Note: Descriptions are shown in the official language in which they were submitted.
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PCTIEP991023135
WO 99152506
Description
PARTICULATE EXCIPIENTS FOR PULMONARY ADMINISTRATION
The invention relates to a dry-formulabls particulate excipient which is
prepared from linear water-insoluble polysaccharides, processes for its
preparation and its use.
In modern pharmaceutical technology, formulations are desirable whose
use form can be administered sparingly and bring a specific influence to
bear on the biodistribution, bioavailability or absorption of a medicament.
Above all, particulate systems, so-called microparticles, which generally
have a particle size in the range of less than 100 pm, have proven of high
quality as administration forms or "drug delivery systems" and serve for
making available active compounds, in particular pharmaceuticals, in the
biological organism. In this context, microparticles in a size range from 1 to
100 Nm have especially proven suitable for parenteral use, e.g.
subcutaneous injection" in which a long-term release of the active
compound can take ptac;e for weeks. Signfficantly smaller microparticles in
the range of less than 0.5 Nm are being intensively investigated in order to
overcome the blood-brain barrier.
Therapeutic substances need an administration form which fulfills the
physiological conditions. (e.g. therapeutic proteins and many others which,
for example, are orally administered, can denature in the stomach or
undergo enzymatic digestion, and are therefore usually only administered
as an injection or in a different invasive manner).
In particular, a specific form of noninvasive administration, the pulmonary
administration form, offers, in the context of inhalation therapy, the
possibility of absorbing therapeutic substances via the lungs with
preservation of the therapeutic, diagnostic or prophylactic properties
(Patton, Chemtech 1997, 27 (12), 34-38) and makes possible systemic and
local treatment and thus Leads to a higher patient compliance. For this, it is
~.n necessary to make available particulate systems in a size and quality such
that: they are able to penetrate into bronchi and alveoli, where on account of
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sensitive membranes an effective absorption of active compound into the
blood stream of the organism takes place.
Previous to this, the pulmonary administration form already on the market
and based on propellant-drNen metered aerosols was especially known,
which is employed almost totally for the therapy of asthmatic disorders. A
disadvantage, however, is the use of ozone-damaging propellants.
At present, great efforts are also being undertaken to transfer the
administration of pharmaceuticals via the lungs to other active compounds
with other indication areas. At the focal point of interest at present are
t 0 powder inhalation systems, so-called "dry powder inhalers" based on a
pharmaceutical dry formulation. This is described in WO 96/32149, and for
this the excipient used far the active compound is human serum albumin
including additive; l-anger et al. in Science 276, 1997, 1868 describe the
use of microparticles of ~polylactic acid, which are loaded to 10-20% with
insulin and achieve comparable results to correspondingly subcutaneously
injected microparticles (Abstract Controlled Release Society Conference,
Sweden 1997). The size of the particles is in the range between 8 and
Vim.
20 In WO 97/35562, active compound-loaded microparticles for pulmonary
administration are described which are prepared from aqueous solution
and disadvantageously can only absorb water-soluble active compounds.
A further publication WO 97144013 discloses porous particles of polylactic-
co-glycolic acid, which have a diameter of 5 to 30 Nm.
In WO 96/32116, microparticles of the order of size of 0.5 to 50 Nm are
described, which consist of nucleic acids or viral vectors and can be
obtained from a suspension by means of hydrophilic oligomeric
polysaccharides with drying, in particular spray drying.
WO 96/32152 describes particles consisting of the active compound
a-1-antitrypsin of size 1-50 Vim, diluents also being allowed.
The publication WO 97/36574 describes hollow particles having a smooth
surface; the size is 0.5-7.0 pm.
In the publications WO 97/36574, WO 96/36314, WO 96/00127 and
WO 96/32096, further particulate systems are described, which consist of
partly synthetic polymers or of the active compound as such, of very
different size.
In the prior art, it is problematic that the depot action of the particles is
relatively limited. In addition, these are water-soluble products mostly
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arising from a chemical synthesis which restricts their biocompatibility
(solvent residues, foreign material etc.). Likewise, inhomogeneous particles
which are uneven in size, surface area and composition are obtained,
which restricts the bioavailability, in particular in pulmonary
administration,
e.g. loss due to deposits outside the target site and adverse effect on the
dispersibility. In addition, problems result in the prior art with respect to
the
loading ability of the carrier.
It is therefore the object of the invention to develop a dry-formulable
excipient of a special quality for the applicators available on the market and
which gets around these disadvantages. In particular, the possibility should
be provided here of establishing a particle technology which is able to
achieve a depot effect in the lung. (t is therefore likewise the object of the
invention to prepare dispersible particulate excipients. An object is likewise
the provision of a process which is as simple and advantageous as
possible for the preparation of dry formulations which can preferably be
used in the context of pulmonary administration. In this regard, the
optimization of the excipient in size, surface area and morphology with
respect to use for inhalation is likewise an object.
The object is achieved in that particulate excipients for pulmonary
administration are made available which contain at least one linear water-
insoluble polysaccharide and whose mean size is less than 10 Nm.
By means of the rrheasures according to the invention additional
advantages are achieved:
Easier loading ability of the excipient with active compound due to the
good suspensibility in various media (e.g. dispersion process, spray
drying);
~ Acceptability and high biocompatibility of the excipient used due to
naturally occurring structures and due to the use of naturally identical
products;
~ The avoidance of an addition of surface-active compounds in the
preparation of the active compound formulation;
~ The aerodynamic diameter, which has a positive effect for the flight
behavior. This is achieved on account of the porous surface nature in
comparison with a smooth sphete and is assisted by the tendency
accompanying the surface roughness for cluster and/or agglomerate
formation.
