DE19533477A1 - Use of di:ethylene-tri:amine-penta:acetic acid cpds. - Google Patents

Abstract

The use of one or more diethylene-triamine-pentaacetic acid (DTPA) cpds. of formula XO2C-CH2-N(C2H4-N(CH2-CO2X)2)2 (I) is claimed for prodn. of immunosuppressants. X = H and/or a metal ion equivalent that has atomic number 3-4, 11-13, 19-20, 25-30, 38 or 56, or is an equiv. of an organic base.

Description

The invention relates to the use of DTPA for the manufacture of pharmaceuticals with an immunosuppressive effect.

Adverse immune reactions are found in hypersensitive patients reactions, autoimmune diseases and organ transplant recipients gladly. Especially in the context of clinical transplants in the recent decades a number of immunosuppressive drugs, such as e.g. B. Cyclosporin (CyA) or Tacrolimus (FK506) developed. The treatment the organ recipient with these drugs led to an overall prolongation lifetimes of the patients. The mode of action of these medicines elements 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 leads to the fact that these cells are no longer interleukin-2 Proliferation and activation can be stimulated. This activation is but for cell-mediated immune defense and thus for central immunology mechanisms of great importance. Unfortunately, the gift of this immune suppressiva the disadvantage that it, due to the systemic oppression immune response, very often serious complications comes from bacterial or viral infections. In organ transplantation infection with the cytomegalovirus (ZMV) is particularly worth mentioning (Koskinen, P.K., et al .: "The correlation between symptomatic CMV infection and CMV antigenemia in heart allograft recipients. "Transplantation, 55: 547, 1993). Infection occurs during immunosuppressive therapy the dose of the immunosuppressive therapeutic must be reduced again increases the risk of graft rejection. Dar In addition, the patient must also be treated with a viral drug (ganciclovir, Foscarnet) are treated. The possible side effects from immune suppressives (e.g. nephrotoxicity, hepatotoxicity) are thus at a high level Dimension expanded (anemia, bone marrow damage, testicular atrophy, etc.).  

Therapeutics are for the treatment of patients after organ transplantation tion, but also for patients with excessive immune reactions of large of great importance. The task therefore was to find agents that were immune Combine suppressive and antiviral properties.

Recent research indicated that the complexation or binding of iron (III) ions to inhibit interleukin-2 receptor expression in lymphocytes (Weinberg et al .: Therapy of graft versus host disease (GVHD), Blood 1986, 68 (S7): 5286). At the same time could be shown that the antioxidant metal complexing agents desferrioxamine (DFO) possess antiviral properties (Cinatl, J., et al .: "In vitro inhibition of human cytomegalovirus replication by desferrioxamine ", Antiviral Res., 25: 73, 1994). In unpublished DE 1 95 18 870.5 it is described that DTPA has antiviral properties.

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 tools for administration poorly soluble ions (e.g. iron),
• - in diagnosis as a contrast medium for radiology, magnetic resonance imaging phie and scintigraphy,
• - in the treatment of radiation accidents for the removal of radionuclides (e.g. ⁹⁹mTc) that have entered the body,
• - As an antidote in the treatment of heavy metal poisoning for chelation flushing and flushing the toxic ions out of the body,
• - in the treatment of iron storage diseases (e.g. (3-thalassemia), for Chelation and flushing of 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-trina  trium-diethylenetriamine-pentaacetate (CaNa₃DTPA) also immunomodulatori effectiveness. It is known that z. B. CaNa₃DTPA because its strongly hydrophilic character does not penetrate the cell and therefore only free, d. H. do not complex metals or ions incorporated in the cell can (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: it is completely new and surprising also that the mechanism of the DTPA effects obviously has nothing to do with the Chelation of the iron (III) ion has to do: An addition of FeCl₃ z. B. the anti-CMV effect of CaNa₃DTPA does not cancel, as is the case with the DFO Case was. Amazingly, even the iron (III) DTPA complex itself is effective sam and also other metal complexes of the DTPA have 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. Possible the DTPA molecule itself works through a direct interaction Receptors on the surface of the membrane from infected cells be formed.

DTPA or its salts or metal complexes correspond to the formula:
(I) XO₂C-CH₂-N [C₂H₄-N (CH₂-CO₂X) ₂] ₂

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) -, NickeI (II) -  Copper (II) or zinc (II) ion. If not all acidic hydrogen atoms in the DTPA can be substituted by the multivalent central ion, one, meh rere or all remaining hydrogen atom (s) by inorganic cations and / or organic bases or amino acids. Suitable anor ganic cations are, for example, the lithium ion, the potassium ion, the Calcium ion, the magnesium ion and especially the sodium ion. Suitable Cations of organic bases include those of primary, sekun där or tertiary amines, such as ethanolamine, diethanolamine Triethanolamine, morpholine, glucamine, N, N, -dimethylglucamine and N-methyl glucamine or 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 virustati contain, bactericidal or immunoregulatory active substances of other classes ten.

The immunomodulatory effect of the characterized in the claims Connections are very clear when using non-toxic concentra ions. The therapeutic index is very high (see DE 1 95 18 870.5) and evaluate the clinical significance accordingly.

