Note: Descriptions are shown in the official language in which they were submitted.
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Powders comprising low molecular dextran and methods of producing
those powders
Field of the invention
The invention relates to the use of low-molecular dextran (Mw: <_ 10,000
Dalton) for the
preparation and stabilisation of powders which contain a pharmaceutical active
substance. The powders are preferably produced by spray-drying. The present
invention also relates to powders, preferably spray-dried powders, which
contain low-
molecular dextran and a pharmaceutical active substance. The present invention
~o relates particularly to protein- or peptide-containing powders and methods
of producing
them.
Background
~5 Active substances/active substance preparations formulated in aqueous
solutions are
in some cases prone to instability which may lead to reduced bioactivity and
increased
incompatibilities. One possible method of stabilisation is offered for example
by spray-
drying, in which the pharmaceutical active substance is dried by spraying in a
current
of hot air. The pharmaceutical active substances are usually sprayed in the
presence
Zo of excipients which on the one hand should maintain the stability of the
active
substances and on the other hand should improve the properties of the spray-
dried
powders.
A crucial factor in stabilising by spray-drying is the immobilisation of the
active
ZS substance in an amorphous matrix. The amorphous state has high viscosity
with low
molecular mobility and low reactivity. The glass transition temperature of a
spray-dried
powder is an important parameter as it indicates the temperature range at
which the
transition from the stable, amorphous state into the less stable rubber-like
state takes
place. Advantageous excipients must be capable of forming an amorphous matrix
with
3o the highest possible glass transition temperature in which the active
substance is
embedded. Substances with a low glass transition temperature can flow even at
low
temperatures and lead to unstable powder formulations. The choice of
excipients thus
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depends particularly on their stabilising qualities. In addition, however,
factors such as
the pharmaceutical acceptance of the excipients and its influence on particle
formation,
dispersibility and flow properties play a decisive role.
Spray-drying is a suitable process for increasing the chemical and physical
stability of
pharmaceutical active substances of the peptide/protein type (cf. Maa et al.,
1998,
Pharmaceutical Research, 15(5), 768-775). Particularly in the field of
pulmonary
treatment spray drying is used to produce peptide/protein-containing powdered
medicaments (US 5,626,874; US 5,972,388; Broadhead et al., 1994, J. Pharm
Pharmacol., 46(6), 458-467). The administration of peptide/proteins by
inhalation is an
alternative to traditional methods of administration in systemic diseases, as
pharmaceutical products taken by inhalation may develop not only a local but
also a
systemic activity (WO 99/07340). The prerequisite for this is that the average
particle
size is in the range from 1-10 Nm, preferably 1-7.5 Nm, so that the particles
can
penetrate deep into the lungs and thus enter the bloodstream. DE-A-179 22 07,
for
example, describes the preparation of corresponding spray dried particles
which are
sufficiently dispersible for medical application (inhalation). In the meantime
a number
of methods of producing inhalable particles have been described (WO 95/31479;
WO
96/09814; WO 96/32096; WO 96/32149; WO 97/41833; WO 97/44013; WO 98/16205;
Zo WO 98/31346; WO 99/66903; WO 00/10541; WO 01/13893; Maa et al., 1998,
supra;
Vidgren et al., 1987, Int. J. Pharmaceutics, 35, 139-144; Niven et al., 1994,
Pharmaceutical Research, 11(8), 1101-1109).
Sugar and alcohols thereof such as, for example, trehalose, lactose,
saccharose or
as mannitol and various polymers have proved suitable as excipients (Maa et
al., 1997,
Pharm. Development and Technology, 2(3), 213-223; Maa et al., 1998, supra;
Dissertation Adler, 1998, University of Erlangen; Costantino, et al., 1998, J.
of Pharm.
Sciences, 87(11 ), 1406-1411 ).
However, the excipients predominantly used have various drawbacks. The
addition of
3o trehalose and mannitol, for example, impairs the flow properties of spray-
drying
formulations (C. Bosquillon et al., 2001 Journal of Controlled Release, 70(3),
329-339).
Moreover, mannitol has a tendency to recrystallise in amounts of more than 20
percent
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by weight (Costantino et al., 1998, supra), as a result of which its
stabilising effects are
dramatically reduced. Lactose, a frequently used excipient, does improve the
flow
properties of spray-drying formulations (C. Bosquillon et al., 2001, supra),
but is
problematic particularly in the formulation of peptide/protein-containing
active
substances, as lactose can enter into destabilising Maillard reactions with
peptides/proteins as a result of its reducing property.
Dextrans with a molecular weight of 40 to 512 kDa are predominantly used in
the
freeze-drying of peptide/protein-containing active substances. They are
amorphous by
nature with a high glass transition at the same time. These high-molecular
dextrans
are only capable of entering into adequate hydrogen bridge bonds with
peptides/proteins to a limited extent because of their rigid skeleton and thus
ensure
adequate stabilisation during freeze-drying. To compensate for this
disadvantage they
are sometimes combined with disaccharides (Allison et al., 2000, J. Pharm.
Sci.,
~s 89(2),199-214). A further disadvantage of high-molecular dextrans resides
in their high
allergenic potential (dextran anaphylaxis).
One aim of the invention was to provide new excipients for the production of
powdered
pharmaceutical preparations. The corresponding powdered preparations should be
ao characterised, among other things, by good stability on storage and, where
possible,
by being inhalable.
A further aim of the present invention was to provide new excipients for the
preparation
of spray-dried pharmaceutical preparations. The corresponding powdered
25 pharmaceutical preparations should again be characterised by good long-term
stability
and, where possible, by being inhalable.
A further aim of the present invention was to provide new excipients for the
preparation
of peptide/protein-containing pharmaceutical formulations, particularly for
those
3o produced by spray-drying. The corresponding peptide/protein-containing
pharmaceutical preparations should again be characterised by good long-term
stability
and, where possible, by being inhalable.
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Another aim of the present invention was to provide pharmaceutical
preparations for
administration by inhalation, either in the form of a dry powder or a
propellant-
containing metered dose aerosol or a propellant-free inhalant solution.
The objectives on which the invention is based are achieved by the embodiments
described below and by the objects/methods recited in the claims.
Summary of the Invention
The present invention relates to powders, preferably spray-dried powders,
which
contain a pharmaceutical active substance and low-molecular dextran with a
molecular
weight between about 500 and 10,000 Dalton (Da), preferably between about 500
and
5,000 Da, and particularly preferably between about 500 and 1,500 Da.
Surprisingly, it
~s was found that the corresponding powders after being spray-dried i) form an
amorphous structure, ii) result in a relatively high yield (of at least 75%
based on the
solid used), iii) have a very high glass transition temperature (up to
65°C) and iv) have
a low tendency to recrystallisation. As another important advantage over e.g.
spray-
dried trehalose corresponding spray-dried powders which contain low-molecular
ao dextran have improved flow properties. Another advantage over the powdered
pharmaceutical preparations described in the prior art, particularly over
known
powdered spray-dried pharmaceutical preparations, resides in the particularly
advantageous process and storage stability of the dextran-containing powders
according to the invention described herein.
as
The amount of low-molecular dextran is preferably in relation to the dry mass
of the
powder between 50 and 99.99 % % by weight (w/w), and according to another
preferred embodiment between 55 and 99.99 % (w/w), preferably between 60 and
99.99% (w/w).
The pharmaceutically active substance is preferably a biological macromolecule
which
may be a polypeptide or a protein, e.g. a growth factor, enzyme or antibody.
The
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invention therefore relates in particular to spray-dried powders containing
(a) a
proportion of 50 to 99.99%, preferably 60 to 99.99% (w/w) of low-molecular
dextran
(relative to the dry mass of the powder) and (b) a biological macromolecule as
pharmaceutical active substance, preferably in a concentration between 0.01
and
40% (w/w), again relative to the dry mass of the powder, the sum of the
percentages
by weight of low-molecular dextran and biological macromolecule being at most
100 % (w/w).
The spray-dried powders according to the invention may contain in addition to
the low-
molecular dextran other excipients, such as for example amino acids, peptides,
proteins or sugars. Particularly advantageous are powders which contain in
addition to
the stabilising low-molecular dextran and the pharmaceutical active substance
at least
one amino acid, a dipeptide, a tripeptide and/or a salt. According to a
preferred
embodiment the present invention relates to spray-dried powders which contain
relative to their dry mass (a) between 60 and 98.99% (w/w) of a low-molecular
dextran,
(b) between 1 and 20% (w/w) of at least one amino acid and/or at least one
peptide as
a further excipient and (c) at least 0.01 % (w/w) of a pharmaceutical active
substance.
Preferably the further excipient is the amino acid isoleucine or a di- or
tripeptide
containing at least one isoleucine group. According to a special embodiment
the
Zo present invention relates to spray-dried powders which contain in relation
to their dry
mass (a) approximately 60 to 89.99% (w/w) of a low-molecular dextran, (b)
approximately 10 to 20% (w/w) of an amino acid, preferably isoleucine and (c)
approximately 0.01 to 30% (w/w) of a pharmaceutical active substance,
preferably a
peptide/protein, for example an antibody. According to another special
embodiment the
25 present invention relates to spray-dried powders which contain in relation
to their dry
mass (a) approximately 60 to 98.99% (w/w) of a low-molecular dextran, (b)
approximately 1 to 20% (w/w) of an isoleucine-containing tripeptide,
preferably
triisoleucine and (c) approximately 0.01 to 39% (w/w) of a pharmaceutical
active
substance, preferably a peptide/protein, for example an antibody.
According to another embodiment the present invention relates particularly to
spray-
dried powders which contain low-molecular dextran and at least one
pharmaceutical
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active substance, the spray-dried powder having a glass transition temperature
of
more than 40°C, preferably more than 45°C, more preferably more
than 50°C, even
more preferably more than 55°C and particularly preferably more than
60°C. The
amount of excipient added, particularly the amount of low-molecular dextran in
the
s powder, is primarily responsible for the corresponding glass transition
temperature.
According to another embodiment the present invention relates to
pharmaceutical
compositions for administration by inhalation, which contain one of the
powders
according to the invention described herein or consist of these powders.
Preferred
~o pharmaceutical compositions for this purpose are those which contain the
powders
according to the invention as propellant-containing metered dose aerosols or
propellant-free inhalable solutions. The spray-dried powders according to the
invention
used to prepare the pharmaceutical composition are characterised according to
another embodiment by a high proportion of inhalable particles with a mean
~5 aerodynamic particle diameter (MMAD) of less than 10 Nm, preferably from
0.5 - 7.5
Nm, more preferably from 0.5 - 5.5 Nm, most preferably from 0.5 - 5.0 Nm.
The invention also provides processes for preparing the corresponding spray-
dried
powders according to the invention, characterised in that a solution or
suspension
ao which contains at least one low-molecular dextran and a pharmaceutical
active
substance is produced and this is sprayed under suitable conditions. The
temperature
for the spraying process is preferably between 50 and 200°C (inflow
temperature) and
30 and 150°C (outflow temperature).
Zs
Description of the Figures
Figure 1 shows the aggregate formation after spray-drying, forced storage and
reconstitution. Aqueous solutions were sprayed, containing a) 10% (w/w) IgG
content,
b) 1 % (w/w) IgG and 9% trehalose content and c) 1 % (w/w) IgG and 9%
dextran~ooo
3o content. The dextran-containing powders are characterised by a low content
of
aggregates.
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Figure 2 shows the aggregate formation after spray-drying, forced storage and
reconstitution. Aqueous solutions were sprayed, containing a) 10% (w/w) IgG
content,
b) 1 % (w/w) IgG, 1 % (w/w) isoleucine and 8% trehalose content and c) 1 %
(w/w) IgG,
1 % (w/w) isoleucine and 8% (w/w) dextran~ooo content. The dextran-containing
powders are characterised by a low content of aggregates.
Figure 3 shows the Mass Mean Aerodynamic Diameter (MMAD) of different powders
produced by spray-drying aqueous solutions containing de~ran~o~, isoleucine
and
IgG. The solutions were prepared as described under EXAMPLES and sprayed. All
the
~o powders have a MMAD of less than 7.5 Nm. The diagram shows the influence of
the
isoleucine content on the MMAD with constant total solids concentrations and
spray
parameters. A higher isoleucine content in the formulation reduces the MMAD.
Total
Solid: proportion of solids in the spray solution. Cyclone I: Buchi Cyclone.
Cyclone II:
Buchi High-performance Cyclone.
Figure 4 shows the Fine Particle Fraction (FPF) with a Cut Off Diameter of
less than 5
Nm for various powders which were prepared by spray-drying aqueous solutions
containing dextran~ooo~ isoleucine and IgG. The solutions were prepared and
sprayed
as described under EXAMPLES. All the powders have a FPF of more than 30 %, or
Zo more than 35 %. Total Solid: proportion of solids in the spray solution.
Cyclone I: Buchi
Cyclone . Cyclone II: Buchi High-performance Cyclone.
Figure 5 shows the Mass Mean Aerodynamic Diameter (MMAD) of various powders
which were prepared by spray-drying aqueous solutions containing dextran~~o,
25 triisoleucine and IgG. The solutions were prepared and sprayed as described
under
EXAMPLES. Both powders have a MMAD of less than 5 Nm, or less than 4 Nm. Total
Solid: proportion of solids in the spray solution. Buchi Cyclone. Cyclone II:
Buchi High-
performance Cyclone.
3o Figure 6 shows the Fine Particle Fraction (FPF) with a Cut Off Diameter of
less than 5
Nm for various powders which were prepared by spray-drying aqueous solutions
containing dextran~ooo, triisoleucine and IgG. The solutions were prepared and
sprayed
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as described under EXAMPLES. Both the powders have a FPF of more than 55%, or
58%. Total Solid: proportion of solids in the spray solution. Buchi Cyclone.
Cyclone II:
Buchi High-performance Cyclone.
s Figure 7 shows the aggregate formation after spray-drying, up to one years
storage at
2-8, 25 and 40°C with subsequent reconstitution. An aqueous solution
was sprayed,
containing 1 % (w/w) IgG, 1 % (w/w) isoleucine and 8% (w/w) dextran~ooo (see
Example
2). The dextran-containing powder is characterised by a low content of
aggregates
after 3, 6, 9 and 12 months storage at 2-8°C, 25°C, 40°C.
Figure 8 shows the aggregate formation after spray-drying, up to one years
storage at
2-8, 25 and 40°C with subsequent reconstitution. An aqueous solution
was sprayed,
containing 0.33% (w/w) IgG, 0.33% (w/w) isoleucine and 2.66% (w/w) dextran~ooo
(see
Example 2). The dextran-containing powder is characterised by a low content of
~s aggregates after one years storage at 2-8°C, 25°C,
40°C.
Figure 9 shows the aggregate formation after spray-drying, up to one years
storage at
2-8, 25 and 40°C with subsequent reconstitution. An aqueous solution
was sprayed,
containing 0.33% (w/w) IgG, 0.33% (w/w) triisoleucine and 2.66% (w/w)
dextran~ooo
Zo (see Example 3). The dextran-containing powder is characterised by a low
content of
aggregates after one years storage at 2-8°C, 25°C, 40°C.
Figure 10 shows the residual monomer content after spray-drying, forced
storage and
reconstitution. Aqueous solutions were sprayed, containing a) 3.33% (w/w)
lysozyme,
25 b) 0.33% (w/w) lysozyme and 3.0% dextran~ooo, c) 0.33% (w/w) lysozyme,
0.33% (w/w)
isoleucine and 2.66% (w/w) dextran~ooo and d) 0.33% (w/w) lysozyme, 0.33%
(w/w)
triisoleucine and 2.66% (w/w) dextran~ooo . The dextran-containing powder is
characterised by a high residual monomer content.
