MXPA05005778A - New use of dextran sulfate - Google Patents

Abstract

The present invention refers to use of dextran sulfate, or a pharmaceutically acceptable derivate thereof, for manufacturing of a medicament for treatment of Instant Blood-Mediated Inflammatory Reaction (IBMIR). In addition, the invention refers to the use of dextran sulfate, or a pharmaceutically acceptable derivate thereof, for manufacturing of a medicament for treatment of morphological disruption of cell transplants and graft-rejection of cell transplants caused by IBMIR. The invention may be applied to patients suffering from type I diabetes, in which porcine islets of Langerhans are transplanted in their portal vein. Administration of dextran sulfate according to the invention inhibits and prevents rejection and destruction of the transplanted islets and makes normoglycemia in the patients possible.

Description

New use of dextran sulfate Technical Field The present invention relates to the new use of dextran sulfate.

Background Today, about 10 million people worldwide suffer from type I diabetes, which is also referred to as insulin-dependent diabetes mellitus. However, it is estimated that the number of people affected will increase drastically and may affect about 25 million by the year 2010. Until today, the research focuses on trying to obtain permanent normoglycemia in patients with type I diabetes by introducing beta cells , producers of insulin. The two main procedures are the transplantation of vascularized pancreatic grafts or that of isolated islets of Langerhans. Although some success has been achieved with vascularized grafts (whole pancreas), problems still persist, mainly due to the risk of surgery as well as post-operative complications. There is also the problem of the shortage of useful pancreatic graft donors. In contrast, the transplantation of isolated pancreatic islets is conventionally carried out by transhepatic injection into the portal vein, with which the islets embolize in the portal tree of the Mgado.

A new protocol for islet transplantation that was recently introduced by Shapiro et al. [1] will undoubtedly be beneficial to a number of patients with type I diabetes. However, even with this new advance, it has been observed that the transplantation of islets from a Pancreas donor in particular is not sufficient to produce normoglycemia in a patient [2]. As a result, the supply of human islets is expected to become a limiting factor in the treatment. They will have to be found, then, alternative sources of insulin-producing cells. One option is the use of islets prepared from animal tissue, with pig islets, being the main candidate.

One of the main obstacles to be solved before isotransplantation is possible, is the harmful inflammatory reaction caused by porcine islets when exposed to human blood in vitro and in vivo [3]. In addition, islets of the human being induce a harmful inflammatory reaction when exposed to ABO-compatible blood from patients at the time of intraportal transplantation [4]. The inflammatory reaction is characterized by a rapid consumption and activation of platelets, which adhere to the surface of the islet promoting the activation of both the coagulation cascade and complement. In addition, the islets are fixed in clots and infiltrated by CDllb + leukocytes, which together generate a destruction of the cell morphology and loss of normoglycemia in patients [3-4]. Likewise, inflammation can accelerate a successful cell-mediated specific immune response at a later stage [5-8]. Consequently, the inhibition of the instantaneous, blood-mediated inflammatory reaction (IBMIR), as the harmful inflammatory reaction is called, appears critical to the success of isotransplantation and alotransplantation.

Two recent studies by Buheler et al. [5] and Cantarovich et al. [6] have shown that adult pig islets are instantaneously destroyed when they are transplanted intraportally to the non-human primate liver even under conditions of extensive immunosuppression of conventional prior art. In these studies, the authors concluded that a powerful innate immune response, IBMIR, which is not affected by immunosuppressive drugs, is linked in the destruction of xenogeneic islets.

Fiorante et al. have studied the use of dextran sulfate for the prevention of hyperacute rejection (Hyperacute Rejection, HAR) of discordant vascularized xenografts [9]. Pig lungs drilled with citrate-anticoagulated human blood experienced HAR at the end of 30 minutes within the xenotransplant model. However, the addition of dextran sulfate at 2 mg / ml prolonged the survival of the lungs by about 200 minutes. The HAR of vascularized complete organs is mediated through the reaction of antibodies in human blood, which identify and bind antigens exposed on the endothelial cells of the blood vessels of transplanted organs. This JHAR antibody-mediated reaction is obtained by components of the complement system [8, 10, 11]. Whenever dextran sulfate is also known to inhibit complement activation [9, 12] the prolongation of lung survival when dextran sulfate is used in the model used for xenotransplantation is believed to derive from this anti-complement effect of dextran sulfate . Nakano et al. Have transplanted isolated syngeneic islets into the livers of STZ-induced diabetic mice in order to investigate the role of hepatocyte growth factor (HGF) in the improvement of hyperglycemia [13]. Dextran sulfate is known to increase the effect of HGF and consequently HGF was administered interperitoneally in the recipient mouse in conjunction with dextran sulfate. Such administration produced normoglycemia in all the mice under investigation. Also the administration of dextran sulfate alone, showed some beneficial effects in some mice, but not when the subcapsular renal space was the site of the islet transplantation.

Treatment of additional anti? GF antibodies to mice administered with dextran sulfate completely abolished the beneficial effects of dextran sulfate, indicating that the effect of dextran sulfate in this model of allogeneic transplantation of islets in mice is mediated via endogenous HGF.

Thomas et al. [14] have shown that soluble dextran sulfate derivatives inhibit complementary activation and mediated complementary damage in vitro. Porcine aortic endothelial cells incubated in human serum resulted in the consumption of complement and deposition of activated fragments C3, C5 and the membrane attack complex C5b-9 in endothelial cells. Addition of 25 mg / ml of CMDB25 dextran sulfate activation of inhibited complement and complex cytolytic deposition on cells. Native Dextran did not have such an effect.

DESCRIPTION.

The present invention overcomes these and other drawbacks of prior art arrangements.

It is a general object of the present invention to provide a treatment for instant blood-mediated inflammatory reaction (Instant Blood-Mediated Inflamatory Reaction, IBMIR).

It is another object of the present invention to provide a morphological disruption treatment of cell transplants caused by the IBMIR.

Yet another object of the present invention is to provide a treatment for graft rejection of cell transplants caused by the IBJMIR.

This and other objects are found by the invention as defined by the complementary claims.

Briefly, the present invention involves the use of dextran sulfate and derivatives thereof for the treatment of the blood-mediated instant inflammatory reaction (Instant Blood-Mediated Inflamatory Reaction, IBMIR). This new characterized form of inflammation is caused when cells or groups of cells are exposed to foreign blood in vitro and in vivo. A very important example of IBJMIR is when xenogeneic or allogeneic cell transplants are transplanted into the body of a recipient mammalian patient, especially a human. The IBMIR will then direct a morphological disruption and destruction of cells or groups of transplanted cells, manifested in loss of structure and form. In addition, IBMIR generally also results in graft rejection in cell transplantation.

