Note: Descriptions are shown in the official language in which they were submitted.
--1--
STABLE PHARMACEUTICAL PREPARATION CONTAINING
GRANULOC~TE COI,ONY STIMUI.ATING FACTOR AND
PROCESS FOR PRODUCING THE SAME
The present invention relates to a pharmaceutîcal
preparation containing a granulocyte colony stimulating
factor. In particular, the present invention relates to a
stabilized pharmaceutical preparation containing a granulo-
cyte colony stimulating factor that is protected against
loss or inactivation of the active component (i.e., granulo-
cyte colony stimulating factor) due to adsorption on thewall of a container in which the preparation is put, or to
association, polymerization or oxidation of said component.
Chemotherapy has been undertaken as one method for
treating a variety of infectious diseases but it has
recently been found that chemotherapy causes some serious
clinical problems such as the generation of drug-resistant
organisms, change of causative organisms, and high side
effects. In order to-avoid these problems associated with
chemotherapy involving the use of therapeutic agents such as
antibiotics and bactericides, attempts are being made to use
a substance that activates the prophylactic capabilities of
the host of an infection-causing organism and thereby pro-
viding a complete solution to the aforementioned problems of
che therapy. Of the various prophylactic capabilities of
25 the host, the phagocytic bactericidal action of leucocytes
is believed to cause the strongest influence in the initial
period of bacterial infection and it is therefore assumed to
be important to enhance the infection protecting capabili-
ties of the host by promoting the growth of neutrophiles and
their differentiation into the mature state. A granulocyte
colony stimulating factor (G-CSF) is one of the very useful
substances that exhibit such actions and the same assignee
of the present invention previously filed a patent applica-
tion on an infection protecting agent using G-CSF (Japanese
Patent Application No. 23777/1985).
As mentioned above, chemotherapy as currently
practiced involves various unavoidable problems and inten-
sive efforts are being made to use a drug substance that is
capable of activating the prophylactic functions of the host
or the person who has been infected.
Needless to say, G-CSF displays by itself the ability
to activate the prophylactic functions of the host and it
has also been found that G-CSF exhibits greater therapeutic
effects in clinical applications if it is used in combina-
tion with a substance that activates the prophylactic
capabilities of the host.
G-CSF is used in a very small amount and a pharma-
ceutical preparation containing 0.1 - 500 ~S (preferably
5 - 50 ~g) of G-CSF is usually administered at a dose rate
of 1 - 7 times a week per adult. However, G-CSF has a
tendency to be adsorbed on the wall of its container such as
an ampule for injection or a syringe. Therefore, if the
drug is used as an injection in such a form as an aqueous
solution, it will be adsorbed on the wall of its container
such as an ampule or a syringe. This either results in the
failure of G-CSF to fully exhibit its activity as a pharma-
ceutical agent or necessitates the incorporation of G-CSF
~ 3--
in a more-than-necessary amount making allowance for its
possible loss due to adsorptionO
In addition, G-CSF is labile and highly s~sceptible
to environmental factors such as temperature, humidity,
oxygen and ultraviolet rays. By the agency of such factors,
G-CSF undergoes physical or chemical changes such as associ-
ation, polymerization and oxidation and suEfers a great loss
in activity. These phenomenon make it difficult to ensure
complete accomplishrnent of a therapeutic act by administer-
ing a very small amount of G-CSF in a very exact manner.
It is therefore necessary to develop a stable
pharmaceutical preparation of G-CSF that is fully protected
against a drop in the activity of its effective component.
This is the principal object of the present invention which
15 provides a stable pharmaceutical preparation of G-CSF.
The present inventors conducted intensive studies
in order to enhance the stability of a G-CSF containing
pharmaceutical preparation and found that this object can
effectively be attained by addition of a pharmaceutically
20 acceptable surfactant, saccharide~ protein or high-molecular
weight compound.
` Therefore~ the stable G-CSF containing pharmaceutical
preparation of the present invention is characterized by
containing both G-CS~ and at least one substance selected
25 from the group of a pharmaceutically acceptable surfactant,
saccharide, protein and high-molecular weight compound.
