Description:TECHNICAL FIELD
[0001] The present disclosure relates to the field of pharmaceuticals. In particular, the present disclosure pertains to a process of preparing hydroquinone liposomal composition. The hydroquinone liposomal composition may be used as skin lightening compositions.

BACKGROUND
[0002] Hydroquinone is a skin-lightening agent commonly used topically to treat conditions such as hyperpigmentation, melasma, and age spots. The existing formulation of Hydroquinone are cream, gel or lotion-based, which are generally formulated in a combination of various other pharmaceutical actives. The most widely manufactured, recommended and used combination of Hydroquinone is the “Triple Combination” which contains Hydroquinone along with a retinol derivative (tretinoin) and a topical steroid (mild or potent steroid).
[0003] While it can be effective, there are certain limitations and potential drawbacks associated with its topical application. Hydroquinone can cause skin irritation, especially in individuals with sensitive skin or to those who use it in high concentrations. Common side effects include redness, dryness, itching, and burning sensation. Also, the prolonged or excessive use of hydroquinone can lead to more severe skin reactions, including dermatitis and contact sensitization. Additionally, there is a possibility of systemic absorption through the skin. Further, hydroquinone can increase the skin’s sensitivity to sunlight and make it more susceptible to sunburn.
[0004] Furthermore, conventional Hydroquinone shows a potent exfoliating and bleaching of the skin which causes mild irritation to the epidermis layer of the skin. To counter the irritation and inflammation caused by Hydroquinone, a mild or potent steroid is generally added to the combination (mostly mometasone or flucinolone). The rationale of adding a steroid in the formulation is to provide an instant relief from the discomfort or inflammation caused by Hydroquinone. As the treatment of hyperpigmentation is carried out for quite a long duration (on an average 3 months), the steroid is feared to cause thinning of the skin (skin atrophy) in this course of time. Hence, it is basically a severe safety threat, especially to individuals with sensitive skin. Also, the molecule is known to get oxidized and gets unstable in the formulation which apparently reduces the therapeutic efficacy of the medicine.
[0005] US 2003/0072724 provides a topical pharmaceutical composition using hydroquinone for skin lightening, which is particularly useful in treating skin hyperpigmentation, together with methods for its use. The composition and methods involve the topical use of an active agent effective in the treatment of skin hyperpigmentation plus a permeation-enhancing base that, in one embodiment, gives the composition a pH of about 8.0 to about 13.0, preferably about 8.0 to 11.5, and most preferably about 8.5 to 10.5.
[0006] US 5279834 relates to a cosmetic or pharmaceutical composition, containing hydroquinone and kojic acid or a derivative thereof, especially the salts or esters thereof. The hydroquinone and/or kojic acid component is in a form which is at least partially incorporated into liposomes.
[0007] Taghavi et al. compared therapeutic effects of topical liposomal hydroquinone with its conventional form on melasma.
[0008] Khoshneviszadeh et al. discloses that the most important parameters describing the liposomal formulation of hydroquinone is encapsulation efficacy. For the efficacy evaluation of hydroquinone trapped in liposomal structure, it first separates liposome from the matrix surrounding it. In this study, the three techniques of centrifuges with and without washing and dialysis were used.
[0009] However, the above disclosed compositions have several disadvantanges as mentioned above. Hence, there exists a necessity within the field to address the drawbacks and limitations inherent in current solutions. This entails delivering improved and enhanced skin-lightening composition using hyrdorquione liposomal compositions.

OBJECTS OF THE PRESENT DISCLOSURE
[0010] An object of the present disclosure is to provide an improved hydroquinone liposomal composition that addresses the disadvantages of the existing compositions.
[0011] Another object of the present disclosure is to provide hydroquinone liposomal composition that provides new and effective approach to treat hyperpigmentation.
[0012] Another object of the present disclosure is to provide hydroquinone liposomal composition that has enhanced ingredient delivery and increased bioavailability.
[0013] Another object of the present disclosure is to provide hydroquinone liposomal composition that has controlled release, targeted delivery, improved stability, and reduced skin irritation.
[0014] Yet another object of the present disclosure is to provide hydroquinone liposomal composition, which is cost-effective, safe and has efficacious treatment of hyperpigmentation with minimal adverse effects and better results.
[0015] Yet another object of the present disclosure is to provide a process or preparing the hydroquinone liposomal composition.

