Note: Descriptions are shown in the official language in which they were submitted.
2 ~
RAN 4100/73
The pcesent invention is concerned with the
stabilization of l~u~vcyte interferon (IFN-a),
; 5 especially in the form of lyophiliza~es, using
; disa~(harides and opti.onally bile acids or bile acid
derivatives.
Under IFN-a thece is to be undHr~tood a body-
-specific pro~;e;n having antiviral and immunoregulatory
~t~livity. The antiviral effect is acli~i.eved not by a direct
i.n~luence on the viruses themselves, but by an activity on
thei.c target cells in the sense of a protecti.on against
the viral. infection. In addition to the ctntiviral
1~ ac~i.vL~y, a-interferon c~trl exert objectifiable effects
on cancer tumors, which makes it suitable for use in
cancer ~:herapy, and it in~luences ~he immune system of the
body in that e.g. it activa~es macrophages and Nl~ cells
~nd increases the exere6sion of various immunologically
si.gnificant cons~.l.uents of the cell membrane.
Thank6 to recombinarlt D~A technology, IFN- can
today be preeared in a microbiological lllarlner ;.n amounts
which hltherto could not be made available by isolation
: 25 ~rom natural material (leucocytes, lym~hocytes) and
ptlrification in ~pite of the greatest efforts.
~, This new technology has opened for the f i.c~t time a
w~y ~or the intensive clinical testing and possible wide
therapelltic use o~ IFN-~ and has made posr,ible an
adequate supply o~ the acl.;.ve sub~l:ance for persons
r3e~3~1ng a treatmerll, with ~,he acti.ve sllb~tctnce.
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I~ has now emerged that IFN-a in pure form is not
completely stable ~ind suffers loss of its biological
aotivity ducing stocage.
Diffecent adjuvants have therefore already been ll~ied
for the stabilization o~ IFN-a. A certain ~itabilization
of IFN-a has been ~t~hieved by the addil;ion of glycine
(r,ee e.g. publ. PCT Application 86/00531 and European
Patent, Publ. No. 82 481), whereby, however, albumin is
prefeeably simultaneously added.
In efforts to achie~e a good stabilization of XFN-a
over the longest possible period while avoiding the use of
high-molecular compounds such a6 ~;e~rum albumin, gelatine,
dextean or starch derivatives, it has bet?n found that this
is successful using d;saccharides and optionally bil~e
acids oe bile acid tleriva~ives.
The presen~ invention is accordingly o~oncecned with
composition~ ;pecially in ~he form of lyophilizates,
whi~h ~on~ain a disacchacide and optionally bile acids or
bile acid derivatives, a~ well as a process for their
manufao~ re which compcises treating an IFN-a solution
with a disaccharide and optionally bile at~id~ oc bile acid
derivatives and, if desired, lyophilizing the sollltion
obtained. The eresen~ invention is al~io concerned with
phaemaceutisal preparations based on the compositions in
accordance with the invention as well as the use of
disaccharides and optionally bile aoid~ or bile acid
derivatives foc the ~itabiliza~ion of IFN-.
The composition6 in accordtlrlce wil;h the inverltion can
be uned for the manlJfacturt? of a ph.lrmaceutical
~ceparation foe the theea~y or proptlylaxis o~ a lacge
3j5 numbec of in~ections and immunoregulatoey anomalies,
especially neoplasms.
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The stabilization can be applied to natural oc
cecombinant IFN-a (r-IFN-). Recomhinant IFN-aA
(r-IFN-~A) is an c~pecially pceferr~d IFN-~ in
connection with the present invention. The content of
IFN-a in the compositions in accordance with the
inverltion is not critical. The concentral:ion range ex~ends
ov~c mor~ than the power of ten and is limited at the
uppee end ~;sentially only by the ~olubility of IFN-a.
Irl the ca~.e of human IFN-a ~:he~e come into
consideration, foc example, concHrl~:cations up to 5~lO8
international units (I.U.)/ml, with a preferred range
being 0.l x lO to l x lO I.U.fml, especially
l x LO to 5 x lO I.U./ml.
The di~accharide, p~eferably lacto6e or sa~harose, is
added ~o the IFN-~ so1llt:ion in a concentration of 0.5%
to L5% (wt./vol.), preferabl~ 5% (wt./vol.).
The bile acids or ~he bile acid deri~atives can be
e.g. cholic acid, deoxycholic acid, glycodeoxycholic acid,
taucodeoxycholic acid, chenodeoxycholic acid,
glycoc~l~nodeoxycholic acid, taurochenodeoxycholic acid,
glycocholic acid or tallrocholic acid, wi~ih glycocholic
acid being especially preeerred. Tt is a~ded to the
IFN-a solution in a concentration Oe O.Ol to 3%
(wt./vol.), preEerably 0.5~ (wt./vol.).
