U.S. patent application number 17/714617 was filed with the patent office on 2022-09-08 for coated silk films, methods for the production thereof and uses thereof.
The applicant listed for this patent is AMSILK GMBH. Invention is credited to Elisa Agostini, Julia Engert, Lin Romer, Ute Slotta, Gerhard Winter.
Application Number | 20220280680 17/714617 |
Document ID | / |
Family ID | 1000006359267 |
Filed Date | 2022-09-08 |
United States Patent
Application |
20220280680 |
Kind Code |
A1 |
Winter; Gerhard ; et
al. |
September 8, 2022 |
COATED SILK FILMS, METHODS FOR THE PRODUCTION THEREOF AND USES
THEREOF
Abstract
The present invention relates to coated silk films. Further, the
present invention relates to pharmaceutical or cosmetic
compositions comprising the coaled silk films. Furthermore, the
present invention relates to coated silk films or pharmaceutical
compositions comprising the coated silk films for use in medicine.
In addition, the present invention relates to methods of producing
the coated silk films.
Inventors: |
Winter; Gerhard; (Penzberg,
DE) ; Agostini; Elisa; (Munich, DE) ; Engert;
Julia; (Munich, DE) ; Romer; Lin; (Ottobrunn,
DE) ; Slotta; Ute; (Munich, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
AMSILK GMBH |
PLANEGG/MARTINSRIED |
|
DE |
|
|
Family ID: |
1000006359267 |
Appl. No.: |
17/714617 |
Filed: |
April 6, 2022 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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15115215 |
Jul 28, 2016 |
11324852 |
|
|
PCT/EP2015/051814 |
Jan 29, 2015 |
|
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17714617 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61L 15/40 20130101;
A61Q 19/00 20130101; A61L 15/44 20130101; A61L 15/32 20130101; A61K
8/0208 20130101; A61K 9/7007 20130101; A61K 8/64 20130101; A61L
2420/02 20130101; A61K 8/987 20130101 |
International
Class: |
A61L 15/32 20060101
A61L015/32; A61K 8/64 20060101 A61K008/64; A61L 15/44 20060101
A61L015/44; A61Q 19/00 20060101 A61Q019/00; A61K 8/02 20060101
A61K008/02; A61L 15/40 20060101 A61L015/40; A61K 9/70 20060101
A61K009/70; A61K 8/98 20060101 A61K008/98 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 4, 2014 |
EP |
14153872.8 |
Claims
1-78. (canceled)
79. A coated silk film comprising (i) an active agent release sheet
which comprises one or more first silk film layers comprising at
least one active agent, wherein the active agent release sheet
comprises (1) a top surface and a base surface, which are opposed
to each other, and (2) at least one side surface connecting the top
and the base surfaces, and (ii) one or more release modifying
layers completely covering the active agent release sheet,
80. The coated silk film of claim 79, wherein the coated silk film
is a self-supporting silk film without carrier element.
81. The coated silk film of claim 79, wherein the active agent
release sheet comprises one or more second silk film layers.
82. The coated silk film of claim 79, wherein the active agent is
selected from the group consisting of a biological agent, a
pharmaceutical agent, a cosmetic agent, a nutrient, and a dietary
supplement.
83. The coated silk film of claim 79, wherein the one or more first
silk film layers comprise at least one silk polypeptide.
84. The coated silk film of claim 83, wherein the at least one silk
polypeptide is a spider silk polypeptide.
85. The coated silk film of claim 81, wherein the one or more
second silk film layers comprise at least one silk polypeptide.
86. The coated silk film of claim 85, wherein the at least one silk
polypeptide is a spider silk polypeptide.
87. The coated silk film of claim 79, wherein the one or more first
silk film layers have a homogenous structure (i.e. are not
porous).
88. The coated silk film of claim 81, wherein the one or more
second silk film layers have a homogenous structure (i.e. are not
porous).
89. The coated silk film of claim 79, wherein the one or more
release modifying layers have a homogenous structure (i.e. are not
porous).
90. A pharmaceutical composition comprising the coated silk film of
claim 79, wherein the active agent is a pharmaceutical agent.
91. A cosmetic composition comprising the coated silk film of claim
79, wherein the active agent is a cosmetic agent.
92. A method of producing a coated silk film comprising the steps
of: (i) providing an active agent release sheet which comprises one
or more first silk film layers comprising at least one active
agent, wherein the active agent release sheet comprises (1) a top
surface and a base surface, which are opposed to each other, and
(2) at least one side surface connecting the top and the base
surfaces, and (ii) completely covering the active agent release
sheet with one or more release modifying layers.
93. The method of claim 92, wherein the coated silk film is a
self-supporting silk film without carrier element.
94. The method of claim 92, wherein the active agent release sheet
comprises one or more second silk film layers.
95. The method of claim 92, wherein the active agent is selected
from the group consisting of a biological agent, a pharmaceutical
agent, a cosmetic agent, a nutrient, and a dietary supplement.
96. The method of claim 92, wherein the one or more first silk film
layers comprise at least one silk polypeptide.
97. The method of claim 96, wherein the at least one silk
polypeptide is a spider silk polypeptide.
98. The method of claim 94, wherein the one or more second silk
film layers comprise at least one silk polypeptide.
99. The method of claim 98, wherein the at least one silk
polypeptide is a spider silk polypeptide.
100. The method of claim 92, wherein the one or more first silk
film layers have a homogenous structure (i.e. are not porous).
101. The method of claim 94, wherein the one or more second silk
film layers have a homogenous structure (i.e. are not porous).
102. The method of claim 92, wherein the one or more release
modifying layers have a homogenous structure (i.e. are not porous).
Description
[0001] The present invention relates to coated silk films. Further,
the present invention relates to pharmaceutical or cosmetic
compositions comprising the coated silk films. Furthermore, the
present invention relates to coated silk films or pharmaceutical
compositions comprising the coated silk films for use in medicine.
In addition, the present invention relates to methods of producing
the coated silk films.
BACKGROUND OF THE INVENTION
[0002] In the past years, sophisticated drug depot systems for
controlled delivery of drugs have been developed, for example to
achieve constant drug levels during therapy. These systems have the
advantage of reducing toxic side effects so that the number of drug
administrations can be decreased, while at the same time improving
cellular uptake and bioavailability. There is also an ongoing quest
to design depot systems which facilitate controlled delivery of
substances other than pharmaceutical compounds such as cosmetic
compounds. In general, the material employed as a carrier for
controlled delivery of a substance should offer control of
structure, morphology and function, while also exhibiting good
mechanical stability. For example, biodegradable and biocompatible
polymers are preferred because of their ability to retain their
properties for a limited period of time before gradually
decomposing into soluble nontoxic degradation products which can be
excreted from the body. Many synthetic polymers (e.g. polyester or
polyurethane) and biopolymers (e.g. polysaccharides or proteins)
have been employed to produce drug depot systems for encapsulation,
incorporation, or binding of active compounds. In particular, the
use of silk as biopolymer for drug depot systems, especially
because of its biocompatibility, non-toxicity and biodegradability,
has been investigated in the art. For example, silk films have been
tested for their ability to deliver drugs and other substances.
Especially, silk derived from spiders or insects has been used.
[0003] The inventors of the present invention studied silk films as
drug release systems. Without wishing to be bound to any theory,
they surprisingly found that drugs released faster through the side
surfaces than through the upper and lower surfaces of silk films,
thus, resulting in a(n) (initial) burst drug release profile.
However, in many applications it is desirable to release the loaded
drug over an extended period of time controllably, constantly, and
sustainably. The inventors of the present invention surprisingly
found that a controlled, constant, and sustained drug release
profile can be achieved with silk films which comprise at least at
their side surfaces a coating layer in order to reduce the rapid
diffusion of compounds through said side surfaces. The favourable
release profile of the coated silk films renders them eminently
suitable for controlled, constant, and sustained delivery of
pharmaceutical as well as cosmetic compounds. The compounds can be
small molecular weight compounds such as drugs and/or proteins. Due
to their stability and biocompatibility under physiological
conditions, the coated silk films are especially suitable for in
vivo applications. The casting of silk films from an aqueous
solution avoids the use of organic solvents.
SUMMARY OF THE INVENTION
[0004] In a first aspect, the present invention relates to a coated
silk film comprising [0005] (i) an active agent release sheet which
comprises one or more first silk film layers comprising at least
one active agent, and [0006] (ii) one or more release modifying
layers covering at least the side surface(s) of the active agent
release sheet.
[0007] In a second aspect, the present invention relates to a
pharmaceutical composition comprising the coated silk film
according to the first aspect, wherein the active agent is a
pharmaceutical agent.
[0008] In a third aspect, the present invention relates to a
cosmetic composition comprising the coated silk film according to
the first aspect, wherein the active agent is a cosmetic agent.
[0009] In a fourth aspect, the present invention relates to a
coated silk film according to the first aspect or pharmaceutical
composition according to the second aspect for use in medicine.
[0010] In a fifth aspect, the present invention relates to a coated
silk film according to the first aspect or pharmaceutical
composition according to the second aspect for controlled and
sustained release of at least one pharmaceutical agent.
[0011] In a sixth aspect, the present invention relates to a coated
silk film according to the first aspect or pharmaceutical
composition according to the second aspect for the treatment of
wounds, skin diseases, or skin defects.
[0012] In a seventh aspect, the present invention relates to the
use of the coated silk film according to the first aspect or
cosmetic composition according to the third aspect for controlled
and sustained release of at least one cosmetic agent.
[0013] In an eight aspect, the present invention relates to a
method of producing a coated silk film comprising the steps of:
[0014] (i) providing an active agent release sheet which comprises
one or more first silk film layers comprising at least one active
agent, and [0015] (ii) covering at least the side surface(s) of the
active agent release sheet with one or more release modifying
layers.
[0016] The following figures are merely illustrative of the present
invention and should not be construed to limit the scope of the
invention as indicated by the appended claims in any way.
BRIEF DESCRIPTION OF THE FIGURES
[0017] FIGS. 1A-1B: Shows in (FIG. 1A) a photograph of a spider
silk film comprising C.sub.16 after casting from an aqueous
solution using a digital camera (DSC-575, Sony Corporation, Tokyo,
Japan) (scale bar=1 cm) and in (FIG. 1B) a photograph of a spider
silk film comprising C.sub.16 after casting from an aqueous
solution with a film applicator using a digital camera (DSC-575,
Sony Corporation, Tokyo, Japan) (scale bar=1 cm). In particular,
the spider silk film comprising C.sub.16 was cast on the plastic
foil A5 22/5B from mtv-messtechnik (Koeln, Germany) at room
temperature using the film applicator Coatmaster 510 (Erichsen,
Hemer, Germany) having a casting knife of 2000 .mu.m and with a
velocity of 1 mm/sec.
[0018] FIG. 2: Shows remote loading of lysozyme as a model protein.
Loading efficiency and loading were determinate for lysozyme
dissolved in high purified water (HPW). For the remote loading of
lysozyme, the loading solution was prepared dissolving 1% w/v
lysozyme in high purified water. Three different films were
weighted, following by incubation in a 6-well plate with 5 ml of
the loading solution at room temperature, at 2 rpm. After 30 min,
films were removed and the loading solution was diluted 1:20 and
analysed photometrically at 280 nm recording its spectra using a
spectrophotometer (Agilent Technologies 8453, Oberhaching,
Germany). A calibration curve was prepared using the original
lysozyme solution. The same method was performed using as
incubation medium of 0.5% and 0.25% of lysozyme.
[0019] FIG. 3: Shows the surface of a spider silk film comprising
C.sub.16 and containing paracetamol as active agent. Before casting
the spider silk film, paracetamol was added and dissolved directly
in a 2.5% w/v C.sub.16 protein solution (direct loading). The
resulting spider silk film comprising C.sub.16 contained 2.5 mg of
paracetamol and optically appeared like unloaded films (FIG. 1). In
particular, said spider silk film showed a smooth, transparent, and
colorless surface. A picture of the film was taken by digital
microscope Keyence VHX-500F (Keyence Corporation, Osaka, Japan).
Scale bar: 250 .mu.m.
[0020] FIGS. 4A-4D: Shows in (FIG. 4A) a schematic cross-sectional
drawing of a silk film comprising an active agent release sheet
which comprises one first silk film layer comprising an active
agent and two second silk film layers (i.e. 3-layer silk film).
(FIG. 4B) Cross section of a C.sub.16 film schematically shown as
silk film in (FIG. 4A) comprising an active agent release sheet
which comprises one first C.sub.16 film layer comprising an active
agent and two second C.sub.16 film layers (Digital Microscope
Keyence VHX-500F) (i.e. 3-layer C.sub.16 film). Scale bar: 50
.mu.m. (FIG. 4C) Schematic drawing of a coated silk film of the
present invention comprising an active agent release sheet which
comprises one first silk film layer comprising an active agent and
two second silk film layers, and one release modifying silk layer
completely covering the active agent release sheet (i.e. 4-layer
silk film). (FIG. 4D) Cross section of a coated C.sub.16 film
schematically shown in (FIG. 4C) comprising an active agent release
sheet which comprises one first C.sub.16 film layer comprising an
active agent and two second C.sub.16 film layers, and one release
modifying C.sub.16 film layer completely covering the active agent
release sheet (i.e. 4-layer C.sub.16 film). Scale bar: 250
.mu.m.
[0021] FIGS. 5A-5C: Shows in (FIG. 5A) a schematic cross-sectional
drawing of a silk film comprising an active agent release sheet
which comprises one first silk film layer comprising an active
agent and six second silk film layers, wherein plasticizer
containing second silk film layers and plasticizer free second silk
film layers are arranged in alteration (i.e. 7-layer silk film).
(FIG. 5B) Cross section of a C.sub.16 film schematically shown as
silk film in (FIG. 5A) comprising an active agent release sheet
which comprises one first C.sub.16 film layer comprising an active
agent and six second C.sub.16 film layers, wherein glycerol
containing second C.sub.16 film layers and glycerol free second
C.sub.16 film layers are arranged in alteration (Digital Microscope
Keyence VHX-500F) (i.e. 7-layer C.sub.16 film). Scale bar: 250
.mu.m. (FIG. 5C) Schematic drawing of a coated silk film of the
present invention comprising an active agent release sheet which
comprises one first silk film layer comprising an active agent and
six second silk film layers, wherein plasticizer containing second
silk film layers and plasticizer free second silk film layers are
arranged in alteration and one release modifying silk layer
completely covering the active agent release sheet (i.e. 8-layer
silk film).
[0022] FIG. 6: Shows the cumulative release of paracetamol,
FITC-dextran 21 kDa (FD) and FITC-BSA from C.sub.16 films.
Paracetamol: Before casting the C.sub.16 films, paracetamol was
added and dissolved directly in 2.5% C.sub.16 protein solution. The
resulting films contained each 2.5 mg of paracetamol. Three film
samples containing paracetamol were incubated in 0.01 M PBS buffer
pH 7.4 at 37.degree. C. At predetermined time points, the buffer
was removed and replaced with fresh medium. The release buffer was
analyzed by NanoDrop 2000 (peqlab, Erlanger, Germany). FITC-BSA and
FITC-dextran (FD): 20 mg/mL of FITC-BSA (MW 66.5 kDa) or
FITC-dextran (FD) (MW 21 kDa) solution in 0.01 M PBS buffer pH 7.4
were directly mixed with the C.sub.16 protein solution. Each film
contained 1.3 mg of FITC-BSA or FD. Films containing the model
drugs (n=3) were incubated in 0.01 M PBS buffer pH 7.4 at
37.degree. C. At predetermined time points the buffer was removed
and replaced with fresh medium. The release buffer was analyzed
using the fluorescence spectrophotometer Cary Eclipse Varian
(Agilent technology, Boblingen, Germany) applying an excitation
wavelength of 490 nm and an emission of 520 nm. The drug which was
not released was quantified by dissolving the film matrix in a 6 M
GdmSCN solution. This solution was subsequently analyzed using a
fluorescence spectrophotometer.
[0023] FIG. 7: Shows the cumulative release of the model drug
FITC-BSA from different C.sub.16 films (n=3). 1% w/v of glycerol
and a 20 mg/mL FITC-BSA solution in 0.01 M PBS buffer pH 7.4 were
directly mixed with the C.sub.16 protein solution. Each film
contained 1.3 mg of FITC-BSA (monolayer with plasticizer glycerol).
Typically, one C.sub.16 film layer containing the model drug
FITC-BSA and the plasticizer glycerol was positioned between two
C.sub.16 film layers. Using the hydraulic press 5T (Maassen GmbH,
Reutlingen, Germany), the film layers were pressed together by
applying 0.2 t for 5 min (3Layers 0.2t); 0.4 t for 5 min (3Layers
0.4t); 1 t for 5 min (3Layers it). During this process, three-layer
films were created, whereby only the middle layer contained the
model drug FITC-BSA. In one case, one C.sub.16 film layer
containing the model drug FITC-BSA and the plasticizer glycerol was
positioned between two C.sub.16 film layers containing the
plasticizer glycerol. The film layers were pressed together using a
hydraulic press by applying 1 t for 5 min (3 Glycerol-layers it).
Three samples of all these different kind of films were incubated
in 0.01 M PBS buffer pH 7.4 at 37.degree. C. At predetermined time
points the buffer was removed and replaced with fresh medium. The
release buffer was analyzed using the fluorescence
spectrophotometer Cary Eclipse Varian (Agilent technology,
Boblingen, Germany) applying an excitation wavelength of 490 nm and
an emission of 520 nm. The drug which was not released, was
quantified by dissolving the film matrix in a 6 M GdmSCN solution.
This solution was subsequently analyzed using a fluorescence
spectrophotometer.
[0024] FIGS. 8A-8B: Shows the effect of glycerol concentration on
the release of FITC-BSA from C.sub.16 films. (FIG. 8A) In one case,
1% w/v of the plasticizer glycerol and a 20 mg/mL FITC-BSA solution
in 0.01 M PBS buffer pH 7.4 were directly mixed with the C.sub.16
protein solution (monolayer with glycerol). In one another case,
the plasticizer glycerol was not included in the casting solution
(monolayer). After casting, each film contained 1.3 mg of FITC-BSA.
Three samples of these two different variants of films were
incubated in 0.01 M PBS buffer pH 7.4 at 37.degree. C. At
predetermined time points, the buffer was removed and replaced with
fresh medium. The release buffer was analyzed using the
fluorescence spectrophotometer Cary Eclipse Varian (Agilent
technology, Boblingen, Germany) applying an excitation wavelength
of 490 nm and an emission of 520 nm. The drug which was not
released, was quantified by dissolving the film matrix in a 6 M
GdmSCN solution. This solution was subsequently analyzed using a
fluorescence spectrophotometer. The monolayer containing glycerol
released the model drug BSA much quicker than the monolayer without
glycerol. (FIG. 8B) Films were cast from a casting solution
containing the model drug FITC-BSA and different concentrations of
the plasticizer glycerol: 1%, 3% and 5% w/v. The release study was
carried out as described in (A). A film containing more glycerol
released the model drug BSA quicker than a film containing low
glycerol. In particular, the film containing the model drug
FITC-BSA and different concentrations of the plasticizer glycerol,
namely 1%, 3% and 5% w/v, was part of a 3-layer C.sub.16 film
(n=3). Said film was positioned between two C.sub.16 film layers.
Thus, said film formed the middle layer of a 3-layer C.sub.16 film.
The middle layer was cast from a casting solution containing the
C.sub.16 protein, the model drug FITC-BSA and different
concentrations of the plasticizer glycerol: 1%, 3% and 5% w/v.
Glycerol and a 20 mg/mL FITC-BSA solution in 0.01 M PBS buffer pH
7.4 were directly mixed with the C.sub.16 protein solution. Each
film contained 1.3 mg of FITC-BSA and the defined concentration of
glycerol (1%, 3% or 5% w/v). The other two C.sub.16 film layers
were cast from a casting solution containing the C.sub.16 protein.
As mentioned above, one C.sub.16 film layer containing the model
drug FITC-BSA and the plasticizer was positioned between two
C.sub.16 film layers. Using the hydraulic press 5T (Maassen GmbH,
Reutlingen, Germany), the film layers were pressed together by
applying 0.2 t for 5 min. During this process, three-layer films
were created, whereby only the middle layer contained the model
drug FITC-BSA and the plasticizer.
[0025] FIG. 9: Shows the cumulative release of FITC-BSA from
different kind of C.sub.16 films (n=3) and a coated C.sub.16 film
(n=3). Monolayer films were prepared by mixing a 20 mg/mL FITC-BSA
solution in 0.01 M PBS buffer pH 7.4 with the C.sub.16 protein
solution. Each film contained 1.3 mg of the model drug FITC-BSA. 3
Layer films were prepared by positioning one C.sub.16 film layer
containing the model drug FITC-BSA and the plasticizer glycerin (1%
w/v) between two C.sub.16 film layers. The film layers were pressed
together using the hydraulic press 5T (Maassen GmbH, Reutlingen,
Germany) and applying 0.2 t for 5 min. Three-layer films were
created where only the middle layer contained the model drug
FITC-BSA. In one case, these sandwich films were coated by dipping
them in a 5% w/v C.sub.16 solution (Coated 3 Layers). 7 Layer films
were prepared by pressing seven film layers together. 0.2 t for 5
min were applied. In particular, one C.sub.16 film layer comprising
the model drug FITC-BSA and the plasticizer glycerol as well as six
C.sub.16 film layers, wherein plasticizer containing C.sub.16 film
layers and plasticizer free C.sub.16 film layers were arranged in
alteration, were pressed together. Three samples of all these
different kind of films were incubated in 0.01 M PBS buffer pH 7.4
at 37.degree. C. At predetermined time points the buffer was
removed and replaced with fresh medium. The release buffer was
analyzed using the fluorescence spectrophotometer Cary Eclipse
Varian (Agilent technology, Boblingen, Germany) applying an
excitation wavelength of 490 nm and an emission of 520 nm. The drug
which was not released, was quantified by dissolving the film
matrix in a 6 M GdmSCN solution. This solution was subsequently
analyzed by fluorescence spectrophotometer.
[0026] FIG. 10: Shows the cumulative release of FITC-BSA from
different kind of coated C.sub.16 silk films (n=3). Monolayer films
were prepared by mixing a 20 mg/mL FITC-BSA solution in 0.01 M PBS
buffer pH 7.4 with the C.sub.16 protein solution. After casting,
each film contained 1.3 mg of the model drug FITC-BSA. Sandwich
films were prepared by positioning one C.sub.16 film layer
containing the model drug FITC-BSA and the plasticizer glycerol (1%
w/v) between two C.sub.16 film layers. The film layers were pressed
together using the hydraulic press 5T (Maassen GmbH, Reutlingen,
Germany). 0.2 t for 5 min were applied. Three-layer films were
created where only the middle layer contained the model drug
FITC-BSA. Finally, monolayer films containing the model drug
FITC-BSA and the sandwiches (FIG. 4A) were coated by dipping them
in a 5% w/v C.sub.16 protein solution. They were dried overnight at
RT in the dark. Three samples of these different kind of films were
incubated in 0.01 M PBS buffer pH 7.4 at 37.degree. C. At
predetermined time points the buffer was removed and replaced with
fresh medium. The release buffer was analyzed by fluorescence
spectrophotometer Cary Eclipse Varian (Agilent technology,
Boblingen, Germany) applying an excitation wavelength of 490 nm and
an emission of 520 nm. The drug which was not released, was
quantified by dissolving the film matrix in a 6 M GdmSCN solution.
This solution was subsequently analyzed by fluorescence
spectrophotometer.
[0027] FIG. 11: Shows the remote loading and the direct loading. In
the case of the remote-loading, C.sub.16 silk films were incubated
in a drug solution and the loading was mainly driven by
electrostatic and hydrophilic/hydrophobic interactions. In the
direct loading approach, drugs were directly incorporated into the
casting C.sub.16 protein solution: the drug was directly dissolved
in the casting C.sub.16 protein solution or, alternatively, the
C.sub.16 protein solution was mixed with a second solution
containing the dissolved drug. The casting mixture loaded with the
drug was cast.
[0028] FIG. 12: Scanning electron micrographs (magnification:
10,000.times.) of a spider silk monolayer film comprising C.sub.16.
The film was cast at room temperature from an aqueous solution
comprising C.sub.16 on a plastic foil A5 22/5B from mtv-messtechnik
(Koeln, Germany) by the solvent evaporation technique. The film was
immobilized on Leit-Tabs (Plano GmbH, Wetzlar, Germany) to the
sample holder. Samples were carbon sputtered under vacuum and
analyzed by a Joel JSM-6500F field emission scanning electron
microscope (Joel Inc., Peabody, USA). The film had a remarkably
uniform and homogenous appearance.
[0029] FIG. 13: FITC-BSA released from a spider silk monolayer film
comprising C.sub.16, the model drug FITC-BSA and 1% w/v glycerol
(black, n=3) and FITC-BSA released from a coated spider silk
monolayer film comprising C.sub.16, the model drug FITC-BSA and 1%
w/v glycerol (grey, n=3). The spider silk monolayer films were cast
at room temperature from an aqueous solution comprising C.sub.16,
the model drug FITC-BSA and 1% w/v glycerol on a plastic foil A5
22/5B from mtv-messtechnik (Koeln, Germany) by the solvent
evaporation technique. The coated spider silk monolayer films
containing C.sub.16, the model drug FITC-BSA and 1% w/v glycerol
were further prepared by dipping them into a 5% w/v C.sub.16
protein solution. They were dried overnight at room temperature
(RT) in the dark. Coating of monolayers containing glycerol with a
highly concentrated spider silk protein solution leads to the
reduction of the release of FITC-BSA compared to uncoated
monolayers containing glycerol.
DETAILED DESCRIPTION OF THE INVENTION
[0030] Although the present invention is described in detail below,
it is to be understood that this invention is not limited to the
particular methodologies, protocols and reagents described herein
as these may vary. It is also to be understood that the terminology
used herein is for the purpose of describing particular embodiments
only, and is not intended to limit the scope of the present
invention which will be limited only by the appended claims. Unless
defined otherwise, all technical and scientific terms used herein
have the same meanings as commonly understood by one of ordinary
skill in the art.
[0031] In the following, the elements of the present invention will
be described. These elements are listed with specific embodiments,
however, it should be understood that they may be combined in any
manner and in any number to create additional embodiments. The
variously described examples and preferred embodiments should not
be construed to limit the present invention to only the explicitly
described embodiments. This description should be understood to
support and encompass embodiments which combine the explicitly
described embodiments with any number of the disclosed and/or
preferred elements. Furthermore, any permutations and combinations
of all described elements in this application should be considered
disclosed by the description of the present application unless the
context indicates otherwise. For example, if in a preferred
embodiment the coated silk film of the present invention comprises
at least one pharmaceutical active agent and if in another
preferred embodiment the one or more first silk film layer of the
coated silk film comprise at least one silk polypeptide, it is a
contemplated preferred embodiment that the coated silk film of the
present invention comprises at least one pharmaceutical active
agent and one or more first silk film layers comprising at least
one silk polypeptide.
[0032] Preferably, the terms used herein are defined as described
in "A multilingual glossary of biotechnological terms: (IUPAC
Recommendations)", Leuenberger, H. G. W, Nagel, B. and Kolbl, H.
eds. (1995), Helvetica Chimica Acta, CH-4010 Basel,
Switzerland).
[0033] The practice of the present invention will employ, unless
otherwise indicated, conventional methods of chemistry,
biochemistry, cell biology, immunology, and recombinant DNA
techniques which are explained in the literature in the field (cf.,
e.g., Molecular Cloning: A Laboratory Manual, 2.sup.nd Edition, J.
Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold
Spring Harbor 1989).
[0034] Throughout this specification and the claims which follow,
unless the context requires otherwise, the word "comprise", and
variations such as "comprises" and "comprising", will be understood
to imply the inclusion of a stated member, integer or step or group
of members, integers or steps but not the exclusion of any other
member, integer or step or group of members, integers or steps. The
terms "a" and "an" and "the" and similar reference used in the
context of describing the invention (especially in the context of
the claims) are to be construed to cover both the singular and the
plural, unless otherwise indicated herein or clearly contradicted
by context. Recitation of ranges of values herein is merely
intended to serve as a shorthand method of referring individually
to each separate value falling within the range. Unless otherwise
indicated herein, each individual value is incorporated into the
specification as if it were individually recited herein. All
methods described herein can be performed in any suitable order
unless otherwise indicated herein or otherwise clearly contradicted
by context. The use of any and all examples, or exemplary language
(e.g., "such as"), provided herein is intended merely to better
illustrate the invention and does not pose a limitation on the
scope of the invention otherwise claimed. No language in the
specification should be construed as indicating any non-claimed
element essential to the practice of the invention.
[0035] Several documents are cited throughout the text of this
specification. Each of the documents cited herein (including all
patents, patent applications, scientific publications,
manufacturer's specifications, instructions, GenBank Accession
Number sequence submissions etc.), whether supra or infra, is
hereby incorporated by reference in its entirety. Nothing herein is
to be construed as an admission that the invention is not entitled
to antedate such disclosure by virtue of prior invention.
[0036] In the following, definitions will be provided which apply
to all aspects of the present invention.
[0037] Residues in two or more polypeptides are said to
"correspond" to each other if the residues occupy an analogous
position in the polypeptide structures. It is well known in the art
that analogous positions in two or more polypeptides can be
determined by aligning the polypeptide sequences based on amino
acid sequence or structural similarities. Such alignment tools are
well known to the person skilled in the art and can be, for
example, obtained on the World Wide Web, e.g., ClustalW
(www.ebi.ac.uk/clustalw) or Align
(http://www.ebi.ac.uk/emboss/align/index.html) using standard
settings, preferably for Align EMBOSS: needle, Matrix: Blosum62,
Gap Open 10.0, Gap Extend 0.5.
[0038] Unless otherwise indicated, the terms "polypeptide" and
"protein" are used interchangeably herein and mean any
peptide-linked chain of amino acids, regardless of length or
post-translational modification.
[0039] The term "silk film", as described herein, refers to a
self-supporting object. A self-supporting object is an object which
has the capacity for supporting itself without the help of
additional materials such as carrier elements. A silk film being
self-supporting preferably has a thickness of at least 0.1
.mu.m.
[0040] The term "silk film", as described herein, also refers to an
object having a first surface and a second surface which are
opposed to each other and having at least one connecting side
surface. The first surface may also be designated as top surface
and the second surface may also be designated as base surface. In
one embodiment, the surface area of the first surface and second
surface is larger than the surface area of the side surface(s). For
example, the first and second surface area is at least 10 fold
larger than the surface area of the side surface(s). The silk film
may have the form of a pancake cylinder having a first surface and
a second surface which are opposed to each other and a connecting
side surface. The silk film may also have a flat rectangular block
shape having a first surface and a second surface which are opposed
to each other and connecting side surfaces. The first and second
surfaces preferably have the same shape and/or dimension.
[0041] The term "coated silk film", as used herein, refers to a
silk film which additionally comprises (a) release modifying
layer(s) covering at least the side surface(s) of the silk film. In
one embodiment, the modifying layer(s) completely cover(s) the silk
film. The modifying layer(s) is (are) not necessarily
self-supporting, but can be self-supporting. The release modifying
layer preferably has a thickness of at least 0.05 .mu.m, e.g. of at
least 0.1 .mu.m.
[0042] The term "coated silk film", as used herein, also refers to
a silk film comprising or consisting of an active agent release
sheet.
[0043] The term "active agent release sheet", as used herein,
refers to a sheet comprising (an) active agent(s). The active agent
release sheet is loaded with said active agent(s). In other words,
the active agent(s) is (are) incorporated into and/or attached to
the active agent release sheet. The active agent release sheet is
designed to release the active agent(s) comprised therein, e.g.
into a physiological environment. Preferably, said release is a
controlled, constant, and/or sustained release. In particular, the
active agent release sheet is designed to release the active
agent(s) at a predetermined rate in order to maintain a constant
active agent concentration for a specific period of time with
minimum side effects.
