DE19533477C2 - Use of diethylenetriaminepentaacetic acid (DTPA) with immunosuppressive effects - Google Patents

Description

The invention relates to the use of DTPA with immunosuppressive effect in organ transplants.

Adverse immune reactions are found in hypersensitive patients reactions, autoimmune diseases and organ transplants capture. Especially in the context of clinical transplants, a number of immunosuppressive drugs in recent decades, such as B. Cyclosporin (CyA) or Tacrolimus (FK506) developed. The Treatment of organ recipients with these drugs resulted in a total to extended patient lifetimes. The mode of action of this drugs mentioned are based on the suppression of the so-called Interleukin-2 receptor on the cell membrane of lymphocytes (Hess, A.D., Colombani, P.M., "Mechanism of action: In vitro studies.", Prog. Allergy, 38: 198 (Karger, Basel), 1986). This causes these cells to stop working Interleukin-2 can be stimulated for proliferation and activation. However, this activation is for the cell-mediated immune defense and thus for central immunological mechanisms of great importance. Unfortunately, the Gabe these immunosuppressants the disadvantage that it, due to the systemic suppression of the immune response, very often too serious complications from bacterial or viral infections. In the Organ transplantation is particularly the infection with the cytomegaly Virus (ZMV) (Koskinen, P.K., et al .: "The correlation between symptomatic CMV infection and CMV antigenemia in heart allograft redpients. "Transplantation, 55: 547, 1993). It occurs in the course of the immune suppressive therapy for infection requires the dose of immunsup  pressive therapeutic agent can be reduced, which in turn increases Risk of graft rejection. In addition, the The patient was additionally treated with a viratic agent (ganciclovir, foscarnet) will. The possible side effects of immunosuppressive drugs (e.g. Nephrotoxicity, hepatotoxicity) are thus expanded to a high degree (Anemia, bone marrow damage, testicular atrophy, etc.). Therapeutics are for the treatment of patients after organ transplantation, as well of great importance for patients with excessive immune reactions.

It was therefore the task of agents with immunosuppressive Provide property.

Recent investigations indicated that the complexation or binding of iron (III) ions led to the inhibition of interleukin-2 receptor expression in lymphocytes (Weinberg et al .: Therapy of graft versus host disease (GVHD), Blood 1986, 68 (57): S286). At the same time it could be shown that the antioxidant metal complexing agents desferrioxamine (DFO) have antiviral properties (Cinatl, J., et al .: "In vitro inhibition of human cytomegalovirus replication by desferrioxamine", Antiviral Res., 25 : 73, 1994). In the unpublished DE 1 95 18870.5 it is described that diethylenetriaminepentaacetate (DTPA) also has antiviral properties.

Using a combination of four chelating agents - CaNa₃ DTPA, calcium gluconate, reduced glutathione and sodium metabi sufflt in fixed proportions for the treatment of viral infection autoimmune diseases caused by viral infections (Multiple sclerosis) is known (WO 94/22438 A1. The effect is on the Zinc-complexing suitability of this mixture is attributed as far as it is concerns virustatic property. It also becomes a hypothetical  immunomodulatory effect derived from that an active CD₈ lymphocyte subpopulation is stimulated, causing a normal CD₄ / CD₈ ratio sets, stating that this effect only on the combination of the four components in the weight described conditions is attributable.

The stabilization of the CD₄ / CD₈ ratio is immunologically of Relevance, but has with immunosuppression, d. H. the prevention of cell-mediated immune response to do nothing.

Furthermore, the use of a compound from a "hapten group" and an "effector group" is known (EP 0419387 A1), um use therapeutic and diagnostic immune reactions. As a hapten can including a chelating agent, especially DTPA ver be applied. The effective effector groups (in the case of diagnostic agents radioactive substances, in the case of therapeutic Effects of cytotoxic compounds) are exerted by the hapten on the The site of action is transported and fixed without the hapten DTPA itself has a corresponding effect.

DTPA as a binder with which different immunogenic groups can be combined and fixed to substrates is known (WO 92/16 838 AI). Free DTPA is not used in these attempts (it would be immunogen ineffective).

