Note: Descriptions are shown in the official language in which they were submitted.
2Q97932
DESCRIPTION
ANTIBODY CONTAINING COMPOSITION
Field ~f the Invention
The present invention relates to a novel antibody
containing medicine which is pharmaceutically useful. More
particularly, it relates to a composition containing the
antibody or its complex such as an immunotoxin complex and the
like.
Backgrou~d of the Invention
Recently, an application of the antibodies to the medicines
is extensively studied. The research and development of
pharmaceutical formulations containing the antibodies in various
fields including the fields of tumor, cardiac and circulatory
system, immunity, allergy and infection have been advanced
extensively. For example, the research for treating cancer with
monoclonal antibodies against tumor-associated antigens is most
active. Such antibodies will be developed as the pharmaceutical
formulations in the near future. At present, some formulations
are subjected to clinical experiments wherein the tumor sites to I -
be treated are covered with a wide range, including blood,
hematopoietic organ, lung, liver, digestive organ, ovarium and
prostate. As a method for treating the cancer with the
monoclonal antibody, an immunotherapy with only the antibody so
that macrophagus and lymphocytes having receptors against Fc
fragments of the antibody are collected at the tumor sites,
thereby the cancer is attacked and a target therapy with the
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immunotoxin complex comprising the monoclonal antibody to which
a toxic substance such as a toxic protein, a radioactive
substance (RI) and an anticancer agent i5 bound are known.
Dosage forms for administrating the monoclonal antibodies
are all injections, because the monoclonal antibodies are
macromolecular protein. Most of the injections are the
injections for intravenous administration. Because the
injection is a troublesome and imposes a burden to a patient,
simpler dosage form is desired. One is an inhalation By the
inhalation having a dosage form for general administration, the
burden to the patient can be lightened and the patient can be
treated at home. On the other hand, when the immunotoxin
complex wherein the monoclonal antibody is bound with, for
example, the toxic protein is intravenously administered, it
circulates through major organs including liver and kidney and
as the result, these organs may be seriously damaged. Thus, the
dosage form in place of the injection is desired. One is the
inhalation for a topical administration in order to treat lung
cancer. It is expected to be effectively used. ~owever, the
inhalation has a defect because of a drug used being
macromolecular.
One of the inhalations is an aqueous inhalation, wherein
the drug in the form of water-soluble microparticles is
administered using a suitable device such as a jet nebulizer and
an ultrasonic nebulizer. The other is a solid inhalation,
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wherein the drug in the form o~ solid particulate powder is
administered using a metered-dose inhaler. The drugs in both
inhalations are mainly low molecular compounds such as ~2-
adrenergic antagonists, steroids and antibiotics.
An attempt for formulating polypeptides as aerosol
preparations is relatively new. For example, a solid aerosol
preparation containing insulin (Lee and Sciarra, J. Pharm., 65,
567, 1976), a solid aerosol preparation containing recombinant
1~antitrypsin (Hubbard et al., Proc. Natl. Acad. Sci. USA, 86,
680, 1989), the administration of an aqueous solution of human
leukocyte interferon- through the jet nebulizer (Kin-nula et
al., J. Interferon Res., 9, 419, 1989) and the administration of
an aqueous solution of human growth hormone through the
nebulizer (JP-A-63-51868) are reported. They describe that the
polypeptide having the molecular weight of 50,000 or less is
used. And, they describe that the preferable particle diameter
is 0.5 to 10 ~m, particularly 5 ~m or less, for adsorbing the -
polypeptides by the inhalation. The effectiveness of certain
drugs as the dosage form for the topical administration has been
also examined.
In case of the aerosol preparation, it is considered that
the absorption sites are determined depending on the particle
size. It is said that smaller particles can reach to alveoli
pulmonum, while larger particles deposit in a nasal or oral
cavity. For reaching to the alveoli pulmonum, the particle
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diameter should be 0.5 to 10 ~. (Porush et al., J. Amer. Pharm.
Ass. Sci, Ed., 49, 70, 1960), preferably 5 ~m or less in
diameter (Newman and Clarks, Thorax, 3~, 881, 1983).
At present, the inhalation of the antibody is
pharmaceutically desired as a simple general administration and
as a topical administration to the alveoli pulmonum. However,
the macromolecular substance such as the monoclonal antibody
having the molecular weight of 150,000 among the polypeptides is
hardly adsorbed through the inhalation. Nevertheless, the
absorption of the macromolecular substance through the
inhalation and the retention thereof in the lung through the
topical administration are important and strongly desired.
Unfortunately, the technic for obtaining the stable preparation
containing the antibody while maintaining its physiological
activities is not well established.
