MXPA04011941A - Method for the protection of endothelial and epithelial cells during chemotherapy. - Google Patents

Abstract

The present invention is directed to the use of a protective oligodeoxyribonucleotide for the treatment of a patient undergoing treatment with an immunosupressant. The invention is further directed to a pharmaceutical composition containing a therapeutically effective dose of an immunosuppressant and of a protective oligodeoxyribonucleotide.

Description

METHOD FOR THE PROTECTION OF ENDOTHELIAL AND EPITHELIAL CELLS DURING CHEMOTHERAPY Field of the invention The invention relates to the field of the use of radiation therapy and / or chemotherapy. More specifically, the invention relates to a method for mitigating side effects associated with that treatment.

STATE OF THE ART Transplantation of non-differentiated allogeneic cells (SCT) is a well-established method for the treatment of hematological malignancies and a growing variety of other malignancies. The SCT consists mainly of two sequential steps: pretransplant conditioning, which typically consists of total body irradiation (TBI) and chemotherapy, which leads to minimal residual disease and immunosuppression of the receptor as a first step, and the transfer of halogenic non-differentiated cells that will finally provide the cure as a second step. However, due to disparities in major histocompatibility (MHC) and (mHAg) antigens, severe inflammatory reactions, including graft-versus-host disease (GvHD), may occur in different post-transplant phases. On the basis of studies carried out by the inventors1 and several other investigators2,3 it is widely accepted that conditioning contributes, via non-specific inflammation, to those complications related to transplantation (CRT). In addition, direct toxicity has been demonstrated, especially of TBI4,5. This has led to a variety of alternative conditioning regimes currently under investigation. In addition, new pretransplant therapies allow the extension of treatment protocols and patient selection. One compound of these novel conditioning concepts is fludarabine, a non-myeloablative immunosuppressant that had originally been used for the treatment of chronic lymphatic leukemia6. Fludarabine in combination with for example BC U and melphalan, cyclophosphamide or other agents can replace TBI or is used together with reduced dose regimens of TBI7,8. The clinical data obtained in this way argue to a large extent the comparatively low lateral effects and a specificity for hematopoietic and immune cells of fludarabine9. However, the influence of this compound on non-hematopoietic cells such as endothelial and epithelial cells has not been investigated yet.

Virtually all CRTs are associated with endothelial dysfunction and damage1. The inventors and others have shown that the endothelium is a target of pretransplant conditioning in vi tro and in vivo. Ionizing radiation induces programmed cell death (apoptosis) in endothelial cells. 11 ~ 14 At the same time the endothelium is activated in terms of expression of adhesion molecules that lead to an increase in leukocyte-endothelial interactions as a prerequisite for inflammatory processes .15,15. These effects are significantly increased by bacterial endotoxins (lipopolysaccharide, LPS) that can be translocated through the barriers of the damaged mucosa from the gastrointestinal tract.17 In addition, LPS has been shown to increase the antigenicity of endothelial cells to allogenic CD8 + cytotoxic T lymphocytes. 18 Clinical results with reduced intensity conditioning regimens (RIC) containing fludarabine obtained show a clear dysregulation of conditioning-related toxicity without affecting immune reconstitution .25 The incidence of acute GvHD in patients receiving RIC is comparable or even lower than in those patients who receive the classic conditioning regimen. However, reports of equally severe or even greater side effects such as osteonecrosis, 21 pulmonary complications, 28 and more cases of chronic GvHD29 clearly demonstrate the potential for serious side effects associated with treatment with fludarabine.

The invention The invention is based on the discovery that fludarabine activates and damages endothelial and epithelial cells. Activation of the cells leads to damage in the treatment situation where fludarabine is used, for example, when treating malignancies using SCT. Epithelial and endothelial cells can be protected from their activation and damage by treatment with defibrotide. This treatment may be concomitant or defibrottide may be given before or after treatment with fludarabine.

Abbreviations and definitions SCT: transplantation of non-differentiated hematopoietic cells. Immunosuppressant: substance that deregulates the immune response of a subject after its administration. Immunosuppressants are used to suppress the immune system of patients undergoing therapy with undifferentiated cells. Examples of immunosuppressants include fludarabine, cyclophosphamide, BCNU, cyclosporine, sirolimus, tacrolimus and melphalan. Preferred within the context of this application is fludarabine (also known as 2-fluoro-9-p-D-arabinofuranosyladenine). Oligodeoxyribonucleotide protector: will mean, within the context of this application, both oligodeoxyribonucleotides as defined in US patent 5,646,268 and polydeoxyribonucleotides as defined in US 5,223,609, which are incorporated herein by reference in their entirety. U.S. Patent 5,646,268 describes a process for producing an oligodeoxyrubonucleotide having the following physicochemical and chemical characteristics: Molecular weight: 4000-10000 h: < 10 A + T / C + G * 1,100-1,455 A + G / C + T * 0.800-1.160 Specific rotation: + 30 ° - + 48 ° * base molar ratio h = hyperchromicity parameter A process for producing that oligodeoxyribonucleotide comprises: precipitating aqueous solutions of 0.8 M sodium acetate from polydeoxyribonucleotide sodium salts at 20 ° C by the addition of an alkyl alcohol selected from the group consisting of ethyl alcohol , propyl and isopropyl. US Pat. No. 5,223,609 discloses a defibrottid which satisfies certain pharmacological and therapeutic properties and is therefore particularly suitable if the nucleotide fractions thereof conform stoichiometrically with the following polydeoxyribonucleotide formula of random sequence: Pi-5, (dAP) 12- 24, (dGp) 10-20, (dTp) i3-26, (dCp) i0-2o where P = phosphoric radical dAp = deoxyadenylic monomer dGp = deoxyguanilic monomer dTp = deoxythymidylic monomer dCp = deoxycytidylic monomer Defibrottid corresponding to this formula also shows the following physicochemical properties: electrophoresis = homogenous anodic mobility; extinction coefficient, ?? cm1% at 260 + 1 nm = 220 +10; extinction ratio, + 0.04; molar extinction coefficient (referred to phosphorus), e (?) = 7.750 + 500; rotating energy [a] D20 ° = 53 ° + 6; reversible hyperchromicity, indicated as% in native DNA, h = 15 + 5. A preferred protective oligodeoxyribonucleotide is Defibrottid (CAS Registry Number: 83712-60-1), a polynucleotide well known to those skilled in the art, which typically identifies a polydeoxyribonucleotide obtained by extraction (US 3,770,720 and US 3,889,481) of an animal and / or plant tissue; this polydeoxyribonucleotide is usually used in the form of an alkali metal salt, generally sodium, and usually has a molecular weight of about 45-50 kDa. Defibrottid is used mainly for its antithrombotic activity (US 3,829,567) although it can be used in different applications, such as, for example, the treatment of acute renal failure (US 4,694,134) and the treatment of acute myocardial ischemia (US 4,693,995). US Patents US 4,985,552 and US 5,223,609 describe a process for the production of defibrottid, which allows a product to be obtained which has constant and well-defined physicochemical characteristics and is also free of any undesirable side effects.

