IE65400B1 - Use of a cholanic acid salt and a lipid - Google Patents

Abstract

Pharmaceutical preparations which contain an immunomodulating protein, for example an interferon, a bile acid derivative and a lipid are described.

Description

Description The present invention is concerned with the use of a cholanic acid salt and a lipid for adsorption prevention, desorption, agglomeration prevention and deagglomeration of proteins having immunomodulatory activity in aqueous solutions.

The expression protein having immunomodulatory activity used here denotes proteins which regulate the maturation, activation and suppression of the different cells of the human and animal immune system. The proteins which come into consideration can be either of natural origin or can be prepared in a recombinant manner.

Examples of such proteins are interferons (IFN) such as IFN-a, IFN-β, IFN-γ; hybrid interferons; interleukins (IL) such as IL-1 (ETAF, LAF), IL-2 (TCG.F), IL-3 (multi-CSF, MCGF), IL-4 (BSF-1, BCGF-1), IL-5 (TRF, BCGF-II), IL-7 (lymphopoeitin 1); lymphotoxin (TNF-β); macrophage inhibitory factor (MIF); thymopoeitin (TPO); transforming growth factor-α (TGF-a); transforming growth factor-β (TGF-β); tumour necrosis factor (TNF-α, cachectin, DIF); uromodulin (Tamm-Horsfall protein); neuroleukin; CD4.

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

It has been found that such proteins can be stabilized using aqueous solutions of cholanic acid derivatives and lipids and that, compared with conventional aqueous solutions, the thus-obtained mixed micelle solutions provide technological advantages, e.g. greater solubilization ability, less adsorption of the proteins to surfaces, a lesser tendency to aggregate and at the same time the ability to produce concentrated preparations, more accurate dosability and better storage stability.

Liposomal solutions which contain interleukin 2, sodium cholate, dimyristoylphosphorylcholine and dimyristoylphosphorylglycine are known from document J. Cell. Biochem. Suppl. 12A, UCLA Symposia 17-30 January 1988, page 255, abstract No. W 100. Document EP-A-56781 describes liposomal medicaments which are composed of phospholipids, cholanic acid derivatives and antigens.

The use in accordance with the invention can be effected in a manner know per se, e.g. according to the methods given in DE-OS 2 730 570.

As cholanic acid derivatives in the preparations in accordance with the invention there come into consideration the salts of cholanic acids or cholanic acid derivatives mentioned in DE-OS 2 730 570, such as cholates, glycocholates and taurocholates, especially the alkali salts such as the sodium salts. Na glycocholate is especially preferred.

Examples of lipids are natural, semi-synthetic or fully synthetic phosphatidylcholines, phosphatidylethanolamine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, sphingomyelin, plasmalogens, cardiolipin, sulphatides, synthetic lecithins having modified side-chains, e.g. those which are described in European Patent Application EP-A-0154977. The mixed micelles can contain as additional components cholesterol (up to about 30 mol% based on the lipid content) and lipids having negatively- or positively-charged groups, such as phosphatidic acid or stearylamine (up to about 10mol% based on the lipid content).

The molar ratio between lipid and cholanic acid salt conveniently lies in the order of 0.1:1 to 2:1. Mixture ratios of 0.8:1 to 1.5:1 are preferred.

The total amount of lipid and cholanic acid salt is preferably about 50-300 mg/ml. The amount of protein per volume unit in the preparations can vary in wide ranges and depends on the biological activity of the particular protein. In general, the protein concentration 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 hydrogen sulphite, sodium pyrosulphite or sodium sulphite can be added to prevent oxidation reactions of the active substance and the carrier materials.

Further additives for pH adjustment, e.g. phosphate, citrate or Tris buffer; for isotonization, e.g. sodium chloride, mannitol, sorbitol or glucose, and for preservation, e.g. 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 with the aid of conventional drying processes such as e.g. lyophilization.