The G-CSF to be contained in the pharmaceutical prep-
aration of the present invention can be obtained by any of
' ~ ' ;, .
~3~
the methods such as those described in the specifications of
Japanese Patent Application Nos. 153273/1984, 269455/1985,
269456/1985, 270838/1985 and 270839/1985. For example, a
human G-CSF can be prepared either by ~ultivating a cell
strain (CNCM Accession Number I-315 or I-483) collected from
tumor cells of patients with oral cavity cancer, or by
expressing a recombinant DNA (which has been prepared by the
agency of a human G-CSF encoding gene) in an appropriate
host cell (e.g. E. coli, C 127 cell or ovary cells of a
Chinese hamster).
Any human G-CSF that has been purified to high degree
may be employed as the G-CSF to be contained in the pharma
ceutical preparation of the present invention. Preferable
human G-CSFs are ones obtained by isolation from the super-
natant of the culture of a human G-CSF producing cell, and a
polypeptide or glycoprotein having the human G-CSF activity
that is obtained by transforming a host with a recombinant
vector haviny incorporated therein a gene coding for a
polypeptide having the human G-CSF activity.
Two particularly preferable examples of humall G-CSF
are shown below:
(1) human G-CSF having the following physicochemical
properties:
i) molecular weight: about 19,000 + 1,000 as measured by
electrophoresis through a sodium
dodecylsulfate - polyacrylamide gel;
ii) isoelectric point: having at least one of the ~hree
isoelectric points, pI = 5.5 + 0.1,
pI = 5.8 + 0.1, and pI = 6.1 ~ 0.1;
~2~ 7
--5--
iii) ultraviolet absorption: having a maximum absorption at
280 nm and a minimum absorption at
250 nm;
iv) amino acid sequence of the 2:L residues from N terminus:
H2N-Thr-~ro-Leu-Gly-Pro-A:La-Ser-Ser-Leu-Pro-Gln-Ser-
Phe-Leu-Leu-Lys-Cys-Leu-GlU-Gln-Val
(2) human G-CSF containing either a polypeptide having the
human granulocyte stimulatillg factor activity which is
represented by all or part oE the amino acid sequence
shown below, or a glycoprotein having both said poly-
peptide and a sugar chain portion:
(Met)nThr Pro Leu Gly Pro Ala Ser Ser Leu Pro
Gln Ser Phe Leu Leu Lys Cys Leu Glu Gln Val
Arg Lys Ile Gln Gly Asp Gly Ala Ala Leu Gln
Glu Lys Leu (Val Ser Glu~mCys Ala Thr Tyr Lys
Leu Cys His Pro Glu Glu Leu Val Leu Leu Gly
His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser
Ser Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly
~ Cys Leu Ser Gln Leu His Ser Gly Leu Phe Leu
Tyr Gln Gly Leu Leu Gln Ala Leu Glu Gly Ile
Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu
Gln Leu Asp Val Ala Asp Phe Ala Thr Thr Ile
Trp Gln Gln Met Glu Glu Leu Gly Met Ala Pro
Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala
Phe Ala Ser Ala Phe Gln Arg Arg Ala Gly Gly
Val Leu Val Ala Ser His Leu Gln Ser Phe Leu
Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala
Gln Pro
(provided that m is 0 or 1; and n is 0 or 1).
- . . ~
,.
' . :.
--6--
For details of the method for preparing these two
types of G-CSF, see the specification of Japanese Patent
Application Nos. 153273/1984, 269455/1985~ 269456/1985,
270838/1985 and 270839/1985/ all having been filed by the
assignee oF the present invention.
Another method that can be employed consists of
perEorming fusion of a G-CSF producing cell with a self-
proliFerating malignant tumor cell and c~31tivating the
res~lting hybridoma in the presence or absence of mytogen.
The human G-CSF containing solution obtained may be
stored in a frozen state after being further purified and
concentrated~ as required, by any known technique. Alterna-
tively, the solution may be stored after being dehydrated by
such means as freeze-drying.
All of the human G-CSFs thus prepared can be
processed as specified by the present invention in order to
attain stable G-CSF containing pharmaceutical preparations.