Administration of dextran sulfate and derivatives thereof abrogate the deleterious effect of IBMIR and effectively prevent graft rejection as well as the morphological disruption of cell transplants. The dextran sulfate according to the invention can have a molecular weight of low molecular weight dextran sulphate (BPM-SD), for example from a few or thousands of Daltons (Da) to high molecular weight dextran sulphate (APM-SD) ), generally with a molecular weight over 500,000 Da. The advantageous effect of dextran sulfate is especially prominent for BPM-SD, but a positive effect is observed by the administration of dextran sulfate with a higher molecular weight. The advantageous effect of longer dextran sulfate molecules on IBJ JIR according to the invention can be increased by increasing the sulfide content, for example the number of sulfate groups per glucosyl residue in the dextran chain. BPM-SD generally has an average molecular weight below 20,000 Da, such as below 10,000 Da and, for example, about 5,000 Da. The average sulfur content for BPM-SD can be from 10 to 25%, such as 15 to 20%, corresponding to about two sulfate groups per glucosyl residue. For dextran sulfate with an average molecular weight greater than 20,000 Da, a longer sulfur content may be employed.

Dextran sulfate and derivatives thereof, can be administered systemically to the IBMIR site or cellular transplant, or it can be administered via direct (locally) to that site. Thus, according to the invention, dextran sulfate and derivatives thereof can be administered orally, intravenously, intraperitoneally, subcutaneously, buccally, rectally, dermally, nasally, tracheally, bronchially, topically, by any other patent means or via inhalation, in the pharmaceutical preparation form comprising the active ingredient in the form of a pharmaceutically acceptable dose.

In therapeutic treatment of mammals, and particularly humans, dextran sulfate and derivatives thereof, can be administered alone, but are generally administered as a pharmaceutical formulation in admixture with a pharmaceutically acceptable adjuvant, diluent or carrier, which can be selected with due consideration to the intended administration route and standard pharmaceutical practice.

The amounts of dextran sulfate or derivatives thereof in the formulation will depend on the severity of the condition as well as the patient to be treated and the current formulation and route of administration employed and can be determined not inventively by the skilled person. The concentration of administered dextran sulfate, or derivatives thereof, according to the present invention should not be so high as to minimize any side effects associated with dextran sulfate. In most clinical situations suitable doses of dextran sulfate, or derivatives thereof, in therapeutic or prophylactic treatments of mammals, particularly humans, patients are those that give a mean blood concentration below 5 mg / ml, probably lower than 2 mg / ml and especially less than 1 mg / ml. A preferred concentration range is between 0.01 mg / ml and 1 mg / ml dextran sulfate, such as, greater than 0.05 mg / ml, more than 0.08 mg / ml or more than 0.01 mg / ml and / or less than 0.08 mg / ml. ml, less than 0.6 mg / ml, less than 0.4 mg / ml or less than 0.02 mg / ml, for example within the concentration range of 0.01 mg / ml and 0.2 mg / ml and / or 0.05 mg / ml and 0.2 mg / ml.

The dextran sulfate according to the present invention is especially suitable for the prevention of graft rejection of transplanted insulin-producing beta cells in patients suffering from type I diabetes. In such patients, islets of Langerhans from other humans or mammals, preferably porcine islets. , can be transplanted by injection of the islets into the portal vein of patients. However, once the islets are exposed to the patient's blood, the IBMIR is prompted and the islet's insulin regulatory function will be destroyed and the islets rejected. Therefore, a therapeutic concentration of dextran sulfate, or derivatives thereof, is preferably obtained, at least locally at the transplant site once the transplantation of cells or cell groups is effected. This can be obtained by administering dextran sulfate prior to the current transplant. Alternatively, the islets can be injected dissolved in a solution comprising dextran sulfate according to the present invention so as to inhibit the IBMIR as well as foresee any destruction of islet rejection, making normoglycemia possible in the patients. The concentration of dextran sulfate in such a cell and the dextran sulfate solution is preferably sufficiently high, such that a therapeutic concentration of dextran sulfate, i. and. preferably less than 5 mg / ml, more preferably 0.01 mg / ml to 1.0 mg / ml, and especially 0.01 mg / ml to 0.2 mg / ml, can be obtained, at least locally, at the transplant site during the first hours after the transplant. The dextran sulfate will then diffuse from the transplant site by lowering the local sulfate concentration. In some applications, additional dextran sulfate is not necessary to inhibit IBMIR, morphological disruption and / or graft rejection of transplanted cells, since a therapeutic concentration of dextran sulfate is probably necessary only for the first 24-48 hours after the transplant.

However, if necessary, additional dextran sulfate can be added, for example intravenously, intraperitoneally, or by any other route of administration. As is understood by a person skilled in the art, the administration of a dextran sulfate solution comprising the cells or cell groups to be administered can also be combined with administration of dextran sulfate or derivatives thereof, prior to the transplant under discussion.

The dextran sulfate and derivatives thereof can also be combined with other therapeutic agents useful in the treatment of graft rejection or in transplanted tissue. Examples suitable but not limited to such immunosuppressive agents that can be used with dextran sulfate for the treatment of graft rejection are cyclosporin, tacrolimus, corticosteroids, rapamycin (sirolimus) and mycophenolate mofetil. In accordance with the present invention, the administration of dextran sulfate can also be coordinated with administration of anti-TF antibodies and / or site-inactivated factor Vlla, which also have some functionality in the inhibition of IBMIR.

BRIEF DESCRIPTION OF THE DRAWINGS The invention together with additional objectives and advantages thereof can be understood by reference to the following description taken in conjunction with the complementary drawings in which ' Figure 1. Is a diagram illustrating the effect of BPM-SD on the generation of C3a during the perfusion of porcine islets with human blood? Figure 2. It is a diagram illustrating the effect of BPM-SD on the generation of sC5b-9 during the perfusion of porcine islets with human blood.

Figure 3. It is a diagram illustrating the direct effect of BPM-SD on the complementary system when human serum is incubated in the presence of BPM-SD, Figure 4. Illustrates the distribution of leukocytes in porcine islets after perfusion with human blood not containing BPM-SD; Figure 5. Illustrates the distribution of leukocytes in porcine silages after perfusion with human blood, containing 1 mg / ml BPM-SD; Figure 6. Illustrates the distribution of platelets in porcine islets after perfusion with human blood without containing BPM-SD; Figure 7. Illustrates the distribution of platelets in porcine islets after perfusion with human blood containing 1 mg / ml BPM-SD; Figure 8. Illustrates the distribution of insulin in porcine silts after intraportal transplantation in athymic diabetic mice without the BPM-SD treatment; Figure 9. Illustrates the distribution of leukocytes in porcine scutes after perfusion with human blood, containing 1 mg / ml BPM-SD; Figure 10. Illustrates the distribution of leukocytes in porcine islets after intra-portal transplantation in athymic diabetic mice without BPM-SD treatment; Y Figure 11. Illustrates the distribution of leukocytes in porcine islets after intraportal transplantation in athymic diabetic mice treated with BPM-SD; CARVED DESCRPTION The present invention generally relates to the novel effect of dextran sulfate on the instant blood-mediated inflammatory reaction (IMIR) and morphological disruption and graft rejection of transplanted cells as a consequence of the IBMIR.

The IBMIR is a relatively identified inflammatory reaction recently activated by the exposure or contact of cells or cell groups with xeno-blood. The IBMIR is characterized by the expression or tissue factor on the cells, activating the local generation of a thrombin. Subsequently, activated platelets adhere to the surface of the cell promoting the activation of both coagulation and complementary systems. In addition to this, the leukocytes are confined and infiltrate the cells. Such combined effects cause a disruption of the morphology of the cells during the first hours of contact with the foreign blood. The IBMIR also accelerates the subsequent cell-mediated specific immune response at a later stage.