DETAILED DESCRIPTION OF THE INVENTION The invention relates to a method for the treatment of a patient undergoing immunosuppressant treatment, comprising the step of administering an effective dose of a protective oligodeoxyribonucleotide to the patient. Treatment with an immunosuppressant preferably occurs during SCT. The immunosuppressant is preferably selected from the group comprising antimetabolites (e.g., 5-fluorouracil (5-FU), methotrexate (MTX), fludarabine, antimicrotubule agents (e.g., vincristine, vinblastine, taxanes (such as paclitaxel and docetaxel), alkylating agents (e.g., cyclophosphamide, melphalan, bischloroethylnitrosurea (BCNU)), platinum agents (e.g., cis-platinum (also called cDDP), carboplatin, oxaliplatin, JM-216, CI-973), anthracyclines (e.g., doxorubicin) , daunorubicin), antibiotic agents, including mitomycin-C, topoisomerase inhibitors (e.g., etoposide, camptothecin), cyclosporine, tacrolimus, sirolimus, and other cytotoxic agents that act to suppress the immune system. frequently used in the therapy of malignant diseases can be found in Gonzales et al., Alergol, Immunol., Clin. 15, 161-181, 2000, which is incorporated herein by reference The preferred immunosuppressants are nucleosides (ie, the glycosides resulting from the removal of the phosphate group from a nucleotide), such as, for example, fludarabine, which is the preferred immunosuppressant for the purposes of the present invention. The protective oligodeoxyribonucleotide can be administered concurrently, simultaneously or together with the immunosuppressant. A preferred combination is the simultaneous administration of defibrotide and fludarabine. The step of administering the protective oligodeoxyribonucleotide preferably occurs concurrently, concomitantly, simultaneously, after or prior to the administration of the immunosuppressant to the patient. In a preferred embodiment of the invention, the step of administering the protective oligodeoxyribonucleotide occurs after the administration of the immunosuppressant to the patient. In a further preferred embodiment, the delay time between the step of administering the shield and administering the immunosuppressant to the patient is from about 1 hour to about 2 weeks. The delay time between the time of administering the protector and administering the immunosuppressant to the patient is preferably from about 2 days to about 7 days. In another preferred embodiment of the invention, the step of administering the protective oligodeoxy ribonucleotide occurs prior to administering the immunosuppressant to the patient. Preferably, the time difference between the step of administering the protector and administering the immunosuppressant to the patient is from about 1 hour to about 2 weeks. Preferably, the time difference between the step of administering the protector and administering the immunosuppressant to the patient is from about 2 hours to about 2 days. The preferred olioligodeoxyribonucleotide protector is defibrotide; however, other substances may be used as mentioned above as protective oligonucleotides. The following embodiments define the preferred doses of defibrottide, however, similar doses may be used when a protective oligodeoxyribonucleotide other than defibrotide is used. The optimal dose for any protective oligodeoxyribonucleotide will be determined by the doctor who pays attention. The experiments described below show the protective effects of defibrottide. The effective dose determined in these experiments can be used as a guide to determine an effective dose for the treatment. Defibrottide is administered, preferably orally or injected intravenously. The preferred dose of defibrotide is chosen so that a blood level of about 100 to 0.1 μ / ta ?? is reached. More preferably, the defibrottid dose is chosen so that a blood level of about 10 μg / mL to about 100 μg / mL is reached. More preferably, the dose of defibrottide is chosen so that a blood level of about 100 μg / mL is reached. In a preferred embodiment of the invention, the dose of defibride administered is about 100 mg / kg of the patient's body weight to about 0.01 mg / kg of body weight. Preferably, the dose of defibride administered is about 20 mg / kg of body weight of the patient to about 0.1 mg / kg of body passage. More preferably, the dose of defibrottide administered is from about 15 mg / kg of the patient's body weight to about 1 mg / kg of body weight. Most preferably, a daily dose of about 12 mg to about 14 mg per kg of body weight of the patient is administered. More preferably, the dose of defibrottide administered is about 12 mg / kg of the patient's body weight. Preferably, the administration of a protective oligodeoxyribonucleotide according to the present invention is capable of protecting endothelial cells and epithelial cells against the effects of the immunosuppressant. The immunosuppressant preferably activates epithelial cells and endothelial cells and induces apoptosis in them. Thus, in a preferred embodiment, the protective oligodeoxynucleotide protects the epithelial and / or endothelial cells from apoptosis and / or activation by the immunosuppressant. The immunosuppressant is preferably fludarabine. The protective oligodeoxyribonucleotide is preferably defibrotide. Activation includes the increased expression of ICAM-1 and MHC molecules of class I. The increase in expression is preferably substantial. Even more preferably, the immunosuppressant induces a proinflammatory activation of endothelial cells and / or epithelial cells in a patient. The cells are preferably human microvascular endothelial cells (HMEC) and / or dermal and / or alveolar epithelial cells. The damage preferably occurs when the patient's endothelial and / or epithelial cells have been exposed to the immunosuppressant for about 1 hour to about 1 week or more. More preferably, the damage occurs when the cells have been exposed from about 5 hours to about 72 hours. Even more preferably, the duration of that exposure is between 20 hours and 72 hours. More preferably, the duration of that exposure is more than 48 hours. Treatment with the immunosuppressant preferably occurs during the transplantation of undifferentiated hematopoietic cells. The transplantation of non-differentiated hematopoietic cells is preferably a transplantation of non-differentiated haematopoietic cells. The invention is also related to. a pharmaceutical composition comprising at least one protective oligodeoxynucleotide, for the treatment of a patient in need thereof, a patient who is being treated with an immunosuppressant. The administration of the pharmaceutical composition alleviates or protects against side effects caused by the immunosuppressant or by the immunosuppressant and a transplant. The transplant is preferably a bone marrow transplant or hematopoietic undifferentiated cells. More preferably, the transplant is a bone marrow transplant or hematopoietic halogenéic non-differentiated cells. The side effects are preferably related to endothelial and / or epithelial cells and / or patient tissues. Preferably, side effects involve apoptosis of the cells, and / or activation of the cells. Activation preferably comprises increased expression of MHC molecules of class I and / or intracellular adhesion molecule 1 (ICAM-1). Side effects damage human microvascular endothelial cells (HMEC) as well as, preferably, dermal and / or alveolar epithelial cell lines after 48 hours of culture, when used in pharmacologically relevant concentrations (range: 10 g / mL · to 1 g / raL). Lateral effects usually include damage to target tissues from complications related to transplantation and stimulated immune responses. Side effects preferably include significant up-regulation of the intercellular adhesion molecule 1 (ICAM-1) and MHC class I molecules in endothelial cells and / or epithelial cells of patients, particularly in alveolar endothelial cells. Lateral effects also include a proinflammatory activity of microvascular endothelial cells. The side effects also preferably include an increase in the lysis of these cells by cytotoxic T lymphocytes restricted by MHC class I allogenic derivatives of the transplant. Administration of the protective oligodeoxynucleotide preferably protects against lateral objects induced by the immunosuppressant, including apoptosis and alloactivation. The pharmaceutical compositions comprising the immunosuppressants of the present invention can be formulated with techniques, excipients and vehicles of the conventional and well-known type, for oral and injection administration, particularly by the intravenous route. The doses of active ingredient in the compositions according to the present invention fluctuate from 50 and 1500 mg for a unit dose, while to achieve the desired results a daily administration of 10 to 40 mg / kg is suggested. Methods for the preparation of defibrotide can be found in US 4,985,552 and US 5,223,609, patents which are hereby incorporated by reference in their entirety. The invention also relates to a pharmaceutical composition containing a therapeutically effective dose of an immunosuppressant and a protective oligodeoxyribonucleotide. The immunosuppressant is preferably fludarabine, the protective oligodeoxyrribonucleotide is preferably defibrotide.