Typical examples of the surfactant that is used to
attain the stable G-CSF containing pharmaceutical prepara-
tion of the present invention are listed below: nonionicsurfactants with HLB of 6 - 18 such as sorbitan aliphatic
acid esters (e.g. sorbitan monocaprylate/ sorbitan mono-
laurate and sorbitan monopalmitate~, glycerin aliphatic acid
esters (e.g. glycerin monocaprylate, glycerin monomyristate,
and glycerin monostearate), polyglycerin aliphatic acid
esters (e.g. decaglyceryl monostearate, decaglyceryl
distearate and decaglyceryl monolinoleate), polyoxyethylene
sorbitan aliphatic acid esters (e.g. polyoxyethylene
--7~
sorbitan monolaurate, polyoxyethylene sorbi~an monooleate,
polyoxyethylene sorbitan monostearate, polyoxyethylene
sorbitan monopalmitate, polyoxyethylene sorbitan trioleate,
and polyoxyethylene sorbitan tristearate), polyoxyethylene
sorbitol aliphatic acid esters (e.g. polyoxyethylene sorbi-
tol tetrastearate and polyoxyethylene sorbitol tetraoleate),
polyethylene glycerin aliphatic acid esters (e.g. polyoxy-
ethylene glyceryl monostearate), polyethylene glycol
aliphatic acid estexs (e.g. polyethylene glycol distearate),
polyoxyethylene alkyl ethers (e.g. polyoxyethylene lauryl
ether), polyoxyethylene polyoxypropylene alkyl ethers (e.g.
polyoxyethylene polyoxypropylene glycol ether, polyoxy-
ethylene polyoxypropylene propyl ether, and polyoxyethylene
polyoxypropylene cetyl ether)! polyoxyethylene alkylphenyl
ethers (e.g. polyoxyethylene nonylphenyl ether), polyoxy-
ethylated castor oil, polyoxyethylated hardened castor oil
(polyoxyethylated hydrogenated castor oil), polyoxyethylated
beeswax derivatives (e.g. polyoxyethylated sorbitol beeswax),
polyoxyethylene lanolin derivatives (e9g. polyoxyethylene
lanolin), and polyoxyethylene aliphatic acid amides (e.g.
polyethylene stearic acid amide); ncnionic surfactants such
as alkyl sulfuric acid salts having a C10 - Cl~ alkyl group
(e.g. sodium cetyl sulfate, sodium lauryl sulfate and sodium
oleyl sulfate), polyoxyethylene alkyl ether sulfuric acid
salts wherein the average molar number of ethylene oxide
addition is 2 - 4 and the alkyl group has 10 - 18 carbon
atoms (e.g. polyoxyethylene sodium lauryl sulfate), salts of
alkyl sulfosuccinate esters wherein the alkyl group has 8 -
--8--18 carbon atoms (e.g. sodium lauryl sulfosuccinate ester);
and natural surfactants such as lecithin, glycerophospho-
lipid, sphingophospholipid (e.g. sphingomyelin), and sucrose
aliphatic acid esters wherein the aliphatic acid has 12 - 18
carbon atoms. These surfactants may of course be used
either independently or in admixture.
The surfactants listed above are preferably used in
amounts of 1 - 10,000 parts by weight per part by weight of
G-CSFo
The saccharide to be used in making the stable G-CSF
containing pharmaceutical preparation of the present inven-
tion may be selected from among monosaccharides, oligo-
saccharides, and polysaccharides, as well as phosphate
esters and nucleotide derivatives thereof so long as they
15 are pharmaceutically acceptable. Typical examples are
listed below: trivalent and higher sugar alcohols such as
glycerin! erythritol, arabitol/ xylitol, sorbitol, and
mannitol; acidic sugars such as glucuronic acid, iduronic
acid, neuraminic acid, galacturonic acid, gluconic acid,
20 mannuronic acid, ketoglycolic acid, ketogalactonic acid and
ketogulonic acid; hyaluronic acid and salts thereof, chon-
droitin sulfate and salts thereo, heparin, inulin, chitin
and derivatives thereof, chitosan and derivatives thereof,
dextrin! dextran with an average molecular weights of 5,000
25 - 150,000, and alginic acid and salts thereof. All of these
saccharides may be used with advantage either independently
or in admixture.
