U.S. patent application number 16/421068 was filed with the patent office on 2019-09-12 for pharmaceutical compositions of lipase-containing products, in particular of pancreatin.
The applicant listed for this patent is ABBOTT PRODUCTS GMBH. Invention is credited to Bernd Boedecker, Peter-Colin Gregory, Siegfried Schaefer, George Shlieout, Bernd Thumbeck.
Application Number | 20190275122 16/421068 |
Document ID | / |
Family ID | 34964300 |
Filed Date | 2019-09-12 |
![](/patent/app/20190275122/US20190275122A1-20190912-M00001.png)
United States Patent
Application |
20190275122 |
Kind Code |
A1 |
Shlieout; George ; et
al. |
September 12, 2019 |
PHARMACEUTICAL COMPOSITIONS OF LIPASE-CONTAINING PRODUCTS, IN
PARTICULAR OF PANCREATIN
Abstract
Orally administrable pharmaceutical compositions of
lipase-containing products, particularly pancreatin and
pancreatin-containing products, or of enzyme products which contain
at least one lipase of non-animal, especially microbial origin,
which improve the lipolytic activity and particularly result in
stabilization of the lipase in the acidic pH range. These oral
pharmaceutical compositions contain a system which includes at
least one surfactant and one co-surfactant and optionally a
lipophilic phase, and are self-emulsifiable on contact with a
hydrophilic and a lipophilic phase. The compositions according to
the invention are suitable for treating or inhibiting maldigestion,
especially maldigestion due to chronic exocrine pancreatic
insufficiency, in mammals and humans.
Inventors: |
Shlieout; George; (Sehnde,
DE) ; Boedecker; Bernd; (Hannover, DE) ;
Schaefer; Siegfried; (Burgwedel/Thoense, DE) ;
Thumbeck; Bernd; (Nordstemmen, DE) ; Gregory;
Peter-Colin; (Hannover, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ABBOTT PRODUCTS GMBH |
Hannover |
|
DE |
|
|
Family ID: |
34964300 |
Appl. No.: |
16/421068 |
Filed: |
May 23, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14308543 |
Jun 18, 2014 |
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16421068 |
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11085073 |
Mar 22, 2005 |
8802087 |
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14308543 |
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60554993 |
Mar 22, 2004 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 38/54 20130101;
A61K 9/1617 20130101; A61K 9/1641 20130101; A61K 38/48 20130101;
A61K 31/745 20130101; A61K 38/465 20130101; A61K 31/22 20130101;
A61P 1/18 20180101; A61K 9/1075 20130101; A61K 2300/00 20130101;
A61K 2300/00 20130101; A61K 38/47 20130101; A61P 1/14 20180101;
A61K 2300/00 20130101; A61K 38/48 20130101; A61K 38/465 20130101;
A61K 38/47 20130101 |
International
Class: |
A61K 38/54 20060101
A61K038/54; A61K 38/48 20060101 A61K038/48; A61K 38/47 20060101
A61K038/47; A61K 38/46 20060101 A61K038/46; A61K 9/16 20060101
A61K009/16; A61K 9/107 20060101 A61K009/107; A61K 31/745 20060101
A61K031/745; A61K 31/22 20060101 A61K031/22 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 22, 2004 |
EP |
04 10 1164.4 |
Claims
1-50. (canceled)
51. A pharmaceutical composition for oral administration, which is
self-emulsifiable on contact with a hydrophilic phase and a
lipophilic phase, said composition comprising: (i) an enzyme or
enzyme mixture comprising pancreatin or a microbial lipase, wherein
the enzyme or enzyme mixture has lipolytic activity and exerts the
lipolytic activity in the digestive tract upon oral administration,
and (ii) a system comprising: (a) at least one surfactant selected
from the group consisting of polyethylene glycol fatty acid esters,
polyethylene glycol glycerol fatty acid esters, polyethylene glycol
alkyl ethers, polyethylene glycol sterol ethers, polyethylene
glycol sorbitan fatty acid esters, sugar esters,
polyoxyethylene-polyoxypropylene block copolymers, sodium oleate,
sodium lauryl sulfate, sodium lauryl sarcosinate, sodium dioctyl
sulfosuccinate, sodium cholate, sodium taurocholate, lauroyl
carnitine, palmitoyl carnitine, myristoyl carnitine, alginate
salts, propylene glycol alginate, alkylsulfates, sodium docusate,
carnitines, and a combination thereof; and (b) at least one
co-surfactant which has a hydrophilic lipophilic balance value
below 10; wherein the pharmaceutical composition does not comprise
any active substances to be absorbed into the bloodstream.
52. The pharmaceutical composition of claim 51, wherein the
hydrophilic phase used to form the final emulsion after ingestion
is supplied by the physiological fluid of the digestive milieu.
53. The pharmaceutical composition of claim 51, wherein the
lipophilic phase used to form the final emulsion in the digestive
tract after ingestion is at least partially supplied by the lipids
present in ingested food.
54. The pharmaceutical composition of claim 51, wherein the system
further comprises a lipidic phase.
55. The pharmaceutical composition of claim 54, wherein (a) the
surfactant comprises at least one agent having a
hydrophilic-lipophilic balance value above 6 and below 18, and (b)
the co-surfactant comprises at least one agent having a
hydrophilic-lipophilic balance value below 10; and wherein said
system comprising surfactant, co-surfactant and lipidic phase has a
hydrophilic-lipophilic balance value of about 4 to 16, and a
melting point of at least 20.degree. C.
56. The pharmaceutical composition of claim 55, wherein said system
has a melting point of at least 25.degree. C.
57. The pharmaceutical composition of claim 54, wherein the system
comprises (a) the surfactant is selected from the group consisting
of polyethylene glycol fatty acid esters, polyethylene glycol
glycerol fatty acid esters, polyethylene glycol alkyl ethers,
polyethylene glycol sterol ethers, polyethylene glycol sorbitan
fatty acid esters, sugar esters, polyoxyethylene-polyoxypropylene
block copolymers, and mixtures thereof; (b) the co-surfactant is
selected from the group consisting of monoacylglycerides,
mono-ethers of glycerol, partial esters of propylenglycol, partial
esters of polyglycerol, partial esters of ethyl diglycol, and
mixtures thereof, and (c) the lipidic phase comprises di- and/or
triacylglycerides.
58. The pharmaceutical composition of claim 57, wherein (a) the
surfactant is selected from the group consisting of polyethylene
glycol fatty acid mono- and/or di-esters with aliphatic
C.sub.6-C.sub.22 carboxylic acids, polyethylene glycol glycerol
fatty acid esters with aliphatic C.sub.6-C.sub.22 carboxylic acids,
polyethylene glycol alkyl mono- and/or di-ethers with aliphatic
C.sub.12-C.sub.18 alcohols, and mixtures thereof; (b) the
co-surfactant is selected from the group consisting of
monoacylglycerides with aliphatic C.sub.6-C.sub.22 carboxylic
acids, mono-ethers of glycerol ethers with aliphatic
C.sub.12-C.sub.18 alcohols, partial esters of propylenglycol with
aliphatic C.sub.6-C.sub.22 carboxylic acids, partial esters of
polyglycerol with aliphatic C.sub.6-C.sub.22 carboxylic acids, and
mixtures thereof; and (c) the lipidic phase comprises di- and/or
triacylglycerides with aliphatic C.sub.6-C.sub.22 carboxylic
acids.
59. The pharmaceutical composition of claim 58, wherein (a) the
surfactant comprises a mixture of polyethylene glycol mono- and
di-esters with aliphatic C.sub.6-C.sub.22 carboxylic acids and/or
polyethylene glycol mono- and di-ethers with aliphatic
C.sub.12-C.sub.18 alcohols, wherein the polyethylene glycol
comprises 6 to 60 ethylene oxide units per molecule, (b) the
co-surfactant comprises monoacylglycerides of aliphatic
C.sub.6-C.sub.22 carboxylic acids and/or monoethers of glycerol
with aliphatic C.sub.12-C.sub.22 alcohols, and (c) the lipidic
phase comprises di- and triacylglycerides of aliphatic
C.sub.6-C.sub.22 carboxylic acids.
60. The pharmaceutical composition of claim 59, wherein the
surfactant is a mixture of polyethylene glycol mono- and di-esters
with aliphatic C.sub.6-C.sub.22 carboxylic acids, wherein the
polyethylene glycol comprises 6 to 40 ethylene oxide units per
molecule, and wherein the co-surfactant comprises
monoacylglycerides of aliphatic C.sub.6-C.sub.22 carboxylic
acids.
61. The pharmaceutical composition of claim 54, wherein the system
comprises 2 to 90% by weight surfactants, 5 to 60% by weight
co-surfactants, and 0 to 70% by weight of the lipidic phase,
wherein the components surfactant, co-surfactant and the lipidic
phase together make up to 100% by weight of the system, and the
system makes up 10% to 95% by weight of the pharmaceutical
composition.
62. The pharmaceutical composition of claim 61, wherein the system
consisting of surfactant, co-surfactant and lipidic phase makes up
10 to 70% by weight of the pharmaceutical composition.
63. The pharmaceutical composition of claim 62, wherein the system
consisting of surfactant, co-surfactant and lipidic phase makes up
20 to 50% by weight of the pharmaceutical composition.
64. The pharmaceutical composition of claim 54, wherein the
composition contains at least one further pharmaceutically
compatible auxiliary, carrier or excipient selected from the group
consisting of polyethylene glycol, glycerol,
C.sub.1-C.sub.4-alcohols, sugars, cellulosics and mixtures
thereof.
65. The pharmaceutical composition of claim 64, wherein said at
least one further pharmaceutically compatible auxiliary, carrier,
or excipient makes up a maximum of 20% by weight of the
composition.
66. The pharmaceutical composition of claim 51, wherein said
composition is a solid pharmaceutical preparation in the form of a
powder, granules, tablets, or pellets.
67. The pharmaceutical composition of claim 66, wherein the enzyme
or enzyme mixtures comprise pancreatin.
68. The pharmaceutical composition of claim 67, wherein the
pancreatin makes up 65-85% of the pharmaceutical composition.
69. The pharmaceutical composition of claim 66, wherein the enzyme
mixture comprises a mixture of at least one microbial lipase and at
least one microbial enzyme selected from the group consisting of
proteases and amylases.
70. The pharmaceutical composition of claim 69, wherein microbial
enzymes make up 5-80% by weight of the pharmaceutical
composition.
71. The pharmaceutical composition of claim 51, wherein the enzyme
or enzyme mixture retains at least 90% of the lipolytic activity
upon exposure to a buffer having pH 5 for 60 to 90 minutes.
72. The pharmaceutical composition of claim 51, wherein the
composition does not contain an enteric coating and wherein the
enzyme or enzyme mixture retains at least 90% of the lipolytic
activity upon exposure to a buffer having pH 5 for 60 to 90
minutes.
73. A pharmaceutical composition for oral administration, which is
self-emulsifiable on contact with a hydrophilic phase and a
lipophilic phase, said composition comprising: (i) pancreatin, and
(ii) a system comprising: (a) at least one surfactant selected from
the group consisting of polyethylene glycol sterol ethers,
polyethylene glycol sorbitan fatty acid esters, sugar esters,
polyoxyethylene-polyoxypropylene block copolymers, sodium oleate,
sodium lauryl sulfate, sodium lauryl sarcosinate, sodium dioctyl
sulfosuccinate, sodium cholate, sodium taurocholate, lauroyl
carnitine, palmitoyl carnitine, myristoyl carnitine, alginate
salts, propylene glycol alginate, alkylsulfates, sodium docusate,
carnitines, and a combination thereof; and (b) at least one
co-surfactant which has a hydrophilic lipophilic balance value
below 10; wherein the pharmaceutical composition does not comprise
any active substances to be absorbed into the bloodstream.
74. The pharmaceutical composition of claim 73, wherein the
hydrophilic phase used to form the final emulsion after ingestion
is supplied by the physiological fluid of the digestive milieu.
75. The pharmaceutical composition of claim 73, wherein the
lipophilic phase used to form the final emulsion in the digestive
tract after ingestion is at least partially supplied by the lipids
present in ingested food.
76. The pharmaceutical composition of claim 73, wherein the system
further comprises a lipidic phase.
77. The pharmaceutical composition of claim 76, wherein (a) the
surfactant comprises at least one agent having a
hydrophilic-lipophilic balance value above 6 and below 18, and (b)
the co-surfactant comprises at least one agent having a
hydrophilic-lipophilic balance value below 10; and wherein said
system comprising surfactant, co-surfactant and lipidic phase has a
hydrophilic-lipophilic balance value of about 4 to 16, and a
melting point of at least 20.degree. C.
78. The pharmaceutical composition of claim 73, wherein said
composition is a solid pharmaceutical preparation in the form of a
powder, granules, tablets, or pellets.
79. The pharmaceutical composition of claim 73, wherein the
pancreatin makes up 65-85% of the pharmaceutical composition.