3o Figure 11 shows the aggregate content after spray-drying, forced storage
and
reconstitution. Aqueous solutions were sprayed, containing a) 3.33% (w/w)
calcitonin,
b) 0.33% (w/w) calcitonin and 3.0% dextran~ooo and c) 0.33% (w/w) calcitonin,
0.33%
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(w/w) isoleucine and 2.66% (w/w) dextran~ooo. The dextran-containing powder is
characterised by a low aggregate content.
Figure 12 shows an inhaler for the administration of dry powdered preparations
by
inhalation.
Detailed description of the invention
Definitions
Terms and designations used within the scope of this specification have the
following
meanings defined below. The details of weight and percentages by weight are
based
on the dry mass of the powders or the solids content of the
solutions/suspensions to
be sprayed, unless stated otherwise.
~s The term "dextran 1" or "dextran ~ooo" refers to a low-molecular dextran
with a mean
molecular weight of about 1.000 Dalton. The molecular weights given in this
patent
specification for dextran in each case relate to the mean molecular weight.
This means
that the dextrans used are generally polymorphous. The mean molecular weight
indicates that at least 50%, preferably 60%, more preferably 70%, even more
Zo preferably 80%, even more preferably 90%, even more preferably 92% , even
more
preferably 94%, even more preferably 96%, even more preferably 98% and even
more
preferably 99% of the dextrans have a molecular weight corresponding to the
numerical value.
25 The term "spray-dried powder formulation" or "dry powder formulation"
refers to
powder formulations which usually contain less than about 10% (w/w) residual
moisture, preferably less than 7% (w/w) residual moisture, most preferably
less than
3% (w/w) residual moisture and even more preferably less than 2% (w/w)
residual
moisture. The residual moisture is essentially dependent on the type and
amount of
3o the pharmaceutical active substance in the powder formulation.
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The term "amorphous" means that the powdered formulation contains less than
10%
crystalline fractions, preferably less than 7%, more preferably less than 5%,
and most
preferably less than 4, 3, 2, or 1 %.
s The word "inhalable" means that the powders are suitable for pulmonary
administration. Inhalable powders can be dispersed and inhaled by means of an
inhaler so that the particles enter the lungs and are able to develop a
systemic activity
optionally through the alveoli. Inhalable particles may have an average
particle
diameter, for example, of between 0.4-10 Nm (MMD = mass median diameter),
usually
between 0.5-5 Nm, preferably between 1-3 Nm and/or an average aerodynamic
particle
diameter (MMAD = mass median aerodynamic diameter) of between 0.5-10 Nm ,
preferably between 0.5-7.5 Nm, more preferably between 0.5-5.5 Nm, even more
preferably 1-5 Nm and particularly preferably between 1-4.5 Nm.
"Mass Median Diameter" or "MMD" is a measurement of the average particle size
distribution as the powders according to the invention are generally
polydispersed. The
results are expressed as diameters of the total volume distribution at 50%
total
throughflow. The MMD values can be determined for example by laser
diffractometry
(cf.: Chapter EXAMPLES, Method), although of course any other conventional
method
Zo may be used (e.g. electron microscopy, centrifugal sedimentation).
The term mean aerodynamic particle diameter (= mass median aerodynamic
diameter
(MMAD)) indicates the aerodynamic particle size at which 50% of the particles
of the
powder normally have a smaller aerodynamic diameter. In cases of doubt the
25 reference method for determining the MMAD is the method specified in this
patent
specification (cf. the chapter EXAMPLES, Method).
The term "fine particle fraction" (FPF) describes the inhalable part of a
powder
consisting of particles with a particle size of 5 5 Nm MMAD. In powder which
is well
3o dispersible the FPF is more than 20%, preferably more than 30%, more
particularly
more than 40%, and more preferably more than 50%, even more preferably more
than
55%. The expression "Cut Off Diameter" used in this context indicates which
particles
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are taken into account when determining the FPF. An FPF of 30% with a Cut Off
Diameter of 5 Nm (FPF 5) means that at least 30% of all the particles in the
powder
have a mean aerodynamic particle diameter of less than 5 Nm.
s The term "spray solution" means aqueous solutions or suspensions in which
the
pharmaceutical active substance together with at least one excipient is
dissolved/suspended.
The term " time of flight" is the name of a standard method of measurements,
as
~o described in more detail in the Chapter EXAMPLES. In a time of flight
measurement
the MMAD and FPF are determined simultaneously (cf.: Chapter EXAMPLES,
Method).
The terms "pharmaceutically acceptable excipients", "carriers" or "matrices"
refer to
~S excipients which may optionally be contained in the formulation within the
scope of the
invention. The excipients may for example be administered to the lungs without
having
any significantly unfavourable toxicological effects on the subjects or on the
subjects'
lungs.
ao The term "pharmaceutically acceptable salts" includes for example the
following salts,
but is not restricted thereto: salts of inorganic acids such as chloride,
sulphate,
phosphate, diphosphate, bromide and nitrate salts. Also, salts of organic
acids, such
as malate, maleate, fumarate, tartrate, succinate, ethylsuccinate, citrate,
acetate,
lactate, methanesulphonate, benzoate, ascorbate, para-toluenesulphonate,
palmoate,
25 salicylate and stearate, and also estolate, gluceptate and lactobianate
salts.
The term "pharmaceutically acceptable rations" includes, without being
restricted
thereto, for example, lithium, sodium, potassium, calcium, aluminium and
ammonium
(including substituted ammonium).
By a "pharmaceutical active substance" " is meant a substance, medicine,
composition
or combination thereof which has a pharmacological, usually positive effect on
an
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organism, an organ and/or a cell if the active substance is brought into
contact with the
organism, organ or cell. When introduced into a patient the effect may be
local or
systemic.
s The term "biological macromolecule" refers to peptides, proteins, fats,
fatty acids or
nucleic acids.
The term "peptide" or "polypeptide" refers to polymers of amino acids
consisting of two
to a hundred amino acid groups. The term "peptide" or "polypeptide" is used as
a
~o pseudonym and includes both homopeptides and heteropeptides, i.e. polymers
of
amino acids consisting of identical or different amino acid groups. Thus, a
"dipeptide"
is made up of two peptidically linked amino acids, a "tripeptide" of three
peptidically
linked amino acids. The term "protein" used here refers to polymers of amino
acids
with more than 100 amino acid groups.
The term "analogues" refers to peptides/proteins in which one or more amino
acids
have been substituted, eliminated (e.g. fragments), added (e.g. derivatives
with a C- or
N-terminal extension) or otherwise modified from the native (wild-type)
sequence. It is
also possible to derivatise the native protein, e.g. by means of sugars,
Zo polyethyleneglycol or the like. Analogues have a bioactivity of at least
10, 20, 30 or
40%, preferably at least 50, 60 or 70% and particularly preferably at least
80, 90, 95
100% or more than 100% bioactivity of the native, non-synthetic protein.
The term "amino acid" denotes compounds which contain at least one amino group
as and at least one carboxyl group. Although the amino group is usually in the
a position
to the carboxyl group any other arrangement in the molecule is also possible.
The
amino acid may also contain other functional groups such as e:g. amino,
carboxamide,
carboxyl, imidazole, thin groups and other groups. Amino acids of natural or
synthetic
origin, racemically or optically active (D- or L-) including various
stereoisomeric ratios
3o are used. For example, the term isoleucine covers both D-isoleucine, L-
isoleucine,
racemic isoleucine and various ratios of the two enantiomers.
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The term "pure protein formulation" refers to spray-dried powders consisting
of one or
more proteins and optionally a suitable buffer (typically 0 to 15% (w/w)
relative to the
weight of the dry powder). The powder basically contains no other excipients,
i.e. the
content of any other excipients is less than 1 % (w/w) relative to the weight
of the dry
powder.
A "surface-active" substance is capable of reducing the surface tension of the
solution
in which it is dissolved. The surface activity is measured for example by the
tensiometer method according to Lecomte du Nouy (Bauer, Fromming, Fuhrer, 6th
edition).
Powders according to the invention
The present invention relates to the preparation of new excipients for
stabilising
pharmaceutical active substances / active substance preparations. Thanks to
the
present invention it is possible to prepare powdered active substance
formulations
which are characterised by particular stability, particularly by a low
aggregate and high
monomer content. Later in the specification these new and surprisingly
superior
stabilising active substances will be described and characterised in more
detail.
zo
Dextran is usually a high-molecular glucose polymer. It may be prepared for
example
by cultivating Leuconostoc Mesenteroides 8512F in the presence of saccharose.
Native dextran can be obtained by partial acid hydrolysis after corresponding
purification steps in desired molecular weight fractions. Dextran is a (1->6)
linked a-D-
25 glucan with side chains bound to the O-3 positions. The degree of branching
is usually
about 5%. The branches are usually 1-2 glucose units long. The dextrans
normally
used have mean molecular weights significantly above 10,000 Da. (usually
40,000,
70,000 or 512,000 Da). The dextran claimed in the Examples of the invention,
on the
other hand, has only an mean molecular weight of up to 10,000 Da, preferably
up to
so 5,000 Da, most preferably up to 1,500 Da. Within the scope of the present
invention it
has been found that dextran with a mean molecular weight of approximately
1,000 Da.
is particularly suitable as a stabiliser in the preparation of particulate
powders.
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Advantages of the pharmaceutical dextrans:
~ USP and Ph. Eur. monographed
~ free from Leuconostoc antigen
~ permitted for i. v. administration
~ fully biodegradable to carbon dioxide and water
~ stable at ambient temperature for 5 years
Special advantage of low-molecular dextran (1,000 Dalton)
~ low antigenicity
The present invention therefore relates to powders, preferably spray-dried
powders,
containing a pharmaceutical active substance and a low-molecular dextran with
a
molecular weight between about 500 and 10,000 Dalton (Da), preferably between
about 500 and 5,000 Da, and particularly preferably between about 500 to 1,500
Da.
According to a particularly preferred embodiment the present invention relates
to
powders, preferably spray-dried powders, which contain in addition to a
pharmaceutical active substance dextran with a mean molecular weight of about
1,000
Da.
ao
Powders which have proved particularly advantageous are those powders,
preferably
spray-dried powders, whose content of low-molecular dextran in relation to the
dry
mass of the powder is between 50 and 99.99% (w/w), preferably between 55 and
99.99% (w/w), more preferably between 60 and 99.99% (w/w), for example 50,
50.1,
as 50.2 50.3, ... 50.7, 50.8, 50.9 etc.; 51, 52, 53, ... 58, 59, 60 etc.; 61,
62, 63, ... 68 .69,
70 etc.; 71, 72, 73, ... 78, 79, 80 etc.; 81, 82, 83, ... 88, 89, 90 etc.; 91,
92, 93, ... 98
etc, 99.1, 99.2, 99.3, ... 99.8, .99.9, etc.; 99.91, 99.92, 99.93, ... 99.98,
99.99 (w/w).
Overall, the amount of low-molecular dextran should be selected so that the
spray-
dried powder is at least partially amorphous, preferably totally amorphous.
The amount
30 of low-molecular dextran may also be reduced to less than 50% (w/w),
provided that
other stabilising excipients are added to the powder in suitable amounts.
Examples of
other stabilising excipients can be found elsewhere in this patent
specification.
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The amount of pharmaceutical active substance in the dry mass of the powder
according to the invention is generally between 0.01 and 50% (w/w), preferably
between 0.33 and 50% (w/w), more preferably between 0.33 and 45% (w/w), even
more preferably between 0.33 and 40% (w/w). According to a another preferred
embodiment the amount of pharmaceutical active substance in the solid content
of the
powder according to the invention is between 0.33 and 35% (w/w), preferably
between
0.33 and 30% (w/w), more preferably between 0.33 and 25% (w/w) and even more
preferably between 0.33 and 10% (w/w). The amount is thus for example 0.01,
0.02,
0.03 ... 0.08, 0.09, etc.; 0.1, 0.2, 0.3, ... 0.8, 0.9 etc.; 1, 2, 3, ... 8,
9, 10 etc.; 11, 12,
13, ... 18, 19, 20 etc.; 21, 22, 23, ... 28, 29, 30 etc.; 31, 32, 33, ... 38,
39, 40 etc.; 41,
42, 43, ... 48, 49, etc; 49.1, 49.2, 49.3, ... 49.8, 49.9, etc.; 49.91, 49.92,
49.93, .. .
49.98, 49.99 (w/w).
~s The invention therefore relates to powders with a ratio of low-molecular
dextran to
active substance of for example 50/50, 51/49, 52/48, 53/47, 54/46, 55/45,
56/44,
57/43, 58/42, 59/41, 60/40, 61 /39, 62/38, 63/37, 64/36, 65/35, 66/34, 67/33,
68/32,
69/31, 70/30, 71 /29, 72/28, 73/27, 74/26, 75/25, 76/24, 77/23, 78/22, 79/21,
80/20,
81 /19, 82/18, 83/17, 84/16, 85/15, 86/14, 87/13, 88/12, 89/11, 90/10, 91 /9,
92/8, 93/7,
zo 94/6, 95/5, 96/4, 97/3, 98/2, 99/1, 99.1 /0.9, 99.2/0.8, 99.3/0.7,
99,4/0.6, 99.5/0.5,
99.6/0.4, 99.66/0.33, 99.7/0.3, 99.8/0.2, 99.9/0.1, 99.99/0.01 (w/w). If the
particular
powder contains one or more additional excipients, either the amount of low-
molecular
dextran, the amount of pharmaceutical active substance or both amounts can be
reduced accordingly, the amount of low-molecular dextran relative to the dry
mass of
ZS the powder preferably having one of the values between 50 and 99.99% (w/w).
Pharmaceutical active substances for the purposes of the invention include, in
addition
to those covered by the general definition, antibiotics, anti-viral active
substances,
antiepileptics, pain-relievers (analgesics), anti-inflammatory active
substances or
3o bronchodilators. They also include active substances which act for example
on the
peripheral nervous system, on adrenergic receptors, cholinergic receptors, the
skeletal
muscles, the cardiovascular system, the smooth muscle, the blood circulatory
system,
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on synaptic points, neuroeffector connecting points, the endocrine system, the
immune
system, the reproductive system, the skeletal system, the autacoid systems,
the
alimentary and excretory systems, the histamine system and the central nervous
system. Suitable active substances also include for example hypnotics and
sedatives,
s psychic energizers, tranquillisers, anti-convulsants, muscle relaxants, anti-
Parkinsons
active substances, pain relievers, anti-inflammatory active substances, muscle
contractants, anti-microbial active substances, hormonal active substances
such as for
example contraceptives, sympathomimetics, diuretics, fat metabolism regulating
active
substances, anti-androgenic active substances, antiparasitics, neoplastics,
antineoplastics and hypoglycaemics.
The term pharmaceutical active substance also includes, for example, active
substances which act on the respiratory system, for example against one of the
following complaints: asthma, chronic obstructive pulmonary diseases (COPD),
~s emphysemic chronic bronchitis, bronchopulmonary dysplasia (BPD), neonatal
Respiratory Distress Syndrome (RDS), bronchiolitis, croup, post-extubation
stridor,
pulmonary fibrosis, pneumonia or cystic fibrosis (CF).
Representative examples of bronchodilators include among others beta-agonists,
Zo anticholinergics or methylxanthine. Examples of anti-inflammatory active
substances
are steroids, cromolyn, nedocromil and leukotriene inhibitors. Examples of
steroids
include beclomethasone, betamethasone, biclomethasone, dexamethasone,
triamcinolone, budesonide, butixocort, ciclesonide, fluticasone, flunisolide,
icomethasone, mometasone, tixocortol and loteprednol. Other examples are
25 budesonide, fluticasone propionate, beclomethasone dipropionate, fometerol
and
triamcinolone acetonide.