It has now surprisingly been found that DTPA is additionally distinct shows immunomodulatory effects in vitro, so that this compound both can be used as immunosuppressions as well as a virustaticum.  

The use of complexing agents or complexes or their salts in medicine has long been known. Examples include:

• Complexing agents as stabilizers of pharmaceutical preparations,
• - Complexes and their salts as aids to administration poor soluble ions (e.g. iron),
• - In diagnosis as a contrast medium for radiology, the nuclear spin tom graphics and scintigraphy,
• - in the treatment of radiation accidents to remove radionukli those (e.g. ⁹⁹mTc) that have entered the body,
• - As an antidote in the treatment of heavy metal poisoning Chelation and flushing of the toxic ions out of the body,
• - in the treatment of iron storage diseases (e.g. β-thalassemia), for chelating and flushing out the excess iron.

Although there is evidence of an effect of DFO on lymphocytes proliferation already existed and it is believed that this substance over the Complexation of the iron (III) ions, it was surprising that the out the therapy of iron storage diseases known chelator calcium trisodium diethylenetriamine pentaacetate (CaNa₃DTPA) also immunomodul has regulatory efficacy. It is known that z. B. CaNa₃DTPA because of its highly hydrophilic character, do not penetrate the cell and therefore only free, d. H. metals or ions not incorporated into the cell can complex (Aisen, P., and I. Listowsky: "Iron transport and storage proteins. ", Annu. Rev. Biochem. 49: 357-393, 1980).

Surprisingly, however, it was found that CaNa₃DTPA and the other Ren in the claims characterized compounds in in vitro experiments  are still effective in cell culture: Completely new and over It is also surprising that the mechanism of the DTPA effects is obvious has nothing to do with the chelation of the ferric ion: an addition of FeCl₃ could, for. B. do not cancel the anti-CMV effect of CaNa₃DTPA, such as this was the case with the DFO. Amazingly, even the iron (III) DTPA complex itself effective and also other metal complexes of DTPA have a more or less strong antiviral effect.

Surprisingly, it can be seen that the effect of the in the claims marked connections is based on a completely new principle: measure inevitable for their effectiveness or ineffectiveness do not seem their own are chelators. On the contrary: iron and other metal-DTPA complexes are themselves effective substances. The DTPA molecule itself may work through a direct change effect with receptors on the surface of the membrane of infected Cells are formed.

DTPA or its salts or metal complexes correspond to the formula:

XO₂C - CH₂ - N [C₂H₄ - N (CH₂ - CO₂X) ₂] ₂ (I)

wherein
X stands for a hydrogen atom and / or a metal ion equivalent of an element of atomic numbers 3-4, 11-13, 19-20, 25-30, 38 or 56 or an equivalent of an organic base.

Suitable polyvalent ions are, for example, magnesium (II) -, aluminum nium (III) -, calcium (II) -, manganese (II) -, iron (II) -, iron (III) -, cobalt (II) -, Nik kel (II) - copper (II) - or zinc (II) ion. If not all acidic hydrogen atoms can be substituted in the DTPA by the polyvalent central ion, several or all remaining hydrogen atom (s) by inorganic cations shear and / or organic bases or amino acids. Suitable inorganic cations are, for example, lithium ion, potassium ion, the calcium ion, the magnesium ion and especially the sodium ion. Suitable cations of organic bases include those from primary, secondary or tertiary amines, such as ethanolamine, Diethanolamine, triethanolamine, morpholine, glucamine, N, N, -dimethyl glucamine and N-methylglucamine or of amino acids, for example that of Lysine, Arginine and Ornithine.

The above DTPA derivatives are mixed with suitable liquid, semi-solid, solid or gaseous carriers and, if appropriate, other auxiliaries substances to oral, nasal, topical, vaginal or parenteral drug processed. Furthermore, the drugs can be other known ones virustatic, bactericidal or immunoregulatory agents of other classes contain.

