AU641718B2 - Pharmaceutical preparations - Google Patents

Abstract

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

Description

S F Ref: 151652 FORM COMMONWEALTH OF AUSTRALIA PATENTS ACT 1952 COMPLETE SPECIFICATION

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FOR OFFICE USE: 641718 Class Int Class

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Complete Specification Lodged: Accepted: Published: Priority: Related Art: Name and Address of Applicant: Address for Service:

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F.Hoffmann-La Roche AG 124 Grenzacherstrasse CH-4002, Basel

SWITZERLAND

Spruson Ferguson, Patent Attorneys Level 33 St Martins Tower, 31 Market Street Sydney, New South Wales, 2000, Australia

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S. S S S Complete Specification for the invention entitled: Pharmaceutical Preparations The following statement is a full description of this invention, including the best method of performing it known to me/us 5845/4 1- RAN 4602/27 Abs tract Pharmaceutical preparations which contain a protein having ixmunomodulatory activity, for examrple an interferon, a cholanic acid derivative and a lipid.

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The invention relates to a method for adsorption prevention, agglomeration prevention, and deagglomeration of proteins having immunomodulatory activity in aqueous solutions, said method comprising solubilizing and/or stabilizing a solution of said protein with an effective amount of a cholanic acid salt and a lipid.

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 used 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-P, IFN-y; o1 hybrid interferons; interleukins such as IL-1 (ETAF, LAF), IL-2 (TCGF), IL-3 (multi-CSF, MCGF), IL-4 (BSF-1, BCGF-2), IL-5 (TRF, BCGF-II), IL-7 (lymphopoietin lymphotoxin (TNF-P); macrophage inhibitory factor (MIF); thymopoietin (TPO); transforming growth factor-a (TGF-ax); transforming growth factor-P (TGF-P); tumour necrosis factor (TNF-ot, cachectin, DIF); uromodulin (Tamm-Horsfall protein); 0 1is 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-I, M-CSF); antibody or antibody-drug conjugates, hybrid proteins such as IL- S 20 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 solubilised and/or stabilised using aqueous solutions of cholanic acid derivatives and lipids and that, compared with S conventional aqueous solutions, the mixed micelle solutions thus obtained have S 25 technological advantages, for example greater solubilisation 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.

The use according to the invention can be accomplished in a manner known per se, for example by the methods indicated in DE-OS 2,730,570.

Suitable cholanic acid derivatives in the preparations according to the invention are the salts of cholanic acids and cholanic 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, semi-synthetic or fully synthetic phosphatidylcholines, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserines, sphingomyelin, plasmalogens, cardiolipin, sulphatides and synthetic lecithins having modified side chains, for example those which are described in European Patent Application A2-0,154,977. The mixed micelles can contain cholesterol (up to s about 30 mol-% relative to the lipid content) and lipids having negatively or positively (Plv2ll001 13:KEH 1 of 3 2 charged groups, such as phosphatidic acid or stearyl-amine (up to about 10 mol-% relative to the lipid content) as additional components.

The molar ratio between lipid and cholanic acid salt is expediently of the order 0. 1: 1 to 2: 1. Mixture 06* 1PrIV21NOO1 13:KEH 3 rations 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 of the preparation may vary within wide limits and depends on the biological activity of the particular protein involved. Generally, the protein will be present in a concentration of 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 compound and the carrier materials.

SFurther auxiliaries for pH adjustment, for *15 example phosphate, citrate or tris buffer; for the a.

.S establishment of isotonicity, for example sodium chloride, mannitol, sorbitol or glucose and for preservation, for example methyl and propyl p-hydroxybenzoate, benzyl alcohol or phenol can also be added.

20 If desired, the mixed micelle-protein solutions can be converted into dry preparations with the aid of conventional drying methods such as, for example, lyophilisation. .aine The preparations according to the invention can be administered parenterally, for example intravenously or interstitially, or enterally, for example, orally, nasally, buccally, rectally or vaginally, or topically.

The examples below illustrate the production of the preparations according to 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-bottomed flask under reduced pressure at 40*C. The film which remains as a residue on the walls of 4 the flask is dispersed in water and the protein is added with stirring. Recombinant interleukin 2 (rIL-2) and recombinant B-ifiterferon (rIFN-p) were added as aqueous buffer solutions (the composition is given in Table 1 below). HisE-p190(1-2) (Plasmodiumn falciparum surface protein) and HIV 22 (HIV 1 fusion protein) were added in lyophilised form. The solutions thus obtained (PC and NaGC concentration 100 niM in each case) were adjusted to pH 7. 1 0. 1 by addintg 0. 1 NaGH, inert gassed with N., sterile-f iltered 22Am M'illipore f ilter) poured into amnpoules and stor~d 4t room temperature.

00 00 Table I Composition of the protein solutions used for the production of the micelle solution.

Protein Protein Concentration [mg/mlI Buffer pH rIL-2 rIFN-p8 3.4 1.19 50 mM acetate/buffer, mg/mi of mannitol 1 mM citric acid 10 *0 0 1 The micelle solutions thus produced were tested for their solubilisation behaviour in comparison to pure aqueous (non-micellar) solutions. For this purpose, these solutions ~were ultracentrifuged (35,000 U/mmn.) at 15 0

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for 1 hour 24 hours after production. The protein content in the supernatant was then measured according to Markwell (Analytical Biochemistry 87: 206-210 (1978)).

The measured values obtained are reproduced in Table II: Table 11 20

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Protein rIL-2 rIFN-P His6p190 (1-2) HIV 22 Initial concer~ trato 1,00 0.20 [irotein ccnttent after ultracentrifugation Micelle Aqueous solution solution 91.0 86.5 75.9 97.0 3.6 (n=3) 2.25 (n=3) 4.24 (n=2) 3.32 (n=4) 78.5 27.3 0.0 55.7 11. 5 (n=3) 2.9 (n=2) 1) 6,3 (n=4) 0.20 1.00 1) of the initial concentration.