SUMMARY OF THE PRESENT DISCLOSURE
[0016] Aspects of the present disclosure relates to a process of preparing hydroquinone liposomal composition comprising the steps of: dissolving phospholipids in water followed by the addition of ethoxydiglycol, propylene glycol, and hydroquinone to form a mixture; stirring the mixture of step a) to form a homogenous lipid paste; adding citric acid and sodium citrate buffering solution to the paste of step b) followed by cooling to form a suspension of lipids; hydrating the suspension at a room temperature to form multilamellar vesicles; preparing hydroquinone liposomes by sonicating it in sonication bath; and storing the prepared hydroquinone liposomes at a temperature between 2-6? followed by aerating with nitrogen gas to form the hydroquinone liposomal composition.
[0017] The present disclosure also relates to the hydroquinone liposomal composition comprising the phospholipids in an amount 1 wt.% to 3 wt.%; water in an amount 5 wt.% to 6 wt.% ; ethoxydiglycol in an amount 8 wt.% to 10 wt.%; propylene glycol in an amount 65 wt.% to 80 wt.%; and hydroquinone in an amount 1.90 wt.% to 2.0 wt.%, and wherein the hydroquinone liposomal composition is aerated with nitrogen gas.

BRIEF DESCRIPTION OF THE DRAWINGS
[0018] The accompanying drawings are included to provide a further understanding of the present disclosure, and are incorporated in and constitute a part of this specification.
[0019] Figure (FIG.) 1: illustrates Cell Viability (%) assessment in keratinocytes (HaCaT cells) through MTT assay;
[0020] FIG. 2 illustrates melanocyte proliferation; and
[0021] FIG. 3 illustrates relative mRNA expression of cyclin D1 in melanocytes.