Examples of antimicrobially active substances are erythromycin, oleandomycin,
troleandomycin, roxithromycin, clarithromycin, davercin, azithromycin,
flurithromycin,
3o dirithromycin, josamycin, spiromycin, midecamycin, leucomycin, miocamycin,
rokitamycin, andazithromycin and swinolide A; fluoroquinolones, for example
ciprofloxacin, ofloxacin, levofloxacin, trovafloxacin, alatrofloxacin,
moxifloxicin,
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norfloxacin, eoxacin, grepafloxacin, gatifloxacin, lomefloxacin, sparfloxacin,
temafloxacin, pefloxacin, amifloxacin, fleroxacin, tosufloxacin,
prulifloxacin, irloxacin,
pazufloxacin, clinafloxacin and sitafloxacin; aminoglycosides such as for
example
gentamicin, netilmicin, paramecin, tobramycin, amikacin, kanamycin, neomycin;
s streptomycin, vancomycin, teicoplanin, rampolanin, mideplanin, colistin,
daptomycin,
gramicidin, colistimethate; polymixins such as for example polymixin B,
capreomycin,
bacitracin, peneme, penicillins including penicillinase-sensitive active
substances wie
penicillin G, penicillin V, penicillinase-resistant active substances such as
methicillin,
oxacillin, cloxacillin, dicloxacillin, floxacillin, nafcillin; active
substances against gram-
negative bacteria such as ampicillin, amoxicillin, hetacillin, cillin and
galampicillin; anti-
pseudomonal penicillins such as carbenicillin, ticarcillin, azlocillin,
mezlocillin,
andpiperacillin; cephalosporins such as cefpodoxime, cefprozil, ceftbuten,
ceftizoxime,
ceftriaxon, cephalothin, cephapirin, cephalexin, cephradrin, cefoxitin,
cefamandol,
cefazolin, cephaloridin, cefaclor cefadroxil, cephaloglycin, cefuroxim,
ceforanid,
cefotaxim, cefatrizin, cephacetril, cefepim, cefixim, cefonizid, cefoperazon,
cefotetan,
cefmetazol, ceftazidim, loracarbef and moxalactam; monobactams such as
aztreonam;
and carbapenems such as for example imipenem, meropenem, pentamidin
isethionate, albuterol sulphate, lidocaine, metaproterenol sulphate,
beclomethasone
dipropionate, triamcinolone acetamide, budesonide acetonide, fluticasone,
ipratropium
Zo bromide, flunisolide, cromolyn sodium, ergotamine tartrate and, where
applicable,
analogues, agonists, antagonists, inhibitors and pharmaceutically usable salt
forms
thereof and the like.
The pharmaceutical active substance is preferably a biological macromolecule
ZS according to another embodiment. In accordance with the definition provided
above
this is intended to include for example peptides, proteins, fats, fatty acids
or nucleic
acids.
Biopharmaceutically important proteins/polypeptides include e.g. antibodies,
enzymes,
3o growth factors, e.g. steroids, cytokines, lymphokines, adhesion molecules,
receptors
and the derivatives or fragments thereof, but are not restricted thereto.
Generally, all
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polypeptides which act as agonists or antagonists and/or have therapeutic or
diagnostic applications are of value.
Suitable peptides or proteins for the purposes of the invention include for
example
s insulin, insulin-like growth factor, human growth hormone (hGH) and other
growth
factors, tissue plasminogen activator (tPA), erythropoietin (EPO), cytokines,
e.g.
interleukines (IL) such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-
9, IL-10, IL-11,
IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, interferon (IFN)-alpha, -
beta, -gamma,
-omega or -tau, tumour necrosis factor (TNF) such as TNF-alpha, -beta or -
gamma,
TRAIL, G-CSF, GM-CSF, M-CSF, MCP-1 and VEGF. Other examples are
monoclonal, polyclonal, multispecific and single chain antibodies and
fragments
thereof such as for example Fab, Fab', F(ab')2, Fc and Fc' fragments, light
(L) and
heavy (H) immunoglobulin chains and the constant, variable or hypervariable
regions
thereof as well as Fv and Fd fragments (Chamov et al., 1999, Antibody Fusion
proteins, Wiley-Liss Inc.). The antibodies may be of human or non-human
origin.
These include for example the classes known in man: IgA, IgD, IgE, IgG and
IgM, with
their various subclasses, for example IgA1, IgA2 and IgG1, IgG2, IgG3 and
IgG4.
Humanised and chimeric antibodies are also possible. Of particular therapeutic
importance and hence a subject of the present invention are powder
formulations
Zo which [contain] antibodies against for example various surface antigens
such as CD4,
CD20 or CD44, various cytokines, for example IL2, IL4 or ILS. Other Examples
are
antibodies against specific classes of immunoglobulin (e.g. anti-IgE
antibodies) or
against viral proteins (e.g. anti-RSV, anti-CMV antibodies, etc.).
Zs Fab fragments (fragment antigen binding = Fab) consist of the variable
regions of both
chains which are held together by the adjacent constant regions. Other
antibody
fragments are F(ab')2 fragments which can be produced by proteolytic digestion
with
pepsin. By gene cloning it is also possible to prepare shortened antibody
fragments
consisting of only the variable region of the heavy (VH) and light chain (VL).
These are
3o known as Fv fragments (fragment variable = fragment of the variable part).
Such
antibody fragments are also referred to as single chain Fv fragments (scFv).
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Examples of scFv antibodies are known and described, cf. for example Huston et
al.,
1988, Proc. Natl. Acad. Sci. USA, 16, 5879ff.
In past years various strategies have been developed for producing multimeric
scFv
derivatives, such as e.g. dia-, tri- and pentabodies. The term diabody is used
in the art
to denote a bivalent homodimeric scFv derivative. Shortening the peptide
linker in the
scFv molecule to 5 to 10 amino acids results in the formation of homodimers by
superimposing VH/VL chains. The diabodies may additionally be stabilised by
inserted
disulphite bridges. Examples of diabodies can be found in the literature, e.g.
in Perisic
et al., 1994 (Structure, 2, 1217ff). The term minibody is used in the art to
denote a
bivalent homodimeric scFv derivative. It consists of a fusion protein which
contains the
CH3 region of an immunoglobulin, preferably IgG, most preferably IgG1, as
dimerisation region. This connects the scFv fragments by means of a hinge
region,
also of IgG, and a linker region. Examples of such minibodies are described by
Hu et
al., 1996, Cancer Res., 56, 3055ff. The term triabody is used in the art to
denote a
trivalent homotrimeric scFv derivative (Kortt et al., 1997, Protein
Engineering, 10,
423ff). The direct fusion of VH-VL without the use of a linker sequence leads
to the
formation of trimers.
2o The fragments known in the art as mini antibodies which have a bi-, tri- or
tetravalent
structure are also derivatives of scFv fragments. The multimerisation is
achieved by
means of di-, tri- or tetrameric coiled coil structures (Pack, P. et al.,
1993,
Biotechnology, 11, 1271ff; Lovejoy, B. et al., 1993, Science, 259, 1288ff;
Pack, P. et
al., 1995, J. Mol. Biol., 246, 28ff).
ZS
A particularly preferred embodiment of the invention relates to a protein from
the class
of antibodies, more precisely type 1 immunoglobulin G. This is a humanised
monoclonal antibody, with 95% human and 5% murine antibody sequences. The
antibody has a molecular weight of about 148 Kilodalton (kDa), consisting of
two light
3o and two heavy chains and a total of four disulphide bridges.
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Particularly advantageous are powders which contain as active substance a
peptide or
protein or a combination of peptide/peptide, peptide/protein or
protein/protein. The
corresponding biological macromolecules may make up between 0.01 to 50% (w/w)
of
the dry mass of the powder. The amount is thus for example 0.01, 0.02, 0.03
... 0.08,
s 0.09, 0.1, 0.2, 0.3 ... 0.8, 0.9 etc.; 1, 2, 3, ... 8, 9, 10 etc.; 11, 12,
13, ... 18, 19, 20
etc.; 21, 22, 23, ... 28, 29, 30 etc.; 31, 32, 33, ... 38, 39, 40 etc.; 41,
42, 43, ... 48, 49,
49.1, 49.2, 49.3, ... 49.8, .49.9 etc.; 49.91, 49.92, 49.93, ... 49.98, 49.99
(w/w).
Particularly advantageous powders according to the invention are powders,
preferably
spray-dried powders, with a ratio of low-molecular dextran to peptide/protein
of 50/50,
51 /49, 52/48, 53/47, 54/46, 55/45, 56/44, 57/43, 58/42, 59/41, 60/40, 61 /39,
62/38,
63/37, 64/36, 65/35, 66/34, 67/33, 68/32, 69/31, 70/30, 71 /29, 72/28, 73/27,
74/26,
75/25, 76/24, 77/23, 78/22, 79/21, 80/20, 81 /19, 82/18, 83/17, 84/16, 85/15,
86/14,
87/13, 88/12, 89/11, 90/10, 91 /9, 92/8, 93/7, 94/6, 95/5, 96/4, 97/3, 98/2,
99/1,
99.1 /0.9, 99.2/0.8, 99.3/0.7, 99.4/0.6, 99.5/0.5, 99.6/0.4, 99.66/0.33,
99.7/0.3, 99.8/0.2,
99.9/0.1, 99.99/0.01 (w/w). If the corresponding powder contains one or more
additional excipients, either the amount of low-molecular dextran, the amount
of
pharmaceutical active substance, or both amounts can be reduced accordingly,
the
amount of low-molecular dextran preferably having one of the values between 50
and
ao 99.99% (w/w).
If the powders according to the invention contain very sma!I proteins/peptides
with a
molecular weight of < 10 kDa, preferably < 5 kDa, such as for example growth
factors,
for example cytokines, the amount is preferably between 0.1 to 10% (w/w), more
25 preferably between 0.2 to 5% (w/w) of the total weight of the powder.
Accordingly,
powders are preferred wherein the amount of cytokines is 0.2, 0.3, 0.4 ...
0.8, 0.9 etc.;
1, 2, 3, ... etc; 4.1, 4.2, 4.3, ... 4.8, 4.9 etc.; 4.91, 4.92, 4.93, .. .
4.98, 4.99 (w/w).
If on the other hand the pharmaceutical active substance is one or more
antibodies or
3o a derivative thereof (preferred embodiment), the proportion of active
substance in the
solid content of the powder is between 0.01 and 50% (w/w), preferably between
0.1
and 50% (w/w), more preferably between 0.33 and 50% (w/w), for example 0.1,
0.2,
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0.3, 0.33, ... 0.66, 0.7, 0.8, 0.9 etc.; 1, 2, 3, ... 8, 9, 10 etc.; 11, 12,
13, ... 18, 19, 20
etc.; 21, 22, 23, ... 28, 29, 30 etc.; 31, 32, 33, ... 38, 39, 40 etc.; 41,
42, 43, ... 48,
49, etc; 49.1, 49.2, 49.3, ... 49.8, 49.9 etc.; 49.91, 49.92, 49.93, ...
49.98, 49.99 (w/w).
According to a particular embodiment the proportion of antibodies in the
solids content
of the powder is between 10 and 50% (w/w), more preferably between 10 and 30%
(w/w), even more preferably between 10 and 20% (w/w). The invention relates,
particularly advantageously, to powders, preferably spray-dried powders, with
a ratio of
low-molecular dextran to antibody of 50/50, 51/49, 52/48, 53/47, 54/46, 55/45,
56/44,
57/43, 58/42, 59/41, 60/40, 61 /39, 62/38, 63/37, 64/36, 65/35, 66/34, 67/33,
68/32,
69/31, 70/30, 71 /29, 72/28, 73/27, 74/26, 75/25, 76/24, 77/23, 78/22, 79/21,
80/20,
81/19, 82/18, 83/17, 84/16, 85/15, 86/14, 87/13, 88/12, 89/11 or 90/10 (w/w).
According to another embodiment the present invention relates to powders,
preferably
spray-dried powders, characterised in that the dry mass of the spray-dried
powder
contains a) at least 50% (w/w), preferably between 55 and 99.99% (w/w), most
preferably between 60 and 99.99% (w/w) of low-molecular dextran and b) up to
30%
(w/w) of a biological macromolecule, the sum of the percentages by weight of
low-
molecular dextran and biological macromolecule being at most 100% (w/w). A
skilled
Zo man is in a position to prepare such powders. Thus, the skilled man knows
that he can
add at most 0.01 % (w/w) of a pharmaceutical active substance relative to the
total
solids content of the solution which is to be sprayed, if the amount of low-
molecular
dextran is to be 99.99% (w/w).
25 The powders according to the invention may also contain other excipients,
such as for
example amino acids, peptides, non-biological or biological polymers, and/or
one or
more sugars. Other excipients known in the art are for example lipids, fatty
acids, fatty
acid esters, steroids (e.g. cholesterol) or chelating agents (e.g. EDTA) as
well as
various cations (see above). Excipients with a high glass transition
temperature, for
3o example above 40°C, preferably above 45°C, or above
55°C, are particularly preferred.
A list of suitable excipients can be found for example in Kippe (Eds.),
"Handbook of
Pharmaceufical Excipient'"' 3rd Ed., 2000.
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Suitable protein-containing excipients include for example albumin (human or
recombinant in origin), gelatine, casein, haemoglobin and the like. The sugars
are
preferably mono-, di-, oligo- or polysaccharides or a combination thereof.
Examples of
s monosaccharides are fructose, maltose, galactose, glucose, d-mannose,
sorbose and
the like. Suitable disaccharides for the purposes of the invention include for
example,
lactose, sucrose, trehalose, cellobiose, and the like. Polysaccharides which
may be
used include in particular raffinose, melecitose, dextrin, starch and the
like. Sugar
alcohols include in addition to mannitol, xylitol, maltitol, galactitol,
arabinitol, adonitol,
lactitol, sorbitol (glucitol), pyranosylsorbitol, inositol, myoinositol and
the like as
excipients. Suitable amino acids include for example alanine, glycine,
arginine,
histidine, glutamate, asparagine, cysteine, leucine, lysine, isoleucine,
valine,
tryptophan, methionine, phenylalanine, tyrosine, citrulline, L-aspartyl-L-
phenylalanine-
methylester (= aspartame), trimethylammonioacetate (= betaine) and the like.
~s Preferably, amino acids are used which act as buffers (e.g. glycine or
histidine) and/or
as dispersing agents. These latter groups include in particular predominantly
hydrophobic amino acids, such as e.g. leucine, valine, isoleucine, tryptophan,
alanine,
methionine, phenylalanine, tyrosine, histidine or proline. Within the scope of
the
present invention it has proved particularly advantageous to use isoleucine in
addition
ao to the low-molecular dextran, preferably in a concentration of 5 to 20%
(w/w), most
preferably from 10 to 20% (w/w), even more preferably from 12 to 20% (w/w). It
is also
particularly advantageous to use di-, tri-, oligo- or polypeptides as further
excipients,
which contain one or more of these predominantly hydrophobic amino acid
groups.
Suitable examples of tripeptides include for example one or more of the
following
25 tripeptides: Leu-Leu-Gly, Leu-Leu-Ala, Leu-Leu-Val, Leu-Leu-Leu, Leu-Leu-
Met, Leu-
Leu-Pro, Leu-Leu-Phe, Leu-Leu-Trp, Leu-Leu-Ser, Leu-Leu-Thr, Leu-Leu-Cys, Leu-
Leu-Tyr, Leu-Leu-Asp, Leu-Leu-Glu, Leu-Leu-Lys, Leu-Leu-Arg, Leu-Leu-His, Leu-
Gly-
Leu, Leu-Ala-Leu, Leu-Val-Leu, Leu-Met-Leu, Leu-Pro-Leu, Leu-Phe-Leu, Leu-Trp-
Leu, Leu-Ser-Leu, Leu-Thr-Leu, Leu-Cys-Leu, Leu-Try-Leu, Leu-Asp-Leu, Leu-Glu-
3o Leu, Leu-Lys-Leu, Leu-Arg-Leu and Leu-His-Leu. It has proved particularly
advantageous to use tripeptides of general formulae: Ile-X-X; X-Ile-X; X-X-
Ile, where
X may be one of the following amino acids: alanine, glycine, arginine,
histidine,
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glutamic acid, glutamine, asparagine, aspartic acid, cysteine, leucine,
lysine, isoleucine
(11e), valine, tryptophan, methionine, phenylalanine, proline, serine,
threonine, tyrosine,
L-aspartyl-L-phenylalanine-methylester (= aspartame), trimethylammonio-
acetate.