The measured values show that rIL-2, rIFK-A, His6p190(1-2) and HIV 22 in mixed micelle solutions remain solubilised in solution at a clearly higher concentration than in non-micellar, conventional aqueous solutions. This 6 01.

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5* solutions. This effect, which can be explained by a lower tendency of the proteins to aggregate and adsorb in micellar solutions, is of considerably practical importance. Proteins having immunomodulatory activity are extremely highly active compounds. The therapeutic use of such active compounds requires a reliable dose. As the data in Table II show, a more accurate dose can clearly be guaranteed more reliably with the solution according to the invention than with conventional solutions. In 0 addition, an immune response (antibody formation) caused by protein aggregates can be reduced or prevented.

Example 2 rIFN-a solutions, which are reconstituted from human serum albumin-containing or -free lyophilisates containing 18 million I.U. of rIFN-a and 3 ml of water for injection or 0.8% benzyl alcohol, show clear agglomerates within a few days, as a rule within 1-2 days at room temperature and within 2-5 days at On the other hand, rIFN-a solutions, which are '0 prepared from the same lyophilisates and a solvent which contains sodium glycocholate and lecithin, remain physically stable at room temperature and at 5°C for up to at least one month after preparation and show no loss of antiviral activity (see Table III).

25 A cytopathological test using MDBK (Madison Darby bovine kidney) cells and VSV (vesicular stomatitis virus) viruses, which has been described by Rubinstein et al.

Viron. 37, 755-758 (1981)] was used to determine the antiviral activity of the r-a-IFN.

Table III Stability of a-interferon solutions from lyophilisates containing 18 million I.U. of rIFN-a and 3 ml of solvent i Solvent Physical stability Antiviral activity of the initial value) cc 0e 15 20 Water for injection clear particle formation (vortex from the bottom) after 1-2 days at RT and 2-5 days at not determined 0.9% benzyl clear particle not determined alcohol formation (vortex from the vial bottom) after 1-2 days at RT and days at Solvent containing clear solution. No 106.1% sodium glyco- particle formation (1 mo./RT) and Cholate and at RT and at 5°C 95.2% lecithin) for up to at least (1 1 month after preparation The solvent used has the following composition: *o Glycocholic acid NaOH 40% Lecithin Benzyl alcohol 1 N NaOH to pH 6.0 Water for injection q.s. to 88.5 mg 19.0 ml 169.0 rng 9.0 mg q.s.

1.0 ml It can be prepared as follows: -8- 8.85 g of glycocholic acid are suspended in 50 ml of N 2 -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 and dissolved by stirring. 0.9 g of benzyl alcohol is added and, after adjusting the pH to with 1 N NaOH, the solution obtained is made up to 100 ml with N 2 -gassed water for injection. This solution is filtered through a membrane filter (0.45 pm), poured into ampoules under aseptic conditions and finally sterilised in an autoclave.

Example 3 Because of a clear adsorption of rIFN-a on the walls of glass lyophilisation flasks, the reconstitution of human serum albumin-free lyophilisates containing 1 15 million I.U. r-IFN-a with the water for injection usually used leads to solutions which show a clearly lower content of r-IFN-a. As can be seen from Table 4, a solvent containing sodium glycocholate and lecithin brings about the desired desorption of r-IFN-a. The administration of accurate doses of r-IFN-a is thus possible without having to resort to the use of the problematical human serum albumin.

If desired, sodium glycocholate and lecithin can be added to the solution of r-IFN-a to be lyophilised or, alternatively, to a ready-to-use ampoule solution of r-IFN-a in order to prevent the adsorption of the protein to the glass walls of the vial in this way.

S! TableIV Antiviral activity of r-IFN-a solutions from human serum albumin-free lyophilisates containing 1 million I.U. of r-IFN-a and 1 ml of solvent.

Solvent Antiviral Activity Water for injection 0.57 million I.U.

Solvent No. 1 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 1 3 dilution (solvent No. 2) with 5% sterile glucose. Per ml, they contain 4.4 mg or 22.1 mg of sodium glycocholate respectively and 8.45 mg or 42.25 mg of lecithin respectively.

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Claims (10)

1. A method for adsorption prevention, agglomeration prevention, and deagglomeration of proteins having immunomodulatory activity in aqueous solutions, said method comprising solubilizing and/or stabilizing a solution of said protein with an effective amount of a cholanic acid salt and a lipid.

2. The method according to claim 1, in which the protein having immunomodulatory activity is a growth or differentiation factor.

3. The method according to claim 2, in which the growth or differentiation factor is a colony stimulating factor.

4. The method according to claim 1, in which the protein having immunomodu'atory activity is a protein for the production of vaccines.

The method according to clainm 1, in which the protein having immunomodulatory activity is a cytokine.

6. The method according to claim 5, in which the cytokine is an interferon or is interleukin.

7. The method according to claim 5, in which the cytokine is r-IFN-a, r-IFN-P or I.-2.

8. The method according to any one of claims 1 to 7 for parenteral and enteral administration. 20

9. A method for adsorption prevention, agglomeration prevention, and deagglomeration of proteins having immunomodulatory activity, said method substantially i as hereinbefore described with reference to any one of the Examples.

10. A solubilized and/or stabilized solution of a protein having immunomodulatory activity, when prepared by the method of any one of claims 1 to 9. S 25 Dated 21 July, 1993 F. Hoffmann-La Roche AG Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON P l21Oi3.tKEH l0 of 3