[0044] The term "active agent release sheet", as used herein, also
refers to an object having a first surface and a second surface
which are opposed to each other and having at least one connecting
side surface. The first surface may also be designated as top
surface and the second surface may also be designated as base
surface. In one embodiment, the surface area of the first surface
and second surface is larger than the surface area of the side
surface(s). For example, the first and second surface area is at
least 10 fold larger than the surface area of the side
surface(s).
[0045] More precisely, the term "active agent release sheet", as
used herein, refers to a sheet which comprises (a) first silk film
layer(s) comprising (an) active agent(s). Said first silk film
layer(s) is (are) loaded with said active agent(s) or the active
agent(s) is (are) incorporated into and/or attached to the first
silk film layer(s). The active agent release sheet can have a
monolayered or multilayered form. In its monolayered form, the
active agent release sheet comprises or consists of a first silk
film layer comprising (an) active agent(s). In its multilayered
form, the active agent release sheet comprises or consists of first
silk film layers comprising (an) active agent(s), or (a) first silk
film layer(s) comprising (an) active agent(s) and (a) second silk
film layer(s). The active agent release sheet preferably has a
thickness of at least 0.1 .mu.m.
[0046] The term "first silk film layer", as used herein, refers to
a film layer comprising or consisting of silk and (an) active
agent(s). The term "second silk film layer", as used herein, refers
to a film layer comprising or consisting of silk. Thus, the second
silk film layer is a layer which does not comprise (an) active
agent(s). In other words, the second silk film layer is active
agent free. The first silk film layer preferably has a thickness of
at least 0.1 .mu.m. In addition, the second silk film layer
preferably has a thickness of at least 0.1 .mu.m.
[0047] The (coated) silk film, particularly the one or more first
silk film layers and/or the one or more second silk film layers,
comprise a silk material (e.g. a silk polypeptide).
[0048] In the coated silk film, the active agent release sheet is
at least at its side surface(s) covered by (a) modifying layer(s).
In one embodiment, the modifying layer(s) completely cover(s) the
active agent release sheet.
[0049] In its broadest sense, the term "coated silk film", as used
herein, refers to a silk film which comprises an active agent
release sheet consisting of a first silk film layer comprising at
least one active agent. Said release sheet is at least at its side
surface(s) covered by a release modifying layer.
[0050] The term "release modifying layer", as used herein, refers
to a layer which is designed to modify or modulate, preferably to
reduce or delay, the release, e.g. by diffusion, of the active
agent(s) from the side surface(s) of the silk film, particularly of
the active agent release sheet, as compared to a silk film,
particularly an active agent release sheet, lacking such a release
modifying layer. In one embodiment, the release modifying layer
completely covers the silk film, particularly the active agent
release sheet, and, thus, modifies or modulates, preferably reduces
or delays, the release, e.g. by diffusion, of the active agent(s)
from any surface of the silk film, particularly of the active agent
release sheet, as compared to a silk film, particularly an active
agent release sheet, lacking such a release modifying layer. For
example, the release modifying layer(s) modify/modifies or
modulate(s), preferably reduce(s) or delay(s), the release of the
active agent(s) from the side surface(s) of the silk film,
particularly of the active agent release sheet, by at least 5, 10,
15, 20, 30, 40, 50, 60, 70, 80, 90, 95, 99, or even 100% compared
to a silk film, particularly an active agent release sheet, lacking
such a release modifying layer. The release modifying layer
preferably has a thickness of at least 0.05 .mu.m, e.g. of at least
0.1 .mu.m. The release modifying layer comprises or consists of a
release modifying material (e.g. a silk material such as a silk
polypeptide).
[0051] The term "release modifying agent", as used herein, refers
to an agent which is able to modify or modulate, preferably to
reduce or delay, the release, e.g. by diffusion, of (an) active
agent(s). Being comprised in the "release modifying layer" or
forming the "release modifying layer", the release modifying agent
particularly modifies or modulates, preferably reduces or delays,
the release of (an) active agent(s) from the side surface(s) of the
silk film, particularly of the active agent release sheet. If the
release modifying layer completely covers the silk film,
particularly the active agent release sheet, the release modifying
agent particularly modifies or modulates, preferably reduces or
delays, the release of (an) active agent(s) from any surface of the
silk film, particularly of the active agent release sheet.
[0052] In the context of the present invention, the term "coating"
refers to a covering that is applied to the silk film, particularly
to the active agent release sheet, to be coated. Said coating
covers or surrounds at least the side surface(s) of the silk film,
particularly of the active agent release sheet. In one embodiment,
said "coating" completely covers or surrounds the silk film,
particularly the active agent release sheet. The coating preferably
has a thickness of at least 0.05 .mu.m, e.g. of at least 0.1 .mu.m.
In particular, the coating of the silk film is achieved with the
release modifying layer(s) as described herein.
[0053] The term "completely covering", as used herein, means that
the silk film, particularly the active agent release sheet, is
covered with a contiguous coating. Said covering may be evenly or
unevenly, preferably evenly.
[0054] The term "active agent (also designated as active
compound)", as used herein, relates to any substance having an
activity when administered to an individual. Said substance may
have a biological, pharmaceutical, or cosmetical activity when
administered to an individual. A pharmaceutical agent may be any
agent with therapeutic, diagnostic, or prophylactic effects, i.e.
any therapeutic agent, diagnostic agent, or prophylactic agent. The
term "active agent", as used herein, further relates to any
substance which is capable of being released from the silk film,
particularly the active agent release sheet. It is preferred that
the active agent is capable of being released upon exposure of the
silk film to physiological conditions, i.e. introducing the silk
film, into a buffer or an aqueous solution. It is further preferred
that the silk film shows a controlled, constant, and/or sustained
release of the loaded active agent. The active agent may be
comprised in the silk film, particularly in the active agent
release sheet, in form of a particle, such as a nano-particle or
micro-particle, or an aggregate.
[0055] The term "release", as used herein, refers to the release of
(an) active agent(s) from the silk film, particularly from the
active agent release sheet, over a period of time.
[0056] The term "sustained (or controlled) release" as used herein"
refers to the gradual release of (an) active agent(s) from the silk
film, particularly from the active agent release sheet, over a
period of time. It is preferred that the release displays
relatively linear kinetics, thereby providing a constant supply of
the active agent over the release period. The release period may
vary from several hours to several months depending upon the
properties of the active agent(s) and its (their) intended use. For
example, it can be desirable that the cumulative release of a
pharmaceutical agent from the silk film over a certain treatment
period be relatively high to avoid the need for excessive loading
of the silk film and consequent waste of unreleased pharmaceutical
agent. The active agent(s) is (are) released into a surrounding
environment. The surrounding environment may be a physiological
environment or a non-physiological environment. The physiological
environment (e.g. the physiological medium) may be a physiological
buffered solution or a body fluid, e.g. blood, lymph or liquor. The
physiological environment may also be an organ (e.g. skin) or a
part of an organ (e.g. tissue). The non-physiological environment
(e.g. the non-physiological medium) may be an aqueous solution such
as water or a buffered aqueous solution, an alcoholic solution, or
an organic solution. It should be further noted that the active
agent(s) can be released into a surrounding intracorporeal or
extracorporeal environment.
[0057] The term "physiological environment", as used herein,
refers, without limitation, to an environment characterized by a
temperature of 37.degree. C. and an atmospheric pressure of 1 bar
(100 kPa). It further refers, without limitation, to an environment
characterized by a temperature of 37.degree. C., an atmospheric
pressure of 1 bar (100 kPa), and a pH of between 4 and 8,
particularly a pH of between 6 and 8. Blood as physiological
environment has usually, for example, a pH of between 7.37 and
7.44, e.g. a pH of 7.40. Skin as physiological environment has
usually, for example, a pH of between 4 and 6.5, e.g. a pH of
5.5.
[0058] As mentioned above, the inventors of the present invention
studied silk films as drug release systems. Without wishing to be
bound to any theory, they surprisingly found that drugs released
faster through the side surfaces than through the upper and lower
surfaces of silk films, thus, resulting in a(n) (initial) burst
drug release profile. However, in many applications it is desirable
to release the loaded drug over an extended period of time
controllably, constantly, and sustainably. The inventors of the
present invention surprisingly found that a controlled, constant,
and sustained drug release profile can be achieved with silk films
which comprise at least at their side surfaces a coating layer in
order to reduce the rapid diffusion of compounds through said side
surfaces. The favourable release profile of the coated silk films
renders them eminently suitable for controlled, constant, and
sustained delivery of pharmaceutical as well as cosmetic compounds.
Due to their stability and biocompatibility under physiological
conditions, the coated silk films are especially suitable for in
vivo applications.
[0059] Thus, in a first aspect, the present invention relates to a
coated silk film comprising or consisting of [0060] (i) an active
agent release sheet which comprises or consists of one or more
first silk film layers comprising at least one active agent, and
[0061] (ii) one or more release modifying layers covering at least
the side surface(s) of the active agent release sheet.
[0062] The coated silk film may comprise an active agent release
sheet comprising 1, 2, 3, or more first silk film layers.
Alternatively or additionally, the coated silk film may comprise 1,
2, 3, 4, 5, or more release modifying layers.
[0063] In one embodiment, the one or more release modifying layers
completely cover the active agent release sheet. Thus, the coated
silk film may comprise or consist of [0064] (i) an active agent
release sheet which comprises or consists of one or more first silk
film layers comprising at least one active agent, and [0065] (ii)
one or more release modifying layers completely covering the active
agent release sheet.
[0066] As mentioned above, the active agent release sheet may
comprise two or more first silk film layers. Each first silk film
layer of the active agent release sheet may comprise the same or
(a) different active agent(s). The incorporation of different
active agents in the first silk film layers of the active agent
release sheet allows the modification of the release profile and
the control of the interaction between different active agents. For
example, if two active agents cannot be incorporated together in
the same first silk film layer due to the negative interaction
between said active agents, each active agent can be loaded in a
different first silk film layer.
[0067] In one another embodiment, the active agent release sheet
comprises one or more second silk film layers. Thus, the coated
silk film may comprise or consist of [0068] (i) an active agent
release sheet which comprises or consists of one or more first silk
film layers comprising at least one active agent and one or more
second silk film layers, and [0069] (ii) one or more release
modifying layers covering at least the side surface(s) of the
active agent release sheet.
[0070] The coated silk film may comprise an active agent release
sheet comprising 1, 2, 3, or more first silk film layers and/or 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more second silk film
layers. Alternatively or additionally, the coated silk film may
comprise 1, 2, 3, 4, 5, or more release modifying layers.
[0071] As mentioned above, in one embodiment, the one or more
release modifying layers completely cover the active agent release
sheet. Thus, the coated silk film may comprise or consist of [0072]
(i) an active agent release sheet which comprises or consists of
one or more first silk film layers comprising at least one active
agent and one or more second silk film layers, and [0073] (ii) one
or more release modifying layers completely covering the active
agent release sheet.
[0074] In one preferred embodiment, the active agent release sheet
comprises or consists of (a) first silk film layer(s) comprising at
least one active agent which is (are) flanked by second silk film
layers. In other word, the first silk film layer(s) comprising at
least one active agent is (are) sandwiched or embedded between
second silk film layers. The inventors of the present invention
found that the number of second silk film layers flanking the first
silk film layer(s) has an influence on the release of the active
agent comprised therein. Thus, an increase of the number of second
silk film layers flanking the first silk film layer(s) reduces the
release of the active agent comprised therein. Thus, the variation
of the number of second silk film layers flanking the first silk
film layer(s) allows to adapt/change the release profile of the
active agents comprised therein.
[0075] In one another preferred embodiment, the one or more first
silk film layers and/or the one or more second silk film layers
comprise at least one plasticizer. The inventors of the present
invention found that the mechanical properties, e.g. the elasticity
and/or flexibility, of the silk film can be improved using
plasticizers. For example, the use of plasticizers, improves,
particularly increases, the film elongation. Further, the inventors
of the present invention found that the presence of plasticizers in
the first silk film layers or in the first and second silk film
layers improves the connection of the first silk film layers or of
the first and second silk film layers with each other, or the
coherence of the first silk film layers or of the first and second
silk film layers to each other. Furthermore, the inventors of the
present invention found that the presence of plasticizers in the
silk film, particularly in the active agent release sheet,
influences the release of active agents from the silk film,
particularly from the active agent release sheet. In particular,
the presence of plasticizers modifies or modulates, preferably
increases or decreases, the release of active agents. For example,
the presence of plasticizers such as glycerol increases the release
of active agents, while the presence of plasticizers such as
2-pyrrolidone decreases the release of active agents. Thus, the
presence of plasticizers in the first and/or second silk film
layer(s) of the active agent release sheet of the silk film or not
and, if yes, the variation of the amount of plasticizers in the
first and/or second silk film layer(s) of the active agent release
sheet of the silk film allows to adapt/change the release profile
of the active agents comprised therein.
[0076] In one further preferred embodiment, the active agent
release sheet comprises plasticizer containing first and/or second
silk film layers and plasticizer free first and/or second silk film
layers in alteration. For example, the active agent release sheet
may comprise one plasticizer containing first silk film layer and
one plasticizer free second silk film layer. Further, the active
agent release sheet may comprise one plasticizer containing first
silk film layer and two plasticizer free second silk film layers,
wherein both plasticizer free second silk film layers are attached
to the plasticizer containing first silk film layer, thereby
forming a sandwich structure. Furthermore, the active agent release
sheet may comprise one plasticizer containing first silk film
layer, one or more plasticizer containing second silk film layers
and one or more plasticizer free second silk film layers, wherein
the plasticizer containing silk film layers and plasticizer free
silk film layers are arranged alternately. The active agent release
sheet may have a structure as described in FIGS. 4A and 4B, and
FIGS. 5A and 5B. The coated silk film may have a structure as
described in FIGS. 4C and 4D and FIG. 5C.
[0077] In one embodiment, the one or more release modifying layers
do not contain a plasticizer. In other words, in one embodiment,
the one or more release modifying layers are plasticizer free. In
one preferred embodiment, the one or more release modifying layers
do not contain a plasticizer which increases the release of active
agents. As mentioned above, the presence of plasticizers such as
glycerol may increase the release of active agents. The release
modifying layers may, however, be layers which are designed to
reduce or delay/sustain the release of active agents at least at
the side surface(s) of the active agent release sheet. In this
case, the presence of plasticizers which increase the release of
active agents in said release modifying layer(s) is not preferred.
In another preferred embodiment, the one or more release modifying
layers contain a plasticizer which decreases the release of active
agents. As mentioned above, the presence of plasticizers such as
2-pyrrolidone may decrease the release of active agents. The
release modifying layers may also be layers which are designed to
reduce or delay/sustain the release of active agents at least at
the side surface(s) of the active agent release sheet. In this
case, the presence of plasticizers which decrease the release of
active agents in said release modifying layer(s) is preferred.
[0078] It is preferred that the amount of the plasticizer in the
one or more first silk film layers and/or one or more second silk
film layers is of between 0.1% (w/w) and 70% (w/w), more preferably
of between 5% (w/w) and 70% (w/w) or of between 15% (w/w) and 70%
(w/w), even more preferably of between 15% (w/w) and 55% (w/w) or
of between 30% (w/w) and 55% (w/w), and most preferably of 30%
(w/w). It is further preferred that the amount of the plasticizer
in the one or more first silk film layers and/or one or more second
silk film layers is of at least 0.1, 1, 5, 10, 15, 20, 25, 30, 35,
40, 45, 50, 55, 60, 65, or 70% (w/w).
[0079] Preferred plasticizers are selected from glycerol,
2-pyrrolidone, polyethylenglycol (PEG), polyvinylalcohol (PVA),
poloxamer, polyvinylpirrolidone (PVP), polyacrylic acid,
polyorthoester, gelatine, collagen, cellulose, cellulose derivates,
and sorbitol. More preferred plasticizers are selected from
glycerol and 2-pyrrolidone.
[0080] It is further preferred that the active agent has a
molecular weight of between 50 Da and 300 kDa, preferably of
between 80 Da and 250 kDa, more preferably of between 100 Da and
200 kDa, and most preferably of between 150 Da and 150 kDa. The
active agent may further have a molecular weight of between 50 Da
and <50 kDa or a molecular weight of between 50 kDa and 150 kDa.
The inventors of the present invention found that the release rate
of active agents depends on their molecular weight. In particular,
they found that the higher the molecular weight of the active
agents, the slower is their release. For example, active agents
having a molecular weight of between 50 kDa and 150 kDa are
released slower from the silk film, particularly from the active
agent release sheet, compared to active agents having a molecular
weight of between 50 Da and <50 kDa.
[0081] Preferred active agents are selected from the group
consisting of a biological agent, a pharmaceutical agent, a
cosmetic agent, a nutrient, and a dietary supplement.
[0082] The term "biological agent (also designated as biological
material)", as used herein, relates to any substance or material
having a biological origin. For example, the term "biological
agent" covers cells (including stem cells), proteins, peptides, or
nucleic acids (DNA or RNA such as mRNA, miRNA, or siRNA).
[0083] The term "pharmaceutical agent (also designated as
pharmaceutical compound)", as used herein, refers to any biological
or chemical substance, particularly pharmacological, metabolic, or
immunological substance, which may be used in the treatment, cure,
prophylaxis, prevention, or diagnosis of a pathological condition,
e.g. a disease or disorder, or which may be used to otherwise
enhance the physical, psychical, or mental well-being. Accordingly,
the term "pharmaceutical agent" envisaged in the context of the
present invention includes any agent with therapeutic, diagnostic,
or prophylactic effects, i.e. any therapeutic agent, diagnostic
agent, or prophylactic agent.
[0084] The pharmaceutical agent may be an agent that affects or
participates in tissue growth, cell growth, cell differentiation,
an agent that is able to invoke a biological action such as an
immune response, or an agent that can play any other role in one or
more biological processes. Preferably, the pharmaceutical agent is
selected from the group consisting of an anti-microbial agent, such
as an antibacterial agent (e.g. an antibiotic), an anti-viral agent
or an anti-fungal agent, an immunosuppressive agent, an
anti-inflammatory agent, an anti-allergic agent, an anti-coagulant,
an anti-rheumatic agent, an anti-psoriatic agent, a sedative agent,
a muscle relaxant, an anti-migraine agent, an anti-depressant, an
insect repellent, a growth factor, a hormone, a hormone antagonist,
an antibody, an adjuvant, e.g. in combination with an immunological
active compound such as an antibody, an antioxidant, a protein,
such as a glycoprotein, lipoprotein, or an enzyme (e.g.
hyaluronidases), a polysaccharide, a free radical scavenger, a
radio-therapeutic agent, a photodynamic therapy agent, a dye such
as a fluorescent dye, a contrast agent, a disinfectant, a
preservative, a vitamin, a tissue substituent, and a blood
substituent. The pharmaceutical agent may also be a small molecule
compound. The term "small molecule compound" refers to a molecule
that can act to affect biological processes. Small molecules can
include any number of therapeutic agents presently known and used,
or can be small molecules synthesized in a library of such
molecules for the purpose of screening for biological function(s).
The small molecule compound usually have a molecular weight less
than about 5,000 daltons (Da), preferably less than about 2,500 Da,
more preferably less than 1,000 Da, most preferably less than about
500 Da. The small molecule compound preferably serves as regulating
molecule of biological processes such as an enzyme substrate, an
antagonist, or an allosterically activating or an allosterically
inhibiting molecule. It is preferred, that the molecule is capable
of binding to another molecule, such as a protein, nucleic acid or
polysaccharide, and acting as an effector, altering the activity of
the other molecule.
[0085] The term "cosmetic agent (also designated as cosmetic
compound)", as used herein, refers to a substance intended mainly
for external use on the body surface of an individual. The body
surface includes skin, hair, nails, and related muscle and glands.
In particular, it is meant that a cosmetic substance is a molecule
which shows a certain predictable effect. Such an effect molecule
can be, for example, a proteinaceous molecule (e.g. an enzyme) or a
non-proteinaceous molecule (e.g. a dye, pigment, photo-protective
agent, vitamin, provitamin, an antioxidant, conditioner, or a
compound comprising metal ions).
[0086] Among the proteinaceous molecules, enzymes are preferred.
Examples for useful enzymes include, but are not limited to,
oxidases, peroxidases, proteases, glucanases, mutanases,
tyrosinases, metal-binding enzymes, lactoperoxidases, lysozymes,
aminoglycosidases, glucose oxidases, super oxide dismutases,
photolyases, proteins binding heavy metals, T4 endonucleases,
catalases, and reductases such as thioredoxin-reductases. Also
preferred are proteinaceous substances which do not possess an
enzymatic function. Examples for non-enzymatic proteinaceous
molecules include, but are not limited to, antimicrobial peptides,
hydrophobins, collagens, keratins, proteins binding heavy metals,
proteins binding odorants, proteins binding cellulose, proteins
binding starch, and proteins binding keratin. Other preferred
proteinaceous molecules are, for example, protein hydrolysates,
e.g. protein hydrolysates of plant or animal sources. Among the
non-proteinaceous molecules, UV-protective agents, antioxidants,
vitamins, provitamins and their precursors and derivatives, dyes,
polysaccharides, or fragrances are preferred. A UV-protective agent
is an organic substance which can absorb specific wavelengths in
the range of UV-wavelengths. The absorbed energy can then emitted
in form of longer wave radiation, e.g. heat. An antioxidant is a
compound that interrupts the photochemical reaction chain triggered
by UV radiation when penetrating into the skin. Typical examples of
antioxidants include, but are not limited to, super oxide
dismutase, catalase, tocopherol (vitamin E), ascorbic acid (vitamin
C), coenzyme Q10 (ubiquinane), and quinione. Examples of vitamins,
provitamins and their precursors include, but are not limited to,
.beta.-carotene (provitamin of vitamin A), ascorbic acid (vitamin
C), tocopherol (vitamin E), the vitamins, provitamins and their
precursors of the vitamin B group encompassing vitamin B.sub.1
(thiamine), vitamin B2 (riboflavin), vitamin B3 (nicotinic acid or
nicotinamid), vitamin B5 (panthothenic acid and panthenol), vitamin
B6 (5-hydroxymethyl-2-methylpyridin-3-ol, also known as pyridoxine,
pyridoasamine or pyridoxal) and vitamin B7 (biotin). Examples of
dyes include, but are not limited to, food dyes, semi-permanent
dyes, permanent dyes, reactive dyes, and oxidation dyes. Useful
dyes are for example described in Rowe Colour Index, 3rd edition,
Society of Dyers and Colourists, Bradford, England, 1971. In
addition, the cosmetic agent may be a protein, particularly a
glycoprotein or lipoprotein, a dye, a fragrance, an
infrared-reflective compound, an infrared-absorbent compound, argan
oil, hyaluronic acid, sea silt extract, gelee royale, gold extract,
medihoney, sacha inchi-oil, or allatonin.
[0087] As used herein, a "nutrient" is a chemical that an organism
needs to live and grow or a substance used in an organism's
metabolism which must be taken in from its environment. Organic
nutrients include carbohydrates, fats, proteins (amino acids), and
vitamins. Inorganic nutrients are dietary minerals, water, and
oxygen. Preferred nutrients are macronutrients such as
carbohydrates, amino acids or proteins and micronutrients such as
vitamins.
[0088] Examples of useful carnohydrates include, but are not
limited to, monosaccharides such as, glyceraldehyde, erythrose,
threose, ribose, arabinose, xylose, lyxose, allose, altrose,
glucose, mannose, gulose, idose, galactose, talose,
dihydroxacetone, erythrulose, ribulose, xylulose, psicose,
fructose, sorbose, tagatose or stereoisomers thereof, amino sugars
such as galactosamine, glucosamine, sialic acid,
N-acetylglucosamine, sulfosugars such as sulfoquinovose,
disaccharides such as sucrose, lactulose, lactose, maltose,
trehalose or maltobiose, and oligosacharides such as
Fructooligosaccharides (FOS), Galactooligosaccharides (GOS) or
Mannan-oligosaccharides (MOS).
[0089] The term "dietary supplement (also designated as food
supplement or nutritional supplement)", as used herein, refers to a
preparation intended to provide nutrients such as vitamins,
minerals, fiber, fatty acids or amino acids, that are missing or
are not consumed in sufficient quantitiy in a person's diet.
Depending on the country dietary supplements are either defended as
foods or as drugs.
[0090] Examples of other dietary supplements include, but are not
limited to, steroids such as dehydroepiandrosterone (DHEA),
pregnenolone, or derivatives thereof, hormones such as melatonin,
and other substances such as hydrazine sulfate, caffeine
(1,3,7-trimethylxanthine), catechins, soy isoflavones, glucosamine,
coenzyme-Q10, or ephedrine-type alkaloids such as ephedrine,
synephrine, norephedrine, or pseudo do ephedrine.
[0091] The term "dye", as used herein, refers to a coloured
substance having affinity to a substrate to which it is being
applied. Dyes are generally applied in aqueous solution. In
contrast, pigments are typically insoluble and possess no affinity
to the substrate. Both dyes and pigments appear to be coloured
because of their ability to absorb specific wavelength of light.
The dye can be a naturally occurring or synthetic organic dye or a
food dye.
[0092] The active agent may be positively or negatively charged.
The active agent may also be electroneutral. Preferably, the active
agent is positively or negatively charged. The terms "positive
charge" and "cationic" as well as "negative charge" and "anionic"
can be used interchangeably. As will be shown in detail in the
examples, especially positively and negatively charged active
agents are well-suited for the remote loading of silk films. For
direct loading, the active agent is preferably electroneutral or
has the same charge as the silk film. As used herein, "positive
charge" means that the active agent possesses at least one
elementary charge of a proton and "negative charge" means that the
active agent possesses at least one elementray charge of an
electron.
[0093] The skilled person knows that the charge of an active agent
is dependent on factors such as the pK.sub.a-value of the active
agent and the pH of the aqueous solvent. As used herein, the term
"pK.sub.a-value", (also known as acidity constant, or
acid-ionization constant) is a quantitative measure of the strength
of an acid in solution. It is derived from the dissociation
constant K.sub.a which describes the equilibrium for a chemical
reaction known as dissociation in the context of an acid-base
reaction. Due to the many orders of magnitude spanned by K.sub.a
values, a logarithmic measure of the acid dissociation constant is
more commonly used in practice. The larger the value pK.sub.a the
smaller the extent of dissociation and the less strong is an acid.
Accordingly, the pK.sub.b value describes the strength of a base in
solution.
[0094] In aqueous solutions the pK.sub.a-value may give an
indication whether an active agent has a positive charge or not,
thus, having, for example, a negative charge. Preferably, the
active agent is positively or negatively charged at the pH used for
film formation.
[0095] Various other methods for determining or measuring the
charge of a compound are known to one of skill in the art. For
example, the charge can typically be measured using electrophoretic
methods. The charge of a molecule in aqueous solution may also be
predicted using suitable software such as ACD/ChemSketch (available
at Advanced Chemistry Development, ACD/labs,
http://www.acdlabs.com).
[0096] The person skilled in the art also knows how to determine
which active agents are suitable for loading silk films, i.e.
whether an active agent of interest possesses, for example, at
least one positive charge at the pH of the aqueous solution which
is used for silk film loading, or which active agents are suitable
for being incorporated into silk films, i.e. whether an active
agent of interest possesses, for example, at least one positive
charged at the pH of the aqueous solution which is used for silk
film casting. Suitable methods include titration methods and the
measurement of the zeta-potential during titration.
[0097] If the active agent is a peptide or a protein or any other
amphiphilic compound, the presence of an overall positive net
charge is dependent on the isoelectric point (pI) value of the
active agent. The isoelectric point, sometimes abbreviated IEP, is
the pH at which a particular molecule or surface carries no net
electrical charge. For example, amphoteric molecules or zwitterions
contain both positive and negative charges depending on the
functional groups present in the molecule. The net charge on the
molecule is affected by pH of their surrounding environment and can
become more positively or negatively charged due to the loss or
gain of protons. The pI the pH value at which the molecule carries
no electrical charge or the negative and positive charges are
equal.
[0098] Methods for determining whether a peptide or protein at a
certain pH has a predominant net charge are known in the art. For
example, suitable tools for calculating the pI value of peptides or
proteins are provided by ExPasyProteomic server (www.expasy.ch).
The program "Compute pI/Mw" is a tool which allows the computation
of the theoretical pI (isoelectric point) and Mw (molecular weight)
for a list of database entries (UniProtKnowledgebase (Swiss-prot or
TrEMBL)) or for user entered sequences. Prediction of pI values are
also described in Bjellqvist et al. (1993) and Gasteider et al.
(2005) (Bjellqvist, B., The focusing positions of polypeptides in
immobilized pH gradients can be predicted from their amino acid
sequences. Electrophoresis 1993, 14, 1023-1031. Gasteiger E.,
Protein Identification and Analysis Tools on the ExPASy Server,
(In) John M. Walker (ed): The Proteomics Protocols Handbook, Humana
Press (2005).
[0099] The inventors of the present invention observed that the use
of an aqueous solution or dispersion having a pH that is lower than
the pI of the silk used for silk film formation results in a
positively charged silk film. In this respect, it is preferred that
the active agent added to the aqueous solution or dispersion is
negatively charged at the pH of the aqueous solution or dispersion
in order to achieve effective incorporation of the active agent
into the silk film. Further, the inventors of the present invention
observed that the use of an aqueous solution or dispersion having a
pH that is higher than the pI of the silk used for silk film
formation results in a negatively charged film. In this respect, it
is preferred that the active agent added to the aqueous solution or
dispersion is positively charged at the pH of the aqueous solution
or dispersion in order to achieve effective incorporation of the
active agent into the silk film. The incorporation of the active
agent into the silk film preferably arises from electrostatic
and/or hydrophobic interactions between the silk material and the
active agent.
[0100] Thus, it is preferred that the active agent comprised in the
silk film is positively charged, if the silk film has a negative
net charge. It is further preferred that the active agent comprised
in the silk film is negatively charged, if the silk film has a
positive net charge.
[0101] The active agent may be released from the silk film,
particularly from the active agent release sheet being part of the
silk film or forming the silk film, by diffusion and/or degradation
upon exposure to a surrounding environment.
[0102] In one embodiment, the surrounding environment is a
physiological environment (e.g. a physiological medium). It is
preferred that the physiological environment (e.g. the
physiological medium) is selected from the group consisting of a
physiological buffered solution or a body fluid, e.g. blood, lymph
or liquor. It is also preferred that the physiological environment
is an organ (e.g. skin) or a part of an organ (e.g. tissue). The
release of the active agent from the silk film, particularly from
the active agent release sheet being part of the silk film or
forming the silk film, can be induced by introducing the silk film
into a physiological buffered aqueous solution or a body fluid such
as blood, lymph or liquor, or by applying the silk film onto an
organ (e.g. skin) or part of an organ (e.g. tissue).
[0103] In one another embodiment, the surrounding environment is a
non-physiological environment (e.g. a non-physiological medium). It
is preferred that the non-physiological environment (e.g. the
non-physiological medium) is selected from the group consisting of
an aqueous solution such as water or a buffered aqueous solution,
an alcoholic solution, and an organic solution.
[0104] It should be further noted that the active agent(s) may be
released from the silk film, particularly from the active agent
release sheet being part of the silk film or forming the silk film,
by diffusion and/or degradation upon exposure to a surrounding
intracorporeal or extracorporeal environment.
[0105] Preferably, the release of the active agent displays
relatively linear kinetics, thereby providing a constant supply of
the active agent over the release period.
[0106] It is preferred that less than 20%, preferably less than
15%, more preferably less than 10%, and most preferably less than
5%, e.g. less than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9,
8, 7, 6, or 5%, of the active agent is released, particularly into
the surrounding environment such as physiological environment or
non-physiological environment (see above), within the first 24
hours.