Further adjuvants for the pH-adjustment, e.g. NaOH,
pH-buffering, e.g. ~hosphate bu~fer and citrate hu~fer,
isotonization, e.g. sodium chloeide, and preservation of
~he ~ceyar-a~lon, e.g. me~hyl p-hydroxybenzoate an~ propyl
p-hydroxybenzoate, a~ well a6 Eor strengthening the
structure oE the lyophiliz-l~e, e.g. glyrine, can also be
added.
Particular~ of the invention are described in the
Eollowirlg ~xamples.
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The r-IF~-A used in the Example6 can be ob~ained in
pure form according to known pcocedures de~cribed in the
lite~ature or according to procedu~e~ obvious to a per60n
skilled in the art.
In order to determine the antiviral activity of the
r-IFN-aA, there was used a cytopathological te~t with MDBK
(Madison Darby Bovine Kidney) cells and VSV (ve~icular
stomatiti6 virus) viruses, ~hich has been described by
Rubins~ein e~ al. [J. Virol. 37, 755-758 (1981)]. MDBK
cells and VSV viruses are gene~ally acce6~ible and can be
obtained e.g. from the American ~ype Culture Collection
(ATCC) (MDBK: ATCC Nos. CCL 21 and CRL 6071: VSV: ATCC
No. VR-1 59).
For the stability testing, solutions with r-IFN-aA
and the additives under inve~tigation were lyophilized and
stored at different temperature~ (5, 25~, 35, 45, 55
and 65C). After fixed time interval6 the antiviral
activity of the IFN-aA still pre~en~ in the
lyophilizates was determined.
The following Examples illustrate the invention
without limitin~ it.
ExamPle 1
3 mio. I.U. of r-IFN-aA were taken up in 1 ml of
water containing hu~an ~erum albumin, glycine or lacto6e
and optionally furthec adjuvants ~see Table 1). The
solutions obtained were sterile-filteced (membrane filter,
0.~ ~m poce size) and loa~ed in 10 ml glass vials in a
3r steam-sterilized freeze-dryer. After free2ing the
solutions at -40C during 4 h the primary drying of the
lyophilization proce~s wa~ carried at about -30C under a
prefisurQ of 0.1 mbar during lg h. Subsequen~ly, the
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secondary drying was carried out under a full vacuum at
~20C during about 4 h. The glass vials were closed wi~h
suitable aluminium caps in the freeze-dryer under a N2
atmosphere. They weEe subsequently subjected to diffeLen~ ::
stability tests, the results of which ace compiled in
Table 1.
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Table 1
ComPoSitlOn of the lyoPhilizates containinq ~-IFN-aA
Ly~philizate from:
1 2 3 4 5
r-IFN-aA (mio. I.U.) 3.0 3.0 3.0 3.0 3.0
Sodium chloride (mg) g.o
10 xuman ~erum albumin (mg) 5.0
Mannitol pyrogen-fcee (mg~ - 20.0
Glycine (mg) - 20~0 ~ lOoO
Lactose (mg) - - 50.0 - - -
NaH2P4 H20 a p q.s. - - q.s.
15 NaOH lN ad pH 7.4 - -q. 6 . q . ~ . _
Water for injec~ion 1.0 ml 1.0 ml 1.0 ~1 1.0 ml 1.0 ml
An~tiviral ac_ivity
(in mio. I.U.) after
manufacture of the 3.0 3.0 Z.7 3.3 Z.3
lyophilizate
2 weeks/ 5C 3.2 3.0 2.8 3.3 1.8
" /25C 3.2 2.7 3.4 3.1 1.5
25" /35C 3.3 2.8 3.3 2.7 1.2
" /45C 2.7 2.0 3.0 2.9 O.9
" /55C 2.8 1.5 2.4 2.1 O.B
" ~65C Z.l O.S - - 0.4
3 month~ 5C 2.7 2.5 3.0 3.6
" /25C 2.5 2.3 ~.1 3.2
" /3SC 2.4 2.1 3.1 2.7 --
5C 1.7 1.2 2.8 1.3
6 months/ 5C 3.5 3.1 3.2 3.1
" /25~C 3.3 3.1 3.2 2.~ -
" /35C 2.6 1.9 3.0 2.~ -
" /45C ~.t. 1.7 3.0 1.1
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12months/ 5C 3 . 7 rl. t . 3 . 0 2 . ~ -
" J25C 2.9 n.t. 2.9 2.5
" /35C 2.1 n.t. 2.7 1.3 - .
" /45C n. t . n. S . 2 . 7 1. 6
n. t . = not te~ted
q . s . = guantum ~atis
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The best results were achieved with the lactose-
-containing lyophilizate (see column 3). This preparation
exhibited an outstanding stability even after long-term
stocage at the stocage temperature of 45C. which is
almost prohibitive for IFN-a. ~his result could also be
confirmed unequivocally in experiments with lyophilizates
containing 1 mio. I.U. of ~-IFN-a~ ~see Table 2).
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Table 2
5 Composition of the lYoPhilizates con~ainina r-IFN-aA
Lyo~hilization from:
10 r-IFN-aA ~mio. I .U. ) O. S 0. 5
Glycine (mg) 10 . O - ~:
Lactose (mg) - 50. 0
NaOH lN ad pH 7.4q.6. 9.S. .