An important example of IBMIR is when cells or cell groups are preferably transplanted into the body of a mammal, and especially human. From the contact with the blood of the recipient patient, the cells activate the IBMIR, which results in the disruption of the morphology of the cells and generally in the graft rejection of the cell transplant. The IBMIR has been detected both in allogeneic cell transplantation, where cells of a donor with ABO-eompatible blood are transplanted into a patient and in xenogeneic cell transplantation, including xenotransplantation of cells and / or pig-to-monkey cell group and pig-a-human.

In the present invention, the term "cell transplantation" generally refers to an independent cell, several independent cells or groups of many cells transplanted into a recipient body of, preferably, mammals and, especially, human patients.

Also included in the term cell transplantation that we use here are long cell groups of non-vascularized tissues. An example of cellular transplants according to the present invention are allogeneic and xenogeneic islets of Langerhans transplanted into the portal vein of the liver of patients affected by type I diabetes. Another example may be transplantation of tissue / embryonic xenogeneic neural cells into the liver. stratium of patients with Parkinson's disease.

As discussed briefly in the section of the state of the art, a promising procedure for obtaining normoglycemia in patients with type I diabetes is to transplant insulin-producing beta cells, for example within the portal vein. Suitable insulin-producing beta cells, for example in the form of Langerhans islets, can be obtained from both allogeneic and xenogeneic donors. Since the islets of various donors are necessary to obtain normoglycemia and the lack of suitable human donors, xenogeneic islets, preferably porcine, can be used. However, both allogeneic and xenogeneic islets cause IBJMIR when exposed to the blood of the recipient patient. As a result, within a few hours after the transplant the cell morphology is broken and destroyed, usually manifested in loss of integrity, structure and shape of the cells. This results in a very pronounced initial release of insulin from the islets of Langerhans followed by a decrease or loss of insulin release. That is, the loss of normoghcemia occurs rapidly after transplantation. Furthermore, IBMIR also causes graft rejection of cell transplants.

The administration of conventional immunosuppressive drugs that prevent the production of antibodies and rejection of organs has no effect on the IBMIR in graft rejection in transplanted cells caused by the IBMIR. This indicates that the main mechanisms of the IBMIR and graft rejection in cell transplantation differ from those of rejection found in transplantation of whole or whole organs and vascularized tissue.

In the following lines a more detailed analysis of the symptoms of the IBMIR continues and particularly, consumption, coagulation and platelet complement activation and leukocyte infiltration. In addition, the effects of dextran sulfate on the respective symptoms are analyzed. For a more detailed study of these effects of dextran sulfate reference is made to the examples section.

Starting with the consumption of platelets, the IBMIR affects the platelet count in the blood exposed to cells or groups of allogeneic or xenogeneic cells. A significant decrease in circulating free platelets can be noticed in blood after blood-cell contact. The platelets are activated and adhered to the cells, originating an aggregate of platelets. After adhesion of the cells, the platelets release various substances including platelet phospholipids, important for clot formation and activation of the coagulation system.

The administration of an effective amount of dextran sulfate according to the invention inhibits the platelet consumption seen as an increase in the platelet count in the blood returning to the value measured in the blood before exposure to foreign cells or cell groups. Also, cell adhesion is considerably diminished by dextran sulfate, although quantities of platelet footprints surrounding the islets can still be seen. The effects of these remaining platelets is not, however, necessarily a disadvantage. Studies of animals have shown that after transplantation at least a week elapses before angiogenesis is detected [15,16]. Platelets contain a number of important growth factors, such as platelet-derived growth factor (Platelet-Derived Growth Factor, PDGF), endotehal vascular growth factor (Vascular Endotehal Growth Factor, VEGF) and fibroblast growth factor (Fibroblast Growth Factor , FGF) [17, 18], which can sustain the revascularization and fungal graft of islet in the patient's body. In the case of clinical islet transplantation, when the islets are embolized within the portal vein, a circular spiral of adhered platelets will similarly support their functioning graft and survive in the liver tissue.

From the contact with the blood, the foreign cells activate the coagulation system, through the expression of factors woven on the cells and through substances released by the adhesion and addition of platelets. Slightly, the tissue factor (TF) produced by the cells complexes with the blood coagulation factor Vlla and acts enzymatically on the X factor to form the activated factor X (FXa). Then there follows a cascade of activation of various factors, which eventually result in the generation of prothrombin thrombin. Thrombin, in turn, produces the polymerization of fibrinogen molecules in the fibrin fibers formed by a fibrin clot around the cells, all of which is well known to a person skilled in the art. Thrombin also activates the intrinsic pathway to initiate blood coagulation, in which factor XII (Factor Hageman) is activated (FXIIa) and instead enzymatically activates factor XI (background of thromboplastin), resulting in FXIa, the activated form of factor XI. Also this path eventually results in the generation of prothrombin thrombin as for the TF-activated extrinsic pathway.

Blood coagulation can be inhibited by antithrombin, a circulating serine protease inhibitor that inactivates FJXIIa, FXIa and thrombin, formed in JXEIa-antithrombin factor (FXIIa-AT), Xla-antithrombin factor (FXIa-AT) and thrombin-antithrombin complexes ( TAT). Additionally, Cl esterase inhibitor is a known inhibitor of FXIa and FXIIa complexes, esterase inhibitor Xla-Cl (FXIa-Cl INH) and esterase inhibitor factor XHa-Cl (FXIIa-Cl INH).

A fibrin clot once formed around the cells or group of cells can be removed by the action of plasmin from the fibrinolytic system. Plasmin degrades the fibrin clot in degraded fibrin products, thus preventing further coagulation. However, the action of plasmin is inhibited by antiplasmin alpha 2, which binds and activates inactive plasmin in serum forming a complex plasmin-alpha 2 antiplasmin (PAP).

IBMIR is characterized by the formation of fibrin clots around cells exposed to foreign blood in vitro and in vivo. In addition, an increase in FXIa-AT, FXIIa-AT, TAT and PAP is detected. The IMBIR has no effect on the amount of FJXIa-Cl INH or on FXIIa-Cl INH. The administration of an effective amount of dextran sulfate according to the invention abrogates the effect of IMBIR on the activation of coagulation, which is manifested in a decrease in the amount of FXIa-AT, FXIIa-AT, TAT and PAP detected in the blood.

The effect of dextran sulfate on coagulation activation can be mediated through the coagulation system per se through the inhibitory effect of dextran sulfate on platelet activation or both Subsequent activation of platelets and coagulation occurs in the IBMIR a cascade complement. One of the components of the complementary system is C3, which when activated is cleaved into the small fragment C3a, a peptide mediator of inflammation and the longest fragment C3b. C3b, on the other hand, binds to other components of the complementary system forming convertase C5, which adheres C5 inside C5a, which spreads away, and the active form C5b, which adheres to the surface of the cell. The C5a link then binds 4 further complementary components forming the membrane attack complex c5b-9. This complex displaces the membrane phospholipids forming long membrane channels, which interrupt the membrane and act on ions and small molecules to spread freely. Therefore, the cell can not maintain its osmotic stability and is lysed by an influx of water and loss of electrolytes.