[0107] It is, alternatively or additionally, preferred that more
than 50%, preferably more than 70%, more preferably more than 80%,
and most preferably 100%, e.g. more than 50, 51, 52, 53, 54, 55,
56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,
73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,
90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, of the active
agent is released, particularly into the surrounding environment
such as physiological environment or non-physiological environment
(see above), within 36 hours. It is, alternatively or additionally,
preferred that more than 50%, preferably more than 70%, more
preferably more than 80%, and most preferably 100%, e.g. more than
50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,
67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,
84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or
100%, of the active agent is released, particularly into the
surrounding environment such as physiological environment or
non-physiological environment (see above), within 48 hours. It is,
alternatively or additionally, preferred that more than 50%,
preferably more than 70%, more preferably more than 80%, and most
preferably 100%, e.g. more than 50, 51, 52, 53, 54, 55, 56, 57, 58,
59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,
76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,
93, 94, 95, 96, 97, 98, 99, or 100%, of the active agent is
released, particularly into the surrounding environment such as
physiological environment or non-physiological environment (see
above), within 72 hours.
[0108] It is, alternatively or additionally, more preferred that
more than 50%, preferably more than 70%, more preferably more than
80%, and most preferably 100%, e.g. more than 50, 51, 52, 53, 54,
55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,
72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,
89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, of the active
agent is released, particularly into the surrounding environment
such as physiological environment or non-physiological environment
(see above), within 7 days. It is, alternatively or additionally,
more preferred that more than 50%, preferably more than 70%, more
preferably more than 80%, and most preferably 100%, e.g. more than
50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,
67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,
84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or
100%, of the active agent is released, particularly into the
surrounding environment such as physiological environment or
non-physiological environment (see above), within 14 days. It is,
alternatively or additionally, more preferred that more than 50%,
preferably more than 70%, more preferably more than 80%, and most
preferably 100%, e.g. more than 50, 51, 52, 53, 54, 55, 56, 57, 58,
59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,
76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,
93, 94, 95, 96, 97, 98, 99, or 100%, of the active agent is
released, particularly into the surrounding environment such as
physiological environment or non-physiological environment (see
above), within 20 days. It is, alternatively or additionally, more
preferred that more than 50%, preferably more than 70%, more
preferably more than 80%, and most preferably 100%, e.g. more than
50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,
67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,
84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or
100%, of the active agent is released, particularly into the
surrounding environment such as physiological environment or
non-physiological environment (see above), within 30 days. It is,
alternatively or additionally, more preferred that more than 50%,
preferably more than 70%, more preferably more than 80%, and most
preferably 100%, e.g. more than 50, 51, 52, 53, 54, 55, 56, 57, 58,
59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,
76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,
93, 94, 95, 96, 97, 98, 99, or 100%, of the active agent is
released, particularly into the surrounding environment such as
physiological environment or non-physiological environment (see
above), within 35 days.
[0109] It is, alternatively or additionally, even more preferred
that more than 50%, preferably more than 70%, more preferably more
than 80%, and most preferably 100%, e.g. more than 50, 51, 52, 53,
54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,
71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,
88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, of the
active agent is released, particularly into the surrounding
environment such as physiological environment or non-physiological
environment (see above), within 5 weeks. It is, alternatively or
additionally, even more preferred that more than 50%, preferably
more than 70%, more preferably more than 80%, and most preferably
100%, e.g. more than 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,
61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,
78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100%, of the active agent is released,
particularly into the surrounding environment such as physiological
environment or non-physiological environment (see above), within 6
weeks. It is, alternatively or additionally, even more preferred
that more than 50%, preferably more than 70%, more preferably more
than 80%, and most preferably 100%, e.g. more than 50, 51, 52, 53,
54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,
71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,
88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, of the
active agent is released, particularly into the surrounding
environment such as physiological environment or non-physiological
environment (see above), within 7 weeks. It is, alternatively or
additionally, even more preferred that more than 50%, preferably
more than 70%, more preferably more than 80%, and most preferably
100%, e.g. more than 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,
61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,
78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100%, of the active agent is released,
particularly into the surrounding environment such as physiological
environment or non-physiological environment (see above), within 8
weeks.
[0110] It is, alternatively or additionally, most preferred that
more than 50%, preferably more than 70%, more preferably more than
80%, and most preferably 100%, e.g. more than 50, 51, 52, 53, 54,
55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,
72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,
89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, of the active
agent is released, particularly into the surrounding environment
such as physiological environment or non-physiological environment
(see above), within 3 months. It is, alternatively or additionally,
most preferred that more than 50%, preferably more than 70%, more
preferably more than 80%, and most preferably 100%, e.g. more than
50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,
67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,
84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or
100%, of the active agent is released, particularly into the
surrounding environment such as physiological environment or
non-physiological environment (see above), within 4 months. It is,
alternatively or additionally, most preferred that more than 50%,
preferably more than 70%, more preferably more than 80%, and most
preferably 100%, e.g. more than 50, 51, 52, 53, 54, 55, 56, 57, 58,
59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,
76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,
93, 94, 95, 96, 97, 98, 99, or 100%, of the active agent is
released, particularly into the surrounding environment such as
physiological environment or non-physiological environment (see
above), within 5 months. It is, alternatively or additionally, most
preferred that more than 50%, preferably more than 70%, more
preferably more than 80%, and most preferably 100%, e.g. more than
50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,
67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,
84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or
100%, of the active agent is released, particularly into the
surrounding environment such as physiological environment or
non-physiological environment (see above), within 6 months. It is,
alternatively or additionally, most preferred that more than 50%,
preferably more than 70%, more preferably more than 80%, and most
preferably 100%, e.g. more than 50, 51, 52, 53, 54, 55, 56, 57, 58,
59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,
76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,
93, 94, 95, 96, 97, 98, 99, or 100%, of the active agent is
released, particularly into the surrounding environment such as
physiological environment or non-physiological environment (see
above), within 7 months. It is, alternatively or additionally, most
preferred that more than 50%, preferably more than 70%, more
preferably more than 80%, and most preferably 100%, e.g. more than
50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,
67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,
84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or
100%, of the active agent is released, particularly into the
surrounding environment such as physiological environment or
non-physiological environment (see above), within 8 months. It is,
alternatively or additionally, most preferred that more than 50%,
preferably more than 70%, more preferably more than 80%, and most
preferably 100%, e.g. more than 50, 51, 52, 53, 54, 55, 56, 57, 58,
59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,
76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,
93, 94, 95, 96, 97, 98, 99, or 100%, of the active agent is
released, particularly into the surrounding environment such as
physiological environment or non-physiological environment (see
above), within 9 months. It is, alternatively or additionally, most
preferred that more than 50%, preferably more than 70%, more
preferably more than 80%, and most preferably 100%, e.g. more than
50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,
67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,
84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or
100%, of the active agent is released, particularly into the
surrounding environment such as physiological environment or
non-physiological environment (see above), within 10 months.
[0111] It is particularly preferred that less than 20%, preferably
less than 15%, more preferably less than 10%, and most preferably
less than 5%, e.g. less than 20, 19, 18, 17, 16, 15, 14, 13, 12,
11, 10, 9, 8, 7, 6, or 5%, of the active agent is released into the
physiological environment such as physiological buffered solution
or body fluid, e.g. blood, lymph or liquor, within the first 24
hours, and/or that more than 50%, preferably more than 70%, more
preferably more than 80%, and most preferably 100%, e.g. more than
50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,
67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,
84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or
100%, of the active agent is released into the physiological
environment such as physiological buffered solution or body fluid,
e.g. blood, lymph or liquor, within 30 days.
[0112] The one or more release modifying layers comprise or consist
of a release modifying material (e.g. a silk material). In one
embodiment, the one or more release modifying layers comprise or
consist of at least one release modifying agent. It is preferred
that the release modifying agent is selected from the group
consisting of a (i) silk polypeptide, (ii) polyester, preferably
polylactide, polyglycolide, polylactic polyglycolic copolymer
(PLGA), and blend of polylactic polyglycolic copolymer (PLGA) and
polylactide, (iii) polyether, preferably polycaprolactone (PCL),
(iv) polyanhydride, (v) polyalkylcyanoacrylate, preferably n-butyl
cyanoacrylate, (vi) polyacrylamide, (vii) polyurethane, and (viii)
polyvinylpirrolidone (PVP). It is more preferred that the release
modifying agent is a silk polypeptide.
[0113] The silk film, particularly the one or more first silk film
layers and/or the one or more second silk film layers, comprise a
silk material. In one embodiment, the silk film, particularly the
one or more first silk film layers and/or the one or more second
silk film layers, comprise at least one silk polypeptide.
[0114] Naturally, the silk polypeptide of the silk film,
particularly of the one or more first silk film layers and/or of
the one or more second silk film layers, possess at least one
negative charge at the carboxyl terminus. The person skilled in the
art also knows how to select appropriate amino acid sequences in
order obtain a silk polypeptide having an overall negative or
positive net charge. For example, this can be achieved by selecting
or generating silk sequences comprising negatively charged amino
acids or positively charged amino acids. A suitable negatively
charged silk polypeptide is, for example, the silk polypeptide
C.sub.16 which comprises 16 repeats of the sequence of module C
(SEQ ID NO: 21) or variants thereof. In addition, a suitable
positively charged silk polypeptide is, for example, the silk
polypeptide C.sup.Kappa.sub.16 which comprises 16 repeats of the
sequence of module C.sup.Kappa (SEQ ID NO: 43) or variants
thereof.
[0115] Preferably, the silk polypeptide consists of between 6 to
1500 amino acids, more preferably of between 200 to 1300 amino
acids, even more preferably of between 250 to 1200 amino acids, and
most preferably of between 500 to 1000 amino acids.
[0116] The silk polypeptide may be expressed in a recombinant, e.g.
microbial, insect, plant, or mammalian expression system, i.e.
separated from its natural milieu, (recombinant silk polypeptide),
or may be harvested from natural sources, e.g. spider, silk worm,
bee, mussel, or fly larvae. The silk polypeptide may be an isolated
or a purified silk polypeptide. In particular, a "purified silk
polypeptide" or an "isolated silk polypeptide" is free or
substantially free of cellular material, production/fermentation
remnants, and/or other contaminating proteins from the cell or
tissue source from which the silk polypeptide is purified or
isolated. The language "substantially free of cellular material"
includes preparations of a silk polypeptide in which the silk
polypeptide is separated from cellular components of the cells from
which it is produced or harvested. A silk polypeptide that is
"substantially free" of cellular material, production/fermentation
remnants, and/or other contaminating proteins from the cell or
tissue source from which the silk polypeptide is purified or
isolated includes preparations of silk polypeptides having less
than about 30%, 20%, 10%, 5%, 1%, or 0.1% (by dry weight) of
contaminating protein and/or less than about 30%, 20%, 10%, 5%, 1%,
or 0.1% (by dry weight) of contaminating lipid, DNA or salt.
[0117] It is preferred that the silk polypeptide is a recombinant
silk polypeptide. It is further preferred that the silk polypeptide
is a (recombinant) spider silk polypeptide, more preferably a
(recombinant) major ampullate silk polypeptide such as a
(recombinant) dragline silk polypeptide, a (recombinant) minor
ampullate silk polypeptide, or a (recombinant) flagelliform silk
polypeptide of an orb-web spider (e.g. Araneidae or Araneoids), an
(recombinant) insect silk polypeptide, a (recombinant) mussel
byssus silk polypeptide, or a mixture thereof. The orb-web spider
may be selected from the group consisting of Araneus diadematus,
Nephila clavipes, and Latrodectus hesperus. The insect silk
polypeptide may be of Lepidoptera, particularly Bombycidae such as
Bombyx mori. The insect silk polypeptide may also be of
Hymenoptera, particularly Apoidea such as Anthophila.
[0118] It is, alternatively or additionally, preferred that the
silk polypeptide comprises or consists of at least two repetitive
units. It should be noted that the terms "repetitive unit" and
"repeat unit" can interchangeable be used in the context of the
present invention.
[0119] The term "repetitive unit", as used herein, refers to a
region which corresponds in amino acid sequence to a region that
comprises or consists of at least one peptide motif (e.g. AAAAAA
(SEQ ID NO: 13) or GPGQQ (SEQ ID NO: 4)) that repetitively occurs
within a naturally occurring silk polypeptide (e.g. MaSpI, ADF-3,
ADF-4, or Flag) (i.e. identical amino acid sequence) or to an amino
acid sequence substantially similar thereto (i.e. variational amino
acid sequence). In this regard "substantially similar" means a
degree of amino acid identity of at least 50%, 51%, 52%, 53%, 54%,
55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%,
68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,
81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99% or even 99.9%, preferably over the
whole length of the respective reference naturally occurring amino
acid sequence.
[0120] A "repetitive unit" having an amino acid sequence which is
"substantially similar" to a corresponding amino acid sequence
within a naturally occurring silk polypeptide (i.e. wild-type
repetitive unit) is also similar with respect to its functional
properties; e.g. a silk polypeptide comprising a "substantially
similar repetitive unit" is still capable of forming a film. The
skilled person can readily assess whether a silk polypeptide
comprising a "substantially similar repetitive unit" is still
capable of forming a film, e.g. by producing an aqueous (buffered)
solution comprising the silk polypeptide comprising the
"substantially similar repetitive unit", casting said solution onto
a solid support and subsequently drying said solution (see
experimental section). Preferably, the produced silk film is a
self-supporting film, i.e. a silk film that has the capacity for
supporting itself without the help of additional materials such as
carrier elements.
[0121] A "repetitive unit" having an amino acid sequence which is
"identical" to the amino acid sequence of a naturally occurring
silk polypeptide can be, for example, a portion of a silk
polypeptide corresponding to one or more peptide motifs of MaSp I
(SEQ ID NO: 35) MaSp II (SEQ ID NO: 36), ADF-3 (SEQ ID NO: 1)
and/or ADF-4 (SEQ ID NO: 2). A "repetitive unit" having an amino
acid sequence which is "substantially similar" to the amino acid
sequence of a naturally occurring silk polypeptide can be, for
example, a portion of a silk polypeptide corresponding to one or
more peptide motifs of MaSpI (SEQ ID NO: 35) MaSpII (SEQ ID NO:
36), ADF-3 (SEQ ID NO: 1) and/or ADF-4 (SEQ ID NO: 2), but having
one or more amino acid substitution(s) at (a) specific amino acid
position(s).
[0122] The term, a "repetitive unit", as used herein, does not
include the non-repetitive hydrophilic amino acid domain generally
thought to be present at the amino terminus and/or carboxyl
terminus of naturally occurring silk polypeptides.
[0123] The term a "repetitive unit", as used herein, further refers
to an amino acid sequence with a length of 3 to 200 amino acids, or
5 to 150 amino acids, preferably with a length of 10 to 100 amino
acids, or 15 to 80 amino acids and more preferably with a length of
18 to 60, or 20 to 40 amino acids. For example, the repetitive unit
according to the present invention can have a length of 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,
41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,
58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,
75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, 100, 105, 110, 115, 120, 125, 130,
135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or
200 amino acids. Preferably, the repetitive unit consists of 3, 4,
5, 6, 7, 8, 9, 10, 12, 15, 18, 20, 24, 27, 28, 30, 34, 35, or 39
amino acids.
[0124] Preferably, the silk polypeptide comprises of between 2 to
100 repetitive units, i.e. at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,
29, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,
45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,
62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,
79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,
96, 97, 98, 99, or 100 repetitive units. The repetitive units in
the silk polypeptide may be the same (identical) or different. It
is preferred that the same (identical) repetitive unit is used in
the silk polypeptide at least 2 times, preferably 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or
more times.
[0125] It is preferred that the silk polypeptide comprises or
consists of at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
preferably at least 95% and most preferably 100% of repetitive
units. It is particularly preferred that the silk polypeptide
comprises or consists of at least 50%, 60%, 65%, 70%, 75%, 80%,
85%, 90%, preferably at least 95% and most preferably 100% of
multiple copies of one identical repetitive unit (e.g. A.sub.2,
Q.sub.6, or C.sub.16, wherein the numerical 2, 6, or 16 represent
the number of repetitive units) or multiple copies of two or more
different repetitive units (e.g. (AQ).sub.24, or
(AQ).sub.12C.sub.16). Said silk polypeptide can further be modified
by adding an artificial tag to facilitate the detection or
purification of said polypeptide (e.g. T7 tag or His Tag).
[0126] The repetitive unit of the silk polypeptide can comprise or
consist of an amino acid sequence of any region that comprises or
consists of at least one peptide motif that repetitively occurs
within a naturally occurring silk polypeptide known to one skilled
in the art. Preferably, the repetitive unit of the silk polypeptide
comprises or consists of an amino acid sequence of a region that
comprises or consists of at least one peptide motif that
repetitively occurs within an arthropod silk polypeptide, more
preferably within a spider silk polypeptide, or an insect silk
polypeptide. The repetitive unit of the silk polypeptide can also
comprise or consist of an amino acid sequence of a region that
comprises or consists of at least one peptide motif that
repetitively occurs within a mussel silk polypeptide.
[0127] It is preferred that the spider silk repetitive unit
comprises or consists of an amino acid sequence of a region that
comprises or consists of at least one peptide motif that
repetitively occurs within a naturally occurring major ampullate
silk polypeptide (MaSp), such as a dragline silk polypeptide, a
minor ampullate silk polypeptide (MiSp), or a flagelliform (FLAG)
silk polypeptide. Most preferably, the repetitive unit comprises or
consists of an amino acid sequence of a region that comprises or
consists of at least one peptide motif that repetitively occurs
within a naturally occurring dragline silk polypeptide or
flagelliform silk polypeptide.
[0128] It is also preferred that the insect silk repetitive unit
comprises or consists of an amino acid sequence of a region that
comprises or consists of at least one peptide motif that
repetitively occurs within a naturally occurring silk polypeptide
of Lepidoptera. More preferably, the insect silk repetitive unit
comprises or consists of an amino acid sequence of a region that
comprises or consists of at least one peptide motif that
repetitively occurs within a naturally occurring insect silk
polypeptide of Bombycidae, most preferably of Bombyx mori.
[0129] Preferably, the silk polypeptide comprises or consists of at
least two repetitive units each comprising at least one, preferably
one, consensus sequence selected from the group consisting of:
[0130] (i) GPGXX (SEQ ID NO: 3), wherein X is any amino acid,
preferably in each case independently selected from A, S, G, Y, P,
and Q; [0131] (ii) GGX, wherein X is any amino acid, preferably in
each case independently selected from Y, P, R, S, A, T, N and Q,
more preferably in each case independently selected from Y, P and
Q; [0132] (iii) A.sub.x, wherein x is an integer from 5 to 10;
[0133] (iv) GGRPSDTYG (SEQ ID NO: 18); and [0134] (v) GGRPSSSYG
(SEQ ID NO: 19).
[0135] The above mentioned silk polypeptide preferably has a
molecular weight of at least 5 kDa, e.g. of at least 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 kDa.
[0136] The term "consensus sequence", as used herein, refers to an
amino acid sequence which contains amino acids which frequently
occur in a certain position (e.g. "G") and wherein, other amino
acids which are not further determined are replaced by the place
holder "X".
[0137] The iterated (peptide) motifs GPGXX (SEQ ID NO: 3) and GGX,
i.e. glycine rich motifs, provide flexibility to the silk
polypeptide and thus, to the film formed from the silk polypeptide
containing said motifs. In detail, the iterated GPGXX (SEQ ID NO:
3) motif forms .beta.-turn spiral structures, which imparts
elasticity to the silk polypeptide. Major ampullate and
flagelliform silks both have a GPGXX (SEQ ID NO: 3) motif. The
iterated GGX motif is associated with a helical structure having
three amino acids per turn and is found in most spider silks. The
GGX motif may provide additional elastic properties to the silk.
The iterated polyalanine A.sub.x (peptide) motif forms a
crystalline .beta.-sheet structure that provides strength to the
silk polypeptide. (WO 03/057727). The GGRPSDTYG (SEQ ID NO: 18) and
GGRPSSSYG (SEQ ID NO: 19) (peptide) motifs have been selected from
Resilin (WO 08/155304). Resilin is an elastomeric protein found in
most arthropods (arthropoda). It is located in specialised regions
of the cuticle, providing low stiffness and high strength (Elvin et
al., Nature (473): 999-1002, 2005).
[0138] Thus, in one preferred embodiment, the silk polypeptide
comprises or consists of repetitive units each comprising at least
one (e.g. 1, 2, 3, 4, 5, 6, 7, 8, or 9), preferably one, amino acid
sequence selected from the group consisting of GPGAS (SEQ ID NO:
5), GPGSG (SEQ ID NO: 6), GPGGY (SEQ ID NO: 7), GPGGP (SEQ ID NO:
8), GPGGA (SEQ ID NO: 9), GPGQQ (SEQ ID NO: 4), GPGGG (SEQ ID NO:
10), GPGQG (SEQ ID NO: 40), and GPGGS (SEQ ID NO: 11). In one
further preferred embodiment of the present invention, the silk
polypeptide comprises or consists of repetitive units each
comprising at least one (e.g. 1, 2, 3, 4, 5, 8, 7, or 8),
preferably one, amino acid sequence selected from the group
consisting of GGY, GGP, GGA, GGR, GGS, GGT, GGN, and GGQ. In one
additionally preferred embodiment of the present invention, the
silk polypeptide comprises or consists of repetitive units each
comprising at least one (e.g. 1, 2, 3, 4, 5, or 6), preferably one,
amino acid sequence selected from the group consisting of AAAAA
(SEQ ID NO: 12), AAAAAA (SEQ ID NO: 13), AAAAAAA (SEQ ID NO: 14),
AAAAAAAA (SEQ ID NO: 15), AAAAAAAAA (SEQ ID NO: 16), and AAAAAAAAAA
(SEQ ID NO: 17).
[0139] In one another preferred embodiment, the silk polypeptide
comprises or consists of repetitive units each comprising at least
one (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20, 21, 22, 23, 24, or 25), preferably one, amino acid
sequence selected from the group consisting of GPGAS (SEQ ID NO:
5), GPGSG (SEQ ID NO: 6), GPGGY (SEQ ID NO: 7), GPGGP (SEQ ID NO:
8), GPGGA (SEQ ID NO: 9), GPGQQ (SEQ ID NO: 4), GPGGG (SEQ ID NO:
10), GPGQG (SEQ ID NO: 40), GPGGS (SEQ ID NO: 11), GGY, GGP, GGA,
GGR, GGS, GGT, GGN, GGQ, AAAAA (SEQ ID NO: 12), AAAAAA (SEQ ID NO:
13), AAAAAAA (SEQ ID NO: 14), AAAAAAAA (SEQ ID NO: 15), AAAAAAAAA
(SEQ ID NO: 16), AAAAAAAAAA (SEQ ID NO: 17), GGRPSDTYG (SEQ ID NO:
18) and GGRPSSSYG (SEQ ID NO: 19).
[0140] More preferably, the silk polypeptide comprises or consists
of repetitive units, which comprise or consist of [0141] (i) GPGAS
(SEQ ID NO: 5), AAAAAA (SEQ ID NO: 13), GGY, and GPGSG (SEQ ID NO:
6) as amino acid sequence, preferably in this order, [0142] (ii)
AAAAAAAA (SEQ ID NO: 15), GPGGY (SEQ ID NO: 7), GPGGY (SEQ ID NO:
7), and GPGGP (SEQ ID NO: 8) as amino acid sequence, preferably in
this order, [0143] (iii) GPGQQ (SEQ ID NO: 4), GPGQQ (SEQ ID NO:
4), GPGQQ (SEQ ID NO: 4) and GPGQQ (SEQ ID NO: 4) as amino acid
sequence, [0144] (iv) GPGGA (SEQ ID NO: 9), GGP, GPGGA (SEQ ID NO:
9), GGP, GPGGA (SEQ ID NO: 9), and GGP as amino acid sequence,
preferably in this order, [0145] (v) AAAAAAAA (SEQ ID NO: 15),
GPGQG (SEQ ID NO: 40), and GGR as amino acid sequence, preferably
in this order, [0146] (vi) AAAAAAAA (SEQ ID NO: 15), GPGGG (SEQ ID
NO: 10), GGR, GGN, and GGR as amino acid sequence, preferably in
this order, [0147] (vii) GGA, GGA, GGA, GGS, GGA, and GGS as amino
acid sequence, preferably in this order, and/or [0148] (viii) GPGGA
(SEQ ID NO: 9), GPGGY (SEQ ID NO: 7), GPGGS (SEQ ID NO: 11), GPGGY
(SEQ ID NO: 7), GPGGS (SEQ ID NO: 11), and GPGGY (SEQ ID NO: 7) as
amino acid sequence, preferably in this order.
[0149] It is further preferred that the repetitive units are
independently selected from module A (SEQ ID NO: 20) or variants
thereof, module C (SEQ ID NO: 21) or variants thereof, module Q
(SEQ ID NO: 22) or variants thereof, module S (SEQ ID NO: 23) or
variants thereof, and module R (SEQ ID NO: 24) or variants thereof.
Modules A (SEQ ID NO: 20) and Q (SEQ ID NO: 22) are based on the
amino acid sequence of ADF-3 of the spider Araneus diadematus.
Module C (SEQ ID NO: 21) is based on the amino acid sequence of
ADF-4 of the spider Araneus diadematus. Modules S (SEQ ID NO: 23)
and R (SEQ ID NO: 24) are based on Resilin (Arthropoda) (WO
2008/155304).
[0150] Thus, in one preferred embodiment, the repetitive units of
the silk polypeptide consist of module A: GPYGPGASAAAAAAGGYGPGSGQQ
(SEQ ID NO: 20), module C: GSSAAAAAAAASGPGGYGPENQGPSGPGGYGPGGP (SEQ
ID NO: 21), module Q: GPGQQGPGQQGPGQQGPGQQ (SEQ ID NO: 22), module
S: PGSSAAAAAAAASGPGQGQGQGQGQGGRPSDTYG (SEQ ID NO: 23), module R:
SAAAAAAAAGPGGGNGGRPSDTYGAPGGGNGGRPSSSYG (SEQ ID NO: 24), or
variants thereof.
[0151] The silk polypeptide may comprise combined repeats of only
one of these modules or of combinations thereof. Preferred
combinations are characterized as follows (the repetitive units are
arranged from N- to C-terminus): XY, wherein X and Y are
independently selected from A, C, Q, R and S or variant thereof and
are each different, i.e. X and Y are not C at the same time.
Preferred combinations that are combined with each other are CA,
AC, CQ, QC, CS, SC, CR, RC, SR, RS, AQ, QA, AS, SA, AR, RA, QS, SQ,
QR, RQ, SR, and RS. In further preferred combinations blocks of
three repetitive units are formed, which follow the following
construction scheme: XYZ, wherein X and Y are independently
selected from A, C, Q, R and S or variant thereof and are each
different and Z is independently selected from A, C, Q, R and S or
variant thereof, is preferably identical to X. Preferred
combinations that are combined with each other are CAA, CAC, CAQ,
CAR, CAS, ACA, ACC. ACQ, ACR, ACS, CQA, CQC, CQQ, CQR, CQS, QCA,
QCC, QCQ, QCR, QCS, CSA; CSC, CSQ, CSR, CSS, SCA, SCC, SCQ, SCR,
SCS, CRA, CRC, CRQ, CRR, CRS, RCA, RCC, RCQ, RCR, RCS, SRA, SRC,
SRQ, SRR, SRS, RSA, RSC, RSQ, RSR, RSS, AQA, AQC, AQQ, AQR, AQS,
QAA, QAC, QAQ, QAR, QAS, ASA; ASC, ASQ, ASR, ASS, SAA, SAC, SAQ,
SAR, SAS, ARA, ARC, ARQ, ARR; ARS, RAA, RAC, RAQ, RAR, RAS, QSA,
QSC, QSQ, QSR, QSS, SQA, SQC, SQQ, SQR, SQS, QRA, QRC, QRQ, QRR,
QRS, RQA, RQC, RQQ, RQR, RQS, SRA. SRC. SRQ, SRE, SRS, RSA, RSC,
RSQ, RSR, and RSS. It is noted that it is in each case possible
that one of the repetitive units is a variant of the respectively
indicated repetitive unit. Accordingly, preferred repetitive units
comprised in the silk polypeptide follow the general structure
X.sub.m, XY.sub.n or XYZ.sub.o, wherein m is between 4 and 100,
i.e. 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,
38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,
55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,
72, 73, 74, 75, 76, 77, 78, 79, 80 or more; n is between 2 and 60,
i.e. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,
36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,
53, 54, 55, 56, 57, 58, 59, 60; and o is between 2 and 40, i.e. 2,
3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,
38, 39, and 40.
[0152] The terms "combined with each other" or "concatenated with
each other", as used herein, mean that the modules (repetitive
units) are directly combined or concatenated with each other, or
mean that the modules (repetitive units) are combined or
concatenated with each other via one or more spacer amino acids.
Thus, in one embodiment, the modules (repetitive units) comprised
in the silk polypeptide are directly combined or concatenated with
each other. In one another embodiment, the modules (repetitive
units) comprised in the silk polypeptide are combined or
concatenated with each other via one or more spacer amino acids,
preferably via 1 to 25 or 1 to 20 spacer amino acids, more
preferably via 1 to 15 or 1 to 10 spacer amino acids, and most
preferably, via 1 to 5 spacer amino acids, e.g. via 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, or 25 spacer amino acids. Said spacer amino acid may be any
amino acid naturally occurring in proteins. Preferably, said spacer
amino acid is not proline. It is preferred that the spacer amino
acid contains a charged group(s). Preferably, the spacer amino acid
containing a charged group(s) is independently selected from the
group consisting of aspartate, glutamate, histidine, and lysine.
Said spacer amino acid should be an amino acid which does not
negatively affect the ability of a silk polypeptide to form a film.
Further, said spacer amino acid should be an amino acid which does
not cause steric hindrance, e.g. an amino acid having a small size
such as lysine and cysteine. In one more preferred embodiments, the
silk polypeptide comprises modules which are directly combined with
each other and modules which are combined with each other via 1 to
25 or 1 to 20 spacer amino acids, more preferably via 1 to 15 or 1
to 10 spacer amino acids, and most preferably, via 1 to 5 spacer
amino acids, e.g. via 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 spacer amino
acids.
[0153] A module A, C, Q, S, or R variant differs from the reference
(wild-type) module A, C, Q, S, or R from which it is derived by up
to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid
changes in the amino acid sequence (i.e. substitutions, additions,
insertions, deletions, N-terminal truncations and/or C-terminal
truncations). Such a module variant can alternatively or
additionally be characterised by a certain degree of sequence
identity to the reference (wild-type) module from which it is
derived. Thus, a module A, C, Q, S, or R variant has a sequence
identity of at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%,
59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%,
72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%,
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99% or even 99.9% to the respective reference (wild-type)
module A, C, Q, S, or R. Preferably, the sequence identity is over
a continuous stretch of at least 10, 15, 18, 20, 24, 27, 28, 30,
34, 35, or more amino acids, preferably over the whole length of
the respective reference (wild-type) module A, C, Q, S, or R.
[0154] It is particularly preferred that the sequence identity is
at least 80% over the whole length, is at least 85% over the whole
length, is at least 90% over the whole length, is at least 95% over
the whole length, is at least 98% over the whole length, or is at
least 99% over the whole length of the respective reference
(wild-type) module A, C, Q, S, or R. It is further particularly
preferred that the sequence identity is at least 80% over a
continuous stretch of at least 10, 15, 18, 20, 24, 28, or 30 amino
acids, is at least 85% over a continuous stretch of at least 10,
15, 18, 20, 24, 28, or 30 amino acids, is at least 90% over a
continuous stretch of at least 10, 15, 18, 20, 24, 28, or 30 amino
acids, is at least 95% over a continuous stretch of at least 10,
15, 18, 20, 24, 28, or 30 amino acids, is at least 98% over a
continuous stretch of at least 10, 15, 18, 20, 24, 28, or 30 amino
acids, or is at least 99% over a continuous stretch of at least 10,
15, 18, 20, 24, 28, or 30 amino acids of the respective reference
(wild-type) module A, C, Q, S, or R. A fragment (or deletion
variant) of module A, C, Q, S, or R has preferably a deletion of up
to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids
at its N-terminus and/or at its C-terminus. The deletion can also
be internally.