80. The pharmaceutical composition of claim 73, wherein the
composition does not contain an enteric coating and wherein at
least 90% of the lipolytic activity of the pancreatin is retained
upon exposure of the composition to a buffer having pH 5 for 60 to
90 minutes.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority from U.S. provisional
patent application No. 60/554,993, filed Mar. 22, 2004, the entire
disclosure of which is incorporated herein by reference. Convention
priority is also claimed based on European patent application no.
04 10 1164.4, filed Mar. 22, 2004, the disclosure of which is
likewise incorporated by reference herein.
BACKGROUND OF THE INVENTION
[0002] The invention relates to novel pharmaceutical compositions
of lipase-containing products for oral administration, in
particular pancreatin and pancreatin-containing products, or of
enzyme products containing at least one lipase of non-animal,
especially microbial origin, in which the pharmaceutical
compositions provide improved lipolytic activity, in particular a
stabilization of the lipase in the acidic pH range. These novel
pharmaceutical compositions contain a system which comprises at
least one surfactant and one co-surfactant, and are
self-emulsifiable on contact with a hydrophilic and a lipophilic
phase. The pharmaceutical compositions of the invention are well
suited for the treatment and/or inhibition of maldigestion, in
particular maldigestion based on chronic exocrine pancreatic
insufficiency, in mammals and humans.
[0003] Maldigestion in mammals and humans is usually based on a
deficiency of digestive enzymes, in particular on a deficiency of
endogenous lipase, but also of protease and/or amylase. The cause
of such a deficiency of digestive enzymes is frequently a
hypofunction of the pancreas (=pancreatic insufficiency), the organ
which produces the most, and the most important, endogenous
digestive enzymes. If the pancreatic insufficiency is pathological,
this may be congenital or acquired. Acquired chronic pancreatic
insufficiency may, for example, be ascribed to alcoholism.
Congenital pancreatic insufficiency may, for example, be ascribed
to the congenital disease cystic fibrosis. Consequences of the
deficiency of digestive enzymes may include severe symptoms of
under-nutrition and malnutrition, which may be accompanied by
increased susceptibility to secondary illnesses.
[0004] Substitution with similarly-acting exogenous digestive
enzymes or mixtures of digestive enzymes has proved effective
treatment for a deficiency in endogenous digestive enzymes. At
present, pharmaceutical preparations (=preparations) which contain
porcine pancreatin (=pancreatin) are frequently used for this
purpose. Such mixtures of digestive enzymes obtained from the pig
pancreas comprise lipases, amylases and proteases, and can be used
effectively for enzyme substitution therapy in humans due to the
great similarity of the enzymes and accompanying substances
contained therein to the contents of human pancreatic juices. For
example, processes are described in U.S. Pat. No. 4,019,958 (=DE 25
12 746) and German Patent Publication No. DE 42 03 315 by which
pancreatin is obtained as a natural enzyme mixture by extraction
from porcine pancreas and subsequently is converted in a known
manner into the desired pharmaceutical form. The pancreatic enzymes
are usually administered orally in the form of solid preparations.
Pancreatin is thus commercially available, for example under the
trade name Kreon.RTM., in the form of granules, pellets or capsules
with enteric-coated micropellets.
[0005] In order that, when taken orally, the administered enzyme
mixtures are not irreversibly denatured in the stomach by gastric
acid and proteolytic enzymes, such as pepsin present there, it is
necessary to provide the enzyme mixtures with an enteric coating.
Such a coating enables the enzyme mixtures to pass intact through
the stomach to their point of action, the duodenum, where, due to
the neutral to slightly alkaline conditions prevailing there, the
protective layer is broken down and the enzymes are released. Like
the endogenous pancreatic enzymes of healthy humans, the orally
supplied enzymes can exert their enzymatic action, in particular
amylolytic, lipolytic and proteolytic activity, there. Such solid
pancreatin formulations which can be coated with an enteric film
are described e.g. in U.S. Pat. No. 4,280,971 (=EP 21, 129).
[0006] U.S. Pat. No. 5,378,462 (=EP 583,726) describes pancreatin
micropellet cores coatable with an enteric film having a pancreatin
content of 65-85%, in particular 75-80%, by weight which have a
bulk density of 0.6 g/ml to 0.85 g/ml, consisting essentially of
pancreatin, polyethylene glycol 4000 and low-viscosity paraffin,
containing per 100 parts by weight pancreatin: 15-50, in particular
20-30, parts by weight polyethylene glycol 4000; and 1.5-5, in
particular 2-3, parts by weight low-viscosity paraffin, and having
a spherical to ellipsoid form, the sphere diameter or the minor
axis being in the range of 0.7-1.4 mm, in particular 0.8-1.2 mm,
and having a particle-size distribution in which at least 80% of
the pancreatin micropellet cores have a ratio of minor axis to
major axis in the range of 1:1 to 1:2.
[0007] Furthermore, U.S. Pat. No. 5,993,806 (=EP 826,375) describes
the use of lecithin as a stabilizing agent added to water-soluble
pharmaceutical preparations of mixtures of digestive enzymes which
contain protease/lipase mixtures, in particular pancreatin, and
which are suitable for the preparation of aqueous solutions for
continuous introduction into the gastrointestinal tract via probes.
The lecithin is added to stabilize the mixtures of digestive
enzymes against a decrease in the lipolytic activity under the
influence of moisture.
[0008] In the case of pharmaceutical formulations not coated with
enteric films, it is known that at the point of action of the
enzymes, in the duodenum, often only a very small proportion of the
lipase contained in the pharmaceutical preparation and taken
therewith is active. Thus in German Patent Publication No. DE 36 42
853 such enzyme deactivation is ascribed to insufficient
neutralization of the gastric acid in the duodenum. Whereas in a
healthy human the postprandial intraduodenal pH value is about 6,
patients with pancreatic insufficiency only have a pH value of
about 4. At this pH value, the lipase contained in the
pharmaceutical preparation has only one fifth of the activity that
it would otherwise have at a pH value of 6.
SUMMARY OF THE INVENTION
[0009] It is therefore an object of the invention to provide
pharmaceutical compositions which contain enzymes or enzyme
mixtures with at least lipolytic activity and have improved
lipolytic activity and in particular show a stabilization of the
lipase activity in the acidic pH range.
[0010] According to the invention, pharmaceutical compositions
intended for oral administration are provided which comprise
enzymes or enzyme mixtures with at least lipolytic activity and a
system comprising at least one surfactant and at least one
co-surfactant. The pharmaceutical compositions of the invention are
characterized in that they form emulsions upon contact with a
hydrophilic phase and a lipophilic phase. Preferably the
hydrophilic phase used to form the final emulsion after ingestion
of the pharmaceutical composition is supplied by the physiological
fluid of the digestive milieu. In a further embodiment of the
present invention, the lipophilic phase used to form the final
emulsion in the digestive tract after ingestion of the
pharmaceutical composition is at least partially supplied by the
lipids present in the food ingested. In particular, to achieve this
object the invention provides pharmaceutical compositions which
contain systems which comprise at least one surfactant, one
co-surfactant and a lipophilic phase.
[0011] Surprisingly, a lipase-containing pharmaceutical composition
containing such a system has improved lipolytic activity and a
lipolytic activity which is stabilized in the acidic pH range. The
use of such a system in pharmaceutical compositions of enzymes or
enzyme mixtures with at least lipolytic activity furthermore has
the advantage that pharmaceutical compositions containing such
enzymes or enzyme mixtures can also be used without enteric
coatings, such as are described for example in U.S. Pat. No.
5,378,462. In the pharmaceutical compositions according to the
invention the reduction in the lipolytic activity during passage
through the stomach is very much less than with pharmaceutical
compositions prepared without the aforementioned system. The system
consisting of surfactant, co-surfactant and optionally a lipophilic
phase, stabilizes the lipolytic activity of the pharmaceutical
compositions according to the invention in the acidic pH range of
the stomach compared with conventional formulations.
[0012] The fact that the use of such enteric-coated polymer films
and softeners which are otherwise necessary for film-coating
various medicament forms (granules, pellets, mini-tablets, tablets
etc.) can be omitted in the preparation of the lipase-containing
compositions according to the invention yields further advantages.
Thus the safety profile of the pharmaceutical composition is
improved by omitting the enteric polymer films and softeners, since
unnecessary taking thereof is avoided. Furthermore, the proportions
of the amount of film-coating material in the medicament forms
provided with an enteric film is approx. 20-30% of the entire
weight of the medicament form. The ability to omit these additives
makes the amount of pharmaceutical composition to be taken smaller,
which results in better acceptance by patients.
[0013] The possibility of omitting enteric coating of the enzymes
or enzyme mixtures furthermore has the advantage that thorough
mixing of the pharmaceutical preparation with the chyme can take
place as early as in the stomach. Thereupon, there forms an
emulsion or micro-emulsion with enlarged surface, on which the
lipase contained in the pharmaceutical composition is distributed
such that it is given optimum possibilities for attack for breaking
down the triglycerides found in the chyme. The formation of
emulsion and microemulsion is further intensified by the lipolytic
breakdown of the triglycerides to form di- and monoglycerides and
free fatty acids. The improved possibilities of attack for the
lipase thus result in intensified breakdown of the triglycerides.
The higher concentration of free fatty acids resulting from the
food provided in this way results in better fat absorption in the
duodenum. In vitro, an increase in the lipolytic activity by about
10% compared with conventional lipase-containing pharmaceutical
preparations was determined for the pharmaceutical composition
according to the invention. The pharmaceutical compositions
according to the invention thus exhibit stabilization of the
lipolytic activity in the stomach as well as in the duodenum.
Additionally, due to the intensified formation of a
(micro)emulsion, the lipolytic activity is increased. The
(micro)emulsion already produced independently in the stomach
results in better activation of the lipase contained in the
pharmaceutical composition.
[0014] Self-emulsifying pharmaceutical compositions in general are
already known from the prior art. Thus, for example, U.S. Pat. No.
6,054,136 (=EP 670,715) describes a perorally administered
composition which is suitable for forming a microemulsion in situ
with the biological liquid of the organism and thus is said to
improve the biological availability of an active substance. Such
pharmaceutical compositions are known under the term SMEDDS.RTM.
(Self Microemulsifying Drug Delivery System) and consist in
principle of a mixture of one or more active substances with a
defined lipophilic phase, a defined surfactant and a defined
co-surfactant, the properties of which are specified such that the
end product is capable of forming a microemulsion on contact with a
given volume of physiological liquid.
[0015] Furthermore, US 2003/021844 (=EP 1,058,540) describes what
is called a SMEDDS.RTM. formulation in a particular pharmaceutical
form, which is referred to as "pellet". These pellets are composed
of an active substance, in particular indomethacin, a binding agent
which is suitable for improving the biological availability of the
active substance, for example Gelucire.RTM. 44/14, and a diluent,
for example lactose, in micronized form.
[0016] The object of prior art systems which automatically form a
microemulsion was, however, always to increase the bioavailability
of mostly lipophilic active substances in that the SMEDDS.RTM.
formulation, as a result of the micelle formation, permits better
absorption of the active substance through the duodenal wall into
the blood circulation. In contrast, the aim of the present
invention is to provide a pharmaceutical composition which does not
contain any lipophilic active substances to be absorbed into the
bloodstream, but which provides as active agent enzymes or enzyme
mixtures with at least lipolytic activity which develop their
action in the gastrointestinal tract. The self-emulsifiable
pharmaceutical compositions according to the invention result in a
surprising increase of the lipolytic activity contained therein and
in an improved stability of the lipase in the acidic pH range. Such
pharmaceutical compositions of lipase-containing enzyme products
which are self-emulsifiable on contact with a hydrophilic phase and
which comprise a system consisting of a surfactant, a co-surfactant
and optionally a lipophilic phase have not been described in the
prior art.
[0017] Subramanian and Wasan describe an assay in which they
demonstrate that the substance Gelucire.RTM. 44/14 in vitro has an
inhibiting effect on the pancreatic lipase activity [Subramanian R.
& Wasan K. M. (2003) "Effect of lipid excipients on in vitro
pancreatic lipase activity" Drug Dev. Ind. Pharm. 29(8): 885-90].
In this experiment, a particular lipid-containing assay buffer with
separate solutions of Gelucire.RTM. 44/14, pancreatic lipase and
co-lipase is mixed, and the influence of Gelucire.RTM. on the
lipase activity is measured. Since the lipase activity decreases,
the authors conclude that Gelucire.RTM. and similar lipidic
additions to pharmaceutical formulations can have an adverse effect
on the in vitro activity of the pancreatic lipase. In contrast, the
present invention shows that self-emulsifiable pharmaceutical
compositions consisting of lipase-containing enzyme mixtures and a
system such as for example Gelucire.RTM. 44/14 result in an
increase in the lipolytic activity contained in the pharmaceutical
formulation.
[0018] Some expressions as used in the context of the present
invention are explained in more detail below.
[0019] The "hydrophilic-lipophilic balance" (=HLB) value is an
empirical parameter commonly used to characterize the relative
hydrophilicity and lipophilicity of non-ionic amphiphilic
compounds. Surfactants or co-surfactants with lower HLB values are
more lipophilic and have greater solubility in oils, whereas
surfactants or co-surfactants with higher HLB values are more
hydrophilic and have greater solubility in aqueous solutions. It
should be kept in mind that for anionic, cationic, or zwitterionic
compounds the HLB scale is not generally applicable.