The immunomodulatory effect of the in the claims drawn compounds is very clear when using non-toxic Concentrations. The therapeutic index is very high (see DE 1 95 18 870.5) and to evaluate the clinical significance accordingly.

Because of the low toxicity of DTPA, it is possible to use the invention Average in daily dosages of up to 0.05 g / kg body weight ap  plicate. Dosages of 1-50 pM / h are required for the desired effect Infusion or 10-200 pM / d for oral administration required per kg body weight. The application can be done 1-3 times a day, however, trials are on Dogs showed that even a larger dose per week is sufficient.

In transplant recipients, their immune system is therapeutically suppressed must be so that there is no immunological rejection reaction comes, all the effects of DTPA shown would have a valuable benefit. The immunosuppressive performance of DTPA in non-toxic concentrations would have the following advantages over current immunosuppressants:

• - DTPA not only suppresses the proliferative activation of Lymphozy but also the molecular requirements for detection of foreign structures on the endothelium of the donor organ. This could be a major advance in immunosuppressive therapy be.
• - In addition to the immunological effects, DTPA is an antioxidant Substance and could therefore be beneficial in weakening oxidative Mechanisms in the context of an inflammatory reaction (e.g. "oxidative burst "). Furthermore, DTPA could be an important component of Organ preservation solutions are established. Currently available Organ preservation already takes into account the effects of Antioxidants. A huge benefit from adding DTPA could be be that in addition to the antioxidant properties the endothelium already is immunologically inhibited before the transplant.  
• - DTPA has an antiviral effect and is probably the only substance so far in addition to the suppression of the immunological mechanisms prevents the multiplication of CMV. Therapeutically, this would be one great advantage for the patient because so far besides the treatment with Immunosuppressive drugs in the event of an infection additionally antiviral Drugs are given. Of course, this also extends that Spectrum of possible side effects. In addition, with one Infection the dosage of the immunosuppressant is decreased which in turn leads to an increased risk of rejection. All in all could potentially cut treatment costs significantly will.
• - DTPA may also work in autoimmune patients Diseases or hypersensitivity reactions are used.

The following examples illustrate the invention without referring to it want to restrict:

example 1 Antiroliferative activity of CaNa DTPA Cells used

Human umbilical vein endothelial cells (human umbilical vein endothelial cells, HUVEC) are used in Iscove's modified Dulbecco's medium (IMDM) 10% addition of fetal calf serum and 20 ng / ml fibroblast growth factor bred.  

Cell cultures of human peripheral lymphocytes (human peripheral blood lymphocytes (PBL) are obtained from buffy coats.

Proliferation test

A standardized test to test the reactivity of lymphocytes is the mitogen stimulation by and on phytohaemagglutinin (PHA) following measurement of proliferation by the radioactive thymidine incorporation test. On the basis of this model, immunomodulatory agents Substances are tested. In our experiments it was the most inhibitory Effect of DTPA at non-toxic concentrations between 10 µM and 100 µM at almost 100% (50 µM inhibited CMV replication in human Fibroblast cultures completely).

Another parameter for determining the degree of activation of the stimulated lymphocyte is the release of interleukin-2 (IL-2). This Cytokine is necessary for a cell-mediated immune response (Hu, J., et al., "Interleukin 2 up-regulates its own production", J. Immunol., 139: 4109, 1987). IL-2 is secreted by PHA-stimulated lymphocytes and can be Cell culture medium using ELISA (enzyme-linked immunosorbent assay) be determined. The degree of secretion is a measure of the degree of Activation. In our experiments, the cultivation suppressed PHA-stimulated Cells with DTPA released the IL-2. For this purpose, Zeitkineti ken created that showed that the suppression was only on the third day after Stimulation occurred. Expression parallel to these experiments of the interleukin-2 receptor on the cells stimulated with PHA FACS quantified. Expression was also suppressed here  on the third day and full suppression on the fourth day after Stimulation.

