Silk Alcohol Formulations
20200270316 · 2020-08-27
Inventors
Cpc classification
A61K9/06
HUMAN NECESSITIES
A61K8/64
HUMAN NECESSITIES
International classification
A61K9/06
HUMAN NECESSITIES
A61K8/64
HUMAN NECESSITIES
Abstract
The present invention relates to an aqueous formulation comprising a structural protein and an alcohol. Further, the present invention relates to a method for producing an aqueous formulation. Furthermore, the present invention relates to a pharmaceutical composition comprising the aqueous formulation comprising a structural protein and an alcohol. In addition, the present invention relates to a cosmetic composition comprising the aqueous formulation comprising a structural protein and an alcohol.
Claims
1. An aqueous formulation comprising a structural protein and an alcohol.
2. The aqueous formulation of claim 1, wherein said formulation has a clear appearance.
3. The aqueous formulation of claim 1 wherein the aqueous formulation comprises between 60 wt % and 90 wt % alcohol, between 0.05 wt % and 5 wt % structural protein, and between 5 wt % and 39.95 wt % water.
4. The aqueous formulation of claim 1, wherein the alcohol is selected from the group consisting of: ethanol, methanol, and isopropanol.
5. The aqueous formulation of claim 1, wherein the structural protein has a molecular weight of between 20 kDa and 140 kDa.
6. The aqueous formulation of claim 1, wherein the aqueous formulation has a complex viscosity of between 0.04 Pa.Math.s and 30 Pa.Math.s.
7. The aqueous formulation of claim 1, wherein the aqueous formulation is a hydrogel.
8. The aqueous formulation of claim 1, wherein the structural protein is a self-assembling protein.
9. The aqueous formulation of claim 1, wherein the structural protein is selected from the group consisting of: a silk protein, keratin, collagen, and elastin.
10. The aqueous formulation of claim 9, wherein the silk protein is a recombinant silk protein.
11. The aqueous formulation of claim 9, wherein the silk protein comprises at least two identical repetitive units.
12. The aqueous formulation of claim 11, wherein the repetitive units are independently selected from the group consisting of: module C having the sequence according to SEQ ID NO: 1 or a variant thereof, and module C.sup.Cys having the sequence according to SEQ ID NO: 2 or a variant thereof.
13. The aqueous formulation of claim 1, wherein the aqueous formulation further comprises a compound.
14. The aqueous formulation of claim 13, wherein the compound is poorly water soluble, water insoluble, lipophilic, or oily.
15. The aqueous formulation of claim 13, wherein the compound is selected from the group consisting of: a pharmaceutical compound, a detergent compound, a cosmetic compound, a chemical compound, and a coloring compound.
16. A method for producing an aqueous formulation comprising a structural protein and an alcohol comprising the steps of: (i) providing an aqueous solution comprising a structural protein and an aqueous solution comprising an alcohol, and (ii) mixing the aqueous solutions, thereby obtaining an aqueous formulation comprising a structural protein and an alcohol.
17. The method of claim 16, wherein the method further comprises subsequent to step (i) a step of: adding the aqueous solution comprising an alcohol to the aqueous solution comprising a structural protein.
18. The method of claim 17, wherein the aqueous solution comprising an alcohol is added to the aqueous solution comprising a structural protein in one motion/at once, or within no more than 10 seconds.
19. The method of claim 17, wherein the mixing is performed by avoiding the application of shear forces.
20. The method of claim 16, wherein the method further comprises subsequent to step (i) a step of: simultaneously bringing together/combining the aqueous solution comprising a structural protein and the aqueous solution comprising an alcohol.
21. The method of claim 20, wherein the aqueous solutions are mixed for no more than 10 seconds.
22. The method of claim 16, wherein the method further comprises subsequent to step (i) a step of: undercoating/underlayering the aqueous solution comprising an alcohol with the aqueous solution comprising a structural protein.
23. The method of claim 22, wherein the aqueous solutions are mixed for no more than 10 seconds.
24. The method of claim 16, wherein the concentration of the structural protein in the aqueous solution provided in (i) is of between 0.05 wt % and 5 wt %.