' ' ' ' ~
.
~,ra~l(D@~';;i'
~9~
The saccharides listed above are preferably used in
amounts of 1 10,000 parts by weight per part by weight of
G~CSF.
Typical examples of the protein to be used in makiny
the stable G~CSF containin~ pharmaceutical preparation of
the present invention include hurnan serum albumin, human
serum globulin, gelatin, acid-treated gelatin (average mol.
wt. = 7,000 - 100,000)! alkali-txeated gelatin (average mol.
wt. = 7,000 - 100,000), and collagen. Needless to say,
these proteins may be used either independently or in
admixture.
The proteins listed above are preferably used in
amounts of 1 - 20,000 parts by weight per part by weight of
G-CSF.
Typical examples of the high-molecular weigh compound
to be used in making the stable C-CSF containing pharmaceu-
tical preparation of the present invention include: natural
polymers such as hydroxypropyl cellulose, hydroxymethyl
cellulose, sodium carboxymethyl cellulose, and hydroxyethyl
cellulose; and synthetic polymers such as polyethylene
glycol (mol. wt. = 300 - 6,000)! polyvinyl alcohol (mol. wt.
= 20,000 - 100,000), and polyvinylpyrrolidone (mol. wt. =
.
20,000 - 100,000). Needless to say, these high-molecular
weight compounds may be used either alone or in combinatlon.
The high-molecular weight compounds listed above are
desirably used in amounts of 1 - 20,000 parts by weight per
part by weight of G-CSF.
.. . . .
' . ' ', :
- . , , . . ~ .
.
- . ~ .
-lU-
In addition to the surfactant, saccharide, protein or
high-molecular weight compound described above, at least one
member selected from the group consisting of an amino acid,
a sulfureous reducing agent and an antioxidant may also be
incorporated in making the G-CSF containing pharmaceutical
preparation of the present invention. Illustrative amino
acids include glycine, threonine, tryptophan, lysine,
hydroxylysine, histidine, arginine, cysteine, cystine, and
methionine. Illustrative sulfureous reducing agents
include: N-acetylcysteine, N-acetylhomocysteine, thioctic
acid! thiodiglycol, thioethanolamine, thioglycerol, thio-
sorbitol, thioglycolic acid and salts thereof, sodium thio-
sulfate, sodium hydrogensulfite, sodium pyrosulfite, sodium
sulfite, thiolactic acid, dithiothreitol, glutathione, and a
15 mild sulfureous reducing agent having a sulfhydryl group
such as a Cl - C7 thioalkanoic acid. Illustrative anti-
oxidants include erythorbic acid, dibutylhydroxytoluene,
butylhydroxyanisole, dQ-a-tocopherol, tocopherol acetate,
L-ascorbic acid and salts thereof, L-ascorbic acid palmitate,
20 L-ascorbic acid stearate, triamyl gallate, propyl gallate
and chelating agents such as disodium ethylenediaminetetra-
acetate (EDTA), sodium pyrophosphate and sodium
metapho~phate.
The above-listed amino acids, sulfureous reducing
25 agents and antioxidants or mixtures thereof are preferably
used in amounts of 1 - 10,000 parts by weight per part by
weight of G-CSE'.
' ''
:
For the purpose of Eorm~lating the stable G-CSF
containing preparation of the present in~ention in a
suitable dosage form~ one or more the following agents may
be incorporated: a diluent, a solubilizing aidl an isotonic
agent, an excipient, a pH modifier, a soothing agent, and a
buffer.
The stabilized G-CSF pharmaceutical preparation o~
the present invention may be formulated either for oral
administratio~ or for parenteral administration such as by
injection applied in various ways' and a variety of dosage
forms may be employed depending upon the specific mode of
administration. Typical dosage forms include~ those
intended for oral administration such as tablets, pills,
J. capsules, granules and suspensions; solutions, suspensions
15 and freeze-dried preparations principally intended for
intravenous injection' intramuscular injection, subcutaneous
injection and intracutaneous injection; and those intended
for transmucosal administration such as rectal supposito-
ries' nasal drugs, and vaginal suppositories.
