NANO-ASSEMBLIES OF CONJUGATES OF RETINOIDS AND AMINO ACIDS

Abstract

The present invention relates to nano-assemblies of conjugates of retinoids and amino acids, to uses thereof, and to methods of making the same.

Claims

1. A nano-assembly comprising conjugates of the following formula (1) ##STR00065## wherein X is selected from the group consisting of ##STR00066## ##STR00067## and/or comprising conjugates of the following formula (4) ##STR00068##

2. The nano-assembly according to claim 1, wherein the nano-assembly comprises conjugates of formula (1).

3. The nano-assembly according to claim 1, wherein the nano-assembly further comprises conjugates of the following formula (5) ##STR00069## conjugates of the following formula (6) ##STR00070## conjugates of the following formula (2) ##STR00071## conjugates of the following formula (3) ##STR00072## and/or combinations of two or more thereof, and wherein R.sub.1 and R.sub.2 are independently selected from the group consisting of H, CH.sub.3, CH.sub.2CH.sub.3, and CH.sub.2CH.sub.2(OCH.sub.2CH.sub.2).sub.mR.sub.3, wherein m is in a range of from 1 to 20, and wherein R.sub.3 is selected from the group consisting of H, OH, NH.sub.2, SH, COOH, and OCH.sub.3.

4. The nano-assembly according to claim 1, wherein the nano-assembly further comprises at least one additional retinoid compound, at least one additional vitamin E compound, at least one additional vitamin D compound, and/or at least one additional vitamin K compound bound to the nano-assembly by non-covalent interactions.

5. The nano-assembly according to claim 4, wherein the additional retinoid compound is all-trans retinoic acid.

6. The nano-assembly according to claim 1, wherein X is selected from the group consisting of ##STR00073##

7. The nano-assembly according to claim 1, comprising a first conjugate having formula (1), and further comprising a second conjugate having formula (1) wherein X of formula (1) is different from X of formula (2).

8. The nano-assembly according to claim 1, comprising conjugates of formula (1) wherein X is ##STR00074## and further comprising conjugates of formula (1) wherein X being is ##STR00075##

9. The nano-assembly according to claim 1, wherein a molar proportion of the conjugates of formula (1) is at least 50 mol % as compared to a total molar amount of conjugates constituting the nano-assembly.

10. (canceled)

11. (canceled)

12. (canceled)

13. A method of producing the nano-assembly of claim 1, the method comprising incubating a mixture of a retinoid compound and an amino acid in a mixture of a polar solvent A and a polar solvent B for a time of from 1 hour to 48 hours, and removing solvent B; wherein solvent A has a higher polarity index as compared to solvent B.

14. The method according to claim 13, wherein a molar ratio of the retinoid compound and the amino acid in the mixture is in a range of from 1:50 to 1:1.

15. The method according to claim 13, wherein the retinoid compound is all trans retinal.

16. The method according to claim 13, wherein the polarity index of solvent A is at least 8.0.

17. The method according to claim 13, further comprising addition of at least one additional retinoid compound to the mixture of the retinoid compound and the amino acid.

18. A method of treating dermatological diseases or conditions and/or pulmonary diseases or conditions, comprising administering a therapeutically effective amount of the nano-assembly according to claim 1 to a patient in need thereof.

19. A pharmaceutical composition comprising the nano-assembly according to claim 1, and a pharmaceutically acceptable carrier.

20. A method for managing or reducing stretch marks on skin (striae), smooth wrinkles, and fine lines comprising administering an effective amount of the nano-assembly according to claim 1 to a person in need thereof.

21. A method for treating androgenetic alopecia comprising administering an effective amount of the nano-assembly according to claim 1 to a person in need thereof.

Description

DESCRIPTION OF THE FIGURES

[0081] FIG. 1 shows the size distribution of different nano-assemblies of the invention. The hydrodynamic diameter (in nm) is shown on the x-axis on logarithmic scale. The size intensity (in percent) is shown on the y-axis. The size distribution was obtained using dynamic light scattering (DLS). The curves shown in FIGS. 1A, 1B and 1C correspond to the nano-assemblies made at a concentration of 0.5, 1 and 2 mg/mL (w/w), respectively.

[0082] FIG. 2 is a cryogenic Transmission Electron microscopy (cryo-TEM) image of nano-assemblies of the invention dispersed in water.

EXAMPLES

[0083] The invention is further explained based on the following examples.