The effects on DNA synthesis and - growth in HFF, HUVEC and PBL cell cultures were also measured using another method: we determined the incorporation of 5-bromo-2-deoxyuridine (BrdU) in the cellular DNA quantitatively with the ELISA test. That is expressed Result by the IC₅₀, that is the concentration that this storage increases 50% suppressed. For CaNa₃DTPA it is 152 µM in HFF cells, 139 µM in HUVEC cells and 240 µM in PBL cells.

Example 2 Influence of DTPA on cell-mediated cytotoxicity

Cell-mediated cytotoxicity is also a standard measurement in Im munology. We used the Europium release test (Volgmann, T., et al .: "A fluorescence-based assay for quantitation of lymphokine-activated killer cell activity ", J. Immunol. Meth., 119: 45, 1989). Cell lines that are sensitive for certain cytotoxic effects (Jurkat cells are sensitive towards cytotoxic cells; K562 versus natural killer cells) are incubated in culture with Europium for a certain period of time. Then the effector cells are added to the cultures, which from the peripheral blood from volunteer donors. The activity of this Effector cells can be administered in vitro prior to co-culture with the target cells amplified by IL-2. Now by killing the target cells also for the release of europium that was previously in the target cells  enriched. The amount of Europium released is a measure of that cytotoxic activity of the cells to be tested. The quantification of Europium in the cell culture medium is based on a photometric method. In these experiments too, DTPA almost completely inhibited the cytotoxic Activity of both cytotoxic and natural killer cells. The Measured values obtained in these experiments for the DTPA-treated Samples always corresponded approximately to the negative control values.

Example 3 Influence of DTPA on the endothelial expression of HLA class I and II antigens

The course of a cell-mediated immune response is part of a Graft rejection to a significant extent from the endothelium of the Graft controlled. Interactions between endothelial cells and Lymphocytes depend on the level of expression of HLA antigens and adhesion molecules. The ability of stimulated endothelial cells Expressing HLA class II molecules is of particular importance (Hirschberg, H., et al .: "Antigen propefties of human vascuiar endothelial cells "J. Exp. Med., 154: 249, 1980). Certain immunological reactions can only be determined by the interaction of T helper cells (subtype of the lymphocyte ten) with cells that are HLA class II positive (so-called antigen presentation cells). Class I HLA molecules are for the interactions responsible with cytotoxic cells. However, they need help from the helper cells. DTPA suppressed basal or cytokine (Interferon-gamma) induced expression of HLA class I and II molecules on the cell membrane of the endothelial cells in vitro by more than 50%  (100 µM). Based on the in vivo situation after organ transplantation this property of DTPA could be an HLA-mediated rejection reaction that is always directed against the endothelium at first suppress or reduce.

Example 4 Influence of DTPA on the endothelial expression of adhesion molecules

According to the importance of the HLA molecules, the role of adhesion to understand molecules. The adhesion molecules we examined are important for closer contact and for the immunological interaction between endothelial cells and lymphocytes (Springer, T.A., et al., "Adhesion receptors of the immune system ", Nature, 346: 425, 1990). The incubation of Endothelial cells with 100 µM DTPA inhibited about 50% of the basal and the IL-1 induced expression of ICAM-1, ELAM-1 and VCAM-1. The ex experiments were carried out several times, as was the case with the determination of the HLA molecule expression flow cytometric and fluorometric in multiple approaches performed and were very reproducible.

Claims (4)

1. Use of at least one compound of the general formula: XO₂C - CH₂ - N [C₂H₄ - N (CH₂ - CO₂X) ₂] ₂worin X for a hydrogen atom and / or a metal ion equivalent of one Elements of atomic numbers 3-4, 11-13, 19-20, 25-30, 38 or 56, or an equivalent of an organic base means to immunosuppres sive therapy for organ transplants. 2. Use according to claim 1, characterized in that CaNa₃ DTPA is used. 3. Use according to claim 1 or 2, characterized in that the Connection in suitable liquid, semi-solid, solid or gaseous Carriers and, if appropriate, other auxiliaries for oral, nasal, topical, vaginal or parenteral application is used. 4. Use according to one of claims 1-3, characterized in that that at the same time other known virustatic, bactericidal or in mun regulating agents of other classes are used.