1. Översiktlig projektbeskrivning

Engelsk titel

ITP from preclinical studies to clinical trials

Sammanfattning av projektet

Background

Idiopathic thrombocytopenic purpura (ITP) is an autoimmune disease where platelets are destroyed prematurely, mainly in the spleen, liver and bone marrow. Bleeding usually commences when the platelet count drops below 30 x 109/l. The frequency of death from hemorrhage in patients with platelet count below 30 x 109/l is estimated to be 1.6-3.9% per patient year. The incidence of ITP is estimated to 1.6-6.6 per 100,000 individuals and year with approximately twice as many women as men affected. ITP is classified as primary or secondary to an underlying disorder, and is also classified as either acute (<6 months) or chronic (>6 months). Acute ITP occurs predominantly in childhood, and in most cases the illness resolves in months, regardless of whether they receive treatment or not. In adults, the disease is chronic, the onset is often insidious and it rarely resolves spontaneously. Primary ITP is an exclusion diagnosis based on isolated thrombocytopenia lasting for more than six months and a platelet count <100 x 109/L, normal bone marrow examination, normal sized spleen, and no other cause for their thrombocytopenia. Secondary forms of the disease occur in association with SLE, the anti-phopholipid syndrome, immune deficiency states (IgA deficiency and common variable hypergammaglobulinemia), lymphoproliferative disorders (chronic lymphocytic leukemia, CLL and large granular lymphocyte leukemia, LGL), infections with HIV and hepatitis C virus, and drug therapy.

The goal of treatment is to raise the platelet count to a hemostatically safe level. The disorder is chronic with a considerable variation in the clinical course, but most patients will eventually, with treatment, attain a safe platelet count. However, a subset of patients suffers from a severe disease, refractory to all forms of treatment, and these patients have increased morbidity and mortality. Persistent platelet counts below 30 x 109/l are associated with an increased incidence of bruising, mucosal bleeding and intracranial hemorrhage. The risk is greatest in the elderly, those with a history of bleeding, and those that do not respond to therapy. In the small subgroup of patients with severe thrombocytopenia, the predicted five-year mortality rate from bleeding range from 2.2% for those younger than 40 years of age to 47.8% for those over 60 years of age indicating a need for long-term treatment for severe disease.

The first pathogenic mechanism was identified in 1951 when Dr William Harrington infused himself and his colleagues with plasma or blood from patients with ITP. Most of them developed a rapid but transient thrombocytopenia, indicating that the cause was of humoral origin. Since then, platelet destruction in ITP has been believed to be mediated by autoantibodies against platelet surface antigens. Once coated with antibodies, platelets undergo sequestration via interaction with Fc receptors on macrophages in the reticuloendothelial system. However, not all blood/plasma from ITP patients induced thrombocytopenia in the recipients in Harringtons experiments. . Secondly, a platelet specific antibody can only be detected in 50–70% of ITP patients. Thirdly, a type 1 T-helper cell profile (Th1), known to promote both cell mediated cytotoxicity and IgG antibody production, has been described in ITP. Fourthly, platelets from ITP patients have been shown to present potential platelet autoantigen by their membrane major histocompatibility complex (MHC) class I molecules, opening the possibility for a cell-mediated mechanism for platelet destruction. Using DNA microarray analysis in combination with flow cytometry and an in house cytotox assay we could show that cytotoxic T-cells mediate lysis of autologous platelets in patients with active ITP but not in patients in remission (Fig 1). These data, published in Nature Medicine, were the first to demonstrate that cell-mediated cytotoxic contributes to the destruction of platelets in ITP. We have also been able to show that activated T-cells from patients with active ITP are resistant to AICD which lead to defective clearance of autoreactive T-cell clones and causes a continued immune destruction of platelets. Conversely, a re-established AICD in ITP patients might be an important mechanism for the achievement of remission. Furthermore, we have been able to show that ITP patients have an accumulation of T-cells in the bone marrow and that this most likely is caused by an increased protein expression of CX3CR1 and VLA-4. These molecules might serve as new treatment targets in ITP. The results were published in Blood (impact factor 10.8). Undersigned and Prof. Hans Wadenvik are patent owners (no. 08 002 029 0) of VLA-4 and CX3CR1 as drugable targets in ITP.