The monoclonal antibody having the physiological activities
such as a binding capacity to cancer cells is easily affected in
a solution system, with respect to its stability and
pharmaceutical activities. For example, it is frequently
deactivated by oxidation, coagulation and thermal denaturation
in the solution system. When its aqueous solution is nebulized
through the jet nebulizer or the ultrasonic nebulizer, a shear
stress is applied to the monoclonal antibody. Therefore, the
powdery preparation is preferable and excellent. Irrespective
of the form (aqueous or solid) of the preparation, the
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selections of a stabilizer and stabilizing conditions are
essential in order to obtain the stable preparation containing
the monoclonal antibody which has higher pharmaceutical
activity.
An object of the present invention is to obtain an antibody
containing composition which is expected to have a
pharmaceutical applicability, as a stable solid aerosol
preparation capable of being inhaled.
Summa~yQ f th~ Invention
The above object will be attained by the present invention.
The present invention relates to a composition containing an
antibody or its complex, which is obtained by freeze-drying a
solution of an antibody or its complex followed by milling and
which has an effective particle diameter of more than 0.5 ~m and
less than 10 ~m.
Br~ gPl~a~ion of Drawings
Fig. 1 shows a serum concentration after inhaling to
rabbits each of the MDI preparations a) and c) which were
prepared in Example 1, as a function of time. - O - is the serum
concentration of human Fab in the rabbits to which the MDI
preparations a) were administered, and - - is that of human IgG
in the rabbits to which the the MDI preparations c) were
administered.
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Fig. 2 shows a serum concentration after inhaling to the
rabbits the MDI preparation e) which was prepared in Example 2,
as a function of time.
~est Mode for car~ying out the InventiQ~
The antibody herein includes immunoglobulins (IgGs), their
fragments or their functionally equivalent molecules, and their
products modified by a genetic engineering technic such as a
chimera antibody in which constant regions in an amino acid
sequence are replaced with other amino acid sequences. As the
antibody fragments, F(ab')2, Fab', Fab and Fv which are produced
by conventional methods are exemplified. The antibody usable in
the present invention is not limited to either polyclonal or
monoclonal antibody. And, an animal species from which the
antibody is derived is not limited.
The antibody having the physiologically activities such as
the binding capacity to the cancer cells can be applied, for
example, to the cancer treatment. When the antibody is
systemicaly administered for treating the cancer, the antibody
used is not limited. When the antibody ls topically
administered to lung, however, only the antibody specific to the
pulmonary cancer is used. For example, SF25 [Cancer Res., 48,
6573-6579 (1988)], XF8, AF20 [Hepatology, ~, 625-634 (1989)],
SWA11 [Br. J. Cancer, 59, 174-178 (1989)], SM1 [Cancer Res., ~,
26S ~1984)] and TFS-4 [Cancer ~es., 47, 826 (1987)] can be used.
As the method for treating the cancer, the immunotherapy is
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mentioned, in which the antibody is singly administered and
thereby, the macrophagus or the lymphocytes which have the
receptors against Fc regions of the antibody are collected on
the tumor sites so as to attack the tumor. Alternatively, the
target therapy is mentioned, in which the complex comprising the
antibody to which the toxic substance (for example, the toxic
protein, the radioactive substance (RI) and anticancer agent) is
bound is used. In the complex comprising the antibody and the
toxic substance, the toxic protein as one of the usable toxic
substances includes A chains of plant toxins (type II) such as
ricin and abrin and active components of plant toxins (type I)
such as luffin, momordin and PAP-S and of bacterial protein
toxin such as P~eudomonas toxin and diphtheria toxin, which
irreversibly terminates a protein synthesis and thereby a high
toxicity is given to the cells. The other preferable toxic
protein includes an enzyme derived from human such as human
RNase, which is incorporated into the cells and thereby lethal
damages can be given to the cells. In the preparation of the
immunotoxin complex by binding the antibody with the toxic
protein, a crosslinking agent is used [Tanpakushitsu-Kakusan-
Koso (PROTEIN NUCLEIC ACID ENZYME), supplement No. 31, p.335-343
(1987)].
For stabilizing the antibody, the addition of a sugar such
as lactose, maltose, sorbitose, tolehalose and xylose and/or a
sugar alcohol such as mannitol, sorbitol and xylitol is
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effective in the present invention. The addition of the protein
such as human serum albumin is also effective for stabilizing
the antibody. The added amount of the sugar or sugar alcohol is
preferably 0.01 to 200 %, preferably 1 to 50 % based on the
total weight of the antibody and the human serum albumin.
The milling of the freeze-dried product is conducted using
a suitable apparatus such as a jet milling apparatus (for
example "Micronizer Mill") and a ball milling apparatus. For
the inhalation, the particles have a particle size distribution
such that more than 50 % or more, preferably 75 % or more of the
total particles have the particle diameter of 0.5 to 5 ~m. The
particle size distribution can be determined by a standard
particle size distribution analyzer (for example, model CAPA-700
of Horiba Ltd. and QCM-Cascade Impactor model PC-2 of California
Measurement Inc.).