[0020] Generally, the HLB value of a surfactant or co-surfactant is
a practical guide used to enable formulation of industrial,
pharmaceutical and cosmetic emulsions. However, for many important
surfactants, including several polyethoxylated surfactants, it has
been reported that HLB values can differ by as much as about 8 HLB
units depending upon the empirical method chosen to determine the
HLB value [Schott, J. Pharm. Sciences, 79(1), 87-88 (1990)].
Likewise, for certain polypropylene oxide containing block
copolymers (poloxamers), the HLB values may not accurately reflect
the true physical/chemical nature of the compounds. Finally,
commercial surfactant and/or co-surfactant products are generally
not pure compounds, but are often complex mixtures of compounds,
and the HLB value reported for a particular compound may more
accurately be characteristic of the commercial product of which the
compound is a major component. Different commercial products having
the same primary surfactant and/or co-surfactant component can, and
typically do, have different HLB values. In addition, a certain
amount of lot-to-lot variability is expected even for a single
commercial surfactant and/or co-surfactant product.
[0021] A "surfactant" in the context of the present invention is a
chemical compound comprising two groups, the first being
hydrophilic and/or polar or ionic and having a high affinity for
water, and the second containing an aliphatic chain of greater or
lesser length and being hydrophobic (lipophilic); i.e., a
surfactant compound must be amphiphilic. These chemical compounds
are intended to cause the formation and stabilization of
oil-in-water emulsions. Surfactants with lower HLB values are more
lipophilic and have greater solubility in oils, whereas surfactants
with higher HLB values are more hydrophilic and have greater
solubility in aqueous solutions. Suitable surfactants in the
context of the present invention have an HLB value above 6 and
below 18, preferably above 8 and below 16. Surfactants can be any
surfactant suitable for use in pharmaceutical compositions.
Suitable surfactants can be anionic, cationic, zwitterionic or
non-ionic. Such surfactants can be grouped into some general
chemical classes as explained below. It should be emphasized that
the invention is not limited to the surfactants indicated herein,
which show representative, but not exclusive, lists of available
surfactants.
[0022] "PEG-Fatty Acid Monoester Surfactants":
[0023] Although polyethylene glycol (PEG) itself does not function
as a surfactant, a variety of PEG-fatty acid esters have useful
surfactant properties. Particulary preferred are PEG-fatty acid
monoesters with aliphatic C.sub.6-C.sub.22 carboxylic acids, in
which the polyethylene glycol comprises 6 to 60 ethylene oxide
units per molecule. Examples of commercially available
polyethoxylated fatty acid monoester surfactants include: PEG-4
laurate, PEG-4 oleate, PEG-4 stearate, PEG-5 stearate, PEG-5
oleate, PEG-6 oleate, PEG-7 oleate, PEG-6 laurate, PEG-7 laurate,
PEG-6 stearate, PEG-8 laurate, PEG-8 oleate, PEG-8 stearate, PEG-9
oleate, PEG-9 stearate, PEG-10 laurate, PEG-10 oleate, PEG-10
stearate, PEG-12 laurate, PEG-12 oleate, PEG-12 ricinoleate, PEG-12
stearate, PEG-15 stearate, PEG-15 oleate, PEG-20 laurate, PEG-20
oleate, PEG-20 stearate, PEG-25 stearate, PEG-32 laurate, PEG-32
oleate, PEG-32 stearate, PEG-30 stearate, PEG 4-100 monolaurate,
PEG 4-100 monooleate, and PEG 4-100 monostearate.
[0024] "PEG-Fatty Acid Diester Surfactants":
[0025] Polyethylene glycol (PEG) fatty acid diesters are also
suitable for use as surfactants in the compositions of the present
invention. Particularly preferred are PEG-fatty acid diesters with
aliphatic C.sub.6-C.sub.22 carboxylic acids in which the
polyethylene glycol comprises 6 to 60 ethylene oxide units per
molecule. Representative commercially available PEG-fatty acid
diesters include: PEG-4 dilaurate, PEG-4 dioleate, PEG-6 dilaurate,
PEG-6 dioleate, PEG-6 distearate, PEG-8 dilaurate, PEG-8 dioleate,
PEG-8 distearate, PEG-10 dipalmitate, PEG-12 dilaurate, PEG-12
distearate, PEG-12 dioleate, PEG-20 dilaurate, PEG-20 dioleate,
PEG-20 distearate, PEG-32 dilaurate, PEG-32 dioleate, and PEG-32
distearate.
[0026] "PEG-Fatty Acid Mono- and Di-ester Mixtures":
[0027] In general, mixtures of surfactants are also useful in the
present invention, including mixtures of two or more commercial
surfactant products. Particularly preferred are mixtures of
PEG-fatty acid mono- and diesters with aliphatic C.sub.6-C.sub.22
carboxylic acids in which the polyethylene glycol comprises 6 to 60
ethylene oxide units per molecule. Several PEG-fatty acid esters
are marketed commercially as mixtures or mono- and diesters.
Representative commercially available surfactant mixtures include:
PEG 4-150 mono, dilaurate; PEG 4-150 mono, dioleate; and PEG 4-150
mono, distearate.
[0028] "Polyethylene Glycol (PEG) Glycerol Fatty Acid Esters":
[0029] In addition, PEG glycerol fatty acid esters suitable as
surfactants in the context of the present invention include: PEG-20
glyceryl laurate, PEG-30 glyceryl laurate, PEG-15 glyceryl laurate,
PEG-40 glyceryl laurate, PEG-20 glyceryl stearate, PEG-20 glyceryl
oleate, and PEG-30 glyceryl oleate. Particularly preferred are PEG
glycerol fatty acid esters with aliphatic C.sub.6-C.sub.22
carboxylic acids in which the polyethylene glycol comprises 6 to 60
ethylene oxide units per molecule.
[0030] "Polyethylene Glycol (PEG) Alkyl Ethers (Mono- and/or
Diethers of Polyethylene Glycol)":
[0031] Ethers of polyethylene glycol and alkyl alcohols also are
suitable surfactants for use in the present invention. Particularly
preferred are PEG-fatty acid mono- and/or diethers with aliphatic
C.sub.12-C.sub.18 alcohols in which the polyethylene glycol
comprises 6 to 60 ethylene oxide units per molecule. Some
commercially available examples of these surfactants include: PEG-2
oleyl ether (oleth-2), PEG-3 oleyl ether (oleth-3), PEG-5 oleyl
ether (oleth-5), PEG-10 oleyl ether (oleth-10), PEG-20 oleyl ether
(oleth-20), PEG-4 lauryl ether (laureth-4), PEG-9 lauryl ether,
PEG-23 lauryl ether (laureth-23), PEG-2 cetyl ether, PEG-10 cetyl
ether, PEG-20 cetyl ether, PEG-2 stearyl ether, PEG-10 stearyl
ether, and PEG-20 stearyl ether.
[0032] "Polyethylene Glycol Sterol Ethers":
[0033] PEG-derivatives of sterols are suitable surfactants for use
in the present invention. Examples of surfactants of this class
include: PEG-24 cholesterol ether, PEG-30 cholestanol, PEG-25
phytosterol, PEG-5 soya sterol, PEG-10 soya sterol, PEG-20 soya
sterol, and PEG-30 soya sterol.
[0034] "Polyethylene Glycol Sorbitan Fatty Acid Esters":
[0035] A variety of PEG-sorbitan fatty acid esters are available
and are suitable for use as surfactants in the present invention.
Examples of these surfactants include: PEG-10 sorbitan laurate,
PEG-20 sorbitan monolaurate, PEG-4 sorbitan monolaurate, PEG-80
sorbitan monolaurate, PEG-6 sorbitan monolaurate, PEG-20 sorbitan
monopalmitate, PEG-20 sorbitan monostearate, PEG-4 sorbitan
monostearate, PEG-8 sorbitan monostearate, PEG-6 sorbitan
monostearate, PEG-20 sorbitan tristearate, PEG-60 sorbitan
tetrastearate, PEG-5 sorbitan monooleate, PEG-6 sorbitan
monooleate, PEG-20 sorbitan monooleate, PEG-40 sorbitan oleate,
PEG-20 sorbitan trioleate, PEG-6 sorbitan tetraoleate, PEG-30
sorbitan tetraoleate, PEG-40 sorbitan tetraoleate, PEG-20 sorbitan
monoisostearate, and PEG sorbitol hexaoleate.
[0036] "Sugar Esters":
[0037] Esters of sugars, in particular mono-esters are suitable
surfactants for use in the present invention. Examples of such
surfactants include: sucrose distearate/monostearate, sucrose
dipalmitate, sucrose monostearate, sucrose monopalmitate, sucrose
monolaurate, and saccharose monolaurate.
[0038] "Polyoxyethylene-Polyoxypropylene Block Copolymers":
[0039] The POE-POP block copolymers are a unique class of polymeric
surfactants. The unique structure of the surfactants, with
hydrophilic POE and lipophilic POP moieties in well-defined ratios
and positions, provides a wide variety of surfactants suitable for
use in the present invention. The generic term for these polymers
is "poloxamer" (CAS 9003-11-6). These polymers have the formula:
HO(C.sub.2H.sub.4O).sub.a(C.sub.3H.sub.6O).sub.b(C.sub.2H.sub.4O).sub.aH,
where "a" and "b" denote the number of polyoxyethylene and
polyoxypropylene units, respectively.
[0040] Furthermore, "amphoteric compounds" such as fatty
acid-amidoalkyl betaines with C.sub.2-C.sub.22 fatty acids are
suitable surfactants.
[0041] The surfactant can also be, or include as a component, an
"ionic surfactant," including cationic, anionic and zwitterionic
surfactants. Preferred anionic surfactants include fatty acid salts
and bile salts. Preferred cationic surfactants include carnitines.
Specifically, preferred ionic surfactants include sodium oleate,
sodium lauryl sulfate, sodium lauryl sarcosinate, sodium dioctyl
sulfosuccinate, sodium cholate, sodium taurocholate; lauroyl
carnitine; palmitoyl carnitine; myristoyl carnitine, alginate
salts; propylene glycol alginate; lecithins and hydrogenated
lecithins; lysolecithin and hydrogenated lysolecithins;
lysophospholipids and derivatives thereof; phospholipids and
derivatives thereof; salts of alkylsulfates; sodium docusate;
carnitines; and mixtures thereof.
[0042] More specifically, examples of preferred ionic surfactants
include lecithin, lysolecithin, phosphatidylcholine,
phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid,
phosphatidylserine, lysophosphatidylcholine,
lysophosphatidylethanolamine, lysophosphatidylglycerol,
lysophosphatidylinositol, lysophosphatidic acid,
lysophosphatidylserine, PEG-phosphatidylethanolamine,
PVP-phosphatidylethanolamine, stearoyl-2-lactylate, stearoyl
lactylate, cholate, taurocholate, glycocholate, deoxycholate,
taurodeoxycholate, chenodeoxycholate, glycodeoxycholate,
glycochenodeoxycholate, taurochenodeoxycholate, ursodeoxycholate,
tauroursodeoxycholate, glycoursodeoxycholate, cholylsarcosine,
N-methyl taurocholate, caproate, caprylate, caprate, laurate,
myristate, palmitate, oleate, ricinoleate, linoleate, linolenate,
stearate, lauryl sulfate, teracecyl sulfate, docusate, lauroyl
carnitines, palmitoyl carnitines, myristoyl carnitines, and salts
and mixtures thereof.
[0043] A "co-surfactant," sometimes also referred to as a
"co-emulsifier", in the context of the present invention is a
chemical compound which has both hydrophobic (lipophilic) and
hydrophilic portions, but with the hydrophobic (lipophilic) nature
predominating. It is intended to make the aqueous and oily phases
in a microemulsion mutually soluble. Suitable co-surfactants in the
context of the present invention have an HLB value below 10,
preferably below 8 and even more preferably below 6. Co-surfactants
can be any partial esters and/or partial ethers of polyhydric
(polyvalent) alcohols, such as glycerol, propylenglycol
(1,2-propanediol; 1,2-dihdroxypropane), ethyl-diglycol or even
polyglycerols (such as diglycerol, triglycerol, tetraglycerol etc.)
with aliphatic carboxylic acids (fatty acids) or aliphatic alcohols
(fatty alcohols).
[0044] Further co-surfactants, which can be grouped into some
general chemical classes, are given below. It should be emphasized
that the invention is not limited to the co-surfactants mentioned
herein, which show representative, but not exclusive, lists of
available co-surfactants.
[0045] "Mono-Glycerides":
[0046] A particularly important class of co-surfactants is the
class of mono-glycerides which are generally lipophilic.