BRIEF DESCRIPTION OF THE FIGURES Figure 1: Fludarabine induces programmed cell death in human microvascular endothelial cells (HMEC). The HMECs were left untreated or incubated with 2-fluoro-9-PD-arabinofuranosyladenine (hereinafter referred to as F-Ara, the metabolised form of fludarabine) at descending concentrations for 48 hours and subjected to flow cytometric analysis (A ) or microscopic analysis of DAPI staining. A: contour graphs of the lateral diffraction image (SSC) (x axis) of propidium iodide negative (PI) cells plotted against the forward diffraction image (y-axis) as a parameter of cell granularity versus size of the cell. B: Quantitative fluorescent microscopy analysis of endothelial cells stained with DAPI. The results are given in% of apoptotic HMEC (% of apoptotic cells) + _ standard deviation (of n = 10 microscopic fields with an average of 70 cells per field). Representative samples of at least five independent experiments are shown. *: p > 0.001 untreated control against cells treated with F-Ara (10.

Figure 2: Defibrottid (D) inhibits F-Ara-induced apoptosis in HMEC, evidence of intracellular antagonism. F-Ara: 10 g / mL. D: 100 μg mL. Flow cytometric analysis of the SSC image of PI negative cells. A: Reproducible induction of apoptosis by F-Ara. B: dose-dependent inhibition of apoptosis induced by F-Ara by D. C: Left graph: incubation of HMEC with F-Ara for 1 hour, subsequent incubation with D for 48 hours after washing. Graph to the right: incubation of HMEC with D for 1 hour, subsequent incubation with F-Ara for 48 hours after washing. For the experimental details see the legend of Figure 1 and the Materials and Methods. One of three independent representative experiments is shown.

Figure 3: F-Ara induces apoptosis in keratinocytes and alveolar epithelial cells, but not in intestinal or bronchial epithelial cells; protective effect of Defibrottid. F-Ara: 10 μg / mL. D: 100 μg / mL. Flow cytometric analysis of the SSC image of PI negative cells (Figure 3A) and analysis of DAPI staining of apoptotic cells (Figure 3B). The results are given in the mean% of apoptotic cells of + standard deviation of three different experiments. HaCaT: human keratinocyte cell line. SW 480: intestine epithelial cell line. A 549: alveolar epithelial lung carcinoma cell line. BEAS-2B: bronchial epithelial cell line. The primary bronchial epithelial cells have been derived from a bronchoscopic scraping procedure. Fig 3 A: *: p = 0.005 of HaCaT cells treated with F-Ara against F-Ara + D. **: p = 0.116 of A 549 cells treated with F-Ara against F-Ara + D. -: without induction of apoptosis. Figure 3B: +: p = 0.02S of HaCat cells treated with F-Ara against F-Ara- + D. ++: p = 0.001 of A 549 cells treated with F-Ara against F-Ara + D.

For the experimental details see the legend of Figure 1 and the Materials and Method. Three representative experiments are summarized for each cell line. Figure 4: Defibrottid (D) does not interfere with the antileukemic and anti-PBMC effect of F-Ara. F-Ara: 10 μg / mL. D: 100 pg / mL. A: Propidium iodide staining of primary acute myeloid leukemia (AML) cells derived from a patient in blast crisis (70% blasts from the total PBMC count). The results are given in the average% of the viability of three independent experiments. *; p = 0.008 of AML cells Control against treated with F-Ara. B: Flow cytometric analysis of the SSC image of negative PBMC PI. A representative sample of five independent experiments with different blood donors is shown.

Figure 5: F-Ara induces expression of ICAM-1 on HMEC, protective effect of Defibrottid (D). Flow cytometric analysis of ICAM-1 positive cells. The HMECs were left untreated or were incubated with F-Ara (10 μg / mL, or descending concentrations in B) in the presence or absence of descending concentrations of D. A: ICAM-1 histogram graph of a representative experiment . Dotted line: Background staining (control with nil); thin line: expression of ICAM-1 from untreated control cells; thick line: ICAM-1 expression of cells treated with F-Ara. B: Induction dependent on the expression dose of ICAM-1 by F-Ara. Summary of three independent experiments. The results are given as the mean% of ICAM-1 positive cells + standard deviation. *: p = 0.075 of F-Ara cells against untreated control. C: dose-dependent inhibition of ICAM-1 expression induced by F-Ara by Defibrotide (D). The results are given as the mean% of ICAM-1 positive cells + standard deviation. **: p = 0.004 of HMEC treated with F-Ara against F-Ara + D. Figure 6: F-Ara increases the allogeneicity of HMEC for CD8 positive cytotoxic T lymphocytes (CTL), protective effect of Defibrotide. A: PBMC were stimulated with HMEC irradiated in the presence of interleukin 2 (50 U / mL) for 7 days and subjected to a 51Cr release assay with untreated HMEC cells (Control) and treated with F-Ara (10 (24 hours of incubation) as target cells Autologous B-LCL: B lymphobastoid cells transformed with autologous EBV (effector) K 562: target cells for natural killer (NK) cells The results are given as% specific lysis according to as described in Materials and Methods. *:% of specific lysis of HMEC treated with F-Ara in the presence of anti-HMC class I antibody w6 / 32. Ratio of E / T: relation of effector / obj ective. : Deregulation of F-Ara-induced allogenicity from HMEC to CD8 positive CTL by Defibrottid (D) CD8 positive PBMCs have been negatively selected (non-depleted CD8 + cells) by separation with magnetic beads For the experimental details see legend of Figure 6A Figure 7: F-Ara decreases the allogenicity of HMEC for NK cells, increased lysis by blocking MHC class I. NK cells have been selected negatively (non-depleted NK cells) by separation with magnetic beads and stimulated with irradiated HMEC in the presence of IL-2 (50 U / mL) for 4 days and then submitted to the 51Cr release assay as described for Figure 6. Table below the graph: Flow cytometric analysis of the effector cell population pre and post-stimulation with HMEC . The NK cells were characterized as CD3- / CD16 + CD56 +. *:% of specific lysis of K562 cells at an E / T ratio of 20: 1.