Particularly preferred are corresponding tripeptides of formula (Ile)2-X, for
example
Ile-Ile-X, Ile-X-Ile, or X-Ile-Ile, where X may again denote one of the amino
acids
listed above. These include for example the tripeptides: Ile-Ile-Gly, Ile-Ile-
Ala, Ile-Ile-
Val, Ile-Ile-Ile, Ile-Ile-Ile, Ile-Ile-Met, Ile-Ile-Pro, Ile-Ile-Phe, Ile-Ile-
Trp, Ile-Ile-Ser, Ile-Ile-
Thr, Ile-Ile-Cys, Ile-Ile-Tyr, Ile-Ile-Asp, Ile-Ile-Glu, Ile-Ile-Lys, Ile-Ile-
Arg, Ile-Ile-His, Ile-
Gly-Ile, Ile-Ala-Ile, Ile-Val-Ile, Ile-Met-Ile, Ile-Pro-Ile, Ile-Phe-Ile, Ile-
Trp-Ile, Ile-Ser-Ile,
Ile-Thr-Ile, Ile-Cys-Ile, Ile-Try-Ile, Ile-Asp-Ile, Ile-Glu-Ile, Ile-Lys-Ile,
Ile-Arg-Ile, Ile-His-
Ile. It is particularly advantageous to use Ile-Ile-Ile.
Suitable polymers include for example the polyvinylpyrrolidones mentioned
above as
excipients, derivatised celluloses, such as hydroxymethyl, hydroxyethyl or
~s hydroxypropylethyl cellulose, polymeric sugars such as Ficoll, starch such
as
hydroxyethyl or hydroxypropyl starch, dextrins such as cyclodextrins (2-
hydroxypropyl-
(3-cyclodextrin, sulphobutylether-f3-cyclodextrin), polyethylenes, glycols
and/or pectins.
The salts may be for example inorganic salts such as chlorides, sulphates,
ao phosphates, diphosphates, hydrobromides and/or nitrate salts. Moreover the
powders
according to the invention may also contain organic salts, such as e.g.
malates,
maleates, fumarates, tartrates, succinates, ethylsuccinates, citrates,
acetates, lactates,
methanesulphonates, benzoates, ascorbates, paratoluenesulphonates, palmoates,
salicylates, stearates, estolates, gluceptates or labionate salts. At the same
time
25 corresponding salts may contain pharmaceutically acceptable cations, such
as for
example sodium, potassium, calcium, aluminium, lithium or ammonium. It is
particularly preferred to use corresponding cations in conjunction with the
stabilisation
of proteins. Consequently, according to another embodiment the present
invention
also relates to powders, preferably spray-dried powders, which contain a
3o pharmaceutically acceptable salt in addition to the low-molecular dextran
and the
pharmaceutical active substance.
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The present invention thus also relates to spray-dried powders which contain
one or
more pharmaceutically acceptable excipients and/or one or more salts in
addition to
the low-molecular dextran and the pharmaceutical active substance. The
excipients
may be, for example, the above-mentioned amino acids, peptides and their
salts,
s sugars, polyols, salts of organic acids and/or polymers.
According to another embodiment the present invention relates to powders,
preferably
spray-dried powders, which contain in addition to the low-molecular dextran
and the
pharmaceutical active substance one or more amino acid(s), preferably one
amino
acid, as a further excipient. In this context the present invention also
relates to
powders which contain in relation to their dry mass at least 50% (w/w),
preferably
between 55 and 98.99% (w/w), most preferably between 60 and 98.99% (w/w) of
low-
molecular dextran, and between 1 and 20% (w/w) of amino acids and between 0.01
and 49% (w/w) of a pharmaceutical active substance, preferably a biological
macromolecule, while the sum of the amounts by weight may be up to at most
100%
(w/w). According to a preferred embodiment the amount of low-molecular dextran
is at
least 60% (w/w), preferably between 70 and 89.99% (w/w) in relation to the dry
mass
of the powder. In a corresponding formulation the amount of amino acids is
preferably
between 10 and 20% (w/w) and the amount of the pharmaceutical active substance
is
Zo between 0.01 to 10% (w/w).
Consequently, according to another embodiment the present invention also
relates to
powders which contain or consist of, for example, 80% (w/w) of low-molecular
dextran/
19% (w/w) amino acid/ 1 % (w/w) pharmaceutical active substance (80/19/1 );
(80/18/2);
25 (80/17/3); (80/16/4); (80/15/5); (80/14/6); (80/13/7); (80/12/8);
(80/11/9); (80/10/10);
(70/19/11 ); (70/18/12); (70/17/13); (70/16/14); (70/15/15); (70/14/16);
(70/13/17);
(70/12/18); (70/11 /19); (70/10/20); (60/20/20); (60/19/21 ); (60/18/22);
(60/17/23);
(60/16/24); (60/15/25); (60/14/26); (60/13/27); (60/12/28); (60/11 /29);
(60/10/30) or
(70/20/10). If the proportion of active substance is reduced from 20% (w/w) to
0.01
30 (w/w), for example to 9.99, ... 9.9, 9.8, 9.7 ... 9.3, 9.2, 9.1 ... 9, 8 7,
6, 5, 4, 3, 2, 1, ...
0.9, 08, 0.7, ... 0.66, ... 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.09, 0.08, 0.07,
0.06, 0.05, 0.04,
0.03, 0.02, 0.01, while the proportion of amino acid remains constant, the
amount of
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low-molecular dextran may be reduced accordingly to, for example, 80.01, ...
80.1,
80.2, 80.3 ... 80.8, 80.9, 81, 82, 83, 84, 85, 86, 87, 88, 89, .. . , 89.1,
89.2, 89.3, ...
89.33, ... 89.4, 89.5, 89.6 , 89.7, 89.8, 89.9, ... 89.91, 89.92, 89.93, ...
89.97,
89.98, 89.99 (w/w), so that the sum of the amounts by weight of the individual
powder
ingredients in relation to the dry mass of the powder is 100% (w/w). By adding
other
excipients or salts the amount of low-molecular dextran, amino acids/peptides
and/or
pharmaceutical active substance can be adjusted/reduced accordingly, so that
the
parts by weight of the individual ingredients add up to a total of 100% (w/w).
If the amino acid added is isoleucine, according to another embodiment the
powders
according to the invention contain an amount of a) low-molecular dextran of at
least
50% (w/w), preferably 55 to 89.99% (w/w), most preferably 60 to 89.99% (w/w),
b) a
proportion of 5 to 20% (w/w) isoleucine and c) at least 0.01 % (w/w),
preferably 0.01 to
at most 45% (w/w) of a pharmaceutical active substance, preferably a
peptide/protein,
according to the invention. Preferably the amount of isoleucine is 10 to 20%
(w/w),
more preferably 12 to 20% (w/w) of the total solids content of the powder.
Here again,
the sum of the % by weight of the individual ingredients does not exceed 100%
(w/w).
The invention also relates to powders of the following composition: 85% (w/w)
of low-
molecular dextran/ 5% amino acid or peptide/ 10% (w/w) of pharmaceutical
active
ao substance (85/5/10), (84/6/10), (83/7/10), (82/8/10), (81/9/10),
(80/10/10); (79/11/10);
(78/12/10); (77/13/10); (76/14/10); (75/15/10); (74/16/10); (73/17/10);
(72/18/10);
(71/19/10); (70/20/10), while the amount of the pharmaceutical active
substance may
also be reduced from 10 to 0.01 % (w/w), for example to 9.99, ... 9.9, 9.8,
9.7 ... 9.3,
9.2, 9.1 ... 9, 8 7, 6, 5, 4, 3, 2, 1, ... 0.9, 08, 0.7, ... 0.66, . . . 0.6,
0.5, 0.4, 0.3, 0.2, 0.1,
ZS 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03 0.02, 0.01 and accordingly the
amount of low-
molecular dextran may be increased to for example 80.01, ... 80.1, 80.2, 80.3
... 80.8,
80.9, 81, 82, 83, 84, 85, 86, 87, 88, 89, ... , 89.1, 89.2, 89.3, :.. 89.33,
... , 89.4, 89.5,
89.6 , 89.7, 89.8, 89.9, ... 89.91, 89.92, 89.93, ... , 89.97, 89.98, 89.99
(w/w), so that
the sum of the parts by weight in relation to the dry mass of the powder makes
up
30 100% (w/w). Therefore, the invention also relates to powders having the
following
composition: 80% (w/w) of low-molecular dextran/ 19% (w/w) of isoleucine / 1 %
(w/w)
of pharmaceutical active substance (80/19/1 ); (80/18/2); (80/17/3);
(80/16/4);
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(80/15/5); (80/14/6); (80/13/7); (80/12/8); (80/11 /9); (80/10/10); (70/19/11
); (70/18/12);
(70/17/13); (70/16/14); (70/15/15); (70/14/16); (70/13/17); (70/12/18); (70/11
/19);
(70/10/20); (60/19/21 ); (60/18/22); (60/17/23); (60/16/24); (60/15/25);
(60/14/26);
(60/13/27); (60/12/28); (60/11/29); (60/10/30). If other excipients or salts
are added the
s amount of low-molecular dextran, isoleucine and/or pharmaceutical active
substance
should be adjusted accordingly so that the amounts by weight of the individual
ingredients add up to 100% (w/w).
Another embodiment of the present invention relates to the use of low-
molecular
dextran and tripeptides for stabilising powders containing a pharmaceutical
active
substance, preferably a peptide, protein, or a mixture thereof. The present
specification
mentions by way of example some tripeptides which may be used together with
low-
molecular dextran to prepare the powders according to the invention. According
to a
particular embodiment the tripeptides are those which contain at least one
isoleucine,
preferably two isoleucines, or according to a particularly advantageous
embodiment,
consist of three isoleucines.
In connection with this, the invention relates to powders containing a) an
amount of
low-molecular dextran of at least 50% (w/w), preferably from 55 to 98.99%
(w/w), most
ao preferably from 60 to 98.99% (w/w), b) a proportion of 1 to 20% (w/w) of a
tripeptide,
preferably triisoleucine and c) 0.01 to at most 49% (w/w) of a pharmaceutical
active
substance, preferably a peptide/protein. Here again, the sum of the individual
solids
cannot add up to more than 100% (w/w). The invention also relates to powders
of the
following composition: 89% (w/w) of low-molecular dextran/ 1 % tripeptide,
preferably
25 an isoleucine-containing tripeptide, most preferably triisoleucine/ 10%
(w/w) of
pharmaceutical active substance (89/1/10); (88/2/10); (87/3/10); (86/4/10);
(85/5/10);
(84/6/10); (83/7/10); (82/8/10); (81/9/10); (80/10/10); (79/11/10);
(78/12/10);
(77/13/10); (76/14/10); (75/15/10); (74/16/10);, (73/17/10); (72/18/10) or
(71/19/10),
while the amount of pharmaceutical active substance can also be reduced from
10 to
30 0.01 % (w/w), for example to 9.99, ... 9.9, 9.8, 9.7 ... 9.3, 9.2, 9.1 ...
9, 8 7, 6, 5, 4, 3,
2, 1, ... 0.9, 08, 0.7, ... 0.66, ... 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.09,
0.08, 0.07, 0.06, 0.05,
0.04, 0.03 0.02, 0.01 % (w/w) and accordingly the amount of low-molecular
dextran
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may increase to for example 80.01, ... 80.1, 80.2, 80.3 ... 80.8, 80.9, 81,
82, 83, 84,
85, 86, 87, 88, 89, ... 89.1, 89.2, 89.3, ... 89.33, ... 89.4, 89.5, 89.6 ,
89.7, 89.8,
89.9, ... 89.91, 89.92, 89.93, ... 89.97, 89.98, 89.99% (w/w), so that the sum
of the
amounts by weight in relation to the dry mass of the powder comes to 100%
(w/w).
Therefore the invention also relates to powders of the following composition:
80%
(w/w) of low-molecular dextran/ 19% (w/w) tripeptide, preferably triisoleucine
/ 1
(w/w) of pharmaceutical active substance (80/19/1 ); (80/18/2); (80/17/3);
(80/16/4);
(80/15/5); (80/14/6); (80/13/7); (80/12/8); (80/11/9); (80/10/10); (70/19/11);
(70/18/12);
(70/17/13); (70/16/14); (70/15/15); (70/14/16); (70/13/17); (70/12/18);
(70/11/19);
io (70/10/20); (60/19/21 ); (60/18/22); (60/17/23); (60/16/24); (60/15/25);
(60/14/26);
(60/13/27); (60/12/28); (60/11/29); (60/10/30), while the amount of
tripeptide,
preferably triisoleucine can also be reduced from 10 to 1 % (w/w), for example
to 9.99,
... 9.9, 9.8, 9.7 ... 9.3, 9.2, 9.1 ... 9, 8 7, 6, 5, 4, 3, 2 .1.9, 1.8, 1.7,
... 1.66, ... 1.6, 1.5,
1.4, 1.3, 1.2, 1.1, 1 % (w/w) and accordingly the amount of pharmaceutical
active
substance, preferably peptide/protein may be increased to for example 30.1,
30.2, 30.3
... 30.8, 30.9, 31, 32, 33, 34, 35, 36, 37, 38, 38.1, 38.2, 38.3, ... 38.33,
... , 38.4, 38.5,
38.6 , 38.7, 38.8, 38.9, ... 39 (w/w), so that the sum of the amounts by
weight in
relation to the dry mass of the powder comes to 100% (w/w). When the amount of
tripeptide is reduced from 10 to 1 (w/w), as shown here, the proportion of low-
ao molecular dextran in the powder can also be increased. When the proportion
of active
substance remains constant at for example 10% (w/w) powders can be produced
with
a dextran content of 80.1, 80.2, 80.3 ... 80.8, 80.9, 81, 82, 83, 84, 85, 86,
87, 88, 88.1,
88.2, 88.3, ... 88.33, ... , 88.4, 88.5, 88.6 , 88.7, 88.8, 88.9 or 89 (w/w).
25 According to another embodiment according to the invention the powders may
additionally contain surfactants such as Tween 20, 40, 60, 80, Brij 35,
Pluronic F 88
and Pluronic F 127 contain. These are preferably used in a concentration of
0.01-0.1
(w/w). Particularly preferred is a spray-dried powder which contains as
excipient low-
molecular dextran and additionally Tween 20, preferably in a concentration of
0.01-
30 0.1 % (w/w), as surfactant.
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According to another embodiment the present invention also relates to
pharmaceutical
compositions containing one of the spray-dried powders described above.
Preparation of the powder according to the invention:
The present invention also provides processes for preparing one of the spray-
dried
powders described above. The process is characterised in that a solution /
suspension
to be sprayed, containing a pharmaceutical active substance and low-molecular
dextran is sprayed below a temperature of 200/120°C (inflow/outflow
temperature)
preferably at 150-185/70-95°C. The process according to the invention
is described
more fully by means of some Examples in the "EXAMPLES" section.
Basically, the powders according to the invention may be prepared by
dissolving the
pharmaceutical active substance, preferably a biological macromolecule in the
form of
a peptide or protein, in an aqueous solution, depending on the solubility
conditions of
the active substance in question. Usually, buffered solutions with a pH of 3-
11,
preferably 3.5-9 are used. When preparing inhalable powders an aqueous
solution with
a pH of 4-7.8 is particularly advantageous. In order to ensure sufficient
solubility, the
pH of the solution should be below the p1 of the peptide/protein. The aqueous
solution
may optionally contain additional water-soluble organic solvents, such as e.g.
acetone,
Zo alcohols or the like. Lower alcohols such as e.g. methanol, ethanol,
propanol, (n or iso-
propanol) or the like are particularly suitable. Mixed solvent systems of this
kind
normally contain between 10-20% (v/v) of a water-soluble organic solvent. The
solid
content in the solution to be sprayed is usually between 0.01-20% (w/w),
preferably
between 0.05-10% (w/w), particularly preferably between 0.1-5% (w/w). Within
the
25 scope of the present invention spray-dried powders were prepared starting
from an
aqueous solution with a solid content of 10% (w/w) or 3.33% (w/wt.%).