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Within the meaning of the invention, particulate excipient designates
particles of a mean size of less than 10 Nm, in particular of 1 to 1 o Nm,
preferably 2 to 6 Nm and particularly preferably 1 to 3 Nm, which as an
essential constituent contain at least one linear water-insoluble
polysaccharide.
Linear water-insoluble polysaccharides within the meaning of the present
invention are polysaccharides, preferably polyglucans, in particular
poly(1,4-alpha-D-glucan), which consist of monosaccharides,
disaccharides, further oligomers thereof or derivatives.
These are always linked to one another in the same manner. Each base
unit defined in this way has exactly two linkages, each one to another
monomer. Excluded therefrom are the two base units, which form the
beginning and the end of the polysaccharide. These base units have only
one linkage to a further monomer. In the case of three or more linkages
(covalent bonds) of a monomer to another group, preferably a further
saccharide unit, branching is referred to. At least three glycosidic bonds
then start from each saccharide unit in the polymer backbone.
According to the invention, branchings do not occur or only occur to such a
small extent that in general they are no longer accessible to the
conventional analytical methods in the existing very small branching
proportions. For example, this is the case if, based on the totality of all
hydroxyl groups present to 100 hydroxyl groups which are not needed for
the synthesis of the linear polysaccharide, at most 5 hydroxyl groups are
occupied by linkages to other saccharide units.
The degree of branching here is maximal (100%) if the free hydroxyl
groups (or other functional groups occurring) have further glycosidic (or
other) bonds to further saccharides in any saccharide unit. The degree of
branching is minimal (0%) if apart from the hydroxyl groups which cause
the linearity of the polymer no further hydroxyl groups on the saccharides
are modified by chemical reaction.
Examples of preferred water-insoluble linear polysaccharides are linear
poly-D-glucans, the nature of the linkage being insignificant as long as
linearity within the meaning of the invention is present. Examples are
poly(1,4-alpha-D-glucan) and poly(1,3-beta-D-glucan), poly(1,4-alpha-D-
glucan) being particularly preferred.
If the base unit has three or more linkages, this is referred to as branching.
The so-called degree of branching results here from the number of hydroxyl
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groups per 100 base units which are not involved in the synthesis of the
linear polymer backbone and which form branchings.
According to the invention, the linear water-insoluble polysaccharides have
5 a degree of branching ~of less than 8%, i.e. they have less than 8
branchings to 100 base units. Preferably, the degree of branching is less
than 4% and in particular at most 1.5%.
If the water~insoluble linear polysaccharide is a polyglucan, e.g. poly(1,4-
1 p alpha-D-glucan), the degree of branching in the 6-position is less than
4%,
preferably at most 2% and in particular at most 0.5°~, and the degree
of
branching in the other positions not involved in the linear linkage, e.g. the
2- or 3-position in the case of the preferred poly(1,4-alpha-D-glucan), is
preferably in each case at most 2% and in particular at most 1 %-
Particularly preferred are polysaccharides, in particular poly-alpha-D-
glucans, which have no branchings, or whose degree of branching is so
minimal that it is no longet detectable using conventional methods,
According to the invention, the prefixes "alpha", Nbeta" or "Dp on their own
relate to the linkages which form the polymer backbone and not to the
branchings.
"Water insolubility" in the sense of the present invention means that no
detectable solubility of the compound exists under normal conditions (room
temperature of 25°C and an air pressure of 101325 pascals or based on
values differing at most 20°~ therefrom).
In the case of the polysaccharides used according to the invention, in
particular of the polyglucans such as poly(1,4-alpha-D-glucan), this means
that at least 98% of the amount employed, preferably an amount of greater
than 99.5%, is insoluble in water. The term insolubility here can also be
explained with the aid of the following observation. If 1 g of the linear
polysaccharide to be investigated is heated to 130°C in 1 I of
deionized
water under a pressurE; of 1 bar, the resulting solution only remains stable
briefly, for a few minutes. On cooling under normal conditions, the
substance reprecipitates. After further cooling and separation using the
centrifuge with inclusion of experimental losses, at least 66% of the amount
employed can be recovered in this way.
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In the context of this invention, linear, water-insoluble polysaccharides are
preferably used which can be obtained with the aid of generally defined
biotechnological or genetic engineering methods. A particularly
advantageous embodiment of the invention described here is the
preparation in a biotechnological process, in particular in a biocatalytic
process or in an enzymatic: process.
Linear polysaccharides prepared by biocatalysis (also: biotransformation) in
the context of this invention means that the linear polysaccharide is
1 p prepared by catalytic reaction of monomeric base units such as oligomeric
saccharides, e.g. of mono- andlor disaccharides, by using a so-called
biocatalyst, customarily an enzyme, under suitable conditions. Preferably,
poly(1,4-alpha-D-glucan) in particular is prepared by means of
polysaccharide synthases and/or starch synthases and/or glycosyl
15 transferases andlor alpha-1,4-glucan transferases andlor glycogen
synthases and/or amylosucrases andlor phosphorylases.
Linear polysaccharides from fem~entation in the usage of the invention are
linear polysaccharides which can be obtained by enzymatic processes
using organisms occurring in nature, such as fungi, algae or
20 microorganisms or using organisms not occurring in nature, which can be
obtained by modification of natural organisms, such as fungi, algae or
microorganisms, by means of genetic engineering methods of general
definition. Moreover, linear polysaccharides for the preparation of the
excipient described in the present invention can be obtained from nonlinear
25 polysaccharides which contain branchings by treating them with an enzyme
and linear polymers thereof can be obtained with cleavage (e.g. by means
of enzymes, such as amylase, iso-amylase, gluconohydrolase, pullulanase,
inter alia) and removal of the branchings.
The obtainment and purification of linear water-insoluble polysaccharides
30 by means of biotechnological and genetic engineering methods from plants
cannot be excluded and is expressly also included.
The particulate excipients can be prepared in particular by the microparticle
technique which is the subject of the patent application (German Patent
Office, ref.: 197 37 461.,6).