Table 1: Anti-endothelial CTL produces a phenotype similar to Tcl Effector IFN-? IL-4 PBMC 319 [+176] 0 CD8 + 524 [+174] 0 The ELISA for the production of interferon gamma (IFN-?) And interleukin 4 (IL-4) in the supernatants of stimulating effector cells (7 days, irradiated HMEC, 50 U / mL of IL-2). The PBMC were left unseparated or negatively selected by CD8 + T cells as given for the experiments in Figure 6. The results are given as the mean pg / mL cytokine + standard deviation of 3 independent experiments.

Example METHODS Cell culture and reagents The human dermal microvascular endothelial cell line CDC / EU. -H EC-1 (also referred to as HMEC) was kindly provided to the Center for Disease Control and Prevention (Atlanta, Georgia, USA) and has been established as described previously.19 HMECs were cultured in MCDB131 medium, supplemented with 15% of fetal sheep serum (FCS), 1 μg mL of hydrocortisone (Sigma, Deisenhofen, Germany), 10 ng / mL of epidermal growth factor (Collaborative Biochemical Products, Bedford, MA, USA) and antibiotics. All cell culture reagents have been purchased from Gibco BRL (Karlsruhe, Germany) unless otherwise stated. 2- Fluoroadenin 9-beta-D-arabinofuranoside (F-Ara) was obtained from Sigma, Deisenhofen, Germany, Defibrotide bottles were obtained from Prociclide ™, Crinos, Como, Italy.

Apoptosis assays An established method was used to detect apoptosis in human endothelial cells as described above20 using flow cytometry (programs and programming systems FACScan ™ and CellQuest ™, Becton Dickinson, Heidelberg, Germany). The endothelial and epithelial cells were left untreated or were incubated with F-Ara in descending concentrations (range: 10 μg / mL to 0.1 ^ g / mL) in the presence or absence of Defibrotide for 48 hours. Subsequently, the cells were washed in 10% PBS and stained with the propidium iodide dye that detects necrosis (PI, 0.2 μg / mL, Sigma, Deisenhofen, Germany). The apoptotic cells were identified by a PI negative stain and by a characteristic diffraction side image distinct from that of non-apoptotic cells. At least three experiments have been carried out per cell type. An alternative method for the detection of apoptosis used microscopic DNA analysis of fluorescently labeled cells. 1 x 105 / plaque of endothelial cells were seeded in 35 mm petri dishes (Nunc, Wiesbaden, Germany). Those cells were treated as previously provided and subsequently fixed with Methanol / Acetone (1: 1) for 2 minutes, washed once in PBS and stained with 4,6-Diamidino-2-phenylindole (DAPI) (0.5μ < 3 / ta1 ·, Sigma, Deisenhofen, Germany), were dissolved in 20% Glycerin / PBS. Samples were mounted and subjected to microscopic analysis. Nuclear condensation according to that revealed by DAPI staining in the absence of trypan blue absorption was considered characteristic of apoptosis as opposed to necrosis21. The quantitative analysis included counting the number of apoptotic cells in relation to all identifiable cells from at least 10 microscopic fields, with an average of 70 cells per field. To clarify the results of handwritten DAPI staining, only the experiments with endothelial cells and HaCaT as well as A 549 cells were presented.

Cell surface analysis Cell surface expression of ICAM-1 (Becton Dickinson / Pharmigen, Heidelberg, Germany) and MHC molecules of class I (w6 / 32, hybridoma supernatant, ATCC, Manassas, VA, USA) on HMEC was evaluated by the indirect immunofluorescence technique and subsequently flow cytometry using the FACScan ™ flow cytometer and the Cell-Quest ™ analysis program (Becton Dickinson, Heidelberg, Germany). Endothelial cells were treated as such and after incubation harvested with trypsin / EDTA (Gibco), washed once in cold PBS / 10% FCS and incubated for 1 hour on ice with 5 μg / mL MoAbs antiadhesive molecule. The cells were washed again and incubated with an F (ab) 2 fragment of goat anti-mouse IgG FITC conjugated antibody (Dako, Hamburg, Germany) for 45 minutes on ice. The cells were then washed in PBS / 10% FCS and subjected to analysis. The viability of the cells was determined by concurrent staining with propidium iodide (0.2 μg / mL, Sigma, Deisenhofen, Germany). The omission of the first antibody served as a negative control to detect non-specific fluorescence. This method, instead of using isotype control antibodies, was justified by previous observations that endothelial cells lack Fe22 receptors. Therefore, a non-specific binding of antibodies through its Fe moiety could be excluded.

Allogeneic peripheral blood cell stimulation with HMEC Peripheral blood mononuclear cells (PBMC) were derived from heparinized blood (Novo Nordisk, Mainz, Germany) from healthy human volunteers or serum coatings from the Bavarian Red Cross according to the standard protocol using centrifugation in Ficoll hypaque density gradient (Pharmacia, Freiburg, Germany). The cells were then stimulated in a ratio of 1: 1 and 2: 1 with irradiated HMEC (20 Gy) for 7 days in the presence of Interleukin 2 (50 U / mL) and 10% of human AB serum (Sigma, Deisenhofen, Germany ). Alternatively, PBMCs were selected from CD8 + T cells and natural killer (NK) cells using cell isolation equipment according to the manufacturer's instructions (MACSMR, Miltenyi Biotech, Bergisch-Gladbach, Germany) based on the elimination of non-CD8 + and non-NK cells, respectively. The stimulation of the selected cells was identical to that of whole PBMC cultures, except for the NK cells which were stimulated for only 3 days.

Cytotoxicity assay Cytotoxicity mediated by T cells or NK cells was evaluated according to a well-established protocol23, using a 51Cr radioisotope assay for 4 hours. The HMECs that had been left untreated or incubated with F-Ara (10 μ9 /? T? _?) Overnight were used as target cells, to be labeled with 0.4 mCi Na251Cr04 for 2 hours. After 3 washing steps, the target cells were adjusted to 104 cells / mL and co-incubated with PBMC, CD8 + or NK effector cells at effector to descending target ratios for another 4 hours. The supernatants were transferred to dry flashing plates and counted in a counter? (all from Canberra Packard, Darmstadt, Germany). Autologous B (effector) B lymphoblastoid cell lines (B-LCL) and K562 sensitive cells as NK were taken as additional control targets. The percentage of specific lysis was calculated as: [(experimental release - spontaneous release) / (maximum release - spontaneous release)] x 100. Spontaneous release in all the experiments was always less than 20%.