Usually, the excipient or a mixture of suitable excipients, as described above
by way of
example, is dissolved in a second container in highly pure water or a suitable
buffer
3o solution with a pH of 3 to 11, preferably 3.5 to 9 and particularly
preferably 4.0 to 7.8
and mixed with the active substance solution in a second step. Then the
solution /
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suspension is adjusted to the desired solid content with pure water or a
suitable buffer
solution with a pH of 3 to 11, preferably 3.5 to 9 and particularly preferably
4.0 to 7.8.
Consequently the present invention relates to a process for preparing a spray-
dried
powder, characterised in that
a) a pharmaceutical active substance is dissolved in an aqueous solution /
suspension;
b) low-molecular dextran is dissolved in an aqueous solution / suspension;
c) if active substance and low-molecular dextran are dissolved in different
solutions / suspension, these are mixed together;
d) the solution / suspension containing low-molecular dextran and the
pharmaceutical active substance is sprayed below a temperature of
200/120°C,
preferably 175/95°C.
The excipient content of low-molecular dextran in the solution/suspension
which is to
be sprayed is between 50% and 99.99% (w/w), preferably between 55% and 99.99
(w/w), most preferably between 60 and 99.99% (w/w) in relation to the solids
content of
the spray solution. The active substance concentration is normally between
0.01 and
50% (w/w), preferably between 0.01 and 40% (w/w), most preferably between 0.01
ao and 30% (w/w) in relation to the solids content of the solution or
suspension which is to
be sprayed. Starting from the powder compositions according to the invention
described above, the skilled man is capable of preparing solutions/suspensions
for
spraying which result in the corresponding powder compositions after spraying.
Zs Consequently the present invention also relates to processes for preparing
a spray-
dried powder, as described above, characterised in that the solids content of
the
solution/suspension which is to be sprayed contains between 50 and 99.99%
(w/w),
preferably between 60 and 99.99% (w/w) of low-molecular dextran. According to
another preferred embodiment the present invention relates to a corresponding
3o process characterised in that the solids content of the solution/suspension
which is to
be sprayed contains between 0.01 and 50% (w/w), preferably between 0.01 and
30%
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(w/w), most preferably between 0.33 and 30% (w/w) of a pharmaceutical active
substance.
According to another embodiment of the present process a spray solution /
suspension
s with a solids content of a) at least 50% (w/w), for example between 60 to
99.99% (w/w)
of a low-molecular dextran and b) at least 0.01 % (w/w), preferably 0.01 to
50% (w/w)
of a pharmaceutical active substance, preferably a biological macromolecule,
is
prepared and sprayed, the sum of the % by weight being at most 100% (w/w).
According to a preferred embodiment a spray solution / suspension with a
solids
content a) of low-molecular dextran of at least 60% (w/w), preferably between
60 to
99.99% (w/w), and b) 0.01 to 40% (w/w) of a pharmaceutical active substance,
preferably a biological macromolecule, is prepared and sprayed, the sum of the
% by
weight of the solution or suspension being at most 100% (w/w).
Corresponding to the powders according to the invention described above,
according
to another embodiment the solution / suspension to be sprayed additionally
contains
one or more pharmaceutically acceptable excipients and/or one or more salts.
The
excipients are preferably amino acids, peptides or their salts, sugars,
polyols, salts of
organic acids and/or polymers.
zo
Preferably the spray solution contains in addition to the pharmaceutical
active
substance and the low-molecular dextran one or more amino acids and/or
peptides or
proteins as other excipients. Consequently the present invention also relates
to a
process for preparing spray-dried powders characterised in that the solution /
Zs suspension to be sprayed contains, in relation to its solids content, a) at
least 50%
(w/w), preferably at least 60% (w/w) of low-molecular dextran, b) between 1
and 20%
(w/w) of at least one amino acid and/or at least one peptide. Examples of
suitable
excipients including pharmaceutically acceptable salts, peptides and amino
acids can
be found under the heading "Powders according to the invention" in this
specification.
According to another preferred embodiment the spray solution also contains in
addition
to low-molecular dextran one or more amino acids as a further excipient. Spray
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solutions /suspensions the solids content of which contains a) at least 50%
(w/w),
preferably 60 to 98.99% (w/w) of low-molecular dextran, b) 1 to 20% (w/w)
amino
acids, c) and at least 0.01 % (w/w) of a pharmaceutical active substance,
preferably a
peptide/protein, such as for example an antibody, are advantageous. The amount
of
pharmaceutical active substance is preferably 0.01 to at most 30% (w/w) while
the sum
of the solids components is at most 100% (w/w). Anyone skilled in the art is
capable of
preparing corresponding powders and adapting the amounts by weight so that the
sum
of the solids components does not exceed 100% (w/w). If the amount (relative
to the
total solids content) of pharmaceutical active substance is for example 30%
(w/w) and
the amount of low-molecular dextran is 60% (w/w) the skilled man knows that he
can
add at most 10% (w/w) of amino acids to the spray solution / suspension.
According to another preferred embodiment the spray solution also contains
isoleucine
as a further excipient in addition to low-molecular dextran. Spray solutions
/suspensions the solids content of which contains a) at least 50% (w/w),
preferably 60
to 89.99% (w/w) of low-molecular dextran, b) 10 to 20% (w/w) of isoleucine, c)
and at
least 0.01 % (w/w) of a pharmaceutical active substance, preferably a
peptide/protein,
such as for example an antibody, are advantageous. The amount of
pharmaceutical
active substance is preferably 0.01 to at most 30% (w/w) while the sum of the
solids
ao components is at most 100% (w/w). Anyone skilled in the art is capable of
preparing
corresponding powders and adapting the amounts by weight so that the sum of
the
solids components does not exceed 100% (w/w). If the amount (relative to the
total
solids content) of pharmaceutical active substance is for example 30% (w/w)
and the
amount of low-molecular dextran is 60% (w/w) the skilled man knows that he can
add
ZS at most 10% (w/w) of isoleucine to the spray solution / suspension.
According to another embodiment the solution to be sprayed contains in
addition to
low-molecular dextran one or more tripeptides, preferably isoleucin-containing
tripeptides, most preferably triisoleucine. Solutions or suspensions for
spraying, the
3o solids content of which contains a) at least 50% (w/w), preferably 60 to
98.99% (w/w)
of low-molecular dextran, b) 1 to 19% (w/w) of a tripeptide, preferably
triisoleucine, and
c) at least 0.01 % (w/w) of a pharmaceutical active substance, preferably a
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peptide/protein such as for example an antibody, are advantageous, while the
sum of
the solids components is at most 100 % (w/w). The amount of pharmaceutical
active
substance is preferably 0.01 to at most 39% (w/w). Anyone skilled in the art
is capable
of preparing corresponding powders and adapting the amounts by weight so that
the
sum of the solids components does not exceed 100% (w/w). If the amount
(relative to
the total solids content) of pharmaceutical active substance is for example
30% (w/w)
and the amount of low-molecular dextran is 60% (w/w) the skilled man knows
that he
can add at most 10% (w/w) of tripeptide, preferably triisoleucine, to the
solution or
suspension which is to be sprayed.
As mentioned previously, it is advantageous to prepare and spray solutions
which are
to be sprayed with a pH of between 3 and 11, preferably 3.5 and 9, most
preferably
between 4.0 and 7.8. Suitable buffer systems are known to the skilled man.
Usually, it
is particularly advantageous to use inorganic or organic salts as the buffer
system.
is
Typically, the optimum excipient and protein content for each protein or
peptide is
determined experimentally. Preferred formulations of the invention may also
contain at
least one other excipient, in order to improve powder characteristics such as
dispersibility and flow properties while retaining superior aggregation-
inhibiting
Zo properties.
The spraying is done in conventional spray driers, for example in apparatus
made by
Messrs Niro A/S (Soeborg, DK), Buchi Labortechnik GmbH (Flawil, CH) or the
like.
The optimum conditions for the spray drying depend in each case on the
25 corresponding formulation and should be determined experimentally. The gas
used is
typically air, but inert gases such as nitrogen or argon are also suitable. In
addition, the
spray drying temperature, i.e. the inlet temperature and outlet temperature,
is
determined in accordance with the temperature sensitivity of the active
substance
used, in each case depending on the stabilisers used. An inlet temperature of
50-
30 200°C is usual, while the outlet temperature is usually 30-
150°C. Within the scope of
the present invention an inlet temperature of approximately 170-185°C
and an outlet
temperature of 80-100°C was used. However, rather higher temperatures
are also
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possible, for example an inlet temperature of up to 200°C, preferably
90-185°C and an
outlet temperature of up to 120°C, preferably 90-105°C,
depending on the amount of
stabiliser. Spraying is generally carried out at a pressure of approximately
20-150 psi,
preferably at about 30 or 40-100 psi, for example at about 30, 40, 50, 60, 70,
80, 90 or
s 100 psi.
With regard to the Buchi sprayer the "Liquid Feed Rate" is normally between
0.1 and
100 ml/min, preferably between 0.1 and 30 ml/min, for example about 3 ml/min.
In
connection with this an Aspirator Flow Rate of 20-40 m3/h, preferably 30-40
m3/h, such
as for example 35 m3/h and an atomising flow rate of 0.3-2.5 m3/h, preferably
about
0.67 m3/h, has proved particularly suitable.
The spray-dried active substance formulations, preferably the powdered protein
formulations, may optionally be subjected to a second gently drying (after-
drying). The
aim is to achieve a uniform residual water content in the formulations of less
than 2%
(w/w), and thereby improve both the active substance stability and also
improve
powder qualities such as the glass transition temperature, flowability and
dispersibility.
The conditions of the after-drying process must be selected such that the
aggregate
formation of the active substances is not significantly increased. This
applies
ao particularly to the use of biological macromolecules, such as for example
the use of
peptides/proteins. The spray-dried powdered active substance formulations are
preferably prepared, processed and stored under dry conditions (at low
relative
humidity). The process of after-drying makes it possible to prepare and decant
the
powders at relatively high humidity levels. Surprisingly, the excipients to
which the
ZS invention relates stabilise the proteins in the preferred formulations even
under non-
optimal processing and storage conditions.
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Properties of the spray-dried dr~powder formulations
The dry powdered protein formulations prepared within the scope of this
invention
have a residual water content of less than 15% (w/w), usually less than 10%
(w/w),
and preferably less than 6% (w/w). More preferably the spray-dried powdered
protein
formulations have a residual water content of less than 3% (w/w), most
preferably less
than 2% (w/w) and most preferably between 0.2 and 2.0% (w/w). Formulations
with a
low residual moisture content generally exhibit improved stability during
unpacking and
storage. Moreover, the dry powdered protein formulations according to the
invention
are predominantly hygroscopic, i.e. they have a tendency to absorb moisture
from their
environment. To avoid this, powders of this kind are usually packaged in
containers
such as blister packs with the exclusion of moisture.
The stabilising effects of the excipients described here are capable of
protecting the
protein from extreme stresses during spray-drying and storage. In the absence
of
excipients spray-dried pure protein formulations form aggregates to a
considerable
degree. Process-related factors such as heat, shear stress and denaturing at
the
air/water interfaces cause aggregation (up to about 3.7% aggregates) during
the
spray-drying and subsequent after-drying (up to about 4.0% aggregates). During
storage massive aggregate formation takes place (from about 11.8 to about 18.9
ao aggregates) as a result of the absence of the stabilising hydrate coat of
the proteins.
The preferred spray-dried formulations of the invention, unlike the pure
protein
formulations, are capable of reducing the formation of aggregates both after
spray-
drying and also keeping it at a very low level under different storage
conditions. As a
2s result of spray-drying only about 1.1 to about 1.4% aggregates are formed
in the
preferred formulations, as against about 4.0% aggregates in pure protein
formulations.
Preferred formulations which are subjected to a second gentle drying, show no
tendency to increased aggregate formation. Under particularly challenging
storage
conditions (40°C, 75% relative humidity) the preferred formulations
(aggregates of ~5.1
to 10.1 %) are clearly superior to pure protein formulations (about 18.9%
aggregates)
and an analogous reference formulation with trehalose as excipient.
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Formulations which have a significant stabilising effect on the incorporated
proteins
even during relatively short storage under particularly destabilising
conditions (1 week
at 40°C, 75% relative humidity) also stabilise proteins for long
periods under far gentler
standard storage conditions (e.g. 1 year, in the dry, at about 25°C).
S
By varying the spray-drying conditions it is possible to produce powders which
preferably have a mean particle size (MMD) of less than 20 Nm, preferably less
than
Nm. According to a particularly preferred embodiment these particles according
to
the invention have a mean particle size of less than 7.5 Nm, preferably less
than 5 pm.
Particularly preferred are particles with a mean particle size of less than 4
Nm and
more preferably less than 3.5 Nm. Generally, it is also possible to prepare
particles with
a mean particle diameter of 0.1-5 Nm, preferably 0.2-4 Nm. In another
embodiment
non-respirable particles, e.g. lactose, with a particle size of at least 40
Nm, preferably
between 40 and 200 Nm, are mixed with the corresponding powders.
Apart from the mean particle size (MMD) the inhalability essentially depends
on the
mean aerodynamic particle diameter (MMAD). The particles according to the
invention
preferably have an MMAD of less than 10 Nm and more preferably less than 7.5
Nm.
Particularly advantageous are powders consisting of particles with an MMAD of
less
Zo than 5.5 Nm, preferably less than 5 pm, even more preferably less than 4.5
Nm. The
powders described in the Examples can be produced with corresponding particle
sizes
by a combination of optimum spray-drying conditions and the choice and
concentration
of excipients according to the invention. In particular the addition of amino
acids and/or
tripeptides leads to an improved particle performance with an increased
proportion of
Zs inhalable particles with an MMAD of less than 7.5, preferably less than
5.5. By the
addition of isoleucine or triisoleucine, inhalable powders with an FPF of more
than
30%, preferably more than 40, more preferably more than 50 and even more
preferably more than 55% can be prepared (see EXAMPLES).
3o The powders according to the invention are also characterised by a glass
transition
temperature of at least 45°C, preferably at least 50°C, more
preferably at least 55°C,
even more preferably at least 60°C. Particularly preferred powders have
a glass
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transition temperature of at least 65°C. In general, the glass
transition temperature of
the dextran-containing powders according to the invention is 60 to
65°C. Accordingly,
the present invention also relates to powders, preferably spray-dried powders,
containing a pharmaceutical active substance and low-molecular dextran,
wherein the
glass transition temperature is 45°C and more, preferably between 45
and 70 °C.
According to another preferred embodiment the glass transition temperature is
55°C or
above, preferably between 55 and 70°C.
Use of the spray-dried powder
The powders according to the invention are suitable for the preparation of a
pharmaceutical composition, preferably for preparing a medicament for
inhalation.
Administration of the powders according to the invention
~s Basically, the powder preparations according to the invention may be
administered
directly as dry powders using so-called dry powder inhalers, or after
reconstitution in
the form of aerosols using so-called nebulisers. The inhalable powders
according to
the invention may be administered using inhalers known from the prior art.
2o Inhalable powders according to the invention may be administered, for
example, by
means of inhalers which deliver a single dose from a supply using a measuring
chamber as described in US 4570630A, or by other means as described in
DE 36 25 685 A. Preferably, the inhalable powders according to the invention
are
packed into capsules (to produce so-called inhalettes) which are used in
inhalers as
Zs described, for example, in WO 94/28958.
