POLYMER MATRIXES FOR DIFFERENT COMPOSITIONS OF MITOCHONDRIALLY TARGETED ANTIOXIDANTS

Abstract

A composition containing amitochodrially-targeted antioxidant of the SkQ type and a polyacrylate, agarose, agar-agar orhyaluronic acid polymer useful for production of pharmaceutical, medicinal and cosmetic compositions having good stability. Such compositions may usefully also include lactic acid.

Claims

1. A composition containing (i) a mitochonrially targeted antioxidant of general formula 1: ##STR00004## wherein: A is a quinone antioxidant having a following structure: ##STR00005## and/or reduced (quinole) form thereof, wherein: m is an integer from 1 to 3: Y is lower alkyl or lower alkoxy L is a linker group, comprising straight or branched hydrocarbon chain which can be optionally substituted by one or more substituents and optionally contains one or more double or triple bonds; and n is integer from 1 to 40; B is a targeting group comprising SkZ, wherein Sk is a lipophilic cation; and Z is a pharmacologically acceptable anion. (ii) and a polymer, wherein the polymer is selected from polyacrylate (carboxyvynil polymer), agarose, agar-agar or hyaluronic acid.

2. A composition according to claim 1 wherein the mitochonrially targeted antioxidant is SkQ1.

3. A composition of the claim 2 of the following content: TABLE-US-00024 Isopentyldiol   5% Aquaxyl (xylitylglucoside and anhydroxylytol and xylitol)  −3% Propyleneglycol   3% Sodium polyacrylate 0.9% Ethylhexylglycerol 0.3% Lactic acid 0.2% SkQ1 − 6 ug/ml.

4. Method of stabilization of SkQ1 in a pharmaceutical or cosmetic composition by addition of lactic acid into such composition

5. Stabilized pharmaceutical or cosmetic composition of SkQ1 containing lactic acid.

Description

DESCRIPTION OF THE INVENTION

[0004] 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:

##STR00001##

wherein: [0005] A is a quinone antioxidant having a following structure:

##STR00002## [0006] and/or reduced (quinole) form thereof, wherein: [0007] m is an integer from 1 to 3; [0008] Y is lower alkyl or lower alkoxy [0009] L is a linker group, comprising straight or branched hydrocarbon chain which can be optionally substituted by one or more substituents and optionally contains one or more double or triple bonds; and [0010] n is integer from 1 to 40; [0011] B is a targeting group comprising SkZ, wherein Sk is a lipophilic cation; and Z is a pharmacologically acceptable anion.

[0012] SkQ 1 is a non-limiting example of SkQ type compounds (SkQs). The formula of SkQ1 (oxidized form) is:

##STR00003##

[0013] 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):

TABLE-US-00001 TABLE 1 Reference in # Polymer or other component this document 1 Carboxyvynil polymer (other names: Exp. Example 1 carbomer, polyacrylate) 2 Lactic acid (we have found that lactic acid is Exp. Example 1 unexpectedly potent stabilizer of SkQs, i.e. SkQ1 stability in a composition can be increased by addition of lactic acid into the composition) 3 pentyleneglycol, glycerol 4 Agarose/Agar-agar Exp. Example 2 5 Polyvynil alcohol, Aquaxyl Exp. Example 2 6 Hyaluronic acid, glycolic acid, mandelic acid, Exp. Example 2 lactobionic acid, aloe juice 7 Algae extract Seafill, beta-glucan Exp. Example 2

[0014] Several compositions provided high SkQ1 stability in our experiments (see experimental example 1). The compositions are:

[0015] Composition 1:

TABLE-US-00002 Propyleneglycol   3% Pentyleneglycol   5% Sodium polyacrylate 0.9% Ethylhexylglycerol 0.3% Lactic acid 0.2% SkQ1-different concentraions, for example 6 ug/ml Water-to 100%

[0016] Stability study is presented in the experimental example

[0017] Composition 2:

TABLE-US-00003 Isopentyldiol   5% Aquaxyl (xylitylglacoside and  −3% anhydroxylytol and xylitol) Propyleneglycol   3% Sodium polyacrylate 0.9% Ethylhexylglycerol 0.3% Lactic acid 0.2% SkQ1-different concentrations, for example 6 ug/ml Water-to 100%

Experimental Example 1. Polyacriclates as Suitable Polymer Matrixes for SkQs

(A) The Study of Stability of Cosmetic Formulations on the Basis of Carbomer 640 and 641

[0018] 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.