Enzyme-linked immunosorbent assays (ELISA) ELISAs for the detection of Interleukin 4 (IL-4, Tc2 response) and Interferon and (IFN- ?, Tcl response), IL-1 and IL-10 in T cell supernatants Allogeneic effectors (see below) were performed exactly according to the instructions of the manufacturer's equipment (R & D Systems, Minneapolis, MN, USA).

Statistical analysis The meaning of the differences between the experimental values was evaluated by means of the Student test.

Example 1 F-Ara induces apoptosis (programmed cell death) in human microvascular endothelial cells (HMEC). To evaluate the influence of F-Ara on the viability of cultured human endothelial cells, HMEC were incubated with descending pharmacologically relevant concentrations (10 μg / mL , at 0.1 μg / mL) of 2-Fluoroadenin 9-beta-D-arabinofuranoside as the metabolized form of fludarabine. The average intracellular level of active fludarabine triphosphate (cytotoxic) in target cells is 20 μ ?, representing a concentration of 5.8 μ9 / t. (SCHERING medac, instructions from the manufacturers). After 48 hours of incubation, the HMEC were subjected to apoptosis tests using cell granularity detection of propidium iodide-negative cells (lateral diffraction image (SSC) in flow cytometry) and microscopic analysis of cells stained with DAPI, respectively. Regardless of the assay system, Figures 1 A and B clearly demonstrate that F-Ara produces apoptosis in HMEC at concentrations of 10 and 5 μ9 / p? 1, whereas 1? 9 / p? 1 was no longer effective. The critical threshold of F-Ara cytotoxicity was between 2 and 3 μg / mL. F-Ara apoptosis was already detected after 24 hours, although to a lesser degree (data not shown).

Example 2 Defibrottid protects HMEC against F-Ara-induced apoptosis HMEC were left untreated or treated with F-Ara in the presence or absence of various concentrations of Defibrotide (100 μ9 / p? _1 at 0.1 μg / mL) and were evaluated for programmed cell death after 48 hours using flow cytometric analyzes of the SSC image as described for Figure 1A. Figure 2A (mean contour plot) shows that Defibrottid alone or as a second control had no influence on the viability of endothelial cells. The apoptotic effect of F-Ara is reproduced in Figure 2A (graph of the right contour), while Figure 2B shows a dose-dependent protection of cell death induced by F-Ara by Defibrottid. To exclude a non-specific artificial extracellular interaction of F-Ara and Defibrotide in vi tro, the HMEC were pretreated with Defibrotido for 1 hour and then, after 3 washing steps, incubated with F-Ara for another 48 hours and vice versa. Figure 2C (graph of the right contour) reveals that the pretreatment of HMEC for 1 hour was sufficient to protect the cells from apoptosis induced by F-Ara. Similarly, pretreatment of HMEC with F-Ara for 1 hour (Figure 2C, left contour plot) and subsequent incubation with Defibrotide did not lead to programmed endothelial cell death.

Example 3 Effect of F-Ara on different epithelial cell lines, protective effect of Defibrotide The skin, the gastrointestinal tract (GIT) and most likely the lung are among the main targets of GvHD. Therefore, it was reasonable to test the influence of F-Ara on cell lines derived from those organs. Cells of keratinocytic (HaCaT), GIT (SW 480), alveolar (A549) and bronchial epithelial (BEAS-2B) cells as well as primary bronchial epithelial cells were incubated with F-Ara (10 μg / mL) as given for Figures 1 and 2 and were tested in flow cytometric apoptosis analysis 48 hours after treatment. Figure 3A summarizes that the intestinal and bronchial epithelial cells appear to be resistant to the apoptotic stimulation of F-Ara while the keratinocytes (HaCaT) and epithelial alveolar cells (A549) showed signs of apoptosis, as determined by flow cytometry of the SSC image (34.0 [+1.0]% of apoptotic cells for HaCaT and 42.9 [+26.7]% for A549, respectively). Again, the protective potential of Defibride (100 μg / mL) was evaluated. The HaCaT (4.3 [+3.0]%) and A 549 (5. [+2.9]%) cells were completely protected against programmed cell death after treatment with F-Ara and Defibrotic, Figure 3A, bar graphs inserted). To confirm these results, apoptosis assays were performed by staining with HaCAT (Figure 3B, columns on the left) and A 549 cells (Figure 3B, right column). As shown for endothelial cells, Defibrottid alone had no influence on the number of apoptotic cells in any cell line (data not shown).

EXAMPLE 4 Defibrottid does not interfere with the antileukemic and anti-PBMC effect of F-Ara In addition to its desirable protective capacity for endothelial and epithelial cells against F-Ara-induced apoptosis, it was important to investigate whether Defibrottid would also interfere with the antileukemic properties of F-Ara. To resolve this issue, acute myeloid leukemia (AML) cells derived from primary peripheral blood were thawed with a blast amount of 70%, maintained in culture for 24 hours and subsequently treated with F-Ara in the presence or absence of Defibride during other 48 hours. Figure 4A shows that almost 50% of the cells died spontaneously of necrotic cell death. However, F-Ara induced cell death in up to 80% of the cells. In contrast to its effect on endothelial and epithelial cells, Defibrottid was not able to protect AML cells from F-Ara-mediated toxicity. It is to be noted that Figure 4A describes the% vitality of the cells, not the% of apoptotic cells, due to the fact that F-Ara directly caused necrosis, rather than apoptosis in AML cells. This could be observed after as early as 24 hours of incubation. Still, Figure 4A clearly shows that Defibrottid does not interfere with the desirable toxicity of F-Ara against leukemic cells.

We then ask whether Defibrottid can modulate the effect of F-Ara against normal hematopoietic cells and perform apoptosis assays (SSC image) with PBMC in normal human blood donors. As could be understood from a representative experiment described in Figure 4B, F-Ara induced apoptosis in 40.1% of the cells compared to 5.1% of apoptotic cells in the untreated control. Again, Defibrottid did not interfere with the apoptotic activity of F-Ara against PBMC (43.1% of apoptotic cells), suggesting that the immunosuppressive properties of F-Ara were not compromised by co-treatment with Defibrotide.

Example 5 F-Ara deregulates the intercellular adhesion molecule 1 (ICAM-1) on HMEC with the antagonistic effects of Defibrotide. On the basis of the previous observations that pretransplant conditioning not only damages, but also leads to proinflammatory activation of endothelial cells in terms of induction of adhesion molecule 15, we next investigate the expression of ICAM-1 under the influence of F-Ara. As described in Figures 5A and B, flow cytometric analyzes demonstrated that F-Ara after 24 hours of incubation significantly improves expression on HMEC in a dose-dependent manner similar to that observed for the induction of apoptosis. . Concentrations of less than 1 9 / p? 1-? of F-Ara were effective to induce ICAM-1. Next we ask if Defibrottid would also function as an F-Ara antagonist in this experimental scenario. The HMECs were treated with F-Ara as such and incubated in the presence or absence of descending concentrations of Defibrottid. Figure 5C summarizes three independent experiments that shows that Defibrott is in effect antagonizing the expression of ICAM-1 induced by F-Ara in concentrations of 100 ^ g / mL and 10 μg / mL. It should be noted that Defibrottid alone did not activate endothelial cells, the expression of ICAM-1 remained unchanged at each concentration tested (data not shown). Since proinflammatory activation of target cells is frequently associated with increased expression of major histocompatibility antigens (MHC) of class I and II, we also analyzed these antigens by flow cytometry after incubation with F-Ara in several concentrations for 24 hours. Despite its well-described immunosuppressive properties, F-Ara induced, surprisingly, MHC class I molecules on HMEC independently of the dose (induction of 1.5 times the intensity of the average fluorescence at 10 μg mL, induction of 1.3 times a 5 μg / mL), while the MHC of Class II remained unchanged (data not shown).