[0155] Additionally, the module A, C, Q, S, or R variant or
fragment is only regarded as a module A, C, Q, S, or R variant or
fragment within the context of the present invention, if the
modifications with respect to the amino acid sequence on which the
variant or fragment is based do not negatively affect the ability
of the silk polypeptide to form a film. The skilled person can
readily assess whether the silk polypeptide comprising a module A,
C, Q, S, or R variant or fragment is still capable of forming a
film, e.g. by producing an aqueous (buffered) solution comprising a
silk polypeptide comprising a module A, C, Q, S, or R variant or
fragment, casting said solution onto a solid support and
subsequently drying said solution (see experimental section).
Preferably, the produced silk film is a self-supporting film, i.e.
a silk film that has the capacity for supporting itself without the
help of additional materials such as carrier elements.
[0156] It is also preferred that the repetitive units are
independently selected from module A.sup.C (SEQ ID NO: 25), module
A.sup.K (SEQ ID NO: 26), module C.sup.C (SEQ ID NO: 27), module
C.sup.K1 (SEQ ID NO: 28), module C.sup.K2 (SEQ ID NO: 29), module
C.sup.KC (SEQ ID NO: 30), and module C.sup.Kappa (SEQ ID NO: 43).
The modules A.sup.C (SEQ ID NO: 25), A.sup.K (SEQ ID NO: 26),
C.sup.C (SEQ ID NO: 27), C.sup.K1 (SEQ ID NO: 28), C.sup.K2 (SEQ ID
NO: 29), C.sup.KC (SEQ ID NO: 30) are variants of the module A
which is based on the amino acid sequence of ADF-3 of the spider
Araneus diadematus and of module C which is based on the amino acid
sequence of ADF-4 of the spider Araneus diadematus (WO
2007/025719). In module A.sup.C (SEQ ID NO: 25) the amino acid S
(serine) at position 21 has been replaced by the amino acid C
(cysteine), in module A.sup.K (SEQ ID NO: 26) the amino acid S at
position 21 has been replaced by the amino acid K (lysine), in
module C.sup.C (SEQ ID NO: 27) the amino acid S at position 25 has
been replaced by the amino acid 25 by C, in module C.sup.K1 (SEQ ID
NO: 28) the amino acid S at position 25 has been replaced by the
amino acid K, in module C.sup.K2 (SEQ ID NO: 29) the amino acid E
(glutamate) at position 20 has been replaced by the amino acid K,
and in module C.sup.Kc (SEQ ID NO: 30) the amino acid E at position
20 has been replaced by the amino acid K and the amino acid S at
position 25 has been replaced by the amino acid C (WO 2007/025719),
and in module C.sup.Kappa (SEQ ID NO: 43), the amino acid E at
position 20 has been replaced by the amino acid K. Thus, in a more
preferred embodiment, the repetitive units in the silk polypeptide
consist of module A.sup.C: GPYGPGASAAAAAAGGYGPGCGQQ (SEQ ID NO:
25), module A.sup.K: GPYGPGASAAAAAAGGYGPGKGQQ (SEQ ID NO: 26),
module C.sup.C: GSSAAAAAAAASGPGGYGPENQGPCGPGGYGPGGP (SEQ ID NO:
27), module C.sup.K1: GSSAAAAAAAASGPGGYGPENQGPKGPGGYGPGGP (SEQ ID
NO: 28), module C.sup.K2: GSSAAAAAAAASGPGGYGPKNQGPSGPGGYGPGGP (SEQ
ID NO: 29), module C.sup.KC: GSSAAAAAAAASGPGGYGPKNQGPCGPGGYGPGGP
(SEQ ID NO: 30), or module C.sup.Kappa:
GSSAAAAAAAASGPGGYGPKNQGPSGPGGYGPGGP (SEQ ID NO: 43.
[0157] The modules A.sup.K, C.sup.C, C.sup.K1, C.sup.K2, C.sup.KC,
and C.sup.Kappa can also be combined with the modules A, C, Q, S,
or R, i.e. module (repetitive unit) A.sup.K can be combined with
module (repetitive unit) C (i.e. combination A.sup.KC), or module
(repetitive unit) C.sup.C can be combined with module (repetitive
unit) C (i.e. combination C.sup.CC), etc., under the proviso that
the silk polypeptide comprises or consists of at least two
repetitive units which are identical. Thus, the silk polypeptide
can also comprise or consist of the modules (AQA.sup.K).sub.n,
(QA.sup.K).sub.n, (QA.sup.KQ).sub.n, (A.sup.KQA).sub.n,
(A.sup.KQA.sup.K).sub.n, (CC.sup.C).sub.n, (CC.sup.CC).sub.n,
(C.sup.CC.sup.CC).sub.n, (CC.sup.CC.sup.C).sub.n, (C.sup.CQ).sub.n,
(QC.sup.C).sub.n, (QC.sup.CQ).sub.n, (C.sup.CQC).sub.n,
(CQC.sup.C).sub.n, (C.sup.CQC.sup.C).sub.n, (CC.sup.K1).sub.n,
(C.sup.K1C).sub.n, (C.sup.K1CC).sub.n, (CC.sup.K1C).sub.n,
(C.sup.KCC.sup.KCC).sub.n, (CC.sup.KCC.sup.KC).sub.n,
(C.sup.KCQ).sub.n, (QC.sup.KC).sub.n, (QC.sup.KCQ).sub.n,
(A.sup.KC.sup.K1Q).sub.n, (QC.sup.K2A.sup.K).sub.n or
(C.sup.K1C.sup.K2C).sub.n, wherein n is at least 2, preferably 4,
5, 6, 7, 8, 10, 12, 16, or 20. As to the terms "combined with each
other" or "concatenated with each other", it is referred to the
definitions provided above. For example, the silk polypeptide
comprises or consists of the modules C.sub.16C.sup.C,
C.sup.CC.sub.16, C.sub.8C.sup.CC.sub.8, C.sub.8C.sup.C.sub.8,
C.sup.C.sub.8C.sub.8, C.sub.4C.sup.C.sub.8C.sub.4,
C.sup.C.sub.4C.sub.8C.sup.C.sub.4, C.sup.C(AQ).sub.24, or
(AQ).sub.24C.sup.C.
[0158] It is more preferred that the repetitive units are
independently selected from module A (SEQ ID NO: 20) or variants
thereof, module C (SEQ ID NO: 21) or variants thereof, module Q
(SEQ ID NO: 22) or variants thereof, module S (SEQ ID NO: 23) or
variants thereof, module R (SEQ ID NO: 24) or variants thereof,
module A.sup.C (SEQ ID NO: 25), module A.sup.K (SEQ ID NO: 26),
module C.sup.C (SEQ ID NO: 27), module C.sup.K1 (SEQ ID NO: 28),
module C.sup.K2 (SEQ ID NO: 29), module C.sup.KC (SEQ ID NO: 30),
and module Kappa (SEQ ID NO: 43).
[0159] The silk polypeptide can further comprise at least one
non-repetitive (NR) unit, e.g. at least 1, 2, 3, 4, 5, 6, or more
NR unit(s), preferably one NR unit. In the context of the present
invention, the term "non-repetitive (NR) unit" refers to a region
of amino acids present in a naturally occurring silk polypeptide
that displays no obvious repetition pattern (non-repetitive unit or
NR unit). Preferably, the amino acid sequence of the non-repetitive
unit corresponds to a non-repetitive amino acid sequence of
naturally occurring dragline polypeptides, preferably of ADF-3 (SEQ
ID NO: 1) or ADF-4 (SEQ ID NO: 2), or to an amino acid sequence
substantially similar thereto. The amino acid sequence of the
non-repetitive unit may also correspond to a non-repetitive amino
acid sequence of black widow. More preferably, the amino acid
sequence of the non-repetitive unit corresponds to a non-repetitive
carboxy terminal amino acid sequence of naturally occurring
dragline polypeptides, preferably of ADF-3 (SEQ ID NO: 1) or ADF-4
(SEQ ID NO: 2), or to an amino acid sequence substantially similar
thereto. Even more preferably, the amino acid sequence of the
non-repetitive unit corresponds to a non-repetitive carboxy
terminal amino acid sequence of ADF-3 (SEQ ID NO: 1) which
comprises amino acids 513 through 636, or of ADF-4 (SEQ ID NO: 2)
which comprises amino acids 302 through 410, or to an amino acid
sequence substantially similar thereto.
[0160] In this regard "substantially similar" means a degree of
amino acid identity of at least 50%, 51%, 52%, 53%, 54%, 55%, 56%,
57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%,
70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,
83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99% or even 99.9%, preferably over 20, 30, 40, 50,
60, 70, 80 or more amino acids, more preferably over the whole
length of the respective reference non-repetitive (carboxy
terminal) amino acid sequence of naturally occurring dragline
polypeptides, preferably of ADF-3 (SEQ ID NO: 1) or ADF-4 (SEQ ID
NO: 2). A "non-repetitive unit" having an amino acid sequence which
is "substantially similar" to a corresponding non-repetitive
(carboxy terminal) amino acid sequence within a naturally occurring
dragline polypeptide (i.e. wild-type non-repetitive (carboxy
terminal) unit), preferably within ADF-3 (SEQ ID NO: 1) or ADF-4
(SEQ ID NO: 2), is also similar with respect to its functional
properties; e.g. a silk polypeptide comprising a "substantially
similar non-repetitive unit" is still capable of forming a film.
The skilled person can readily assess whether a silk polypeptide
comprising a "substantially similar non-repetitive unit" is still
capable of forming a film, e.g. by producing an aqueous (buffered)
solution comprising the silk polypeptide comprising the
"substantially similar non-repetitive unit", casting said solution
onto a solid support and subsequently drying said solution (see
experimental section). Preferably, the produced silk film is a
self-supporting film, i.e. a silk film that has the capacity for
supporting itself without the help of additional materials such as
carrier elements.
[0161] Most preferably, the non-repetitive (NR) unit is selected
from NR3 (SEQ ID NO: 31) or variants thereof, NR4 (SEQ ID NO: 32)
or variants thereof, NR5 (SEQ ID NO: 33) or variants thereof, and
NR6 (SEQ ID NO: 34) or variants thereof. The NR3 (SEQ ID NO: 31)
unit is based on the amino acid sequence of ADF-3 of the spider
Araneus diadematus and the NR4 (SEQ ID NO: 32) unit is based on the
amino acid sequence of ADF-4 of the spider Araneus diadematus (WO
2006/008163). In addition, the NR5 (SEQ ID NO: 33) unit and the NR6
(SEQ ID NO: 34) unit is derived from Latrodectus hesperus.
[0162] A NR3, NR4, NR5, or NR6 unit variant differs from the
reference NR3 (SEQ ID NO: 31), NR4 (SEQ ID NO: 32), NR5 (SEQ ID NO:
33), or NR6 (SEQ ID NO: 34) unit from which it is derived by up to
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
20, 25, or 30 amino acid changes in the amino acid sequence (i.e.
exchanges, insertions, deletions, N-terminal truncations and/or
C-terminal truncations). Such a NR3, NR4, NR5, or NR6 unit variant
can alternatively or additionally be characterised by a certain
degree of sequence identity to the reference NR3, NR4, NR5, or NR6
unit from which it is derived. Thus, a NR3, NR4, NR5, or NR6 unit
variant has a sequence identity of at least 50%, 55%, 60%, 65%,
70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,
87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or
even 99.9% to the respective reference NR3, NR4, NR5, or NR6 unit.
Preferably, the sequence identity is over a continuous stretch of
at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or more amino acids,
preferably over the whole length of the respective reference NR3,
NR4, NR5, or NR6 unit.
[0163] It is particularly preferred that the sequence identity is
at least 80% over the whole length, is at least 85% over the whole
length, is at least 90% over the whole length, is at least 95% over
the whole length, is at least 98% over the whole length, or is at
least 99% over the whole length of the respective reference NR3,
NR4, NR5, or NR6 unit. It is further particularly preferred that
the sequence identity is at least 80% over a continuous stretch of
at least 20, 30, 40, 50, 60, 70, or 80 amino acids, is at least 85%
over a continuous stretch of at least 20, 30, 40, 50, 60, 70, or 80
amino acids, is at least 90% over a continuous stretch of at least
20, 30, 40, 50, 60, 70, or 80 amino acids, is at least 95% over a
continuous stretch of at least 20, 30, 40, 50, 60, 70, or 80 amino
acids, is at least 98% over a continuous stretch of at least 20,
30, 40, 50, 60, 70, or 80 amino acids, or is at least 99% over a
continuous stretch of at least 20, 30, 40, 50, 60, 70, or 80 amino
acids of the respective reference NR3, NR4, NR5, or NR6 unit.
[0164] A fragment (or deletion variant) of a NR3, NR4, NR5, or NR6
unit has preferably a deletion of up to 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30,
35, 40, 45, 50, 55, or 60 amino acids at its N-terminus and/or at
its C-terminus. The deletion can also be internally.
[0165] Additionally, the NR3, NR4, NR5, or NR6 unit variant or
fragment is only regarded as a NR3, NR4, NR5, or NR6 unit variant
or fragment within the context of the present invention, if the
modifications with respect to the amino acid sequence on which the
variant or fragment is based do not negatively affect the ability
of a silk polypeptide to form a film. The skilled person can
readily assess whether the silk polypeptide comprising a NR3, NR4,
NR5, or NR6 unit variant or fragment is still capable of forming a
film, e.g. by producing an aqueous (buffered) solution comprising a
silk polypeptide comprising a NR3, NR4, NR5, or NR6 unit variant or
fragment, casting said solution onto a solid support and
subsequently drying said solution (see experimental section).
Preferably, the produced silk film is a self-supporting film, i.e.
a silk film that has the capacity for supporting itself without the
help of additional materials such as carrier elements.
[0166] It is even more preferred that the silk polypeptide is
selected from the group consisting of ADF-3 (SEQ ID NO: 1) or
variants thereof, ADF-4 (SEQ ID NO: 2) or variants thereof, MaSp I
(SEQ ID NO: 35) or variants thereof, MaSp II (SEQ ID NO: 36) or
variants thereof, (C).sub.m, (C).sub.mNR.sub.z, NR.sub.z(C).sub.m,
(C.sup.Kappa).sub.m, (C.sup.Kappa).sub.mNR.sub.z,
NR.sub.z(C.sup.Kappa).sub.m, (AQ).sub.n, (AQ).sub.nNR.sub.z,
NR.sub.z(AQ).sub.n, (QAQ).sub.o, NR.sub.z(QAQ).sub.o, and
(QAQ).sub.oNR.sub.z, wherein m is an integer of 4 to 64, i.e. 4, 5,
6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,
41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,
58, 59, 60, 61, 62, 63, or 64, n is an integer of 6 to 40, i.e. 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or
40, o is an integer of 8 to 40, i.e. 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,
33, 34, 35, 36, 37, 38, 39, or 40, z is an integer of 1 to 3, i.e.
1, 2, or 3, and NR is in each case independently a non-repetitive
unit, preferably a NR3, NR4, NR5, or NR6 non-repetitive unit or a
variant thereof.
[0167] The above mentioned formulas are defined by one of the
following: In the formula [0168] (i) (C).sub.m, a "m" number of C
modules, namely 4 to 64 C modules, represented by the amino acid
sequence according to SEQ ID NO: 21, are combined with each other
[0169] (ii) (C).sub.mNR.sub.z, a "m" number of C modules, namely 4
to 64 C modules, represented by the amino acid sequence according
to SEQ ID NO: 21, are combined with each other, wherein said C
modules are further combined with a "z" number of non-repetitive
(NR) units, namely 1 to 3 non-repetitive (NR) units, e.g. the
non-repetitive (NR) units NR3 represented by the amino acid
sequence according to SEQ ID NO: 31, NR4 represented by the amino
acid sequence according to SEQ ID NO: 32, NR5 represented by the
amino acid sequence according to SEQ ID NO: 33, or NR6 represented
by the amino acid sequence according to SEQ ID NO: 34, [0170] (iii)
NR.sub.z(C).sub.m, a "z" number of non-repetitive (NR) units,
namely 1 to 3 non-repetitive (NR) units, e.g. the non-repetitive
(NR) units NR3 represented by the amino acid sequence according to
SEQ ID NO: 31, NR4 represented by the amino acid sequence according
to SEQ ID NO: 32, NR5 represented by the amino acid sequence
according to SEQ ID NO: 33, or NR6 represented by the amino acid
sequence according to SEQ ID NO: 34, is present (z=1) or are
combined with each other (z=2 or 3), wherein said non-repetitive
(NR) unit(s) is (are) further combined with a "m" number of C
modules, namely 4 to 64 C modules, represented by the amino acid
sequence according to SEQ ID NO: 21, [0171] (iv) (AQ).sub.n, a "n"
number of A and Q module combinations, namely 6 to 40 A and Q
module combinations, wherein module A is represented by the amino
acid sequence according to SEQ ID NO: 20 and module Q is
represented by the amino acid sequence according to SEQ ID NO: 22,
are combined with each other, [0172] (v) (AQ).sub.nNR.sub.z, a "n"
number of A and Q module combinations, namely 6 to 40 A and Q
module combinations, wherein module A is represented by the amino
acid sequence according to SEQ ID NO: 20 and module Q is
represented by the amino acid sequence according to SEQ ID NO: 22,
are combined with each other, and wherein said A and Q module
combinations are further combined with a "z" number of
non-repetitive (NR) units, namely 1 to 3 non-repetitive (NR) units,
e.g. the non-repetitive (NR) units NR3 represented by the amino
acid sequence according to SEQ ID NO: 31, NR4 represented by the
amino acid sequence according to SEQ ID NO: 32, NR5 represented by
the amino acid sequence according to SEQ ID NO: 33, or NR6
represented by the amino acid sequence according to SEQ ID NO: 34,
[0173] (vi) NR.sub.z(AQ).sub.n, a "z" number of non-repetitive (NR)
units, namely 1 to 3 non-repetitive (NR) units, e.g. the
non-repetitive (NR) units NR3 represented by the amino acid
sequence according to SEQ ID NO: 31, NR4 represented by the amino
acid sequence according to SEQ ID NO: 32, NR5 represented by the
amino acid sequence according to SEQ ID NO: 33, or NR6 represented
by the amino acid sequence according to SEQ ID NO: 34, is present
(z=1) or are combined with each other (z=2 or 3), wherein said
non-repetitive (NR) unit(s) is (are) further combined with a "n"
number of A and Q module combinations, namely 6 to 40 A and Q
module combinations, wherein module A is represented by the amino
acid sequence according to SEQ ID NO: 20 and module Q is
represented by the amino acid sequence according to SEQ ID NO: 22,
[0174] (vii) (QAQ).sub.o, a "o" number of Q, A and Q module
combinations, namely 8 to 40 Q, A and Q module combinations,
wherein module Q is represented by an amino acid sequence according
to SEQ ID NO: 22 and module A is represented by the amino acid
sequence according to SEQ ID NO: 20, are combined with each other,
[0175] (viii) (QAQ).sub.oNR.sub.z, a "o" number of Q, A and Q
module combinations, namely 8 to 40 Q, A and Q module combinations,
wherein module Q is represented by an amino acid sequence according
to SEQ ID NO: 22 and module A is represented by the amino acid
sequence according to SEQ ID NO: 20, are combined with each other,
and wherein said Q, A and Q module combinations are further
combined with a "z" number of non-repetitive (NR) units, namely 1
to 3 non-repetitive (NR) units, e.g. the non-repetitive (NR) units
NR3 represented by the amino acid sequence according to SEQ ID NO:
31, NR4 represented by the amino acid sequence according to SEQ ID
NO: 32, NR5 represented by the amino acid sequence according to SEQ
ID NO: 33, or NR6 represented by the amino acid sequence according
to SEQ ID NO: 34, and [0176] (ix) NR.sub.z(QAQ).sub.o, a "z" number
of non-repetitive (NR) units, namely 1 to 3 non-repetitive (NR)
units, e.g. the non-repetitive (NR) units NR3 represented by the
amino acid sequence according to SEQ ID NO: 31, NR4 represented by
the amino acid sequence according to SEQ ID NO: 32, NR5 represented
by the amino acid sequence according to SEQ ID NO: 33, or NR6
represented by the amino acid sequence according to SEQ ID NO: 34,
is present (z=1) or are combined with each other (z=2 or 3),
wherein said non-repetitive (NR) unit(s) is (are) further combined
with a "o" number of Q, A and Q module combinations, namely 8 to 40
Q, A and Q module combinations, wherein module Q is represented by
an amino acid sequence according to SEQ ID NO: 22 and module A is
represented by the amino acid sequence according to SEQ ID NO:
20.
[0177] It is most preferred that the silk polypeptide is selected
from the group consisting of C.sub.8, C.sub.8NR4, NR4C.sub.8,
C.sub.8NR3, NR3C.sub.8, NR4C.sub.8NR4, NR3C.sub.8NR3,
NR4C.sub.8NR3, NR3C.sub.8NR4, C.sub.16, C.sub.16NR4, NR4C.sub.16,
C.sub.16NR3, NR3C.sub.16, NR4C.sub.16NR4, NR3C.sub.16NR3,
NR4C.sub.16NR3, NR3C.sub.16NR4, C.sub.32, C.sub.32NR4, NR4C.sub.32,
C.sub.32NR3, NR3C.sub.32, NR4C.sub.32NR4, NR3C.sub.32NR3,
NR4C.sub.32NR3, NR3C.sub.32NR4, C.sup.Kappa.sub.16,
C.sup.Kappa.sub.16NR4, NR4C.sup.Kappa.sub.16,
C.sup.Kappa.sub.16NR3, NR3C.sup.Kappa.sub.16,
NR4C.sup.Kappa.sub.16NR4, NR3C.sup.Kappa.sub.16NR3,
NR4C.sup.Kappa.sub.16NR3, NR3C.sup.Kappa.sub.16NR4, (AQ).sub.12,
(AQ).sub.12NR3, NR3(AQ).sub.12, (AQ).sub.12NR4, NR4(AQ).sub.12,
NR3(AQ).sub.12NR3, NR4(AQ).sub.12NR4, NR3(AQ).sub.12NR4,
NR4(AQ).sub.12NR3, (AQ).sub.24, (AQ).sub.24NR3, NR3(AQ).sub.24,
(AQ).sub.24NR4, NR4(AQ).sub.24, NR3(AQ).sub.24NR3,
NR4(AQ).sub.24NR4, NR3(AQ).sub.24NR4, NR4(AQ).sub.24NR3,
(QAQ).sub.8, and (QAQ).sub.16.
[0178] An ADF-3, ADF-4, MaSp I or MaSp II variant differs from the
reference (wild-type) ADF-3 (SEQ ID NO: 1), ADF-4 (SEQ ID NO: 2),
MaSp I (SEQ ID NO: 35) or MaSp II (SEQ ID NO: 36) polypeptide from
which it is derived by up to 150 (up to 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50,
55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, or
150) amino acid changes in the amino acid sequence (i.e.
substitutions, insertions, deletions, N-terminal truncations and/or
C-terminal truncations). Such a variant can alternatively or
additionally be characterised by a certain degree of sequence
identity to the reference (wild-type) polypeptide from which it is
derived. Thus, an ADF-3, ADF-4, MaSp I or MaSp II variant has a
sequence identity of at least 50%, 51%, 52%, 53%, 54%, 55%, 56%,
57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%,
70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,
83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99% or even 99.9% to the respective reference
(wild-type) ADF-3, ADF-4, MaSp I or MaSp II polypeptide.
Preferably, the sequence identity is over a continuous stretch of
at least 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 120, 150,
180, 200, 250, 300, 350, 400, or more amino acids, preferably over
the whole length of the respective reference (wild-type) ADF-3,
ADF-4, MaSp I or MaSp II polypeptide.
[0179] It is particularly preferred that the sequence identity is
at least 80% over the whole length, is at least 85% over the whole
length, is at least 90% over the whole length, is at least 95% over
the whole length, is at least 98% over the whole length, or is at
least 99% over the whole length of the respective reference
(wild-type) ADF-3, ADF-4, MaSp I or MaSp II polypeptide. It is
further particularly preferred that the sequence identity is at
least 80% over a continuous stretch of at least 20, 30, 50, 100,
150, 200, 250, or 300 amino acids, is at least 85% over a
continuous stretch of at least 20, 30, 50, 100, 150, 200, 250, or
300 amino acids, is at least 90% over a continuous stretch of at
least 20, 30, 50, 100, 150, 200, 250, or 300 amino acids, is at
least 95% over a continuous stretch of at least 20, 30, 50, 100,
150, 200, 250, or 300 amino acids, is at least 98% over a
continuous stretch of at least 20, 30, 50, 100, 150, 200, 250, or
300 amino acids, or is at least 99% over a continuous stretch of at
least 20, 30, 50, 100, 150, 200, 250, or 300 amino acids of the
respective reference (wild-type) ADF-3, ADF-4, MaSp I or MaSp II
polypeptide.
[0180] A fragment (or deletion variant) of the ADF-3 (SEQ ID NO: 1)
polypeptide has preferably a deletion of up to 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,
85, 90, 95, 100, 120, 150, 170, 200, 220, 250, 270, 300, 320, 350,
370, 400, 420, 450, 470, 500, 520, 550, 570, 600, or 610 amino
acids at its N-terminus and/or at its C-terminus. The deletion can
also be internally.
[0181] A fragment (or deletion variant) of the ADF-4 (SEQ ID NO: 2)
polypeptide has preferably a deletion of up to 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,
85, 90, 95, 100, 120, 150, 170, 200, 220, 250, 270, 300, 320, 330,
340, 350, 360, 370, 380, or 390 amino acids at its N-terminus
and/or at its C-terminus. The deletion can also be internally.
[0182] A fragment (or deletion variant) of the MaSp I (SEQ ID NO:
35) polypeptide has preferably a deletion of up to 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,
80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550,
600, 620, 640, 660, 670, 680, or 690 amino acids at its N-terminus
and/or at its C-terminus. The deletion can also be internally.
[0183] A fragment (or deletion variant) of the MaSp II (SEQ ID NO:
36) polypeptide has preferably a deletion of up to 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,
80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 520,
540, 560, or 570 amino acids at its N-terminus and/or at its
C-terminus. The deletion can also be internally.
[0184] Additionally, the ADF-3, ADF-4, MaSp I or MaSp II variant or
fragment is only regarded as an ADF-3, ADF-4, MaSp I or MaSp II
variant or fragment within the context of the present invention, if
the modifications with respect to the amino acid sequence on which
the variant or fragment is based do not negatively affect the
ability of the silk polypeptide to form a film. The skilled person
can readily assess whether the silk polypeptide comprising a ADF-3,
ADF-4, MaSp I or MaSp II variant or fragment is still capable of
forming a film, e.g. by producing an aqueous (buffered) solution
comprising a silk polypeptide comprising a ADF-3, ADF-4, MaSp I or
MaSp II variant or fragment, casting said solution onto a solid
support and subsequently drying said solution (see experimental
section). Preferably, the produced silk film is a self-supporting
film, i.e. a silk film that has the capacity for supporting itself
without the help of additional materials such as carrier
elements.
[0185] The silk polypeptide may further comprise an amino terminal
and/or a carboxy terminal TAG selected from the group consisting of
(i) TAG.sup.CYS1 consisting of the amino acid sequence
GCGGGGGGSGGGG (SEQ ID NO: 37), (ii) TAG.sup.CYS2 consisting of the
amino acid sequence GCGGGGGG (SEQ ID NO: 38), (iii) TAG.sup.CYS3
consisting of the amino acid sequence GCGGSGGGGSGGGG (SEQ ID NO:
39), (iv) TAG.sup.LYS1 consisting of the amino acid sequence
GKGGGGGGSGGGG (SEQ ID NO: 41), and (v) TAG.sup.LYS2 consisting of
the amino acid sequence GKGGGGGG (SEQ ID NO: 42). Said amino
terminal and/or a carboxy terminal TAGs allow the coupling (by
forming covalent bounds) of active agents to the silk polypeptide
and, thus, to the silk film.
[0186] In one embodiment, the one or more first silk film layers
have a thickness of at least 0.1, 0.5, 1, 2, 3, 4, 5, 10, 15, 20,
25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140,
150, 160, 170, 180, 190, 200, 300, 400, 500, 600, 700, 800, 900
.mu.m, 1, 2, 3, 4, 5, 6, 7, 8, 9 mm, or 1 cm. In one further
embodiment, the one or more first silk film layers have a thickness
of between 0.1 .mu.m and 1 cm, preferably of between 1 .mu.m and 5
mm or of between 1 .mu.m and 1 mm, more preferably of between 1
.mu.m and 200 .mu.m or of between 1 .mu.m and 100 .mu.m, and most
preferably of between 1 .mu.m and 50 .mu.m or of between 1 .mu.m
and 40 .mu.m. In this respect, it should be noted that the
thickness of the first silk film layers may be varied depending on
the amount of the active agent to be incorporated. For example, an
increased thickness of the first silk film layers allows the
incorporation of a higher amount of the active agent.
[0187] In one another embodiment, the one or more release modifying
layers have a thickness of at least 0.05, 0.1, 0.5, 1, 2, 3, 4, 5,
10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120,
130, 140, 150, 160, 170, 180, 190, 200, 300, 400, or 500 .mu.m. In
one further embodiment, the one or more release modifying layers
have a thickness of between 0.05 .mu.m and 500 .mu.m or of between
0.1 .mu.m and 500 .mu.m, preferably of between 0.5 .mu.m and 200
.mu.m or of between 0.5 .mu.m and 100 .mu.m, even more preferably
of between 0.5 .mu.m and 80 .mu.m or of between 0.5 .mu.m and 50
.mu.m, and most preferably of between 0.5 .mu.m and 40 .mu.m or of
between 0.5 .mu.m and 20 .mu.m. In this respect, it should be noted
that the thickness of the release modifying layers may be varied
depending on the desired release of the active agent. For example,
an increased thickness of the release modifying layers allows a
slower release of the active agent.
[0188] In one alternative or additional embodiment, the one or more
second silk film layers have a thickness of at least 0.1, 0.5, 1,
2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90,
100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400,
500, 600, 700, 800, 900 .mu.m, 1, 2, 3, 4, 5, 6, 7, 8, 9 mm, or 1
cm. In one further embodiment, the one or more second silk film
layers have a thickness of between 0.1 .mu.m and 1 cm, preferably
of between 1 .mu.m and 5 mm or of between 1 .mu.m and 1 mm, more
preferably of between 1 .mu.m and 200 .mu.m or of between 1 .mu.m
and 100 .mu.m, and most preferably of between 1 .mu.m and 50 .mu.m
or of between 1 .mu.m and 40 .mu.m. In this respect, it should be
noted that the thickness of the second silk film layers may be
varied depending on the desired retardation of the active agent.
For example, an increased thickness of the second silk film layers
allows a better retardation of release of the active agent.
[0189] In one further embodiment, the coated silk film is
water-soluble or water-insoluble. As to the production of
water-insoluble coated silk films, it is referred to the eighth
aspect of the present invention.
[0190] In a second aspect, the present invention relates to a
pharmaceutical composition comprising or consisting of the coated
silk film according to the first aspect, wherein the active agent
is a pharmaceutical agent. As to the preferred pharmaceutical
agents, it is referred to the first aspect of the present
invention.
[0191] The coated silk film of the present invention may be
administered in the form of any suitable pharmaceutical
composition. Said pharmaceutical compositions include, but are not
limited to, wound closure or coverage systems, such as plaster,
patches, and wound dressings, protection systems, such as bandages,
skin substitution systems, such as skin grafts like autografts,
implants, contact lenses, and depot systems. The contact lenses may
comprise the coated silk film of the first aspect. The
pharmaceutical agents comprised therein may be anti-inflammatory
agents. The depot systems may comprise contraceptive agents.
[0192] The pharmaceutical composition may further comprise one or
more pharmaceutically acceptable carriers, diluents and/or
excipients. Said pharmaceutically acceptable carriers, diluents
and/or excipients may already be comprised in the aqueous solution
or dispersion used for silk film formation or may subsequently be
added to or loaded into the silk film. The term "pharmaceutically
acceptable", as used herein, refers to the non-toxicity of a
material which does not interact with the action of the active
component of the pharmaceutical composition. The term "carrier", as
used herein, relates to one or more compatible solid or liquid
fillers or diluents, which are suitable for an administration to an
individual. The term "carrier" relates to a natural or synthetic
organic or inorganic component which is combined with an active
agent in order to facilitate the application of the active agent.