Water for injection 1.0 ml 1.0 ml
:~
Ant_iral activity
(in mio. I.U. ) afte~
manuf acture of the
lyophiliza~e 0.4 0.4
2 week~/ 5C û. 5 0 . 4
" t25C o . ~ o. 4
" /35C O . ~ O . 4
" /45C 0. 3 O. 3
" /65C: O . 2 O. 3
3 month~J 5C 0. 5 O. 5
" /25~ 0.4 0.4
" /35C O . 3 0 . 4
" /45C O . 2 0 . 4
6 mon~h~/ 5C 0. 4 O. S
" /25C o . 4 o . ~
" /3~oC 0. 2 0 . 4
" /45C O . 05 0 . 3
2 ~
-- 10 --
~xam~le 2
The stability o~ lyophilizates containing r-IFN-aA
and sacchaeose was investigated in an analogou& mannee to
Example 1. It was established that the s~abilization of
r-lFN-aA obtained with lacto~e can also be achieved with
saccharose (see Table 3).
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Table 3
Com~osition of the lyo~hilizates containinq r-IFN-aA
1 2 3
I.Yophilizate flom:
r-IFN-aA tmio. I.U.~ 3 3 3
10 Sodium chloride (mg~ 9.0
Human seru~ albumin 5.0 - -
Glycine - 20.0 10.0
Saccharose (mg) - - - 50.0
NaHzPO4 H20 ad pH 7.4 - q-S-
15 NaOH lN ad pH 7.4 _ q~s
Water for injection
ad 1. 0 ml1. 0 ml1. 0
20 Antiviral activit~
(in mio. I.U.~ after
manuSacsure of the
lyophilizate 2.7 3.0 3.4
3 month6~ 5C 2.7 2.5 3.9
" /25C 2.5 2.3 3.6
" /35C 2.4 2.1 3.7
" /45C 1.7 1.2
6 months/ 5C 3.5 3.1 4.0
" /25C 3.3 3.1 3.7
" /35~C ~.6 l.g 3.7
' /45C - 1.7
7, ~
Exam le 3
The stability of lyophilizates containing r-IPM-aA
in high concentrations and lactose or saccharose was
investigated in an analogous manner to Example 1. It was
established ~ha~ the stabilization oE IFN-a produced
using disaccharides can also be achile~ed in the ca~e of
high rIFN-aA concentrat:ions (see Table 4).
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Table 4
Composition of l~oPhili-zate~ containinq r-IFN-aA
Lyophilized from: 1 2 3 4
r-IFN-aA ~mio. I.U.)9.0 9.0 18.0 18.0
Lacto~e 50,0 m5 - 50.0 mg
Saccharo~e - 50.0 mg - 50.0 mg
Glycine 10.0 mg 10.0 mg 10.0 mg 10.0 mg
NaOH 1~ ad pH 7.4 - - - q.s.
Water for injection
ad 1.0 ml 1.0 ml 1.0 ml 1.0 ml
Antiviral activit~
(in ~iQ. I.~.) af~er
manufacture of the
lyophiliæate 8.~ 8.9 18.3 19.9
3 month~/ 5C 9.9 9.6 17.8 Z3.4
" /25C 10.0 10.5 19.1 23.Z
" /35C 10.3 10.1 20.5 n.t.
" /45C 10.4 10.9 22.0 n.t.
6 months/ 5C 9.7 n.t. 17.B 19.1
" /25C 9.9 n.t. 21.1 21.g
/35C 8.g n.t. 16.6 n.t.
" /45C 8.4 n.t. 16.1 n.~.
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Exam~e 4
The stability o~ lyophilizate6 containing ~-IFN-aA,
lactQse and glycocholic acid was investigated in an
analogous mann~c to Example 1. The results obtained a~e
compiled in Table S.
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Table 5
Compo~ll.;on of the lyophilizates containinq r-IFN-aA `-
lyophilizate from:
r-IFN-a~ (mio. I.U.j 1 3 g 18
Glycocholic acid (mg) 5.0 5.0 5.0 5.0
Lactose (mg) 50.050.0 50.0 50.0
10 Sodillm chloride (mg) Z.5 2.5 Z.5 2.5
NaOH lN ad ~H 7.4 q.~.q.s. q.6. q.6.
Water for injeo-ti.on
ad 1.0 ml 1.0 ml 1.0 ml 1.0 ml
An~iviral activity
(in mio, I.U.~ after
manufacture of the
20 lyo~hilizate 0.962.97 8.56 18.8
14 dayst 5C 0.85
" / RT 0.92 - - -
" /35C 0.85
" /45C 0.83
6 months/ 5C 1.022.89 9.2 18.7
/ RT 1.12.8Z 8.7 18.7
" /35C 1.06 3.11 8.9 18.3
30 " /45C 0.99 Z.71 8.94 n.t.
36
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