25. The method of claim 16, wherein the concentration of the alcohol in the aqueous solution added in step (i) is of between 50 wt % and 90 wt %.
26. The method of claim 16, wherein the aqueous solution comprising a structural protein is homogenous.
27. The method of claim 16, wherein said aqueous formulation is a hydrogel.
28. The method of claim 16, wherein the alcohol is selected from the group consisting of: ethanol, methanol, and isopropanol.
29. The method of claim 16, wherein the structural protein has a molecular weight of between 20 kDa and 140 kDa.
30. The method of claim 16, wherein the method further comprises the step of: adding a compound to: the aqueous solution comprising a structural protein provided in step (i), the aqueous solution comprising an alcohol provided in step (i), and/or the mixture in step (ii).
31. The method of claim 30, wherein the compound is poorly water soluble, water insoluble, lipophilic, or oily.
32. The method of claim 30, wherein the compound is selected from the group consisting of: a pharmaceutical compound, a detergent compound, a cosmetic compound, a chemical compound, and a coloring compound.
33. The method of claim 16, wherein the structural protein is a self-assembling protein.
34. The method of claim 16, wherein the structural protein is selected from the group consisting of: a silk protein, keratin, collagen, and elastin.
35. The method of claim 34, wherein the silk protein is a recombinant silk protein.
36. The method of claim 34, wherein the silk protein comprises at least two identical repetitive units.
37. The method of claim 36, wherein the repetitive units are independently selected from the group consisting of: module C having the sequence according to SEQ ID NO: 1 or a variant thereof, and module C.sup.Cys having the sequence according to SEQ ID NO: 2 or a variant thereof.
38. An aqueous formulation comprising a structural protein and an alcohol produced by the method of claim 16.
39. A method for producing an article comprising the steps of: (i) providing an aqueous formulation comprising a structural protein and an alcohol according to claims claim 1, and (ii) forming an article out of/from the said aqueous formulation.
40. The method of claim 39, wherein the method further comprises the step of: adding a compound to the aqueous formulation provided in step (i) or to the article formed in step (ii).
41. The method of claim 40, wherein the compound is poorly water soluble, water insoluble, lipophilic, or oily.
42. The method of claim 40, wherein the compound is selected from the group consisting of: a pharmaceutical compound, a detergent compound, a cosmetic compound, a chemical compound, and a coloring compound.
43. An article produced by the method of claim 39.
44. A pharmaceutical composition comprising: the aqueous formulation comprising a structural protein and an alcohol according to claim 1.
45. A cosmetic composition comprising: the aqueous formulation comprising a structural protein and an alcohol according to claim 1.
46. (canceled)
47. A method of protecting a compound, the method comprising the step of: utilizing the aqueous formulation comprising a structural protein and an alcohol according to claim 1 to protect the compound.
48. The method of claim 47, wherein the compound is selected from the group consisting of: a pharmaceutical compound, a detergent compound, a cosmetic compound, a chemical compound, and a coloring compound.
49. A method of providing sustained or controlled release of a compound, the method comprising the step of: utilizing the aqueous formulation comprising a structural protein and an alcohol according to claim 1 to provide sustained or controlled release of the compound.
50. The method of claim 49, wherein the compound is selected from the group consisting of: pharmaceutical compound, a detergent compound, a cosmetic compound, a chemical compound, and a coloring compound.
51. A method of prolonging the retention time of a compound, the method comprising the step of: utilizing the aqueous formulation comprising a structural protein and an alcohol according to claim 1 to provide prolongation of the retention time of the compound.
52. The method of claim 51, wherein the compound is selected from the group consisting of: a pharmaceutical compound, a detergent compound, a cosmetic compound, a chemical compound, and a coloring compound.
53. A method of formulating a poorly water soluble, a water insoluble, a lipophilic, or an oily compound, the method comprising the step of: Use of utilizing the aqueous formulation comprising a structural protein and an alcohol according to claim 1 in the formulation of the poorly water soluble, a water insoluble, a lipophilic, or an oily compound.