TABLE-US-00004 No Description Code Composition 1 Carbomer 641 C088-060819-03 Propylene glycol-8% based hydrogel Ethylhexylglycerin-0.3% Carbomer 641-0.9% Lactic acid-0.2% SkQ1 50 uM water pH 5.5 2 Carbomer 641 C088-060819-04 Propylene glycol-8% based hydrogel Ethylhexylglycerin-0.3% Carbomer 641-0.9% SkQ1 50 uM water pH 5.5 3 Carbomer 641 C088-060819-05 Propylene glycol-8% based hydrogel Carbomer 641-0.9% Lactic acid-0.2% SkQ1 50 uM water pH 5.5 4 Carbomer 640 C088-060819-06 Propylene glycol-8% based hydrogel Ethylhexylglycerin-0.3% Carbomer 640-0.9% Lactic acid-0.2% SkQ1 50 uM water pH 5.5
Results of analysis after +60° C. incubation

TABLE-US-00005 Time at C., ug/g 60 C., Sample date C088- C088- C088- C088- days collection 060819-03-60 060819-04-60 060819-05-60 060819-06-60  0 Aug. 8, 2019 40.28471 28.01799 57.00258 58.10061  4 Aug. 12, 2019 14.16035 0.92242 8.879284 24.65781  8 Aug. 16, 2019 8.680195 0.62347 15.21795 19.59364 11 Aug. 19, 2019 6.12771 0.03129 8.886293 12.3145 15 Aug. 23, 2019 8.013269 5.85823 9.427738 18 Aug. 26, 2019 5.256037 14.1804 3.436147 22 Aug. 30, 2019 2.787277 13.86665 2.92139 25 Sep. 2, 2019 1.947372 6.041945 1.049452 29 Sep. 6, 2019 1.720692 9.569733 0.467825 32 Sep. 9, 2019 1.257592 11.77894 0.410404

Compositions 03 and 05 demonstrated pronounced degradation (with biphasic kinetics).

TABLE-US-00006 Initial concentration No of −k′ d − 1 of the linear Degradation rate for 365 days of storage, % composition 60 C. section, ug/g 60 C. 25 C. 8 C. 4 C. 2 C. C088-060819- −0.08505 19.59137 100.0% 93.1% 56.1% 46.4% 41.9% 03-60 C088-060819- −0.55671 28.01799 100.0% 100.0% 99.5% 98.3% 97.1% 04-60 C088-060819- −6.82E−04 10.05812 21.6% 2.1% 0.7% 0.5% 0.4% 05-60 C088-060819- −0.1577  58.10061 100.0% 99.3% 78.3% 68.6% 63.5% 06-60

[0019] A certain stability is observed only for the composition C088-060819-05-60. provided the initial concentration is not more than 10 micrograms.

The Study of the Stability of the New Composition of Plastic0 Package

[0020] 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.

[0021] Final dilution ratio: 8 times.

[0022] The injection volume is 100 μl.

[0023] Test polyacrylate composition:

TABLE-US-00007 SkQ1 bromide  6.2 μg Propylene glycol 0.03 ml Pentyleneglycol 0.05 ml Sodium polyacrylate  0.9 mg Ethylhexylglycerol  0.3 mg Lactic acid  0.2 mg Water up to 1 ml

[0024] 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).

[0025] Analysis results:

TABLE-US-00008 C., time, d ug/ml 60 C. 5.88  0 5.78  3 5.69  7 5.54 10 4.99 14 4.72 17 4.46 21 4.21 24 4.23 28 4.17 31

[0026] Extrapolation of kinetic constants to actual storage conditions

TABLE-US-00009 −k′ d − 1 Degradation rate for 365 days of storage, % 60 C. 60 C. 25 C. 8 C. 4 C. 2 C. Test −1.30E−02 99.0% 33.7% 11.9% 9.1% 8.0% polyacrylate composition

[0027] 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.