The preparations obtainable in accordance with the invention can be administered parenterally, e.g. intravenously or interstitially, or enterally, e.g. orally, nasally, buccally, rectally or vaginally, or topically.

The following Examples illustrate the use in accordance with the invention.

Example 1 Lecithin (PC) and Na glycocholate (NaGC) are dissolved in the molar ratio 1:1 in chloroform/methanol (1:1 parts by vol.). The solvents are evaporated in a rotating round flask under reduced pressure at 40°C. The film which remains as a residue on the walls of the flask is dispersed in water and treated with the protein while stirring. Recombinant interleukin 2 (rlL-2) and recombinant β-interferon (rlFN-β) were added as aqueous buffer solutions (the composition is given in Table 1 below). His6-pl90(l-2) (Plasmodium falciparum surface protein) and HIV 22 (HIV 1 fusion protein) were added in lyophilized form. The. thus-obtained solutions (PC and NaGC concentration 100 mM in each case) were adjusted to pH 7.1 ± 0.1 by adding 0.1N NaOH, inert gassed with N2, filtered sterile (0.22 mm Millipore filter), filled into ampoules and stored at room temperature.

TabieJ Composition of the protein solutions used for the production of the micelle solution.

Protein Protein Concentration [mg/ml] Buffer pH rlL-2 3.4 50 mM acetate/buffer, 50 mg/ml of mannitol 3.5 rlFN-β 1.19 1 mM citric acid The thus-produced micelle solutions were tested for their solubilization behaviour in comparison to pure aqueous (nonmicellar) solutions. For this purpose, these solutions were ultracentrifuged (35000 r/min.) for 1 hour at 15°C 24 hours after production. Thereafter, the protein content in the supernatant was measured according to Markwell (Analytical Biochemistry 87. 206-210 (1978). The measured values obtained are compiled in Table II: Table H Protein Initial concen- tration [mg/ml] Protein content after u tracentrifugation Micelle solution [%]’) Aqueous solution [%]1> rlL-2 1,00 91.0 ± 3.6 (n=3) 78.5 ±11.5 (n=3) rlFN-β 0.20 86.5 ± 2.25 (n=3) 27.3 ± 2.9 (n=2) His6- p190 (I-2) 0.20 75.9 ± 4.24 (n=2) 0.0 (n=1) HIV 22 1.00 97.0 ± 3.32 (n=4) 55.7 ± 6.3 (n=4) 1) % of the initial concentration.

The measured values show that rlL-2, rlFN-β, His6-pl90(1-2) and HIV 22 in mixed micelle solutions remain solubilized in solution in a clearly higher concentration than in non-micellar, conventional aqueous solutions. This effect, which can be explained by a lower tendency of the proteins to aggregate and adsorb in micellar solutions, is of considerable practical significance. Proteins having immunmodulatory activity are extremely highly active active substances. The therapeutic use of such active substances presupposes a reliable dosage. As the data in Table II show, an accurate dosage with the solutions in accordance with the invention can clearly be guaranteed more reliably than with conventional solutions. Furthermore, an immune response (antibody formation) caused by protein aggregates can be reduced or prevented.

Example 2 rlFN-α solutions, which are reconstituted from human serum albumin-containing or -free lyophilizates having 18 Mio.

I.U. of rlFN-α and 3 ml of water for injection or 0.9% benzyl alcohol, show clear agglomerates within a few days, as a rule within 1-2 days at RT and within 2-5 days at 5°C.

On the other hand. rIFN-tx solutions, which are prepared from the same lyophilizates and a solvent which contains sodium glycocholate and lecithin, remain physically stable at RT and at 5°C for up to at least 1 month after preparation and show no loss of antiviral actvity (see Table III).

A cytopatholical test using MDBK (Madison Darby Bovine Kidney) cells and VSV (vescicular stomatitis virus) viruses, which has been described by Rubinstein et al. [J. Virol 37, 755-758 (1981)] was used to determine the antiviral activity of the r-a-IFN.