Preferably, carrier components are sterile liquids such as water or
oils, including those which are derived from mineral oil, animals,
or plants, such as peanut oil, soy bean oil, sesame oil, sunflower
oil, etc. Salt solutions and aqueous dextrose and glycerin
solutions may also be used as aqueous carrier compounds. The term
"diluent", as used herein, relates a diluting and/or thinning
agent. Moreover, the term "diluent" includes any one or more of
fluid, liquid or solid suspension and/or mixing media. The term
"excipient", as used herein, is intended to indicate all substances
in a pharmaceutical composition which are not active ingredients
such as binders, lubricants, thickeners, surface active agents,
preservatives, emulsifiers, buffers, flavoring agents, or
colorants.
[0193] Preferably, the pharmaceutical composition is useful for
treating or reducing the severity of a disease or medical condition
by administering said pharmaceutical composition to an individual.
The pharmaceutical composition can be administered locally or
systemically, preferably locally. Said treatment includes, but is
not limited to, therapeutical treatment, prophylactic treatment,
and preventive treatment.
[0194] The terms "individual" and "subject" are used herein
interchangeably. They preferably refer to a human or another mammal
(e.g. mouse, rat, rabbit, dog, cat, cattle, swine, sheep, horse or
primate). Unless otherwise stated, the terms "individual" and
"subject" do not denote a particular age and, thus, encompass
adults, elderlies, children, and newborns.
[0195] The term "disease (also designated as disorder)", as used
herein, refers to an abnormal condition that affects the body of an
individual. A disease is often construed as a medical condition
associated with specific symptoms and signs. A disease may be
caused by factors originally from an external source, such as
infectious disease, or it may be caused by internal dysfunctions.
In humans, a disease is often used more broadly to refer to any
condition that causes pain, dysfunction, distress, social problems,
or death to the individual afflicted, or similar problems for those
in contact with the individual. In this broader sense, it sometimes
includes injuries, disabilities, disorders, syndromes, infections,
isolated symptoms, deviant behaviors, and atypical variations of
structure and function, while in other contexts and for other
purposes these may be considered distinguishable categories.
Diseases usually affect individuals not only physically, but also
emotionally, as contracting and living with many diseases can alter
one's perspective on life, and one's personality.
[0196] Preferably, the disease is selected from the group
consisting of an infectious disease, such as herpes, an autoimmune
disease, such as psoriasis, a cutaneous disease, such as acne,
rosacea, dermatitis or eczema, and a gastro-intestinal disease,
such as dyspepsia or indigestion. The term "infectious disease", as
used herein, refers to any disease which can be transmitted from
individual to individual or from organism to organism, and is
caused by a microbial agent. Infectious diseases are known in the
art and include, for example, a viral disease, a bacterial disease,
or a parasitic disease, which diseases are caused by a virus, a
bacterium, and a parasite, respectively. The term "cutaneous
disease", as used herein, refers to any disease that affects the
integumentary system, the organ system that encloses the body and
includes skin, hair, nails, and related muscle and glands.
[0197] Preferably, the medical condition is selected from the group
consisting of burns, skin lesions, and wounds, such as superficial
or inner wounds.
[0198] The term "therapeutic treatment", as used herein, relates to
any treatment which improves the health status and/or prolongs
(increases) the lifespan of an individual. Said treatment may
eliminate the disease in an individual, arrest or slow the
development of a disease in an individual, inhibit or slow the
development of a disease in an individual, decrease the frequency
or severity of symptoms in an individual, and/or decrease the
recurrence in an individual who currently has or who previously has
had a disease. The terms "prophylactic treatment" or "preventive
treatment", as used herein, relate to any treatment that is
intended to prevent a disease from occurring in an individual. The
terms "prophylactic treatment" or "preventive treatment" are used
herein interchangeably.
[0199] As mentioned above, the pharmaceutical composition may be
administered to an individual in need thereof. The term
"administration", as used herein, refers to the manner in which the
pharmaceutical composition is presented to an individual. The
pharmaceutical composition according to the invention may be
administered to an individual using several ways. Preferred forms
of administration include, but are not limited to, intradermal,
transdermal, and topical administrations, particularly to the skin
or tissue.
[0200] Preferably, the pharmaceutical composition is useful for
controlled and sustained delivery of the pharmaceutical agent. Due
the constant release profile of the coated silk film, the
pharmaceutical composition comprising or consisting of the coated
silk film is capable of releasing the loaded, attached, and/or
incorporated pharmaceutical agent over a period of time, e.g.
several days or weeks.
[0201] It is also possible to apply the coated silk film comprised
in the pharmaceutical composition to several preferential objects
including, but not limited to, meshes, scaffolds, patches, and
nonwovens.
[0202] In a third aspect, the present invention relates to a
cosmetic composition comprising or consisting of the coated silk
film according to the first aspect, wherein the active agent is a
cosmetic agent. As to the preferred cosmetic agents, it is referred
to the first aspect of the present invention.
[0203] The coated silk film of the present invention may be
administered in the form of any suitable cosmetic composition. Said
cosmetic compositions include, but are not limited to, skin care
products, such as skin masks, cleaning wipes, cleaning pads,
creams, lotions or gels, and hair care products, such as
conditioners or shampoos.
[0204] The cosmetic composition may further comprise one or more
cosmetically acceptable carriers, diluents and/or excipients. Said
cosmetically acceptable carriers, diluents and/or excipients may
already be comprised in the aqueous solution used for silk film
formation or may subsequently be added to or loaded into the silk
film. The term "cosmetically acceptable", as used herein, refers to
the non-toxicity of a material which does not interact with the
action of the active component of the cosmetic composition.
[0205] The cosmetic composition according to the invention may be
administered to an individual using several ways. A preferred form
of administration is the topical administration, particularly to
the skin.
[0206] Preferably, the cosmetic composition is useful for
controlled and sustained delivery of the cosmetic agent. Due the
constant release profile of the coated silk film, the cosmetic
composition comprising or consisting of the coated silk film is
capable of releasing the loaded, attached, and/or incorporated
cosmetic agent over a period of time, e.g. several days or
weeks.
[0207] It is also possible to apply the coated silk film comprised
in the cosmetic composition to several preferential objects
including, but not limited to, meshes, scaffolds, patches, and
nonwovens.
[0208] In a fourth aspect, the present invention relates to a
coated silk film according to the first aspect or pharmaceutical
composition according to the second aspect for use in medicine.
Preferably, the coated silk film according to the first aspect or
the pharmaceutical composition according to the second aspect is
for the treatment of a disease or medical condition.
[0209] It is preferred that the coated silk film comprises an
active agent such as a biological agent, pharmaceutical agent, or
cosmetic agent, preferably a pharmaceutical agent. As to the
preferred biological agents, pharmaceutical agents, or cosmetic
agents, it is referred to the first aspect of the present
invention.
[0210] It is further preferred that the disease is selected from
the group consisting of an infectious disease, such as herpes, an
autoimmune disease, such as psoriasis, a cutaneous disease, such as
acne, rosacea, dermatitis or eczema, and a gastro-intestinal
disease, such as dyspepsia or indigestion. It is also preferred
that the medical condition is selected from the group consisting of
burns, skin lesions, and wounds such as superficial wounds or inner
wounds.
[0211] In a fifth aspect, the present invention relates to a coated
silk film according to the first aspect or pharmaceutical
composition according to the second aspect for controlled and
sustained release of at least one pharmaceutical agent. As to the
preferred pharmaceutical agents, it is referred to the first aspect
of the present invention.
[0212] The pharmaceutical agent may be released from the coated
silk film or pharmaceutical composition comprising or consisting of
the coated silk film by diffusion and/or degradation upon exposure
to a physiological environment (e.g. a physiological medium). The
physiological environment (e.g. the physiological medium) may be a
physiological buffered solution or a body fluid, e.g. blood, lymph
or liquor. The release of the pharmaceutical agent from the coated
silk film or pharmaceutical composition comprising or consisting of
the coated silk film may be induced by introducing the coated silk
film or pharmaceutical composition comprising or consisting of the
coated silk film into a body fluid such as blood, lymph or liquor,
or by applying the coated silk film or pharmaceutical composition
comprising or consisting of the coated silk film onto an organ
(e.g. skin) or a part of an organ (e.g. tissue).
[0213] Preferably, the release of the pharmaceutical agent displays
relatively linear kinetics, thereby providing a constant supply of
the pharmaceutical agent over the release period.
[0214] It is preferred that less than 20%, preferably less than
15%, more preferably less than 10%, and most preferably less than
5%, e.g. less than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9,
8, 7, 6, or 5%, of the pharmaceutical agent is released,
particularly into the physiological environment, within the first
24 hours.
[0215] It is, alternatively or additionally, preferred that more
than 50%, preferably more than 70%, more preferably more than 80%,
and most preferably 100% of the pharmaceutical agent is released,
particularly into the physiological environment, within 36 hours.
It is, alternatively or additionally, preferred that more than 50%,
preferably more than 70%, more preferably more than 80%, and most
preferably 100% of the pharmaceutical agent is released,
particularly into the physiological environment, within 48 hours.
It is, alternatively or additionally, preferred that more than 50%,
preferably more than 70%, more preferably more than 80%, and most
preferably 100% of the pharmaceutical agent is released,
particularly into the physiological environment, within 72
hours.
[0216] It is, alternatively or additionally, more preferred that
more than 50%, preferably more than 70%, more preferably more than
80%, and most preferably 100% of the pharmaceutical agent is
released, particularly into the physiological environment, within 7
days. It is, alternatively or additionally, more preferred that
more than 50%, preferably more than 70%, more preferably more than
80%, and most preferably 100% of the pharmaceutical agent is
released, particularly into the physiological environment, within
14 days. It is, alternatively or additionally, more preferred that
more than 50%, preferably more than 70%, more preferably more than
80%, and most preferably 100% of the pharmaceutical agent is
released, particularly into the physiological environment, within
20 days. It is, alternatively or additionally, more preferred that
more than 50%, preferably more than 70%, more preferably more than
80%, and most preferably 100% of the pharmaceutical agent is
released, particularly into the physiological environment, within
30 days. It is, alternatively or additionally, more preferred that
more than 50%, preferably more than 70%, more preferably more than
80%, and most preferably 100% of the pharmaceutical agent is
released, particularly into the physiological environment, within
35 days.
[0217] It is, alternatively or additionally, even more preferred
that more than 50%, preferably more than 70%, more preferably more
than 80%, and most preferably 100% of the pharmaceutical agent is
released, particularly into the physiological environment, within 5
weeks. It is, alternatively or additionally, even more preferred
that more than 50%, preferably more than 70%, more preferably more
than 80%, and most preferably 100% of the pharmaceutical agent is
released, particularly into the physiological environment, within 6
weeks. It is, alternatively or additionally, even more preferred
that more than 50%, preferably more than 70%, more preferably more
than 80%, and most preferably 100% of the pharmaceutical agent is
released, particularly into the physiological environment, within 7
weeks. It is, alternatively or additionally, even more preferred
that more than 50%, preferably more than 70%, more preferably more
than 80%, and most preferably 100% of the pharmaceutical agent is
released, particularly into the physiological environment, within 8
weeks.
[0218] It is, alternatively or additionally, most preferred that
more than 50%, preferably more than 70%, more preferably more than
80%, and most preferably 100% of the pharmaceutical agent is
released, particularly into the physiological environment, within 3
months. It is, alternatively or additionally, most preferred that
more than 50%, preferably more than 70%, more preferably more than
80%, and most preferably 100% of the pharmaceutical agent is
released, particularly into the physiological environment, within 4
months. It is, alternatively or additionally, most preferred that
more than 50%, preferably more than 70%, more preferably more than
80%, and most preferably 100% of the pharmaceutical agent is
released, particularly into the physiological environment, within 5
months. It is, alternatively or additionally, most preferred that
more than 50%, preferably more than 70%, more preferably more than
80%, and most preferably 100% of the pharmaceutical agent is
released, particularly into the physiological environment, within 6
months. It is, alternatively or additionally, most preferred that
more than 50%, preferably more than 70%, more preferably more than
80%, and most preferably 100% of the pharmaceutical agent is
released, particularly into the physiological environment, within 7
months. It is, alternatively or additionally, most preferred that
more than 50%, preferably more than 70%, more preferably more than
80%, and most preferably 100% of the pharmaceutical agent is
released, particularly into the physiological environment, within 8
months. It is, alternatively or additionally, most preferred that
more than 50%, preferably more than 70%, more preferably more than
80%, and most preferably 100% of the pharmaceutical agent is
released, particularly into the physiological environment, within 9
months. It is, alternatively or additionally, most preferred that
more than 50%, preferably more than 70%, more preferably more than
80%, and most preferably 100% of the pharmaceutical agent is
released, particularly into the physiological environment, within
10 months.
[0219] In a sixth aspect, the present invention relates to a coated
silk film according to the first aspect or pharmaceutical
composition according to the second aspect for the treatment of
wounds, skin diseases, or skin defects. The coated silk film
according to the first aspect preferably comprises a pharmaceutical
agent. As to the preferred embodiments of the pharmaceutical agent,
it is referred to the first aspect of the present invention.
[0220] The term "wound", as used herein, includes damages to any
organ, particularly tissue, in an individual. The wound may be
comprised on the surface of the body of an individual, (i.e. a
superficial wound), or may be comprised within the body of an
individual (i.e. an internal wound). The wound may have been caused
by any means including, but not limited to, infections,
inflammations, surgical interventions, external components such as
sharp objects, e.g. scalpels, knifes or nails, and external
circumstances, such as accidents, e.g. bicycle, motor vehicle, or
auto accidents.
[0221] The wound may be comprised on the surface (e.g. skin) of the
body of an individual (i.e. a superficial wound), or may be
comprised within the body of an individual (i.e. an internal
wound). The wound may have been caused by any means including, but
not limited to, infections, inflammations, surgical interventions,
external components such as sharp objects, e.g. scalpels, knifes or
nails, and external circumstances, such as accidents, e.g. bicycle,
motor vehicle, or auto accidents.
[0222] It is particularly preferred that the wound is selected from
the group consisting of a topical wound, deep wound, gaping wound,
stab wound, puncture wound, penetration wound, surgical incision,
laceration, and cut. The term "topical wound", as used herein,
refers to a wound on the tissue surface. The term "puncture wound",
as used herein, refers to a wound caused by an object puncturing
tissue(s), e.g. multiple (different) tissues, particularly an
organ, e.g. skin, such as a nail or needle. The term "penetration
wound", as used herein, refers to a wound caused by an object
entering and coming out from the tissue(s), e.g. multiple
(different) tissues, particularly an organ, e.g. skin, such as a
nail or needle. The term "surgical incision", as used herein,
refers to a wound caused by a sharp object, e.g. a scalpel or
knife, during surgery.
[0223] The wound may be located in or on the surface of a tissue.
The tissue may be selected form the group consisting of connective
tissue, muscle tissue, nervous tissue, epithelial tissue, and
combinations thereof, e.g. multiple (different) tissues. An organ,
e.g. stomach, small intestine, large intestine, bowel, rectum,
oesophagus, lung, spleen, brain, heart, kidney, liver, skin, glands
such as lymph and thyroid glands, eye, or pancreas, is, for
example, comprised of multiple (different) tissues. Thus, the wound
may also be located in an organ, particularly encompassing multiple
(different) tissues or tissue layers. Particularly, the wound is a
skin lesion.
[0224] Preferably, the coated silk film according to the first
aspect or pharmaceutical composition according to the second aspect
is for the topical treatment of a wound site and/or for the
treatment of an internal wound site, e.g. in case of deeper wounds
or during surgical procedures.
[0225] It is preferred that the skin disease is selected from the
group consisting of an infectious disease, such as herpes, an
autoimmune disease, such as psoriasis, a cutaneous disease, such as
acne, rosacea, dermatitis or eczema, and a gastro-intestinal
disease, such as dyspepsia or indigestion. It is further preferred
that the skin defect is selected from the group consisting of
burns, skin lesions, and wounds, such as superficial or inner
wounds.
[0226] In a seventh aspect, the present invention relates to the
use of the coated silk film according to the first aspect or
cosmetic composition according to the third aspect for controlled
and sustained release of at least one cosmetic agent. As to the
preferred embodiments of the cosmetic agent, it is referred to the
first aspect of the present invention.
[0227] The cosmetic agent may be released from the coated silk film
or cosmetic composition comprising or consisting of the coated silk
film by diffusion and/or degradation upon exposure to a
physiological environment. The physiological environment may be
skin. The release of the cosmetic agent from the coated silk film
or cosmetic composition comprising or consisting of the coated silk
film can be induced by applying the coated silk film or cosmetic
composition comprising or consisting of the coated silk film onto
skin.
[0228] Preferably, the release of the cosmetic agent displays
relatively linear kinetics, thereby providing a constant supply of
the cosmetic agent over the release period.
[0229] It is preferred that less than 20%, preferably less than
15%, more preferably less than 10%, and most preferably less than
5%, e.g. less than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9,
8, 7, 6, or 5%, of the cosmetic agent is released, particularly
into the physiological environment, within the first 24 hours.
[0230] It is, alternatively or additionally, preferred that more
than 50%, preferably more than 70%, more preferably more than 80%,
and most preferably 100% of the cosmetic agent is released,
particularly into the physiological environment, within 36 hours.
It is, alternatively or additionally, preferred that more than 50%,
preferably more than 70%, more preferably more than 80%, and most
preferably 100% of the cosmetic agent is released, particularly
into the physiological environment, within 48 hours. It is,
alternatively or additionally, preferred that more than 50%,
preferably more than 70%, more preferably more than 80%, and most
preferably 100% of the cosmetic agent is released, particularly
into the physiological environment, within 72 hours.
[0231] It is, alternatively or additionally, more preferred that
more than 50%, preferably more than 70%, more preferably more than
80%, and most preferably 100% of the cosmetic agent is released,
particularly into the physiological environment, within 7 days. It
is, alternatively or additionally, more preferred that more than
50%, preferably more than 70%, more preferably more than 80%, and
most preferably 100% of the cosmetic agent is released,
particularly into the physiological environment, within 14 days. It
is, alternatively or additionally, more preferred that more than
50%, preferably more than 70%, more preferably more than 80%, and
most preferably 100% of the cosmetic agent is released,
particularly into the physiological environment, within 20 days. It
is, alternatively or additionally, more preferred that more than
50%, preferably more than 70%, more preferably more than 80%, and
most preferably 100% of the cosmetic agent is released,
particularly into the physiological environment, within 30 days. It
is, alternatively or additionally, more preferred that more than
50%, preferably more than 70%, more preferably more than 80%, and
most preferably 100% of the cosmetic agent is released,
particularly into the physiological environment, within 35
days.
[0232] It is, alternatively or additionally, even more preferred
that more than 50%, preferably more than 70%, more preferably more
than 80%, and most preferably 100% of the cosmetic agent is
released, particularly into the physiological environment, within 5
weeks. It is, alternatively or additionally, even more preferred
that more than 50%, preferably more than 70%, more preferably more
than 80%, and most preferably 100% of the cosmetic agent is
released, particularly into the physiological environment, within 6
weeks. It is, alternatively or additionally, even more preferred
that more than 50%, preferably more than 70%, more preferably more
than 80%, and most preferably 100% of the cosmetic agent is
released, particularly into the physiological environment, within 7
weeks. It is, alternatively or additionally, even more preferred
that more than 50%, preferably more than 70%, more preferably more
than 80%, and most preferably 100% of the cosmetic agent is
released, particularly into the physiological environment, within 8
weeks.
[0233] It is, alternatively or additionally, most preferred that
more than 50%, preferably more than 70%, more preferably more than
80%, and most preferably 100% of the cosmetic agent is released,
particularly into the physiological environment, within 3 months.
It is, alternatively or additionally, most preferred that more than
50%, preferably more than 70%, more preferably more than 80%, and
most preferably 100% of the cosmetic agent is released,
particularly into the physiological environment, within 4 months.
It is, alternatively or additionally, most preferred that more than
50%, preferably more than 70%, more preferably more than 80%, and
most preferably 100% of the cosmetic agent is released,
particularly into the physiological environment, within 5 months.
It is, alternatively or additionally, most preferred that more than
50%, preferably more than 70%, more preferably more than 80%, and
most preferably 100% of the cosmetic agent is released,
particularly into the physiological environment, within 6 months.
It is, alternatively or additionally, most preferred that more than
50%, preferably more than 70%, more preferably more than 80%, and
most preferably 100% of the cosmetic agent is released,
particularly into the physiological environment, within 7 months.
It is, alternatively or additionally, most preferred that more than
50%, preferably more than 70%, more preferably more than 80%, and
most preferably 100% of the cosmetic agent is released,
particularly into the physiological environment, within 8 months.
It is, alternatively or additionally, most preferred that more than
50%, preferably more than 70%, more preferably more than 80%, and
most preferably 100% of the cosmetic agent is released,
particularly into the physiological environment, within 9 months.
It is, alternatively or additionally, most preferred that more than
50%, preferably more than 70%, more preferably more than 80%, and
most preferably 100% of the cosmetic agent is released,
particularly into the physiological environment, within 10
months.
[0234] In a further aspect, the present invention relates to an
active agent delivery device or active agent depot system
comprising the coated silk film according to the first aspect. The
active agent depot system comprising the coated silk film according
to the first aspect may comprise conceptive agents.
[0235] In another aspect, the present invention relates to a
coating comprising the coated silk film according to the first
aspect.
[0236] In an eight aspect, the present invention relates to a
method of producing a coated silk film comprising the steps of:
[0237] (i) providing an active agent release sheet which comprises
or consists of one or more first silk film layers comprising at
least one active agent, and [0238] (ii) covering at least the side
surface(s) of the active agent release sheet with one or more
release modifying layers.
[0239] Due to the covering of at least the side surface(s) of the
active agent release sheet with one or more release modifying
layers, the coated silk film is obtained. Said coated silk film
comprises or consists of an active agent release sheet which
comprises or consists of one or more first silk film layers
comprising at least one active agent, and one or more release
modifying layers covering at least the side surface(s) of the
active agent release sheet.
[0240] The active agent release sheet may comprise 1, 2, 3, or more
first silk film layers. Alternatively or additionally, at least the
side surface(s) of the active agent release sheet may be covered
with 1, 2, 3, 4, 5, or more release modifying layers.
[0241] In one embodiment, the active agent release sheet is
completely covered with one or more release modifying layers. Thus,
the method of producing a coated silk film may comprise the steps
of: [0242] (i) providing an active agent release sheet which
comprises or consists of one or more first silk film layers
comprising at least one active agent, and [0243] (ii) completely
covering the active agent release sheet with one or more release
modifying layers.
[0244] Due to the complete covering of the active agent release
sheet with one or more release modifying layers, the coated silk
film is obtained. Said coated silk film comprises or consists of an
active agent release sheet which comprises or consists of one or
more first silk film layers comprising at least one active agent,
and one or more release modifying layers completely covering the
active agent release sheet.
[0245] In one another embodiment, the active agent release sheet
comprises one or more second silk film layers. Thus, method of
producing a coated silk film may comprise the steps of: [0246] (i)
providing an active agent release sheet which comprises or consists
of one or more first silk film layers comprising at least one
active agent and one or more second silk film layers, and [0247]
(ii) covering at least the side surface(s) of the active agent
release sheet with one or more release modifying layers.
[0248] The active agent release sheet may comprise 1, 2, 3, or more
first silk film layers and/or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, or more second silk film layers. Alternatively or additionally,
at least the side surface(s) of the active agent release sheet may
be covered with 1, 2, 3, 4, 5, or more release modifying
layers.
[0249] As mentioned above, in one embodiment, the active agent
release sheet is completely covered with one or more release
modifying layers. Thus, the method of producing a coated silk film
may comprise the steps of: [0250] (i) providing an active agent
release sheet which comprises or consists of one or more first silk
film layers comprising at least one active agent and one or more
second silk film layers, and [0251] (ii) completely covering the
active agent release sheet with one or more release modifying
layers.
[0252] In one preferred embodiment, the active agent release sheet
comprises or consists of (a) first silk film layer(s) comprising at
least one active agent which is (are) flanked by second silk film
layers. In other word, the first silk film layer(s) comprising at
least one active agent is (are) sandwiched or embedded between
second silk film layers.
[0253] It one another preferred embodiment, the one or more first
silk film layers and/or the one or more second silk film layers
comprise at least one plasticizer.
[0254] In one further preferred embodiment, the active agent
release sheet comprises plasticizer containing first and/or second
silk film layers and plasticizer free first and/or second silk film
layers in alteration. For example, the active agent release sheet
may comprise one plasticizer containing first silk film layer and
one plasticizer free second silk film layer. Further, the active
agent release sheet may comprise one plasticizer containing first
silk film layer and two plasticizer free second silk film layers,
wherein both plasticizer free second silk film layers are attached
to the plasticizer containing first silk film layer, thereby
forming a sandwich structure. Furthermore, the active agent release
sheet may comprise one plasticizer containing first silk film
layer, one or more plasticizer containing second silk film layers
and one or more plasticizer free second silk film layers, wherein
the plasticizer containing silk film layers and plasticizer free
silk film layers are arranged alternately. The active agent release
sheet may have a structure as described in FIGS. 4A and 5A. The
advantages of the use of plasticizers are described with respect to
the first aspect of the present invention.
[0255] Preferably, the one or more release modifying layers does
not contain a plasticizer. In other words, it is preferred that the
one or more release modifying layers are plasticizer free.
[0256] It is preferred that the amount of the plasticizer in the
one or more first silk film layers and/or one or more second silk
film layers is of between 0.1% (w/w) and 70% (w/w), more preferably
of between 5% (w/w) and 70% (w/w) or of between 15% (w/w) and 70%
(w/w), even more preferably of between 15% (w/w) and 55% (w/w) or
of between 30% (w/w) and 55% (w/w), and most preferably of 30%
(w/w). It is further preferred that the amount of the plasticizer
in the one or more first silk film layers and/or one or more second
silk film layers is of at least 0.1, 1, 5, 10, 15, 20, 25, 30, 35,
40, 45, 50, 55, 60, 65, or 70% (w/w).
[0257] Preferred plasticizers are selected from glycerol,
2-pyrrolidone, polyethylenglycol (PEG), polyvinylalcohol (PVA),
poloxamer, polyvinylpirrolidone (PVP), polyacrylic acid,
polyorthoester, gelatine, collagen, cellulose, cellulose derivates,
and sorbitol. More preferred plasticizers are selected from
glycerol and 2-pyrrolidone.
[0258] It is further preferred that the active agent has a
molecular weight of between 50 Da and 300 kDa, preferably of
between 80 Da and 250 kDa, more preferably of between 100 Da and
200 kDa, and most preferably of between 150 Da and 150 kDa. The
active agent may further have a molecular weight of between 50 Da
and <50 kDa or a molecular weight of between 50 kDa and 150
kDa.
[0259] Preferred active agents are selected from the group
consisting of a biological agent, a pharmaceutical agent, a
cosmetic agent, a nutrient, and a dietary supplement. As to the
preferred embodiments of a biological agent, pharmaceutical agent,
cosmetic agent, nutrient, or dietary supplement, it is referred to
the first aspect of the present invention.
[0260] The active agent may be positively or negatively charged.
The active agent may also be electroneutral. Preferably, the active
agent is positively or negatively charged. The silk film comprising
or consisting of an active agent release sheet may have a positive
or negative net charge. Thus, it is preferred that the active agent
comprised in the first silk film layer(s) of the active agent
release sheet is positively charged, if the silk film has a
negative net charge. It is further preferred that the active agent
comprised in the first silk film layer(s) of the active agent
release sheet is negatively charged, if the silk film has a
positive net charge.
[0261] The one or more release modifying layers comprise or consist
of a release modifying material (e.g. a silk material). In one
embodiment, the one or more release modifying layers comprise or
consist of at least one release modifying agent. It is preferred
that the release modifying agent is selected from the group
consisting of a (i) silk polypeptide, (ii) polyester, preferably
polylactide, polyglycolide, polylactic polyglycolic copolymer
(PLGA), and blend of polylactic polyglycolic copolymer (PLGA) and
polylactide, (iii) polyether, preferably polycaprolactone (PCL),
(iv) polyanhydride, (v) polyalkylcyanoacrylate, preferably n-butyl
cyanoacrylate, (vi) polyacrylamide, (vii) polyurethane, and (viii)
polyvinylpirrolidone (PVP). It is more preferred that the release
modifying agent is a silk polypeptide.
[0262] The silk film, particularly the one or more first silk film
layers and/or the one or more second silk film layers, comprise a
silk material. In one embodiment, the silk film, particularly the
one or more first silk film layers and/or the one or more second
silk film layers, comprise at least one silk polypeptide.
[0263] It is preferred that the silk polypeptide is a recombinant
silk polypeptide. It is further preferred that the silk polypeptide
is a (recombinant) spider silk polypeptide, more preferably a
(recombinant) major ampullate silk polypeptide such as a
(recombinant) dragline silk polypeptide, a (recombinant) minor
ampullate silk polypeptide, or a (recombinant) flagelliform silk
polypeptide of an orb-web spider (e.g. Araneidae or Araneoids), an
(recombinant) insect silk polypeptide, a (recombinant) mussel
byssus silk polypeptide, or a mixture thereof. The orb-web spider
may be selected from the group consisting of Araneus diadematus,
Nephila clavipes, and Latrodectus hesperus. The insect silk
polypeptide may be of Lepidoptera, particularly Bombycidae such as
Bombyx mori. The insect silk polypeptide may also be of
Hymenoptera, particularly Apoidea such as Anthophila.
[0264] It is, alternatively or additionally, preferred that the
silk polypeptide comprises or consists of at least two repetitive
units.
[0265] Preferably, the silk polypeptide comprises or consists of at
least two repetitive units each comprising at least one, preferably
one, consensus sequence selected from the group consisting of:
[0266] (i) GPGXX (SEQ ID NO: 3), wherein X is any amino acid,
preferably in each case independently selected from A, S, G, Y, P,
and Q; [0267] (ii) GGX, wherein X is any amino acid, preferably in
each case independently selected from Y, P, R, S, A, T, N and Q,
more preferably in each case independently selected from Y, P and
Q; [0268] (iii) A.sub.x, wherein x is an integer from 5 to 10;
[0269] (iv) GGRPSDTYG (SEQ ID NO: 18); and [0270] (v) GGRPSSSYG
(SEQ ID NO: 19).
[0271] The above mentioned silk polypeptide preferably has a
molecular weight of at least 5 kDa, e.g. of at least 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 kDa.
[0272] It is preferred that the repetitive units are independently
selected from module A (SEQ ID NO: 20) or variants thereof, module
C (SEQ ID NO: 21) or variants thereof, module Q (SEQ ID NO: 22) or
variants thereof, module S (SEQ ID NO: 23) or variants thereof, and
module R (SEQ ID NO: 24) or variants thereof.
[0273] It is further preferred that the repetitive units are
independently selected from module A.sup.C (SEQ ID NO: 25), module
A.sup.K (SEQ ID NO: 26), module C.sup.C (SEQ ID NO: 27), module
C.sup.K1 (SEQ ID NO: 28), module C.sup.K2 (SEQ ID NO: 29), module
C.sup.KC (SEQ ID NO: 30), and module C.sup.Kappa (SEQ ID NO:
43).
[0274] It is more preferred that the repetitive units are
independently selected from module A (SEQ ID NO: 20) or variants
thereof, module C (SEQ ID NO: 21) or variants thereof, module Q
(SEQ ID NO: 22) or variants thereof, module S (SEQ ID NO: 23) or
variants thereof, module R (SEQ ID NO: 24) or variants thereof,
module A.sup.C (SEQ ID NO: 25), module A.sup.K (SEQ ID NO: 26),
module C.sup.C (SEQ ID NO: 27), module C.sup.K1 (SEQ ID NO: 28),
module C.sup.K2 (SEQ ID NO: 29), module C.sup.KC (SEQ ID NO: 30),
and module C.sup.Kappa (SEQ ID NO: 43).