54. The method claim 53, wherein the compound is selected from the group consisting of: a pharmaceutical compound, a detergent compound, a cosmetic compound, a chemical compound, and a coloring compound.
Description
BRIEF DESCRIPTION OF THE FIGURES
[0366] The following figures and 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.
[0367]
[0368]
[0369]
EXAMPLES
[0370] The examples given below are for illustrative purposes only and do not limit the invention described above in any way.
Example 1
Preparation of C.SUB.8., C.SUB.16., C.SUB.32 .and C.SUB.48 .Silk Hydrogels
[0371] a) Preparation of C.sub.8, C.sub.16, C.sub.32 and C.sub.48 protein:
[0372] The C.sub.16 protein (SEQ ID NO: 3) was prepared as described in WO 2006/008163. C.sub.8 (SEQ ID NO: 6, C.sub.32 protein (SEQ ID NO: 4) and C.sub.48 (SEQ ID NO: 5) protein have been prepared analogous to the same process.
[0373] b) Preparation of an aqueous C.sub.8, C.sub.16, C.sub.32 and C.sub.48 protein solution:
[0374] For the preparation of the protein solutions, the silk proteins were dissolved in 6 M GdmSCN and 50 mM Tris/HCl, pH 8.0. In order to remove the GmdSCN, the protein solution was either dialyzed against 5 mM Tris/HCl, pH 8.0 using a Spectra/Por Dialysis Membrane with a MWCO of 6000-8000. After dialysis, the protein solution was filtered via crossflow filtration (VIVAFLOW 200, Hydrosat, 10 kDa) in order to further remove the GmdSCN and to concentration the protein in the solution.
[0375] When the volume of the protein solution was >500 mL, the GmdSCN can be removed and the protein solution concentrated without dialysis using a crossflow unit (Sartorius AG, Gttingen) with SARTOCON Slice Cassettes (Filter material: Hydrosat with 10 kDa cut off). The C.sub.8, C.sub.16, C.sub.32 and C.sub.48 protein concentrations were determined by measuring the absorbance at 276 nm using the UV/Vis spectroscopy (Beckman Coulter). The final protein concentrations of the C.sub.8, C.sub.16, C.sub.32 and C.sub.48 protein solution were between 3.75% and 6.65% (w/w).
[0376] c) Preparation of C.sub.8, C.sub.16, C.sub.32 and C.sub.48 silk hydrogels in 70% EtOH (Option 1):
[0377] For the preparation of silk hydrogels with a final ethanol concentration of 70%, deinonized water and 99.5% EtOH were mixed to obtain an aqueous solution with the respective EtOH concentration. This aqueous EtOH solution was added to a first beaker glass. Aqueous protein solutions (C.sub.8, C.sub.16, C.sub.32 and C.sub.48), prepared as described above, were added to a second beaker glass. The aqueous EtOH solution (first beaker glass) was added in one motion/at once to the aqueous protein solution in the second beaker glass and promptly mixed by agitating and subsequently slewing the mixture. The addition of the aqueous EtOH/deinonized water solution had to be carried out within no more than 5 seconds.
[0378] The final concentrations of C.sub.8 silk hydrogels were 1.35% (w/w), 1.5% (w/w), 1.625% (w/w) and 1.75% (w/w) in a final concentration of 70% EtOH. Silk hydrogels with a protein concentration of up to 1.625% (w/w) result in a flowable hydrogel.
[0379] The final concentrations of C.sub.16 silk hydrogels were 0.5% (w/w), 1.0% (w/w), 1.25% (w/w), 1.5% (w/w) and 2.0% (w/w) in a final concentration of 70% EtOH. Silk hydrogels with a protein concentration up to 1.25% (w/w) result in a flowable hydrogel. Silk hydrogels with protein concentrations of 1.5% (w/w) and 2.0% (w/w) result in a non-flowable hydrogel.