It is preferable to add any surfactant to the freeze-dried
and milled product for improving the dispersibility of the
particles. The surfactant used in the present invention is at
least one of sorbitan trioleate (Span 85), oleyl alcohol, soya
lecitin and hydrogenated castor oil derivatives (HCO60). The
addition amount of the surfactant is 0.001 to 5 %, preferably
0.05 to 2 % based on the total weight of the antibody and the
human serum albumin.
Optionally, mineral ions which are necessary to keep an
osmotic pressure intrinsically present in an organism, such as
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20~7932
calcium, magnesium, sodium, potassium, chloride and phosphate
ions may be added. If necessary, a surfactant derived from
alveoli pulmonum may be added for controlling a stimulating
property to the organism.
The thus-prepared antibody containing composition is as
such or dispersed in a nebulizing gas such as compressed air and
compressed carbon oxide gas or in a suitable propellant in order
to inhale through the oral or nasal cavity. The usable .
propellant includes chlorofluorocarbon (furon 11, 12, 114 etc.),
hydrochlorofluorocarbon (furon 123, 124, 141b) and fluorocarbon
(furon 125, 134a).
Exa~les
The following examples will more fully illustrate the
present invention, but they are not intended to be limiting of
the present invention.
Exam.~?le 1
Powders were prepared by freeze-drying 20 ml of a solution
con~aining 0.75 mg/ml of a freeze-dried powder of human
immunoglobulin IgG (ex. PAESEL) or human immunoglobulin Fab (ex.
CAPPEL), 15.0 mg/ml of human serum albumin, 2.0 mg/ml of
sorbitol, 0.375 mg/ml of phosphate buffer and 1.5 mg/ml of
sodium chloride. Then, each of the freeze-dried solid powders
was collected followed by milling in Sturtevant jet milling
apparatus (500 ml capacity), thereby a milled powder was
obtained. The resultant milled powder was analyzed using the
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2097932
particle size distrlbution analyzer (model CAPA-700 of Horiba
Ltd.). The milled powder comprising IgG had the average
particle diameter of 5.20 i 1.86 ~m, the proportion of the
particles with the particle diameter of 5.45 ~m or less being
55.0 %. While, the milled powder comprising Fab had the average
particle diameter of 1.95 i 1.05 ~m, the proportion of the
particles with the particle diameter of 4.00 ~m or less being
97.9 %.
Next, 50 to 100 mg of the powder was placed in a glass vial
(10 ml capacity), to which oleyl alcohol or sorbitan trioleate
(Span 85) was added such that it is contained in an amount of
0.25 % under nitrogen atmosphere. After a metered-dose valve
was attached on the glass vial, furon 12 was introduced therein
so that the total amount was 10 ml. Thus, four MDI (metered -
dose inhalation) preparations a), b), c) and d) were obtained.
A composition of each preparation was shown in Table 1.
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2097 932
Table 1MDI preparations comprising human immunoglobulin IgG and
human immunoglobulin Fab'
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com~ o~ition and d~ ~e _ _
MDI preparation compo~ition amount weight do~e
. (w/v ~) (~) (Uq/time)
a) Fab powder* 0.5 0.050 9.55
Fab oleyl alcohol 0.25 0.025
(oleyl alcohol) CFC_12 99.25 13.27
b~ Fab powder*1.0 0.10 19.1
Fab SPAN 85 O.25 O.025
(SPAN 85) CFC 12 98.75 _ 13.20
c) IgG 1.0 0.10 500
IgG oleyl alcohol 0.25 0.025
(oleyl alcohol) CFC 12 9a.25 13.20
d) IgG 1.0 0.10 500
IgG SPAN 85 O.25 O.025
(SPAN 85) CFC 12 98.25 _13.20
* Fab powder contained the following ingredients per 100 mg:
Fab powder : 3.82 mg
HSA : 76.4 mg
phosphate : 1.91 mg
NaCl : 7.6 mg
D-sorbitol : 10.2 mg
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Using a metered~dose inhaler, the human immunoglobulin Fab
or IgG in a dose shown in Table l was inhaled to rabbits. Three
NZW male rabbits of body weight of 3.0 to 3.5 kg were used per
each group. Before the administration to the rabbit, the MDI
preparation was forcedly sprayed in a bellows-type spacer five
times. Then, an adapter was attached, through which the MDI
preparation was administered to the lung of the rabbit by a
spontaneous inhalation for one minute. For each of the MDI
preparations c), d), a) and b), the above procedure was repeated
~wo times, four times, six times and six times, respectively.