[0275] The silk polypeptide can further comprise at least one
non-repetitive (NR) unit, e.g. at least 1, 2, 3, 4, 5, 6, or more
NR unit(s), preferably one NR unit. It is preferred that the
non-repetitive (NR) unit is selected from NR3 (SEQ ID NO: 31) or
variants thereof, NR4 (SEQ ID NO: 32) or variants thereof, NR5 (SEQ
ID NO: 33) or variants thereof, or NR6 (SEQ ID NO: 34) or variants
thereof.
[0276] It is even more preferred that the silk polypeptide is
selected from the group consisting of ADF-3 (SEQ ID NO: 1) or
variants thereof, ADF-4 (SEQ ID NO: 2) or variants thereof, MaSp I
(SEQ ID NO: 35) or variants thereof, MaSp II (SEQ ID NO: 36) or
variants thereof, (C).sub.m, (C).sub.mNR.sub.z, NR.sub.z(C).sub.m,
(C.sup.Kappa).sub.m, (C.sup.Kappa).sub.mNR.sub.z,
NR.sub.z(C.sup.Kappa).sub.m, (AQ).sub.n, (AQ).sub.nNR.sub.z,
NR.sub.z(AQ).sub.n, (QAQ).sub.o, NR.sub.z(QAQ).sub.o, and
(QAQ).sub.oNR.sub.z, wherein m is an integer of 4 to 64, i.e. 4, 5,
6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,
41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,
58, 59, 60, 61, 62, 63, or 64, n is an integer of 6 to 40, i.e. 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or
40, o is an integer of 8 to 40, i.e. 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,
33, 34, 35, 36, 37, 38, 39, or 40, z is an integer of 1 to 3, i.e.
1, 2, or 3, and NR is in each case independently a non-repetitive
unit, preferably a NR3, NR4, NR5, or NR6 non-repetitive unit or a
variant thereof.
[0277] It is most preferred that the silk polypeptide is selected
from the group consisting of C.sub.16, C.sub.16NR4, NR4C.sub.16,
C.sub.16NR3, NR3C.sub.16, NR4C.sub.16NR4, NR3C.sub.16NR3,
NR4C.sub.16NR3, NR3C.sub.16NR4, C.sub.32, C.sub.32NR4, NR4C.sub.32,
C.sub.32NR3, NR3C.sub.32, NR4C.sub.32NR4, NR3C.sub.32NR3,
NR4C.sub.32NR3, NR3C.sub.32NR4, C.sup.Kappa.sub.16,
C.sup.Kappa.sub.16NR4, NR4C.sup.Kappa.sub.16,
C.sup.Kappa.sub.16NR3, NR3C.sup.Kappa.sub.16,
NR4C.sup.Kappa.sub.16NR4, NR3C.sup.Kappa.sub.16NR3,
NR4C.sup.Kappa.sub.16NR3, NR3C.sup.Kappa.sub.16NR4, (AQ).sub.12,
(AQ).sub.12NR3, NR3(AQ).sub.12, (AQ).sub.12NR4, NR4(AQ).sub.12,
NR3(AQ).sub.12NR3, NR4(AQ).sub.12NR4, NR3(AQ).sub.12NR4,
NR4(AQ).sub.12NR3, (AQ).sub.24, (AQ).sub.24NR3, NR3(AQ).sub.24,
(AQ).sub.24NR4, NR4(AQ).sub.24, NR3(AQ).sub.24NR3,
NR4(AQ).sub.24NR4, NR3(AQ).sub.24NR4, NR4(AQ).sub.24NR3,
(QAQ).sub.8, and (QAQ).sub.16.
[0278] The silk polypeptide may further comprise an amino terminal
and/or a carboxy terminal TAG selected from the group consisting of
(i) TAG.sup.CYS1 consisting of the amino acid sequence
GCGGGGGGSGGGG (SEQ ID NO: 37), (ii) TAG.sup.CYS2 consisting of the
amino acid sequence GCGGGGGG (SEQ ID NO: 38), (iii) TAGCYS.sup.3
consisting of the amino acid sequence GCGGSGGGGSGGGG (SEQ ID NO:
39), (iv) TAG.sup.LYS1 consisting of the amino acid sequence
GKGGGGGGSGGGG (SEQ ID NO: 41), and (v) TAG.sup.LYS2 consisting of
the amino acid sequence GKGGGGGG (SEQ ID NO: 42). Said amino
terminal and/or a carboxy terminal TAGs allow the coupling (by
forming covalent bounds) of active agents to the silk polypeptide
and, thus, to the silk film.
[0279] As to further preferred embodiments of the silk polypeptide,
it is referred to the first aspect of the present invention.
[0280] In one embodiment, the one or more first silk film layers
have a thickness of at least 0.1, 0.5, 1, 2, 3, 4, 5, 10, 15, 20,
25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140,
150, 160, 170, 180, 190, 200, 300, 400, 500, 600, 700, 800, 900
.mu.m, 1, 2, 3, 4, 5, 6, 7, 8, 9 mm, or 1 cm. In one further
embodiment, the one or more first silk film layers have a thickness
of between 0.1 .mu.m and 1 cm, preferably of between 1 .mu.m and 5
mm or of between 1 .mu.m and 1 mm, more preferably of between 1
.mu.m and 200 .mu.m or of between 1 .mu.m and 100 .mu.m, and most
preferably of between 1 .mu.m and 50 .mu.m or of between 1 .mu.m
and 40 .mu.m. In this respect, it should be noted that the
thickness of the first silk film layers may be varied depending on
the amount of the active agent to be incorporated. For example, an
increased thickness of the first silk film layers allows the
incorporation of a higher amount of the active agent.
[0281] In one another embodiment, the one or more release modifying
layers have a thickness of at least 0.05, 0.1, 0.5, 1, 2, 3, 4, 5,
10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120,
130, 140, 150, 160, 170, 180, 190, 200, 300, 400, or 500 .mu.m. In
one further embodiment, the one or more release modifying layers
have a thickness of between 0.05 .mu.m and 500 .mu.m or of between
0.1 .mu.m and 500 .mu.m, preferably of between 0.5 .mu.m and 200
.mu.m or of between 0.5 .mu.m and 100 .mu.m, even more preferably
of between 0.5 .mu.m and 80 .mu.m or of between 0.5 .mu.m and 50
.mu.m, and most preferably of between 0.5 .mu.m and 40 .mu.m or of
between 0.5 .mu.m and 20 .mu.m. In this respect, it should be noted
that the thickness of the release modifying layers may be varied
depending on the desired release of the active agent. For example,
an increased thickness of the release modifying layers allows a
slower release of the active agent.
[0282] In one alternative or additional embodiment, the one or more
second silk film layers have a thickness of at least 0.1, 0.5, 1,
2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90,
100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400,
500, 600, 700, 800, 900 .mu.m, 1, 2, 3, 4, 5, 6, 7, 8, 9 mm, or 1
cm. In one further embodiment, the one or more second silk film
layers have a thickness of between 0.1 .mu.m and 1 cm, preferably
of between 1 .mu.m and 5 mm or of between 1 .mu.m and 1 mm, more
preferably of between 1 .mu.m and 200 .mu.m or of between 1 .mu.m
and 100 .mu.m, and most preferably of between 1 .mu.m and 50 .mu.m
or of between 1 .mu.m and 40 .mu.m. In this respect, it should be
noted that the thickness of the second silk film layers may be
varied depending on the desired retardation of the active agent.
For example, an increased thickness of the second silk film layers
allows a better retardation of release of the active agent.
[0283] It is preferred that the method further comprises the step
of drying the one or more release modifying layers. The drying can
be carried out, for example, by drying in the air, baking, using a
heat chamber, a vacuum chamber, laminar flow (e.g. of a gas such as
nitrogen or carbon dioxide), radiation, or a fan (at low
temperatures, at room temperature or at elevated temperatures).
[0284] It is further preferred that the method further comprises
the step of applying pressure to the one or more release modifying
layers. The inventors of the present invention found that the
application of pressure to the one or more release modifying layers
increases and improves the connection of the release modifying
layer(s) to the active agent release sheet of the silk film, which,
in turn, results in an increase of the packing density of the
coated silk film. The inventors of the present invention further
found that an increased packing density of the coated silk film has
an influence on the release of active agents from said film.
Particularly, an increased packing density of the coated silk film
reduces the release of active agents from said film. Thus, the
application of pressure to the release modifying layer(s) or not
and, if yes, the variation of the pressure applied to the release
modifying layer(s) allows to adapt/change the release profile of
the active agents from the coated silk film. The application of
pressure to the one or more release modifying layer(s) may further
improve, particularly increase, the film elongation. Preferably,
the pressure applied to the one or more release modifying layers
corresponds to a weight force of at least 0.1, 0.2, 0.3, 0.4, 0.5,
0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8,
1.9, or 2 t/m.sup.2. A weight force of at least 0.1, 0.2, 0.3, 0.4,
0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,
1.8, 1.9, or 2 t/m.sup.2 corresponds to 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 kN/m.sup.2,
respectively.
[0285] It is, alternatively or additionally, also preferred that
the method further comprises the step of applying water vapour to
the one or more release modifying layers. The inventors of the
present invention found that the application of water vapour allows
the transformation of a water-soluble coated silk film into a
water-insoluble coated silk film. Thus, a coated silk film, which
initially was water-soluble, can be processed with water vapour
leading to water-insolubility. The conversion of a water-soluble
coated silk film into a water-insoluble coated silk film is
attributable to, on a structural basis, an increased .beta.-sheet
structure content.
[0286] It is preferred that the water vapour has a temperature of
between 1.degree. C. and 121.degree. C., preferably of between
1.degree. C. and 80.degree. C., more preferably of between
4.degree. C. and 50.degree. C., even more preferably of between
15.degree. C. and 40.degree. C., and most preferably of between of
between 19.degree. C. or 20.degree. C. (room temperature) and
35.degree. C., e.g. a temperature of 19, 20, 21, 22, 23, 24, 25,
26, 27, 28, 29, 30, 31, 32, 33, 34, or 35.degree. C. It is,
alternatively or additionally, preferred that the water vapour has
a relative humidity of between 50% and 100%, preferably of between
50% and 90%, more preferably of between 60% and 90%, and most
preferably of between 70% and 80%, e.g. a relative humidity of 70,
71, 72, 73, 74, 75, 76, 77, 78, 79, or 80%.
[0287] Thus, the method may further comprise the steps of (i)
drying the one or more release modifying layers and applying
pressure to the one or more release modifying layers, (ii) drying
the one or more release modifying layers and applying water vapour
to the one or more release modifying layers, (iii) drying the one
or more release modifying layers, applying pressure to the one or
more release modifying layers and applying water vapour to the one
or more release modifying layers, or (iv) drying the one or more
release modifying layers, applying water vapour to the one or more
release modifying layers, and applying pressure to the one or more
release modifying layers.
[0288] In one embodiment, the active agent release sheet is at
least at its side surface(s) covered with one release modifying
layer by [0289] (i) immersing the active agent release sheet at
least at its side surface(s) into an aqueous solution or dispersion
comprising or consisting of a release modifying material (e.g. at
least one release modifying agent), or spraying an aqueous solution
or dispersion comprising or consisting of a release modifying
material (e.g. at least one release modifying agent) on at least
the side surface(s) of the active agent release sheet, [0290] (ii)
forming one release modifying layer which covers at least the side
surface(s) of the active agent release sheet, particularly by
drying the aqueous solution or dispersion comprising or consisting
of a release modifying material (e.g. at least one release
modifying agent), and [0291] (iii) optionally applying pressure
and/or water vapour to the one release modifying layer. Preferably,
steps (i) and (ii) and optionally step (iii) are repeated one or
more times to form further release modifying layers on the release
modifying layer formed in step (ii).
[0292] As mentioned above, in one embodiment, the active agent
release sheet is completely covered with one or more release
modifying layers. In this case, the active agent release sheet is
completely covered with one release modifying layer by [0293] (i)
completely immersing the active agent release sheet into an aqueous
solution or dispersion comprising or consisting of a release
modifying material (e.g. at least one release modifying agent), or
spraying an aqueous solution or dispersion comprising or consisting
of a release modifying material (e.g. at least one release
modifying agent) on the active agent release sheet, [0294] (ii)
forming one release modifying layer which completely covers the
active agent release sheet, particularly by drying the aqueous
solution or dispersion comprising or consisting of a release
modifying material (e.g. at least one release modifying agent), and
[0295] (iii) optionally applying pressure and/or water vapour to
the one release modifying layer. Preferably, steps (i) and (ii) and
optionally step (iii) are repeated one or more times to form
further release modifying layers on the release modifying layer
formed in step (ii).
[0296] The drying can be carried out, for example, by drying in the
air, baking, using a heat chamber, a vacuum chamber, laminar flow
(e.g. of a gas such as nitrogen or carbon dioxide), radiation, or a
fan (at low temperatures, at room temperature or at elevated
temperatures).
[0297] If pressure is applied to the release modifying layer(s), it
is preferred that the pressure corresponds to a weight force of at
least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2,
1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2 t/m.sup.2. A weight force
of at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1,
1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2 t/m.sup.2 corresponds
to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, or 20 kN/m.sup.2, respectively.
[0298] If water vapour is applied to the release modifying
layer(s), it is further preferred that the water vapour has a
temperature of between 1.degree. C. and 121.degree. C., preferably
of between 1.degree. C. and 80.degree. C., more preferably of
between 4.degree. C. and 50.degree. C., even more preferably of
between 15.degree. C. and 40.degree. C., and most preferably of
between of between 19.degree. C. or 20.degree. C. (room
temperature) and 35.degree. C., e.g. a temperature of 19, 20, 21,
22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35.degree.
C. It is, alternatively or additionally, also preferred that the
water vapour has a relative humidity of between 50% and 100%,
preferably of between 50% and 90%, more preferably of between 60%
and 90%, and most preferably of between 70% and 80%, e.g. a
relative humidity of 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or
80%.
[0299] It is preferred that the release modifying agent is
comprised in the aqueous solution or dispersion in a concentration
of between 0.5 mg/ml and 15 mg/ml, preferably in a concentration of
between 1 mg/ml and 10 mg/ml, and more preferably in a
concentration of between 1 mg/ml and 5 mg/ml. Preferably, the
release modifying agent is a silk polypeptide.
[0300] It is further preferred that the aqueous solution or
dispersion mentioned above comprises at least one plasticizer.
Preferably, the plasticizer is comprised in the aqueous solution or
dispersion in a concentration of between 0.1% (w/v) and 10% (w/v),
more preferably of between 0.5% (w/v) and 5% (w/v) or between 1%
(w/v) and 5% (w/v), even more preferably of between 1% (w/v) and 3%
(w/v) or between 1% (w/v) and 2% (w/v), and most preferably of
between 3% (w/v). Preferred plasticizers are selected from
glycerol, 2-pyrrolidone, polyethylenglycol (PEG), polyvinylalcohol
(PVA), poloxamer, polyvinylpirrolidone (PVP), polyacrylic acid,
polyorthoester, gelatine, collagen, cellulose, cellulose derivates,
and sorbitol. More preferred plasticizers are selected from
glycerol and 2-pyrrolidone.
[0301] The aqueous solution or dispersion may be a buffered aqueous
solution or dispersion, preferably TRIS buffer-HCl (pH 8),
particularly 5 mM TRIS puffer-HCl (pH 8).
[0302] The active agent release sheet of the silk film provided in
step (i) of the method of producing a coated silk film may be
produced in several ways which are described below: In one
embodiment, the active agent release sheet is produced by the steps
of: [0303] (a) providing an aqueous solution or dispersion
comprising or consisting of a silk material (e.g. at least one silk
polypeptide) and at least one active agent, and [0304] (b) forming
a first silk film layer comprising at least one active agent from
the aqueous solution or dispersion comprising or consisting of a
silk material (e.g. at least one silk polypeptide) and at least one
active agent, particularly by applying the aqueous solution or
dispersion comprising or consisting of a silk material (e.g. at
least one silk polypeptide) and at least one active agent on a
support material and drying said aqueous solution or dispersion,
and [0305] (c) optionally applying water vapour to the first silk
film layer. In one embodiment, the method of producing the active
agent release sheet further comprises the steps of: [0306] (d)
providing an aqueous solution or dispersion comprising or
consisting of a silk material (e.g. at least one silk polypeptide)
and at least one active agent, or providing an aqueous solution or
dispersion comprising or consisting of a silk material (e.g. at
least one silk polypeptide), [0307] (e) forming a further first
silk film layer comprising or consisting of at least one active
agent from the aqueous solution or dispersion comprising or
consisting of a silk material (e.g. at least one silk polypeptide)
and at least one active agent on the first silk film layer formed
in step (b), particularly by applying the aqueous solution or
dispersion comprising or consisting of a silk material (e.g. at
least one silk polypeptide) and at least one active agent on said
first silk film layer and drying said aqueous solution or
dispersion, or forming a second silk film layer from the aqueous
solution or dispersion comprising or consisting of a silk material
(e.g. at least one silk polypeptide) on the first silk film layer
formed in step (b), particularly by applying the aqueous solution
or dispersion comprising or consisting of a silk material (e.g. at
least one silk polypeptide) on said first silk film layer and
drying said aqueous solution or dispersion, and [0308] (f)
optionally applying pressure and/or water vapour to the first silk
film layer or second silk film layer. Preferably, steps (d) and (e)
and optionally step (0 are repeated one or more times (e.g. 1, 2, 3
4, 5, 6, 7, 8, 9, 10, or more times) to form further first and/or
second silk film layers on the first silk film layer formed in step
(b).
[0309] In one another embodiment, the active agent release sheet is
produced by the steps of: [0310] (a) providing an aqueous solution
or dispersion comprising or consisting of a silk material (e.g. at
least one silk polypeptide), and [0311] (b) forming a second silk
film layer from the aqueous solution or dispersion comprising or
consisting of a silk material (e.g. at least one silk polypeptide),
particularly by applying the aqueous solution or dispersion
comprising or consisting of a silk material (e.g. at least one silk
polypeptide) on a support material and drying said aqueous solution
or dispersion, and [0312] (c) optionally applying water vapour to
the second silk film layer. In one embodiment, the method of
producing the active agent release sheet further comprises the
steps of: [0313] (d) providing an aqueous solution or dispersion
comprising or consisting of a silk material (e.g. at least one silk
polypeptide) and at least one active agent, or providing an aqueous
solution or dispersion comprising or consisting of a silk material
(e.g. at least one silk polypeptide), [0314] (e) forming a first
silk film layer comprising or consisting of at least one active
agent from the aqueous solution or dispersion comprising or
consisting of a silk material (e.g. at least one silk polypeptide)
and at least one active agent on the second silk film layer formed
in step (b), particularly by applying the aqueous solution or
dispersion comprising or consisting of a silk material (e.g. at
least one silk polypeptide) and at least one active agent on said
second silk film layer and drying said aqueous solution or
dispersion, or forming a further second silk film layer from the
aqueous solution or dispersion comprising or consisting of a silk
material (e.g. at least one silk polypeptide) on the second silk
film layer formed in step (b), particularly by applying the aqueous
solution or dispersion comprising or consisting of a silk material
(e.g. at least one silk polypeptide) on said second silk film layer
and drying said aqueous solution or dispersion, and [0315] (f)
optionally applying pressure and/or water vapour to the first silk
film layer or second silk film layer. Preferably, steps (d) and (e)
and optionally step (f) are repeated one or more times (e.g. 1, 2,
3 4, 5, 6, 7, 8, 9, 10, or more times) to form further first and/or
second silk film layers on the second silk film layer formed in
step (b).
[0316] In this respect, it should be noted that any variations and
arrangements of silk film layers are possible, provided that the
active agent release sheet comprises at least one first silk film
layer.
[0317] The support material may be any solid support material such
as polystyrene, glass, polytetrafluoroethylene, or silane. The
application of the aqueous solution or dispersion comprising or
consisting of a silk material (e.g. at least one silk polypeptide)
and at least one active agent or the aqueous solution or dispersion
comprising or consisting of a silk material (e.g. at least one silk
polypeptide) may take place by casting, spraying, or dropping said
aqueous solution or dispersion on the support material or on the
already existing/formed first and/or second silk film layers.
[0318] It is preferred to produce with the above described methods
an active agent release sheet which comprises or consists of (a)
first silk film layer(s) comprising at least one active agent which
is (are) flanked by second silk film layers. In other word, the
first silk film layer(s) comprising at least one active agent is
(are) sandwiched or embedded between second silk film layers.
[0319] The technique in which the at least one active agent is
directly comprised in the aqueous solution or dispersion can be
designated as "direct loading technique".
[0320] In one alternative embodiment, the active agent release
sheet is produced by the steps of: [0321] (a) providing an aqueous
solution or dispersion comprising or consisting of a silk material
(e.g. at least one silk polypeptide), [0322] (b) forming a silk
film layer from the aqueous solution or dispersion comprising or
consisting of a silk material (e.g. at least one silk polypeptide),
particularly by applying the aqueous solution or dispersion
comprising or consisting of a silk material (e.g. at least one silk
polypeptide) on a support material and drying said aqueous solution
or dispersion, and [0323] (c) loading at least one active agent
into and/or onto the silk film layer, thereby producing a first
silk film layer comprising or consisting of at least one active
agent, and [0324] (d) optionally applying water vapour to the first
silk film layer. In one embodiment, the method of producing the
active agent release sheet further comprises the steps of: [0325]
(e) providing an aqueous solution or dispersion comprising or
consisting of a silk material (e.g. at least one silk polypeptide),
[0326] (f) forming a second silk film layer from the aqueous
solution or dispersion comprising or consisting of a silk material
(e.g. at least one silk polypeptide) on the first silk film layer
produced in step (c), particularly by applying the aqueous solution
or dispersion comprising or consisting of a silk material (e.g. at
least one silk polypeptide) on said first silk film layer and
drying said aqueous solution or dispersion, and [0327] (g)
optionally applying pressure and/or water vapour to the second silk
film layer. Preferably, steps (e) and (0 and optionally step (g)
are repeated one or more times to form further second silk film
layers on the first silk film layer produced in step (c).
[0328] The loading of the at least one active agent into the silk
film layer may take place by introducing/placing the silk film
layer into an aqueous solution or dispersion comprising the at
least one active agent.
[0329] The technique in which the at least one active agent is
loaded into the (already produced/existing) silk film (layer) can
be designated as "remote loading technique".
[0330] The support material may be any solid support material such
as polystyrene, glass, polytetrafluoroethylene, or silane. The
application of the aqueous solution or dispersion comprising or
consisting of a silk material (e.g. at least one silk polypeptide)
may take place by casting, spraying, or dropping said aqueous
solution or dispersion on the support material or on the already
existing/formed first and/or second silk film layers.
[0331] It is preferred to produce with the above described method
an active agent release sheet which comprises or consists of (a)
first silk film layer(s) comprising at least one active agent which
is (are) flanked by second silk film layers. In other word, the
first silk film layer(s) comprising at least one active agent is
(are) sandwiched or embedded between second silk film layers.
[0332] Within the context of the present invention, the loading and
the loading efficiency of the silk film, particularly of the active
agent release sheet of the silk film, can be calculated on basis of
the following equations:
loading .times. ( w / w .times. % ) = amount .times. active .times.
agent .times. in .times. the .times. silk .times. film amount
.times. of .times. the .times. si1k .times. film .times. 100
##EQU00001##
For example the "loading" is calculated to be 80% with the
following data: amount of active agent non-covalently bound to the
surface of the silk film and/or incorporated into the silk film:
0.8 g, amount of active agent initially added: 1.0 g.
loading .times. ( w / w .times. % ) = 0.8 g 10 .times. g .times.
100 = 80 .times. % ##EQU00002## loading .times. efficiency .times.
( w / w .times. % ) = amount .times. of .times. active .times.
agent .times. in .times. the .times. silk .times. film active
.times. agent .times. in .times. the .times. aqueous .times.
solution / dispersion .times. 100 ##EQU00002.2##
For example, the "encapsulation efficiency" is calculated to be 66%
with the following data: amount of active agent non-covalently
bound to the surface of the silk film and/or incorporated into the
silk film: 0.1 g, amount of active compound initially added: 0.15
g.
loading .times. efficiency .times. ( w / w .times. % ) = 0.1 g 0.15
g .times. 100 = 66 .times. % ##EQU00003##
The loading and the loading efficiency of the silk film,
particularly of the active agent release sheet of the silk film,
can be determined using UV-Vis spectroscopy.
[0333] In one another alternative embodiment, the active agent
release sheet is produced by the steps of: [0334] (a) providing two
or more first silk film layers comprising or consisting of at least
one active agent and preferably one or more second silk film
layers, or providing one or more first silk film layers comprising
or consisting of at least one active agent and one or more second
silk film layers, [0335] (b) stacking up the two or more first silk
layers comprising or consisting of at least one active agent and
preferably the one or more second silk film layers, or the one or
more first silk film layer comprising or consisting of at least one
active agent and the one or more second silk film layers, and
[0336] (c) applying pressure to the stacked layers in order to
combine or connect said layers with each other.
[0337] It is preferred that the first silk film layer used above is
produced by: providing an aqueous solution or dispersion comprising
or consisting of a silk material (e.g. at least one silk
polypeptide) and at least one active agent, forming a first silk
film layer comprising or consisting of at least one active agent
from the aqueous solution or dispersion comprising or consisting of
a silk material (e.g. at least one silk polypeptide) and at least
one active agent, particularly by applying the aqueous solution or
dispersion comprising or consisting of a silk material (e.g. at
least one silk polypeptide) and at least one active agent on a
support material and drying said aqueous solution or dispersion,
and optionally applying water vapour to the first silk film
layer.
[0338] Alternatively, it is preferred that the first silk film
layer is produced by: providing an aqueous solution or dispersion
comprising or consisting of a silk material (e.g. at least one silk
polypeptide), forming a silk film layer from the aqueous solution
or dispersion comprising or consisting of a silk material (e.g. at
least one silk polypeptide), particularly by applying the aqueous
solution or dispersion comprising or consisting of a silk material
(e.g. at least one silk polypeptide) on a support material and
drying said aqueous solution or dispersion, loading at least one
active agent into and/or onto the silk film layer, thereby
producing a first silk film layer comprising or consisting of at
least one active agent, and optionally applying water vapour to the
first silk film layer.
[0339] It is preferred that the second silk film layer is produced
by:
providing an aqueous solution or dispersion comprising or
consisting of a silk material (e.g. at least one silk polypeptide),
forming a second silk film layer from the aqueous solution or
dispersion comprising or consisting of a silk material (e.g. at
least one silk polypeptide), particularly by applying the aqueous
solution or dispersion comprising or consisting of a silk material
(e.g. at least one silk polypeptide) on a support material and
drying said aqueous solution or dispersion, and optionally applying
water vapour to the second silk film layer.
[0340] The application of the aqueous solution or dispersion
comprising or consisting of a silk material (e.g. at least one silk
polypeptide) and at least one active agent or the aqueous solution
or dispersion comprising or consisting of a silk material (e.g. at
least one silk polypeptide) may take place by casting, spraying, or
dropping said aqueous solution or dispersion on a support material.
The support material may be any solid support material such as
polystyrene, glass, polytetrafluoroethylene, or silane.
[0341] It is preferred to produce with the above described method
an active agent release sheet which comprises or consists of (a)
first silk film layer(s) comprising at least one active agent which
is (are) flanked by second silk film layers. In other word, the
first silk film layer(s) comprising at least one active agent is
(are) sandwiched or embedded between second silk film layers.
[0342] The drying can be carried out, for example, by drying in the
air, baking, using a heat chamber, a vacuum chamber, laminar flow
(e.g. of a gas such as nitrogen or carbon dioxide), radiation, or a
fan (at low temperatures, at room temperature or at elevated
temperatures).
[0343] If pressure is applied to the first and/or second silk film
layer(s) or to the stacked first and/or second silk film layers, it
is preferred that the pressure corresponds to a weight force of at
least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2,
1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2 t/m.sup.2. A weight force
of at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1,
1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2 t/m.sup.2 corresponds
to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, or 20 kN/m.sup.2, respectively.
[0344] If water vapour is applied to the first and/or second silk
film layer(s) or to the silk film layer, it is further preferred
that the water vapour has a temperature of between 1.degree. C. and
121.degree. C., preferably of between 1.degree. C. and 80.degree.
C., more preferably of between 4.degree. C. and 50.degree. C., even
more preferably of between 15.degree. C. and 40.degree. C., and
most preferably of between of between 19.degree. C. or 20.degree.
C. (room temperature) and 35.degree. C., e.g. a temperature of 19,
20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or
35.degree.. It is, alternatively or additionally, also preferred
that the water vapour has a relative humidity of between 50% and
100%, preferably of between 50% and 90%, more preferably of between
60% and 90%, and most preferably of between 70% and 80%, e.g. a
relative humidity of 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or
80%.
[0345] It is preferred that the aqueous solution or dispersion
mentioned above comprises at least one plasticizer. Preferably, the
plasticizer is comprised in the aqueous solution or dispersion in a
concentration of between 0.1% (w/v) and 10% (w/v), more preferably
of between 0.5% (w/v) and 5% (w/v) or between 1% (w/v) and 5%
(w/v), even more preferably of between 1% (w/v) and 3% (w/v) or
between 1% (w/v) and 2% (w/v), and most preferably of between 3%
(w/v). Preferred plasticizers are selected from glycerol,
2-pyrrolidone, polyethylenglycol (PEG), polyvinylalcohol (PVA),
poloxamer, polyvinylpirrolidone (PVP), polyacrylic acid,
polyorthoester, gelatine, collagen, cellulose, cellulose derivates,
and sorbitol. More preferred plasticizers are selected from
glycerol and 2-pyrrolidone.
[0346] The aqueous solution or dispersion may be a buffered aqueous
solution or dispersion, preferably TRIS buffer-HCl (pH 8),
particularly 5 mM TRIS puffer-HCl (pH 8).
[0347] It is further preferred that the concentration of the at
least one silk polypeptide in the aqueous solution is of between
0.1% (w/v) and 30% (w/v), preferably of between 0.5% (w/v) and 20%
(w/v), and more preferably of between 1% (w/v) and 10% (w/v).
[0348] It is, alternatively or additionally, also preferred that
the active agent is comprised in the aqueous solution or dispersion
in a concentration of between 0.5 mg/ml and 15 mg/ml, preferably in
a concentration of between 1 mg/ml and 10 mg/ml, and more
preferably in a concentration of between 1 mg/ml and 5 mg/ml.
[0349] In this respect, the following should be noted: The
inventors of the present invention observed that the use of an
aqueous solution or dispersion having a pH that is lower than the
pI of the silk material (e.g. at least one silk polypeptide) used
for silk film formation results in a positively charged silk film.
In this respect, it is preferred that the active agent added to the
aqueous solution or dispersion is negatively charged at the pH of
the aqueous solution or dispersion in order to achieve effective
incorporation of the active agent into the silk film. Further, the
inventors of the present invention observed that the use of an
aqueous solution or dispersion having a pH that is higher than the
pI of the silk material (e.g. at least one silk polypeptide) used
for silk film formation results in a negatively charged film. In
this respect, it is preferred that the active agent added to the
aqueous solution or dispersion is positively charged at the pH of
the aqueous solution or dispersion in order to achieve effective
incorporation of the active agent into the silk film. The
incorporation of the active agent into the silk film preferably
arises from electrostatic and/or hydrophobic interactions between
the silk material (e.g. at least one silk polypeptide) and the
active agent.
[0350] For example, the spider silk polypeptide C.sub.16 has a pI
of 3.48 and the spider silk polypeptide C.sup.Kappa.sub.16 has a pI
of 9.7. Thus, the use of an aqueous solution or dispersion
comprising the spider silk polypeptide C.sub.16 and having a pH of
<3.48 results in a positively charged spider silk polypeptide
C.sub.16 film. Accordingly, the active agent added to this aqueous
solution or dispersion should be negatively charged at this pH in
order achieve effective loading of the silk film. In contrast
thereto, the use of an aqueous solution or dispersion comprising
the spider silk polypeptide C.sub.16 and having a pH of >3.48
results in a negatively charged spider silk polypeptide C.sub.16
film. Accordingly, the active agent added to this aqueous solution
or dispersion should be positively charged at this pH in order
achieve effective loading of the silk film.
