Note: Descriptions are shown in the official language in which they were submitted.
2377 CANADA
BIOLOGICALLY STA~LE
INTERFERON COMPOSITIONS
The present invention relates to pharmaceutical
compositions comprising interferon and in particular to
such compositions which are substantially resistant to
microorganism contamination and growth during storage at
room temperature.
By the present invention, thimerosal (i.e.
sodium ethylmercurithiosalicylate, also known as
merthiolate) is employed in the compositions as an
antimicrobial preservative.
Formulations of the present invention are
use~ul in preparing stable interferon solutions suitable
for injectable, ophthalmic and nasal products.
Interferons have qreat potential as drugs for
the treatment of a variety disease states, e.~. various
types of viral infections and certain cancers.
As used herein, the term "interferon" includes
natural and recombinant alpha (leucocyte) and beta
~fibroblast) interferons, but alpha interferons are
preferred. As used herein, the term "alpha interferon"
means a natural or recombinant interferon exhibiting
biological properties similar to those of human leucocyte
interferon. It should be noted tha~ a number of alpha
interferon species are known, usually designated by a
numeral after the Greek letter, and all are contemplated
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--2--
for use in this invention. Also included within the
scope of this invention are the so-called alpha hybrid
interferons wherein fragments of two or more native alpha
interferon species are joined (see for instance, EP
51873). Preferred forms of alpha interferon for use in
the formulations of the present invention are alpha-l and
alpha-2 interferon. Particularly preferred for use in
the formulations of the present invention is alpha-2
interferon. Alpha-2 interferon may be prepared by
recombinant-DNA methods, for example those disclosed by
Nagata et al., Nature, Vol. 284, pages 316-320 (1980),
and by Weissmann, European Patent 32,13~.
Biologically stable interferon compositions
are known. See Kwan U.S. Patent 4,496,537, which claims
a method of improving biological stability of an inter-
feron formulation comprising the addition of glycine or
alanine to an alpha interferon formulation prior to
lyophilization. While such lyophilized powders are not
generally susceptible to microbiological contamination
since interferon formulations are commonly prepared under
aseptic conditions, reconstituted solutions of these
lyophilized formulations are susceptible to microbio-
logical contamination and growth. This makes reconsti-
tuted solutions unsuitable for multiple dose appli-
cations, e.g. nasal or ophthalmic applications, where the
solution will not be used all at once.
Varlous preservatives and preservative
combinations have been tested, but in general have been
Eound to cause physical instability such as precipitation
or turbidity in the reconstituted solution. Some
preservatives are also ineffective in preventing
microbial contamination and/or reduce interferon
activity.
z~
We have surprisingly found that of the many
preservatives tested, only thimerosal is effective in
preventing microbial contamination in a reconstituted
interferon solution for at least four weeks when the
reconstituted solution is stored at room temperature or
under refrigsration. Furthermore, such reconstituted
solutions are stable with respect to antiviral activity,
pH and physical appearance under those conditions.
Thimerosal can be incorporated with the interferon in the
lyophilized powder or can be added in the diluent for
reconstitution of lyophilized interferon powder. The
concentration of thimerosal in the solution may be in the
ranqe of 0.005 to 1 mg/ml, with 0.02 to 0.05 mg/ml being
preferred, and 0.02 mg/ml being most preferred.
A particular advantage of the present invention
is the ability to store the reconstituted solution at
room temperature safely and effectively for prolonged
periods. This advanta~e is evident especially for
ophthalmic and nasal preparation where a non-hospitalized
patient will be self-administerin~ the formulations for
an extended time period, e.g. 1-3 weeks, and a portable
and easily stored formulation is desirable. Another
advantage is the increased comfort of the patient in
being able to use such formulations as eye drops or nasal
sprays at room temperature.
The various preservatives and preservative
combinations compared to thimerosal were tested by
reconstituting lyophilized alpha interferon formulations
with a vehicle comprising the preservative or
preservative combination. Reconstituted solutions stored
at room temperature and under refriqeration were
evaluated periodically for one month for physical
stability and appearance; interferon activity was
determined initially, after 7 days, 14 days and at the
end of the study by the standard method of the inhibition
~2~5~
of cvtopathic effect (CPE) of a virus. Samples which
were stable for at least 7 days at room temperature were
further tested using the USP XX Antimicrobial
Preservative Effectiveness (APE) Test and the British
Pharmacopeia (BP) test for antimicrobial preservative
effectiveness.