1. Nano-Assemblies Based on Conjugates of All Trans Retinal and Glycine

[0084] All trans retinal and glycine were mixed in polar solvent consisting of ethanol (EtOH) and water. The molar ratio of retinal to glycine was 1:1.1. The volume ratio of EtOH to H.sub.2O was 6:4 (v/v). Sodium carbonate was added to the mixed solution at a molar ratio of retinal to sodium carbonate of 1:10. The reaction was carried out at room temperature for 24 h.

##STR00063##

[0085] The nano-assemblies were formed spontaneously in water upon removal of EtOH.

[0086] The conjugate can be isolated using the following procedure. Following the removal of EtOH, the conjugate is extracted with an excess amount of chloroform and dried over magnesium sulfate (MgSO.sub.4). Chloroform is then removed under reduced pressure at room temperature, and the residue solvent is then dried under reduced pressure at room temperature for 12 h. The conversion yield was 100%, while the isolated yield was higher than 95%. The conjugate can be then dissolved in polar solvent consisting of ethanol (EtOH) and water. The volume ratio of EtOH to H.sub.2O was 6:4 (v/v). The nano-assemblies formed spontaneously in water upon removal of EtOH.

[0087] The loading weight percentage of retinoid was 78.3%.

[0088] Hydrodynamic diameter, polydispersity index (PDI) and zeta-potential of the nano-assemblies was determined using dynamic light scattering (DLS, NanoBrook, Brookhaven Instruments, Holtsville, NY; the device is equipped with a 40 mW diode laser scattering angle of 90? at 25? C.). The results are summarized in the following table as mean?standard deviation.

TABLE-US-00001 Nano-assemblies made at the concentration Size zeta-potential (mg/mL) (nm) PDI (mV) 1.0 290.8 ? 1.1 0.05 ? 0.00 ?13.8 ? 0.2

[0089] The low PDI shows that there was a homogeneous size distribution.

2. Nano-Assemblies Based on Conjugates of All Trans Retinal and Taurine

[0090] All trans retinal and taurine were mixed in polar solvent consisting of ethanol (EtOH) and water. The molar ratio of retinal to taurine was 1:1.1. The volume ratio of EtOH to H.sub.2O was 6:4 (v/v). Sodium carbonate was added to the mixed solution at a molar ratio of retinal to sodium carbonate of 1:10. The reaction was carried out at room temperature for 24 h.

##STR00064##

[0091] The nano-assemblies were formed spontaneously in water upon removal of EtOH.

[0092] The conjugate can be isolated using the following procedure. Following the removal of EtOH, the conjugate is extracted with an excess amount of chloroform and dried over magnesium sulfate (MgSO.sub.4). Chloroform is then removed under reduced pressure at room temperature, and the residue solvent is then dried under reduced pressure at room temperature for 12 h. The isolated yield was higher than 95%. The conjugate can be then dissolved in polar solvent consisting of ethanol (EtOH) and water. The volume ratio of EtOH to H.sub.2O was 6:4 (v/v). The nano-assemblies formed spontaneously in water upon removal of EtOH.

[0093] The loading weight percentage of retinoid was 68.2%.

[0094] Hydrodynamic diameter, polydispersity index (PDI) and zeta-potential of the nano-assemblies was determined using dynamic light scattering. The results are summarized in the following table as mean?standard deviation.

TABLE-US-00002 Size zeta-potential Sample (nm) PDI (mV) 1 203.6 ? 0.5 0.069 ? 0.044 ?23.0 ? 1.6 2 260.6 ? 0.9 0.144 ? 0.022 3 466.4 ? 6.8 0.157 ? 0.015

[0095] Samples 1, 2 and 3 show the characteristics of the nano-assemblies made at a concentration of 0.5, 1 and 2 mg/mL (w/w), respectively. The concentration of the nano-assemblies is referred to as the concentration in water. The concentration was varied by adding an additional amount of the polar solvent consisting of ethanol (EtOH) and water prior to the removal of EtOH.

[0096] The low PDI shows that there was a homogeneous size distribution.

[0097] The size distribution of samples 1 to 3 are shown in FIGS. 1A, 1B, and 1C, respectively.

[0098] A cryo-TEM image of nano-assemblies dispersed in water is shown in FIG. 2. The retinoid loading weight percentage of the nano-assemblies of FIG. 2 was 68.2%.