According to the present invention, at least oné
substance selected from the group consisting of a surfac-
tant, a saccharide! a protein or a high-molecular weight
compound is added to a G-CSF containing pharmaceutical
preparation so that it is prevented from being adsorbed on
25 the wall of its container or a syringe while at the same
time, it remains stable over a prolonged period of time.
The detailed mechanism by which the substances
mentioned above stabilized G-CSF or prevent it from being
: : ' ' .,
. - -, .
. ' ` ' ~" ' ' : .
.
~7'~'7
-12-
adsorbed is yet to be clarified. In the presence of a
surfactant, the surface of G-CSF which is a hydrophobic
protein would be covered with the surfactant to become
solubilized so that the G-CSF present in a trace amount is
effectively prevented from being adsorbed on the wall of
its container or a syringe. A saccharide or hydrophilic
high-molecular weight compound would form a hydrated layer
between G-CSF and the adsorptive surface of the wall of
its container or a syringe, thereby preventing adsorption
of G-CSF in an effective manner. A protein would compete
with G-CSF for adsorption on the wall of its container or
a syringe, thereby effectively inhibiting adsorption of
G-CSF.
Besides the prevention of ~-CSF adsorption! the
substances mentioned above would also contribute to the
prevention of association or polymerization of the molecules
of G-CSF. In the presence of a surfactant9 saccharide,
protein or high-molecular weight compound, the individual
molecules of G-CSF are dispersed in these substances and
the interaction between the G CSF molecules is sufficiently
reduced to cause a significant decrease in the probability
of their association or polymerization. In addition, these
substances would retard the autoxidation of G-CSF that is
accelerated under high temperature or humidity or prevent
G-CSF from being associated or polymerized as a result of
its autoxidation. These effects of retarding autoxidation
of G-CSF or preventing it from being associated or polyme-
rized would be further enhanced by addition of an amino
acid, a sulfureous reducing agent or an antioxidant.
The problems described above are particularly notice-
able in solutions for injection and in suspensions but they
also occur during the process of formulating G-CSF in other
dosage forms such as tablets~ The addition of surfactants,
saccharides, proteins or high-molecular weight compounds is
also effective in this latter case.
Through the addition of at: least one substance
selected from the group consisting of a surfactant, saccha-
ride, protein and a high-molecular weight compound, G-CSF is
highly stabilized and maintains its activity for a prolonged
period of time, as will be demonstrated in the examples that
follow. To attain these results, the amount of each of
these substances, in particular its lower limit, is critical
15 and the following ranges are desirable: 1 - 10~0~0 parts by
weight of surfactant, 1 - 10,000 parts by weight of saccha-
ride, 1 - 20,000 parts by weight of proteinl and 1 20,000
parts by weight of high-molecular weight compound, per 1
part by weight of G-CSF.
According to the present invention, a surfactant,
a saccharide, a protein and/or a high-molecular weight
compound is used in a specified concentration and this is
effective not only in preventing G-CSF from being adsorbed
on the wall of its container or a syringe but also in
enhancing the stability of a G-CSF containing pharmaceutical
preparation. As a result, it becomes possible to ensure
the administration of a small but highly precise dose of
G-CSF to patients; since G-CSF is costly, its efficient
`~ : - - , : , .
:
utilization will lead to lower costs for the production of
G-CSF con'aining pharmaceutical preparations.
The follo~ing examples are provided for the purpose
of further illustrating the present invention but are in no
sense to be taken as limiting. Xn these examples, the
residual activity of G-CSF was determined by one of the
following methods.
(a) Soft agar method usina mouse bone marrow cells:
A horse serum (0.4 ml), 0"1 ml of the sample, 0.1 ml
of a C3H/He (female) mouse bone marrow cell suspension (0.5
- 1 x 105 nuclear cells), and 0.4 ml of a modified McCoy's
5A culture solution containing 0.75% of agar were mixed,
poured into a plastic dish for tissue culture (35 mm0),
coagulated, and cultured for 5 days at 37C in 5% CO2/95%
air and at 100% humidity. The number of colonies formed was
counted (one colony consisting of at least 50 cells) and the
activity was determined with one unit being the activity for
forming one colony~
The modified McCoy's 5A culture solution used in the
method ~a) was prepared by the followiny procedures.