35 In addition to the dry formulation, inhalations based on dispersions and
suspensions of the e~;cipient are conceivable, which are expressly also
included here. A particularly suitable and preferred embodiment, however,
is to be seen as inhalation based on the dry formulation. In this connection,
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this is a dispersible dry powder which can be administered to the airways
by means of dry powder inhalers.
it is essential to the invention that microparticles are made available in the
dry formulation as descr<bed in the patent application (German Patent
Office, ref.: 197 37 481.6). In this connection, these are monoparticles
(Figure 1 and 2) which have a particularly suitable aerodynamic diameter
which has an advantageous effect on an inhalation (e.g. decreased
deposition on walls). The clusters and/or aggregates andlor agglomerates
formed from the monoparticles exhibit particularly advantageous
aerodynamic diameters for the purposes of the invention. Likewise, an
improved active compound distribution and lung penetration (also:
respiratory depth) is achieved on account of the mixing of these particles
taking into account the anatomical conditions of the lungs (see Examples
on Andersen Impactor).
For the definition of the aerodynamic diameter mentioned, the invention
makes use of the definition on the basis of the spec'rfication WO 97/36574,
this being determined by the multiplication of the geometric diameter, which
determines the spatial extent of a spherical structure (e.g. a spherical
particle) by the distance from surface to surface through the center point of
the spherical structure, with the route of the particle density.
A cluster and/or an aggregate and/or agglomerate is to be understood as
meaning an accumulation of monoparticles which results due to the build-
up of noncovalent forces. A cap-like and/or raspberry-like andlor spherical
structure, for example, acts particularly advantageously here, as shown in
Figure 3 and Figure 4. Noncovalent forces can be, for example, bipolar
interactions, van-der-Waals forces, hydrogen bridges or alternatively steric
interactions, the latter as generally defined also being designated as the
key-lock principle.
The molecular weights May of the linear polysaccharides used according to
the invention can vary within a wide range from 103 glmol to i 0~ g/mol,
preferably molecular weights MW in the range from 104 g/mol to 105 g/mol,
in particular 5 x 103 g/mol to 5 x 104 g/mol. For the linear polysaccharide
poly(1,4-alpha-D-glucan) preferably used, corresponding ranges of the
molecular weights are used.
"Biocompatible" within the meaning of this invention means that the
polysaccharides employed may be subject to complete biological degrada-
tion and very substantially no concentration takes place in the body.
Biological degradation is regarded here as any process occurring in vivo
which leads to degradation or destruction of the polymer. In particular,
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hydrolytic or enzymatic processes likewise fall in this area. For the
biocompatibility of the polysaccharide and its degradation products
(metabolites), even the naturally identical character of the polysaccharides
employed is not in the end of great importance. Therefore the
polysaccharides in question are particularly suitable for therapeutic,
diagnostic or prophylactic use.
1n addition, the term "pharmaceutically acceptable" within the meaning of
this invention adds that a carrier for an active compound, an auxiliary or
even a so-called excipient can be absorbed by a living being without
1 p significant side effects resulting for the organism. In the particular
case of
pulmonary administration" this means that the auxiliary can be absorbed by
the lungs and is degraded, dissolved and further transported by the
endogenous mechanisms or else deposits occur which also do not lead to
disadvantageous effects for the living being due to accumulation.
15 "Controlled release of active compound" is understood as meaning that the
active compound is released after a specific time and/or period of time in a
dose which is advantageous for the biological organism with acceptance of
a statistical deviation corresponding to the circumstances.
20 This definition also includes extremes. On the one hand, the spontaneous
release of all actrVe compounds present in the formulation within a period of
time approximating to the value zero. On the other hand, the minimal
necessary amount/dose for the attainment of a therapeutic effect over a
long, even infinite period of time, at least a period of time which is
25 necessary to release all active compounds present in the formulation.
For the dry formulation present here, therefore, reference is synonymously
made to a depot formulation or formulation having delayed release.
"Dry formulation" and/or "powder" within the meaning of this invention
means a composition which consists of fine solid particles of the respective
30 phamnaceutical composition. These particles are free-flowing and
dispersible in their entir~sty.
In the particular case of use by inhalation, dry formulation andlor powder
additionally means that they are used in an apparatus for the inhalation of a
35 dry powder such that a therapeutic effect is discernible. In this
connection,
the term "utilizable for the pulmonary transport of active compounds" or
"inhalable", "breathable" or "respirable" is also used.
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In this connection, in the context of this invention "dry" is understood as
meaning a water content of less than 25%, a water content of less than
15% being preferred. A water content of 5% to 10% is particularly
preferred.
"Therapeutic effect" within the meaning of this invention means that a
therapeutically effective amount of an active compound reaches the
desired target site, displays 'tts action there, and causes a physiological
reaction. the palliative andlor curative effect is included.
t 0 "Dispersibilit~' within the meaning of this invention means that the dry
formulation obtained by external mechanical forces can be atomi2ed or
fluidized such that this state is so stable for a determined short period of
time (solid in gaseous or solid in liquid) that a continuing process can be
initiated which gives a greater benefit, In the context of this invention
pulmonary use, i.e. active compound absorption via the lungs, serves as an
outstanding example, but other active compound transport phenomena are
also not excluded.
For pulmonary administration, the generally accessible inhalers found on
the market, in particular so-called "dry powder inhalers" can be employed
and equipment already described in patents and publications can be used
(manufacturer: 3M Manufacturing Minnesota Mining, Inc., Inhale
Therapeutic Systems, Inc., Dura Pharmaceuticals, Aerogen and Aradigm
Corporation, WO 94/16756, WO 96/30068, WO 96/13292, WO 96/33759,
WO 96113290, WO 96J32978, WO 94/08552, WO 96/~~, WO 95128192).