Other examples of suitable inhalers may be found interalia in US 5,458,135; US
5,785,049 or WO 01/00263. Other suitable inhalers are known from WO 97/41031;
US 3,906,950 and US 4,013,075. Other dispersion inhalers for dry powder
3o preparations are described in EP 129 985; EP 472 598; EP 467 172 and US
5,522,385.
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The inhalable powders according to the invention may for example be
administered
using the inhaler known by the name Turbuhaler~ (AstraZeneca LP) or with
inhalers
as disclosed for example in EP 237 507 A. Other suitable inhalers are the
Rotahaler~
or the Discus~ (both made by GIaxoSmithKline Corp.), the Spiros~" inhaler
(Dura
Pharmaceuticals) and the Spinhaler~ (Fiscon).
A particularly preferred inhaler for administering the pharmaceutical
combination in
inhalettes according to the invention is shown in Figure 12. This inhaler
(Handyhaler)
for inhaling powdered pharmaceutical compositions from capsules is
characterised by
a housing 1 containing two windows 2, a deck 3 in which there are air inlet
ports and
which is provided with a screen 5 secured via a screen housing 4, an
inhalation
chamber 6 connected to the deck 3 on which there is a push button 9 provided
with
two sharpened pins 7 and movable counter to a spring 8, and a mouthpiece 12
which
is connected to the housing 1, the deck 3 and a cover 11 via a spindle 10 to
enable it
~s to be flipped open or shut, as well as air through-holes 13 for adjusting
the flow
resistance.
If the inhalable powders according to the invention are to be packed into
capsules
(inhalettes) for the preferred use described above, the quantities packed into
each
ao capsule should be 1 to 30mg.
The powders according to the invention may also be administered as propellant-
containing inhalable aerosols. For this, the powders according to the
invention are
reconstituted in an aqueous solution. Suitable solutions are known in the art.
For
25 example, it is advantageous to reconstitute the powders in physiological
solutions with
a pH of 3-11, preferably 4-9. Reconstitution in an aqueous solution with a pH
of 5.5-
7.8 is particularly advantageous. The solution for reconstituting the powders
according
to the invention may also contain further excipients in the form of
stabilisers,
emulsifiers, surfactants or water-soluble organic solvents. Corresponding
substances
3o are known to the skilled man and described for example in Bauer, Lehrbuch
der
Pharmazeutischen Technologie, Wissenschaftl. Verlagsgesellschaft mbH,
Stuttgart,
178-184; Adler, 1998, Journal of Pharmaceutical Sciences, 88(2), 199-208.
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Corresponding inhalable aerosols which are prepared by reconstituting the
powders
according to the invention are also a subject of the present invention.
The propellant gases which may be used to prepare the inhalation aerosols
according
to the invention are also known from the prior art. Suitable propellant gases
are
selected from among hydrocarbons such as n-propane, n-butane or isobutane and
halohydrocarbons such as preferably chlorinated and fluorinated derivatives of
methane, ethane, propane, butane, cyclopropane or cyclobutane. The propellant
gases mentioned above may be used on their own or in mixtures thereof.
Particularly
preferred propellant gases are halogenated alkane derivatives selected from
TG11,
TG12, TG134a (1,1,1,2-tetrafluoroethane), TG227 (1,1,1,2,3,3,3-
heptafluoropropane)
and mixtures thereof, the propellant gases TG134a, TG227 and mixtures thereof
being
preferred.
The inhalation aerosols containing propellant gas according to the invention
may
contain up to 5 % (w/w) of active substance. Aerosols according to the
invention
contain, for example, 0.002 to 5 wt.-%, 0.01 to 3 wt.-%, 0.015 to 2 wt.-%, 0.1
to
2 wt.-%, 0.5 to 2 wt.-% or 0.5 to 1 wt.-% of the pharmaceutical active
substance.
Inhalable aerosols with an active substance concentration in this range may be
Zo prepared by controlled reconstitution of the powders according to the
invention in a
corresponding amount of solvent.
The propellant-driven inhalation aerosols according to the invention mentioned
above
may be administered using inhalers known in the art (MDIs = metered dose
inhalers).
ZS Reference may be made here to the Ventolin~ (Ventolin Pharmacy) or the
inhalers
described in US 5,32,094 or US 5,672,581. Accordingly, in another aspect, the
present
invention relates to pharmaceutical compositions in the form of propellant-
driven
aerosols as hereinbefore described combined with one or more inhalers suitable
for
administering these aerosols. In addition, the present invention relates to
inhalers
3o which are characterised in that they contain the propellant gas-containing
aerosols
described above according to the invention.
The present invention also relates to cartridges which are fitted with a
suitable valve
and can be used in a suitable inhaler and which contain one of the above-
mentioned
propellant gas-containing inhalation aerosols according to the invention.
Suitable
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cartridges and methods of filling these cartridges with the inhalable aerosols
containing
propellant gas according to the invention are known from the prior art.
The powders according to the invention may also be reconstituted in propellant-
free
inhalable solutions or suspensions. Corresponding propellant-free inhalable
solutions
s contain for example aqueous or alcoholic, preferably ethanolic solvents,
optionally
ethanolic solvents mixed with aqueous solvents. In the case of
aqueous/ethanolic
solvent mixtures the relative proportion of ethanol compared with water is not
limited
but the maximum is preferably up to 70 percent by volume, more particularly up
to 60
percent by volume of ethanol. The remainder of the volume is made up of water.
Co-
solvents and/or other excipients as described above may be added to the
propellant-
free inhalable solutions according to the invention. Preferred co-solvents are
those
which contain hydroxyl groups or other polar groups, e.g. alcohols -
particularly
isopropyl alcohol, glycols - particularly propyleneglycol, polyethyleneglycol,
polypropyleneglycol, glycolether, glycerol, polyoxyethylene alcohols and
~s polyoxyethylene fatty acid esters. The terms excipients and additives in
this context
denote any pharmacologically acceptable substance which is not an active
substance
but which can be formulated with the active substance or substances in the
pharmacologically suitable solvent in order to improve the qualitative
properties of the
active substance formulation. Preferably, these substances have no
pharmacological
Zo effect or, in connection with the desired therapy, no appreciable or at
least no
undesirable pharmacological effect. The excipients and additives include, in
addition
to those described above, for example, surfactants such as soya lecithin,
oleic acid,
sorbitan esters, such as polysorbates, polyvinylpyrrolidone, other
stabilisers,
complexing agents, antioxidants and/or preservatives which guarantee or
prolong the
25 shelf life of the finished pharmaceutical formulation, flavourings,
vitamins and/or other
additives known in the art. The additives also include pharmacologically
acceptable
salts such as sodium chloride as isotonic agents. The preferred excipients
include
antioxidants such as ascorbic acid, for example, provided that it has not
already been
used to adjust the pH, vitamin A, vitamin E, tocopherols and similar vitamins
and
3o provitamins occurring in the human body. Preservatives may be used to
protect the
formulation from contamination with pathogens. Suitable preservatives are
those
which are known in the art, particularly cetyl pyridinium chloride,
benzalkonium chloride
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or benzoic acid or benzoates such as sodium benzoate in the concentration
known
from the prior art. The preservatives mentioned above are preferably present
in
concentrations of up to 50 mg/100 ml, more preferably between 5 and 20 mg/100
ml.
Accordingly, the present invention also includes propellant-free inhalable
aerosols
which are prepared by reconstituting the powders according to the invention.
The propellant-free inhalable solutions according to the invention are
administered in
particular using inhalers of the kind which are capable of nebulising a small
amount of
a liquid formulation in the therapeutic dose within a few seconds to produce
an aerosol
suitable for therapeutic inhalation. Within the scope of the present
invention, preferred
inhalers are those in which a quantity of less than 100~,L, preferably less
than 50~L,
more preferably between 10 and 30~.L of active substance solution can be
nebulised in
preferably one spray action to form an aerosol with an average particle size
of less
than 20wm, preferably less than 10~.m, such that the inhalable part of the
aerosol
~s corresponds to the therapeutically effective quantity.
An apparatus of this kind for propellant-free delivery of a metered quantity
of a liquid
pharmaceutical composition for inhalation is described for example in
International
Patent Application WO 91/14468 and also in WO 97/12687 (cf. in particular
Figures 6a
and 6b). Reference is specifically made within the scope of the present
invention to the
Zo corresponding Figures 6a and 6b of WO 97/12687 including the associated
parts of the
description. The nebulisers (devices) described therein are also known by the
name
RespimatO (Boehringer Ingelheim Pharma). Because of its cylindrical shape and
handy size of less than 9 to 15 cm long and 2 to 4 cm wide, this device can be
carried
at all times by the patient. The nebuliser sprays a defined volume of the
Zs pharmaceutical formulation using high pressures through small nozzles so as
to
produce inhalable aerosols.
The preferred atomiser essentially consists of an upper housing part, a pump
housing,
a nozzle, a locking mechanism, a spring housing, a spring and a storage
container,
characterised by
30 - a pump housing which is secured in the upper housing part and which
comprises at one end a nozzle body with the nozzle or nozzle arrangement,
- a hollow plunger with valve body,
- a power takeoff flange in which the hollow plunger is secured and which is
located in the upper housing part,
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- a locking mechanism situated in the upper housing part,
- a spring housing with the spring contained therein, which is rotatably
mounted
on the upper housing part by means of a rotary bearing,
- a lower housing part which is fitted onto the spring housing in the axial
direction.
The hollow plunger with valve body corresponds to a device disclosed in
WO 97/12687. It projects partially into the cylinder of the pump housing and
is axially
movable within the cylinder. Reference is made in particular to Figures 1 to
4,
especially Figure 3, and the relevant parts of the description. The hollow
plunger with
valve body exerts a pressure of 5 to 60 MPa (about 50 to 600 bar), preferably
10 to
60 MPa (about 100 to 600 bar) on the fluid, the measured amount of active
substance
solution, at its high pressure end at the moment when the spring is actuated.
Volumes
of 10 to 50 microlitres are preferred, while volumes of 10 to 20 microlitres
are
particularly preferred and a volume of 15 microlitres per spray is most
particularly
~s preferred.
The valve body is preferably mounted at the end of the hollow plunger facing
the valve
body.
2o The nozzle in the nozzle body is preferably microstructured, i.e. produced
by
microtechnology. Microstructured nozzle bodies are disclosed for example in
WO-94/07607; reference is hereby made to the contents of this specification,
particularly Figure 1 disclosed therein and the associated description. The
nozzle body
consists for example of two sheets of glass and/or silicon firmly joined
together, at
Zs least one of which has one or more microstructured channels which connect
the nozzle
inlet end to the nozzle outlet end. At the nozzle outlet end there is at least
one round
or non-round opening 2 to 10 microns deep and 5 to 15 microns wide, the depth
preferably being 4.5 to 6.5 microns while the length is preferably 7 to 9
microns. In the
case of a plurality of nozzle openings, preferably two, the directions of
spraying of the
3o nozzles in the nozzle body may extend parallel to one another or may be
inclined
relative to one another in the direction of the nozzle opening. In a nozzle
body with at
least two nozzle openings at the outlet end the directions of spraying may be
inclined
at an angle of 20 to 160° to one another, preferably 60 to 150°,
most preferably 80 to
100°. The nozzle openings are preferably arranged at a spacing of 10 to
200 microns,
35 more preferably at a spacing of 10 to 100 microns, most preferably 30 to 70
microns.
Spacings of 50 microns are most preferred.
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The directions of spraying will therefore meet in the vicinity of the nozzle
openings.
The liquid pharmaceutical preparation strikes the nozzle body with an entry
pressure of
up to 600 bar, preferably 200 to 300 bar, and is atomised into an inhalable
aerosol
s through the nozzle openings. The preferred particle or droplet sizes of the
aerosol are
up to 20 microns, preferably 3 to 10 microns.
The locking mechanism contains a spring, preferably a cylindrical helical
compression
spring, as a store for the mechanical energy. The spring acts on the power
takeoff
flange as an actuating member the movement of which is determined by the
position of
a locking member. The travel of the power takeoff flange is precisely limited
by an
upper and lower stop. The spring is preferably biased, via a power step-up
gear, e.g. a
helical thrust gear, by an external torque which is produced when the upper
housing
part is rotated counter to the spring housing in the lower housing part. In
this case, the
upper housing part and the power takeoff flange have a single or multiple V-
shaped
gear.
The locking member with engaging locking surfaces is arranged in a ring around
the
power takeoff flange. It consists, for example, of a ring of plastic or metal
which is
inherently radially elastically deformable. The ring is arranged in a plane at
right
angles to the atomiser axis. After the biasing of the spring, the locking
surfaces of the
ao locking member move into the path of the power takeoff flange and prevent
the spring
from relaxing. The locking member is actuated by means of a button. The
actuating
button is connected or coupled to the locking member. In order to actuate the
locking
mechanism, the actuating button is moved parallel to the annular plane,
preferably into
the atomiser; this causes the deformable ring to deform in the annular plane.
Details
25 of the construction of the locking mechanism are given in WO 97/20590.
The lower housing part is pushed axially over the spring housing and covers
the
mounting, the drive of the spindle and the storage container for the fluid.
When the atomiser is actuated the upper housing part is rotated relative to
the lower
housing part, the lower housing part taking the spring housing with it. The
spring is
3o thereby compressed and biased by means of the helical thrust gear and the
locking
mechanism engages automatically. The angle of rotation is preferably a whole-
number fraction of 360 degrees, e.g. 180 degrees. At the same time as the
spring is
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biased, the power takeoff part in the upper housing part is moved along by a
given
distance, the hollow plunger is withdrawn inside the cylinder in the pump
housing, as a
result of which some of the fluid is sucked out of the storage container and
into the
high pressure chamber in front of the nozzle.
s If desired, a number of exchangeable storage containers which contain the
fluid to be
atomised may be pushed into the atomiser one after another and used in
succession.
The storage container contains the aqueous aerosol preparation according to
the
invention.
The atomising process is initiated by gently pressing the actuating button. As
a result,
the locking mechanism opens up the path for the power takeoff member. The
biased
spring pushes the plunger into the cylinder of the pump housing. The fluid
leaves the
nozzle of the atomiser in atomised form.
Further details of construction are disclosed in PCT Applications WO 97/12683
and
WO 97/20590, to the contents of which reference is hereby made.
The components of the atomiser (nebuliser) are made of a material which is
suitable
for its purpose. The housing of the atomiser and, if its operation permits,
other parts
as well are preferably made of plastics, e.g. by injection moulding. For
medicinal
purposes, physiologically safe materials are used.
2o Figures 6 a/b of WO 97/12687, including the associated description to which
reference
is hereby made once more, show a corresponding nebuliser (Respimat~). This is
particularly suitable for administering the propellant-free inhalable aerosols
according
to the invention.
25 Figure 6 a of WO 97/12687 shows a longitudinal section through the atomiser
with the
spring under tension, Figure 6 b of WO 97/12687 shows a longitudinal section
through
the atomiser with the spring released. The upper housing part (51 ) contains
the pump
housing (52), on the end of which is mounted the holder (53) for the atomiser
nozzle.
In the holder is the nozzle body (54) and a filter (55). The hollow piston
(57) fixed in the
3o power take-off flange (56) of the locking clamping mechanism projects
partly into the
cylinder of the pump housing. At its end the hollow piston carries the valve
body (58).
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The hollow piston is sealed off by the gasket (59). Inside the upper housing
part is the
stop (60) on which the power take-off flange rests when the spring is relaxed.
Located
on the power take-off flange is the stop (61 ) on which the power take-off
flange rests
when the spring is under tension. After the tensioning of the spring, the
locking
s member (62) slides between the stop (61 ) and a support (63) in the upper
housing
part. The actuating button (64) is connected to the locking member. The upper
housing
part ends in the mouthpiece (65) and is closed off by the removable protective
cap
(66). The spring housing (67) with compression spring (68) is rotatably
mounted on
the upper housing part by means of the snap-fit lugs (69) and rotary bearings.