[0380] The final concentrations of C.sub.32 silk hydrogels were 0.5% (w/w), 0.75% (w/w), 1.0% (w/w) and 1.25% (w/w) in a final concentration of 70% EtOH. Silk hydrogels with a protein concentration up to 0.75 (w/w) result in a flowable hydrogel. Silk hydrogels with protein concentrations of 1.0% (w/w) and 1.25% (w/w) result in a non-flowable hydrogel.
[0381] The final concentrations of C.sub.48 silk hydrogels were 0.25% (w/w), 0.5% (w/w), 0.75% (w/w), 1.0% (w/w) and 1.165% (w/w) in a final concentration of 70% EtOH. Silk hydrogels with a protein concentration up to 0.5% (w/w) result in a flowable hydrogel. Silk hydrogels with protein concentrations of 0.75% (w/w), 1.0% (w/w) and 1.165% (w/w) result in a non-flowable hydrogel.
[0382] The complex viscosities of the hydrogels are shown in
[0383] An increase of the molecular weight of the protein result in an increase of viscosity.
[0384] The examples show that lower protein concentrations result in a non-flowable hydrogel the higher the molecular weight of the protein is. A person skilled in the art can determine the respective concentration in order to obtain a flowable or a non-flowable hydrogel.
Example 2
Determination of the Complex Viscosity of the Silk Hydrogels:
[0385] The complex viscosities of the silk hydrogels produced in Example 1 have been determined in a cone-plate measuring system (Modular Compact Rheometer Manufacturer: Anton Paar Type: MCR 102, Measurement cone: CP25-1, d: 25 mm, angle: 1 (Serial No.: 31081) according to the manufactures manual with the following parameters:
[0386] Value: Shear deformation (oscillating)
[0387] Profile: ramp logarithmic
[0388] Start value: 0.01%
[0389] End value: 100%
[0390] Value: (rad/s) circle-frequency
[0391] Profile: constant
[0392] Value: 10 rad/s
[0393] Sample measurement temperature (Plate): 15 C.
[0394] Measurement gap: 50 m
[0395] Evaluate parameter for description the silk gel viscosity: shear deformation at 1% (LVE)->G (Pa).
[0396] The complex viscosities of the C.sub.8, C.sub.16, C.sub.32 and C.sub.48 silk hydrogels have been determined in triplicate. The mean values G (Pa) at 1% (LVE) versus different protein contents (%) of C.sub.8, C.sub.16, C.sub.32 and C.sub.48 silk hydrogels are shown in
[0397] C.sub.16 protein correspond to molecular weight of 47.7 kDa. C.sub.32 protein correspond to molecular weight of 93.8 kDa and C.sub.48 protein correspond to a molecular weight of 139.9 kDa. The higher the complex viscosity of the protein the lower protein concentration result in a non-flowable hydrogel. The lower the complex viscosity of the protein the higher protein concentration result in a non-flowable hydrogel. This means that higher concentrations of proteins with lower molecular weight can be formed into a flowable hydrogel than with higher molecular weight proteins respectively that flowable hydrogels with higher concentrations can be achieved with proteins of lower molecular weight/lower complex viscosity than with proteins of higher molecular weight/higher complex viscosity.
[0398] A person skilled in the art can determine the respective concentrations of a protein needed in order to obtain a flowable or non-flowable hydrogel considering the molecular weight respectively the complex viscosity of the protein. Alternatively the respective concentrations of a protein needed in order to obtain a flowable or non-flowable hydrogel can be determined empirically for example by a dilution series of the respective protein concentration. In order to determine the complex viscosity of a protein the sequence of the amino acids of the protein has to be considered as well as the content of hydrophilic or hydrophobic amounts in the protein.