The active compounds which can be administered to the airways as a
pharmaceutical composition by means of the excipient described are
subject to no restrictions whatsoever.
In particular, the pharmaceutical composition is efficacious for syndromes
which are either genetically caused or else have been acquired in the
course of Irfe.
The release of the active compound can either take place systemically or
locally. A palliative or curative effect can be demanded.
The active compounds employed with the present invention can be either
water-soluble or water-insoluble. The active substances or active com
pounds employed in the pharmaceutical sector can either be
therapeutically or diagnostically active, but also prophylactically active.
The active compounds employed can apply to all sorts of indications.
Those to be mentioned are, for example:
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asthma, cystic fibrosis, general lung diseases, diabetes (hypoglycemic
reaction), cancer, mucoviscidosis, renal anemia, hemophilia, stimulation of
ovulation, neutropenias, Gaucher's disease, hypernephroma, hair cell
leukemia, carcinoma, multiple sclerosis, chronic granulomatosis,
myocardial infarct, thromboses, pituitary hyposomia, treatment of heparin-
associated thrombocytopenia (HAT) type II, vaccinations, prevention of
hepatitis B, stimulation of growth of cellular elements, bronchitis, chronic
airway disorders, lung diseases and chest infections.
The particulate excipients are subject to no restrictions whatsoever with
1 p respect to the stability of the active compounds. The naturally identical
polysaccharides used a.re chemically inert substances, so that even
sensitive active compounds, such as peptides and proteins can be
administered. In this context, peptides and proteins are particularly of
interest which are constructed of the twenty natural amino acids. The
partial replacement of natural amino acids by amino acids which do not
occur naturally, however, has no effect on the utility of the invention
described here (e.g. cetrorelix). Furthermore, the administration of
oligonucleotides or viral vectors is also possible.
In the case of the proteins and peptides, compounds derived from nature
20 can be employed or even those which can be prepared, in particular, by
biotechnological and/or genetic engineering process steps and are
generally to be incorporated under the designation of recombinant active
compounds. In particular, therapeutics or vaccines are distinguished here,
as are human DNAse (dornase alpha), erythropoietin alpha (epoetin alpha),
25 erythropoietin beta (epoetin beta), factor VII (eptacog alpha), factor
VIII,
follitropin alpha, follitropin beta, G-CSF, glycosyfated (lenograstim), G-CSF
(filgrastim), GM-CSF (molgramostim), glucagon, glucocerebrosidase
(alglucerase), IL-2 (aldesleukin), interferon alpha-2a, interferon alpha-2b,
interferon beta-1 b, interferon gamma-1 b, insulin (human insulin, lispro),
30 t-PA (alteplase), r-P,A (reteplase), human growth hormone (HGH,
somatropin), hirudin, hepatitis B-antigen, hepatitis AIB combination
vaccine, MPIF-1 (myeloid progenitor inhibitor factor-1), KGF-2 (keratinocyte
growth factor).
35 All in aH, active compaunds from the following group and classes of these
active compounds can also be advantageously used, especially in
pulmonary administration, with the technology presented here:
calcitonin, felbamate, AZT, DDI, GCSF, lamotrigin, gonadotropin releasing
factor (hormone) (GNRH, GHRH and GHRH analogs), luteinizing hormone
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releasing hormone (LHRM) and LHRH analogs (both antagonists and
agonists, e.g. leuprolide acetate, leuprolide, lupron or lupron depot,
buserelin, ramorelix, cetrorelix), TRH (thyreotropin releasing hormone),
adenosine deaminase, argatroban, a1-antitrypsin, albuterol, amiloride,
terbutalin, isoproterenol, metaproteranol, pirbuterol, fluticasone propionate,
budesonide, beclomethasone dipropionate, cromoglycic acid, disodium
cromoglycate, nedocromil, sultanol, beclomethasone, bambuterol,
mometasone, triacetonide, isoproterenol, salmeterol, salmeterol xinotate,
formotorol, triamcinolone acetonide, flunisolide, fluticasone, salbutamol,
1 o fenoterol, ipratropium brornide, tachykinin, tradykinin, furosemide,
nafarelin,
albuterol sulfate, metaproterenol sulfate, somatostatin, oxytocin,
desmopressin, ACTH analogs, secretin glucagon, codeine, morphine,
diltiazem, ketotifen, cephalosporin, pentamidine, fluticasone, tipredan,
noscapine, isoetharine, amiloride, ipratropium, oxitropium, cortisone,
prednisolone, aminophylline, theophylline, methapyrilene.
Analgesics, anginal preparations, antiallergics, antihistamines,
antiinflammatories, bronchodilators, bronchospasmolytics, diuretics,
anticholinergics, antiadhesion molecules, cytokine modulators, biologically
actwe endonucleases, recombinant human DNases, neurotransmitters,
leukotrine inhibitors, vasoactive intestinal peptide, endothelin antagonists,
analeptics, analgesics, local anesthetics, anesthetics, antiepileptics,
anticonwlsants, antiparkinson agents, antiemetics, compounds regulating
or stimulating the hormone system, compounds regulating or stimulating
the cardiovascular system, compounds regulating or stimulating the
respiratory tract system" vitamins, trace elements, antioxidants, cytostatics,
antimetabolites, antiinfectives, immunomodulators, immunosuppressants,
antibiotics, proteins, peptides, hormones, growth hormones, growth factors,
xanthines, vaccines, steroids, ~2 mimetics.
Either the free acids and bases, or pharmaceutically active and acceptable
salts of these compounds and susbtance classes, as well as their
hydrolyzates or fragments or metabolites thereof can be used.
Likewise, customary auxiliaries and additives can be used for the present
formulation.
The following examples serve for further explanation of the invention,
without restricting it to products and embodiments described in the
examples.
