Use of a bile acid salt and a lipid.

The present invention relates to pharmaceutical preparations which contain an immunomodulatory protein, a bile acid derivative and a lipid.

The term "immunomodulatory protein" as used herein means proteins that regulate the maturation, activation and suppression of the various cells of the human and animal immune system. The proteins used can either be of natural origin or produced by a recombinant route.

Examples of such proteins are interferons (IFN), such as IFN-α, IFN-β, IFN-γ hybrid interferons; Interleukins (IL), such as IL-1 (ETAF, LAF), IL-2 (TCGF), IL-3 (Multi-CSF, MCGF), IL-4 (BSF-1, BCGF-1), IL-5 ( IRF, BCGF-II), IL-7 (lymphopoietin 1); Lymphotoxin (TNF-ß); Macrophage inhibitory factor (MIF); Thymopoietin (TPO); Transforming growth factor-α (TGF-α); Transforming growth factor-β (TGF-β); Tumor necrosis factor (TNF-α, cachectin, DIF); Uromodulin (Tamm-Horsfall protein); Neuroleukin; CD4.

Further examples of proteins with an effect on immune functions are growth and differentiation factors, such as granulocyte colony stimulating factor (G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF, CSF-2), macrophage colony stimulating factor (CSF-1, M-CSF); Antibodies or antibody-drug conjugates, hybrid proteins such as IL-2 diphtheria toxin and proteins for the production of vaccines against AIDS, malaria, hepatitis, herpes, influenza, poliomyelitis and other infectious diseases.

It has been found that such proteins can be solubilized and / or stabilized with aqueous solutions of bile acid derivatives and lipids and that the mixed micelle solutions obtained in this way offer application-related advantages over conventional aqueous solutions, for example Greater solubilization capacity, less adsorption of the proteins on surfaces, less tendency to aggregate and thus the manufacturability of concentrated preparations, more precise dosing and better storage stability.

The invention thus also relates to the use of a bile acid salt and a lipid for the production of solutions and dry preparations of immunomodulatory proteins and the use for the prevention of adsorption, desorption, agglomeration prevention and deagglomeration of immunomodulatory proteins in aqueous solutions.

The preparations according to the invention can be prepared in a manner known per se, for example by the methods specified in DE-OS 2730 570.

Suitable bile acid derivatives in the preparations according to the invention are the salts of bile acids or bile acid derivatives mentioned in DE-OS 2 730 570, such as cholates, glycocholates and taurocholates, in particular the alkali metal salts, such as the sodium salts. Na glycocholate is particularly preferred.

Examples of lipids are natural, semisynthetic or fully synthetic phosphatidylcholines, phosphatidylethanolamine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserines, sphingomyelin, plasmalogens, cardiolipin, sulfatides, synthetic lecithins with modified side chains, for example, those described in European patent application A2-015. The mixed micelles can contain as additional components cholesterol (up to about 30 mol% based on the lipid portion) and lipids with negatively or positively charged groups, such as phosphatidic acid or stearylamine (up to about 10 mol% based on the lipid portion).

The molar ratio between lipid and bile acid salt is expediently on the order of 0.1: 1 to 2: 1. Mixing ratios of 0.8: 1 to 1.5: 1 are preferred.

The total amount of lipid and bile acid salt is preferably about 50-300 mg / ml. The amount of protein per unit volume in the preparations can vary widely and depends on the biological activity of the protein in question. In general, the concentration of the protein will be about 0.001 mg / ml to 10 mg / ml, preferably about 0.01 to 1 mg / ml.

Antioxidants such as tocopherols, ascorbyl palmitate, sodium ascorbate, sodium bisulfite, sodium pyrosulfite or sodium sulfite can be added to prevent oxidation reactions of the active ingredient and the carrier materials.

Other auxiliaries for pH adjustment, for example phosphate, citrate or Tris buffers; for isotonicization, for example sodium chloride, mannitol, sorbitol or glucose and for preservation, for example methyl- and propyl-p-hydroxybenzoic acid, benzyl alcohol or phenol can also be added.

If desired, the mixed micelle-protein solutions can be converted into dry preparations using conventional drying processes, such as lyophilization.

The preparations according to the invention can be used parenterally, for example intravenously or interstitially, or enterally, for example orally, nasally, buccally, rectally or vaginally, or topically.