Modified Mc~oy's_SA culture solution (double concentrationL
Twelve grams of McCoy's 5A culture solution (Gibco),
2.55 g of MEM amino acid-vitamin medium (Nissui Seiyaku ~o.,
Ltd.), 2~18 g of sodium bicarbonate and 50,000 uniks of
potassium penicillin G were dissolved twice in 500 ml of
distilled water and the solution was aseptically filtered
through a Millipore filter (0~22 ~m)O
3~
~15-
(b) Reverse-phase high-perEormance liquid chromatoqraphy:
Using a reverse-phase C8 column (4.6 mrn x 300 mm;
5 ~m) and an n-propanol/trifluoroacetic acid mixture as a
mobile phase, the residual activity of G-CSF (injected in an
amount equivalent to 1 ~g) was determined under the follow-
ing gradient conditions:
Time (secL Solvent (A) Solvent (B) Gradient
0 100% o%
3 linear
0% 100%
. linear
100% o%
Solvent (A): 30% n-propanol and 0.1% trifluoroacetic acid
Solvent (B): 60% n-propanol and 0.1% trifluoroacetic acid
Detection was conducted at a wavelength of 210 nm and
the percentage of the residual G-CSF activity was calculated
by the following formula:
the residual amount of G-CSF
Residual G-CSF _ after the lapse of a given time x 100
activity (%3 the initial amount of G-CSF
The residual amount of G CSF as determined by this
method correlated very well with the result attained in
measurement by the soft agar method (a~ using mouse bone
marrow cells.
Example l
To 5 ~9 of G-CSF/ one of the stabilizing agents
listed in Table 1 was added and the mixture was aseptically
: dissolved in a 20 mM buffer solution (containing 100 mM
sodium chloride; pH 7.4) to make a pharmaceutical prepara-
tion containing 5 ~g of G-CSF per ml! which was then
freezer-dried. The time-dependent change in G-CSF activity
~7(31~
-16-
was measured by method (a) and the results are shown in
Table 1. The term "activity (~)" in the table represents
the residual activity of G-CSF relative to the initial unit
and is defined by the following :Eormula:
activity unit after
Activity (%) = inlitPal actiavgty u-nit e x 100
Freeze-drying was conducted by the following
procedures:
l'he G-CSF solution containing a stabili~ing agent was
put into a sterile sulfa-treated glass vial, frozen at -40C
or below for 4 hours, subjected to primary drying by heating
from -40C to 0C over a period of 48 hours with the pres-
sure increased Erom 0.03 to 0.1 torr, then to secondary
during by heating from 0C to 20C for a period of 12 hours
with the pressure increased from 0.03 to 0.08 torr; there-
after, the interior of the vial was Eilled with a sterile
15 dry nitrogen gas to attain an atmospheric pressure and the
vial was plugged with a freeze-drying rubber stopper, then
sealed with an alum.inum cap.
-17-
Table l
.. _ -- .
Activ. ty (%)
Stabilizing agent (parts by After After
weight) storage at 37C for
6 months 1 month
___ , .. __ .. _I
xylitol 10,000 92 86
. ~ _
mannitol 10,000 91 85
. ~ .. . . _
glucuronic acid 10,000 86 82
__ ......... . ~_ _ .
hyaluronic acid 2,000 92 89
.. . _. . __ .
dextran (m.w. 40,000) 2,000 95 90
_ _ ._ ~ .. _ _ _
heparin 5,000 85 80
... _~ .~ _ _
chitosan 2,000 93 91
. _ _ . _ .. ._ ..
alginic acid 2,000 90 90
_ . . _ . . _ . . _
human serum albumin 1,000 98 99
. ._ . _ . . ._ __
human serum globulin 1,000 98 95
_ _ .___ .. _ . .. __
acid-treated gelatin 2,000 97 95
. ~ .
alkali-treated gelatin 1,000 99 g6
.. ..__
collagen 2,000 95 90
... . _ . ____
polyethylene glycol 10,000 94 90
. ~ . .__ . __ . _
hydroxypropyl cellulose 1,000 98 94
_ __ ................................... _
sodium carboxymethyl 1,000 88 80
. _ - ~ .... _ __~ ._
hydroxymethyl cellulose 5,000 92 90
_ _ ___ _
(m.w. 50~000) 2~000 96 95
. ._
(m.w. 50,000) 2,000 95 94
. . ___ .. _ .
human serum albumin2 t
mannitol 2,000 100 97
cystelne ~00 _ _ _ _ _
. ,.