The
lower housing part (70) is pushed over the spring housing. Inside the spring
housing is
the replaceable storage container (71 ) for the fluid (72) which is to be
atomised. The
storage container is closed off by the stopper (73), through which the hollow
piston
projects into the storage container and dips its end into the fluid (supply of
active
substance solution). The spindle (74) for the mechanical counter is mounted on
the
outside of the spring housing. The drive pinion (75) is located at the end of
the spindle
facing the upper housing part. On the spindle is the slider (76).
If the formulation according to the invention is nebulised using the method
described
above (Respimat~), the mass expelled, in at least 97%, preferably at least 98%
of all
ao the actuations of the inhaler (puffs), should correspond to a defined
quantity with a
range of tolerance of not more than 25%, preferably 20% of this quantity.
Preferably,
between 5 and 30 mg, more preferably between 5 and 20 mg of formulation are
delivered as a defined mass per puff.
ZS However, the formulation according to the invention can also be nebulised
using
inhalers other than those described above, for example jet-stream inhalers or
other
stationary nebulisers.
Accordingly, in another aspect, the present invention relates to
pharmaceutical
3o compositions in the form of propellant-free inhalable solutions or
suspensions as
hereinbefore described in conjunction with a device suitable for administering
these
formulations, preferably in conjunction with the Respimat~. Preferably the
present
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invention is directed to propellant-free Inhalable solutions or suspensions,
containing
one of the powders according to the invention, in conjunction with the device
known as
a Respimat~. Moreover the present invention relates to the above-mentioned
devices
for inhalation, preferably the Respimat~, characterised in that they contain
the
propellant-free inhalable solutions or suspensions according to the invention
as
described above.
According to the invention, inhalable solutions containing one of the powders
according to the invention as described herein in a single preparation are
preferred.
i0
The propellant-free inhalable solutions or suspensions according to the
invention may
take the form of concentrates or sterile inhalable solutions or suspensions
ready for
use, as well as the above-mentioned solutions and suspensions designed for use
in
the RespimatO. Formulations ready for use may be produced from the
concentrates,
~s for example, by the addition of isotonic saline solutions. Sterile
formulations ready for
use may be administered using energy-operated fixed or portable nebulisers
which
produce inhalable aerosols by means of ultrasound or compressed air by the
Venturi
principle or other principles.
2o Accordingly, in another aspect, the present invention relates to
pharmaceutical
compositions in the form of propellant-free inhalable solutions or suspensions
as
described hereinbefore which take the form of concentrates or sterile
formulations
ready for use, combined with a device suitable for administering these
solutions,
characterised in that the device is an energy-operated free-standing or
portable
25 nebuliser which produces inhalable aerosols by means of ultrasound or
compressed
air by the Venturi principle or other methods.
Other suitable nebulisers for inhaling reconstituted aerosols are the AERxT""
(Aradigm),
Ultravent~ (Mallinkrodt) and Aconll~ (Maquest Medical Products).
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Examples:
Equipment and Methods
Materials:
A humanised monoclonal antibody with a molecular weight of about 148 kDa was
used
as IgG1. The antibody is derived from a murine antibody in which the
complementarity-
determining regions of the murine antibody have been transferred to a human
immunoglobulin structure. A chimeric antibody has been produced with 95% human
content and 5% murine content. The antibody is expressed by murine myeloma
cell
lines. The cells are removed by Tangential Flow Microfiltration and the cell-
free
solution is purified by various chromatographic methods. Other steps include
nuclease
treatment, treatment at a low pH and nanofiltration. The bulk solution
containing the
antibodies contains 25 mM histidine and 1.6 mM glycine as buffer and has been
concentrated to approx. 100 mg/ml by diafiltration, for the preparation of the
solution
for spray drying. The bulk for the preparation of the spray solution contained
0.8%
aggregates. The finished drug can be stored at 2-8°C for at least 2
years. Low
molecular dextran1 or dextran~ooo with a mean molecular weight of about 1000
Da
obtained from Amersham Biosciences AB, Uppsalla, Sweden. Trehalose is obtained
Zo from Georg Breuer GmbH, Germany. L-isoleucine was obtained from Sigma-
Aldrich
Chemie GmbH, Germany. Triisoleucine was obtained from Iris Biotech GmbH,
Germany. Chicken albumin lysozyme (lysozyme), 135500 U/mg, was obtained from
SERVA Electrophoresis GmbH, Germany. Synthetic salmon calcitonin (calcitonin)
was
obtained from Biotrend Chemikalien GmbH, Germany.
Sara -y drying with Buchi 8-290
The spray-drying was done using a Buchi Mini Spray Dryer B-290 made by Messrs
Buchi Labortechnik (AG, CH). The spray-drying of the formulations was carried
out
chiefly as described in the "Spray Drying Handbook", 5th Edition., K. Masters,
John
3o Wiley and Sons , Inc., NY, NY (1991 ):
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The spray drier is made up of a heating system, a filter, an aspirator, a
drying tower, a
cyclone, temperature sensors for measuring the inlet and outlet temperature
and a
collecting vessel. The solution to be sprayed is pumped into the two-substance
nozzle
by means of a peristaltic pump. There, the solution is atomised into small
drops using
s compressed air. The drying in the spray tower is done using heated air which
is
aspirated through the spray tower by the direct current method by means of the
aspirator. The product is collected in the collecting vessel after passing
through the
cyclone.
Two different cyclones were used:
Cyclone I: Buchi Cyclone (product number 4189)
~ Cyclone II: Buchi High-performance Cyclone (product number 46369)
The solid content of the spray solutions was 10% (w/v) in 50 ml, 3.33% in 300
ml and
3.33% in 600 ml. The inlet temperature was about 170 to 185°C, the
liquid feed rate
approx. 3 ml/min, the aspirator flow rate 35 m3/h and the atomiser flow rate
0.67 m3/h.
This produced an outlet temperature of about 80-95°C.
X-Ray diffractometry (wide-angle X ray diffractometry (WARS)):
In order to determine the crystallinity of the dried samples the samples were
investigated with a Seifert X-ray diffractometer XRD 3000 TT (Messrs Seifert,
zo Ahrensburg, DE) in a chamber at a controlled temperature of 22 °C.
The X-ray tube Cu
anode, Cu-Ka radiation with ~. = 0.15418 mm (Ni primary filter), was operated
at an
anode voltage of 40 kV and a current strength of 30 mA. After the sample dish
had
been placed in the apparatus the sample was measured in the range from 5 to
40° at a
scan rate of 2 0 = 0.05° with 2 sec measuring time at each angle.
ZS
The powder diftractograms were taken with the ScanX- Rayflex application,
Version
3.07 device XRD 3000 (Scan), or the Rayflex Version 2.1, 1996 (Analysis) on
the SC
1000 V detector.
Size exclusion chromatocr~ aphy (SEC-HPLC):
a) Soluble IgG1 protein aggregates
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SEC-HPLC was used to quantify IgG1-protein aggregates in the reconstituted
powders. The SEC-HPLC was carried out with a HP1090 made by Messrs Agilent.
The column used for separation was a TSK3000SWXL column (300 x 7.8mm) made
by Messrs Tosoh Biosep (Tosoh Bioscience, Stuttgart, DE). The eluant used was
a
s buffer consisting of 0.1 M di-sodium hydrogen phosphate-dehydrate, 0.1 M
sodium
sulphate which was dewatered and adjusted to pH 6.8 with ortho-phosphoric acid
85%.
The amount of sample put in was 25N1 at a protein concentration of 2-10 mg/ml.
The
protein was detected using a diode array detector made by Messrs Agilent at
280nm.
The chromatographs were evaluated using the Chemstation software made by
Agilent.
b) Soluble calcitonin protein aggregates
In order to quantify calcitonin-protein aggregates in the reconstituted
powders SEC-
HPLC was carried out. The SEC-HPLC was carried out using an HP1100 made by
Messrs Agilent. The column used for separation was a TSK3000SWXL column (300 x
is 7.8mm) made by Messrs Tosoh Biosep (Tosoh Bioscience, Stuttgart, DE). The
eluant
used was a buffer consisting of 0.25 sodium sulphate with a pH of about 6
(Windisch et
al. 1997). The amount of sample put in was 20p1 at a protein concentration of
0.5-2
mg/ml. The protein was detected using a UV detector made by Messrs Agilent at
210nm. The chromatographs were evaluated using the HP-Chemstation software
Zo made by Messrs Agilent.
c) Lysozyme residual monomer content
In order to quantify the lysozyme residual monomer content in the
reconstituted
lysozyme formulations a modified SEC-HPLC was carried out (van de Weert,
2000).
25 The SEC-HPLC was carried out using an HP1100 made by Messrs Agilent. The
column used for separation was a TSK2000SWXL column (300 x 7.8mm) made by
Messrs Tosoh Biosep (Tosoh Bioscience, Stuttgart, DE). The eluant used was a
buffer
consisting of 0.05 M disodium hydrogen phosphate-dehydrate and 0.2 M sodium
chloride, adjusted to pH 7.0 with ortho-phosphoric acid 85%. The amount of
sample
3o put in was 25N1 at a protein concentration of 2-10 mg/ml. The protein was
detected
using a UV detector made by Messrs Agilent at 280nm. The chromatographs were
evaluated using the Agilent Chemstation software made by Messrs Agilent.
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In order to evaluate the formulations, the soluble monomer remaining was
quantified
by the following method. First, a calibrating line was drawn using lysozyme
standard
solutions with concentrations of 2.5 mg/ml, 5.0 mg/ml and 10 mg/ml. The AUC of
the
monomer peaks was studied in relation to the corresponding lysozyme
concentrations
in the standard solution under investigation.
The residual monomer content of the various lysozyme formulations under
investigation was calculated using the calibrating line. The higher the
residual
monomer content of a formulation, the better the protein stability.
Determining the particle size (MMD):
The Mass Median Diameter or the mean particle size of the particles was
determined
using the Sympatech Helos made by Messrs Sympatech GmbH (Clausthal-Zellerfeld,
DE). The measuring principle is based on laser diffraction, using a helium
neon laser.
1-3 mg of powder are dispersed with an air pressure of 2 bar, and passed
through a
parallel laser beam in front of the Fourier lens (50 mm). The particle size
distribution is
evaluated using a Fraunhofer model. Two measurements were carried out on each
powder.
Mass Median Diameter (MMAD) and fine particle fraction (FPF)
Zo For the measurements, 12-18mg of powder were transferred into hard gelatine
capsules (size 3) and placed in the HandiHaler (powder inhaler made by Messrs
Boehringer Ingelheim). Using an adapter the HandiHaler was coupled to the USP
EP/throat of the impactor inlet of the measuring device and the powder was
delivered
at a rate of 39.0 I/min with an intake time of 6.15 sec. The air throughput
was
25 controlled by means of an external controlling wall. At least three
capsules were
measured for each powder.
The APS 3321 of Messrs TSI Inc., MN, USA is used in conjunction with the
Impactorinlet 3306 to simultaneously measure the aerodynamic particle size
(MMAD)
by measuring the time of flight and the fine particle fraction (FPF) using a
one-step
3o impactor (effective cut off diameter at 39L/min: S.Opm). After being
expelled through
the EP/USP Throat or Sample Induction Port the powder reaches a thin capillary
where 0.2% of the powder can be removed under isokinetic conditions in order
to
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measure the time of flight. The time of flight is measured after passing the
capillary
through 2 laser beams which detect the time of flight for a defined distance
analogously to a light barrier. As a result, a numerical distribution is
obtained which is
then converted into a mass distribution and thus into the Mass Median
Aerodynamic
Diameter (MMAD).
The remaining 99.8% of the powder population which has travelled past the
capillary is
then separated off using the one-step impactor. The fraction larger than 5.0
Nm is
deposited on a baffle plate in the impactor as a result of mass inertia. The
fine particle
fraction (FPF) follows the air current and is finally deposited on a deep
filter. The fine
particle fraction is determined by gravimetry. The fine particle fraction is
calculated
from the amount of powder deposited on the filter relative to the total amount
of
powder used, i.e. the powder weighed out for each capsule.
Residual water content:
~s The residual water content in the dried products was determined by
coulometric
titration (Metrohm 737 KF Coulometer with 703 titration stand, Germany). For
the
measurement, powder was dissolved or dispersed in methanol (Hydranal -
Methanol
dry, VWR / Merck Eurolab). The measuring solution (Hydranal -Coulomat
solution,
VWR / Merck Eurolab) of the Metrohm Coulometer was adjusted at the start of
the
Zo measurements, i.e. the measuring solution was calibrated to a zero content
of water.
The sample was injected into the titration cell and measured.
Detennining stability:
The powders were investigated for different stabilities after spray-drying. In
the case of
as IgG1 and calcitonin the percentage amount of protein aggregates was used as
the
measurement of stability of the formulations. In the case of lysozyme the
percentage
amount of the residual monomer content was used as the measurement of
stability of
the formulations. The innovative excipients described in the invention were
compared
with the pure protein formulation and optionally an analogous trehalose
formulation as
3o reference. Analysis to detect any aggregates was carried out with a
validated size
exclusion chromatography (SEC-HPLC) with UV detection (DAD). For this the
pretreated powders were first reconstituted in highly purified water (pH 6 to
8).
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Selected formulations were investigated for their stability after one weeks
open storage
at about 40°C and about 75% relative humidity (40°C, 75% rh) in
open glass vials
(forced storage stability).
Selected formulations were stored after spray-drying and vacuum drying under
nitrogen in sealed glass vials at 2-8°C, 25°C and 40°C.
The formulations were
removed after one, three, six and twelve months and tested for their stability
(stability
over 1 year).
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Example 1
Spray-drying a 10 % (w/v) IgG1 formulation
Pure IgG1 in a concentration of about 109 mg/ml, formulated in a glycine
histidine
buffer, pH 6 (see Materials), was diluted with demineralised water (pH about
7.5) to a
content of 100mg/ml and spray-dried in the absence of any other excipients as
described above using the Cyclone I. The volume of the solution was 50 ml. The
~o content of aggregates was investigated as described above. After forced
storage the
solution of the reconstituted powder contained about 18.9% aggregates
Spray-drying a formulation containing 9% (w/v) trehalose 1 % (w/v) IgG1
~s 4.5 g trehalose was dissolved in about 40 ml of demineralised water (pH
about 7.5).
Next, about 4.6 ml of pure IgG1 with a concentration of about 109 mg/ml,
formulated in
a glycine histidine buffer pH 6 (see Materials), was added and diluted to a
volume of
50 ml with demineralised water (pH about 7.5). The solution thus obtained
contains
about 9% (w/v) excipient or matrix and 1 % (w/v) protein and was spray-dried
as
Zo described above using the Cyclone I. The content of aggregates was
investigated as
described above. After forced storage the solution of the reconstituted powder
contained about 12.6% aggregates.
Spray-drying a formulation containing 9% (w/v) dextran~ooo 1% (w/v) IgG1
zs
4.5 g dextran~ooo was dissolved in about 40 ml of demineralised water (pH
about 7.5).
Next, about 4.6 ml of pure IgG1 with a concentration of about 109 mg/ml,
formulated in
a glycine histidine buffer pH 6 (see Materials), was added and diluted to a
volume of
50 ml with demineralised water (pH about 7.5). The solution thus obtained
contains
so about 9% (w/v) excipient or matrix and 1 % (w/v) protein and was spray-
dried as
described above using the Cyclone I. The content of aggregates was
investigated as
described above. The following aggregate contents were obtained for the
storage
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stability. After forced storage the solution of the reconstituted powder
contained about
5.1 % aggregates.
Spray-drying a formulation containing 2.00% (w/v) dextran~ooo 1.33% (w/v) IgG1
S
3.0 g dextran~ooo was dissolved in about 120 ml of demineralised water (pH
about 7.5).