Specific aims

(i)To evaluate the use of Tysabri in treatment of patients with ITP through a phase I/II clinical trial.
(ii)To establish 3 different mouse models for primary chronic ITP, specifically (a) overexpression of the heavy and light chain of platelet specific antibodies, (b) generation of CD4+ T-helper and (c) CD8+ cytotoxic T-cell models, in order to study both antibody and T-cell mediated destruction of platelets.

Workplan

I. Evaluation of treatment with natalizumab (Tysabri®) in ITP
The literature suggests that autoimmune diseases are not identical in modus operandi but have a lot in common. Our findings of T-cell mediated cytotoxicity in both ITP and MS further support this. In mice with experimental autoimmune encephalomyelitis (EAE), administration of antibodies against VLA-4 inhibits the adhesion of leukocytes to vessels in the brain stem and spinal cord and thus prevents the normal paralysis of hind legs. This was later repeated in patients with MS where treatment with natalizumab (Tysabri®), which is a humanized monoclonal antibody of IgG4 subclass against VLA-4 (interin-4α), markedly decreased the number of relapses compared to placebo. The effect of Natalizumab has also been acknowledged in Crohn’s disease where it is now an FDA approved treatment.Our findings of increased mRNA and protein expression of VLA-4 in peripheral blood T-cells and increased protein expression of VLA-4 in T-cells in bone marrow, which is one of the organs in ITP where platelets are destroyed, together with increased number of T-cells in bone marrow of in patients with ITP suggest that alterations in T-cell trafficking may be a feature that is shared among autoimmune diseases. To test if VLA-4 is of pathogenic importance in ITP we will perform a phase I clinical trial. Twenty patients with active ITP will receive an infusion of 300 mg natalizumab once monthly for three months. The inclusion criteria for this study are: 1) unequivocal chronic ITP and a stable platelet count <30 x 109/l or bleeding symptoms, 2) no immunosuppressive treatment within the last 3 months, 3) acceptable pregnancy prevention, 4) age ≥ 18 years, and 5) signed consent form. The primary endpoints are improvement of platelet count and bleeding tendency. The secondary endpoints are: 1) degree of T-cell accumulation in the bone marrow and change in the number of activated T-cells, regulatory T-cells and antigens involved in homing. We will analyze peripheral blood and bone marrow by flow cytometry before and after treatment with natalizumab and determine the number of total T-cells (CD3) and helper T-cells (CD3+/CD4+), cytotoxic T-cells (CD3+/CD8+), T-regs (CD4+/CD25+), Fas, KIR receptors, HLA-DR, CD69 and CX3CR1, 2) degree of T-cell mediated cytotoxicity before and after treatment with natalizumab. We will analyze the degree of apoptosis induced in platelets by mixing autologous platelets with T-cells. Apoptosis will be assayed by Annexin V and propidium iodide staining, and flow cytometry. 3) Changes in serum cytokines will be analysed by ELISA, to test if this treatment induces a shift from Th1 (Interferon-γ) to Th2 (IL-4 and IL-10) or Th3 (TGFβ1) profile.