3~3
-18-
Table 1 (cont'd)
_ _ _ _ Activity (%)
Stabilizing agent mo ntAfter After
weight) storage at 37C for
6 months 1 month
. .._ .
human serum albumin 2,000
polyoxyethylene 100 99 96
sorbitan monolaurate
mannitol 2,000
. ~ . _
human serum albumin2,000
hydroxypropyl cellulose 500 98 92
dextran (m.w. 40,000)2,000
. .__ _ , __
polyoxyethylene 100
sorbitan monolaurate 98 96
sorbitol 2,000 _
polyoxyethylated 100
hardened castor oil
94 92
dextran (m.w. 40,000)2,000
.. ___ . .. _ _ . _
not added _ 74 58
Example 2
To 10 ~g of G-CSF, one of the stabilizing agents
listed in Table 2 was added and the mixture was aseptically
dissolved in a 20 mM phosphate buffer solution tcontaining
5 100 mM sodlum chloride; pH, 7.4) to make a pharmaceutical
preparation containing 10 ~g of G-CSF per ml. The prepara- !
tion was aseptically charged into a sulfa~treated glass vial
and sealed to make a G~CSF solution. The time-dependent
change in the activity o~ G-CSF in this solution was
10 measured by the same method as used in Example 1 and the
results are shown in Table 2.
~2~7~
--19--
Table 2
_ _--
Activity (%)
Amount After After After
Stabilizing agent (parts storage storag~ storage
weight) for ; for 2 forRl
days months month
. .__ .____ _ _
mannitol - 5,000 91 87 82
. .. __ _ . . __ . _
hyaluronic acid 2,000 93 87 70
. ~ . . . __ . . .
dextran (m.w. 40,000) 2,000 96 95 85
. .. .. _ .. __ . _
glycerin 10,000 90 90 88
_ . . _ _ . _~
neuraminic acid 5,000 93 91 84
.. __ _ _ . _ . _ _ . ._
chitin 2,000 95 92 86
_ . _ __ . . ._ __
dextrin 2,000 90 92 87
... ._ _ .... _ .. . _ _
human serum albumin 1,000 99 95 92
_ ._ .. ._ . __
human serum globulin1,000 98 94 90
. _ _ . _ _ __
acid-treated gelatin2/000 97 96 87
~~ ..... ._ . _
alkali-treated gelatin 500 99 95 92
_ . . .. _ . _ . . . __
collagen 2,000 99 94 88
. __ . .. .__
: (m.w. 4,000) 10!000 94 89 90
, . _ _ , . __ _ _
hydroxypropyl cellulose 2,000 98 95 92
_ . _ . . . _ . __
~ ~ sodium carboxymethyl 2,000 92 91 80
. .. .__ _ ..
: hy~roxyethyl cellulose 4,000 92 94 90
_ - __ _.. ~_ __ _ __
(olyvinyl alcohol 4~000 97 93 90
. ~. , _ . _.
(m.w. 50,000) 4~000 95 95 92
. . ~ . _ __._ ~
sorbitan monolaurate 400 37 96 95
. __ ._ ~_ _~ .. __
sorbitan monolaurate __ 96 ___ _
,
.