Example 6 F-Ara increases the antigenicity of endothelial cells towards allogeneic peripheral blood cells, protection by Defibrotide The induction of MHC molecules of class I on HMEC by F-Ara encouraged us to examine whether F-Ara would also increase the ability of HMEC to stimulate alocitotoxic responses. Peripheral blood mononuclear cells (PBMC) as effectors were derived from heparinized blood from healthy human volunteers or from serum coating preparations, stimulated with irradiated HMEC (20 Gy) in the presence of 50 U / mL of interleukin 2 (IL-2). ) for 7 days and then subjected to standard 51Cr release test (for details see materials and methods). At day 1 fresh HMEC cells as target were left unstimulated or incubated with F-Ara (10 μg / mL) in the presence or absence of a neutralizing anti-MHC antibody class I (w6 / 32). B lymphoblastoid cell lines transformed with autologous effector Epstein-Barr virus (B-LCL) and K562 as targets of classical natural killer (NK) cells served as controls. Figure 6A demonstrates that F-Ara actually increased the antigenicity of HMEC against allogeneic PBMC at all E / T ratios tested. The absence of specific lysis of K 562 and autologous effector B-LCL verified the involvement of cytotoxic T lymphocytes (CTL) restricted by MHC. In addition, the lysis of HMEC untreated or treated with F-Ara could be blocked almost completely after coincubation of those cells with the anti-MHC class I antibody w6 / 32 (Figure 6A, *) To better confirm that the CD8 + CTLs were responsible for the cytotoxic antiendothelial activity, PBMCs were selected by CD8 + and CD4 + T cells (non-CD8 and non-CD4 depleted PBMC, respectively) using magnetic bead separation with MACSMR bead kits. The purity of the preparations was _ > 93% in all cases with a complete absence of the other cell population (not shown). Separated T cells were stimulated with HMEC and IL-2 exactly as described for unselected PBMCs (see above) · As shown in Figure 6B, the lysis of HMEC treated by F-Ara by CD8 + CTL was again , significantly greater than that of HMEC control. In addition, the pretreatment of white HMEC with F-Ara and Defibrottid (F-Ara + D) deregulated the specific lysis even below control levels, suggesting that Defibrottid also protects endothelial cells against the lysis of allogeneic effector lymphocytes. The CD4 + T cells stimulated with HMEC showed no sign of cytotoxic activity in this experimental scenario (data not shown). Flow cytometric analyzes of HMEC treated with F-Ara against F-Ara + D resulted in a significant deregulation of MHC class I molecules by Defibrottid, suggesting that MHC expression of class I is the critical element in the regulation of the cytotoxic response induced by F-Ara (data not shown).

Example 7 Endothelial anti-CTL has a phenotype similar to that of T0-l. To have information about the nature of endothelial anti-CTL, PBMC and CD8 + T cells were stimulated as above, and supernatants were collected for the evaluation of interferon. gamma (IFN-?) and interleukin 4 (IL-4) using ELISA analysis.

As described in Table 1, stimulation with the HMEC and IL-2 obviously led to increased growth of T01-like T cells as could be said of the only expression of IFN-α, while no IL-4 was produced.

