Patent Application
20230038070
This invention relates to pharmaceutics, medicine and cosmetics. The invention describes several stable compositions comprising a mitochondrially targeted antioxidant, a polymer matrix and optionally additional components.
Mitochondrially targeted antioxidants of SkQ type (SkQs, see general formula I) are not stable in compositions comprising many ingredients common in pharmaceutical or cosmetic compositions. This makes SkQs not compatible (in the view of stability required for some reasonable storage duration) with many typical ingredients of pharmaceuticals and\or cosmetics.
This invention provides several reasonably stable compositions containing mitochondrially targeted antioxidants of SkQ type, wherein mitochondrially targeted antioxidants of SkQ type have general formula 1:
wherein:
SkQ 1 is a non-limiting example of SkQ type compounds (SkQs). The formula of SkQ1 (oxidized form) is:
SkQ1 was used in the following experiments in a firm of bromide or chloride. In our experiments we demonstrated that many common components of pharmaceuticals and cosmetics are not compatible with SkQs because these components destabilize SkQs thus making the storage time of corresponding compositions short. However, we have found that the following polymers and other components do not destabilize significantly SkQs when combined with the mitochondrial targeted antioxidant in a composition (see table 1):
Several compositions provided high SkQ1 stability in our experiments (see experimental example 1). The compositions are:
Composition 1:
Stability study is presented in the experimental example
Composition 2:
Sample preparation: 1 V sample+1 V NaBr 0. 5 M, mix, 15 min on a shaker at 70 C,+9 V ethanol 96% fractional by 1 V, centrifuge. To 500 mcl of super add 500 mcl 0, 1 M of phosphoric acid in water, centrifuge and transfer to a vial. The final dilution of 22 times
Injection volume: 100 μl, detection at 260 nm.
Results of analysis after +60° C. incubation
Compositions 03 and 05 demonstrated pronounced degradation (with biphasic kinetics).
A certain stability is observed only for the composition C088-060819-05-60. provided the initial concentration is not more than 10 micrograms.
Method of sample preparation: Place 200 mg of the test polyacrylate composition (exact weight) in Eppendorf test tube, add 2 volumes of water (about 400 μl), and heat for 30 minutes (on a thermoshaker at 70 C). Add 1 volume of NaBr 1 M in water (about 200 μl), heat while mixing for another 30 minutes (on a thermoshaker at 70 C). Add 2 volumes of 96% ethanol (about 400 μl), mix on a vortex, and centrifuge. Select 900 μl of the supernatant and add 300 μl of the phosphoric acid solution in ethanol, mix, and centrifuge.
Final dilution ratio: 8 times.
The injection volume is 100 μl.
Test polyacrylate composition:
Accelerated study of stability was performed at +60° C. Samples were prepared as described above and analyzed for SkQ1 concentration by HPLC method (injected volume 100 ul, detection at 260 nm).
Analysis results:
Extrapolation of kinetic constants to actual storage conditions
Conclusion: SkQ1 in the polyacrylate composition demonstrate acceptable stability.
(B) Stability study of carbomer-based gels 980 (10 uM and 50 mM SkQ1 concentration) in the presence of glycerol.
Stability curves at +60° C.:
Kinetics of SkQ1 degradation at 60 C in the composition of carbomer 980-based gels.
Extrapolation of the average rates at actual storage conditions.
Conclusion: The rate of degradation slightly depends on the initial concentration, which indicates the absence of an autocatalytic degradation mechanism. The required stability is not observed.
In the next experiment, we investigated the stability of SkQ1 in compositions at elevated temperatures. Together with the control compositions, the following composition (code MitoVitan—1-3) was studied:
Extrapolation of kinetic constants to actual storage conditions:
The MitoVitan-1-3 composition demonstrates acceptable stability in a model experiment of accelerated storage (at elevated temperature). Further experiments at +37 C and room temperature showed greater stability of SkQ in this composition compared to the calculated one.
Additional conclusions from experiments above:
Comparison of the stability of compositions C088-060819-03 and C088-060819-04 demonstrates that addition of lactic acid stabilizes SkQ1 . These experiments also revealed an unexpected destabilizing effect of surfactants (ethylhexylglycerol) present in the composition of C088-060819-05 and having dramatically worse stability compared to the same composition without ethylhexylglycerol (C0884)60819-03).
Based on the results of our experiments, we can conclude that various polyacrylates are well compatible with SkQ1 and can serve as a polymer base for cosmetic and pharmaceutical compositions of mitochondrial-targeted antioxidants. At the same time, the best compatibility (stability of SkQ1) was demonstrated by carbomers 640, 641 and 974. Unexpectedly, carbomer 980 was worse than the above.