-20-
Table 2 (cont'd)
. .__ _ . _ . ___
Activity (%)
Amount After After After
Stabilizing agent (part storage storage storage
weight) for ; for 2 ~aOtrRl
days months month
_ _ _
polyoxyethylene 400 98 97
polyoxyethylene
polyoxypropylene 400 100 94 93
glycol ether __ __
polyoxyethylated 400 99 98 90
.... __ . .__ . _ .
sodium lauryl sulfate 2,000 97 93 87
._ ~ .. ._ _ _
lecithin 2,000 97 94 90
._
human serum albumin 2,000
mannitol 2~000 100 99 97
,cysteine 100 _ ~_ __
human serum albumin 2,000
polyoxyethylene 100 99 97 95
mannitol 2,000 __ _ __ _
human serum albumin 1,000
hydroxypropyl cellulose 500 99 97 95
dextran ~(m.w. 40,000)2,000 _
polyoxyethylene 100
sorbitan monopalmitate 96 96 93
sorbitol 2,000
.___ _. - . _ __ .. .
polyoxyethylated 10 n
hardened castor oil 95 92 92
dextran (m.w. 40~,000) 2,000 _ _ ___
not added 72 61 47
'
- :
.
-21
Example 3
To 10 llg of G-CSF, one of the stabilizing ayents
listed in Table 3 was added and the mixture was aseptically
dissolved in a 20 mM phosphate buffer solution (containing
100 mM sodium chloride; pH, 7.4) to make a pharmaceutical
preparation containing 10 ~g of G-CSF per ml. One milli-
liter of the preparation was charged into a sulfa-treated
silicone-coated glass vial and left at 4C. The effective-
ness of each stabilizing agent in preventing G-CSF adsorp-
tion was evaluated by measuring the residual activity of
G-CSF in the solution after 0.5, 2 and 24 hours. The
measurement was conducted by method (b) using reverse-phase
high~performance liquid chromatography. The results are
shown in Table 3.
- . ' , ' :
' . . '
1~970CD~d
~~2-
Table 3
_ __ _
Amount Residual activity (~)
Stabilizing agent (parts
welght) initial 0.5 h ~ h 24 h
. _ _
monnitol 5,000 100 93 90 91
_ _
hyaluronic acid 2,000 100 97 92 92
_ _ . _
dextran (m.w. 40,000) 2,000 100 98 95 96
... , .. ... _ .. __ .
glycerin 10,000 100 94 91 90
_ .... _ _ ~ _
heparin 2,000 100 92 90 90
. . _ .. _ . . __ . __ __
glucuronic acid 5,000 100 96 90 91
. . __ . __ _ .
ketoglycolic acid 5,000 100 92 88 90
human serum albumin 1,000 100 100 101 99
human serum globulin 1,000 100 98 100 98
. . _ __. . _ ,~ . _
alkali-treated gelatin 500 100 99 98 99
. ___ _ . __ r __
acid-treated yelatin 2,000 100 99 97 97
.___ ... . .
collagen 2,000 100 100 98 99
(m.w. 4,000) 10,000 100 100 100 99
. . . _
hydroxypropyl cellulose 2,000 100 100 100 99
_ __ ____ _ _ _ ____- . __. _. ._ _____ .
sodium carboxymethyl 2,000 100 98 96 95
.~ ._ ..~ __
hydroxyethyl cellulose 4,000 100 96 93 92
_. __ ....... _ _
p(olyvinyl alcohol 4,000 100 99 100 98
_ . _ . . .. .
(m.w. 50,000) 4,000 100 98 98 96
.. ___.
sorbitan monocaprylate 400 100 100 100 98
. .. ____ . _ _
sorbitan monostearate 400 100 100 98 100
. .__ _
hardened castor oil 400 100 99 101 99
.
'
-23-
Table 3 (cont'd)
.. __~ _ .
Amount Residual activity (%)
Stabilizing agent (parts
weight) inltial 0.5 h 2 h 24 h
sodium lauryl sulfate2!000 100 100 . 97
lecithin 2,000 100 99 100 98
h~man serum albumin2,000
mannitol 2,000 100 100 100 101
cysteine 100
__ . ... _ _ _ ..
human serum albumin 2,000
polyoxyethylene 100 100 100 98 99
mannitol 2,000
_ _ __ . . . _ .. _
human serum albumin 1,000
hydroxypropyl cellulose500 100 101 99 100
dextran (m.w. 40r000)2,000 _ __
polyoxyethylene 100
sorbitan monolaurate
100 100 99 99
sorbitol ~ 2,000 _
polyoxyethylated 100
hardened castor oil
100 100 98 97
dextran (m.w. 40,000) 2,000
__ ,. ....
not added ___ 100 91 72 73
.
.
,