Particulary preferred are mixtures of monoglycerides with aliphatic
C.sub.6-C.sub.22 carboxylic acids. Examples of this class of
co-surfactants include: monopalmitolein (C16:1), monoelaidin
(C18:1), monocaproin (C6), monocaprylin, monocaprin, monolaurin,
glyceryl monomyristate (C14), glyceryl monooleate (C18:1), glyceryl
monooleate, glyceryl monolinoleate, glyceryl ricinoleate, glyceryl
monolaurate, glyceryl monopalmitate, glyceryl monostearate,
glyceryl monopalmate, glycerol monostearate, glyceryl caprylate,
and glyceryl caprate as well as mixtures thereof.
[0047] "Polyglycerized Fatty Acids":
[0048] Polyglycerol esters of fatty acids, in particular
Polyglycerol mono-esters, are also suitable co-surfactants for the
present invention. Particulary preferred are mixtures of
polyglycerol esters with aliphatic C.sub.6-C.sub.22 carboxylic
acids. Examples of suitable commercially available polyglyceryl
esters include: polyglyceryl-2 stearate, polyglyceryl-2 oleate,
polyglyceryl-2 isostearate, polyglyceryl-3 oleate, polyglyceryl-4
oleate, polyglyceryl-4 stearate, polyglyceryl-6 oleate,
polyglyceryl-2 dioleate and polyglyceryl-6 dioleate.
[0049] "Propylene Glycol Fatty Acid Esters":
[0050] Partial esters of propylene glycol and fatty acids, in
particular mono-esters, are suitable co-surfactants for use in the
present invention. Particularly preferred are mixtures of propylene
glycol esters with aliphatic C.sub.6-C.sub.22 carboxylic acids.
Examples of co-surfactants of this class include: propylene glycol
monocaprylate, propylene glycol monolaurate, propylene glycol
oleate, propylene glycol myristate, propylene glycol monostearate,
propylene glycol hydroxy stearate, propylene glycol ricinoleate,
propylene glycol isostearate, propylene glycol monooleate,
propylene glycol dicaprylate/dicaprate, propylene glycol
dioctanoate, propylene glycol caprylate/caprate, propylene glycol
dilaurate, propylene glycol distearate, propylene glycol
dicaprylate, and propylene glycol dicaprate.
[0051] A "lipophilic phase" in the context of the present invention
is understood to mean a water-immiscible liquid. The lipophilic
phase may also be referred to as a lipidic phase. For compositions
of the present invention in which the system also includes a
lipophilic component, the lipophilic component is preferably a
triglyceride or a mixture of a triglyceride and a diglyceride.
Suitable lipophilic phases are preferably di- and triacylglycerides
of aliphatic carboxylic acids (fatty acids) with 4 to 22 carbon
atoms, in particular with 6 to 22 carbon atoms, and also mixtures
thereof.
[0052] Preferred "di-glycerides" in the context of the present
invention are mixtures of diglycerides with aliphatic
C.sub.6-C.sub.22 carboxylic acids. Examples include: glyceryl
dioleate, glyceryl dipalmitate, glyceryl dilaurate, glyceryl
dilinoieate, glyceryl dicaprylate, glyceryl dicaprate, glyceryl
caprylate/caprate, glyceryl distearate, glyceryl
stearate/palmitate, glyceryl oleate/linoleate and glyceryl
dimyristate.
[0053] Preferred "triglycerides" are those which solidify at
ambient room temperature, with or without addition of appropriate
additives, or those which in combination with particular
surfactants and/or co-surfactants and/or active ingredients
solidify at room temperature. Examples of triglycerides suitable
for use in the present invention include: aceituno oil, almond oil,
araehis oil, babassu oil, beeswax, black currant seed oil, borage
oil, buffalo ground oil, candlenut oil, canola oil, castor oil,
Chinese vegetable tallow oil, cocoa butter, coconut oil, coffee
seed oil, corn oil, cottonseed oil, crambe oil, cuphea species oil,
evening primrose oil, grapeseed oil, groundnut oil, hemp seed oil,
illipe butter, kapok seed oil, linseed oil, menhaden oil, mowrah
butter, mustard seed oil, oiticica oil, olive oil, palm oil, palm
kernel oil, peanut oil, poppy seed oil, rapeseed oil, rice bran
oil, safflower oil, sal fat, sesame oil, shark liver oil, shea nut
oil, soybean oil, stillingia oil, sunflower oil, tall oil, tea seed
oil, tobacco seed oil, tung oil (China wood oil), ucuhuba, vernonia
oil, wheat germ oil, hydrogenated castor oil, hydrogenated coconut
oil, hydrogenated cottonseed oil, hydrogenated palm oil,
hydrogenated soybean oil, hydrogenated vegetable oil, hydrogenated
cottonseed and castor oil, partially hydrogenated soybean oil,
partially hydrogenated soy and cottonseed oil, glyceryl mono-, di-,
tri-behenate, glycerol tributyrate, glyceryl tricaproate, glyceryl
tricaprylate, glyceryl tricaprate, glyceryl triundecanoate,
glyceryl trilaurate, glyceryl trimyristate, glyceryl tripalmitate,
glyceryl tristearate, glyceryl triarchidate, glyceryl
trimyristoleate, glyceryl tripalmitoleate, glyceryl trioleate,
glyceryl trilinoleate, glyceryl trilinolenate, glyceryl
tricaprylate/caprate, glyceryl tricaprylate/caprate/laurate,
glyceryl tricaprylate/caprate/linoleate, glyceryl
tricaprylate/caprate/stearate, glyceryl
tricaprylate/laurate/stearate, glyceryl 1,2-caprylate-3-linoleate,
glyceryl 1,2-caprate-3-stearate, glyceryl 1,2-laurate-3-myristate,
glyceryl 1,2-myristate-3-laurate, glyceryl
1,3-palmitate-2-butyrate, glyceryl 1;3-stearate-2-caprate, glyceryl
1,2-linoleate-3-caprylate.
[0054] Fractionated triglycerides, modified triglycerides,
synthetic triglycerides, and mixtures of triglycerides are also
within the scope of the invention. Preferred triglycerides include
vegetable oils, fish oils, animal fats, hydrogenated vegetable
oils, partially hydrogenated vegetable oils, medium and long-chain
triglycerides, and structured triglycerides.
[0055] Furthermore, the following compounds may be suitable as
"lipophilic phase": low-viscosity and high-viscosity aliphatic
hydrocarbons, and also in particular oleic acid oleyl ester,
isooctyl stearate, lauric acid hexyl ester, di-n-butyl adipate,
isopropyl myristate, isopropyl palmitate and isopropyl stearate,
oleyl alcohol, ethereal oils, isopropyl caprylate, isopropyl
caprinate and isopropyl laurate.
[0056] "Complete Systems Composed of Surfactant, Co-Surfactant and
Lipophilic Phase"
[0057] Several commercial surfactant and/or co-surfactant
compositions contain small to moderate amounts of di- and
triglycerides, typically as a result of incomplete reaction of a
triglyceride starting material in, for example, a
transesterification reaction. Such commercial surfactant and/or
co-surfactant compositions, while nominally referred to as
"surfactants" and/or "co-surfactant", may be suitable to
provide--in addition to the surfactant and/or co-surfactant part of
the system--all or part of the lipophilic component, i.e. the di-
and triglyceride component, for the compositions of the present
invention.
[0058] Still other commercial surfactant and/or co-surfactant
compositions having significant di- and triglyceride content are
known to those skilled in the art. It should be appreciated that
such compositions, which contain di- and triglycerides as well as
surfactants and/or co-surfactants, may be suitable to provide the
complete system composed of surfactant, co-surfactant and
lipophilic phase, of the compositions of the present invention.
Typical examples for such kind of systems are so-called
macrogolglycerides (or polyoxyethylated glycerides) with different
kinds of fatty acids. Macrogolglycerides are mixtures of
mono-esters, di-esters and tri-esters of glycerol and mono-ester
and di-ester of PEG (=polyethylene glycol, macrogol,
polyoxyethlene, polyethylene oxide, polyglycol) with fatty acids,
whereby the molecular mass of the PEG may be defined as well as the
nature of the fatty acids. Macrogolglycerides can be obtained by a
partial hydrolyzis/esterification reaction of triglycerides using
the respective macrogol. Alternatively, macrogolglycerides can be
obtained by esterification of glycerol and the macrogol and the
corresponding free fatty acids. As triglycerides a variety of
natural and/or hydrogenated oils can be used. Most commonly used
oils are castor oil or hydrogenated castor oil or an edible
vegetable oil such as corn oil, olive oil, peanut oil, palm kernel
oil, apricot kernel oil, or almond oil, or the corresponding
hydrogenated vegetable oil.
[0059] Typically, such transesterification products of oils and
polyethylenglycol (or other polyalcohols) are named by there
educts: PEG-20 castor oil, PEG-23 castor oil, PEG-30 castor oil,
PEG-35 castor oil, PEG-38 castor oil, PEG-40 castor oil, PEG-50
castor oil, PEG-56 castor oil, PEG-7 hydrogenated castor oil,
PEG-10 hydrogenated castor oil, PEG-20 hydrogenated castor oil,
PEG-25 hydrogenated castor oil, PEG-30 hydrogenated castor oil,
PEG-40 hydrogenated castor oil, PEG-45 hydrogenated castor oil,
PEG-50 hydrogenated castor oil, PEG-60 hydrogenated castor oil,
PEG-80 hydrogenated castor oil, PEG-8 corn oil, PEG-20 corn oil,
PEG-20 almond oil, PEG-25 trioleate, PEG-40 palm kernel oil, PEG-60
corn oil, PEG-60 almond oil, PEG-8 caprylic/capric glycerides,
lauroyl macrogol-32 glyceride (=PEG-32 hydrogenated palm kernel
oil, e.g. Gelucire.RTM. 44/14), stearoyl macrogol glyceride (e.g.
Gelucire.RTM. 50/13)
[0060] Examples of commercial co-surfactant compositions of
mono-glycerides, in addition containing di- and triglycerides
include some members of the co-surfactant families Maisines.RTM.
(Gattefosse) and Imwitors.RTM. (Huls). These commercial
compositions may be used for providing the co-surfactant and the
lipophilic phase in one composition. Specific examples of these
compositions are: Maisine.RTM. 35-I (linoleic glycerides) and
Imwitor.RTM. 742 (caprylic/capric glycerides).
[0061] "Aliphatic Carboxylic Acids with 6 to 22 Carbon Atoms":
[0062] In the context of the present invention, aliphatic
carboxylic acids with 6 to 22 carbon atoms are understood to be
aliphatic C.sub.6-C.sub.22 carboxylic acids. Thus preferably
carboxylic acids selected from the group containing caproic acid
(C6), caprylic acid (C8), capric acid (C10), lauric acid (C12),
myristic acid (C14), palmitic acid (C16), stearic acid (C18),
arachidic acid (C20), and behenic acid (C22), as well as the
corresponding unsaturated carboxylic acids, such as palmitoleic
acid (C16), oleic acid (C18), linoleic acid (C18), linolenic acid
(C18), eicosenoic acid (C20), individually or as a mixture, are
used. Particularly preferably, the saturated carboxylic acids are
selected.
[0063] "Aliphatic Alcohols with 12 to 18 Carbon Atoms":
[0064] In the context of the present invention, aliphatic alcohols
with 12 to 18 carbon atoms are understood to be aliphatic
C.sub.12-C.sub.18 alcohols. Preferably alcohols selected from the
group consisting of lauryl alcohol (C12), myristyl alcohol (C14),
cetyl alcohol (C16), stearyl alcohol (C18), oleyl alcohol (C18),
linoleyl alcohol (C18) and linolenyl alcohol (C18), individually or
as a mixture, are used. Particularly preferably, the saturated
alcohols are selected.
[0065] "Aliphatic Alcohols with 12 to 22 Carbon Atoms":
[0066] In the context of the present invention, aliphatic alcohols
with 12 to 22 carbon atoms are understood to be aliphatic
C.sub.12-C.sub.22 alcohols. Preferably alcohols selected from the
group consisting of lauryl alcohol (C12), myristyl alcohol (C14),
cetyl alcohol (C16), stearyl alcohol (C18), arachidyl alcohol
(C20), behenyl alcohol (C22), oleyl alcohol (C18), linoleyl alcohol
(C18) and linolenyl alcohol (C18), individually or as a mixture,
are used. Particularly preferably, the saturated alcohols are
selected.
[0067] The "hydrophilic phase" in the context of the present
invention is understood in particular to mean an aqueous phase
which is preferably supplied by the physiological liquid of the
digestion medium and/or by an aqueous liquid ingested in parallel
with the food and/or the pharmaceutical preparation.
[0068] "Enzymes or enzyme mixtures with at least lipolytic
activity" in the context of the present invention are understood to
mean physiologically acceptable enzyme mixtures which contain at
least one lipase. The enzymes or enzyme mixtures may, however, also
have proteolytic activity in addition to the lipolytic activity,
i.e. contain at least one protease, and/or amylolytic activity,
i.e. contain at least one amylase.