The stabilizing effect of pentylene glycol and glycerol was also revealed.
(A) Compositions Based on Agarose and Agar-Agar. Stability Study at 60 C
From 300 to 500 mg of the composition (exact weight) was placed in a test tube after incubation at +60° C. temperature. Collected sample was melted on a thermoshaker at 70 C for 10 minutes and a volume of about 300-500 μl of acetonitrile is added (a volume is equal to the exact weight). The mixture was thoroughly mixed and centrifuged at 14000 rpm for 10 minutes, then 400 μl of the supernatant was transferred into a 1.5 ml tube, 600 μl of acetonitrile was added, mixed and thoroughly centrifuged at 14000 rpm for 30 minutes. Finally 400 μl of the supernatant was transferred into a chromatographic vial, 600 μl of water was added and the mixture was thoroughly mixed. 3 samples were taken for each time point.
Measurement results in different time points:
Extrapolation of the initial section of the kinetic curve to actual storage conditions:
Conclusion: The initial phase of degradation is replaced by the second phase where concentration is constant The ointment is very likely not to have the necessary stability at 2-8 C storage. It is likely that for this type of polymer it is not fully correct to extrapolate the data obtained during storage at +60 C to significantly lower temperatures (25 C, 2-8 C) due to the features of the polymer used. It is necessary to conduct a long experiment at lower temperatures (see below).
(B) Compositions Based on Agarose. Storage at +25° C.
Sampling: 3 eppendorfs per point. 25 C 1 time a month.
Sample preparation (sample weight 300-500 mg):
The test tube contains from 300 to 500 mg of the composition (exact weight). After incubation at +25° C., the sample is melted on a thermoshaker at 70 C for 10 minutes and a volume of about 300-500 μl of acetonitrile is added (volume equal to the exact weight). Mixture is thoroughly mixed and centrifuged at 14000 rpm for 10 minutes. 400 μl of the supernatant is transferred into a 1.5 ml tube, 600 μl of acetonitrile is added, mixed and thoroughly centrifuged at 14000 rpm for 30 minutes. 400 μl of the supernatant is transferred into a chromatographic vial and 600 μl of water is added followed by mix. The final dilution is 12.5 times, HPLC injection volume of 20 μl, detection at 260 nm.
Results of measurement of selected samples:
Conclusion: Degradation during 6 months at 25 C was not detected. Similar results were obtained at 25° C. when agarose was replaced with agar-agar.
(B) Study of the stability of SkQ1 in multi-active cosmetic masks.
Sample preparation for all masks: 1 V of the sample (about 200 mg)+1 V 0.5M of sodium bromide, heat for 15 minutes while mixing, take 100 μl of the resulting solution and add gradually 900 μl of 96% ethanol, mix after each addition of ethanol, centrifuge. Take 500 ml. of supernatant, add 500 ml of phosphoric acid in water, centrifuge, transfer to vials. The final dilution is 40 times and the HPLC injection volume is 100 μl. Detection at 260 nm.
Degradation curves (kinetics of degradation. of SkQ1 at 60° C.):
Extrapolation of kinetic constants to actual storage conditions.
Conclusion: Compositions No 2 and No 3 have the best stability.
Further experiments confirmed the stability of SkQ1 in compositions C088-140219-02 n C088-140219-03 when stored at +37 C (no sums of SkQ1 degradation were detected for 6 months).
The following compositions were also tested (with a pH close to neutral):
Composition C088-1604194)7:
Composition C088-160419-08:
Results of studying the stability of SkQ1 at +60 C (SkQ1 concentrations are shown in μg/ml):
Predicted degrees of degradation:
Stability at +37 C was studied for the composition C088-160419-07:
Sample preparation: 1 V of the sample+1 V 0.5 M of sodium bromide, heat for 15 minutes while mixing, +9 V 96% ethanol, mix, centrifuge. To 500 ml of super add 500 ml of 0.1 M phosphoric acid in water, centrifuge, transfer to vials. The final dilution of 22 times. HPLC sample volume is 100 μl, detection at 260 nm.
Based on the above data, you the following stability was predicted for the composition during long-term storage:
Conclusion: the composition stability is acceptable, especially for storage at 2-8 C. The result may improve after analyzing the data at 25 C.
Also, in our experiments, we obtained data demonstrating acceptable stability of SkQ1 in compositions that include polyvinyl alcohol or the Aquaxyl.
This application claims the benefit to U.S. Provisional Patent Application No. 62/945,939, filed Dec. 10, 2019, the contents of which are incorporated herein by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US20/64334 | 12/10/2020 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 62945939 | Dec 2019 | US |