In the first clinical studies of 2000 patients, receiving treatment with natalizumab against MS and Crohn’s disease, three cases of multifocal leukoencephalopathy (PML) were identified by reactivation of JC virus infection. Two of these were fatal. All three PML cases had a combination treatment with other immunosuppressive drugs e.g. interferon-beta1. However, currently there are over 30 000 patients with MS and Chron´s disease that are being treated with natalizumab and there has not been any additional PML cases. This is most likely due to a change in protocol where natalizumab treatment is not given in combination with other immunosuppressants. Furthermore, exclusion of ongoing JC virus infection prior to the start of natalizumab treatment has most likely contributed. To ensure the safety of the participants in our study: 1) All patients will have their medical history and previous treatments for ITP recorded; 2) Physical examination, blood pressure, pulse rate, laboratory variables such as blood cell count, electrolytes, and liver function tests will be performed once weekly; 3) Pregnancy test of women in fertile age, will be performed before start of treatment; 4) A MRI scan of the brain will be undertaken before the start of natalizumab treatment, to rule out an ongoing JC virus infection.

An application has been sent to both the regional ethics committee and the Swedish Medical Products Agency.

II. Overexpression of platelet specific antibodies
During my Ph.D. student days within the field of endocrinology and metabolism I worked with the generation and evaluation of different transgenic mouse models. We used both transgenic overexpression and knockout models and performed advanced analyses such as glucose tolerance tests, telemetri for heart rate and blood pressure, behavioural studies, lipid analysis, resting metabolic rate, dual X-ray energy analysis (DEXA) among a few. Therefore, we have the competence to perform the animal studies described below.

Autoreactive antibodies have been known to cause ITP since the classical experiment of Dr. William Harrington. We will screen all available platelet-specific mouse monoclonal antibodies where the hybridoma cells are available. The heavy and light chain of the positive clones will be cloned and sequenced. We will then generate double transgenic mice overexpressing both the heavy and light chain in tandem as has previously been performed in mouse models for autoimmune haemolytic anaemia by Watanabe et. al. The mice will as usual first be confimed by PCR alternatively Southern blot and then the platelet count will be followed. Only the mice developing thrombocytopenia will be used.
III. Generation of mouse models with autoreactive CD4+ T-helper and CD8+ cytotoxic T-cell clones.
We have shown that cytotoxic T-cells can directly destroy platelets in patients with ITP. Furthermore, it is well known that in order to generate a humoral immune response by B-cells a specific and costimulatory CD4+ T-helper response is needed. Both autoreactive CD4+ T-helper and CD8+ cytotoxic T-cell clones contribute to EAE, a mouse model for multiple MS. We plan to follow the protocols similar to those used for generation of EAE mice and experimental models of rheumatoid arthritis. In these protocols cell specific proteins such as myelin basic protein (MBP) in EAE or collagen type II in experimental autoimmune arthritis or whole tissue such as spinal cord in EAE were used in combination with complete Freund’s adjuvans. We intend to both use whole platelets and platelet specific protein such as GPIIb/IIIa or GP Ib/IX in combination with complete Freund’s adjuvans. The evaluation of the models is in our case very easy by monitoring the platelet count. In the mice where the platelet count drops we will isolate cells from the lymph nodes and expand them in vitro with the same type of stimulation as used in vivo i.e. whole platelets or platelet specific proteins. The autoreactive clones will subcloned and once more expanded. Once expanded, these cells will be transferred into healthy mice to verify that they induce thrombocytopenia.

IV. Evaluation of current treatments for ITP in mouse models for ITP.
The current first line treatment in ITP is the use of corticosteroids. It increases the platelet count to a hemostatically effective level in 75% of the patients and usually within the first three weeks. However, only 10-20% of the responses are sustained, and most patients will eventually relapse. Intravenous immunoglobulin treatment (IVIG) is used in patients with ongoing bleeding, when the platelet count is below 5 x 109/l or prior to splenectomy if the platelet count is below 30 x 109/l in patients unresponsive to glucocorticoids. Approximately 80% respond within a week, but sustained remission is rare, and the cost is considerable. Splenectomy is appropriate for most adults with relapse who do not respond to corticosteroids or require high doses to maintain a platelet count >30 x 109/l. Results of numerous studies indicate that approximately two thirds of the patients respond and usually within days.