DETAILED DESCRIPTION OF THE PRESENT DISCLOSURE
[0022] The following is a detailed description of embodiments of the disclosure. The embodiments are in such details as to clearly communicate the disclosure. However, the amount of detail offered is not intended to limit the anticipated variations of embodiments; on the contrary, the intention is to cover all the modifications, equivalents, and alternatives falling within the scope of the present disclosures as defined by the appended claims.
[0023] Embodiments explained herein relate to economical and reliable solution for producing the hydroquinone liposomal composition and the hydroquinone liposomal composition that may be used as the skin lightening composition.
[0024] The term “liposomes” in the context of the present disclosure is defined as the spherical structures made up of phospholipids and other materials similar to cell membranes. They are used in a variety of applications such as drug delivery, cosmetics etc.
[0025] The phrase “hydroquinone liposomal composition” in the context of the present disclosure is defined as hydroquinone in liposomes formulation. The hydroquinone is encapsulated in liposomes. Liposomes act as carriers, protecting the ingredients from degradation and facilitating their penetration into the deeper layers of the skin. The said phrase is abbreviated as “HLC” in the context of the present disclosure.
[0026] The phrase “improved hydroquinone liposomal composition” in the context of the present disclosure is defined as a cost-effective, safe and efficacious treatment of hyperpigmentation, which shows minimal adverse effects and better results than the conventional hydroquione composition.
[0027] The concentration of each component of the hydroquinone liposomal composition is defined in weight (wt.) percentage (%).
[0028] The present disclosure relates to a hydroquione liposomal composition that may be used as a skin lightening composition. The hydroquinone liposomal composition of the present disclsoure comprises phospholipids, water, ethoxydiglycol, propylene glycol, and hydroquinone.
[0029] In another embodiment of the present disclosure, the phospholipids are selected from the group consisting of 1 wt.% to 3 wt.%. These examples do not intend to limit the scope of the present disclosure. This embodiments also includes the examples of the phospholipids well-known in the art that have similar characteristics.
[0030] Another embodiment of the present disclosure, an amount of water used in the present disclosure is in the range of 5 wt.% to 6 wt.%. The amount is not limited by this embodiment. The optimization of the amount as known by the person skilled in the art is covered with in the scope of the present disclosure.
[0031] In an embodiment of the present disclosure, water may be Distilled water (DI).
[0032] Another embodiment of the present disclosure, an amount of ethoxydiglycol used in the present disclosure is in the range of 8 wt.% to 10 wt.%. The amount is not limited by this embodiment. The optimization of the amount as known by the person skilled in the art is covered with in the scope of the present disclosure.
[0033] Another embodiment of the present disclosure, an amount of propylene glycol used in the present disclosure is in the range of 65 wt.% to 80 wt.%. The amount is not limited by this embodiment. The optimization of the amount as known by the person skilled in the art is covered with in the scope of the present disclosure.
[0034] Another embodiment of the present disclosure, an amount of hydroquione used in the present disclosure is in the range of 1.90 wt.% to 2.0 wt.%. The amount is not limited by this embodiment. The optimization of the amount as known by the person skilled in the art is covered with in the scope of the present disclosure.
[0035] In another embodiment of the present disclosure, the pH of the hydroquinone liposomal composition is with in the range of 4 to 5.
[0036] It is noted that the conventional liposomes are very sensitive formulations. The stability of liposomes is affected by the various factors. For example, liposomes lose their stability at high temperatures. Therefore, liposomes should be stored between 4–8 ºC (39–47 ºF). It is affected by the exposure to direct sunlight and fluorescent or ultraviolet lights, which can break down lipids or can alter the structure of liposomes. Further, variations in pH can affect liposome stability. Another factor is the oxidation of phospholipid components that can occur during the purification, sterilization, or storage. Moreover, lipids are prone to hydrolytic degradation. The organic components in natural seawater, such as amino acids and fatty acids, can interact with the membrane and change the properties of liposomes. Therefore, the hydroquione liposomal composition overcomes these problems and provide improved composition.
[0037] The improved hydroquinone composition is attributed to a novel technology that involves nitrogen gas flushing in the manufacturing procedure. The nitrogen flushing helps in drying down the organic part of the liposomes which makes them more stable and oxidation-free.
[0038] The present disclosure also relates to the process of preparing the hydroquinone liposomal compositions. The process comprises the steps of: dissolving phospholipids in water followed by addition of ethoxydiglycol, propylene glycol, and hydroquinone to form a mixture; stirring the mixture to form a homogenous lipid paste; adding citric acid and sodium citrate buffering solution to the paste of step b) followed by cooling to form a suspension of lipids, hydrating the suspension at a room temperature to form multilamellar vesicles, preparing hydroquinone liposomes by sonicating liposomes in sonication bath; and storing the prepared hydroquinone liposomes.
[0039] In another embodiment of the present disclosure, the hydroquione liposomes is aerated with nitrogen gas to form the improved hydroquinone liposomal composition.
[0040] In an embodiment of the present disclosure, an amount of the phospholipids is in the range of 1 wt. % to 3 wt.%. The amount is not limited by this embodiment. The optimization of the amount as known by the person skilled in the art is covered with in the scope of the present disclosure.
[0041] In an embodiment of the present disclosure, an amount of ethoxydiglycol is in the range of 8 wt.% to 10 wt. %. The amount is not limited by this embodiment. The optimization of the amount as known by the person skilled in the art is covered with in the scope of the present disclosure.
[0042] In yet another embodiment of the present disclosure, an amount of the water is in the range of 5 wt.% to 6 wt.%. The amount is not limited by this embodiment. The optimization of the amount as known by the person skilled in the art is covered with in the scope of the present disclosure.
[0043] In an embodiment of the present disclosure, an amount of propylene glycol is in the range of 65 wt. % to 80 wt.%. The amount is not limited by this embodiment. The optimization of the amount as known by the person skilled in the art is covered with in the scope of the present disclosure.
[0044] In yet another embodiment of the present disclosure, an amount of hydroquinone is in the range of 1.90 wt.% to 4 wt.% The amount is not limited by this embodiment. The optimization of the amount as known by the person skilled in the art is covered with in the scope of the present disclosure.
[0045] In yet another embodiment of the present disclosure, the stirring to form the homogenous liquid paste is carried out at a temperature of 50°C to 70°C.
[0046] In yet another embodiment of the present disclosure, the sodium and citrate in the sodium citrate buffering solution is in the ratio of 1:1 to 1:2. The ratio is not limited by this embodiment. The optimization of the ratio as known by the person skilled in the art is covered with in the scope of the present disclosure.
[0047] In yet another embodiment of the present disclsosure, the pH of the process is maintained at 4.0 to 5.0. The pH is not limited by this embodiment. The the optimization of the pH as known by the person skilled in the art is covered with in the scope of the present disclosure.
[0048] In another embodiment of the present disclosure, the hydration of the suspension to form the multilamellar vesicles is carried out for a time period in the range 46 hours to 48 hours. The optimization of the time period as known by the person skilled in the art is covered with in the scope of the present disclosure.
[0049] In yet another embodiment of the present disclosure, storing of the prepared hydroquinone liposomes at a temperature between 2-6 ? for 2-4 days. The temperature may vary as known by the person skilled in the art is covered with in the scope of the present disclosure.
[0050] Further, in another embodiment of the present disclosure, the sonication is carried out for a time period in the range of 3 minutes to 4 minutes. The time period may vary as known by the person skilled in the art is covered with in the scope of the present disclosure. The sonication is carried out with ultrasonic frequencies range from 20 kHz to 25kHz .
[0051] It is noted that stabilization of Hydroquinone is a big challenge due to the highly oxidizing nature of the molecule. Therefore, stabilizing agents like citric acid is used to prevent against deterioration in color and odor, but the systemic absorption of hydroquinone and irritating & skin sensitizing nature can not be achieved by mere stabilization of hydroquinone. Therefore, aeration using nitrogen is required to addresses the disadvantages of the conventional composition.
[0052] In an embodiment of the present disclosure, the nitrogen gas is supplied in an amount of 1.0 barg to 3.0 barg, preferabaly 2.0 barg.
[0053] The present disclosure also relates to the use of the improved hydroquinone liposomal composition as skin lightening agent and to treat hyperpigmentation.