Example 8 F-Ara deregulates the lysis of HMEC by allogenic NK cells Another interesting question was how the F-Ara-induced modulations of MHC expression of class I affect the cytolytic response of natural killer (NK) cells against endothelial cells . PBMCs from healthy individuals were negatively selected by NK cells (non-depleted NK cells) and stimulated for 4 days with irradiated HMEC in the presence of IL-2, as described for the experiment in Figure 6B. At day 4, the HMECs as target cells had been left untreated or incubated with F-Ara (10 μ9 /? -.) For 24 hours and subjected to a standard 51 Cr release assay with the NK cells stimulated as effectors. Figure 7 demonstrates that F-Ara significantly deregulated the allogenicity of HMEC towards NK cells. As a positive control of the activity of the NK cells, a lysis of MHC-negative K 562 cells of class I could be observed (Figure 7, *). The pretreatment of HMEC stimulated with F-Ara with the anti-MHC class I antibody w6 / 32 completely abolished the effect of F-Ara and led to a specific lysis of almost 100% of HMEC (Figure 7), suggesting that MHC class I over the surface of HMEC is, again, the critical switch of the regulation of the cytotoxic response of NK cells. The role of killer cell inhibitory receptors (KIR) that has been found to be negatively regulated by the high expression levels of MHC class I24 molecules may be responsible for the decreased cytolytic response of NK cells. DISCUSSION The clinical results with reduced intensity conditioning regimens (RIC) containing fludarabine obtained in this way show a clear dysregulation of the conditioning-related toxicity without affecting the immune reconstitution.25 The incidence of GvHD in patients receiving RIC is comparable or even less than in those patients who receive the classic conditioning regimen26. Nevertheless, there are reports of late effects that are equally severe or greater, such as osteonecrosis27, pulmonary complications, 28 and more cases of chronic GvHD.29. Despite its well-documented immunosuppressive properties, fludarabine, in our study, has reactivated and damaged endothelial and epithelial cells. This observation may, at least in part, explain the undesirable clinical side effects described above, since osteonecrosis is an expression of endothelial dysfunction, and fludarabine appears to be toxic to alveolar epithelial cells. It is interesting to note that the dangerous effects of fludarabine on lung cells appear to be compartment specific, since bronchial epithelial cells did not suffer apoptosis in response to this immunosuppressant. The fact that the keratinocyte cell line (HaCaT) was also sensitive to fludarabine suggests that it may also be involved in skin disorders after SCT. Because the pathogenesis of late complications is multifunctional and can also be influenced by the increased age of patients with SCT and the use of peripheral non-differentiated cells, further evaluation in clinical analysis of pulmonary and dermatological complications is necessary. Since in many pretransplant protocols fludarabine is used in combination with ionizing radiation it was important to test whether these two compounds would cooperate to affect endothelial cells. Interestingly, we could not find any increase in cell death induced by radiation by fludarabine or vice versa (data not shown). This suggests differential mechanisms of how the apoptotic death signal is transferred to the endothelial cells. The precise mechanism of how fludarabine induces apoptosis in endothelial and epithelial cells remains undetermined. It is likely that fludarabine-as a purine analog-is integrated into DNA and thus produces mutations that lead to genetic suppression as previously reported30. It has also been suggested that fludarabine may cooperate with cytochrome c and apoptosis protein activating factor 1 (APAF-1) in the activation of the apoptotic caspase pathway 31. Fludarabine increases the allogenicity of white cell endothelial CD8 + T cells. In contrast, fludarabine demodulates significantly endothelial lysis by allogenic NK cells. MHC expression of class I appears to be critical for the regulation of any of these immune responses, since a class I blockade completely approved CTL lysis and tremendously deregulated lysis by NK cells. These opposite effects of fludarabine, taken together with the clinical observation that fludarabine shows a less acute toxicity or equal to or even more chronic than the classical conditioning regimen that gives rise to speculation that NK and CTL cells might be active in different phases of the pathophysiology of GvHD, meaning that NK cells would act mainly at the beginning (suppressed by fludarabine) and CTLs in the final phase (augmented by fludarabine) after treatment. With respect to the nature of endothelial anti-CTL, there is an interesting question as to whether these CTLs are endothelial or simply alloespecific. The existence of specific endothelial effector lymphocytes has been previously described32. In contrast to the CTL that we characterize as having a phenotype similar to Tcl, many of the reported CTL clones show little, if any, IFN-? and unusually express CD40 ligand at rest which may increase cytolytic activity33. But these data do not exclude the existence of additional allogenic CTL with a specificity for non-hematopoietic targets. Defibrotide is a well-tolerated drug successfully used for the treatment of veno-occlusive disease as a major hepatic complication after SCT34. In addition, there is an increasing number of preclinical and clinical reports that show its efficacy in the treatment of ischaemia / reperfusion injury and atherosclerosis, as well as recurrent thrombotic thrombocytopenic purpura.35"37 It is known that defibrotide acts directly on endothelial cells without being It requires additional metabolism38, and therefore, could be used in our in vi tro studies.Fibibrotic completely protected endothelial and epithelial cells from fludarabine-mediated apoptosis Additional experiments are needed to evaluate the precise protection mechanism by which defibrotide antagonizes fludarabine, but one can imagine a role for defibrotic in an inhibition of the integration of fludarabine DNA or caspase activation mentioned above In addition to its antiapoptotic effects, defibrotide was able to regulate the endothelial anti-CTL response regulating the MHC expression of Class I. In contrast, defibrotide did not affect the desirable antileukemic effect of fludarabine, as shown by the absence of protection of AML cells. Another important observation was that defibrottid can not block the fludarabine-mediated apoptosis of PBMC. This suggests that the immunosuppressive effect of the fludarabine required for conditioning is not influenced by a cotreatment with Defibrottid. It should be noted that Defibrottid was not protective against radiation-induced cellular damage, suggesting that its effect is specific for fludarabine-mediated cellular changes (data not shown). On the basis of these results with respect to their few, if any, side effects, 39 we conclude from our study that defibrottide is a good candidate used in combination with fludarabine during conditioning prior to SCT, especially in patients at risk of VOD Studies that analyze endothelial protection against additional conditioning agents will help clarify whether defibrottide can be used as a broad protective agent.

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Microangiopathy in patients on cyclosporine pro-phylaxis who developed acute graft-versus-host disease after HLA-identical bone marrow transplantation. Blood. 1989; 73: 2018-2024. 11. Eissner G, Kohlhuber F, Grell M et al.

Critical involvement of transmembrane TNF-a in endothelial programmed cell death mediated by ionizing radiation and bacterial endotoxin. Blood. nineteen ninety five; 86: 4184-4193. 12. Lindner H, Holler E., Ertl B et al. Peripheral blood mononuclear cells induces pro-grammed cell death in human endothelial cells and may prevent repair-role of cytokines. Blood. 1997; 89: 1931-1938. 13. Haimovitz-Friedman A, Balaban N, Mcloughlin et al. Protein kinase C mediates basic fibroblast gro th factor protection of endothelial cells against radiation-induced apoptosis. Cancer Res. 1994; 54: 2591-2597. 14. Fuks Z, Persaud RS, Alfieri A et al. Bacic fibroblast growth factor protects endo-thelial cells against radiation-induced programmed cell death in vitro and in vivo. Cancer Res. 1994; 54: 2582-2590. 15. Eissner G, Lindner H, Behrends U et al. Influence of bacterial endotoxin on radiation-induced activation of human endothelial cells in vitro and in vivo, protective role of IL-10. Transplantation nineteen ninety six; 62: 819-827. 16. Lindner H, Holler E, Gerbitz A, Johnson JP, Bornkamm GW, Eissner G. Influence of bacterial endotoxin on radiation-induced activation of human endothelial cells in vitro and in vivo: 2. IL-10 protects against transendothelial migration. Transplantation. 1997; 64: 1370-1973. 17. Beelen DW, Haralambie E, Brandt H et al. Evidence that increased the suppression of intestinal anaerobe bacteria reduces the risk of graft-versus-host disease after sibling marrow transplantation. Blood. 1992; 80: 2668-2676. 18. Eissner G, Multhoff G, Holler E. Influence of bacterial endotoxin on the allogenicity of human endothelial cells. Bone Marrow Transplant. 1998; 21: 1286-1287. 19. Ades EW, Candal FJ, Swerlick RA et al, HMEC-1: establishment of an immortal-ized human microvascular endothelial cell line. J Invest Dermatol. 1992; 99: 683-690. 20. Cotter TG, LennonSV, Glynn JM, Green DR. Microfilament-disrupting agents prevent the formation of apoptotic bodies in tumor cells undergoing apoptosis. Cancer Res. 1992; 52: 997-1005. 21. Lee A, Whyte MK, Haslett C. Inhibition of apoptosis and prolongation of neutrophil functional longevity by inflammatory mediators. J Leukoc Biol. 1993; 54: 283-288. 22. Westphal JR, Tax J, Willems HW, Koene RA, DJ Ruiter, De-Waal RM. Accessory function of endothelial cells in anti-CD3-induced T-cell proliferatio: synergism with monocytes. Scand J Immunol. 1992; 35: 449-457. 23. MacDonald HR, Engers HD, Cerrottini JC, Brunner KT. Generation of cytotoxic T lymphocytes in vitro. J Exp Med. 1974; 140: 718-722. 24. Long E. Regulation of immune responses through inhibitory receptors. Annu Rev Immunol. 1999; 17: 875-904. 25. Nagler A, Aker M, Or R etal. Low-intensity conditioning is sufficient to ensure engraftment in matched unrelated bone marrow transplantation. Exp Hemato. 2001; 29: 362-370. 26. Michallet M, Bilger K, Garban F et al. Allogeneic hematopoietic stem-cell tranplantation after nonmyeloablative preparative regimens: impact of pretransplantation and posttransplantation factors on outcome. J ClinOncol. 2001; 19: 3340-3349. 27. Holler E, communication personnel 28. Hildebrandt G, Bertz H, Mestan A et al. Analysis of pulmonary function after alio- genic bone in blood transplantation (BMT) or blood stem cell transplantation (PBSCT) using conditioning regimens with total body irradiation (TBI) and conventional intensity compared to regimens without TBI with reduced intensity [ab-stract] . Bone Marrow Trans lant. 2001; 27 (Suppl 1): S216. 29. Bornhauser M, Thiede C, Schuler U et al. Dose-reduced conditioning for allogeneic blood stem cell transplantation: durable engraftment without antithymocyte globulin. Bone Marrow Transplant. 2000; 26: 119-125.

