DE19957341A1 - Synergistic compositions containing thalidomide compound and phosphodiesterase IV inhibitor useful for treatment of inflammation, autoimmune diseases, chronic infection and tumors - Google Patents

Abstract

Compositions containing a thalidomide compound and a phosphodiesterase IV (PDE IV) inhibitor are new. Compositions containing a thalidomide compound and a phosphodiesterase IV (PDE IV) inhibitor are new. The thalidomide compound is selected from thalidomide itself (I), EM12 and their alpha -methyl compounds.

Description

The present invention relates to a medicament agents especially for the treatment of diseases, by the formation of the pro-inflammatory cyto kins IL-12 can be caused.

IL-12 is a heterodimeric molecule that consists of a covalently linked p35 and p40 chain. The Molecule is derived from antigen-presenting cells (Mono cytocytes / macrophages, dendritic cells, B-lymphocytes) educated. The formation of IL-12 by mono Cytes / macrophages are identified by either microbial products such as lipopolysaccharide (LPS), Lipo peptides, bacterial DNA or in interaction with activated T-lymphocytes triggered (Trinchieri. 1995. Ann. Rev. Iminunol. 13: 251). IL-12 has a central one immunoregulatory importance and is responsible for the development of inflammation of the TH1 reactives vitäten. In the presence of a TH1 immune response against Self-antigens lead to the appearance of severe cranes fluctuations.

The importance of inflammatory cytokines like IL-12 for the development and course of inflammation There are numerous diseases or autoimmune diseases animal experiment and the first clinical sub searches clearly documented. In different animal motifs dents for diseases such as rheumatoid arthritis, mul tiple sclerosis, diabetes mellitus as well as inflammatory Bowel, skin and mucous membrane diseases show up the pathophysiological significance of IL-12 (Trembleau et al. 1995. Immunol. Today 16: 383; Müller et al. 1995th  J. Immunol. 155: 4661; Neurath et al. 1995. J. Exp. Med. 182: 1281; Segal et al. 1998. J. Exp. Med. 187: 537). By applying IL-12, the respective Er trigger disease or after neutralization of endoge The IL-12 showed a weakened disease run to an animal healing. The application of antibodies against IL-12 in humans is currently available before.

To sum up, there is an excess of IL-12 the pathophysiology of a variety of inflammatory Diseases caused. Approaches to normalizing the IL-12 mirrors therefore have a great therapeutic value Potential.

The immunomodulatory properties of thalidomide are already used for a number of clinical pictures such as Erythema nodosum leprosum (Sampaio, et all 1993. J. Infect. Dis. 168: 408), cutaneous systemic lupus erythematosus (Atra and Sato. 1993. Clin. Exp. Rheumatol. 11: 487), Behcet's disease (Hamuryudan, et al. 1998. Ann. Intern. Med. 128: 443) and stomatitis aphthosa (Grinspan, et al. 1989. Am. Acad. Dermatol. 20: 1060) used therapeutically. The underlying is Mechanism of action has not yet been clarified However, it has been shown sufficiently that thalidomide pro-inflammatory cytokines TNFα (Sampaio, et al. 1991. J. Exp. Med. 173: 699) and in particular IL-12 (Moller, et al. 1997. J. Immunol. 159: 5157) to inhibit ren, which is essential to the pathogenesis of auto immune diseases and inflammatory reactions contribute.

Changes in intracellular concentrations cyclic nucleotides such as cyclic adenosine mono phosphate (cAMP) have a pronounced effect on the Cytokine production in inflammatory cells (Sinha et al. 1995. Eur. J. Immunol. 25: 147). At the dismantling of the cAMP in  Inflammatory cells are primarily phosphodiesterases Type IV (PDE IV) involved. By inhibiting them there is an increase in the cAMP level, which in inhibition of proinflammatory cytokines TNFα, IL-1β and IL-12 as well as from IFN-γ re results (Marriott et al. 1997. DDT 2: 273). The type TV Phosphodiesterase is predominant in monocytes (Torphy and others. 1991. Thorax 46: 512) and therefore adjusts preferred target for the suppression of cAMP-sensi functions in this cell type. A speci The inhibitor of PDE IV is rolipram (Semmler et al. 1993. Int. J. Immunopharmacol. 15: 409). In some animal It was able to model inflammatory diseases Efficacy shown: In a rat model for experimental allergic encephalomyelitis (EAE) Vermin occurred after administration of rolipram change in TNFα level and to alleviate the Er disease (Sommer et al. 1995. Nat. Med. 1: 244). The Efficacy at EAE was found in non-human primates (Marmoset monkeys) confirmed (Genain et al. 1995. Proc. Natl. Acad. Sci. U.S.A. 92: 3601). Furthermore took the survival rate of advice after receiving rolipram with acute "Respiratory distress syndrome" clearly to (Turner, et al. 1994. J. Am. Med. Assoc. 273: 934). In had acute and chronic inflammatory reactions Rolipram protective effect (Sekut et al. 1995. Clin. Exp. Immunol. 100: 1926). It also humiliated Rolipram in a murine model for septic shock LPS-induced serum IL-12 and IFN-γ levels (Haskó et al. 1998. Eur. J. Immunol. 28: 468).