The majority of platelet consumption has already occurred before effects mediated by complement of the IBMIR can be detected, suggesting that the coagulation reaction can induce complement activation. IBMIR causes significant complement activation when measured by a C3a increase and soluble c5b-9 membrane attack complex in the blood. The administration of an effective amount of dextran sulfate according to the invention reduces the amount of these complement components in the blood in a dose-dependent manner.

IBJMIR is characterized by the infiltration of leukocytes into cells or groups of cells. The infiltration of CDII + polymorphonuclear cells and monocytes into the cells is clearly detected by immunohistochemical labeling. Anahsis immunohistochemicals showed that leukocyte infiltration was completely abrogated by the administration of dextran sulfate.

According to a first aspect of the invention there is provided the use of dextran sulfate or a pharmaceutically acceptable derivative thereof in, in the manufacture of a medicament for the treatment of the IBMIR.

According to another aspect of the invention, the use of dextran sulfate or a pharmaceutically acceptable derivative thereof is provided in the manufacture of a medicament for the treatment of morphological disruption of transplanted transplanted cells. Also the use of dextran sulfate or a pharmaceutically acceptable derivative thereof, in the manufacture of a medicament for the treatment of graft rejection of cell transplants is within the scope of the present invention. These two effects, disruption of cell morphology and graft rejection, in transplanted cells, groups of cells or non-vascularized tissue in mammals, preferably humans, are due to the deleterious effect of the IBMIR. The IBMIR-mediated effect on cell transplantation occurs both in human-to-human transplantation with ABO compatible donors and used other mammalian donors, preferably pigs. Thus, dextran sulfate has an advantageous therapeutic effect both in allogeneic and xenogeneic cell transplants.

To avoid confusion, as used herein, the term 'treatment' includes the therapeutic and / or prophylactic treatment of IBMIR. 'Pharmaceutically acceptable derivatives' includes salts and solvents.

The dextran sulfate, or derivatives thereof, used in accordance with the invention may have a molecular weight from low molecular weight dextran sulfate (BPM-SD), v. g. from a few hundred or thousands of Daltons (Da), to high molecular weight dextran sulfate (APM-SD), generally with a molecular weight greater than 50000 Da, v. g. > 1 000 000 Da The advantageous effect of dextran sulfate is especially prominent for BPM-SD, but a positive effect is also noted by the administration of dextran sulfate with a higher molecular weight. However, longer dextran sulfate molecules can activate FJXII causing some side effects, which is discussed in more detail below. The advantageous effect of longer dextran sulfate molecules on the IBMIR according to the invention can be raised by increasing the sulfide content, i. and. the number of sulfate groups per glucosyl residue in the dextran chain. BPM-SD generally has an average molecular weight of less than 20,000 Da, such as below 10,000 Da v. g. about 5,000 Da. The average sulfur content for BPM-SD can be approximately 10 to 20%, such as 15 to 20% corresponding to approximately 2 sulfate groups per glucosyl residue. For dextran sulfate with an average molecular weight greater than 20,000 Da, a higher sulfur content may be employed.

According to a further aspect of the present invention there is provided an IBMIR treatment method which comprises administering a therapeutically effective amount of dextran sulfate or a pharmaceutically acceptable derivative thereof to a patient in need of such treatment.

Additional aspects of the invention are a method for the treatment of graft rejection of cell transplants, which comprises administering a therapeutically effective amount of dextran sulfate, or a pharmaceutically acceptable derivative thereof, to a patient in need of such treatment, and a method of treating morphological disruption of transplantation of transplanted cells, which comprises therapeutically administering an effective amount of dextran sulfate or a pharmaceutically acceptable derivative thereof, to a patient in need of such treatment.

The dextran sulfate, and derivatives thereof, may be administered systemically to the site of the IBMIR or cell transplant or may be administered directly (locally) to that site using an appropriate means of administration that is known to the skilled person.

Therefore, according to the invention, dextran sulfate, and derivatives thereof, can be administered orally, intravenously, intraperitoneally, subcutaneously, buccally, rectally, dermally, nasally, tracheally, bronchially, topically by any other patent route or via inhalation. in the form of a pharmaceutical preparation comprising the active ingredient in the form of a pharmaceutically acceptable dose. Depending on the form of cell transplantation, the place of transplantation and the patient to be treated, as well as the route of administration, the compositions may be administered in varying doses.

In therapeutic treatment of mammals, and especially humans, dextran sulfate, and derivatives thereof, can be administered alone, but will generally be administered as a pharmaceutical formulation in admixture with a pharmaceutically acceptable adjuvant, diluent or carrier, which can be selected with due relationship to the intended administration route and standard pharmaceutical practice.

The amounts of dextran sulfate, or derivatives thereof, in the formulation will depend on the severity of the condition, and on the patient to be treated, as well as the current formulation and route of administration employed and can be determined non-inventively by the person skilled in the art. . The concentration of administered dextran sulfate, or derivatives thereof, according to the present invention should not be so high in order to minimize any side effects associated with dextran sulfate. In most clinical situations appropriate doses of dextran sulfate, or derivatives thereof, in the prophylactic and / or therapeutic treatment of mammals, especially humans, patients are those that give a level of concentration in the blood below 5 mg / ml , probably less than 2 mg / ml and particularly less than 1 mg / ml. A preferred concentration range is between 0.01 mg / ml and 1 mg / ml dextran sulfate, such that it is greater than 0.05 mg / ml, more than 0.08 mg / ml or more than 0.1 mg / ml and / or less than 0.8 mg / ml. ml, less than 0.6 mg / ml, less than 0.4 mg / ml or less than 0.2 mg / ml, v. g. within the concentration range of 0.01 mg / ml and 0.2 mg / ml and / or 0.05 mg / ml and 0.2 mg / ml. These concentrations have proven to be sufficient to prevent or inhibit the IBMIR and morphological disruption and graft rejection of cell transplants, but remain low enough to minimize any side effects commonly associated with the administration of dextran sulfate. In addition, islets cultured in BPM-SD had no adverse effect on islet function in the range of 0.01 to 1 mg / ml. In any event, the physicist or trained person will be able to determine the current dose, which will be more appropriate for a particular patient and same that will vary with the age, weight and response of the patient in particular. The doses identified above are examples or average dose of the average number of cases. However, there may be individual instances that will merit higher or lower dose ranges, which are within the scope of the present invention.

Today, dextran sulfate has already been used in clinical studies for antiviral therapy against HIV, treatment of acute cerebral infarction in combination with urokinase as well as in anti-hyperhpidemic therapy, where dextran sulfate is coupled to a solid phase. In both types of study the injection rate was approximately 45 mg / hour, which was maintained for a period of 2 to 3 weeks by continuously injecting dextran sulfate (MW 8 000 Da) and the blood concentration was found to be approximately 0.01 mg / ml. In all these patients thrombocytopenia (sometimes associated with hemorrhage) was observed after 3 days of treatment, and alopecia was reported in almost half of the patients. However, both effects were reversible. It is estimated that the administration of dextran sulfate for inhibition of the IBMIR, morphological disruption and / or graft rejection of cell transplants is usually effected from 1-2 or a few more days. Consequently, the side effects identified above will be greatly attenuated during such a short administration period (a few days compared to 2 or 3 weeks).