. Huang P, Siciliano MJ, Plunkett. Gene deletion, a mechanism of induced mutation by arabinosyl nucleosides. Mutat Res. 1989; 210: 291-301. 31. Genini D, Budihardjol, Plunkett W et al. Nucleotide requirements for the in vitro activation of apoptosis protein-activating factor-1-mediated caspase path ay. J Biol Chem. 2000; 275: 29-34. 32. Biederman BC, Pober JS. human vascular endothelial cells favorclonal expansion of unusualalloreactive CTL. J Immunol. 1999; 162: 7022-7030. 33. Briscoe D, Alexander Al, Lichtman AH. Interactions between T lymphocytes and endothelial cells in allograft rejection. Curr Op Immunol. 1998; 10: 525-531. 34. Pegram AA, Kennedy LD. Prevention and treatemnt of veno-occlusive disease. Ann Pharmacother. 2001; 35: 935-942. 35. Rossini G, Pompilio G, Biglioli P et al. Protectant activity of defibrotide in cardioplegia followed by idchemia / (repersion injury in the isolated rat heart J Card Surg 1999, 14: 334-341 36. Rossini G, Berti F, Trento F et al. Defibrotide normalizes cardiovascular function hampered by establishedatherosclerosis in the rabbit, Thromb Res. 2000; 97: 29-38, 37. Pogliani EM, Perseghin P, Parma M, Pioltelli P, Corneo G. Defibrotide in recurrent thrombotic thrombocytopenic purpura, Clin Appl Thromb Hemost., 2000; 6 : 69-70 38. San G, Moini H, Emerk K, Bilsel S. Protective effect of defibrotide on perfusion induced endothelial damage, Throm Res. 2000; 99: 335-341 39. Chopra R, Eaton JD, Grassi A et al., Defibrotide for the treatment of hepatic veno-occlusive disease: results of the European compassionate-use study, Br J Haematol, 2000; 111: 1122-1129.

Claims (28)

1. CLAIMS 1. The use of a protective oligodeoxyribonucleotide for the preparation of a drug for the treatment of a patient undergoing treatment with an immunosuppressant.
2. 2. The use of a protective oligodeoxyribonucleotide for the preparation of a drug to protect epithelial and / or endothelial cells from the effects of an immunosuppressant.
3. 3. The use of a protective oligodeoxyribonucleotide for the preparation of a medicament for protecting epithelial and / or endothelial cells from apoptosis and / or activation induced by the administration of an immunosuppressant.
4. 4. The use according to claims 1-3, wherein the immunosuppressant is a nucleoside.
5. 5. The use according to claims 1-3, wherein the immunosuppressant is selected from the groups comprising fludarabine, cyclophosphamide, BCNU, melphalan.
6. 6. The use according to claims 1-3, wherein the immunosuppressant is fludarabine.
7. 7. The use according to claims 1-3, wherein the protective oligodeoxyribonucleotide is defibrotide.
8. 8. The use according to claims 1-7, wherein the step of administering the protective oligodeoxyribonucleotide occurs concomitantly, simultaneously, after or before the administration of the immunosuppressant to the patient.
9. The use according to claim 8, wherein the step of administering the protective oligodeoxyribonucleotide occurs after administering the immunosuppressant to the patient.
10. The use according to claim 9, wherein the delay time between the step of administering the protective oligodeoxyribonucleotide and administering the immunosuppressant to the patient is from about 1 hour to about 2 weeks, preferably from about 2 days to about 7 days.
11. The use according to claim 8, wherein the step of administering the protective oligodeoxyribonucleotide occurs prior to administering the immunosuppressant to the patient.
12. The use according to claim 11, wherein the time difference between the step of administering the protective oligodeoxyribonucleotide and that of administering the immunosuppressant to the patient is from about one hour to about two weeks, preferably from about two hours to about two days.
13. 13. The use according to claims 1-12, wherein the dose of defibrottide administered is chosen so that an assay level of approximately 100 μg / G is achieved. to about 0.1 μ9 / p ?? -, up to about 0.1 μ9 /? 1, preferably from about 10 μg / mL to about 100 μ9 / ??? -.
14. The use according to claim 13, wherein the dose of defibride administered is chosen so that a blood level of about 10 μ / ml is reached.
15. 15. The use according to claims 1-14, wherein the dose of defibride administered is about 100 mg / kg of body weight of the patient up to about 0.1 mg / kg of body weight, preferably about 20 mg / kg of body weight of the patient. patient up to approximately 0.1 mg / kg of body weight.
16. The use according to claim 15, wherein the dose of defibride administered is about 15 mg / kg of body weight of the patient to about 1 mg / kg of body weight, preferably about 12 mg / kg of body weight of the patient.
17. 17. The use according to any of the preceding claims wherein the activation includes increasing the expression of ICAM-1.
18. 18. The use according to any of the preceding claims wherein treatment with an immunosuppressant occurs during the transplantation of undifferentiated cells.
19. 19. The use according to claim 18, wherein the transplantation of undifferentiated cells is the transplantation of non-differentiated allogeneic cells.
20. 20. A pharmaceutical composition containing a therapeutically effective dose of an immunosuppressant and a protective oligodeoxyribonucleotide.
21. 21. A pharmaceutical composition according to claim 20 constituted by two formulations separately administrable, different, one containing the immunosuppressant and the other the protective oligodeoxyribonucleotide.
22. 22. A pharmaceutical composition according to claim 20 as a combined preparation for simultaneous use, sequentially separated.
23. 23. A pharmaceutical composition according to claims 20-22 wherein the immunosuppressant is a nucleoside.
24. 24. A pharmaceutical composition according to claims 20-22 wherein the immunosuppressant is selected from the group comprising fludarabine, cyclophosphamide, BCNU, melphalan.
25. 25. A pharmaceutical composition according to claims 20-22 wherein the immunosuppressant is fludarabine.
26. 26. A pharmaceutical composition according to claims 20-22 wherein the protective oligodeoxyribonucleotide is defibrotide.
27. 27. A pharmaceutical composition according to any of the preceding claims, characterized in that it also contains the customary excipients and / or adjuvants. 28. "A pharmaceutical composition according to any of the preceding claims, characterized in that it is injectable intravenously.