Next, about 19.5 ml of pure IgG1 with a concentration of about 102.8 mg/ml,
formulated in a glycine histidine buffer pH 6 (see Materials), was added and
diluted to
a volume of 150 ml with demineralised water (pH about 7.5). The solution thus
to obtained contains about 2.0% (w/v) excipient or matrix and 1.33% (w/v)
protein and
was spray-dried as described above using the Cyclone II. The content of
aggregates
was investigated as described above. The following aggregate contents were
obtained
for the storage stability. After forced storage the solution of the
reconstituted powder
contained about 11.1 % aggregates.
Example 2
Spray-drying a formulation containing 8% (w/v) trehalose 1 % (w/v) L-
isoleucine
1% (w/v) IgG1
Zo 4 g trehalose and 0.5 g L-isoleucine were dissolved in an ultrasound bath
in about 40
ml of demineralised water (pH about 7.5). Next, about 4.6 ml of pure IgG1 with
a
concentration of about 109 mg/ml, formulated in a glycine histidine buffer pH
6 (see
Materials), was added and diluted to a volume of 50 ml with demineralised
water (pH
about 7.5). The solution thus obtained contains about 9% (w/v) excipient or
matrix and
1 % (w/v) protein and was spray-dried as described above using the Cyclone I.
The
content of aggregates was investigated as described above. After forced
storage the
solution of the reconstituted powder contained about 22.2% aggregates.
Spray-drying a formulation containing 8% (w/v) dextran~ooo 1 % (w/v) L-
isoleucine
1 % (w/v) IgG1
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4 g dextran~ooo and 0.5 g L-isoleucine were dissolved in an ultrasound bath in
about 40
ml of demineralised water (pH about 7.5). Next, about 4.6 ml of pure IgG1 with
a
concentration of about 109 mg/ml, formulated in a glycine histidine buffer pH
6 (see
Materials), was added and diluted to a volume of 50 ml with demineralised
water (pH
about 7.5). The solution thus obtained contains about 9% (w/v) excipient or
matrix and
1 % (w/v) protein and was spray-dried as described above using the Cyclone I.
The
content of aggregates was investigated as described above. After forced
storage the
solution of the reconstituted powder contained only about 10.1 % aggregates.
In a
second larger mixture an analogous formulation (20g solid and 200 ml volume)
was
spray-dried under the same conditions. The content of aggregates was
investigated as
described above. After 12 months storage at 40°C (1 years stability)
the solution of the
reconstituted powder contained about 2.5% aggregates. After 3 months storage
at
25°C (3 months stability) the solution of the reconstituted powder
contained about
2.2% aggregates. After 3 months storage at 2-8°C (3 months stability)
the solution of
the reconstituted powder contained about 2.0% aggregates. The powder obtained
was
measured for MMD, MMAD and FPF. The MMD of the powder was determined as
described above. The MMD of the powder was 5.11 Nm. The MMAD and FPF of the
powder were determined as described above. The MMAD was 6.8 Nm and the Fine
Particle Fraction was 34.8 % relative to the weight of powder placed in the
capsule.
zo
Spray-drying a formulation containing 2.833% (w/v) dextran~ooo, 0.166% (w/v)
L-isoleucine, 0.33% (w/v) IgG1
8.5 g dextran~ooo and 0.5 g L-isoleucine were dissolved in an ultrasound bath
in about
ZS 280 ml of demineralised water (pH about 7.5). Next, about 9.7 ml of pure
IgG1 with a
concentration of about 102.8 mg/ml, formulated in a glycine histidine buffer
pH 6 (see
Materials), was added and diluted to a volume of 300 ml with demineralised
water (pH
about 7.5). The solution thus obtained contains about 3% (w/v) excipient or
matrix and
0.33% (w/v) protein and was spray-dried as described above using the Cyclone
II.
The content of aggregates was investigated as described above. After forced
storage
the solution of the reconstituted powder contained about 6.3% aggregates. The
MMD
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of the powder was determined as described above. The MMD of the powder was
2.98
Nm. The MMAD and FPF of the powder was determined as described above. The
MMAD was 5.3 Nm and the Fine Particle Fraction was 35.2 % relative to the
weight of
powder placed in the capsule.
Spray-drying a formulation containing 2.66% (w/v) dextran~ooo , 0.33% (w/v)
L-isoleucine, 0.33% (w/v) IgG1
8.0 g dextran~ooo and 1 g L-isoleucine were dissolved in an ultrasound bath in
about
280 ml of demineralised water (pH about 7.5). Next, about 9.7 ml of pure IgG1
with a
concentration of about 102.8 mg/ml, formulated in a glycine histidine buffer
pH 6 (see
Materials), was added and diluted to a volume of 300 ml with demineralised
water (pH
about 7.5). The solution thus obtained contains about 3% (w/v) excipient or
matrix and
0.33% (w/v) protein and was spray-dried as described above using the Cyclone
II.
The content of aggregates was investigated as described above. After forced
storage
the solution of the reconstituted powder contained about 7.1 % aggregates.
After 12
months storage at 40°C (1 years stability) the solution of the
reconstituted powder
contained about 3.3% aggregates. After 12 months storage at 25°C (1
years stability)
2o the solution of the reconstituted powder contained about 2.3% aggregates.
After 12
months storage at 2-8°C (1 years stability) the solution of the
reconstituted powder
contained about 1.9% aggregates. The MMD of the powder was determined as
described above. The MMD of the powder was 2.75 Nm. The MMAD and FPF of the
powder was determined as described above. The MMAD was 5.3 Nm and the Fine
as Particle Fraction was 39.2 % relative to the weight of powder placed in the
capsule.
Spray-drying a formulation containing 2.33% (w/v) dextran~ooo, 0.66% (w/v)
L-isoleucine, 0.33% (w/v) IgG1
7.0 g dextran~ooo and 2 g L-isoleucine were dissolved in an ultrasound bath in
about
280 ml of demineralised water (pH about 7.5). Next, about 9.7 ml of pure IgG1
with a
concentration of about 102.8 mg/ml, formulated in a glycine histidine buffer
pH 6 (see
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Materials), was added and diluted to a volume of 300 ml with demineralised
water (pH
about 7.5). The solution thus obtained contains about 3% (w/v) excipient or
matrix and
0.33% (w/v) protein and was spray-dried as described above using the Cyclone
II.
The content of aggregates was investigated as described above. After forced
storage
the solution of the reconstituted powder contained about 10.6% aggregates. The
MMD
of the powder was determined as described above. The MMD of the powder was
2.71
Nm. The MMAD and FPF of the powder was determined as described above. The
MMAD was 5.1 Nm and the Fine Particle Fraction was 36.4 % relative to the
weight of
powder placed in the capsule.
Example 3
~s Spray-drying a formulation containing 2.66% (w/v) dextran~ooo , 0.33% (w/v)
triisoleucine and 0.33% (w/v) IgG1
16.0 g dextran~ooo and 2 g triisoleucine were dissolved in an ultrasound bath
in about
560 ml of demineralised water (pH about 7.5). Next, about 20.7 ml of pure IgG1
with a
ao concentration of about 96.55 mg/ml, formulated in a glycine histidine
buffer pH 6 (see
Materials), was added and diluted to a volume of 600 ml with demineralised
water (pH
about 7.5). The solution thus obtained contains about 3% (w/v) excipient or
matrix and
0.33% (w/v) protein and was spray-dried as described above using the Cyclone
I.
The content of aggregates was investigated as described above. After 3 months
ZS storage at 40°C (3 months stability) the solution of the
reconstituted powder contained
about 3.2% aggregates. After 3 months storage at 25°C (3 months
stability) the
solution of the reconstituted powder contained about 1.2% aggregates. After 3
months
storage at 2-8°C (3 months stability) the solution of the reconstituted
powder contained
about 0.9% aggregates. After 12 months storage at 40°C (1 years
stability) the solution
30 of the reconstituted powder contained about 7.3% aggregates. After 12
months
storage at 25°C (1 years stability) the solution of the reconstituted
powder contained
about 2.0% aggregates. After 12 months storage at 2-8°C (1 years
stability) the
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solution of the reconstituted powder contained about 1.3% aggregates. The MMAD
and FPF of the powder was determined as described above. The MMAD was 4.6 Nm
and the Fine Particle Fraction was 55.7% relative to the weight of powder
placed in the
capsule.
Spray-drying a formulation containing 2.66% (w/v) dextran» , 0.33% (w/v)
triisoleucine and 0.33% (w/v) IgG1
8.0 g dextran~ooo and 1 g triisoleucine were dissolved in an ultrasound bath
in about
280 ml of demineralised water (pH about 7.5). Next, about 10.36 ml of pure
IgG1 with
a concentration of about 96.55 mg/ml, formulated in a glycine histidine buffer
pH 6 (see
Materials), was added and diluted to a volume of 300 ml with demineralised
water (pH
about 7.5). The solution thus obtained contains about 3% (w/v) excipient or
matrix and
0.33% (w/v) protein and was spray-dried as described above using the Cyclone
II.
The stability was not measured as the formulation is exactly the same as that
described immediately above. The use of a different cyclone has no effect on
the
protein stability. The MMD of the powder was determined as described above.
The
MMD of the powder was 2.96 Nm. The MMAD and FPF of the powder was determined
as described above. The MMAD was 3.9 pm and the Fine Particle Fraction was
58.4
2o relative to the weight of powder placed in the capsule.
Example 4: Preparation of other powders according to the invention
Zs Spray-drying a formulation containing 3.33% (w/v) lysozyme
5 g iysozyme is dissolved in about 140 ml of demineralised water (pH about
7.5) and
diluted to a volume of 150m1 with demineralised water (pH about 7.5). The
solution
thus obtained is spray-dried as described above using the Cyclone II. The
residual
3o monomer content was investigated as described above. After forced storage
the
solution of the reconstituted powder contained a residual monomer content of
35.3%.
The MMD of the powder was determined as described above. The MMD of the powder
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was 3.23 Nm. The MMAD and FPF of the powder was determined as described above.
The MMAD was 4.0 Nm and the Fine Particle Fraction was 70.4 % relative to the
weight of powder placed in the capsule.
Spray-drying a formulation containing dextranlooo 3.00% (w/v) lysozyme 0.33%
(w/v)
formulation
9.0 g dextran~ooo is dissolved in the ultrasound bath in about 280 ml
demineralised
water (pH about 7.5). Next, 1 g lysozyme is added, and the mixture is diluted
with
demineralised water (pH about 7.5) to a volume of 300m1. The solution thus
obtained is
spray-dried as described above using the Cyclone II.
The residual monomer content was investigated as described above. After forced
storage the solution of the reconstituted powder contained a residual monomer
content
of 49.8%. The MMD of the powder was determined as described above. The MMD of
the powder was 2.82 Nm. The MMAD and FPF of the powder was determined as
described above. The MMAD was 4.2 Nm and the Fine Particle Fraction was 34.7
relative to the weight of powder placed in the capsule.
zo
Spray-drying a formulation containing 2.66% (w/v) dextran»o , 0.33 % (w/v)
isoleucine
and 0.33% (w/v) lysozyme
8.0 g dextran~ooo and 1 g isoleucine are dissolved in the ultrasound bath in
about 280
Zs ml demineralised water (pH about 7.5). Next, 1 g lysozyme is added, and the
mixture is
diluted with demineralised water (pH about 7.5) to a volume of 300 ml. The
solution
thus obtained is spray-dried as described above using the Cyclone II.
The residual monomer content was investigated as described above. After forced
storage the solution of the reconstituted powder contained a residual monomer
content
30 of 50.7%. The MMD of the powder was determined as described above. The MMD
of
the powder was 3.01 Nm. The MMAD and FPF of the powder was determined as
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described above. The MMAD was 4.2 Nm and the Fine Particle Fraction was 36.6
relative to the weight of powder placed in the capsule.
Spray-drying a formulation containing 2.66% (w/v) dextran~ooo, 0.33 % (w/v)
triisoleucine and 0.33% (w/v) lysozyme
8.0 g dextran~ooo and 1 g triisoleucine are dissolved in the ultrasound bath
in about 280
ml demineralised water (pH about 7.5). Next, 1 g lysozyme is added, and the
mixture is
diluted with demineralised water (pH about 7.5) to a volume of 300 ml. The
solution
thus obtained is spray-dried as described above using the Cyclone II.
The residual monomer content was investigated as described above. After forced
storage the solution of the reconstituted powder contained a residual monomer
content
of 43.9%. The MMD of the powder was determined as described above. The MMD of
~s the powder was 2.53 Nm. The MMAD and FPF of the powder was determined as
described above. The MMAD was 3.2 Nm and the Fine Particle Fraction was 58.6
relative to the weight of powder placed in the capsule.
2o Spray-drying a formulation containing 3.33% (w/v) calcitonin
1 g calcitonin is dissolved in about 25 ml demineralised water (pH about 7.5)
and
diluted with demineralised water (pH about 7.5) to a volume of 30 ml. The
solution thus
obtained is spray-dried as described above using the Cyclone II.
25 The content of aggregates was investigated as described above. After forced
storage
the solution of the reconstituted powder contained about 32.6 % aggregates.
The
MMAD and FPF of the powder was determined as described above. The MMAD was
3.9 Nm and the Fine Particle Fraction was 59.0 % relative to the weight of
powder
placed in the capsule.
Spray-drying a formulation containing 3.166% (w/v) dextran~ooo and 0.166%
(w/v)
calcitonin
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4.750 g dextran~ooo is dissolved in about 140 ml demineralised water (pH about
7.5) in
the ultrasound bath. Then 0.250 g calcitonin is added and diluted with
demineralised
water (pH about 7.5) to a volume of 150 ml. The solution thus obtained is
spray-dried
s as described above using the Cyclone II.
The content of aggregates was investigated as described above. After forced
storage
the solution of the reconstituted powder contained about 27.5 % aggregates.
The MMD
of the powder was determined as described above. The MMD of the powder was
3.00
Nm. The MMAD and FPF of the powder was determined as described above. The
MMAD was 4.6 Nm and the Fine Particle Fraction was 42.6 % relative to the
weight of
powder placed in the capsule.
Spray-drying a formulation containing 2.833% (w/v) dextran~ooo , 0.33 % (w/v)
isoleucine and 0.166% (w/v) calcitonin
is
4.250 g dextran~ooo and 0.50 g isoleucine are dissolved in about 140 ml
demineralised
water (pH about 7.5) in the ultrasound bath. Then 0.250 g calcitonin is added
and
diluted with demineralised water (pH about 7.5) to a volume of 150 ml. The
solution
thus obtained is spray-dried as described above using the Cyclone II.
ao The content of aggregates was investigated as described above. After forced
storage
the solution of the reconstituted powder contained about 23.2 % aggregates.
The MMD
of the powder was determined as described above. The MMD of the powder was
2.86
Nm. The MMAD and FPF of the powder was determined as described above. The
MMAD was 4.5 Nm and the Fine Particle Fraction was 57.1 % relative to the
weight of
Zs powder placed in the capsule.
Spray-drying a formulation containing 2.866% (w/v) dextran~ooo , 0.33 % (w/v)
triisoleucine and 0.166% (w/v) calcitonin
4.250 g de~ctran~ooo and 0.50 g triisoleucine are dissolved in about 140 ml
demineralised water (pH about 7.5) in the ultrasound bath. Then 0.250 g
calcitonin is
CA 02548132 2006-06-O1
WO 2005/055976 PCTBP2004/013938
-61 -
added and diluted with demineralised water (pH about 7.5) to a volume of 150
ml. The
solution thus obtained is spray-dried as described above using the Cyclone II.
The MMD of the powder was determined as described above. The MMD of the powder
was 2.60 Nm. The MMAD and FPF of the powder was determined as described above.
The MMAD was 3.7 Nm and the Fine Particle Fraction was 62.5% relative to the
weight
of powder placed in the capsule.