[0069] Enzymes or enzyme mixtures may be used which exhibit (i)
purely lipolytic; or (ii) lipolytic and proteolytic; or (iii)
lipolytic and amylolytic; or (iv) lipolytic, proteolytic and
amylolytic activity. Suitable enzymes or enzyme mixtures may be of
any animal or microbiological origin. The enzyme mixtures with at
least lipolytic, and optionally also proteolytic and/or amylolytic
activity used in the context of the invention may therefore be of
purely microbial origin or of purely animal origin, or
alternatively represent a mixture of enzymes of animal and
microbial origin.
[0070] Lipase-containing enzyme products of non-animal origin as
well as preparations thereof, are enzyme mixtures comprising at
least one lipase and optionally also at least one protease and/or
amylase. These enzymes may be plant-derived or of fungal or
bacterial origin. These lipases, proteases and/or amylases may, for
example, be obtained by fermentation of optionally recombinant
bacteria or fungi. The lipase-containing enzyme products may be
composed of purely microbial derived enzyme preparations (i.e.
enzymes obtained from fungi or bacteria) or enzyme preparations
obtained from plants, but also of synthetic mixtures of enzyme
preparations from plants, bacteria and/or fungi, optionally
produced recombinantly in a microbial system. Furthermore, the
recombinantly produced enzyme may be an enzyme variant or a mutated
enzyme which is functionally equivalent or which has structural
features similar to a naturally occurring enzyme.
[0071] By "recombinantly produced microbial enzyme", in particular
"recombinantly produced lipase, amylase or protease", is meant an
enzyme produced by recombinant DNA-technology, the enzyme being of
microbial origin, i.e. obtained from fungi or bacteria. In the
context of this invention suitable lipases include recombinantly
produced microbial lipases that possess lipolytic activity,
preferably at relatively low pH. In the context of this invention
suitable proteases include recombinantly produced microbial
proteases that possess proteolytic activity, preferably at
relatively low pH. In the context of this invention suitable
amylases include recombinantly produced microbial amylases that
possess amylolytic activity, preferably at relatively low pH.
[0072] The recombinantly produced microbial enzyme, i.e. the
lipase, amylase or protease, may be an enzyme variant or a mutated
enzyme which is functionally equivalent or which has structural
features similar to a naturally occurring enzyme.
[0073] Preferred recombinantly produced microbial lipases include
lipases derived from fungi, e.g. from Humicola, Rhizomucor,
Rhizopus, Geotrichum or Candida species, in particular Humicola
lanuginosa (Thermomyces lanuginosa), Rhizomucor miehei, Rhizopus
javanicus, Rhizopus arrhizus, Rhizopus oryzae, Rhizopus delamar,
Candida cylindracea, Candida rugosa or Geotrichum candidum; or may
be derived from bacteria, e.g. from Pseudomonas, Burkholderia or
Bacillus species, in particular Burkholderia cepacia. Most
preferred are lipases derived from a strain of Humicola lanuginosa
(Thermomyces lanuginosa) or Rhizomucor miehei.
[0074] Lipases of microbial origin which can be used in the context
of the present invention and their production by e.g. recombinant
technology are described, for example, in U.S. Pat. No. 5,614,189
(=EP 600,868), U.S. Pat. No. 5,766,912 (=EP 238,023), U.S. Pat. No.
5,536,661 (=EP 305,216), U.S. Pat. No. 6,051,220 (=EP 828,509),
U.S. Pat. No. 5,849,296 (=EP 550,450), US Patent Publication No.
2001/046493 (=EP 1,261368), U.S. Pat. No. 6,140,475 (=EP 973,878)
and U.S. Pat. No. 5,489,530 (=EP 592,478), which publications are
each hereby incorporated by reference.
[0075] Preferred recombinantly produced microbial amylases include
amylases derived from fungi, e.g. from Aspergillus or Rhizopus
species, in particular Aspergillus niger or Aspergillus oryzae; or
may be derived from bacteria, e.g. from Bacillus species, in
particular Bacillus subtilis. Amylases derived from a strain of
Aspergillus oryzae are most preferred.
[0076] Amylases of microbial origin which can be used in the
context of the present invention and their production by
recombinant technology are described in e.g. U.S. Pat. No.
6,051,220 (=EP 828,509) which publication is hereby incorporated by
reference.
[0077] Preferred recombinantly produced microbial proteases include
proteases derived from fungi, e.g. from Aspergillus or Rhizopus
species, in particular Aspergillus melleus, Aspergillus oryzae,
Aspergillus niger, or Rhizopus oryzae; or may be derived from
bacteria, e.g. from Bacillus species, in particular Bacillus
subtilis. Most preferred are proteases derived from a strain of
Aspergillus melleus.
[0078] Proteases of microbial origin which can be used in the
context of the present invention are described in e.g. U.S. Pat.
No. 6,767,729 (=EP 1,186,658), which is hereby incorporated by
reference, and in Pariza M. W. & Johnson E. A., "Evaluating the
safety of microbial enzyme preparations used in food processing:
update for a new century." Regul Toxicol Pharmacol. 2001 April;
33(2):173-86. (Review).
[0079] The recombinantly produced microbial enzyme, i.e. lipase,
amylase or protease, preferably the recombinantly produced lipase,
may be obtained by fermentation of a fungal cell, e.g. belonging to
the genus Aspergillus, such as A. niger, A. oryzae, or A. nidulans;
a yeast cell, e.g. belonging to a strain of Saccharomyces, such as
S. cerevisiae, or a methylotrophic yeast from the genera Hansenula,
such as H. polymorpha, or Phichia, such as P. pastoris; or a
bacterial cell, e.g. belonging to a strain of Bacillus, such as B.
subtilis, or B. lentus; the cell being transformed with the gene
encoding the microbial lipase. Most preferred host organisms are
members of Aspergillus oryzae.
[0080] An enzyme variant or mutated enzyme is obtainable by
alteration of the DNA sequence of the parent gene or its
derivatives. The enzyme variant or mutated enzyme may be expressed
and produced when the DNA nucleotide sequence encoding the
respective enzyme is inserted into a suitable vector in a suitable
host organism. The host organism does not necessarily have to be
identical to the organism from which the parent gene originated.
The methods for introducing mutations into genes are well known in
the art, see, for example, U.S. Pat. No. 5,658,871 (=EP 407, 225),
the disclosure of which is incorporated by reference.
[0081] Preferred lipase variants or mutated lipases are obtainable
from parent microbial lipases. In a preferred embodiment the parent
lipase is derived from a fungus, e.g. a strain of Humicola or
Rhizomucor, preferably a strain of Humicola lanuginosa or a strain
of Rhizomucor miehei. In another preferred embodiment the parent
lipase is derived from yeast, e.g. from a strain of Candida. In a
further preferred embodiment the parent lipase is derived from a
bacterium, e.g. from a strain of Pseudomonas. More preferred lipase
variants or mutated lipases are lipase variants of parent lipases
comprising a trypsin-like catalytic triad including an active
serine residue located in a predominantly hydrophobic, elongated
binding pocket of the lipase molecule, in which the electrostatic
charge and/or hydrophobicity of a lipid contact zone comprising
residues located in the vicinity of the lipase structure containing
the active serine residue, which residues may participate in the
interaction with the substrate at or during hydrolyzis, has been
changed by deleting or substituting one or more negatively charged
amino acid residues by neutral or positively charged amino acid
residue(s), and/or by replacing one or more neutral amino acid
residues with positively charged amino acid residue(s), and/or by
deleting or substituting one or more hydrophobic amino acid
residues by hydrophobic amino acid residue(s).
[0082] Pharmaceutically compatible auxiliaries, carriers and/or
excipients useful in the context of the present invention are
preferably selected from the group consisting of free polyethylene
glycols having an average molecular weight of about 200 to about
6000; glycerol; lower alcohols, in particular straight-chain or
branched C.sub.1-C.sub.4-alcohols such as 2-propanol; sugars, such
as lactose, sucrose or dextrose; polysaccharides, such as
maltodextrin or dextrates; starches; cellulosics, such as
microcrystalline cellulose or microcrystalline cellulose/sodium
carboxymethyl cellulose; inorganics, such as dicalcium phosphate,
hydroxyapitite, tricalcium phosphate, talc, or titania; and
polyols, such as mannitol, xylitol, sorbitol or cyclodextrin; and
mixtures of the aforementioned substances.
[0083] The present invention includes pharmaceutical compositions
for oral administration, which are self-emulsifiable on contact
with a hydrophilic phase and a lipophilic phase, the compositions
comprising: [0084] (i) enzymes or enzyme mixtures with at least
lipolytic activity, and [0085] (ii) a system comprising [0086] at
least one surfactant, [0087] at least one co-surfactant, and [0088]
optionally a lipophilic phase.
[0089] Preferably the pharmaceutical composition according to the
invention comprises enzymes or enzyme mixtures with at least
lipolytic activity and a system comprising [0090] at least one
surfactant having an HLB value above 6 and below 18, [0091] at
least one co-surfactant having an HLB-value below 10, and [0092] a
lipidic (lipophilic) phase, [0093] wherein the system comprising
surfactant, co-surfactant and lipophilic phase has an HLB value of
about 4 to 16, and a melting point of at least 20.degree. C.,
preferably of at least 25.degree. C.
[0094] The surfactant of the system is preferably selected from the
group consisting of polyethylene glycol fatty acid esters;
polyethylene glycol glycerol fatty acid esters; polyethylene glycol
alkyl ethers, polyethylene glycol sterol ethers, polyethylene
glycol sorbitan fatty acid esters, sugar esters,
polyoxyethylene-polyoxypropylene block copolymers, ionic
surfactants and mixtures thereof. Even more preferred, the
surfactant is selected from the group consisting of polyethylene
glycol (PEG) fatty acid mono- and/or di-esters with aliphatic
C.sub.6-C.sub.22 carboxylic acids; polyethylene glycol (PEG)
glycerol fatty acid esters with aliphatic C.sub.6-C.sub.22
carboxylic acids; polyethylene glycol (PEG) alkyl mono- and/or
di-ethers with aliphatic C.sub.12-C.sub.18 alcohols, and mixtures
thereof. In particular, the surfactant used is represented by a
mixture of polyethylene glycol (PEG) mono- and di-esters with
aliphatic C.sub.6-C.sub.22 carboxylic acids and/or polyethylene
glycol (PEG) mono- and di-ethers with aliphatic C.sub.12-C.sub.18
alcohols, wherein the polyethylene glycol (PEG) comprises 6 to 60
ethylene oxide units per molecule (PEG-6 to PEG-60, also named as
PEG 300 to PEG 3000). Preferably the surfactant is a mixture of
polyethylene glycol mono- and di-esters with aliphatic
C.sub.6-C.sub.22 carboxylic acids, wherein the polyethylene glycol
comprises 6 to 40 ethylene oxide units per molecule.
[0095] The co-surfactant of the system is preferably selected from
the group consisting of mono-acylglycerides, mono-ethers of
glycerol, partial esters of propylenglycol, partial esters of
polyglycerol, partial esters of ethyl diglycol and mixtures
thereof. Even more preferred is a co-surfactant selected from the
group consisting of mono-acylglycerides with aliphatic
C.sub.6-C.sub.22 carboxylic acids, mono-ethers of glycerol ethers
with aliphatic C.sub.12-C.sub.18 alcohols, partial esters of
propylenglycol with aliphatic C.sub.6-C.sub.22 carboxylic acids,
partial esters of polyglycerol with aliphatic C.sub.6-C.sub.22
carboxylic acids, and mixtures thereof. Particularly preferred
co-surfactants are monoacylglycerides of aliphatic C.sub.6-C.sub.22
carboxylic acids and/or monoethers of glycerol with aliphatic
C.sub.12-C.sub.22 alcohols, especially monoacylglycerides of
aliphatic C.sub.6-C.sub.22 carboxylic acids.
[0096] The "lipophilic phase" is preferably represented by di-
and/or triacylglycerides, especially preferably by di- and/or
triacylglycerides with aliphatic C.sub.6-C.sub.22 carboxylic
acids.
[0097] In one preferred embodiment, the system which is part of the
pharmaceutical composition comprises [0098] as surfactant a mixture
of polyethylene glycol (PEG) mono- and di-esters with aliphatic
C.sub.6-C.sub.22 carboxylic acids and/or polyethylene glycol (PEG)
mono- and di-ethers with aliphatic C.sub.12-C.sub.18 alcohols, in
which the polyethylene glycol (PEG) comprises 6 to 60 ethylene
oxide units per molecule, preferably a mixture of polyethylene
glycol mono- and di-esters with aliphatic C.sub.6-C.sub.22
carboxylic acids, in which the polyethylene glycol comprises 6 to
40 ethylene oxide units per molecule; [0099] as co-surfactant
monoacylglycerides of aliphatic C.sub.6-C.sub.22 carboxylic acids
and/or monoethers of glycerol with aliphatic C.sub.12-C.sub.22
alcohols, preferably monoacylglycerides of aliphatic
C.sub.6-C.sub.22 carboxylic acids, and [0100] as lipophilic phase
di- and triacylglycerides of aliphatic C.sub.6-C.sub.22 carboxylic
acids.