It is well known that dextran sulfate induces hypotension via the release of bradykinin resulting from the activation of plasma kallikrein. However, this observation was first made when APM-SD has been immobilized in plasmapheresis columns for the treatment of hyperhidemia and not during the injection of dextran sulfate. This effect is a consequence of the direct activation of FJXII to FJXIIa. However, in this document it is argued that factor XII is not directly activated by BPM-SD. As mentioned above, the levels of FXIIa-AT and PAP are elevated when the cells are exposed to external blood without BPM-DS. However, these high levels are normalized when BPM-SD is added.

In order to prevent IBMIR subsequent to cell transplantation and / or morphological disruption and graft rejection of cell transplants, a therapeutic concentration of dextran sulfate, or derivatives thereof, is preferably obtained, at least locally, at the site of transplant, once the cell transplant has been performed. This can be obtained by administering dextran sulphate prior to the transplant in turn. Alternatively, the cells or groups of cells to be transplanted into a patient can be injected dissolved in a solution comprising the dextran sulfate according to the present invention. The concentration of dextran sulfate in such a cell and the dextran sulfate solution is preferably less than 5 mg / ml, and more preferably between 0.01 mg / ml, can be obtained (locally) at the transplant site during the first hours after transplant. As a person skilled in the art will understand the current concentration of dextran sulfate, or derivatives thereof, may be temporarily greater than the optimum concentration in the blood of the patient when the cells or groups of cells are transplanted dissolved in a solution with dextran sulfate. Subsequently, dextran sulfate will spread from the transplant site decreasing the local concentration of dextran sulfate. In some applications, additional dextran sulfate is not required to inhibit IBMIR, morphological disruption and / or graft rejection of cell transplants, since a concentration of probably will only be required up to the first 24-48 hours after transplant. In any case, additional dextran sulfate may be added whenever necessary. g. intravenously, intraperitoneally or by any other route of administration identified above. As a person skilled in the art understands, administration of a dextran sulfate solution comprising the cells or groups of cells to be transplanted can also be combined with administration of dextran sulfate, or derivatives thereof, prior to the transplantation in question.

According to a further aspect of the invention, a pharmaceutical preparation is provided for use in the treatment of IBJMIR comprising an effective amount of dextran sulfate or a pharmaceutically acceptable derivative thereof.

The present invention also relates to a pharmaceutical preparation for use in the treatment of graft rejection of cell transplants comprising an effective amount of dextran sulfate, or a pharmaceutically acceptable derivative thereof, and a pharmaceutical preparation for the treatment of morphological disruption of cell transplants comprising an effective amount of dextran sulfate, or a pharmaceutically acceptable derivative thereof.

Dextran sulfate, and derivatives thereof, can also be combined with other therapeutic agents useful in the treatment of graft rejection of transplanted tissue. Suitable but not limited examples of such immunosuppressive agents that can be used together with dextran sulfate for treatment of graft rejection are cyclosporin, tacrolimus, corticosteroids, rapamycin (sirolimus) and mycophenolate mofetil. Administration of dextran sulfate, or derivatives thereof, according to the invention can also be coordinated with administration of anti-TF antibodies and / or site-inactivated Vlla factor, which has been shown to have some functionality in the inhibition of IBMIR.

EXAMPLES Reagents Low molecular weight dextran sulfate (BPM-SD) with an average molecular weight of 5,000 Da and a sulfur content of approximately 17% was obtained from Sigma Chemicals (St. Louis MO, USA). High molecular weight dextran sulfate (APM-SD) having an average molecular weight of > 1 000 000 Da and a sulfur content of 16-19% was purchased from Amersham Bioscience (Uppsala, Sweden). Low molecular weight dextran sulfate (BPM-SD, MW 5 000 Da) and high molecular weight dextran (HMW-D, MW> 1 000 000 Da) was obtained from Fluka Chemicals (Buchs, Switzerland) and Sigma Chemicals (St Louis MO USA), respectively.

Treatment of Heparin All materials that were in contact with whole blood were supplied with a Corline heparin cover (Corline Systems AB, Uppsala, Sweden) according to the manufacturer's recommendation. The surface concentration of heparin was 0.5 μg / cm2, corresponding to approximately 0.1 U / cm2, with an antithrombin binding capacity of 2-4 pmol / cm2.

Preparation of blood. Fresh human blood was collected from healthy volunteers who had not received medication for at least 14 days in heparinized 60-ml syringes (18 gauge, Microlance, Becton Dickinson, Franklin Lakes, NJ.). The cannula of the syringes were connected to the heparinized silicone tubes. During the analysis, the syringes were rotated on their axis, continuously.

Animals Congenitally nude male mice (a Black-6 BomMice) from Bomholt Gaard Breeding & Research Center, Ltd. (Ry. DenmarJk), 22-25 g, were employed as recipients. All the animals had access to a standard diet of food and water.

Isolation of islets. Adult pig islets (API) were isolated from the Swedish Landrace sow pancreas (200 to 300 kg) by means of a mechanical and enzymatic pancreatic digestion procedure followed by filtration and separation of the islets on Ficoll gradients as suggested by Ricordi et. to the. [19-20]. The islets were suspended in M199 culture medium (GIBCO, BRL, Life Technologies Ltd., Paisley, Scotland) supplemented with 10% porcine serum (GIBCO, BRL), 1 mM calcium nitrate, 0.02 μM selenium, 20 mM nicotinamide , 25 mM HEPES, Fungizone (500 μ g / 1) and gentamicin (50 mg / l) and cultivated in 250-ml bottles at 37 ° C in 5% CO2 and humidified air, for 1 to 4 days. The culture medium was changed on day 1 and then every other day. The volume and purity of the islets was determined under an inverted microscope after staining with a dithizone (Sigma Chemicals, St. Louis, MO).

Induction of athymic mice to diabetes. Athymic mice were induced to diabetes by intravenous injection (i.v.) of streptozotocin (steptozotocin) from Sigma Chemicals (Palo Alto CA, USA) according to Wennberg et al. [twenty]. The dose of streptozotocin was 250 mg / kg body weight for athymic mice. An animal was considered diabetic if its blood glucose (S-glucose) exceeded 20 mmol / l (> 360 mg / dl) for 2 or more consecutive days.

Adult pig islet transplants (API) in athymic diabetic mice treated with or without BPM-SD After being cultured for four days, 5 μl API (-5000 IEQs) of 5 isolates were transplanted into the liver, via portal vein , of 11 congenitally athymic male mice themselves that were anesthetized with isoflurane. Five mice were treated i. v. with BPM-SD. 0.15 mg of BPM-SD was injected 10 minutes before transplantation and an additional 0.3 mg was injected 6 hours after transplantation. Subsequently, BPM-SD was administered twice a day on days 1-2, 3-4 and 5-6 after transplantation at declined doses of 1, 0.5 and 0.25 mg respectively. Six untreated mice were injected in the same way with an equivalent volume of saline.