Accordingly, the expected final dose of the immunoglobulin was 5
mg, lO mg, 287 ~g and 573 ~g, respectively.
After 0.5, l, 3, 6, 9 and 12 hours from the administration
of the MDI preparation, a blood was sampled from an ear
auricular vein of each rabbit. A serum in a blood sample was
separated so as to prepare a sample for determining a serum
concentration of human immunoglobulin IgG or Fab. The
determination is conducted according to the EIA method.
Firstly, an immunoplate (ex. NUNC) was coated with lO0 ~l of a
lO ~g/ml solution of a sheep anti-human F(ab')2 antibody (ex.
CAPPEL) in PBS(-) at 4C overnight. After washing with 250 ~l
of PBS(-), the plate was blocked with 1% BSA/PBS(-) at 4C
overnight followed by washing with each of 250 ~l of a washing
solution (0.05% Tween 20/PBS(-)) three times. Then, a human
immunoglobulin IgG standard (ex. ZYMED), a human immunoglobulin
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Fab standard (ex. CAPPEL) and a sample were added and the
reaction was continued at room temperature for 1 hour. After
washing with each of 250 ~1 of the washing solution three times,
100 ~1 of a horseraddish peroxidase (HRP) standard goat anti-
human IgG (H + L) chains antibody (ex. ZYMED) which was
previously diluted 12,000 times was reacted at room temperature
for 1 hour. Thereafter, the washing was repeated. A substrate
for HRP was added in order to develop a color. After the
reaction was terminated, an absorption at 490 nm was detected
using an immuno reader.
The results showed that in both preparations comprising the
human immunoglobulins IgG and Fab, the preparations comprising
oleyl alcohol showed higher absorption efficiency, as compared
with those comprising SPAN 85. Thus, the change in serum
concentration after the inhalation of the preparation (a) or (c)
comprising oleyl alcohol was determined. The results are shown
in Fig. 1. After 30 minutes from the inhalation, the serum
concentration was reached to a maximum value in both
preparations a) and c). In the preparation a), it was 1.18 % of
the dose. In the preparation c), it was 0.98 % of the dose.
Further, Fab showed the higher serum concentration as compared
with IgG.
xample 2
A IgG-Abrin A chain complex as the immunotoxin complex was
prepared from human immunoglobulin IgG (ex. PAESEL) and abrin A
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chain using SPDP (N-succinimidyl 3-(2-pyridyl-dithio)propionate)
of the crosslinking agent. For the purification of the complex,
blue Sepharose column and a gel filtration column were used.
A powder was prepared by freeze-drying 20 ml of a solution
containing 0.7 mg/ml of IgG-abrin A chain complex, 15.0 mg/ml of
human serum albumin, 2.0 mg/ml of sorbitol, 0.75 mg/ml of
phosphate buffer and 1.5 mg/ml of sodium chloride. The freeze-
dried powder was milled in Sturtevant jet milling apparatus (500
ml capacity), thereby a milled powder was obtained. The
resultant milled powder was analyzed using the particle size
distribution analyzer (model CAPA-700 of Horiba Ltd.). The
milled powder had the average particle diameter of 4.65 i 1.58
~m, the proportion of the particles with the particle diameter
of 5.00 ~m or less being 63.6 %.
One hundred mg of the powder was placed in a glass vial (10
ml capacity), to which oleyl alcohol was added such that it is
contained in an amount of 0.25 % under nitrogen atmosphere.
After a metered-dose valve was attached on the glass vial, furon
12 was introduced therein so that the total amount was 10 ml.
Thus, MDI preparations e) was obtained.
Using the metered-dose inhaler, the complex in a dose of
500 mg/time was inhaled to three NZW male rabbits of body weight
of 3.0 to 3.5 kg per each group. Before the administration to
the rabbit, the MDI preparation was forcedly sprayed in a
bellows-type spacer five times. Then, an adapter was attached,
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through which the MDI preparation was a~ninistered to the lung
of the rabbit by a spontaneous inhalation for one minute. The
,,above procedure was repeated two times. Accordingly, the
expected final dose of the complex was 5 mg.
After 0.5, 1, 3, 6, 9 and 12 hours from the administration
of the MDI preparation, a blood was sampled from an ear
auricular vein of each rabbit. A serum in a blood sample was
separated so as to prepare a sample for determining a serum
concentration of the complex. The determination is conducted
according to the EIA method.
The change in serum concentration after the inhalation is
shown in Fig. 2. After 30 minutes from the inhalation, the
serum concentration was reached to a maximum value. It was
confirmed to be 0.42 % of the dose.
Utiliz,~a,,,bility in Industries
Because the antibody containing composition of the present
invention is the stable solid composition, it is suitable as the
composition for the inhalation of the àntibody or its complex
which is pharmaceutically effective.