[0101] In the pharmaceutical composition according to the invention
the system preferably comprises: [0102] 2 to 90% by weight
surfactants as defined above, [0103] 5 to 60% by weight
co-surfactants as defined above, and [0104] 0 to 70% by weight of
the lipophilic phase as defined above, [0105] in which the
components surfactant, co-surfactant and the lipophilic phase
together make up to 100% by weight of the system, and in which the
system consisting of surfactant, co-surfactant and the lipophilic
phase makes up 10% to 95% by weight of the pharmaceutical
composition.
[0106] Preferably, the system consisting of surfactant,
co-surfactant and lipophilic phase makes up 10 to 70% by weight,
preferably 20 to 50% by weight, more preferably 25 to 40% by
weight, of the pharmaceutical composition.
[0107] In a further embodiment of the pharmaceutical composition
according to the invention, the system comprises: [0108] 40 to 90%
by weight, preferably 60 to 85% by weight, surfactants, [0109] 5 to
40% by weight, preferably 15-30% by weight, co-surfactants, and
[0110] 0 to 40% by weight, preferably 15-30% by weight, of the
lipophilic phase, [0111] and the total of co-surfactants and the
lipophilic phase together is at least 10% by weight, preferably
between 15 and 40% by weight of the system.
[0112] In the context of the present invention, the pharmaceutical
compositions may furthermore contain conventional pharmaceutically
compatible auxiliaries, carriers and/or excipients as defined
hereinafter.
[0113] In particular, the pharmaceutically compatible auxiliaries,
carriers and/or excipients may be selected from the group
consisting of free polyethylene glycols having an average molecular
weight of about 200 to about 6000; glycerol; lower alcohols, in
particular straight-chain or branched C.sub.1-C.sub.4-alcohols such
as 2-propanol; sugars, such as lactose, sucrose or dextrose;
cellulosics, such as microcrystalline cellulose or microcrystalline
cellulose/sodium carboxymethyl cellulose; and mixtures of the
aforementioned substances.
[0114] In a preferred embodiment, the proportion of the
pharmaceutically compatible auxiliaries and/or excipients
furthermore contained therein is at most 20% by weight of the
pharmaceutical composition.
[0115] In a further preferred embodiment, the pharmaceutical
composition according to the invention comprises a
macrogolglyceride mixture representing the system consisting of
surfactant, co-surfactant and lipophilic phase, in which the
macrogolglycerides are a mixture of mono-, di- and
tri-acylglycerides and polyethylene glycol (PEG) mono- and
di-esters of aliphatic C.sub.6-C.sub.22 carboxylic acids, and
optionally also small proportions of glycerol and free polyethylene
glycol.
[0116] The polyethylene glycol (PEG) contained in the
macrogolglyceride mixtures is preferably a PEG which has on average
6 to at most 40 ethylene oxide units per molecule or a molecular
weight of between 200 and 2000.
[0117] One further aspect of the invention provides for the
pharmaceutical composition to comprise a system consisting of
surfactant, co-surfactant and lipophilic phase, the system having
an HLB value of at least 10 and a melting point of at least
30.degree. C. In a preferred embodiment, the system has an HLB
value of 10 to 16, preferably of 12 to 15, and has a melting point
of between 30 and 60.degree. C., preferably between 40 and
50.degree. C.
[0118] In particular, the system characterized by HLB value and
melting point is a mixture of mono-, di- and triacylgylcerides and
mono- and diesters of polyethylene glycol (PEG) with aliphatic
carboxylic acids with 8 to 20 carbon atoms, in which the
polyethylene glycol preferably contains about 6 to about 32
ethylene oxide units per molecule, and the system optionally
contains free glycerin and/or free polyethylene glycol. The HLB
value of such a system is preferably regulated by the chain length
of the PEG. The melting point of such a system is regulated by the
chain length of the fatty acids, the chain length of the PEG and
the degree of saturation of the fatty-acid chains, and hence by the
starting oil used for the preparation of the macrogolglyceride
mixture.
[0119] The term "aliphatic C.sub.5-C.sub.18 carboxylic acids"
designates mixtures in which caprylic acid (C8), capric acid (C10),
lauric acid (C12), myristic acid (C14), palmitic acid (C16) and
stearic acid (C18) are contained in a significant and variable
proportion, if these acids are saturated, and the corresponding
unsaturated C.sub.5-C.sub.18 carboxylic acids. The proportions of
these fatty acids may vary depending on the starting oils.
[0120] Such a mixture of mono-, di- and triacylgylcerides and mono-
and diesters of polyethylene glycol (PEG) with aliphatic carboxylic
acids with 8 to 18 carbon atoms can, for example, be obtained by a
reaction between a polyethylene glycol with a molecular weight of
between 200 and 1500 and a starting oil, the starting oil
consisting of a triglyceride mixture with fatty acids which are
selected from the group consisting of caprylic acid, capric acid,
lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid
and linolenic acid, individually or as a mixture. Optionally, the
product of such a reaction may also contain small proportions of
glycerin and free polyethylene glycol.
[0121] Such a mixture is commercially available, for example, under
the trade name Gelucire. One advantageous embodiment of the
invention provides that, of the products known under the trade name
Gelucire.RTM., in particular "Gelucire 50/13" and/or "Gelucire.RTM.
44/14" represent suitable mixtures for use in the pharmaceutical
preparations according to the invention.
[0122] Gelucire.RTM. 50/13 is a mixture in which mono-, di- and
triacylglycerides and mono- and diesters of polyethylene glycol,
with palmitic acid (C16) and stearic acid (C18) at 40% to 50% and
48% to 58%, respectively, making up the major proportion of bound
fatty acids. The proportion of caprylic acid (C8) and capric acid
(C10) is less than 3% in each case, and the proportion of lauric
acid (C12) and myristic acid (C14) in each case is less than
5%.
[0123] A preferred embodiment of the present invention provides a
pharmaceutical composition which comprises a system containing a
mixture of mono-, di- and triacylglycerides and polyethylene glycol
mono- and diesters of aliphatic C.sub.8-C.sub.18 carboxylic acids
and optionally also small proportions of glycerin and free
polyethylene glycol, the system having a melting point between
46.degree. C. and 51.degree. C. and an HLB value of around 13.
[0124] Gelucire.RTM. 44/14 is a mixture of mono-, di- and
triacylgylcerides and mono- and diesters of polyethylene glycol,
the respective proportions of palmitic acid (C16) being 4 to 25%,
stearic acid (C18) 5 to 35%, caprylic acid (C8) less than 15%,
capric acid (C10) less than 12%, lauric acid (C12) 30 to 50% and
myristic acid (C14) 5 to 25%. Gelucire.RTM. 44/14 can, for example,
be prepared by an alcoholysis/esterification reaction using palm
kernel oil and polyethylene glycol 1500.
[0125] One preferred embodiment of the present invention provides a
pharmaceutical composition which comprises a system containing a
mixture of mono-, di- and triacylglycerides and polyethylene glycol
mono- and diesters of aliphatic C.sub.8-C.sub.18 carboxylic acids
and optionally also small proportions of glycerin and free
polyethylene glycol, the system having a melting point between
42.degree. C. and 48.degree. C. and an HLB value of around 14.
[0126] In an alternative embodiment of the pharmaceutical
composition of the invention, an ionic surfactant is used as
surfactant. Preferably, the ionic surfactant is selected from the
group consisting of lecithin, lysolecithin, phosphatidylcholine,
phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine,
lysophosphatidylcholine, lysophosphatidylethanolamine,
lysophosphatidylglycerol, lysophosphatidylinositol,
lysophosphatidic acid, lysophosphatidylserine, and mixtures
thereof. Lysophosphatidylcholine is especially preferred.
[0127] One particularly preferred pharmaceutical composition
according to the invention comprises a system containing [0128] as
surfactant lysophosphatidylcholine, [0129] as co-surfactant a
mixture of mono-acylglycerides with aliphatic saturated and/or
unsaturated C.sub.16-C.sub.20 carboxylic acids, preferably with
oleic and/or linoleic acid, and [0130] a lipophilic phase of di-
and/or triacylglycerides with aliphatic C.sub.16-C.sub.20
carboxylic acids, preferably with oleic and/or linoleic acid.
[0131] As a commercially available mixture of mono-, di- and
triacylglycerides with aliphatic saturated and/or unsaturated
C.sub.16-C.sub.20 carboxylic acids Maisine.RTM. (Gattefosse) can be
used.
[0132] Preferably, the pharmaceutical composition is one in which
the system comprises 2 to 10%, preferably about 5%, by weight
lysophosphatidylcholine, 28 to 51% by weight mono-acylglycerides
mainly of oleic acid and linoleic acid, and 36 to 54% by weight
di-acylglycerides and 4 to 20% by weight tri-acylglycerides mainly
of oleic acid and linoleic acid, in which the system consisting of
surfactant, co-surfactant and the lipophilic phase together makes
up 10% to 30%, preferably about 20%, by weight of the
pharmaceutical composition.
[0133] For the pharmaceutical preparations according to the
invention, preferably solid orally administered dosage forms may be
selected, for example powders, pellets, granules, tablets, or
microspheres, which if desired may be filled into capsules or
sachets or may be compressed to form tablets. Granules are
preferably produced by melt granulation. Tablets are usually made
from a powder or from melt granules. Pellets can be produced either
by exploiting the thermoplastic properties of the auxiliaries in a
heavy-duty mixer (melt pelletization) or by traditional methods
e.g. extrusion (e.g. melt extrusion or wet extrusion) and
spheronisation. If individual enzyme types are present and are
obtained separately, such as a lipase, a protease or an amylase
from microbial origin, these may either be mixed together or
spatially separated from each other. If the individual enzymes are
not spatially separated from each other, dry processing and/or
storage is preferred.
[0134] The pharmaceutical compositions according to the invention,
which are self-emulsifiable on contact with a hydrophilic phase and
optionally a lipophilic phase, contain enzymes or enzyme mixtures
with at least lipolytic activity as active substance. In a
preferred variant of the present invention, the enzymes or enzyme
mixtures may however, in addition to the lipolytic activity, also
have proteolytic activity, i.e. contain at least one protease,
and/or amylolytic activity, i.e. contain at least one amylase.
[0135] In one preferred variant of the present invention, the
lipolytic activity of the enzymes or enzyme mixtures is provided by
a microbial lipase.
[0136] In another embodiment, the pharmaceutical composition
contains enzymes or enzyme mixtures which comprise pancreatin
and/or are pancreatin-like, preferably pancreatin-containing
mixtures of digestive enzymes. Preferably, the pancreatin and/or
pancreatin-like mixtures of digestive enzymes make up 65-85%, in
particular 75-80%, by weight, of the pharmaceutical
composition.
[0137] Alternatively, the enzyme mixture used is a mixture of at
least one microbial lipase and one or more microbial enzymes
selected from the group consisting of proteases and amylases.
[0138] In one variant of the invention, the enzyme mixture used is
purely of microbial origin. Examples of such physiologically
acceptable bacterial and/or fungal enzymes have already been
described in the prior art, together with procedures for obtaining
these enzymes and for their use in the treatment of maldigestion.
For example, such synthetic mixtures of lipase, protease and
amylase, each of which are microbially obtained, and also
pharmaceutical preparations containing these mixtures are described
in US Patent Publication No. 2004/057944 (=WO 02/060474) and U.S.
Pat. No. 6,051,220 (=EP 828,509).
[0139] Preferably, the pharmaceutical composition contains
microbial enzymes making up 5-80%, in particular 20-60%, by weight
of the pharmaceutical composition.
[0140] In the context of the invention, most preferred are those
mixtures of digestive enzymes with lipolytic, proteolytic and
amylolytic activity, the properties of which are close to those of
pancreatin. Pancreatin-containing mixtures of digestive enzymes and
also in particular pancreatin itself are therefore preferred in the
context of the present invention as disclosed above. However it is
possible, if desired, to add to the pancreatin or the
pancreatin-containing mixtures of digestive enzymes one or more
microbial enzymes, i.e. lipases, proteases and/or amylases obtained
from microbial sources.
[0141] Suitable microbial enzymes for use as the sole enzyme
mixture or as an addition to pancreatin or the
pancreatin-containing mixtures of digestive enzymes include, in
particular, bacterial or fungal enzymes, such as from the species
Bacillus or Pseudomonas, or from fungal cultures, such as from the
species Aspergillus, Humicola or Rhizomucor. Preferably, the
microbial enzymes, in particular the microbial lipase, are
recombinantly produced. In a further preferred variant of the
present invention, the microbial lipase is a lipase variant or a
mutated lipase.
[0142] The present invention furthermore relates to the use of a
system comprising [0143] at least one surfactant, [0144] at least
one co-surfactant, and [0145] optionally a lipophilic phase for
stabilizing the lipolytic activity in the acidic pH range and/or
for improving the lipolytic activity of solid pharmaceutical
preparations containing enzymes or enzyme mixtures with at least
lipolytic activity, preferably pancreatin or pancreatin-like
mixtures of digestive enzymes.
[0146] The possible further configurations of the system to be
used, consisting of surfactant, co-surfactant and lipophilic phase,
correspond to the embodiments described above for the
self-emulsifiable pharmaceutical preparation according to the
invention, which comprises such a system.