Statistic analysis. All the data are given as mean ± SEM (Standard Error of the Mean) and compared using ANOVA (Analysis of Variance) (Table 1), paired Student t test (Table 3), the test of the signed Wilcoxon ranges (Table 1). 4) following the post hoc Scheffe test (Table 5). In the morphological study of transplanted islets, the frequency of clot formation and the infiltration intensity of leukocytes were evaluated using the Wilcoxon rank-sum test. Values-P <; 0.05 were considered statistically significant.

Quality of Islets A constant glucose stimulation test was performed as a functional test for API. 15 islets were carefully selected and carefully agitated Kerbs-Ringer bicarbonate containing 1.6 mM glucose at 37 ° C for 60 minutes. Then, the glucose concentration was modified to 16.7 mM for 60 more minutes. The floating leftovers were collected and stored at -20 ° C until analysis. The insulin content in the floaters was analyzed by ELISA (DAKO Diagnostics, Ltd., Ely, UK). The stimulation index was calculated as a radius of insulin concentration in low and high glucose, respectively. The purity of APIs used in this study ranged from 81 to 95% (average, 88.5 ± 2.2%). The stimulation index in the constant glucose stimulation test was between 0.8 and 7.8 (3.4 + 1.2), and the average insulin content was 85.5 + 6.2 pmol / μg DNA.

Additionally, the ADP / ATP rate was measured to evaluate the viability of cultured APIs, using the ApoGlow ™ kit (LumiTech, Ltd., Nottingham, UK). In summary, 75 equivalent islets (IEQ) of API were rinsed in PBS and then mixed with 100 μl nucleotide-bearing reagent for 10 minutes at room temperature. Subsequently, 20 μl of monitor-nucleotide reagent was added to the solution, and ATP levels were measured using a luminometer (Luminometer FB 12, Berthold DEtection Systems GmbH, Pforzheim, Germany) and expressed as the number of relative light units. Subsequently, the ADP / ATP rate in APIs was calculated as suggested by Bradbury et al. [twenty-one]. The rate of insulin DNA in the APIs was measured according to Wennberg et al. [22] and expressed as pinol / μg The surviving rate of cultured APIs was calculated as a percentage of IEQ values obtained on day 3 compared to day 0.

Any possible toxicity of BPM-SD was assessed by culturing API from three different pancreas in the presence (0.01, 01 or 1 mg / ml) or absence of BPM-SD for 3 days. The result of the survival rate, stimulation index, ADP / ATP rate and insulin / DNA rate for control examples and for examples with the three different concentrations of BPM-SD are presented in Table 1 below. BPM-SD showed no adverse effects on the function, viability or survival rate of API in any of the concentrations tested. In addition, there was no difference between the morphology of API BPM-SD-treated and that of API cultured in the absence of BPM-SD.

TABLE 1 API Cultured API Cultured API Cultured API Cultivated without BPM-SD with BPM-SD with BPM-SD with BPM-SD (O.Olmg / ml) (0.1 mg / ml) (l mg / ml) Survival Rate 57.0 ± 5.2 43.6 ± 6.7 58.4 ± 4.3 68.9 ± 2.6 Stimulation index In SGS test 1.77 ± 0.17 2.26 0.51 3.22 ± 0.89 1.42 ± 0.35 ADP / ATP Rate 0.11 ± 0.03 0.08 ± 0.03 0.10 ± 0.03 0.11 ± 0.01 Insulin / DNA ratio (pmolug) 79.0 ± 11.4 66.1 ± 5.8 69.1 ± 9.6 71.6 ± 7.4 Clotting time Blood was collected from four healthy volunteers in Vacutainer ™ tubes containing citrate. Whole blood (980 μl) was incubated with 2 μl API at 37 ° C in cups for polypropylene samples in a ReoRox ™ rheometer (Global Haemostasis International, Gothenburg, Sweden). The coagulation reaction was initiated by adding 20 μl of 1 M CaC in the presence or absence of various types of dextran (BMP-SD, APM-SD, BPM-D and APM-D). Every 6 seconds, the sample cup was placed within free torsional oscillation around its vertical axis, and the damping and frequency of the oscillation was recorded. The coagulation time was identified as the point of maximum cushioning.

The results obtained from the coagulation time experiments are presented below in Table 2. The APIs incubated in citrated human blood, induced a rapid coagulation at an average of 6.1 ± 0.3 minutes after calcification. Clot formation was completely abrogated in the presence of BMP-SD at all doses tested, whereas APM-SD inhibited clot formation at only 0.1 mg / ml. Both BPM-SD and APM-SD extended the coagulation time to only one degree less, compared to control samples (non-additives). Therefore, sulfation of dextran sulfate appears to be crucial for the inhibitory capacity observed.

TABLE 2 Experiment 1 Experiment 1 Experiment 1 Experiment 1 No additives 5.6 6.3 6.9 5.5 BPM-SD (0.01 mg / ml) > 60 > 60 > 60 > 60 BPM-SD (0.1 mg / ml) > 60 > 60 > 60 > 60 APM-SD (0.01 mg / ml) 15.8 36.9 21.0 38.3 APM-SD (0.1 mg / ml) > 60 > 60 > 60 > 60 BPM-S (0.01 mg / ml) 19.2 11.3 BPM-S (0.1 mg / ml) 25.2 25.8 APM-S (0.01 mg / ml) 19.8 13.8 APM-S (0.1 mg / ml) 14.2 33.2 Inhibition of IBMIR by LMW-DS (BPM-SD) As a test model of the effect of BPM-SD on IBJMIR and pig-to-human xenotransplantation, adult porcine islet perfusion was used in heparinized PVC tubular rings. This protocol is basically carried out as already described [4, 23] with some modifications. In general terms, PVC rings (6.3 mm in diameter and 390 mm in length) whose internal surface has been coated with immobilized heparin were used. The tube was held together with a specially designed heparinized connector. A circular ring was formed when the connector terminals were pushed forcefully into the lumen of the tube terminals.

An oscillation apparatus set at 37 ° C in an incubator was used to generate a bloodstream within the rings. The rings were oscillated in a setting that prevented blood from coming in contact with the connectors.

Seven 60-minute, islet experiments were performed using API (adult pig islets) from different pigs. BPM-SD dissolved in saline was tested at 0, 0.01, 0.1 and 1 mg / ml (final concentration). For each experiment, a ring containing fresh human blood and saline without API were included as a control. In two experiments, one ring containing fresh human blood and 1 mg / ml non-sulfated BPM-D was tested in 5 experiments. In each experiment 7 ml of fresh human blood from the same donor was added to each ring. The rings were then placed in the oscillating device for 5 minutes with either BPM-SD or saline. Then, the rings were opened and 100 μl of saline with or without 5 μl of API (approximately 5,000 IEQ) were added to the rings, followed by another 60 minutes of incubation on the oscillation device at 37 ° C. The glucose levels in the blood were measured with a glucometer (Glucometer ehte, Bayer Diagnostics, Leverkusen Germany) before perfusion.

After each perfusion, the contents of the rings were filtered through 70μm diameter filters (Filcons, Cuptype, DAKO, Denmark).