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Examples
The following examples relate in particular to the preparation of
microparticies, as described in the patent application (German Patent
Office, ref.: 197 37 481.6), to which reference is expressly made. In
addition, a particularly advantageous method for the preparation of
poly(1,4-alpha-D-glucan) is described in WO 95/31553.
Moreover, examples for illustrating pulmonary administration are
mentioned, in particular with the aid of the dry formulation in the context of
powder inhalation.
Example 1
In-vitro production of poly{1,4-a-D-gluCan) in a biocatalytic process with the
aid of the enzyme amylosucrase
10 l of a 20% strength sucrose solution are added to a sterilized (steam
sterilization) 151 vessel. The enzyme extract comprising amylosucrase
obtained by means of fermentation is added to the sucrose solution in one
portion. The enzyme activity is 16 units (1 unit corresponds to the reaction
of 1 Vmol of sucrose per minute per mg of enzyme). The apparatus is
provided with a KPG stirrer, which is also sterilized. The vessel is sealed
and kept at 40°C and stirred. After some time, a white precipitate
forms.
The reaction is ended after a period of time of 180 hours. The precipitate is
filtered off and washed a number of times to remove low molecular weight
sugars. The residue remaining in the filter is dried at temperatures between
and 40°C in a dryinc,~ oven with application of a vacuum with the aid
of a
membrane pump (Vacuubrand GmbH & Co, CVC 2). The mass is 685 g
(yield 69%).
Example 2
Characterization of the poly(1,4-a-D-glucan) synthesized with amylo-
sucrase from Example 1 by means of gel permeation chromatography
2 mg of the poly(t ,4-~-D-glucan) from Example 1 are dissolved in dimethyl
sulfoxide (DMSO, p. a. from Riedel-de-Haen) at room temperature and
filtered (2 mm filter). One part of the solution is injected into a gel
permeation chromatography column, DMSO is used as an eluent. The
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signal intensity is measured by means of an RI detector and evaluated
against pullulan standards (Polymer Standard Systems). The flow rate is
1.0 ml per minute.
The result of a measurement is indicated below: numerical average of the
molecular weight (Mn) of 3200 glmol and a weight average of the molecular
weight (MW) of 9300 glmol. This corresponds to a dispersity of 2.8.
Example 3
Preparation of microparticles of poly(1,4-a-D-glucan)
400 g of poly(1,4-a~D-glucan) are dissolved in 2 I of dimethyl sulfoxide
(DMSO, p.a. from Riedel-de-Haen) at 40°C in the course of 1.5 h. The
solution is then stirred at room temperature for one hour. The solution is
added to 201 of double-distilled water with stirring through a dropping
funnel over a period of time of 2 h. The mixture is stored at 6°C for
40 h. A
fine suspension is formed. The particles are separated off by first decanting
off the supernatant. The sediment is scurried and centrifuged in small
portions (ultracentrifuge RCSC: 5 minutes each at 5000 revolutions per
minute). The solid residue is slurried with double-distilled water and
centrifuged again a tot<~I of three times. The solids are collected and the
suspension of about 1000 ml is freeze-dried (Christ Delta 1-24 KD). 283 g
of white solid are isolated (Example 3a: yield 71 °~), The collected
supernatants are kept .at a temperature of 18°C overnight. Working up
is
carried out as described. A further 55 g of the white solid are isolated
(Example 3b: yield 14°~). The total yield is 85%.
Example 4
Desulfurization of the microparticles from Example 3
To remove dimethyl sulfoxide remaining in the particles, the procedure is
as follows. 100 g of the amylose particles from Example 9 are added to
1000 ml of deionized water. The mixture is left by itself for 24 h with slight
swirling. The particNes are removed as described in Example 9
(ultracentrifuge RCSC: 15 minutes each, 3000 rpm). After freeze-drying, a
final weight of 98.3 g results (98% yield). Sulfur determination by elemental
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14
analysis gives the following values (test method combustion and 1R
detection):
sulfur content of the particles from Example 2: 6% +/- 0.1
sulfur content of the particles from Example 3: < 0.01 °~
Example 5
Investigations of the microparticles from Example 3 by means of electron
microscopy
To characterize the particles, scanning electron micrographs (SEMs)
(Camscan S-4) are carried out. The figures (1=figure 1 and Figure 2) show
plots of the particles which illustrate that they are spherical, very uniform
particles with respect to shape, size and surface roughness.
The figures (Figure 3 and Figure 4) show plots of the clusters andlor
agglomerates formed.
Example 6
Investigations of the size distributions of the particles from Example 3
To characterize the size distributions of the particles from Examples 1 and
9, investigations with a Mastersizer were carried out (Malvern Instruments).
The investigation was carried out in the Fraunhofer mode (evaluation:
multimodal, number) with a density of 1.080 glcm3 and volume
concentration in the range from 0.012% to 0.014%.
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Table 1:
Characterization of the particle diameters of the microparticles from
Example 3
5
Example I Diameter ~ Particle distribution
n*, ~*2 dw / d (10%)*4 d (50%)*5 d (90%)'6
No. d
(mm) (mm) do*3 (mm) (mm) (mm)
3a I 1.664 , 4.184 I 2.541 I 0.873 ~ 1.504 ~ 2.624
3b I 0.945 I 2.34"a '2.481 I 0.587 ~ 0.871 ~ 1.399
~~ dn: Number average of the diameter
~2 dw: Weight average of the diameter
f 3 dw / dn: Dispersity of the particle diameters
10 ~4 d(10%): 10% of all particles have a smaller diameter than the value
indicated
~5 d(50°~): 500 of all particles have a smaller diameter than the value
indicated
~6 d(90%): 90% of al) particles have a smaller diameter than the value
indicated
Example 7
Determination of the flight distance of microparticles of poly(1,4-a-D-
glucan)
An Andersen impactor (Andersen Samplers Inc., 4215 Wendell Drive,
Atlanta, GA, USA and cf. Pharmacopoeia Europaeum, Stuttgart, Deutsche
Apothekerverlag 1997, Chap. 2.9.18; here: appliance D) consists of a
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16
cascade of plates separated from one another, which are equipped with
successively smaller holes (pores). Depending on the aerodynamic particle
size, deposition takes place on the individual plates.
