CA2033714A1 - Pharmaceutical preparations - Google Patents

Abstract

Abstract Pharmaceutical preparations which contain a protein having immunomodulatory activity, for example an interferon, a cholanic acid derivative and a lipid.

Description

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F. HOFFMANN-LA ROCHE AG, sasle/Switzerland The prPsent invention relates to pharmaceutical preparations which contain a protein having Lmmuno-modulatory activity, a cholanic acid derivative and a lipid.
The expression "protein having immunomodulatory activity~' used here denotes proteins which regulate the maturation, activation and suppression of the di~ferent cells of the human and anLmal Lmmune system. The prote:Lns used can be either of natural origin or can be prepared in a recombinant manner.
Examples of such proteins are interferons (IFN), such as I~N-~, IFN-~, IFN-~; hybrid interferons;
interleukins (IL), such as IL-1 [ETAE, LAF), IL-2 (TCGF), IL-3 (multi-CSF, MCGF), IL-4 (BSF-1, BCGF-2), IL-5 (TRE, BCGF-II), IL 7 llymphopoietin 1); lymphotoxin (TNF~
macxophage inhibitory factor (MIF); thymopoietin tTPO);
transforming growth factor-~ (TGF-~); transforming gxowth factor-~ (TGF-~); tumour necrosis faator ~TNF~
cachectin, DIF); uromodulin (Tamm-Horsfall protein);
neuroleukin; C~4.
Further examples of proteins having activity on immune functions are growth and diferentiation faotors such a8 ~ranulocyte colony stimulating factor (G-CSF), granuloa~te-maaxop~age colony stimulat:Lng factor (GNWCSF, CSF-2), ma~rophage colony st~mulating factor (CSF-I, M-C~F); ~ntibod~ or antibod~-drllg con~ugates, hybrid protein~ suah a~ IL-2 diphtheria toxin and proteins ~or the preparAtion of vaccines agalnst ~IDS r malaria, hepatitis, herpeH, in1uenza, poliomyelltifi and other in~ectiou~ di~ease~.
It has been ~ound that such proteins can be solubilised and/or stabilised using aqueous solutions of :. ~, :. ... .

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cholanic acid derivatives and lîpids and that, compared with convent~onal agueous solutions, the mixed micelle solukions thus obtained have technological advantages, for example greater solubilisation abili~y, less adsorption of the proteins to surfaces, a lesser tendency to aggregate and a~ the same time ~he ability to produce concentrated preparationsy more accurate dos-ability and bekter ~torage s~ability.
The invention thus also relates to the use of a cholanic acid salt and a lipid for the production of solutions and dry preparations of proteins having immuno-modulatory activity and use for adsorption prevention, desorption, agglomeration prevention and deagglomeration of proteins having immunomodulatory activity in aqueous solutions.
The preparations according to the invention can be prepared in a manner known per sel for example by khe methods indicated in DE-OS 2,730,570.
Suitabl~ cholanic acid derivatives in the prepar-ations according to the invention are the salts ofcholanic acids and cholanic acid deriva$ives 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 i8 particularly pre-ferred.
Examples of lipid~ are natural, semi-synthetic or iully s~nthetic phosphatidylcholines, phosphakidyl-ethanolamine, pho~phatidylinositol, phosphatidylserines r sphingom~elin, pla~malogens, cardiolipinl sulphaticles and synthetic lecithin~ havlng modi~ied ~ide chains, ior example those which are de~cribed in European Patent Appllcation A2-0,154,977. ~he mixed micelles can contain cholesterol (up to about 30 mol-% relative to tha lipid content) and lipids having nega~ively or po~itively 3~ charged groups, such as phosphatidic acid or s~earyl-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 o~ the order of 0.1:1 ko 2:1. Mixture ` ' ` ' ' -' ': ~, ' `