The following examples illustrate the preparation of the preparations according to the invention.

example 1

Lecithin (PC) and Na glycocholate (NaGC) are dissolved in a molar ratio of 1: 1 in chloroform / methanol (1: 1 parts by volume). The solvents are evaporated in a rotating round bottom flask under reduced pressure at 40 ° C. The film remaining on the flask wall as a residue is dispersed in water and the protein is added with stirring. Recombinant interleukin 2 (rIL-2) and recombinant β-interferon (rIFN-β) were added as aqueous buffer solutions (the composition is shown in Table 1 below). His6-p190 (I-2) (Plasmodium falciparum surface protein) and HIV 22 (HIV 1 fusion protein) were added in lyophilized form. The solutions thus obtained (PC and NaGC concentration each 100 mM) were brought to pH 7.1 by adding 0.1 N NaOH<u style="single">+</u> 0.1 set, inert gassed with N2, sterile filtered (0.22 µm Millipore filter), filled into ampoules and stored at room temperature.<tables id="tabl0001" num="0001"><img file="EP0440100A1_D0001.tif" /></tables>

The micell solutions produced in this way were tested for their solubilization behavior in comparison to purely aqueous (non-micellar) solutions. For this purpose, these solutions were ultracentrifuged at 35 ° C. (35,000 rpm) 24 hours after preparation for 1 hour. The protein content was then measured in the supernatant according to Markwell (Analytical Biochemistry 87: 206-210 (1978). The measured values ​​obtained are shown in Table II:<tables id="tabl0002" num="0002"><img file="EP0440100A1_D0002.tif" /></tables>

The measured values ​​show that rIL-2, rIFN-β, His6-p190 (1-2) and HIV 22 remain solubilized in solution in mixed micell solutions in a significantly higher concentration than in non-micellar, conventional aqueous solutions. This effect, which can be explained by the lower tendency of proteins to aggregate and adsorb in micellar solutions, is of considerable practical importance. Immunomodulatory proteins are extremely highly active substances. The therapeutic use of such active ingredients requires a reliable dosage. As the data in Table II show, more precise dosing can be ensured with the solution according to the invention clearly more reliably than with conventional solutions. Furthermore, an immune response (antibody formation) caused by protein aggregates can be reduced or prevented.

Example 2

rIFN-α solutions, which from human serum albumin containing resp. -free lyophilisates with 18 million IU rIFN-α and 3 ml water for injections respectively. 0.9% benzyl alcohol reconstituted, show clear agglomerates within a few days, usually within 1-2 days at room temperature and within 2-5 days at 5 ° C.

In contrast, rIFN-α solutions, which are prepared from the same lyophilisates and a solvent containing sodium glycocholate and lecithin, remain physically stable for at least 1 month after preparation at room temperature and at 5 ° C and show no loss of antiviral activity (see Table III ).

To determine the antiviral activity of the r-α-IFN, a cytopathological test using MDBK (Madison Darby Bovine Kidney) cells and VSV (vesicular stomatitis virus) viruses was used, which was described by Rubinstein et al. [J.Virol.<u style="single">37</u>, 755-758 (1981)].<tables id="tabl0003" num="0003"><img file="EP0440100A1_D0003.tif" /></tables>

It can be made as follows.

8.85 g of glycocholic acid are suspended in 50 ml of N2-fumigated water for injection purposes and dissolved with the help of 1.9 ml of NaOH 40%. 16.9 g of finely cut lecithin are added and dissolved by stirring. 0.9 g of benzyl alcohol are added and, after adjusting the pH with NaOH 1 N to 6.0, the solution obtained is made up to 100 ml with N2-gassed water for injection purposes. This solution is filtered through a membrane filter (0.45 µm), filled into ampoules under aseptic conditions and finally sterilized in an autoclave.

Example 3

Because of a clear adsorption of rIFN-α on the wall of glass lyophilization vials, the reconstitution of human serum albumin-free lyophilisates with 1 million IU r-IFN-α: with the commonly used water for injections leads to solutions with a significantly lower content rIFN-α: show. As can be seen from Table 4, a solvent with sodium glycocholate and lecithin effects the desired desorption of rIFN-α. The administration of precise doses of rIFN-α is thus possible without having to resort to the problematic human serum albumin.

If desired, sodium glycocholate and lecithin can be added to the solution of rIFN-α: to be lyophilized or to a ready-to-use ampoule solution of rIFN-α, in order to prevent the adsorption of the protein on the glass wall of the vials.<tables id="tabl0004" num="0004"><img file="EP0440100A1_D0004.tif" /></tables>