Because of the low toxicity of DTPA, it is possible to use the ones according to the invention Average in daily dosages of up to 0.05 g / kg body weight apply. Dosages of 1-50 µM / h are required for the desired effect Infusion or 10-200 µM / d required for oral administration 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.

For transplant recipients whose immune system is therapeutically suppressed must be done so that there is no immunological rejection reaction, all the effects of DTPA shown would have a valuable benefit. The immune suppressive performance of DTPA in non-toxic concentrations would have been Compared to the current immunosuppressive drugs, the following advantages:

• - DTPA not only suppresses the proliferative activation of lymphocytes, but also the molecular requirements for the detection of foreign structures on the endothelium of the donor organ. This could be a to be a major advance in immunosuppressive therapy.
• - In addition to the immunological effects, DTPA is an antioxidative substance and could therefore be beneficial in weakening oxidative mechanisms men in the context of an inflammatory reaction (eg "oxidative burst"). Furthermore, DTPA could be an important part of organ preservation solutions are established. Organ preservation currently available already take into account the effects of antioxidants. An enormous advantage by adding DTPA it could be that in addition to the antioxidant egg properties of the endothelium immunologically before the transplantation is inhibited.
• - DTPA has an antiviral effect and is probably the only substance that suppression of the immunological mechanisms Increase in CMV prevented. This would be a great therapeutic advantage for the patient, since in addition to treatment with immunosuppressive drugs in the event of an infection, additional antiviral drugs are given. Of course, this also extends the spectrum of possible side effects. In addition, the dosage of the immunosuppressant must be in an infection are reduced, which in turn increases the risk of rejection leads. Overall, the treatment costs could potentially be significant be lowered.
• - DTPA may also affect patients with autoimmune diseases cations or hypersensitivity reactions are used.

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

example 1 Antiproliferative activity of CaNa₃DTPA 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 attempt to test the reactivity of lymphocytes is mitogen stimulation by phytohaemagglutinin (PHA) and the subsequent proliferation measurement by the radioactive thymidine incorporation test. Using this model, substances with an immunomodulatory effect can be used be tested. In our experiments, the inhibitory effect of DTPA was at non-toxic concentrations between 10 µM and 100 µM 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 for a cell-mediated immune response is necessary (Hu, J., et al., "Interleukin 2 up-regulates its own production ", J. Immunol., 139: 4109, 1987). IL-2 is stimulated by PHA Lymphocytes are secreted and can be in the cell culture medium by means of ELISA (enzyme-linked immunosorbent assay) can be determined. The measure of the Sezer Nation is a measure of the degree of activation. In our experiments below the cultivation of PHA-stimulated cells with DTPA suppressed the IL-2 release. For this purpose, time kinetics were created, which showed that the suppression did not take place until the third day after stimulation. In parallel to these experiments was the expression of the interleukin-2 receptor on the stimulated with PHA Cells quantified using FACS. Here too there was a suppression of  Expression on the third day and complete 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 storage of 5-bromo-2-deoxyuridine (BrdU) in the cell Quantitative DNA with the ELISA test. The result is expressed through the IC₅₀, that is the concentration that this storage is 50% below presses. 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 sensitive to certain cytotoxic effects are (Jurkat cells are sensitive to cytotoxic cells; K562 versus natural killer cells) are in culture incubated with Europium for a certain period of time. After that, the Kul the effector cells added, which from the peripheral blood voluntarily Donors are won. The activity of these effector cells can be demonstrated in vitro the coculture with the target cells can be enhanced by administration of IL-2. By killing the target cells now also releases Europium, that has previously accumulated in the target cells. The amount of released Europium is a measure of the cytotoxic activity of the cells to be tested. The Quantification of europium in the cell culture medium is based on a photome trical method. In these experiments too, DTPA inhibited almost completely 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 carried out as part of a trans implant rejection to a significant extent from the endothelium of the trans plantats controlled. Interactions between endothelial cells and lymphocytes depend on the degree of expression of HLA antigens and adhesion ion molecules. The ability of stimulated endothelial cells, molecules of the Expressing HLA class II is of particular importance (Hirschberg, H., et al .: "Antigen properties of human vascular endothelial cells" J. Exp. Med., 154: 249, 1980). Certain immunological reactions can only be achieved through the Interactions of helper T cells (subtype of lymphocytes) with cells that have HLA Class II are positive (so-called antigen-presenting cells). Class I HLA molecules are for interactions with cytotoxic cells responsible. However, these need the help of the helper cells. DTPA suppressed the basal or those induced by cytokines (interferon-gamma) Expression of HLA class I and II molecules on the cell membrane of the En dothelial cells in vitro by more than 50% (100 µM). Based on the in vivo situation after organ transplantation, this property of DTPA could be a HLA-mediated rejection, initially always against the endothelium may 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 also the case when determining 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 for the preparation of means for immunosuppressive therapy. 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 with suitable liquid, semi-solid, solid or gaseous Carriers and, if appropriate, other auxiliaries for oral, nasa len, topical, vaginal or parenteral drugs is processed. 4. Use according to one of claims 1-3, characterized in that that the drug other known virustatic, bactericidal or in contains regulatory substances of other classes.