TABLE-US-00010 No Manic Code Composition 1 Pharmaceutical C088- SkQ1 (50 uM), lactic acid, sodium lactate, glycerol composition for external 230119-01 1%, carbomer 980-, benzalkonium chloride 0.01% use based on carbomer 980 (50 uM) 2 Pharmaceutical C088- SkQ1 (10 uM), lactic acid, sodium lactate, glycerol composition for external 230119-02 1%, carbomer 980-, benzalkonium chloride 0.01% use based on carbomer 980 (10 uM)
Stability curves at +60° C.:

[0028] Kinetics of SkQ1 degradation at 60 C in the composition of carbomer 980-based gels.

Extrapolation of the average rates at actual storage conditions.

TABLE-US-00011 No of −k′ d − 1 Degradation rate for 365 days of storage, % composition 60 C. 60 C. 25 C. 8 C. 4 C. 2 C. C088- −0.02127 99.9% 48.8% 18.6% 14.5% 12.7% 230119-02 (10 uM) C088- −0.03519 100.0% 67.0% 28.9% 22.8% 20.1% 230119-01 (50 uM)

[0029] 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.

[0030] 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: [0031] Isopentyldiol 5% [0032] Aquaxyl (xylitylglucoside and anhydroxylytol and xylitol)—3% [0033] Propylenegycol 3% [0034] Sodium polyacrylate 0.9% [0035] Ethylhexylglycerol 0.3% [0036] Lactic acid 0.2% [0037] SkQ1—6 ug/ml [0038] Water up to 100%.

[0039] Extrapolation of kinetic constants to actual storage conditions:

TABLE-US-00012 −k′ d − 1 Degradation rate for 365 days of storage, % 60 C. 60 C. 25 C. 8 C. 4 C. 2 C. MitoVitan-1- −0.02233 100.0% 50.5% 19.4% 15.1% 13.3% 3

[0040] 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:

[0041] 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).

[0042] 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.

[0043] The stabilizing effect of pentylene glycol and glycerol was also revealed.

Experimental Example 2. Agar-Agar and Agarose as Suitable Natural Polymer Matrixes for SkQs

[0044] (A) Compositions Based on Agarose and Agar-Agar. Stability Study at 60 C

Composition:

[0045] Agar (or agarose)—5%, [0046] Lactic acid (sodium lactate)—1 %, [0047] propylene glycol—20%. [0048] SkQ1—50 microns. [0049] Water
Sample preparation (sample weight 300-500 mg):

[0050] 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:

TABLE-US-00013 Time at SkQ1 StDev, 60 C., μg/g μg/g days 306.4  116.2   0 171.5  37.0   3 129.2  1.0  6 103.7  6.2  9 87.4  6.7 13 82.2  3.8 16 86.9  1.9 20 89.5  7.2 23 63.0  1.4 27 77.7  0.5 30

[0051] Extrapolation of the initial section of the kinetic curve to actual storage conditions:

TABLE-US-00014 Temperature No of −k′ d − 1 Degradation rate for 12 mes of storage, % composition 60 C. 60 C. 37 C. 25 C. 8 C. 4 C. 2 C. C088- −0.10905 100 100 97 66 56 51 070918-02

[0052] 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.

Composition:

[0053] Agar (or agarose)—5%, [0054] Lactic acid (sodium lactate)—1%, [0055] propylene glycol—20%. [0056] SkQ1—50 microns, [0057] Water.

[0058] Sampling: 3 eppendorfs per point. 25 C 1 time a month.

Sample preparation (sample weight 300-500 mg):

[0059] 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:

TABLE-US-00015 Time at SkQ1 StDev, 60 C., μg/g μg/g days 251.5 39.9  0 250.5 30.2  1 224.2 8.0 2 264.0 22.9  3 260.4 16.3  4 242.8 39.7  5 267.2 62.7  6

[0060] 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.