[0351] In a ninth aspect, the present invention relates to the use
of the coated silk film according to the first aspect for
controlled and sustained release of at least one active agent.
Preferably, the use is a non-therapeutic use. As to the definition
of the active agent, it is referred to the first aspect of the
present invention.
[0352] The active agent may be released from the silk film,
particularly from the active agent release sheet being part of the
silk film or forming the silk film, by diffusion and/or degradation
upon exposure to a surrounding environment.
[0353] In one embodiment, the surrounding environment is a
physiological environment (e.g. a physiological medium). It is
preferred that the physiological environment (e.g. the
physiological medium) is selected from the group consisting of a
physiological buffered solution or a body fluid, e.g. blood, lymph
or liquor. It is also preferred that the physiological environment
is an organ (e.g. skin) or a part of an organ (e.g. tissue). The
release of the active agent from the coated silk film can be
induced by introducing the coated silk film into a physiological
buffered aqueous solution or a body fluid such as blood, lymph or
liquor, or by applying the coated silk film onto an organ (e.g.
skin) or part of an organ (e.g. tissue).
[0354] In one another embodiment, the surrounding environment is a
non-physiological environment (e.g. a non-physiological medium). It
is preferred that the non-physiological environment (e.g. the
non-physiological medium) is selected from the group consisting of
an aqueous solution such as water or a buffered aqueous solution,
an alcoholic solution, and an organic solution.
[0355] It should be further noted that the active agent(s) may be
released by diffusion and/or degradation upon exposure to a
surrounding intracorporeal or extracorporeal environment.
[0356] Preferably, the release of the active agent displays
relatively linear kinetics, thereby providing a constant supply of
the active agent over the release period.
[0357] It is preferred that less than 20%, preferably less than
15%, more preferably less than 10%, and most preferably less than
5%, e.g. less than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9,
8, 7, 6, or 5%, of the active agent is released, particularly into
the surrounding environment, within the first 24 hours.
[0358] It is, alternatively or additionally, preferred that more
than 50%, preferably more than 70%, more preferably more than 80%,
and most preferably 100% of the active agent is released,
particularly into the surrounding environment, within 36 hours. It
is, alternatively or additionally, preferred that more than 50%,
preferably more than 70%, more preferably more than 80%, and most
preferably 100% of the active agent is released, particularly into
the surrounding environment, within 48 hours. It is, alternatively
or additionally, preferred that more than 50%, preferably more than
70%, more preferably more than 80%, and most preferably 100% of the
active agent is released, particularly into the surrounding
environment, within 72 hours.
[0359] It is, alternatively or additionally, more preferred that
more than 50%, preferably more than 70%, more preferably more than
80%, and most preferably 100% of the active agent is released,
particularly into the surrounding environment, within 7 days. It
is, alternatively or additionally, more preferred that more than
50%, preferably more than 70%, more preferably more than 80%, and
most preferably 100% of the active agent is released, particularly
into the surrounding environment, within 14 days. It is,
alternatively or additionally, more preferred that more than 50%,
preferably more than 70%, more preferably more than 80%, and most
preferably 100% of the active agent is released, particularly into
the surrounding environment, within 20 days. It is, alternatively
or additionally, more preferred that more than 50%, preferably more
than 70%, more preferably more than 80%, and most preferably 100%
of the active agent is released, particularly into the surrounding
environment, within 30 days. It is, alternatively or additionally,
more preferred that more than 50%, preferably more than 70%, more
preferably more than 80%, and most preferably 100% of the active
agent is released, particularly into the surrounding environment,
within 35 days.
[0360] It is, alternatively or additionally, even more preferred
that more than 50%, preferably more than 70%, more preferably more
than 80%, and most preferably 100% of the active agent is released,
particularly into the surrounding environment, within 5 weeks. It
is, alternatively or additionally, even more preferred that more
than 50%, preferably more than 70%, more preferably more than 80%,
and most preferably 100% of the active agent is released,
particularly into the surrounding environment, within 6 weeks. It
is, alternatively or additionally, even more preferred that more
than 50%, preferably more than 70%, more preferably more than 80%,
and most preferably 100% of the active agent is released,
particularly into the surrounding environment, within 7 weeks. It
is, alternatively or additionally, even more preferred that more
than 50%, preferably more than 70%, more preferably more than 80%,
and most preferably 100% of the active agent is released,
particularly into the surrounding environment, within 8 weeks.
[0361] It is, alternatively or additionally, most preferred that
more than 50%, preferably more than 70%, more preferably more than
80%, and most preferably 100% of the active agent is released,
particularly into the surrounding environment, within 3 months. It
is, alternatively or additionally, most preferred that more than
50%, preferably more than 70%, more preferably more than 80%, and
most preferably 100% of the active agent is released, particularly
into the surrounding environment, within 4 months. It is,
alternatively or additionally, most preferred that more than 50%,
preferably more than 70%, more preferably more than 80%, and most
preferably 100% of the active agent is released, particularly into
the surrounding environment, within 5 months. It is, alternatively
or additionally, most preferred that more than 50%, preferably more
than 70%, more preferably more than 80%, and most preferably 100%
of the active agent is released, particularly into the surrounding
environment, within 6 months. It is, alternatively or additionally,
most preferred that more than 50%, preferably more than 70%, more
preferably more than 80%, and most preferably 100% of the active
agent is released, particularly into the surrounding environment,
within 7 months. It is, alternatively or additionally, most
preferred that more than 50%, preferably more than 70%, more
preferably more than 80%, and most preferably 100% of the active
agent is released, particularly into the surrounding environment,
within 8 months. It is, alternatively or additionally, most
preferred that more than 50%, preferably more than 70%, more
preferably more than 80%, and most preferably 100% of the active
agent is released, particularly into the surrounding environment,
within 9 months. It is, alternatively or additionally, most
preferred that more than 50%, preferably more than 70%, more
preferably more than 80%, and most preferably 100% of the active
agent is released, particularly into the surrounding environment,
within 10 months.
[0362] In a tenth aspect, the present invention relates to a method
of producing a water insoluble silk article comprising the steps
of: [0363] (i) providing a silk article, and [0364] (ii) applying
water vapour to the silk article, thereby rendering the silk
article water insoluble.
[0365] Silk articles, e.g. silk films or fibers, produced, e.g.
cast or spun, from an aqueous solution or a dispersion display a
predominantly .alpha.-helical secondary structure. The inventors of
the present invention found that the application of water vapour
results in a transition to a .beta.-sheet rich structure. While the
silk articles, e.g. silk films or fibers, provided in step (i) can
be dissolved in water, processed .beta.-sheet rich silk articles,
e.g. silk films or fibers, are water-insoluble.
[0366] For example, water-insoluble silk films are known in the
art. However, these silk films have been rendered water-insoluble
by treating them with methanol or ethanol, or by casting them from
formic acid. Although methanol, ethanol, or formic acid is
evaporated after silk film treatment/formation, residues of these
substances might remain in the silk film. Thus, such
water-insoluble silk films might have harmful side effects when
administered to an individual. The silk articles, e.g. silk films
or fibers, produced with the above method have the advantage that
they have been rendered water-insoluble by simply applying water
vapour. They do not comprise harmful compounds. Thus, they can be
basis for a large number of innovative products in the cosmetical,
medical, or pharmaceutical market. It is further possible to apply
the water-insoluble silk articles, e.g. silk films or fibers, to
several preferential objects including, but not limited to, meshes,
scaffolds, patches, and nonwovens.
[0367] In one embodiment, the water vapour has a temperature of
between 1.degree. C. and 121.degree. C., preferably of between
1.degree. C. and 80.degree. C., more preferably of between
4.degree. C. and 50.degree. C., even more preferably of between
15.degree. C. and 40.degree. C., and most preferably of between of
between 19.degree. C. or 20.degree. C. (room temperature) and
35.degree. C., e.g. a temperature of 19, 20, 21, 22, 23, 24, 25,
26, 27, 28, 29, 30, 31, 32, 33, 34, or 35.degree. C. and/or the
water vapour has a relative humidity of between 50% and 100%,
preferably of between 50% and 90%, more preferably of between 60%
and 90%, and most preferably of between 70% and 80%, e.g. a
relative humidity of 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or
80%.
[0368] In one another embodiment, the silk article comprises at
least one active agent. Preferably, the active agent is selected
from the group consisting of a biological agent, a pharmaceutical
agent, a cosmetic agent, a nutrient, and dietary supplement. As to
the definition of the term "active agent" and as to the preferred
embodiments of the biological agent, pharmaceutical agent, cosmetic
agent, nutrient, or dietary supplement, it is referred to the first
aspect of the present invention.
[0369] The silk article comprises a silk material. Preferably, the
silk article comprises at least one silk polypeptide. Said silk
polypeptide comprises at least two repetitive units. As to the
definitions with respect to the silk polypeptide and as to the
preferred embodiments of the silk polypeptide, it is referred to
the first aspect of the present invention.
[0370] Preferably, the silk article is selected from the group
consisting of a silk film, a silk particle, a silk fiber, a silk
foam, a silk mesh, and a silk coating. It is preferred that the
silk article, e.g. silk film or fiber, provided in step (i) is
produced by the steps of: [0371] (a) providing an aqueous solution
or dispersion comprising or consisting of a silk material (e.g. at
least one silk polypeptide) and optionally at least one active
agent, [0372] (b) forming a silk article, e.g. silk film or fiber,
from the aqueous solution or dispersion comprising or consisting of
a silk material (e.g. at least one silk polypeptide) and optionally
at least one active agent.
[0373] In one embodiment, the silk article is a silk film and said
silk film is formed in step (b) by applying the aqueous solution or
dispersion comprising or consisting of a silk material (e.g. at
least one silk polypeptide) and optionally at least one active
agent on a support material and drying said aqueous solution or
dispersion.
[0374] The application of the aqueous solution or dispersion
comprising or consisting of a silk material (e.g. at least one silk
polypeptide) and optionally at least one active agent may take
place by casting, spraying, or dropping said aqueous solution or
dispersion on a support material. The support material may be any
solid support material such as polystyrene, glass,
polytetrafluoroethylene, or silane.
[0375] The drying can be carried out, for example, by drying in the
air, baking, using a heat chamber, a vacuum chamber, laminar flow
(e.g. of a gas such as nitrogen or carbon dioxide), radiation, or a
fan (at low temperatures, at room temperature or at elevated
temperatures).
[0376] In one another embodiment, the silk article is a silk fiber
and said silk fiber is formed in step (b) by spinning the silk
fiber from the aqueous solution or dispersion comprising or
consisting of a silk material (e.g. at least one silk polypeptide)
and optionally at least one active agent. The spinning technique
preferably is selected from the group consisting of wet spinning
and electrospinning.
[0377] It is further preferred that the concentration of the at
least one silk polypeptide in the aqueous solution is of between
0.1% (w/v) and 30% (w/v), preferably of between 0.5% (w/v) and 20%
(w/v), and more preferably of between 1% (w/v) and 10% (w/v).
[0378] The aqueous solution or dispersion may be a buffered aqueous
solution or dispersion, preferably TRIS buffer-HCl (pH 8),
particularly 5 mM TRIS puffer-HCl (pH 8).
[0379] Various modifications and variations of the invention will
be apparent to those skilled in the art without departing from the
scope of invention. Although the invention has been described in
connection with specific preferred embodiments, it should be
understood that the invention as claimed should not be unduly
limited to such specific embodiments. Indeed, various modifications
of the described modes for carrying out the invention which are
obvious to those skilled in the art in the relevant fields are
intended to be covered by the present invention.
[0380] The following examples are merely illustrative of the
present invention and should not be construed to limit the scope of
the invention as indicated by the appended claims in any way.
EXAMPLES
Example 1: Production of Silk Films
[0381] (A) Spray-dried C.sub.16 proteins were dissolved in 6 M
guanidinium thiocyanate and subsequently dialyzed against 5 mM
Tris/HCl buffer, pH 8 at 4.degree. C. The pH was measured using a
pH meter. The protein solution was centrifuged for 15 min at 10000
rpm and filtered through a 0.45 .mu.m cellulose acetate filter. The
protein concentration was determined photometrically. The protein
solution was finally diluted with Tris buffer to 2.5% w/v. Films
(2.5.times.3 cm) were cast on the plastic foil A5 22/5B from
mtv-messtechnik (Koeln, Germany) and dried in a laminar flow
cabinet at room temperature overnight at 45% relative humidity (see
FIG. 1A).
[0382] (B) Spray-dried C.sub.16 proteins were dissolved in 6 M
guanidinium thiocyanate and subsequently dialyzed against 5 mM
Tris/HCl buffer, pH 9 at 4.degree. C. The pH was settled using a pH
meter. The protein solution was firstly centrifuged at 4.degree. C.
for 15 min at 10000 rpm and secondly filtered through a 0.45 .mu.m
cellulose acetate filter. Afterwards, the protein solution was
dialyzed against 5 mM Tris/HCl buffer, pH 9 containing 10% of PEG
20 kDa at 4.degree. C. The protein concentration was determined
photometrically. The protein concentration of the casting solution
was finally adjusted to 5% w/v. Films were cast on the plastic foil
A5 22/5B from mtv-messtechnik (Koeln, Germany) at room temperature
using the film applicator Coatmaster 510 (Erichsen, Hemer, Germany)
having a casting knife of 2000 .mu.m and with a velocity of 1
mm/sec. 2 ml of the casting solution were used to cast each film.
During the whole film casting procedure, the film applicator was
placed under the laminar flow cabinet. The films were dried at room
temperature overnight in the laminar flow cabinet which was closed
and switched off. The final films were obtained cutting samples of
ca. 2.5.times.3 cm using a scalpel (see FIG. 1B).
Example 2: Remote Loading of Silk Films
[0383] A) Low molecular weight drugs (tetracain hydrochloride,
ethacridine monohydrate lactate, nipagin and phenol red) were
dissolved at a concentration of 0.5 mg/ml in highly purified water
(HPW), Tris 5 mM pH 7, and formate buffer 5 mM, pH 3, respectively.
After dissolving the drugs, all solutions were filtered through a
0.45 .mu.m cellulose acetate filter.
[0384] For the remote loading process, the previously cast C.sub.16
films were cut into pieces, weighed and placed into 24-well plates.
At least four C.sub.16 film samples were incubated with 1 ml of
each drug solutions for 30 min at gently agitation of 2 rpm at room
temperature. After removing the C.sub.16 films, the quantities of
the loaded drugs were calculated indirectly by measuring the
remaining loading solutions photometrically. Finally, the loading
and the loading-efficiency for each experimental condition were
calculated (see Table 1).
[0385] For the remote loading of the model protein lysozyme, the
loading solution was prepared by dissolving 1% w/v lysozyme in HPW.
As before, three different C.sub.16 films were weighed. Afterwards,
C.sub.16 films were incubated in a 6-well plate with 5 ml of the
loading solution at gently agitation of 2 rpm at room temperature.
After 30 min, C.sub.16 films were removed and the loading solution
was analysed photometrically at 280 nm. Spectra were recorded using
a spectrophotometer. At physiological pH, spider silk films are
negatively charged.
[0386] As mentioned above, the remote loading of the model protein
Lysozyme was also considered. Lysozyme is positively charged at
physiological pH. The remote loading of C.sub.16 films with
lysozyme performed in HPW demonstrated that it is possible to load
high molecular weight molecules by remote loading (see Table
1).
TABLE-US-00001 TABLE 1 Remote loading of different low molecular
weight (LMW) drugs and the model protein lysozyme. Loading
efficiency (LE) and Loading (L) were determinate at different pH
and different ionic strength. HPW stand for highly purified water.
pH 7 pH 3 HPW LE (%) L (%) LE (%) L (%) LE (%) L (%) LMW drugs
Tetracaine HCl 24.3 .+-. 7.7 2.5 .+-. 0.7 0.0 .+-. 0.0 0.0 .+-. 0.0
35.1 .+-. 8.3 3.8 .+-. 1.9 Ethacridine lact. 45.8 .+-. 2.7 3.5 .+-.
1.0 16.5 .+-. 5.4 2.4 .+-. 0.5 54.2 .+-. 3.2 3.3 .+-. 0.3 Nipagin
0.0 .+-. 0.0 0.0 .+-. 0.0 5.3 .+-. 2.3 0.5 .+-. 0.2 9.6 .+-. 4.3
0.8 .+-. 0.3 Phenol red 7.2 .+-. 2.0 0.7 .+-. 0.2 17.2 .+-. 5.0 1.8
.+-. 0.7 2.8 .+-. 1.3 0.3 .+-. 1.3 Model Protein Lysozyme -- -- --
-- 22.8 .+-. 0.7 39.8 .+-. 1.8
[0387] B) For the remote loading of Lysozyme, the loading solution
was prepared dissolving 1% w/v lysozyme in high purified water
(HPW). Three different C.sub.16 films were weighted, followed by
incubation in a 6-well plate with 5 ml of the loading solution at
gently agitation of 2 rpm at room temperature. After 30 min,
C.sub.16 films were removed and the loading solution was diluted
1:20 and analyzed photometrically at 280 nm. In particular, a
spectra of this solution was subsequently recorded using a
spectrophotometer (Agilent Technologies 8453, Oberhaching,
Germany). A calibration curve was prepared using the original
lysozyme solution. The same method was performed using an
incubation medium with 0.5% and 0.25% of Lysozyme. The results are
shown in FIG. 2.
Example 3: Cumulative Release of Paracetamol, FITC-BSA and
FITC-Dextran (FD) from C.sub.16 Films Cast from an Aqueous
Solution
[0388] A) Spray-dried C.sub.16 proteins were dissolved in 6 M
guanidinium thiocyanate and subsequently dialyzed against 5 mM
Tris/HCl buffer, pH 8 at 4.degree. C. The pH was measured using a
pH meter. The protein solution was centrifuged at 10000 rpm for 15
min and filtered through a 0.45 .mu.m cellulose acetate filter. The
protein concentration was determined photometrically. The protein
solution was finally diluted with Tris buffer to 2.5% w/v. Before
casting the films, paracetamol was added and dissolved directly in
the protein solution. Films (2.5.times.3 cm) were cast on the
plastic foil A5 22/5B from mtv-messtechnik (Koeln, Germany) and
dried in a laminar flow cabinet at room temperature overnight at
45% relative humidity. The resulting films contained 2.5 mg of
drug. Three film samples containing paracetamol were incubated in
0.01 M PBS buffer, pH 7.4 at 37.degree. C. At predetermined time
points, the buffer was removed and replaced with fresh medium. The
release buffer was analysed using a UV-Vis spectrophotometer.
[0389] A 20 mg/ml FITC-BSA or FITC-dextran (FD) solution in 0.01 M
PBS buffer, pH 7.4 was directly mixed with the C.sub.16 protein
solution. Films were cast as described above. Every film contained
25 mg of C.sub.16 and 1.3 mg of FITC-BSA or FD, respectively. Films
containing the substances were incubated in 0.01 M PBS buffer, pH
7.4 at 37.degree. C. At predetermined time points, the buffer was
removed and replaced with fresh medium. The release buffer was
analysed using a fluorescence spectrophotometer applying an
excitation wavelength of 490 nm and an emission of 520 nm. The
substance which was not released was quantified by dissolving the
film matrix in a 6M GdmSCN solution. This solution was subsequently
analysed using a fluorescence spectrophotometer. The sum of the
non-released substance and the released amount of substance was
calculated as 100%. The results showed that the release depends on
the molecular weight of the substances. Spider silk films showed to
be an excellent drug carrier for small molecules, capable to
completely release paracetamol and FITC-dextran (FD) in a short
time period. The release of FITC-BSA, which is six times larger
than the used dextran in terms of molecular weight, was observed to
be slower. After direct loading with FITC-BSA, C.sub.16 films
released almost 65% of the model protein within one week.
Interesting, after a burst release of approx. 30% on day 1, spider
silk films released the protein BSA in a constant amount per day.
The cumulative release of paracetamol, FITC-dextran 21 kDa (FD) and
FITC-BSA from C.sub.16 films casted from an aqueous solution is
shown in FIG. 6.
[0390] B) Spider silk films comprising C.sub.16 and containing
paracetamol as active agent were prepared as described in Example
3A. The resulting spider silk films contained 2.5 mg of dissolved
paracetamol and optically appeared like unloaded films showing a
smooth, transparent, and colorless surface (see FIG. 3).
Example 4: Cumulative Release of FITC-BSA from Different Types of
Spider Silk Films
[0391] Spray-dried C.sub.16 proteins were dissolved in 6 M
guanidinium thiocyanate and subsequently dialyzed against 5 mM
Tris/HCl buffer, pH 8 at 4.degree. C. The pH was measured using a
pH meter. The protein solution was centrifuged at 10000 rpm for 15
min and filtered through a 0.45 .mu.m cellulose acetate filter. The
protein concentration was determined photometrically. The protein
solution was finally diluted with Tris buffer to 2.5% w/v.
[0392] Monolayer films were prepared by mixing a 20 mg/mL FITC-BSA
solution in 0.01 M PBS buffer, pH 7.4 with the aqueous C.sub.16
solution. Films (2.5.times.3 cm) were cast on the plastic foil A5
22/5B from mtv-messtechnik (Koeln, Germany) and dried in a laminar
flow cabinet at room temperature overnight at 45% relative
humidity. After casting, each film contained 1.3 mg of the model
drug.
[0393] 3-Layer films were prepared as follows: In a first step, a
C.sub.16 film layer containing FITC-BSA and glycerol was prepared
by adding 1% w/v of glycerol directly to the casting solution
comprising C.sub.16 and by subsequently adding a 20 mg/ml FITC-BSA
solution in 0.01 M PBS buffer, pH 7.4 to said casting solution. The
film layer was cast as described above. The film layer contained
1.3 mg of FITC-BSA. In a second step, the C.sub.16 film layer
containing FITC-BSA and the plasticizer glycerol was positioned
between two C.sub.16 film layers. Said three film layers were
pressed together applying 2000 N for 5 min. During this process,
3-Layer films were created where only the middle layer contained
the model drug FITC-BSA.
[0394] In one case, sandwich films were additionally coated by
dipping them in a 5% w/v C.sub.16 solution (Coated 3-Layer films,
sealed films). They were dried overnight at room temperature (RT).
The 5% w/v C.sub.16 solution was prepared by dialysis of the
protein solution obtained by the method described above (Example
1).
[0395] 7-Layer films were prepared pressing seven film layers
together. 2000 N for 5 min were applied. In particular, one
C.sub.16 film layer comprising FITC-BSA and glycerol and six
C.sub.16 film layers, wherein glycerol containing C.sub.16 film
layers and glycerol free C.sub.16 film layers were arranged in
alteration, where pressed together.
[0396] Three samples of all these different kind of films were
incubated in 0.01 M PBS buffer, pH 7.4 at 37.degree. C. At
predetermined time points, the buffer was removed and replaced with
fresh medium. The release buffer was analysed using a fluorescence
spectrophotometer applying an excitation wavelength of 490 nm and
an emission of 520 nm. The drug which was not released was
quantified by dissolving the film matrix in a 6 M GdmSCN solution.
This solution was subsequently analysed using a fluorescence
spectrophotometer. The sum of the non-released substance and the
released amount of substance was calculated as 100%. The results
are shown in FIG. 9.
Example 5: Cumulative Release of FITC-BSA from Different Kinds of
Coated Spider Silk Films (n=3)
[0397] Spray-dried C.sub.16 proteins were dissolved in 6 M
guanidinium thiocyanate and subsequently dialyzed against 5 mM
Tris/HCl buffer, pH 8 at 4.degree. C. The pH was measured using a
pH meter. The protein solution was centrifuged at 10000 rpm for 15
min and filtered through a 0.45 .mu.m cellulose acetate filter. The
protein concentration was determined photometrically. The protein
solution was finally diluted with Tris buffer to 2.5% w/v.
[0398] Monolayer films were prepared by mixing a 20 mg/ml FITC-BSA
solution in 0.01 M PBS buffer, pH 7.4 with the aqueous C.sub.16
solution. Films (2.5.times.3 cm) were cast on the plastic foil A5
22/5B from mtv-messtechnik (Koeln, Germany) and dried in a laminar
flow cabinet at room temperature overnight at 45% relative
humidity. After cast, each film contained 1.3 mg of FITC-BSA.
[0399] Sandwich films were prepared as following: In a first step,
a C.sub.16 film layer containing FITC-BSA and glycerol was prepared
by adding 1% w/v of glycerol directly to the casting solution
comprising C.sub.16 and by subsequently adding a 20 mg/mL FITC-BSA
solution in 0.01 M PBS buffer, pH 7.4 to said casting solution. The
film layer was cast as described above. The film layer contained
1.3 mg of FITC-BSA. Typically, one C.sub.16 film layer containing
FITC-BSA and the plasticizer glycerol was positioned between two
C.sub.16 film layers. Said three film layers were pressed together
by applying 2000 N for 5 min. During this process three-layer films
were created, where only the middle layer contained the model drug
FITC-BSA.
[0400] Finally, monolayer films containing the model drug FITC-BSA
and the sandwiches were coated by dipping them in a 5% w/v C.sub.16
solution. They were dried overnight at room temperature (RT). The
5% w/v C.sub.16 solution was prepared by a second dialysis of the
protein solution obtained by the method described above (Example
1).
[0401] The release of FITC-BSA from these two different systems was
subsequently investigated. Three samples were incubated in 0.01 M
PBS buffer, pH 7.4 at 37.degree. C. At predetermined time points
the buffer was removed and replaced with fresh medium. The release
buffer was analyzed using a fluorescence spectrophotometer applying
an excitation wavelength of 490 nm and an emission of 520 nm. The
drug which was not released was quantified by dissolving the film
matrix in a 6 M GdmSCN solution. This solution was subsequently
analyzed using a fluorescence spectrophotometer. The sum of the
non-released substance and the released amount of substance was
calculated as 100%.
[0402] By coated C.sub.16 films, a release of the model drug
FITC-BSA within 40 days with a zero kinetic order was achieved. The
cumulative release of the model drug FITC-BSA from a coated
monolayer and a coated sandwich is shown in FIG. 10.
Example 6: Cumulative Release of FITC-BSA from Different C.sub.16
Films
[0403] In another experiment, the cumulative release of the model
drug FITC-BSA from different C.sub.16 films (n=3) has been
evaluated. 1% w/v of glycerol and a 20 mg/mL FITC-BSA solution in
0.01 M PBS buffer pH 7.4 were directly mixed with the C.sub.16
protein solution. Each film contained 1.3 mg of FITC-BSA (monolayer
with plasticizer glycerol). Typically, one C.sub.16 film layer
containing the model drug FITC-BSA and the plasticizer glycerol was
positioned between two C.sub.16 film layers. Using the hydraulic
press 5T (Maassen GmbH, Reutlingen, Germany), the film layers were
pressed together by applying 0.2 t for 5 min (3Layers 0.2t), 0.4 t
for 5 min (3Layers 0.4t) and 1 t for 5 min (3Layers 1t). During
this process, three-layer films were created, whereby only the
middle layer contained the model drug FITC-BSA. In one case, one
C.sub.16 film layer containing the model drug FITC-BSA and the
plasticizer glycerol was positioned between two C.sub.16 film
layers containing the plasticizer glycerol. The film layers were
pressed together using a hydraulic press by applying 1 t for 5 min
(3 Glycerol-layers 1t). Three samples of all these different kind
of films were incubated in 0.01 M PBS buffer pH 7.4 at 37.degree.
C. At predetermined time points the buffer was removed and replaced
with fresh medium. The release buffer was analyzed using the
fluorescence spectrophotometer Cary Eclipse Varian (Agilent
technology, Boblingen, Germany) applying an excitation wavelength
of 490 nm and an emission of 520 nm. The drug which was not
released, was quantified by dissolving the film matrix in a 6 M
GdmSCN solution. This solution was subsequently analyzed using a
fluorescence spectrophotometer. FIG. 7 illustrates the cumulative
release of the model drug FITC-BSA from different C.sub.16
films.
Example 7: Effect of Glycerol Concentration on the Release of
FITC-BSA from C.sub.16 Films
[0404] In another experiment, the effect of glycerol concentration
on the release of FITC-BSA from C.sub.16 films is shown. In one
case, 1% w/v of the plasticizer glycerol and a 20 mg/mL FITC-BSA
solution in 0.01 M PBS buffer pH 7.4 were directly mixed with the
C.sub.16 protein solution (monolayer with glycerol). In one another
case, the plasticizer glycerol was not included in the casting
solution (monolayer). After casting, each film contained 1.3 mg of
FITC-BSA. Three samples of these two different variants of films
were incubated in 0.01 M PBS buffer pH 7.4 at 37.degree. C. At
predetermined time points, the buffer was removed and replaced with
fresh medium. The release buffer was analyzed using the
fluorescence spectrophotometer Cary Eclipse Varian (Agilent
technology, Boblingen, Germany) applying an excitation wavelength
of 490 nm and an emission of 520 nm. The drug which was not
released, was quantified by dissolving the film matrix in a 6 M
GdmSCN solution. This solution was subsequently analyzed using a
fluorescence spectrophotometer. The monolayer containing glycerol
released the model drug BSA much quicker than the monolayer without
glycerol. The results of this experiment are illustrated in FIG.
8A.
[0405] Films were further cast from a casting solution containing
the model drug FITC-BSA and different concentrations of the
plasticizer glycerol: 1%, 3% and 5% w/v. The release study was
carried out as described above (see also FIG. 8A). A film
containing more glycerol released the model drug BSA quicker than a
film containing low glycerol. In particular, the film containing
the model drug FITC-BSA and different concentrations of the
plasticizer glycerol, namely 1%, 3% and 5% w/v, was part of a
3-layer C.sub.16 film (n=3). Said film was positioned between two
C.sub.16 film layers. Thus, said film formed the middle layer of a
3-layer C.sub.16 film. The middle layer was cast from a casting
solution containing the C.sub.16 protein, the model drug FITC-BSA
and different concentrations of the plasticizer glycerol: 1%, 3%
and 5% w/v. Glycerol and a 20 mg/mL FITC-BSA solution in 0.01 M PBS
buffer pH 7.4 were directly mixed with the C.sub.16 protein
solution. Each film contained 1.3 mg of FITC-BSA and the defined
concentration of glycerol (1%, 3% or 5% w/v). The other two
C.sub.16 film layers were cast from a casting solution containing
the C.sub.16 protein. As mentioned above, one C.sub.16 film layer
containing the model drug FITC-BSA and the plasticizer was
positioned between two C.sub.16 film layers. Using the hydraulic
press 5T (Maassen GmbH, Reutlingen, Germany), the film layers were
pressed together by applying 0.2 t for 5 min. During this process,
three-layer films were created, whereby only the middle layer
contained the model drug FITC-BSA and the plasticizer. The results
of this experiment are illustrated in FIG. 8B.
Example 8: Remote and Direct Loading
[0406] Using the remote loading method, eADF4 (C.sub.16) films were
incubated in a drug solution. The loading of the drug into the
C.sub.16 films was mainly driven by electrostatic and
hydrophilic/hydrophobic interactions. An alternative way of loading
drugs is represented by the direct loading method. Using the direct
loading method, the drug was directly dissolved in the casting
spider silk solution or, alternatively, the spider silk solution
was mixed with a second solution containing the dissolved drug.
Subsequently, the films were cast. The advantage of the direct
loading method is the one-step process and the known amount of the
drug directly incorporated, which leads to the theoretical 100% of
drug loading. FIG. 11 illustrates the remote and direct loading
method.
Example 9: Cumulative Release of FITC-BSA from Monolayer Silk Films
and Coated Monolayer Silk Films
[0407] A further experiment has been performed where FITC-BSA was
released from a spider silk monolayer film comprising C.sub.16, the
model drug FITC-BSA and 1% w/v glycerol (black, n=3) and FITC-BSA
was released from a coated spider silk monolayer film comprising
C.sub.16, the model drug FITC-BSA and 1% w/v glycerol (grey, n=3).