Example 3
Alternative Preparation of C.SUB.16., Silk Hydrogels in 70% EtOH
[0399] a) Preparation of a C.sub.16 Silk hydrogel with a protein concentration of 0.75% (w/w) and 1.5% in 70% EtOH via simultaneous mixing in a mixing chamber (Option 2):
[0400] The C.sub.16 protein (SEQ ID NO: 3) and the aqueous C.sub.16 protein solution were prepared as described in Example 1. An aqueous EtOH solution (99.5% EtOH) was added to a first reaction vessel and an aqueous C.sub.16 protein solution with 3.3% or 6.6% (w:w) protein respectively was added to a second reaction vessel. Both solutions were simultaneously combined in a mixing chamber and mixed with a magnetic stirrer so that a hydrogel with a protein concentration of 0.75% (w/w) or 1.5% (w/w) was formed. The reaction vessels were connected with the mixing chamber by flexible tubes. The aqueous EtOH solution was fed to the aqueous protein solution in the mixing chamber in a mixing ratio of 4.3:1 (EtOH solution:protein solution).
[0401] Silk hydrogels with a protein concentration of 0.75% (w/w) result in a flowable hydrogel.
[0402] Silk hydrogels with a protein concentration of 1.5% (w/w) result in a non-flowable hydrogel.
[0403] b) Preparation of a C.sub.16 Silk hydrogel with a protein concentration of 0.75 (w/w) and 1.5% in 70% EtOH via two-phase liquid system (Option 3):
[0404] The C.sub.16 protein (SEQ ID NO: 3) and the aqueous C.sub.16 protein solution were prepared as described in Example 1. In order to obtain a two-phase liquid system, an aqueous EtOH solution (99.5% EtOH) was added to a reaction tube with a stirrer and then gently underlaid with an aqueous C.sub.16 protein solution with 3.3% or 6.6% (w:w) protein respectively. The resulting two-phase liquid system consisting of an aqueous EtOH phase and an aqueous protein phase was mixed with the stirrer so that a silk hydrogel with a protein concentration of 0.75% (w/w) or 1.5% (w/w) was formed.
[0405] Silk hydrogels with a protein concentration of 0.75% (w/w) result in a flowable hydrogel.
[0406] Silk hydrogels with a protein concentration of 1.5% (w/w) result in a non-flowable hydrogel.
Example 4
Sustained Release of a Compound from a Composition Comprising an Aqueous Formulation of a Structural Protein and an Alcohol
[0407] In order to show the sustained release of a compound, a fragrance (Phenetylethanol) as exemplary poorly water soluble compound was added to an aqueous composition comprising a structural protein and an alcohol. The sustained release of the fragrance was compared to aqueous solutions without structural protein and aqueous solutions comprising the fixative Dipropylenglycol (Carl Roth, Karlsruhe, Germany) or Tegosoft M (Franken Chemie, Wendelstein Germany). Therefore 5% Phenetylethanol (Carl Roth, Karlsruhe, Germany) was added to aqueous solutions with C.sub.16 protein resulting in a concentration of 0.25% C.sub.16 protein (SSP), 70% EtOH or to aqueous solutions with Dipropylenglycol resulting in a concentration of 0.25% Dipropylenglycol, 70% EtOH (Dipro), to aqueous solutions with Tegosoft M resulting in a concentration of 0.25% Tegosoft M, 70% EtOH (Tego). An aqueous solution with 70% EtOH without structural protein or fixative served as a negative control (Neg.). 100 l of each composition containing the structural C.sub.16 protein (SSP), Dipropylenglycol (Dipro), Tegosoft M (Tego) and the negative control (Neg.) were applied to a teststrip (Rotilabo-Riechstreifen, Carl Roth, Karlsruhe, Germany).
[0408] 26 test persons determined the release of the fragrance by estimating the sent intensity of the fragrance 10 min, 20 min, 30 min, 40 min, 60 min and 80 min after application of the fragrance to the test strip. The sent represents the top note of a perfume which is a highly volatile scent quickly released by the medium. The sent intensity of the released fragrance Phenetylethanol in relation to the release time of the fragrance is shown in
[0409] The use of the inventive protein-alcohol solution allows the sustained release of fragrances without the help of fixatives. In addition less amount of fragrance is needed to obtain a sustained and long lasting release profile for fragrances.