Secondary treatments include the use of Rituximab, anti-D immunoglobulin, vinca alkaloids, danazol, azathioprine, cyclophosphamide, cyclosporine A and dapsone. Rituximab is a humanized monoclonal antibody against CD20 a protein specifically expressed on B-cells and treatment with this substance completely ablates all B-cells. The response rate in small series of refractory ITP patients ranges from 50-75%. However, the usefulness of Rituximab has not been evaluated in any large studies insofar. Anti-D immunoglobulin treatment is efficacious in Rh-positive patients where it elevates platelet counts by 70-90%. The mechanism of action is believed to be mediated through destruction of Rhesus (D)-positive red cells which are preferentially removed by the reticuloendothelial system, thus sparing autoantibody-coated platelets. Anti-D treatment is suitable for Rhesus (D)-positive patients who are not splenectomised, but is not recommended for refractory patients following splenectomy. There are only limited data for the use of these other agents.

Currently animal models for ITP are completely lacking and in our three different disease models, autoreactive antibodies, CD4+ T-helper and CD8+ cytotoxic T-cell clones, different treatment strategies will be evaluated. This will enable us to better understand the mechanisms behind the different treatment strategies and gain insights to how they work. We will also be able to better understand the role of innate immunity such as NK-cells and macrophages in the pathogenesis of ITP. Furthermore, these disease models will assist us when evaluating new targets and pharmaceuticals for ITP.

Importance

Autoimmune diseases result from the failure of normal self-tolerance mechanisms, and, collectively, they affect approximately 5-7 % of the population, often with debilitating effects. Patients within a specific autoimmune disease can be very heterogeneous in the response to treatment indicating that different disease mechanisms may coexist. We were the first group to demonstrate conclusive evidence that T-cell mediated cytotoxicity is involved in the pathogenesis of both ITP and MS. This indicates that T-cell mediated cytotoxicity may be a common mechanism in autoimmunity. This opens for new treatment strategies in order to combat this previously neglected disease mechanism. Furthermore, this paves the way for the generation of novel pharmaceutical drugs that specifically target this mechanism. However, utmost care must be taken to avoid inducing general immunosuppression or cytokine storms that may occur when activating co-stimulatory molecules. Therefore, targets for modulating T-cell activation, T-cell mediated cytotoxicity and T-cell trafficking specifically in autoimmune diseases needs to be identified.

Typ av projekt

Forskningsprojekt

MeSH-termer för att beskriva ämnesområdet

Inlagda MeSH-termer

Purpura, Thrombocytopenic, Idiopathic

Thrombocytopenia occurring in the absence of toxic exposure or a disease associated with decreased platelets. It is mediated by immune mechanisms, in most cases IMMUNOGLOBULIN G autoantibodies which attach to platelets and subsequently undergo destruction by macrophages. The disease is seen in acute (affecting children) and chronic (adult) forms.

Projektets delaktighet i utbildning

Avhandling
D-uppsats / Magisterexamen
C-uppsats / Kandidatexamen
ST-läkarutbildning
Annan utbildning
Ej del i utbildning

3. Processen och projektets redovisning

Pågående aktiviteter

Planering och förberedelse före datainsamling
Datainsamling pågår
Analys av insamlade data pågår
Författande av skriftlig redovisning / publikation pågår
En eller flera publikationer från projektet är publicerade
Slutfört och inget mer görs inom ramen för detta projekt

Projektstart (när planeringen påbörjas och börjar dokumenteras skriftligt)

2009-01-01

Datum för påbörjande av datainsamling

2009-09-01

Datum då projektet är slutrapporterat

2012-12-31

Tillämpning av resultat - tidsaspekt (projektledarens bedömning)

Resultaten kommer sannolikt att tillämpas inom 5 år från projektslut.

Tillämpning av resultat - genomslag (projektledarens bedömning)

Internationellt (i flera länder)