Table 2: Characteristics of the Andersen impactor
in "inches".
The Andersen impactor experiments proceed as follows: 100 mg of the
substance (in this case: three different samples of microparticles and
agglomerates (clusters) of poly(1,4-a-D-glucan); prepared according to
Ex. 3 and 4) are added to the mixture opening of the impactor and sucked
into the filter cascade using the pump belonging to the equipment (pump
output: 35 Umin.). After one minute, the apparatus is stopped and the mass
of the individual fitters. (or impact plates) is determined. The masses of
microparticles are plotted in the following figure. fn this case, the good
flow
behavior with minimal pump output is especially clear, which is reflected in
the high mass on higher impact plates (filters). The residue remaining at
the starting point can Ide minimized by keeping the particles in suspension
by mechanical vibration at the mixture opening of the impactor
(cf. Sample 2).
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7
The behavior of various samples of poly(1,4-a-D-9lucan) in the Anderson
impactor is shown in Figure 5.
Example 8
Determination of the flight distance of powders of further polysaccharides,
[lacuna] conventional inhalers and microparticles of synthetic polymers
(comparison examples)
0 The comparison examples with other, partly water-soluble polysaccharides,
material from conventional inhalers and microparticles of synthetic, but
biodegradable polymers (comparison examples) were carried out
analogously to Example 8. The polysaccharides are a potato starch of the
type Toffena (Sudst~rke) and a rice starch of the type Remygel (REMIT
~ 5 with particle sizes up to 100 mm and around 4 mm. The inhalers are the
rolizer Inhaler 1, Ciba Geigy) and the Atemur Diskus (inhaler 2,
Ae (
Cascan). The microparticles of polylactide-co-glycolide (PLGA) were
prepared by means of spray drying (material PLGA 65:35-d, l of Medisorb)
and have approx. diameters between 2 and 15 mm.
The behavior of other, partly water-soluble polysaccharides in the
Andersen impactor (comparison examples) is shown in Figure 6.
The behavior of material from conventional inhalers and microparticles from
synthetic, biodegradable polymers in the Andersen impactor
(comparison examples) is shown in Figure 7.
Example 9
Determination of the flight distance of microparticles of various geometries
of poly(1,4-a-D-glucan) and agglomerates thereof
The flight or deposition behavior of microparticles of various size and
surface roughness is investigated in a curved tube. The experiments form
the basis for estimation of an optical morphology of particles and
agglomerates for administration in a dry powder inhaler. The model is
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18
based on the following assumptions: passage through which air flows
nsi rfl =1.2 kglm3; air viscosity h =1.81 x 10 5 Pa s;
d = 20 mm; air de tY
flow rate vf~ = 5 mls; radius of curvature R = 20 (200) mm; angle of
curvature a = 45°; particle diameter x =1 - 5 mm; solid density
rs -1550 kglm~; form factor Y =1 and differing.
First, the behavior of spherical and nonspherical particles is compared with
one another. For nonspherical particles, a structure for the description of
the geometry is prespecified. The settling rates in the gravitational field in
air and the radial deflection of such a particle when flowing through a bend
1p in a tube are sought. With the aid of the calculated deflection, it can be
estimated how many particles touch the wall and remain adhered, i.e. are
deposited, when flowing through the bend in the tube.
The deviation of the particle form from the sphere can be described with
the aid of the form factor ~'. It is defined as the ratio of the surface of a
5 sphere with equal volume to the actual surface of the particle. For spheres,
~Y =1, for nonspherical particles 'Y is < 1. The smaller the form factor, all
the more a particle follows the air flow and all the less it is deposited by
watt
adhesion in tube curves. The aerodynamic diameter behaves reciprocally
to the form factor. A value of 'Y = 0.8 is, for example, comparable with a
20 decrease in the solid density from 1550 kglm~ to 1400 kg/m~.
In a first observation, a smooth sphere was compared with a sphere with a
rough surface. The roughness was approximated by relatively small
spherical sections (spherical caps) which were mounted on a base sphere.
The sections were on the one hand directed outward (raspberry structure)
25 and on the other hand inward (crater landscape). For outwardly directed
sections, the form factor turned out to be '~ = 0.958 and for inwardly
directed sections to be ~ = 0.946. Furthermore, agglomerates of particles
were observed. The observed agglomerates (bodies) ideally form the
following arrangements in the arrangement of a very tight spherical packing
30 (for tetrahedrons see also Figure 2). The bodies are modeled from
agglomerates of individual spheres and have the following structure: a) a
tetrahedron constructed from spheres having 4layers. The edges are
formed of 4 spheres in each case. All in all, the tetrahedron consists of
20 individual spheres; b) bodies formed from 3 spherical layers having 3, 7
35 and again 3 individual spheres (small double cone); c) bodies formed from
5 layers having a sph~:re number of 1, 3, 7, 3 and 1 per layer (large double
cone).
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19
The table summarizes the results of the calculations. For comparability,
onl the deviation from the spherical form is considered as a parameter in
v
the case of a prespecified volume-equivalent particle diameter of 5 Nm. It is
to be noted that the diameter of the individual spheres for the
abovementioned geometries is correspondingly lower. The settling rate
decreases with increasing deviation from the spherical form. Accordingly,
the degree of deposition when flowing through a bend in the tube also
decreases. In comparison with the sphere having the diameter of 5 Nm, the
de ree of deposition is reduced to 66% for the particle forms constructed
9
from agglomerates. The results of these model experiments confirm the
advantageousness of the present invention with respect to the surface
roughness, and also the agglomerates, which are organized in the sense of
a very tight spherical packing. In particular, it is seen that with a
decreasing
form factor ~Y, i.e. toward zero, the aerodynamic diameter improves. The
better the aerodynamic diameter, the deeper the lung penetration
(respiratory depth).
