a superiority of derived type APL peptides upon original peptides.
In
It is possible to appreciate in this graph, that from day 12 after the induction of the disease, the animals of groups I, II and XI began to show the characteristic clinical signs of RA. Many animals also presented extra-articular manifestations like nodules in the ears, in the tail and in the extremities. The most severe manifestations of the RA were observed around day 20 for the three ill animal groups. In the case of animals treated with E18-12, in day 21 we could not appreciate significant differences with respect to the control of the disease induction group. Nevertheless, around day 35 after the induction of the disease, clinical signs in animals of this group were significantly inferior to those of group XI (p<0.001). These results indicate that protective effect of the E18-12 peptide is shown around day 35. In this sense, it would be recommendable to administer this peptide in earlier phases of the disease in order to obtain better results at day 21. However, in the case of animals of groups II the clinical signs of inflammation were evidently smaller in comparison with groups I and XI (p<0.001), which indicates that this peptide exerts a potent protective effect in the animals where the disease was induced.
| Number | Date | Country | Kind |
|---|---|---|---|
| CU2004-0207 | Sep 2004 | CU | national |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 11663603 | Mar 2008 | US |
| Child | 12649687 | US |