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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 activ-ity of the particular protein invslved., Generally, the protein will be present in a concentration of about 0,001 mg/ml to lO mg/ml, preferably about 0,01 to l mg/ml.
Antioxidants, such as tocopherols, a~corbyl 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.
Further auxiliaries for pH adjustment, for lS example phosphate, citrate or tris buffer; for the establishment of isotonicity, for example sodium chloride, mannitol, ~orbitol or glucose and for preser-vation, for example methyl and propyL p-hydro~ybenzoate, benzyl alcohol or phenol can also be added.
I desired, the mixed micelle-protein solutions can be con~erted into dry oreparations with the aid of conventional drying methods such as, for example r lyophilisation.
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 ~he preparation~ according to the inven~ion.
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Lecithin (PC) and Na glycocholate (NaGC) are dissolved in the molar ratio 1:1 in chloro~o.rm/methanol (l:1 par~s by vol.). q'he solvents are evaporated in a rotating round-bottomed ~lask under reduced pressure at 40C. The film which remains as a re~idue on the walls of , . :. : : : . :

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the flask is dispersed in water and the pro~ein is added with stirring. R~com~inant interleukin 2 (rIL-2) and recombinant ~-interferon (rIFN-~) were added as aqueous buffer solutions (the composition is given in Table l below). His6-pl90(I-2~ (plasmodium falciparum surfac~
protein) and HIV 22 (HIV l fusion protein) were added in lyophilised form. The solu~ions thus obtained (PC and NaGC concentration l00 mM in each case) were adjusted to pH 7.l ' 0.l by adding 0.l NaOH, iner~ gassed with Nz, sterile-filtered (0.22 ~m Millipore filter) r poured into ampoules and stored at room temperature.

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Table I
Composition of the protein solutions used for the production of the micelle solution.
Protein Protein Buffer ._ Concentration [~g~ml ]
_ _ _ _ . .
rIL-2 3.4 50 mM acetate/buffer, 3.5 50 mg/ml of mannitol rIFN ~ 1.19 1 mM citric acid The micelle solutions thus produced were tested for their solubilisation behaviour in comparison to pure aqueous (non-micellar).~olutions. For this purpose, these solutions were ultracentrifuged (35,000 U/min.~ at 15C
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 i.n Table II:

Table II
.
Protein Initial Proteincontent afterultracentrifugation concen-tratio~ Nicelle Aqueou~
[mg/ml] ~ol-ltion solution t%ll) [%]l) , ' _ _~ _ rIL-2 1,00 91.0 ~ 3.6 ~n-3) 78.S ~ 11.5 (n=3) 2S rIFN~p 0.20 86.5 ~ 2.2$ (n~3) 27.3 ~ 2.g (n~2) ~1~6 plgO(I-2) 0.20 75.9 ~ 4.~4 (n~2) 0,0 (n-1~
HIV 22 1.00 _ 97.0 ~ 3.32 (n~4) S5.7 ~ 6,3 (n=4) 1) ~ o~ the initial concenkra~ion.

The mea~ured values show that rIL-2, rIFN-a, His6-pl90(1~2) and HIV 22 in mixed micelle solution~ remain solubilised in solution at a clearly higher concentration than in non-micellar, conventional agueou~ solution~. This : .~ . . . ... ... . . .
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solutions. This eff~ct, which can be explained by a lower tendency of the proteins to aggregate and adsorb in micellar solutions, is of considerably practical Lmpor-tance. Proteins having Lmmunomodulatory activity are extremely highly active compounds. The therapeutic use o~
such ac~ive compounds requires a reliable dos~. 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 addition, an immune response (antibody formation) caused by protein aggregates can be reduced or prevented.

Example 2 rIFN-~ solutions, which are reconstituted from human serum albumin-containing or -free lyophilisates containing 18 million I.U. of rIFN-~ 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 ~emperature and within 2-5 days at 5~C.
On the other hand, rIFN-~ solutions, which are prepared from the same lyophilisates and a solvent which contains sodium glycocholate and lecithin, remain physically stable at room temperature and at 5C for up to at least one month after preparation and show no 105s of antiviral activity (see Table III)u A cytopathological test using MDB~ (Madison Darby bovine kidney) cells and VSV (vesicular stomatitis virus) viruses, which has been described by Rubinstein et al.
lJ. Viron. 37, 755-758 (1981)~ was used to determine the an~iviral activity of the r-~-IFN~