It is known to use thalidomide in combination with others anti-inflammatory, especially steroidal or non-steroidal active ingredients for the therapy of rheu matoid arthritis (WO 95/04553) and the inhibition of Use angiogenesis (WO 98/19649).  

Surprisingly, it has now been found that one Combination therapy of thalidomide, EM12 or their α-methyl compounds only with a phosphodiesterase (PDE) type IV inhibitor the IL-12 inhibition synergi is strengthened. An unspecific PDE inhibitor like pentoxifylline is ineffective in this regard.

The invention accordingly relates to a station wagon nation preparation containing thalidomide, EM12 or their α-methyl compounds and a phosphodiesterase type IV Inhibitor, preferably rolipram.

In addition to the preferred thalidomide, the Kombina tion therapy, the subject of the present inven is preferably also α-methyl-thalidomide (EN978) used as a thalidomide compound.

The active ingredients of the combination preparation according to the invention rates can be as a common wording or separately be administered.

Combination preparations according to the invention contain in addition the active substances usually carrier materials, filling substances, solvents, diluents, dyes and / or binders. The selection of excipients as well the amounts to be used depend on whether he Medicaments according to the invention orally, rectally, ophthalmically (intravitreal, intracameral), nasal, topical (a finally buccal or sublingual), vaginal or parenterally (including subcutaneously, intramuscularly, intravenous, intradermal, intratracheal or epidural) should be applied.

Preparations in are suitable for oral administration Form of tablets, chewable tablets, coated tablets, capsules, Granules, drops, juices or syrups for which parenteral, topical and inhalation application solutions,  Suspensions, easily reconstitutable dryness preparations and sprays. Combination according to the invention in a depot in dissolved form, a carrier film or a plaster, optionally with the addition of agents that promote skin penetration are examples for suitable percutaneous application forms. From oral or percutaneously applicable preparation forms can Compounds according to the invention released with a delay become. Ophthalmic forms of application include Trop ointments, ointments and gels.

The amount of active ingredient to be administered to patients varies depending on the weight of the patient Application type, the indication and the severity of the Illness. Usually 1 to 150 mg / kg become one applied combination according to the invention.

The combination preparations according to the invention are suitable for the therapy and / or prophylaxis of diseases, where an excessive IL-12 production for the Pathogenesis is held responsible (inter alia skin, mucous membranes, vessels and Autoimmune diseases). The synergistic effect of Thalidomide / analogues with a PDE IV inhibitor such as Roli pram is characterized by an optimal dosage almost complete inhibition of IL-12 production by LPS-activated monocytes. Even when combined Suboptimal dosing will result in higher levels of inhibition achieved than with the individual doses. The Concentrations of different inhibitors that are therefore necessary for combined use lower than the required individual concentrations, resulting in far fewer side effects of the individual Inhibitors can be expected. Furthermore, due to the syner visible over a wide dose range gistic effect depending on the severity of the  Suitable dose can be determined, the is therapeutically effective.

The active ingredients are used to treat the above-mentioned diseases in a dose range that includes serum concentrations from 10 times below the EC ₅₀ to 100 times above the EC ₅₀ . EC ₅₀ values for thalidomide / analogs are 1 to 2 µM, the EC ₅₀ values for the PDE IV inhibitor Roli pram used as an example is 10 nM.

The diseases of the above-mentioned groups of forms include including inflammation of the skin (e.g. atopic Dermatitis, psoriasis, eczema, scleroderma), inflammation respiratory tract (e.g. bronchitis, pneumonia, asthma bronchial ARDS (adult respiratory distress syndrome), Sarcoidosis, silicosis / fibrosis), inflammation of the gastro intestinal tract (e.g. gastroduodenal ulcers, disease Crohn, ulcerative colitis), also diseases such as Hepatitis, pancreatitis, appendicitis, peritonitis, Nephritis, aphthosis, conjunctivitis, keratitis, Uveitis, retinopathy, rhinitis.

The autoimmune diseases include e.g. B. Diseases of the arthritic form (e.g. rheumatoid ar thritis, HLA-B27 associated diseases), also mul Triple sclerosis, adolescent diabetes or lupus erythematosus.

Other indications are sepsis, bacterial meningi tis, chronic bacterial and chronic viral infections cations (e.g. HIV / AIDS, hepatitis), cachexia, trans plantat rejection, graft versus host reak tion, atherosclerosis and the reperfusion syn drom / heart failure and tumor diseases.