Table 2: Results of the calculation for the particle forms described above
Form Agglomerate Settling rate in Radial Degree of
factor densit~l the gravitational deflection deposition
[kglrrr~) field [mm1 [
[m/s)
Sphere 1 1550 0.001165 0.466 2.33
Sph~re 0.946 1550 0.001134 0.453 2.26
(caps on the
inside)
Sphere 0.958 1550 0.001141 0.456 2.28
(caps on the
outside) _
Agglomerate 0.49 1430 0.000753 0.301 1.50
Tetrahedron
Agglomerate 0.56 1390 ~ 0.000782 ~ 0.3t 3 ~ 1.56
double cone
3 planes
Agglomerate 0.52 1405 I 0.000762 ~ 0.305 ~ 1.52
double cone
5 planes
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Example 10
din of the particles with actrve compound by means of a suspension
Loa 9
process
The microparticles, or agglomerates, of poly(1,4-a D-glucan) (preparation
in Examples 3 and 4) are loaded with active compound by means of a
sus ension process. 250 mg of busereliri are dissolved in 10 ml of distilled
P
water. 100 mg of particles are added. The suspension is stirred for 3 h. The
sus ension is centrifuged. The centrifugate is washed with water. The
P
articulate solid is separated off by centrifuge (3000 rpm) and the
P
centrifugate is freeze-dried. By dissolution of an exact amount of the
particles in water~dimethyl sulfoxide and spectroscopic measurement in a
UV-Vis spectrometer, the loading with buserelin can be calculated to be
3.28% by means of a calibration curve, based on the total mass of the
particles. By means of the modification of the solvent for the active
compound, e.g. alcohols, the solubility and thus the loading of the particles
with active compound can be influenced.
(' 5-0xo-L-prolyl-L-histidyl~L-tryptophyl-L-Beryl-L-tyrosyl-O-tart-butyl-D-
seryl-L-leucyl-L-arginyl-N-ethyl-L-prolinamide)
Example 11
Loading of the particles with active compound by means of spray drying
The microparticles are suspended in distilled water, or a mixture of water
and a readily volatile component such as acetone or ethanol. To do this,
10 g of the solid are suspended in 1000 ml of the solvent. 0.5 g of
theophylline was dissolved in the solvent beforehand. The spray drier (mini
spray drier 191 from f~uchi) is operated as follows:
Atomization air flow '700 liters per hour, inlet temperature 200°C,
nozzle
cooling switched on, nozzle diameter 0.5 mm, aspirator 70°/°,
pump 10%.
The spectroscopic investigation of the loading (description see example
above) shows a degree of loading of 4.8°l°. This value agrees
with the
theoretically achievable value of 5.0% within the limits of error.
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Example 12
Determination of the solubility of polysaccharides
100 mg of poly(1,4-a-D-glucan) are added to 5 ml of double-distilled water.
The reaction vessel is slowly heated with stirring (magnetic stirrer). It is
ted in a step program with intervals of twenty degrees and observed
hea
' h the a e. No changes are to be observed at temperatures of 40°C,
wrt Y
60°C, 80°C and 100°G. According to these observations,
the compound
1 p can be assigned the characteristic "water-insoluble".
Example 13
Determination of the solubility of polysaccharides and classification
~ 5 according to the German Pharmacopeia (GP)
564 mg of poly(1,4-a-D-glucan) are heated in about 0.51 of double-distilled
water in an autoclave at 1.3 bar and 130°C for 1.5 hours (Certociav
apparatus). The weight of the reaction vessel has been measured before-
20 hand. The pressure in the apparatus is then released and it is cooled to
room temperature. The contents are weighed. They correspond to
501.74 g. After a further 24 hours, the mixture is centr'rfuged and the
supernatant is decanted. The solid residue is dried and weighed: 468 mg. A
dissolved fraction of 96 mg is calculated therefrom. Based on the solvent
25 employed, it is calculated therefrom that for 1 mg of poly(1,4-a-D-glucan)
5226 mg of water are necessary, According to the classification in the
German Pharmacopeia, the classification results therefrom that this
substance is "very poorly soluble", since between 1000 and 10,000 parts of
solvent are necessary in order to bring 1 part of the substance into solution.
3o Of the 7 classes for the classification of solubility (from "very readily
soluble" (Class 1 ) to "virtually insoluble" (Class 7)), this is Class Number
6.
Example 14
35 Determination of the solubility of polysaccharides and dass'rfication
according to the German Pharmacopeia (GP)
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22
he ex riment is tattled out as in F-~cample 13. the only difference is a
T pe
lln rocess which is inserted after the autoclave treatment and cooling
coo g P
to room temperature. The substance mixture is stored at 5°C for 3
hours.
6 m of poly(1,4-a-D-glucan) are weighed into about 480 ml of double-
s 52 9
istilled water. After the heat treatment, a final weight of 468.09 g results.
d
The dried sediment amounts to 488 mg. 38 mg of the poly(1,4-a-D-glucan)
have therefore dissolved. This corresponds to a ratio of 1 mg of substance
to 12,318 parts of solvent. The substance is therefore to be assigned to
lass Number T as spec'rfied in the GP according to this treatment method
C
and accordingly to be classified as virtually insoluble, because more than
10,000 parts of solvent are needed for one part of substance.