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Table III
Stability of ~-interferon solutions from lyophilisates con~aining 18 million I.U. of rIFN-u and 3 ml of solvent Solvent Physical s~ability Antiviral activity ~ of the initial value~ .
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Water for clear particle not determined injection formation (vortex from the bottom) after 1-2 days at T and 2-5 days at O.9~ benzyl cle~r particle not determined alcohol formation ~vortex from the vial bottom) after 1-2 days at RT and 2-5 days at 5C
Solvent containing clear solution. No 106.1%
sodium glyco- particle formation ~l mo./RT) and Cholate and at RT and at 5C 95.2%
lecithinl) for up to at least (1 mo./5C) 1 month after _ _ preparation _ _ ~he ~olvent used has the ~ollowing composition:

Glycoaholic acid88.5 mg NaOH ~0~ 19.0 ml Lecithin 16g.0 mg Benzyl alcohol 9.0 mg 1 N NaO~ to pH 6.0 q.8.
Water ~or in~ectlon q.s. to 1.0 ml It can be prepared as follows:

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B.85 g of glycocholic acid are suspended in 50 ml of N2-gassed water for injection and dissolved wlth 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, af~er adjusting the pH to 6.0 with 1 N NaOH, the solution obtained is made up to 100 ml with Nz-gas~ed water for injection. This solution is filtered through a membrane filter (O.45 ~m~, poured into ampoules under aseptic conditions and finally sterilised in an autoclave.
Example 3 Because of a clear adsorption of rIFN-~ on the walls of ~lass lyophilisation 1asks, the reconstitution of human serum al~umin-free lyophilisates containing 1 million I.U. r~ with the water for injection usually used leads to solutions which show a clearly lower content of r-IFN-~. As can be ~een from ~able 4, a sol~en~ containing sodium glycocholate and lecithin brings about thQ desired desorption of r-IFN-~. The admini~tration of accurate doses of r-IFN-~ is thu~
possible without having to resort to the use of the pro~lematical human serum albumin.
If desired, sodium glycocholate and lecithin can be added to the solution of r-IFN-~ to be lyophilised or, alternatively, to a ready-to-use ampoule solution of r-IF~-~ in order to prevent the adsorption of the protein to the glass walls of the vial in this way.
TableIV
Antiviral activi~ of r-I~N-~ ~olution~ from 30 human serum albumin-free lyophilisa~es containing milllon I.U. o r IFN-~ and 1 ml a~ solvent.

~olvent Antiviral Activitr Wa~er or in~ectiwl 0.57 million I.U.
Solvent No. 11) 0.91 mlllion I.V.
Solvenk No. ~ 0.96 million I.U.

1) The 2 solvent~ were prepared from the solvent des-cribed under I'~ble III by 1 + 19 dilution (solvent No. 1) .

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g and 1 + 3 dilution (solvent No. 2) with 5% sterile glucose. Per ml, they contain 4.4 ~g or 22.1 mg of sodium glycocholate respectively and B.45 mg or 42.25 mg of lecithin respectively.

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Claims (11)

1. Pharmaceutical preparations containing a protein having immunomodulatory activity, a cholanic acid deriva-tive and a lipid.

2. Preparation according to Claim 1, in which the protein having immunomodulatory activity is a growth or differentiation factor.

3. Preparation according to Claim 2, in which the growth or differentiation factor is a colony stimulating factor.

4. Preparation according to Claim 1, in which the protein having immunomodulatory activity is a protein for the production of vaccines.

5. Preparation according to Claim 1, in which the protein having immunomodulatory activity is a cytokine.

6. Preparation according to Claim 5, in which the cytokine is an interferon or interleukin.

7. Preparation according to Claim 5, in which the cytokine is r-IFN-.alpha., r-IFN-.beta. or IL-2.

8. Preparation according to Claims 1 - 7 for parent-eral and enteral administration.

9. Use of a cholanic acid salt and a lipid for the production of solutions or dry preparations of proteins having immunomodulatory activity.

10. Use of a cholanic acid salt and a lipid for adsorption prevention, desorption, agglomeration preven-tion and deagglomeration of proteins having immuno-modulatory activity in aqueous solutions.

11. The invention as described hereinbefore especially with reference to the Examples.