Both blood clots and tissue recovered on the filters were frozen in isopentane for immunohistochemistry. The remaining filtered blood was collected in 4.1 mM EDTA (final concentration) and used for hematological analyzes (platelets, lymphocytes, monocytes and granulocytes) and coagulation activation assays (fator thrombin-antithrombin [TAT], Xla-antithrombin factor complexes [FXIa] -AT], and Xlla-antithrombin factor complexes [FXIIa-AT]), activation of fibrinolysis (plasmin-alpha 2 antiplasmin [PAP] complexes), complement activation, (C3a and s5C-9) and inhibition of activation of Cl esterase [FXIIa-Cl INH]. Examples taken at 0, 15 and 30 minutes were also analyzed. In the 0 min examples, the blood was not added to the rings, but was immediately transferred to the tubes containing EDTA. The blood samples were centrifuged at 4 ° C at 3290 x g for 20 minutes and the plasma was collected and stored at -70 ° C until anaharzed. Blood glucose levels before perfusion of API ranged from 4.8 to 6.2 mmol / L.

Platelet count and differential white blood cell count were analyzed on a Coulter ACT-diff analyzer (Beckman Coulter, FL, USA), using blood treated with EDTA (EDTA, Ethylenediaminetetraacetic Acid). TAT and PAP were quantified using available commercial enzyme immunoassay (EIA) kits (Enzygnost TAT, Behringswerke, Marburg, Germany, PAP Imuclone®, America Diagnostica Inc., Greenwich USA). FXIa-AT, FXIIa-AT, FXIa-Cl INH and FJXIIa-cl IJNH were ana- lyzed according to the method of Sánchez et al [24]. C3a was analyzed as previously described by Nilsson Ekdahl et al. [25] and sC5b "9 was analyzed using an EIA modification described by Mollnes et al. [25,26].

In tubular rings containing fresh human blood without API, the counting and coagulation of blood cells as well as complement parameters were altered only slightly as can be seen in Table 3. All these alterations are considered as normal changes of the background, resulting from the interaction of the blood with the surface of the pipeline as well as the interference of the air fluid.

BPM-SD prevented macroscopic coagulation, consumption of blood-inhibited cells and reduced both coagulation and complement activation in a dose-dependent fashion (see Table 3). A significant increase in platelets in blood treated with BPM-SD can be seen at a concentration of 0.01 mg / ml of BPM-SD, demonstrating a restoration of the blood cell count close to normal levels, only in this low concentration of BPM-SD. SD. The TAT, FXIa-AT and FJXIIa-AT products of coagulation activation were suppressed at 0.01 mg / ml, but FXIa-AT again increased slightly in doses ranging from 0.1 to 1 mg / ml BPM-SD.

BMP-SD reduces complement activation, as practiced by the generation of C3a as well as by the membrane attack complex sC5b-9, as observed in Table 3. Fig. 1 illustrates in more detail the effect of BPM- SD in the generation of C3a during 60 minutes of the perfusion of API with fresh human blood, in the model of tubular rings. In the examples where the BPM-SD was added, the dextran sulfate was previously incubated with fresh human blood for 5 minutes before perfusion with API with the blood. Black circles represent 0.01 mg / ml BMP-SD and black and white squares correspond to 0.1 mg / ml and 1 mg / ml of BPM-SD, respectively. A corresponding diagram of the effect of BPM-SD on the generation of sC5b-9 is found in Fig. 2. As can be clearly seen from the diagrams of Figs. 1 and 2, the main activation of the main complement occurred approximately 30 minutes after API perfusion. The administration of 0.1 mg / ml and 1 mg / ml of BPM-SD totally inhibits the generation of both C3a as well as the membrane attack complex sC5b-9, while the lowest concentration of BPM-SD (0.01 mg / m) ]) significantly reduces the generation of C3a.

FXIa-Cl IJNH was not generated in any of the tubular rings tested during the 60-minute infusion (data not shown). FJ IIa-Cl INH did not change either in the presence or absence of BPM-SD.

PAP was increased in the absence of BPM-SD, while it was significantly suppressed in 0.01 mg / ml of BPM-SD.

Table 3 The effect of low molecular weight dextran (BPM-dextran) on the blood cell count and on the coagulation and complement parameters after 60 minutes of adult porcine islet (API) perfusion with fresh human blood was investigated using the tubular ring model similar to BPM-SD, as discussed above. A comparison between BPM-D and BPM-SD on the symptoms of the IBMIR can be found in Table 4. BPM-dextran, which is not sulfated, only has a marginal effect on the IBMIR. These data indicate that sulphation seems to be crucial for the inhibitory effect of dextran on the IBMIR caused by the APIs (islets of adult porcine).

TABLE 4 BPM-SD (1 mg / ml, n = 5) BPM-D (1 mg / ml, n = 5) Platelets (xl09D 181.1 ± 16.3 * 44.9 ± 18.7 Lymphocytes (xl09D 1.94 ± 0.12 2.14 ± 0.32 Monoliths (xl091) 0.38 ± 0.10 0.29 ± 0.08 Granulocytes (xl09F 3.23 ± 0.21 * 1.90 ± 0.43 TAT (ug / ml) 12.8 + 4.9 * 4429.2 ± 2002.5 FJ Ia-AT (umol / D 1.56 ± 0.82 1.09 ± 0.08 C3a (mg / ml) 171.5 ± 72.4 1490.3 ± 406.4 sC5b-9 (AU / D 41.6 + 11.5 * 157.3 + 19.1 Direct effect of BPM-SD on the complementary system in human serum.

The direct effect of BPM-SD on the complement cascade was investigated by incubation of human serum in the polypropylene tuna. Serum (l ml) was added to each tube along with BPM-SD in a final concentration of 0.01 or 0.1 mg / ml. At 5, 10, 15, 30, 45 and 60 minutes after incubation of serum at 37 ° C, 100 μl of serum was transferred to tubes containing 10 Mm EDTA. These samples were stored at -70 ° C before the analysis of the complement components C3a and sC5b * 9.

Fig. 3 illustrates the effect of BPM-SD on the presence of C3a and sC5b-9 of the complement system in human serum. The values are represented as percentages of the amount of C3a and sC5b-9 in control samples (without BPM-SD). The full bars represent the generation of C3a and the empty ones correspond to sC5b-9. At 0.01 mg / ml, an activation of activated complement was reflected in an increased generation of both C3a and sC5b-9, but at 1 mg / ml an inhibitory effect was observed. Although the effects on whole blood and serum can not be directly compared, BPM-SD by itself probably induces complement activation at the lowest doses of BPM-SD applied. At a higher concentration, the inhibitory effect remains.

Graft survival in diabetic nude mice. Blood glucose levels were measured in blood obtained from container tails using a glucose measuring instrument, Ehte® Glucometer (Bayer AB, Gothenburg, Sweden). The measurements were taken daily before 12 am and expressed as mmol / l (1 mmol / l ~ 18 mg / dl). A loss of graft function was considered to occur if the glucose levels exceeded 11.1 mmol / L (> 200 mg / dL) for two or more consecutive days. The duration of post-transplant normoghcemia (< 200 mg / dl) was defined as the graft survival period.