[0147] The invention also relates to a process for the preparation
of solid pharmaceutical preparations containing enzymes or enzyme
mixtures with at least lipolytic and optionally also proteolytic
and/or amylolytic activity, preferably pancreatin and/or
pancreatin-like mixtures of digestive enzymes. According to the
invention, the enzymes or enzyme mixtures are converted into a
suitable medicament form together with a system comprising [0148] a
surfactant selected from the group consisting of polyethylene
glycol fatty acid esters, polyethylene glycol glycerol fatty acid
esters, polyethylene glycol alkyl ethers, polyethylene glycol
sterol ethers, polyethylene glycol sorbitan fatty acid esters,
sugar esters, polyoxyethylene-polyoxypropylene block copolymers,
ionic surfactants, and mixtures thereof; [0149] a co-surfactant
selected from the group consisting of mono-acylglycerides,
mono-ethers of glycerol, partial esters of propylenglycol, partial
esters of polyglycerol, partial esters of ethyl diglycol, and
mixtures thereof, and [0150] a lipophilic phase, which is
represented by di- and/or triacylglycerides, and also optionally
conventional, pharmaceutically compatible auxiliaries, carriers
and/or excipients.
[0151] The further possible configurations of the system consisting
of surfactant, co-surfactant and lipophilic phase used in the
preparation process correspond to the embodiments described above
for the self-emulsifiable pharmaceutical preparation according to
the invention, which comprises such a system.
[0152] The following examples are intended to illustrate the
invention in further detail without limiting its scope:
EXAMPLE 1: PREPARATION OF PANCREATIN-CONTAINING COMPOSITIONS
ACCORDING TO THE INVENTION AND COMPARISON OF THE LIPOLYTIC ACTIVITY
OF A CONVENTIONAL PANCREATIN FORMULATION AND A PANCREATIN
FORMULATION ACCORDING TO THE INVENTION COMPRISING A SYSTEM
CONSISTING OF A SURFACTANT, CO-SURFACTANT AND LIPOPHILIC PHASE
a) Conventional Preparation (Pellets) not According to the
Invention:
[0153] The conventional formulation was prepared according to the
process disclosed in U.S. Pat. No. 5,378,462 (=EP 583,726). 120 g
pancreatin and 30 g PEG 4000 were initially dry-mixed and then
moistened with 20 g isopropanol. The moist mixture was extruded and
then rounded in a suitable rounder with the aid of paraffin oil.
The resulting pellets were then dried.
b) Preparation According to the Invention (Pellets) (Example
1A)
[0154] 350 g Gelucire.RTM. 50/13 was melted in a beaker in a water
bath at a temperature of 52.degree. C. The molten mass was mixed
with 650 g pancreatin in a dual-jacket mixer for 10 min. The
homogenous mixture was placed in a melt extruder for extrusion.
Then the extrudate was rounded in a suitable rounder or
spheroniser. The resulting pellets had a diameter of 1.0-1.6
mm.
c) Preparation According to the Invention (Granules) (Example
1B)
[0155] 300 g Gelucire.RTM. 44/14 was melted in a beaker in a water
bath at a temperature of 48.degree. C. The molten mass was mixed
with 700 g pancreatin in a dual-jacket mixer for approximately 15
minutes and then cooled (melt granulation).
[0156] The activity of the lipase as a function of the pH value and
the time-dependent change in lipase activity were determined in
accordance with the method of the "Federation Internationale
Pharmaceutique/European Pharmacopeia" (abbreviated hereinafter as
FIP/Ph.Eur.). In this standard analysis method, the hydrolytic
activity of the lipase in the sample to be investigated is
determined with the substrate olive oil. The free fatty acids
cleaved off from the triglycerides of the olive oil are titrated
with sodium hydroxide solution at a constant pH of 9.0. The lipase
activity of the sample is determined by comparing the rate at which
the sample hydrolyzes an olive oil emulsion with the rate at which
a suspension of a standard pancreas reference powder hydrolyzes the
same substrate under the same conditions.
[0157] The absolute and relative lipolytic activity of the
conventional preparation and the preparation 1A according to the
invention (pellets) determined in each case in accordance with
FIP/Ph.Eur. are summarized in the following Table 1. The absolute
and relative lipolytic activity of the conventional preparation and
the preparation 1B according to the invention (granules) determined
in each case in accordance with FIP/Ph.Eur. are summarized in the
following Table 2:
TABLE-US-00001 TABLE 1 Absolute and relative lipolytic activity of
the standard preparation and the preparation according to the
invention (pellets with Gelucire .RTM. 50/13) Theoretically present
Lipase lipase Measured activity activity absolute Relative starting
in the lipase lipase pancreatin formulation activity activity
Sample [U/g] [U/g] [U/g] [%] Example 1A: 92620 60203 63173 104
Pellets Conventional 92620 74096 69650 94 formulation
TABLE-US-00002 TABLE 2 Absolute and relative lipolytic activity of
the standard preparation and the preparation according to the
invention (granules with Gelucire .RTM. 44/14) Theoretically
present Lipase lipase Determined activity activity absolute
Relative starting in the lipase lipase pancreatin formulation
activity activity Sample [U/g] [U/g] [U/g] [%] Example 1B: 84787
59351 67653 114 Granules Conventional 84132 67306 64950 97
formulation
[0158] It is apparent from the data that the addition of systems
comprising at least one surfactant, at least one co-surfactant and
a lipophilic phase to pharmaceutical preparations of enzymes and
enzyme mixtures with at least lipolytic activity, preferably
pancreatin and/or pancreatin-like mixtures of digestive enzymes,
contributes to improved lipolytic activity compared with
conventional formulations of pancreatin known in the prior art.
[0159] The absolute lipolytic activity of the respective
pharmaceutical preparation determined in accordance with
FIP/Ph.Eur. is expressed with reference to the total lipolytic
activity theoretically present in the sample in the form of a
relative activity, in order to take account of the different
concentrations of pancreatin in the formulations. Comparison of the
relative lipase activities determined shows that the relative
lipase activity of the preparations according to the invention is
approximately 10% higher than those of the conventional
formulations. Accordingly, the pharmaceutical preparations
according to the invention have increased lipolytic activity
compared with conventional pancreatin formulations.
[0160] Furthermore, with reference to the value of the relative
lipase activity of the preparation according to the invention of
more than 100%, it is apparent that the system consisting of
surfactant, co-surfactant and optionally lipophilic phase added to
the preparations according to the invention exerts an activating
effect on the lipase.
EXAMPLE 2: COMPARISON OF THE STABILITY OF LIPOLYTIC ACTIVITY OF A
CONVENTIONAL PANCREATIN FORMULATION AND A PANCREATIN FORMULATION
ACCORDING TO THE INVENTION COMPRISING A SYSTEM CONSISTING OF A
SURFACTANT, CO-SURFACTANT AND LIPOPHILIC PHASE AT DIFFERENT PH
VALUES
[0161] In order to compare the stability of lipolytic activity of a
conventional pancreatin formulation and a pharmaceutical
formulation according to the invention comprising an enzyme mixture
with at least lipolytic activity and a system consisting of at
least one surfactant, at least one co-surfactant, and a lipophilic
phase, the activity of such a conventional pancreatin formulation
was compared with the activity of a mixture of Gelucire.RTM. and
pancreatin incubated for up to 2 hours at different pH values (pH
6, pH 5 and pH 4).
a) Standard Preparation (Pellets):
[0162] The conventional formulation was prepared according to the
process disclosed in U.S. Pat. No. 5,378,462. 120 g pancreatin and
30 g PEG 4000 were initially dry-mixed and then moistened with 20 g
isopropanol. The moist mixture was extruded and then rounded in a
suitable rounder with the aid of paraffin oil. The resulting
pellets were then dried.
b) Preparation According to the Invention (Pellets)--Example 2
[0163] 300 g Gelucire.RTM. 44/14 was melted in a beaker in a water
bath at a temperature of 48.degree. C. The molten mass was mixed
with 700 g pancreatin in a dual-jacket high-speed mixer (melt
pelletization).
[0164] The activity of the lipase as a function of the pH value and
also the time-dependent change in lipase activity were determined
in accordance with the method of the FIP/Ph.Eur. as described
above.
[0165] To determine the release behavior of the lipase in the
conventional preparation and the preparation according to the
invention at different pH values, the samples were incubated in a
decomposition apparatus for 2 hours at 37.degree. C. in phosphate
buffer solution (pH 6, pH 5, pH 4). Samples were taken at intervals
of 15 minutes, and the lipolytic activity in the samples was
determined in accordance with the FIP/Ph.Eur. method described
above.
[0166] 600 ml buffer (67 mM phosphate, 34 mM NaCl, pH 6.0, pH 5.0,
pH 4.0) was heated to a constant temperature of 37.degree. C. in a
1 liter beaker in the decomposition tester. Once the constant
temperature had been reached, 2 g of sample was added to the beaker
and the decomposition tester was set in motion. The pH value of the
phosphate buffer was kept constant during the testing time. Samples
were taken at intervals of 15 minutes in each case, and the
lipolytic activity in the samples was determined in accordance with
FIP/Ph.Eur.
[0167] The relative lipolytic activities of the conventional
preparation and the preparation according to the invention
determined after 15, 30, 45, 60, 75, 90, 105 and 120 minutes in
accordance with FIP/Ph.Eur. are summarised in the following Table
3. Details are given in % of the activity of the respective sample
compared with a standard pancreas reference powder in accordance
with FIP/Ph.Eur.
TABLE-US-00003 TABLE 3 pH-dependency of the relative lipolytic
activity of a conventional pancreatin formulation and a pancreatin
preparation according to the invention pH 6 5 4 Time Conventional
Conventional Conventional [min] formulation Example 2 formulation
Example 2 formulation Example 2 15 93 98 89 100 30 55 30 90 101 82
99 19 39 45 84 97 77 95 13 31 60 81 89 72 95 11 27 75 79 82 71 94 8
23 90 76 76 68 92 7 19 105 72 71 65 89 6 18 120 69 66 61 86 5
17
[0168] It can be seen from this data that the addition of systems
consisting of at least one surfactant, at least one co-surfactant,
and a lipophilic phase to pharmaceutical preparations of enzymes
and enzyme mixtures having at least lipolytic activity, preferably
pancreatin and/or pancreatin-like mixtures of digestive enzymes,
contributes to stabilizing the lipolytic activity in the acidic pH
range. At a pH value of 6, comparison of the lipolytic activity of
a conventional pancreatin preparation and a pancreatin preparation
according to the invention over a time of 120 minutes shows that
the lipolytic activity in both preparations over time decreases
only relatively slightly, with the lipolytic activity of the
preparation according to the invention increased by approximately
10% compared with the conventional formulation again being observed
within the first hour. However, a pH value of 6 is known not to
have any great influence on the lipolytic activity. On the other
hand, at a pH value of 5 the lipolytic activity of the conventional
preparation decreases much more quickly compared with the
preparation according to the invention. Whereas the preparation
according to the invention has lost less than 10% of the lipolytic
activity after 90 minutes, the conventional preparation has only a
lipolytic activity of less than 70% remaining compared with a
pancreas reference powder in accordance with FIP/Ph.Eur. In
particular at a pH value of 4, the preparation according to the
invention has a markedly greater lipolytic (residual) activity than
the conventional preparation. Accordingly, it can be seen that the
pharmaceutical preparations according to the invention have a
substantially increased lipolytic activity in an acidic pH
medium.
EXAMPLE 3: DOSAGE DEPENDENCE OF A PANCREATIN FORMULATION ACCORDING
TO THE INVENTION COMPRISING A SYSTEM CONSISTING OF A SURFACTANT,
CO-SURFACTANT AND LIPOPHILIC PHASE ON DIGESTIBILITY OF A HIGH FAT
DIET IN THE PANCREATIC EXOCRINE DEFICIENT MINIPIG
[0169] The efficacy of a pelleted pharmaceutical formulation
according to the invention comprising an enzyme mixture with at
least lipolytic activity and a system consisting of at least one
surfactant, at least one co-surfactant, and a lipophilic phase to
improve digestion and absorption of fat in minipigs, in which the
pancreatic duct has been ligated to induce a complete pancreatic
exocrine insufficiency, was analyzed in pigs fed a high (32%) fat
diet.
a) Preparation According to the Invention (Pellets)
[0170] 250 g Gelucire.RTM. 44/14 (Gattefosse) was melted in a
beaker in a water bath at a temperature of 48.degree. C. The molten
mass was mixed with 750 g pancreatin in a dual-jacket high-speed
mixer (melt pelletization). The pellet size of this formulation was
similar to that of the commercially available pancreatin
product.
Determination of the Activity of Lipase
[0171] Studies were performed in 6 minipigs (Ellegaard, female
Gottingen minipigs) with induced pancreatic exocrine insufficiency,
weighing 20-30 kg at surgery. The pigs were prepared as previously
described by Tabeling R., Gregory P., Kamphues J., 1999: Studies on
nutrient digestibilities (pre-caecal and total) in pancreatic
duct-ligated pigs and the effects of enzyme substitution. J. Anim.