The followinq Table l gives the results of the
testinq of physical stability of the interferon
~ormulations to which various preservatives and
preservative combinations were added:
TABLE 1
Compatibility with
Preservative Concentration % alpha-Interferon*
Benzalkonium Chloride 0.005 P
0.02 P
Benzyl Alcohol 0.9 CR
1.8 P
Chlorhexidine Gluconate 0.005 P
0.01 P
Chlorohutanol 0.5 CR
Methylparaben/Propylparaben 0.12/0.012 CR
Phenylmercuric Acetate 0.004 CR
Phenylethyl Alcohol 0.5 P
Phenol 0.25 P
Benzyl Alcohol + 0.9 CR
Methylparaben ~ 0.08
Propylparaben 0.01
Benzyl Alcohol + 0-9 CR
TABLE 1 (continued)
Chlorobutanol 0.3
Chlorhexidine Gluconate ~ 0.005 P
Methylparaben ~ 0.08
Propylparaben 0.01
Phenylethyl Alcohol + 0.5 P
Methylparaben + 0.08
Propylparaben 0.01
Chlorhexidine Gluconate + 0.005 P
Chlorohutanol 0.5
Chlorobutanol ~ 0-5 P
Methylparaben + 0.08
Propylparaben 0.01
Thimerosal 0.002 C
0.005 C
* C - compatible for at least 7 days at
refriqeration (REF) and room temperature (RT)
CR - compatible for at least 7 days at REF, but not
at RT
P - turbidity and/or precipitation occurs within 7
days at REF and RT
Alpha interferon solutions prepared from
thimerosal-containing lyophilized powders and from
thimerosal-containin~ diluents passed physical stability,
APE and CPE tests. Alpha interferon solutions prepared
from thimerosal-containing diluents also passed the BP
test for antimicrobial preservative effectiveness.
Alpha interferon compositions tested comprised
1 x 107, 2.5 x 107 and 5 x 107 International Units (I.U.)
interferon/ml, although it is contemplated that
compositions of the present invention may contain a ran~e
of 2 x 103 to 5x 108 I.U./ml, Preferably 1 x 104 to 2 x
108 I.U. alpha interferon/ml. The specific activity of
the alpha interferon used in the compositions of the
present invention should be at least 5 x 107 I.U./mg
total protein, preferably at least 1 x 108 I.U./mq total
protein. In addition to alpha interferon and thimerosal,
the pharmaceutical solutions of the present invention
contain 0-2 mg albumin/ml and 5-25 mg qlycine/ml in a
compatible buffer to maintain the pH at 6.5 to 8Ø
Preferably the compositions comprise l mq albumin/ml and
20 mg glycine/ml in a sodium dibasic phosphate and sodium
monobasic phosphate buffer system at a pH of 7.0 to 7.4.
When thimerosal is added to the alpha
interferon solution before lyophilization, the diluent
for reconstitution may be purified water. When
thimerosal is added to alpha interferon lyophilized
powder, the diluent may be water, but is preferably an
aqueous solution of a compatible buffer to maintain the
pH at a level which will prevent decomposition of
thimerosal. The buffer system may be the same as that
used in the alpha interferon lyophilized powder, e.~. pH
6.5 to 7.5 sodium phosphate buffer, but preferably is a
pH 3.5 to 5.5 citric acid buffer prepared from citric
acid tmonohydrate or anhydrous) and sodium citrate
(dihydrate or anhydrous).
Following are examples of alpha-2 interferon
formulations wherein thimerosal is present in the diluent
and wherein thimerosal is present in the lyophilized
powder.
EXAMPLE l
Lyophilized Powder* mg/ml
Alpha-2 Interferon 2 x 103 - 2 x 108 I.U.
Sodium Phosphate Dibasic 1 - 5
Sodium PhosPhate Monobasic 0.2 - 1.0
Aminoacetic Acid 5 - 25
Human Albumin 0.5 - 2
: .
.
7--
Preserved Diluent for
Reconstitution (A) mq/ml
Thimerosal 0.005 - 0.1
Sodium Phosphate Dibasic 0.2 - 1.0
Sodium Phosphate Monobasic 0.4 - 2.0
Purified Water q.s. ad 1.0 ml
Preserved Diluent for
Reconstitution tB ? mg/ml
Thimerosal 0.005 - 0.1
Sodium Citrate Dihydrate 0.1 - 0.5
Citric Acid Monohydrate 0.1 - 0.5
Purified Water q.s. ad 1.0 ml
EXAMPLE 2
Lyophilized Powder* mg/ml
Alpha-2 Interferon 2 x 103 - 2 x 108I.U.
Sodium Phosphate Dibasic 1 - 5
Sodium Phosphate Monobasic 0.2 - 1.0
Aminoacetic Acid 5 - 25
Human Albumin 0.5 - 2
Thimerosal 0.005 - 0.1
*Lyophilized powder may contain other stabilizers to
improve/enhance Interferon stability.
Lyophilized powders are prepared by combining
and lyophilizing the inqredients for the powder
formulations using standard techniques.
Diluents are prepared by combining the
in~redients of the diluents usinq standard techniques.
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