Examples

Table 1

Immunomodulators used

Stimulation of human monocytes with lipopolysaccharide Secretion of IL-12

Human monocytes were isolated from peripheral blood mononuclear cells (PBMC) obtained from a heparinized whole blood using a Ficoll density gradient centrifugation. For this purpose, the PBMC were incubated with a monoclonal antibody which is directed against the monocyte-specific surface molecule CD14 and which are coupled to the superparamagnetic microbeads (Miltenyi Biotech, Bergisch Gladbach). For positive selection of the labeled monocytes from the cell mixture of the PBMC, the entire cell suspension was applied to a column with a ferromagnetic carrier matrix and placed in a magnetic field. As a result, the cells loaded with microbeads were bound to the support matrix, unlabelled cells passed through the column and were discarded. After removing the matrix from the magnetic field, the antibody-loaded cells were eluted by rinsing the now demagnetized column with buffer. The purity of this CD14 positive monocyte population thus obtained was about 95 to 98%. These monocytes were incubated at a density of 10 ⁵ cells / ml culture medium (RPMI, supplemented with 10% foal calf serum) with the test substances dissolved in DMSO for one hour at 37 ° C. and 5% CO ₂ . Then 20 µg / ml LPS from E. coli was added. After 24 hours, cell-free culture supernatants were taken and tested for the IL-12 content.

The concentration of IL-12 in the cell culture supernatants which was used by means of sandwich ELISAs two anti-IL-12 monoclonal antibodies (Biosource Europe, Fleurus, Belgium). A reference standard Standard curve with human IL-12 was included. The Detection limit of the IL-12 ELISA was 10 pg / ml.  

Table 2

Influence of thalidomide and rolipram, individually and in combination, on the IL-12 production of LPS-activated monocytes

Table 3

Influence of EM 978 and Rolipram, individually and in combination, on the TL-12 production of LPS-activated monocytes

Table 4

Influence of EM 12 and rolipram, individually and in combination, on the IL-12 production of LPS-activated monocytes

Table 5

Influence of EL 1010 and rolipram, individually and in combination, on the IL-12 production of LPS-activated monocytes

Table 6

Influence of thalidomide and pentoxifylline, individually and in combination, on the IL-12 production of LPS-activated monocytes (comparative example)

The results shown in Tables 2 to 5 show those of thalidomide, EM 12, EM 978 and EL 1010 outgoing dose-dependent, inhibitory effect the IL-12 production of LPS-stimulated monocytes. Also Rolipram inhibits the formation of IL-12. surprisingly wise, however, can be combined by a simultaneous Administration of rolipram and thalidomide / analogues clearly increased inhibition can be observed. The climb inhibition is both at optimal and suboptimal concentrations of both inhibitor classes see. An almost complete inhibition of IL-12 is through a combination in the area of higher Thalidomide / analog concentrations and higher raw to achieve pram concentrations.  

It is interesting that by using the α-methyl verb of thalidomide, the EM 978, is 10 times lower Concentration than used with thalidomide / EM 12 can to achieve the same effects.

In comparison, thalidomide in Kombina tion with pentoxifylline (non-specific phosphodiester ase inhibitor) no synergistic effect was observed be (Table 6). Both at optimal as well were suboptimal concentrations of inhibitors when at all, only minimal increases in IL-12 inhibi tion to watch. This makes it clear that only by combining thalidomide / analogs with one specific phosphodiesterase type IV inhibitor to develop synergistic enhancement of IL-12 inhibition len is.

By combining two different doses Classes of immunomodulators, thalidomide, EM 12, EM 978 or EL 1010 together with a PDE IV inhibitor such as On the one hand, Rolipram succeeds in strengthening the IL-12 Inhibition, and also a reduction in inhibi tor concentrations, which are individually for each substance is required. Combination therapy thus enables on the one hand a much more efficient inhibition of the inflammatory mediator IL-12, on the other hand by reducing the individual dose required for inhibition reduce unwanted side effects expect.

Claims (7)

1. Combination preparation containing a thalidomide ver binding from the thalidomide itself, EM12 and their group comprising α-methyl compounds and a phosphodiesterase type IV inhibitor. 2. Combination preparation according to claim 1, wherein as Thalidomide compound thalidomide or α-methyl-thali domid (EM978) is included. 3. Combination preparation according to one of claims 1 to 2, being a phosphodiesterase type IV inhibitor Rolipram is included. 4. Combination preparation according to one of claims 1 to 3, with the active ingredients as a common formulation available. 5. Combination preparation according to one of claims 1 to 3, the active substances in separate formulation available. 6. Combination preparation according to one of claims 1 to 5, wherein the active ingredients are contained in a dose range which includes serum concentrations from 10 times below to 100 times above the respective EC ₅₀ . 7. Combination preparation according to one of claims 1 to 6 for use in therapy and / or prophylaxis of inflammatory and autoimmune diseases, chronic infectious diseases and tumor diseases.