All the diabetic induced streptozotocin nude mice were severely hyperglycemic before transplantation, with no difference with the glucose levels seen among the various groups of recipients. Glucose levels were reduced immediately after transplantation in all diabetic recipients implanted intraportally with API. However, untreated mice remained normoghemic only for a limited time, see Table 5. Glucose levels increased again during the first 3 days after transplantation in four of six untreated mice. In contrast, normoghcemia was sustained for a significantly longer period in mice treated with BPM-SD than in untreated mice (8.8 ± 1.9 days vs. 3.5 ± 1.2 days, p = 0.045, Table 5). All the APIs used in the present study were shown to cure athymic diabetic mice when equivalent quantities were transplanted under the subcapsular renal space (removal of the graft from the graft bearing quickly resulted in an elevated level of blood glucose) TABLE 5 Implant site Treatment n Graft survival Survival medium Individual (days) Saline 6 1, 1, 2, 3, 6, 8 3.5 ± 1.2 * Liver BPM-SD 5 4, 6, 8, 12, 14 8.8 ± 1.9 * Kidney suppository 5 > 56 (x5) > 56 * 3 Immunohistochemical experiments Islets and macroscopic clots were recovered in filters after 60 minutes of perfusion with blood and with BPM-SD (0.1 mg / ml and 1 mg / ml) or without BPM-SD (control), then collected in medium and frozen lace in isopentane. The islets were sectioned and subsequently stained with horseradish peroxidase (HRP) conjugated mouse CD41a anti-human (R & D Systems, Abigdon, UK) and anti-CDllb + (Clone 2LPM 19c DAKO, Carpinteria, CA, USA). In the in vivo study, mouse livers containing API were recovered 10 days after transplantation with guinea pig anti-insulin (DAKA, Carpintería, CA, USA) and CDllb + anti-mouse, rat (Serotec LTD, Scandinavia, Oslo , Norway).

After 60 minutes, the islets recovered from the control rings without treatment were consistently found to be embedded in clots. Immunohistochemical staining showed a fibrin capsule and platelets surrounding the islets. FIG. 4 illustrates the infiltration of pohformonuclear CDllb + cells and monocytes into the control islets. In contrast, a complete inhibition of clot formation was observed and the number of infiltrated CDllb + cells decreased considerably when 1 mg / ml of BPM-SD was added during the incubation illustrated in figure 5. A similar effect was also observed. observed 0.1 mg / ml. The control samples also had a thicker layer of platelets adhering to the cells as seen in Figure 6. A much thinner and thinner layer of platelets adhering to the islets was observed in the samples treated with BPM-SD, illustrated in figure 7. Control islets not exposed to blood were negative in all the staining used.

Most of the islets recovered from untreated mice were encased in clots as shown in figure 8. In this figure 8, the arrow represents thrombus formation with the islets of enclosed porcine. However, only a few islets of mice treated with BPM-SD were enclosed, which is illustrated in Figure 9. Immunohistochemical staining showed a filtration of CDlb + leukocytes (JMAC-1 +) within the islets recovered from untreated mice, as shown in Figure 10. In contrast, there is markedly less leakage of CDllb + cells (MAC-1 +) in mice treated with BPM-SD, illustrated in Figure 11. The frequency of clot formation and leukocyte intensity was significantly lower in containers treated with BPM-SD than in untreated containers (p = 0.034). Figures 4 to 9 are amps at 200x and figures 10 and 11 at 100x.

A person skilled in the art will understand that various modifications and changes can be made to the present invention without departing from its scope, which is defined by the appended claims.

Claims (22)

1. Use of dextran sulfate, or an acceptable pharmaceutical derivative thereof, for the manufacture of a medicament for the treatment of instant blood-mediated inflammatory reaction (IBMIR).

2. The use of the dextran sulfate according to claim 1, characterized in that such an IBMIR is caused by the exposure of a cellular transplant to the blood after transplantation of said cellular transplant in a foreign container body.

3. Use of the dextran sulfate or an acceptable pharmaceutical derivative thereof for the manufacture of a medicament for the treatment of the morphological disruption of transplantation of transplanted cells.

4. The use of dextran sulfate according to claim 3, characterized in that such morphological disruption of cell transplantation is caused by an instant blood-mediated inflammatory reaction (IBMIR).

5. Use of the dextran sulfate or an acceptable pharmaceutical derivative thereof for the manufacture of a medicament for the treatment of transplant rejection of cell transplants.

6. The use of dextran sulfate, according to claim 5, characterized in that such graft rejection of cellular transplantation is due to the blood-mediated instantaneous inflammatory reaction (IBMIR).

7. The use of dextran sulfate according to any of claims 2 to 6, characterized in that such a cell transplant is transplanted into a human container body.

8. The use of dextran sulfate according to any of claims 2 to 7, characterized in that such a cellular transplant is selected from a list of: - allogeneic cell transplantation; or - xenogeneic cell transplantation

9. The use of dextran sulphate according to any of claims 2 to 8, characterized in that such a cellular transplant is selected from a list of: - individual cell - cell groups - non-vascularized tissue

10. The use of dextran sulfate according to any of claims 2 to 9, characterized in that such a cellular transplant is islets of Langerhans.

11. The use of the dextran sulfate according to any of the preceding claims, characterized in that such dextran sulfate has a molecular weight of less than 20,000 Da, preferably less than 10,000 Da.

12. The use of dextran sulfate according to any of the preceding claims, characterized in that such dextran sulfate has a sulfur content of 10-25%, preferably 15-20%.

13. A blood-mediated instant inflammatory reaction (IBMIR) treatment method, which comprises administering a therapeutically effective amount of dextran sulfate, or an acceptable pharmaceutical derivative thereof, to a patient in need of such treatment.

14. The method according to claim 13, characterized in that such a patient is a human patient and said IBMIR is caused by the exposure of a cellular transplant to foreign blood after transplantation of said cellular transplant in the recipient body of said patient.

15. A method of cell transplantation graft rejection treatment, same comprising administering a therapeutically effective amount of dextran sulfate, or an acceptable pharmaceutical derivative thereof, to a patient in need of such treatment.

16. The method according to claim 15, characterized in that said patient is a human patient and that such graft rejection of cell transplantation is due to a blood-mediated instantaneous inflammatory reaction (IBMIR).

17. A method of treating the morphological disruption of transplanted cell transplantation, same comprising administering a therapeutically effective amount of dextran sulfate, or an acceptable pharmaceutical derivative thereof, to a patient in need of such treatment.

18. The method according to claim 17, characterized in that said patient is a human patient and that such morphological disruption of cell transplantation is due to a blood-mediated instantaneous inflammatory reaction (IBMIR).

19. The method according to any of claims 13 to 18, characterized in that such a therapeutically effective amount of dextran sulfate results in a concentration of dextran sulfate in the blood of said patient of less than 5 mg / ml, preferably 0.01 - 1 mg / ml, and preferably not greater than 0.05 - 0.2 mg / ml dextran sulfate.

20. The method according to claim 14, 15 or 17, characterized in that said dextran sulfate is administered by injecting said dissolved cell transplantation said dextran sulfate.

21. The method according to any of claims 13 to 20, characterized in that such dextran sulfate has a molecular weight of less than 20,000 Da, preferably less than 10,000 Da.

22. The method according to any of claims 13 to 21, characterized in that said dextran sulfate has a sulfur content of 10-25%, preferably 15-20%.