TABLE-US-00016 Summary table: The initial real SkQ1 concentration No Name/Code Composition μg/g 1 Base neutral mask/ SkQ1, agar-agar, lactic acid, sodium  8.339 C088-140219-01 lactate, sodium hyaluronate, glycerin 2 Base acid mask/ SkQ1, agar-agar, lactic acid, hyaluronic  10.726 C088-140219-02 acid, glycerin 3 Full acid mask/ SkQ1, agar-agar, lactic acid, hyaluronic  16.168 C088-140219-03 acid, glycerin, glycolic acid, mandelic acid, lactobionic acid, aloe juice 4 Full neutral mask/ SkQ1, agar-agar, lactic acid, sodium  4.754 C088-140219-04 lactate, sodium hyaluronate, glycerin. aloe juice, algae extract, tremella extract, xylitol, ectoin, beta-glucan.

[0061] 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.

Stability at +60° C.

[0062] Degradation curves (kinetics of degradation. of SkQ1 at 60° C.):

[0063] Extrapolation of kinetic constants to actual storage conditions.

TABLE-US-00017 No of −k′ d − 1 Degradation rate for 365 days of storage, % composition 60 C. 60 C. 25 C. 8 C. 4 C. 2 C. C088- −0.07215 100.0% 89.7% 50.3% 41.1% 36.9% 140219-01 C088- −0.0148 99.5% 37.2% 13.4% 10.3% 9.0% 140219-02 C088- −0.01522 99.6% 38.1% 13.7% 10.6% 9.3% 140219-03 C088- −0.02787 100.0% 58.4% 23.7% 18.5% 16.3% 140219-04

[0064] Conclusion: Compositions No 2 and No 3 have the best stability.

[0065] 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).

[0066] The following compositions were also tested (with a pH close to neutral):

[0067] Composition C088-1604194)7:

TABLE-US-00018   SkQ1-50 μM agar-agar 0.25% Sodium lactate-5% Glycerin-10% aloe juice-4% algae extract (Seafill)-2% beta-glucan-1%

[0068] Composition C088-160419-08:

TABLE-US-00019   SkQ1-50 μM agar-agar 0.25% Sodium lactate-5% Glycerin-10% aloe juice-10% algae extract (Seafill)-2% beta-gluucan-1% Easyliance-2% SLMW hyaluronate-0.05 Biotin-100 μM

[0069] Results of studying the stability of SkQ1 at +60 C (SkQ1 concentrations are shown in μg/ml):

TABLE-US-00020 Time at Composition Composition +60° C., d C088-160419-07 C088-160419-08  0 26.74 28.52  4 28.05 29.91  8 26.47 22.20 11 28.88 19.69 15 25.94 13.22 18 25.94 11.15 22 17.40  9.05 25 14.46  7.93 29 10.20  6.94 32  8.75  4.43

[0070] Predicted degrees of degradation:

TABLE-US-00021 No of −k′ d − 1 Degradation rate for 365 days of storage, % composition 60 C. 60 C. 25 C. 8 C. 4 C. 2 C. C088- −0.03693 100.0% 68.7% 30.1% 23.7% 21.0% 160419-07 C088- −0.05487 100.0% 82.2% 41.2% 33.1% 29.6% 160419-08

[0071] Stability at +37 C was studied for the composition C088-160419-07:

[0072] 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.

Results:

[0073]

TABLE-US-00022 Storage 37 C., C., No of sample 24 hours μg/ml C088-160419-07-37-0  0 29.77 C088-160419-07-37-1  14 27.88 C088-160419-07-37-2  28 27.22 C088-160419-07-37-3  42 26.44 C088-160419-07-37-4  56 24.71 C088-160419-07-37-5  70 24.58 C088-160419-07-37-6  84 24.56 C088-160419-07-37-7  98 24.05 C088-160419-07-37-8 112 25.29 C088-160419-07-37-9 126 22.89

[0074] Based on the above data, you the following stability was predicted for the composition during long-term storage:

TABLE-US-00023 No of −k′ d − 1 Degradation rate for 365 days of storage, % composition 37 C. 37 C. 25 C. 8 C. 4 C. 2 C. C088- −0.00169 45.1% 23.0% 7.7% 5.9% 5.2% 160419-07

[0075] Conclusion: the composition stability is acceptable, especially for storage at 2-8 C. The result may improve after analyzing the data at 25 C.

[0076] Also, in our experiments, we obtained data demonstrating acceptable stability of SkQ1 in compositions that include polyvinyl alcohol or the Aquaxyl.