Note: Descriptions are shown in the official language in which they were submitted.
2~337~
F. HOFFMANN-LA ROCHE AG, sasle/Switzerland
RAN 4602/27
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.
3~ me~
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~ .
.~ . , .. .......... _.. , .. _ . ___ . ..
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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