3. All Trans Retinoic Acid (ATRA) Loaded Nano-Assemblies Based on Conjugates of All Trans Retinal and Glycine

[0099] The conjugates of all trans retinal and glycine that were synthesized and isolated in Example 1 were mixed with ATRA at the weight ratio of ATRA to the conjugates of 1:3 (w/w). The mixture was then dissolved in polar solvent consisting of ethanol (EtOH) and water. The volume ratio of EtOH to H.sub.2O was 6:4 (v/v).

[0100] The ATRA loaded nano-assemblies based on conjugates of all trans retinal and glycine were formed spontaneously in water upon removal of EtOH.

[0101] Hydrodynamic diameter, polydispersity index (PDI) and zeta-potential of the nano-assemblies was determined using dynamic light scattering (DLS, NanoBrook, Brookhaven Instruments, Holtsville, NY; the device is equipped with a 40 mW diode laser scattering angle of 90? at 25? C.). The results are summarized in the following table as mean?standard deviation.

TABLE-US-00003 ATRA loaded nano- assemblies made at the concentration Size zeta-potential (mg/mL) (nm) PDI (mV) ~0.48 208.5 ? 3.4 0.08 ? 0.02 ?9.3 ? 0.5

[0102] The low PDI shows that there was a homogeneous size distribution.

[0103] The following table summarizes the loading weight and loading weight percentage of retinoids

TABLE-US-00004 Total concentration in water Total (including Retinoids ATRA Retinal Retinoids Glycine) loading weight [?g/mL] [?g/mL] [?g/mL] [?g/mL] percentage 100 300 400 ~480 ~83.5%

[0104] The loading weight percentage of retinoid was about 83.5%.

[0105] This example shows that ATRA could be loaded in the nano-assemblies based on conjugates of all trans retinal and glycine using a facile method. The loading weight percentage of retinoids can be further tuned compared to Example 1 and Example 2 by adding an additional amount of ATRA.

[0106] Furthermore, the loading efficiency of ATRA was significantly high at 100%, which was analyzed by high-performance liquid chromatography (HPLC). The loading efficiency was determined as follows. The ATRA loaded nano-assemblies were completely solubilized in methanol and used in HPLC analysis. The mass of ATRA in the nano-assemblies was detected using HPLC, and the loading efficiency was determined using the following equation (II).

[00003]$\begin{array}{cc}\mathrm{ATRA}\mathrm{loading}\mathrm{efficiency}\left(\%\right)=\frac{\mathrm{ATRA}{\mathrm{mass}}_{\mathrm{detected}}}{\mathrm{ATRA}{\mathrm{mass}}_{\mathrm{beginning}}}?100& \left(\mathrm{II}\right)\end{array}$

wherein the ATRA mass.sub.detected is the ATRA mass loaded in the nano-assemblies determined using HPLC, and the ATRA mass.sub.beginning is the initial ATRA mass used in the loading procedure.

4. pH Dependent Release Study

[0107] The nano-assemblies from Example 2 were diluted at 10% (w/v) in acetate buffer consisting of sodium acetate and acetic acid (pH 4.5, 150 mM) at 37? C. At 2 h post-incubation, the suspension was centrifuged at 15000 g for 30 min, the supernatant was removed, and the pellet was collected and freeze dried overnight. The dried compound was then dissolved in chloroform-d for proton NMR analysis.

[0108] The complete cleavage of the imine bond in the nano-assemblies was confirmed by proton NMR spectrum after 2 h incubation. In details, the corresponding proton peaks of imine bond in the conjugates typically from 8.5 ppm to 9.5 ppm disappeared, and the typical peaks of aldehyde group in retinal appeared from 9.51 ppm to 10.5 ppm. Conclusively, the complete disappearance of imine peaks confirmed the complete (100%) cleavage of the imine bond at pH 4.5 after 2 h incubation.

5. Light Stability Study

[0109] The nano-assemblies from Example 1 were diluted at 30% (w/v) in distilled water, and the solution was exposed to simulated sunlight for 2 h under the accumulated irradiation energy of 16.56 Joule (J). Afterwards, the nano-assemblies solution was analyzed using dynamic light scattering (DLS, NanoBrook, Brookhaven Instruments, Holtsville, NY; the device is equipped with a 40 mW diode laser scattering angle of 90? at 25? C.). The results are summarized in the following table as mean?standard deviation.