Physiol. a. Anim. Nutr. 82, 251-263. The pancreatic duct was
ligated under halothane anaesthesia following a mid-line
laparotomy; after which the pigs were chronically fitted with an
ileo-caecal re-entrant fistula which was exteriorized on the right
flank.
[0172] The success of the pancreatic duct ligation was confirmed by
a faecal chymotrypsin test before starting the digestibility
studies, which began at least 4 weeks after the pigs had recovered
from the surgery.
[0173] The pigs were fed two 250 g meals/day (08.00 and 20.00 h) of
a high fat diet (containing: 180 g double-milled Altromin 9021
[modified], 70 g soya oil [Roth]; overall contents are 99% dry
matter, 4% crude ash, 32% crude fat, 16% crude protein, 28% starch,
3% crude fiber) plus 0.625 g Cr.sub.2O.sub.3 per meal, mixed with 1
liter water. The meals plus enzymes were carefully mixed together
immediately before offering to the pigs. The meals were generally
consumed within 5 minutes.
[0174] During the study the pigs received zero, 28,000 or 336,000
FIP lipase units per meal as a formulation according to the
invention for 14 days, with a complete collection of faeces for the
last 5 days. The faeces (and the feed) were frozen at -20.degree.
C., freeze dried, and a Weender analysis was performed [Naumann C,
Bassler R. 1993: Die chemische Untersuchung von Futtermitteln. 3.
Aufl. VDLUFA-Verlag, Darmstadt] to determine content of dry matter
(drying at 103.degree. C. for 8 hours), and crude fat (determined
gravimetrically after boiling for 30 minutes with conc. HCl,
followed by a 6 hours extraction with petroleum ether).
Cr.sub.2O.sub.3 was oxidized to chromate, and chromium content was
calculated via extinction at 365 nm [Petry H, Rapp W. 1970: Zur
Problematik der Chromoxidbestimmung in Verdauungsversuchen. Z.
Tierphysiol. Tierernahrung and Futtermitellkunde 27, 181-189].
[0175] Based on the content of fat and chromium determined per 100
g dry matter feed and faeces (see above), the digestibility of fat
(CFA) was calculated according to the formula:
% fat digestibility = 100 - ( [ % Cr 2 O 3 in feed ] [ % Cr 2 O 3
in faeces ] .times. [ % fat in faeces ] [ % fat in feed ] .times.
100 ) ##EQU00001##
[0176] The efficacy to improve digestion and absorption of fat in
minipigs, in which the pancreatic duct has been ligated to induce a
complete pancreatic exocrine insufficiency, measured in the % fat
digestibility is given for the preparation according to the
invention for different amounts of lipase activity added (given in
FIP/Ph.Eur. units).
TABLE-US-00004 TABLE 4 % fat digestibility in minipigs receiving a
pancreatin preparation according to the invention 28,000 336,000
FIP FIP 0 Lipase lipase lipase Substitution Units U/meal U/meal No
enzymes added 31.66 .+-. 13.78 Formulation 61.98 .+-. 11.60 * 79.25
.+-. 7.00 ** according to the invention *; ** Results are mean .+-.
S.D.
[0177] It can be seen that the formulation according to the
invention caused a very strong and dose-dependent improvement in
fat digestibility, already showing a highly efficient improvement
at the lower dose tested.
EXAMPLE 4: COMPARISON OF THE STABILITY OF LIPOLYTIC ACTIVITY OF A
CONVENTIONAL PANCREATIN POWDER AND A PANCREATIN FORMULATION
ACCORDING TO THE INVENTION COMPRISING A SYSTEM CONSISTING OF A
SURFACTANT, CO-SURFACTANT AND LIPOPHILIC PHASE AT DIFFERENT PH
VALUES
[0178] Further preparations according to the invention were
prepared and analyzed with regard to their lipolytic activity in
comparison to pancreatin powder at different acidic pH values (pH
6, pH 5 and pH 4).
a) Preparation for Comparison not According to the Invention:
[0179] Pancreatin powder
b) Preparation According to the Invention--Example 4A
[0180] 700 g Pancreatin powder
[0181] 200 g Gelucire.TM. 44/14 (Gattefosse)
[0182] 100 g Labrasol.TM. (Gattefosse)
[0183] The Gelucire.RTM. 44/14 and the Labrasol.RTM. were mixed and
melted in a beaker in a water bath at a temperature of 48.degree.
C. The molten mass was mixed with 700 g pancreatin in a dual-jacket
high-speed mixer (melt granulation).
c) Preparation According to the Invention--Example 4B
[0184] 800 g Pancreatin powder
[0185] 190 g Maisine (Gattefosse)
[0186] 10 g LPC (Lysophosphatidylcholine)
[0187] The Maisine.RTM. and the Lysophosphatidylcholine were mixed
and melted in a beaker in a water bath at a temperature of
48.degree. C. The molten mass was mixed with 800 g pancreatin in a
dual-jacket high-speed mixer (melt granulation).
[0188] The activity of the lipase as a function of the pH value and
also the time-dependent change in lipase activity were determined
as described in Example 2.
[0189] The release behavior of the lipase at different pH values in
the pancreatin powder and the preparation according to the
invention was measured as described above for Example 2.
[0190] The relative lipolytic activity determined after 15, 30, 45,
60, 75, 90, 105 and 120 minutes of the pancreatin powder and the
preparations "Example 4A" and "Example 4B" according to the
invention in accordance with FIP/Ph.Eur. are summarized in the
following Tables 5A and 5B. Details are given in % of the activity
of the respective sample compared with a standard pancreas
reference powder in accordance with FIP/Ph.Eur.
TABLE-US-00005 TABLE 5A pH-dependency of the relative lipolytic
activity of a standard pancreatin powder and the pancreatin
preparation "4A" according to the invention pH 6 5 4 Time
Pancreatin Pancreatin Pancreatin [min] Powder Example 4A Powder
Example 4A Powder Example 4A 15 55.7 90.4 61.5 93.8 19.3 49.0 30
54.9 105.1 53.3 99.0 12.5 32.4 45 48.3 102.9 51.8 95.8 8.4 25.5 60
43.1 97.6 48.1 92.7 6.5 23.1 75 39.0 91.7 41.4 93.2 5.6 19.9 90
35.4 87.2 39.9 91.2 4.3 18.8 105 33.0 82.3 44.8 88.1 3.9 17.8 120
30.4 79.2 39.1 85.6 3.6 16.5
TABLE-US-00006 TABLE 5B pH-dependency of the relative lipolytic
activity of a standard pancreatin powder and the pancreatin
preparation "4B" according to the invention pH 6 5 4 Time
Pancreatin Pancreatin Pancreatin [min] Powder Example 4B Powder
Example 4B Powder Example 4B 15 55.7 91.9 61.5 83.2 19.3 27.1 30
54.9 87.4 53.3 88.0 12.5 16.4 45 48.3 81.2 51.8 86.3 8.4 13.0 60
43.1 73.7 48.1 83.5 6.5 9.9 75 39.0 69.8 41.4 83.8 5.6 8.9 90 35.4
63.8 39.9 80.0 4.3 7.8 105 33 57.1 44.8 78.4 3.9 6.5 120 30.4 53.0
39.1 74.0 3.6 4.9
[0191] From these data it can be concluded that the addition of
systems consisting of at least one surfactant, at least one
co-surfactant, and a lipophilic phase to pharmaceutical
preparations of enzymes and enzyme mixtures with at least lipolytic
activity, preferably pancreatin and/or pancreatin-like mixtures of
digestive enzymes, contributes to stabilizing the lipolytic
activity in the acidic pH range.
EXAMPLE 5: DETERMINATION OF THE LIPOLYTIC ACTIVITY OF A FORMULATION
ACCORDING TO THE INVENTION COMPRISING A LIPASE OF MICROBIAL ORIGIN
AND A SYSTEM CONSISTING OF A SURFACTANT, CO-SURFACTANT AND
LIPOPHILIC PHASE AND DETERMINATION OF STABILITY AT DIFFERENT PH
VALUES
[0192] In order to determine the lipolytic activity and to show the
improved stability at acidic pH of a pharmaceutical formulation
according to the invention comprising an enzyme mixture with at
least lipolytic activity, in which the lipolytic activity is
provided by a microbial, optionally recombinantly produced lipase,
and a system consisting of at least one surfactant, at least one
co-surfactant, and a lipophilic phase, the activity of a
pharmaceutical formulation consisting of a mixture of Gelucire.TM.
and a microbial lipase is determined at different pH values (pH 6,
pH 5, pH 4 and 3) and compared to a lipase preparation which has
not been stabilized.
a) Preparation According to the Invention (Granulate)
[0193] 562.5 g Gelucire.RTM. 44/14 was melted in a beaker in a
water bath at a temperature of 48.degree. C. 937.5 g of a microbial
lipase preparation (the active lipase protein representing about 50
to 60% (w/w) of the dry matter of the preparation) were provided in
a dual-jacket mixer at 46.degree. C., then the molten Gelucire was
added and the compounds were mixed, first at low speed for 3 min,
then for approx. 15 min. at high speed, and finally cooled (melt
granulation).
b) Comparison Preparation (not According to the Invention)
[0194] A microbial lipase preparation was prepared by using common
spray dry technique.
[0195] The activity of the lipase was determined in accordance with
the method of the "Federation Internationale Pharmaceutique"
(abbreviated hereafter to FIP) for microbial lipases, except that
the concentration of bile salts is 10 mM.
[0196] Using this standard analysis method, the hydrolytic activity
of the lipase in the sample to be investigated is determined using
olive oil as a substrate. Released free fatty acids are titrated
with sodium hydroxide solution at a constant pH of 7.0. The lipase
activity of the sample is determined by comparison of the rate at
which the sample hydrolyzes an olive oil emulsion with the rate at
which a suspension of a microbial lipase reference powder
hydrolyzes the same substrate under the same conditions.
[0197] To determine the pH-stability of the lipase at different pH
values in an unstabilized preparation and in the preparation
according to the invention, the samples were incubated in a
decomposition apparatus for 2 hours at 37.degree. C. in buffer
solution (pH 5, pH 4 and pH 3). Samples were taken at intervals of
15 minutes, and the lipolytic activity in the samples was
determined in accordance with the FIP method.
[0198] 100 mg of lipase were incubated in 100 ml buffer (0.1 M
malonic acid buffer, 1 mM calcium chloride pH 3, 4 and 5) at
37.degree. C. Samples were drawn every 15 mM for a total duration
of 2 hours and the lipolytic activity of the samples was determined
as follows: An olive oil suspension was prepared by mixing 175 g
olive oil with 630 ml of a solution of 700 g of gum arabic and 94.4
g calcium chloride di-hydrate in 5,900 ml water for 15 minutes in a
food mixer at maximal speed. The emulsion was cooled to 37.degree.
C., and the pH was adjusted to pH 6.8 with sodium hydroxide
solution. Three reference solutions were prepared by extracting an
appropriate amount of FIP microbial lipase standard with an
ice-cold 1% (m/v) solution of sodium chloride such that reference
solutions with 50 FIP-U/ml, 65 FIP-U/ml and 80 FIP-U/ml were
obtained. Sample solutions were prepared by extracting an amount of
sample corresponding to app. 6,500 units activity for 15 minutes
with a total of 100 ml ice-cold 1% (m/v) solution of sodium
chloride. The samples were further diluted in ice-cold 1% (m/v)
solution of sodium chloride such that the titration rate was within
the range of the titration rates obtained with the reference
solutions.
[0199] The titration rates of the reference and sample solutions
were determined by combining in a thermostated vessel 19 ml of
olive oil suspension with 10 ml of a solution of 492 mg lipase
activating mixture (FIP) in 500 ml of water. The combined solutions
were thermostated to 37.degree. C., and the pH was adjusted to pH
7.0. One ml of reference solution or sample solution were added,
and the released fatty acids titrated under pH stable conditions
with 0.1 M sodium hydroxide solution for a duration of 5 minutes.
The titration rate was calculated by linear regression from at
least 9 measurement points between the 60th and the 300th second of
titration.
[0200] From the titration rates of the reference solutions a
calibration function was calculated by linear regression. The
calibration function takes the form y=mx+b where y: titration rate;
m: slope; x: FIP-units of the reference solution; and b: axis
intercept. Using the values thus determined for m and b, the
lipolytic activity x was calculated for each sample solution using
the formula x=(y-b)/m.
[0201] The relative lipolytic activities determined after 0, 15,
30, 45, 60, 75, 90, 105 and 120 minutes of an un-stabilized
microbial lipase preparation and the preparation according to the
invention in accordance with FIP are determined. A comparison of
the results obtained can show the improved lipolytic activity and
the increased stability within the acidic pH range of the
formulation according to the invention comprising a microbial
lipase preparation over the un-stabilized lipase preparation.
[0202] The foregoing description and examples have been set forth
merely to illustrate the invention and are not intended to be
limiting. Since modifications of the described embodiments
incorporating the spirit and substance of the invention may occur
to persons skilled in the art, the invention should be construed
broadly to include all variations within the scope of the appended
claims and equivalents thereof.
* * * * *