Table III Stability of α-interferon solutions from lyophilizates having 18 Mio. I.U. of rlFN-α and 3 ml of solvent Solvent Physical stability Antiviral activity (% of the initial value) 20 Water for injection clear particle formation (vortex from the bottom) after 1-2 days at RT and 2-5 days at 5°C not determined 25 0.9% benzyl alcohol clear particle formation (vortex from the vial bottom) after 1-2 days at RT and 2-5 days at 5°C not determined 30 Solvent containing sodium glycocholate and lecithin1) clear solution. No particle formation at RT and at 5°C for up to at least 1 month after preparation 106.1% (1 mo./RT) and 95.2% (1 mo./5°C) 1) The solvent used has the following composition: Glycocholic acid NaOH 40 % Lecithin Benzyl alcohol NaOH 1 Nad pH 6.0 Water for injection q.s. ad 88.5 mg 19.0 ml 169.0 mg 9.0 mg q.s. 1.0 ml It can be prepared as follows. 8.85 g of glycocholic acid are suspended in 50 ml of N2-gassed water for injection and dissolved with the aid of 1.9 ml of 40% NaOH. 16.9 g of finely divided lecithin are added thereto and dissolved by stirring. 0.9 g of benzyl alcohol is added and, after adjusting the pH value to 6.0 with 1N NaOH, the solution obtained is made up to 100 ml with N2-gassed water for injection. This solution is filtered through a membrane filter (0.45 mm), filled into ampoules under aseptic conditions and finally sterilized in an autoclave.

Example 3 Because of a clear adsorption of rlFN-α to the wall of glass lyophilization flasks, the reconstitution of human serum albumin-free lyophilizates having 1 Mio. I.U. of r-IFN-α with water for injection usually used leads to solutions which show a clearly lower content of rlFN-α. As will be evident from Table 4, a solvent containing sodium glycocholate and lecithin brings about the desired desorption of rlFN-α. The administration of accurate doses of rlFN-α is accordingly possible without the need of resorting to the use of the problematical human serum albumin If desired, sodium glycocholate and lecithin can be added to the solution of rlFN-α to be lyophilized or also added to a readyfor-use ampoule solution of rlFN-α in order in this manner to prevent the adsorption of the protein to the glass wall of the vials.

Table IV Antiviral activity of rlFN-α solutions from human serum 5 albumin-free lyophilizates having 1 Mio. I.U. of rlFN-α and 1 ml of solvent Solvent Antiviral Activity Water for injection 0.57 million I.U. Solvent No. 11) 0.91 million I.U. Solvent No. 22) 0.96 million I.U. 1) The 2 solvents were prepared from the solvent described under Table III by 1 + 19 dilution (solvent No. 1) and, respectively, 1 + 3 dilution (solvent No. 2) with 5% sterile glucose. They contain per ml 4.4 mg and, respectively, 22.1 mg of sodium glycocholate and 8.45 mg and, respectively, 42.25 mg of lecithin.

Claims (9)

Claims

1. Use of a cholanic acid salt and a lipid for adsorption prevention, desorption, agglomeration prevention and deagglomeration of proteins having immunomodulatory activity in aqueous 5 solutions.

2. Use according to claim 1, wherein the protein having immunomodulatory activity is a growth or differentiation factor.

3. Use according to claim 2, wherein the growth or differentiation factor is a colony stimulating factor. 10

4. Us© according to claim 1, wherein the protein having immunomodulatory activity is a protein for the production of vaccines.

5. Use according to claim 1, wherein the protein having immunomodulatory activity is a cytokine. 15

6. Use according to claim 5, wherein the cytokine is an interferon or interleukin.

7. Use according to claim 5, wherein the cytokine is rIFN-a, rlFN-β or IL-2.

8. Use according to claims 1-7 for parenteral and enteral 20 administration.

9. Use according to claim 1, substantially as hereinbefore described.