TABLE-US-00005 Samples Size (nm) PDI Nano-assemblies based on conjugates of 290.8 ? 1.1 0.05 ? 0.00 all trans retinal and glycine - BEFORE exposure to sunlight Nano-assemblies based on conjugates of 273.9 ? 10.3 0.20 ? 0.05 all trans retinal and glycine - AFTER exposure to sunlight

[0110] There were only slight changes in size and PDI of the nano-assemblies based on conjugates of all trains retinal and glycine after 2 h exposure to sunlight. This means that the nano-assemblies were stable under the tested conditions.

6. Oxidation Stability Study

[0111] The all trans retinoic acid (ATRA) loaded nano-assemblies based on conjugates of all trans retinal and glycine made in Example 3 were first diluted in distilled water at 50% (w/v). The ATRA loaded nano-assemblies solution was then mixed with H.sub.2O.sub.2 solution (30% in water) at the volume ratio of nano-assemblies solution to H.sub.2O.sub.2 solution of 9:1 (v/v). The mixture was kept for 30 min or 60 min at room temperature. The ATRA amount loaded in the nano-assemblies before and after mixing with H.sub.2O.sub.2 was analyzed using HPLC, and the data is expressed in percentages and summarized in the following table as mean?standard deviation.

TABLE-US-00006 Percentage (%) of ATRA amount loaded in the Samples nano-assemblies ATRA loaded nano-assemblies based 100 ? 0 on conjugates of all trans retinal and glycine - BEFORE mixing with H.sub.2O.sub.2 ATRA loaded nano-assemblies based 100 ? 0 on conjugates of all trans retinal and glycine - 30 min AFTER mixing with H.sub.2O.sub.2 ATRA loaded nano-assemblies based 100 ? 0 on conjugates of all trans retinal and glycine - 60 min AFTER mixing with H.sub.2O.sub.2

[0112] The percentage of ATRA amount loaded in the nano-assemblies is calculated using the equation (III)

[00004]$\begin{array}{cc}\mathrm{Percentage}\mathrm{of}\mathrm{ATRA}\mathrm{amount}\mathrm{loaded}\mathrm{in}\mathrm{the}\mathrm{nano}-\mathrm{assemblies}=\frac{\mathrm{ATRA}{\mathrm{mass}}_{\mathrm{detected}}}{\mathrm{ATRA}{\mathrm{mass}}_{\mathrm{before}\mathrm{mixing}\mathrm{with}{H}_2{O}_2}}?100\left(\%\right)& \left(\mathrm{III}\right)\end{array}$

wherein the ATRA mass detected is the ATRA mass loaded or remained in the nano-assemblies determined using HPLC, and the ATRA mass before mixing with H.sub.2O.sub.2 is the initial ATRA mass loaded in the nano-assemblies which was determined in Example 3. The percentage of ATRA amount loaded in the nano-assemblies before mixing with H.sub.2O.sub.2 is always 100%.

[0113] After mixing with the strong oxidant H.sub.2O.sub.2 for 30 min and 60 min, the percentage of ATRA loaded in the nano-assemblies in both cases stayed stable at 100%. This indicates that the loading of ATRA in the nano-assemblies protected ATRA from H.sub.2O.sub.2.

7. Stability at Storage Conditions (Room Temperature (25-30? C.) and Ambient Pressure)

[0114] The nano-assemblies based on conjugates of all trans retinal and glycine were produced according to the procedure reported in Example 1 and then diluted in PBS to the concentration of 50 microgram/mL. The samples were then kept at room temperature (25-30? C.) and ambient pressure. The characteristics of the nano-assemblies, including hydrodynamic diameter, and polydispersity index (PDI) were determined at designated time points, 3 h, 7 d, 15 d, 21 d, and 28 d after the sample preparation, using dynamic light scattering (DLS, NanoBrook, Brookhaven Instruments, Holtsville, NY; the device is equipped with a 40 mW diode laser scattering angle of 90? at 25? C.). The results are summarized in the following table as mean?standard deviation.

TABLE-US-00007 Time after sample preparation Size (nm) PDI 3 h 297.0 ? 6.5 0.04 ? 0.00 7 d 240.0 ? 1.7 0.12 ? 0.02 15 d 219.1 ? 3.9 0.10 ? 0.05 21 d 217.9 ? 8.7 0.12 ? 0.02 28 d 229.3 ? 4.0 0.17 ? 0.01

[0115] Despite slight changes, the characteristics of the nano-assemblies in aqueous media at room temperature and ambient pressure stayed stable over the study period.