The spider silk monolayer films were cast at room temperature from
an aqueous solution comprising C.sub.16, the model drug FITC-BSA
and 1% w/v glycerol on a plastic foil A5 22/5B from mtv-messtechnik
(Koeln, Germany) by the solvent evaporation technique. The coated
spider silk monolayer films containing C.sub.16, the model drug
FITC-BSA and 1% w/v glycerol were further prepared by dipping them
into a 5% w/v C.sub.16 protein solution. They were dried overnight
at room temperature (RT) in the dark. Coating of monolayers
containing glycerol with a highly concentrated spider silk protein
solution leads to the reduction of the release of FITC-BSA compared
to uncoated monolayers containing glycerol. The cumulative release
of the model drug FITC-BSA from a monolayer with glycerol and a
coated monolayer with glycerol is shown in FIG. 13.
Sequence CWU 1
1
431636PRTAraneus diadematusPEPTIDE(1)..(636)ADF-3 1Ala Arg Ala Gly
Ser Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly1 5 10 15Gln Gln Gly
Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala 20 25 30Ala Ala
Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ser Gly Gln Gln Gly 35 40 45Pro
Ser Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro 50 55
60Tyr Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly65
70 75 80Pro Gly Ser Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly
Pro 85 90 95Gly Ser Ser Ala Ala Ala Ala Ala Ala Gly Gly Asn Gly Pro
Gly Ser 100 105 110Gly Gln Gln Gly Ala Gly Gln Gln Gly Pro Gly Gln
Gln Gly Pro Gly 115 120 125Ala Ser Ala Ala Ala Ala Ala Ala Gly Gly
Tyr Gly Pro Gly Ser Gly 130 135 140Gln Gln Gly Pro Gly Gln Gln Gly
Pro Gly Gly Gln Gly Pro Tyr Gly145 150 155 160Pro Gly Ala Ser Ala
Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly 165 170 175Ser Gly Gln
Gly Pro Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr 180 185 190Gly
Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro 195 200
205Gly Ser Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly
210 215 220Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala
Ala Ala225 230 235 240Ala Ala Gly Gly Tyr Gly Pro Gly Tyr Gly Gln
Gln Gly Pro Gly Gln 245 250 255Gln Gly Pro Gly Gly Gln Gly Pro Tyr
Gly Pro Gly Ala Ser Ala Ala 260 265 270Ser Ala Ala Ser Gly Gly Tyr
Gly Pro Gly Ser Gly Gln Gln Gly Pro 275 280 285Gly Gln Gln Gly Pro
Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ser 290 295 300Ala Ala Ala
Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ser Gly Gln Gln305 310 315
320Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly
325 330 335Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala
Ala Ala 340 345 350Ala Ala Gly Gly Tyr Gly Pro Gly Ser Gly Gln Gln
Gly Pro Gly Gln 355 360 365Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln
Gln Gly Pro Gly Gln Gln 370 375 380Gly Pro Gly Gln Gln Gly Pro Gly
Gln Gln Gly Pro Gly Gln Gln Gly385 390 395 400Pro Gly Gln Gln Gly
Pro Gly Gly Gln Gly Ala Tyr Gly Pro Gly Ala 405 410 415Ser Ala Ala
Ala Gly Ala Ala Gly Gly Tyr Gly Pro Gly Ser Gly Gln 420 425 430Gln
Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln 435 440
445Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly
450 455 460Pro Gly Gln Gln Gly Pro Tyr Gly Pro Gly Ala Ser Ala Ala
Ala Ala465 470 475 480Ala Ala Gly Gly Tyr Gly Pro Gly Ser Gly Gln
Gln Gly Pro Gly Gln 485 490 495Gln Gly Pro Gly Gln Gln Gly Pro Gly
Gly Gln Gly Pro Tyr Gly Pro 500 505 510Gly Ala Ala Ser Ala Ala Val
Ser Val Gly Gly Tyr Gly Pro Gln Ser 515 520 525Ser Ser Val Pro Val
Ala Ser Ala Val Ala Ser Arg Leu Ser Ser Pro 530 535 540Ala Ala Ser
Ser Arg Val Ser Ser Ala Val Ser Ser Leu Val Ser Ser545 550 555
560Gly Pro Thr Lys His Ala Ala Leu Ser Asn Thr Ile Ser Ser Val Val
565 570 575Ser Gln Val Ser Ala Ser Asn Pro Gly Leu Ser Gly Cys Asp
Val Leu 580 585 590Val Gln Ala Leu Leu Glu Val Val Ser Ala Leu Val
Ser Ile Leu Gly 595 600 605Ser Ser Ser Ile Gly Gln Ile Asn Tyr Gly
Ala Ser Ala Gln Tyr Thr 610 615 620Gln Met Val Gly Gln Ser Val Ala
Gln Ala Leu Ala625 630 6352410PRTAraneus
diadematusPEPTIDE(1)..(410)ADF-4 2Ala Gly Ser Ser Ala Ala Ala Ala
Ala Ala Ala Ser Gly Ser Gly Gly1 5 10 15Tyr Gly Pro Glu Asn Gln Gly
Pro Ser Gly Pro Val Ala Tyr Gly Pro 20 25 30Gly Gly Pro Val Ser Ser
Ala Ala Ala Ala Ala Ala Ala Gly Ser Gly 35 40 45Pro Gly Gly Tyr Gly
Pro Glu Asn Gln Gly Pro Ser Gly Pro Gly Gly 50 55 60Tyr Gly Pro Gly
Gly Ser Gly Ser Ser Ala Ala Ala Ala Ala Ala Ala65 70 75 80Ala Ser
Gly Pro Gly Gly Tyr Gly Pro Gly Ser Gln Gly Pro Ser Gly 85 90 95Pro
Gly Gly Ser Gly Gly Tyr Gly Pro Gly Ser Gln Gly Ala Ser Gly 100 105
110Pro Gly Gly Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala Ala Ala Ala
115 120 125Ala Ser Gly Pro Gly Gly Tyr Gly Pro Gly Ser Gln Gly Pro
Ser Gly 130 135 140Pro Gly Ala Tyr Gly Pro Gly Gly Pro Gly Ser Ser
Ala Ala Ala Ala145 150 155 160Ala Ala Ala Ala Ser Gly Pro Gly Gly
Tyr Gly Pro Gly Ser Gln Gly 165 170 175Pro Ser Gly Pro Gly Val Tyr
Gly Pro Gly Gly Pro Gly Ser Ser Ala 180 185 190Ala Ala Ala Ala Ala
Ala Gly Ser Gly Pro Gly Gly Tyr Gly Pro Glu 195 200 205Asn Gln Gly
Pro Ser Gly Pro Gly Gly Tyr Gly Pro Gly Gly Ser Gly 210 215 220Ser
Ser Ala Ala Ala Ala Ala Ala Ala Ala Ser Gly Pro Gly Gly Tyr225 230
235 240Gly Pro Gly Ser Gln Gly Pro Ser Gly Pro Gly Gly Ser Gly Gly
Tyr 245 250 255Gly Pro Gly Ser Gln Gly Gly Ser Gly Pro Gly Ala Ser
Ala Ala Ala 260 265 270Ala Ala Ala Ala Ala Ser Gly Pro Gly Gly Tyr
Gly Pro Gly Ser Gln 275 280 285Gly Pro Ser Gly Pro Gly Tyr Gln Gly
Pro Ser Gly Pro Gly Ala Tyr 290 295 300Gly Pro Ser Pro Ser Ala Ser
Ala Ser Val Ala Ala Ser Val Tyr Leu305 310 315 320Arg Leu Gln Pro
Arg Leu Glu Val Ser Ser Ala Val Ser Ser Leu Val 325 330 335Ser Ser
Gly Pro Thr Asn Gly Ala Ala Val Ser Gly Ala Leu Asn Ser 340 345
350Leu Val Ser Gln Ile Ser Ala Ser Asn Pro Gly Leu Ser Gly Cys Asp
355 360 365Ala Leu Val Gln Ala Leu Leu Glu Leu Val Ser Ala Leu Val
Ala Ile 370 375 380Leu Ser Ser Ala Ser Ile Gly Gln Val Asn Val Ser
Ser Val Ser Gln385 390 395 400Ser Thr Gln Met Ile Ser Gln Ala Leu
Ser 405 41035PRTArtificial SequencesyntheticREPEAT(1)..(5)consensus
peptide motif 3Gly Pro Gly Xaa Xaa1 545PRTAraneus
diadematusVARIANT(4)..(4)Q at position 4 may also be alanine,
serine, glycine, tyrosine, proline, or glutamineVARIANT(5)..(5)Q at
position 5 may also be alanine, serine, glycine, tyrosine, proline,
or glutamine 4Gly Pro Gly Gln Gln1 555PRTAraneus
diadematusREPEAT(1)..(5)peptide motif (ADF-3) 5Gly Pro Gly Ala Ser1
565PRTAraneus diadematusREPEAT(1)..(5)peptide motif (ADF-3) 6Gly
Pro Gly Ser Gly1 575PRTAraneus diadematusREPEAT(1)..(5)peptide
motif (ADF-4) 7Gly Pro Gly Gly Tyr1 585PRTAraneus
diadematusREPEAT(1)..(5)peptide motif (ADF-4) 8Gly Pro Gly Gly Pro1
595PRTNephila clavipesREPEAT(1)..(5)peptide motif (flagelliform
protein) 9Gly Pro Gly Gly Ala1 5105PRTUnknownrepetitive polypeptide
motif found in phylum ArthropodaREPEAT(1)..(5)peptide motif
(resilin) 10Gly Pro Gly Gly Gly1 5115PRTNephila
clavipesREPEAT(1)..(5)peptide motif (flagelliform protein) 11Gly
Pro Gly Gly Ser1 5125PRTArtificial
SequencesyntheticREPEAT(1)..(5)Ax peptide motif 12Ala Ala Ala Ala
Ala1 5136PRTAraneus diadematusREPEAT(1)..(6)Ax peptide motif (ADF
3) 13Ala Ala Ala Ala Ala Ala1 5147PRTAraneus
diadematusREPEAT(1)..(7)Ax peptide motif (ADF-4) 14Ala Ala Ala Ala
Ala Ala Ala1 5158PRTAraneus diadematusREPEAT(1)..(8)Ax peptide
motif (ADF-4) 15Ala Ala Ala Ala Ala Ala Ala Ala1 5169PRTArtificial
SequencesyntheticREPEAT(1)..(9)Ax peptide motif 16Ala Ala Ala Ala
Ala Ala Ala Ala Ala1 51710PRTAraneus diadematusREPEAT(1)..(10)Ax
peptide motif (ADF-4) 17Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala1 5
10189PRTUnknownelastomeric protein (resilin) found commonly in
phylum ArthropodaREPEAT(1)..(9)peptide motif (based on resilin)
18Gly Gly Arg Pro Ser Asp Thr Tyr Gly1 5199PRTUnknownelastomeric
protein (resilin) found commonly in phylum
ArthropodaREPEAT(1)..(9)peptide motif (based on resilin) 19Gly Gly
Arg Pro Ser Ser Ser Tyr Gly1 52024PRTArtificial
SequencesyntheticDOMAIN(1)..(24)Module A (ADF-3) 20Gly Pro Tyr Gly
Pro Gly Ala Ser Ala Ala Ala Ala Ala Ala Gly Gly1 5 10 15Tyr Gly Pro
Gly Ser Gly Gln Gln 202135PRTArtificial
SequencesyntheticDOMAIN(1)..(35)Module C (ADF-4) 21Gly Ser Ser Ala
Ala Ala Ala Ala Ala Ala Ala Ser Gly Pro Gly Gly1 5 10 15Tyr Gly Pro
Glu Asn Gln Gly Pro Ser Gly Pro Gly Gly Tyr Gly Pro 20 25 30Gly Gly
Pro 352220PRTArtificial SequencesyntheticDOMAIN(1)..(20)Module Q
(ADF-3) 22Gly Pro Gly Gln Gln Gly Pro Gly Gln Gln Gly Pro Gly Gln
Gln Gly1 5 10 15Pro Gly Gln Gln 202334PRTArtificial
SequencesyntheticDOMAIN(1)..(34)Module S (Resilin) 23Pro Gly Ser
Ser Ala Ala Ala Ala Ala Ala Ala Ala Ser Gly Pro Gly1 5 10 15Gln Gly
Gln Gly Gln Gly Gln Gly Gln Gly Gly Arg Pro Ser Asp Thr 20 25 30Tyr
Gly2439PRTArtificial SequencesyntheticDOMAIN(1)..(39)Module R
(Resilin) 24Ser Ala Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly Gly Gly
Asn Gly1 5 10 15Gly Arg Pro Ser Asp Thr Tyr Gly Ala Pro Gly Gly Gly
Asn Gly Gly 20 25 30Arg Pro Ser Ser Ser Tyr Gly 352524PRTArtificial
SequencesyntheticDOMAIN(1)..(24)Module Ac 25Gly Pro Tyr Gly Pro Gly
Ala Ser Ala Ala Ala Ala Ala Ala Gly Gly1 5 10 15Tyr Gly Pro Gly Cys
Gly Gln Gln 202624PRTArtificial
SequencesyntheticDOMAIN(1)..(24)Module Ak 26Gly Pro Tyr Gly Pro Gly
Ala Ser Ala Ala Ala Ala Ala Ala Gly Gly1 5 10 15Tyr Gly Pro Gly Lys
Gly Gln Gln 202735PRTArtificial
SequencesyntheticDOMAIN(1)..(35)Module Cc 27Gly Ser Ser Ala Ala Ala
Ala Ala Ala Ala Ala Ser Gly Pro Gly Gly1 5 10 15Tyr Gly Pro Glu Asn
Gln Gly Pro Cys Gly Pro Gly Gly Tyr Gly Pro 20 25 30Gly Gly Pro
352835PRTArtificial SequencesyntheticDOMAIN(1)..(35)Module Ck1
28Gly Ser Ser Ala Ala Ala Ala Ala Ala Ala Ala Ser Gly Pro Gly Gly1
5 10 15Tyr Gly Pro Glu Asn Gln Gly Pro Lys Gly Pro Gly Gly Tyr Gly
Pro 20 25 30Gly Gly Pro 352935PRTArtificial
SequencesyntheticDOMAIN(1)..(35)Module Ck2 29Gly Ser Ser Ala Ala
Ala Ala Ala Ala Ala Ala Ser Gly Pro Gly Gly1 5 10 15Tyr Gly Pro Lys
Asn Gln Gly Pro Ser Gly Pro Gly Gly Tyr Gly Pro 20 25 30Gly Gly Pro
353035PRTArtificial SequencesyntheticDOMAIN(1)..(35)Module Ckc
30Gly Ser Ser Ala Ala Ala Ala Ala Ala Ala Ala Ser Gly Pro Gly Gly1
5 10 15Tyr Gly Pro Lys Asn Gln Gly Pro Cys Gly Pro Gly Gly Tyr Gly
Pro 20 25 30Gly Gly Pro 3531124PRTArtificial Sequencebased on
ADF-3DOMAIN(1)..(124)NR3 (ADF-3) 31Gly Ala Ala Ser Ala Ala Val Ser
Val Gly Gly Tyr Gly Pro Gln Ser1 5 10 15Ser Ser Ala Pro Val Ala Ser
Ala Ala Ala Ser Arg Leu Ser Ser Pro 20 25 30Ala Ala Ser Ser Arg Val
Ser Ser Ala Val Ser Ser Leu Val Ser Ser 35 40 45Gly Pro Thr Asn Gln
Ala Ala Leu Ser Asn Thr Ile Ser Ser Val Val 50 55 60Ser Gln Val Ser
Ala Ser Asn Pro Gly Leu Ser Gly Cys Asp Val Leu65 70 75 80Val Gln
Ala Leu Leu Glu Val Val Ser Ala Leu Val Ser Ile Leu Gly 85 90 95Ser
Ser Ser Ile Gly Gln Ile Asn Tyr Gly Ala Ser Ala Gln Tyr Thr 100 105
110Gln Met Val Gly Gln Ser Val Ala Gln Ala Leu Ala 115
12032109PRTArtificial Sequencebased on ADF-4DOMAIN(1)..(109)NR4
(ADF-4) 32Gly Ala Tyr Gly Pro Ser Pro Ser Ala Ser Ala Ser Val Ala
Ala Ser1 5 10 15Arg Leu Ser Ser Pro Ala Ala Ser Ser Arg Val Ser Ser
Ala Val Ser 20 25 30Ser Leu Val Ser Ser Gly Pro Thr Asn Gly Ala Ala
Val Ser Gly Ala 35 40 45Leu Asn Ser Leu Val Ser Gln Ile Ser Ala Ser
Asn Pro Gly Leu Ser 50 55 60Gly Cys Asp Ala Leu Val Gln Ala Leu Leu
Glu Leu Val Ser Ala Leu65 70 75 80Val Ala Ile Leu Ser Ser Ala Ser
Ile Gly Gln Val Asn Val Ser Ser 85 90 95Val Ser Gln Ser Thr Gln Met
Ile Ser Gln Ala Leu Ser 100
10533136PRTartificialsyntheticdomain(1)..(136)derived from
Latrodectus hesperus 33Met Gly Gln Ala Asn Thr Pro Trp Ser Ser Lys
Ala Asn Ala Asp Ala1 5 10 15Phe Ile Asn Ser Phe Ile Ser Ala Ala Ser
Asn Thr Gly Ser Phe Ser 20 25 30Gln Asp Gln Met Glu Asp Met Ser Leu
Ile Gly Asn Thr Leu Met Ala 35 40 45Ala Met Asp Asn Met Gly Gly Arg
Ile Thr Pro Ser Lys Leu Gln Ala 50 55 60Leu Asp Met Ala Phe Ala Ser
Ser Val Ala Glu Ile Ala Ala Ser Glu65 70 75 80Gly Gly Asp Leu Gly
Val Thr Thr Asn Ala Ile Ala Asp Ala Leu Thr 85 90 95Ser Ala Phe Tyr
Gln Thr Thr Gly Val Val Asn Ser Arg Phe Ile Ser 100 105 110Glu Ile
Arg Ser Leu Ile Gly Met Phe Ala Gln Ala Ser Ala Asn Asp 115 120
125Val Tyr Ala Ser Ala Gly Ser Gly 130
13534137PRTartificialsyntheticdomain(1)..(137)derived from
Latrodectus hesperus 34Met Gly Gln Ala Asn Thr Pro Trp Ser Ser Lys
Glu Asn Ala Asp Ala1 5 10 15Phe Ile Gly Ala Phe Met Asn Ala Ala Ser
Gln Ser Gly Ala Phe Ser 20 25 30Ser Asp Gln Ile Asp Asp Met Ser Val
Ile Ser Asn Thr Leu Met Ala 35 40 45Ala Met Asp Asn Met Gly Gly Arg
Ile Thr Gln Ser Lys Leu Gln Ala 50 55 60Leu Asp Met Ala Phe Ala Ser
Ser Val Ala Glu Ile Ala Val Ala Asp65 70 75 80Gly Gln Asn Val Gly
Ala Ala Thr Asn Ala Ile Ser Asp Ala Leu Arg 85 90 95Ser Ala Phe Tyr
Gln Thr Thr Gly Val Val Asn Asn Gln Phe Ile Thr 100 105 110Gly Ile
Ser Ser Leu Ile Gly Met Phe Ala Gln Val Ser Gly Asn Glu 115 120
125Val Ser Tyr Ser Ser Ala Gly Ser Gly 130 13535747PRTAraneus
diadematusPEPTIDE(1)..(747)MaSp I 35Gln Gly Ala Gly Ala Ala Ala Ala
Ala Ala Gly Gly Ala Gly Gln Gly1 5 10 15Gly Tyr Gly Gly Leu Gly Gly
Gln Gly Ala Gly Gln Gly Gly Tyr Gly 20 25 30Gly Leu Gly Gly Gln Gly
Ala Gly Gln Gly Ala Gly Ala Ala Ala Ala 35 40 45Ala Ala Ala Gly Gly
Ala Gly Gln Gly Gly Tyr Gly Gly Leu Gly Ser 50 55 60Gln Gly Ala Gly
Arg Gly Gly Gln Gly Ala Gly Ala Ala Ala Ala Ala65 70 75 80Ala Gly
Gly Ala Gly Gln Gly Gly Tyr Gly Gly Leu Gly Ser Gln Gly 85 90 95Ala
Gly Arg Gly Gly Leu Gly Gly Gln Gly Ala
Gly Ala Ala Ala Ala 100 105 110Ala Ala Ala Gly Gly Ala Gly Gln Gly
Gly Tyr Gly Gly Leu Gly Asn 115 120 125Gln Gly Ala Gly Arg Gly Gly
Gln Gly Ala Ala Ala Ala Ala Ala Gly 130 135 140Gly Ala Gly Gln Gly
Gly Tyr Gly Gly Leu Gly Ser Gln Gly Ala Gly145 150 155 160Arg Gly
Gly Leu Gly Gly Gln Gly Ala Gly Ala Ala Ala Ala Ala Ala 165 170
175Gly Gly Ala Gly Gln Gly Gly Tyr Gly Gly Leu Gly Gly Gln Gly Ala
180 185 190Gly Gln Gly Gly Tyr Gly Gly Leu Gly Ser Gln Gly Ala Gly
Arg Gly 195 200 205Gly Leu Gly Gly Gln Gly Ala Gly Ala Ala Ala Ala
Ala Ala Ala Gly 210 215 220Gly Ala Gly Gln Gly Gly Leu Gly Gly Gln
Gly Ala Gly Gln Gly Ala225 230 235 240Gly Ala Ser Ala Ala Ala Ala
Gly Gly Ala Gly Gln Gly Gly Tyr Gly 245 250 255Gly Leu Gly Ser Gln
Gly Ala Gly Arg Gly Gly Glu Gly Ala Gly Ala 260 265 270Ala Ala Ala
Ala Ala Gly Gly Ala Gly Gln Gly Gly Tyr Gly Gly Leu 275 280 285Gly
Gly Gln Gly Ala Gly Gln Gly Gly Tyr Gly Gly Leu Gly Ser Gln 290 295
300Gly Ala Gly Arg Gly Gly Leu Gly Gly Gln Gly Ala Gly Ala Ala
Ala305 310 315 320Ala Gly Gly Ala Gly Gln Gly Gly Leu Gly Gly Gln
Gly Ala Gly Gln 325 330 335Gly Ala Gly Ala Ala Ala Ala Ala Ala Gly
Gly Ala Gly Gln Gly Gly 340 345 350Tyr Gly Gly Leu Gly Ser Gln Gly
Ala Gly Arg Gly Gly Leu Gly Gly 355 360 365Gln Gly Ala Gly Ala Val
Ala Ala Ala Ala Ala Gly Gly Ala Gly Gln 370 375 380Gly Gly Tyr Gly
Gly Leu Gly Ser Gln Gly Ala Gly Arg Gly Gly Gln385 390 395 400Gly
Ala Gly Ala Ala Ala Ala Ala Ala Gly Gly Ala Gly Gln Arg Gly 405 410
415Tyr Gly Gly Leu Gly Asn Gln Gly Ala Gly Arg Gly Gly Leu Gly Gly
420 425 430Gln Gly Ala Gly Ala Ala Ala Ala Ala Ala Ala Gly Gly Ala
Gly Gln 435 440 445Gly Gly Tyr Gly Gly Leu Gly Asn Gln Gly Ala Gly
Arg Gly Gly Gln 450 455 460Gly Ala Ala Ala Ala Ala Gly Gly Ala Gly
Gln Gly Gly Tyr Gly Gly465 470 475 480Leu Gly Ser Gln Gly Ala Gly
Arg Gly Gly Gln Gly Ala Gly Ala Ala 485 490 495Ala Ala Ala Ala Val
Gly Ala Gly Gln Glu Gly Ile Arg Gly Gln Gly 500 505 510Ala Gly Gln
Gly Gly Tyr Gly Gly Leu Gly Ser Gln Gly Ser Gly Arg 515 520 525Gly
Gly Leu Gly Gly Gln Gly Ala Gly Ala Ala Ala Ala Ala Ala Gly 530 535
540Gly Ala Gly Gln Gly Gly Leu Gly Gly Gln Gly Ala Gly Gln Gly
Ala545 550 555 560Gly Ala Ala Ala Ala Ala Ala Gly Gly Val Arg Gln
Gly Gly Tyr Gly 565 570 575Gly Leu Gly Ser Gln Gly Ala Gly Arg Gly
Gly Gln Gly Ala Gly Ala 580 585 590Ala Ala Ala Ala Ala Gly Gly Ala
Gly Gln Gly Gly Tyr Gly Gly Leu 595 600 605Gly Gly Gln Gly Val Gly
Arg Gly Gly Leu Gly Gly Gln Gly Ala Gly 610 615 620Ala Ala Ala Ala
Gly Gly Ala Gly Gln Gly Gly Tyr Gly Gly Val Gly625 630 635 640Ser
Gly Ala Ser Ala Ala Ser Ala Ala Ala Ser Arg Leu Ser Ser Pro 645 650
655Gln Ala Ser Ser Arg Val Ser Ser Ala Val Ser Asn Leu Val Ala Ser
660 665 670Gly Pro Thr Asn Ser Ala Ala Leu Ser Ser Thr Ile Ser Asn
Val Val 675 680 685Ser Gln Ile Gly Ala Ser Asn Pro Gly Leu Ser Gly
Cys Asp Val Leu 690 695 700Ile Gln Ala Leu Leu Glu Val Val Ser Ala
Leu Ile Gln Ile Leu Gly705 710 715 720Ser Ser Ser Ile Gly Gln Val
Asn Tyr Gly Ser Ala Gly Gln Ala Thr 725 730 735Gln Ile Val Gly Gln
Ser Val Tyr Gln Ala Leu 740 74536627PRTAraneus
diadematusPEPTIDE(1)..(627)MaSp II 36Pro Gly Gly Tyr Gly Pro Gly
Gln Gln Gly Pro Gly Gly Tyr Gly Pro1 5 10 15Gly Gln Gln Gly Pro Ser
Gly Pro Gly Ser Ala Ala Ala Ala Ala Ala 20 25 30Ala Ala Ala Ala Gly
Pro Gly Gly Tyr Gly Pro Gly Gln Gln Gly Pro 35 40 45Gly Gly Tyr Gly
Pro Gly Gln Gln Gly Pro Gly Arg Tyr Gly Pro Gly 50 55 60Gln Gln Gly
Pro Ser Gly Pro Gly Ser Ala Ala Ala Ala Ala Ala Gly65 70 75 80Ser
Gly Gln Gln Gly Pro Gly Gly Tyr Gly Pro Arg Gln Gln Gly Pro 85 90
95Gly Gly Tyr Gly Gln Gly Gln Gln Gly Pro Ser Gly Pro Gly Ser Ala
100 105 110Ala Ala Ala Ser Ala Ala Ala Ser Ala Glu Ser Gly Gln Gln
Gly Pro 115 120 125Gly Gly Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gly
Tyr Gly Pro Gly 130 135 140Gln Gln Gly Pro Gly Gly Tyr Gly Pro Gly
Gln Gln Gly Pro Ser Gly145 150 155 160Pro Gly Ser Ala Ala Ala Ala
Ala Ala Ala Ala Ser Gly Pro Gly Gln 165 170 175Gln Gly Pro Gly Gly
Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gly Tyr 180 185 190Gly Pro Gly
Gln Gln Gly Pro Ser Gly Pro Gly Ser Ala Ala Ala Ala 195 200 205Ala
Ala Ala Ala Ser Gly Pro Gly Gln Gln Gly Pro Gly Gly Tyr Gly 210 215
220Pro Gly Gln Gln Gly Pro Gly Gly Tyr Gly Pro Gly Gln Gln Gly
Leu225 230 235 240Ser Gly Pro Gly Ser Ala Ala Ala Ala Ala Ala Ala
Gly Pro Gly Gln 245 250 255Gln Gly Pro Gly Gly Tyr Gly Pro Gly Gln
Gln Gly Pro Ser Gly Pro 260 265 270Gly Ser Ala Ala Ala Ala Ala Ala
Ala Ala Ala Gly Pro Gly Gly Tyr 275 280 285Gly Pro Gly Gln Gln Gly
Pro Gly Gly Tyr Gly Pro Gly Gln Gln Gly 290 295 300Pro Ser Gly Ala
Gly Ser Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly305 310 315 320Gln
Gln Gly Leu Gly Gly Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gly 325 330
335Tyr Gly Pro Gly Gln Gln Gly Pro Gly Gly Tyr Gly Pro Gly Ser Ala
340 345 350Ser Ala Ala Ala Ala Ala Ala Gly Pro Gly Gln Gln Gly Pro
Gly Gly 355 360 365Tyr Gly Pro Gly Gln Gln Gly Pro Ser Gly Pro Gly
Ser Ala Ser Ala 370 375 380Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly
Gly Tyr Gly Pro Gly Gln385 390 395 400Gln Gly Pro Gly Gly Tyr Ala
Pro Gly Gln Gln Gly Pro Ser Gly Pro 405 410 415Gly Ser Ala Ser Ala
Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly Gly 420 425 430Tyr Gly Pro
Gly Gln Gln Gly Pro Gly Gly Tyr Ala Pro Gly Gln Gln 435 440 445Gly
Pro Ser Gly Pro Gly Ser Ala Ala Ala Ala Ala Ala Ala Ala Ala 450 455
460Gly Pro Gly Gly Tyr Gly Pro Ala Gln Gln Gly Pro Ser Gly Pro
Gly465 470 475 480Ile Ala Ala Ser Ala Ala Ser Ala Gly Pro Gly Gly
Tyr Gly Pro Ala 485 490 495Gln Gln Gly Pro Ala Gly Tyr Gly Pro Gly
Ser Ala Val Ala Ala Ser 500 505 510Ala Gly Ala Gly Ser Ala Gly Tyr
Gly Pro Gly Ser Gln Ala Ser Ala 515 520 525Ala Ala Ser Arg Leu Ala
Ser Pro Asp Ser Gly Ala Arg Val Ala Ser 530 535 540Ala Val Ser Asn
Leu Val Ser Ser Gly Pro Thr Ser Ser Ala Ala Leu545 550 555 560Ser
Ser Val Ile Ser Asn Ala Val Ser Gln Ile Gly Ala Ser Asn Pro 565 570
575Gly Leu Ser Gly Cys Asp Val Leu Ile Gln Ala Leu Leu Glu Ile Val
580 585 590Ser Ala Cys Val Thr Ile Leu Ser Ser Ser Ser Ile Gly Gln
Val Asn 595 600 605Tyr Gly Ala Ala Ser Gln Phe Ala Gln Val Val Gly
Gln Ser Val Leu 610 615 620Ser Ala Phe6253713PRTArtificial
SequencesyntheticDOMAIN(1)..(13)TAG cys1 37Gly Cys Gly Gly Gly Gly
Gly Gly Ser Gly Gly Gly Gly1 5 10388PRTArtificial
SequencesyntheticDOMAIN(1)..(8)TAG cys2 38Gly Cys Gly Gly Gly Gly
Gly Gly1 53914PRTArtificial SequencesyntheticDOMAIN(1)..(14)TAG
cys3 39Gly Cys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly1 5
10405PRTUnknownrepetitive polypeptide motif found in phylum
ArthropodaREPEAT(1)..(5)peptide motif (resilin) 40Gly Pro Gly Gln
Gly1 54113PRTArtificial SequencesyntheticDOMAIN(1)..(13)TAG lys1
41Gly Lys Gly Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly1 5
10428PRTArtificial SequencesyntheticDOMAIN(1)..(8)TAG lys2 42Gly
Lys Gly Gly Gly Gly Gly Gly1 54335PRTArtificial
sequencesyntheticdomain(1)..(35)Ckappa 43Gly Ser Ser Ala Ala Ala
Ala Ala Ala Ala Ala Ser Gly Pro Gly Gly1 5 10 15Tyr Gly Pro Lys Asn
Gln Gly Pro Ser Gly Pro Gly Gly Tyr Gly Pro 20 25 30Gly Gly Pro
35
* * * * *
References