Example 15
preparation of microparticles from an autoclaved poly(1,4-alpha-D-glucan)
suspension
3.5 g of poly(1,4-alpha-D-glucan) powder are washed three times with
water at 60°C. It is then suspended in 200 ml of deionized water. The
suspension is placed in a laboratory autoclave (Pressure Vessels; type
452 HCT 316; from Parr instruments Deutschland GmbH). The chamber is
flushed with nitrogen so that a pressure of 1.5 bar is reached.
The autoclave is heated to 130°C with stirring. The autoclaving time
at this
temperature is 30 min. A pressure of about 5 bar is generated.
After cooling of the autoclave, the suspension is removed and immediately
sprayed into a spray dryer. During this the suspension is continuously
stirred with the aid of a magnetic stirrer.
The spray dryer (Mini Spray Dryer 191, from Buchi, Switzerland) is
operated as follows: atomizing nitrogen stream 7001iters per hour, inlet
temperature 220°C, nozzle cooling switched on, nozzle diameter 0.5 mm,
aspirator 70%, pump 70%.
The spray-dried mafierial is removed from the collecting vessel of the
cyclone and stored in the dry. The size of more than 90% of the
microparticles is in the range below 10 ~m (analysis of electron
micrographs).
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23
Example 16
Preparation of microparticles from poly(1,4-alpha~D-glucan)
The ossibility of using polyglucan resulting directly from biotransformation
P
for re aring respirable padicles is investigated. This is done by preparing
P P
a 20°~ strength suspension by suspending 500 g of poly(1,4-alpha
p- lucan) from Example 1 in 2:51 of water. The suspension is spray dried.
9
This is done by operating the spray dryer (Mini Spray Dryer B-191 from
Biichi) as follows: atomizing air stream 650 Titers per hour, inlet
temperature 140°C, no nozzle cooling, nozzle diameter 0.7 mm, aspirator
70%, pump 30°!°. 197.5 g of white solid are taken from the
product
collecting vessel. The yield is 40°!°. A proportion of more than
90°!° of the
particles have their diameter in the range 1-'10 Pm.
The utilizability as excipient for administration as dry powder in the
administration of an active substance by inhalation is determined using the
Andersen impactor apparatus described in Example 7. The results show in
Figure 8 the respirability in principle of the particles obtained by spray
drying (see also Figures 5-7 relating to Examples 7 and 8).
Example 17
Characterization of the flowability of microparticles and powders of poly-
(1,4-alpha-p-glucan) in accordance with List et al., Arzneiformenlehre,
WVG, Stuttgart, 1985; section 2.10.1.
100 g of powder from Ex. 3 and 4 and Ex. 1 are packed into a flow funnel
(flowability tester from Engelsmann, Ludwigshafen) keeping the outflow
orifice closed during this. The outflow orifice is opened and the powder is
mixed with the agitating blade by actuating a crank while it is running out.
After resting for a period of 2 minutes, the diameter and height of the
powder cone are measured and the angle of repose is determined there
from. If the bulk volume is too high, only 50 g of powder are employed
instead of 100 g.
Table: the following applies: tan a = h/r according to List (supra, page 53 et
seq.)
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24
Hei h' ne Radius of An le
cone of
p binder
r in cm a in
Unit h in cm 37
0
Microparticles 6.4 8.50 .
from
Ex. 3, 4
25 29.9
6
Powder from Ex. 3.b . 3
~ 31
Powder from Ex. 4.1 6.75 .
1
Powders with an angle of repose <_ 30° are particularly suitable.
Corres
ndin reference measurements with materials known to flow well, such
po 9
s EMDEX~, Avicel~, Toffena~. reveal angles of repose around 30° (~.
a
Bauer, Froming, Fuhrer, Pharmazeutische Technologic, Stuttgart, 1989,
p. 345).
Example 1 B:
>_oading of microparticles prepared from an autoclaved poly(1,4-alpha-
D-glucan) suspension
3.5 g of poly(1,4-alpha-D-glucan) powder are washed three times with
~ 5 water at 60°C. It is then suspended in 200 ml of deionized water.
The
suspension is placed in a laboratory autoclave (Pressure Vessels; type
452 HCT 316; from Parr Instruments Deutschland GmbH). The chamber is
flushed with nitrogen s~o that a pressure of 1.5 bar is reached.
The autoclave is heated to 130°C with stirring. The autoclaving time
at this
2p temperature is 30 min. A pressure of about 5 bar is generated.
After cooling of the autoclave, the suspension is removed, 175 mg of
theophylline (corresponding to a 5% loading) are dissolved therein, and the
active substance-containing suspension is immediately sprayed into a
spray dryer, During thus the suspension is continuously stirred with the aid
25 of a magnetic stirrer.
The spray dryer (Mini Sprilhtrockner 191, from Buchi, Switzerland) is
operated as follows: atomizing nitrogen stream 7001iters per hour, inlet
temperature 220°C, nozzle cooling switched on, nozzle diameter 0.5 mm,
aspirator 70%, pump 70%.
34 The spray-dried material (yield at least 25% based on the amount of solid
used in the autoclave) is removed from the collecting vessel of the cyclone
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d in the dry The size of more than 90% of the microparticles is in
and store
the range below 10 pm (analysis of electron micrographs).
din of the microparticles with theophylline is checked by
The loa g
. This is done by dissolving 50 mg of the sample in 100 ml of
photometry
I sulfoxide at 60°C and measuring at 271 nm (Lambda 20
dimethy
Photometer, Perkin Elmer',l. The degree of loading is at least 95%.
of the active substance-loaded microparticles are packed into the
20 mg
terin chamber of a commerciaNy available dry powder inhaler
me 9
erolizer~ from Ciba-Geigy), and the flight distance is measured with the
(A
10 Andersen impactor as described in Example ?.
CORRECTED SHEET (RULE 91)
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N
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cil c~o~ N~ ~~ ~~ ~~ °~ a
c~~ c'~~c~ ~~ a~ °'~°~ of o~ o
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CA 02340340 2000-10-06