ADVANTAGES OF THE PRESENT DISCLOSURE
[0054] The improved hydroquinone liposomal composition has the following advantages:

• Enhanced ingredient delivery: The hydroquione liposomal composition may transport both hydrophilic (water-loving) and lipophilic (fat-loving) ingredients. Liposomes act as carriers, protecting the ingredients from degradation and facilitating their penetration into the deeper layers of the skin.
• Increased bioavailability: The hydroquione liposomal composition may improve the bioavailability of active ingredients by enhancing their absorption and reducing wastage. By encapsulating the ingredients, liposomes protect them from enzymatic degradation and other external factors, ensuring that a higher concentration of the active compounds reaches the targeted skin cells.
• Controlled release: Liposomes are designed to release their contents gradually, providing a sustained release of Hydroquinone over an extended period. This controlled release mechanism allows for a longer-lasting effect of the skin cream, ensuring that the active compounds remain available to the skin for an extended duration.
• Targeted delivery: Liposomes of present disclosure are prepared to selectively target specific skin layers or cells. This targeted delivery approach is particularly advantageous when addressing specific skin concerns, such as blemishes, uneven skin tone and hyperpigmentation.
• Improved stability: The hydroquione liposomal composition may act as a protective barrier, shielding the encapsulated Hydroquinone and increasing its stability. This ensures that the skin formulation retains its potency and effectiveness for a longer period.
• Reduced skin irritation: Conventional Hydroquinone is irritating to the sensitive skin, especially when used in high concentrations. Liposomal encapsulation can help minimize skin irritation by reducing the direct contact of these ingredients with the skin. The liposome acts as a buffer, allowing for a more gentle and tolerable application of the skin cream.
• The hydroquione liposomal composition may offer a cost-effective, safe and efficacious treatment of hyperpigmentation, which shows minimal adverse effects and better results over conventional composition.

The invention is further illustrated by the following examples, which is provided to be exemplary of the invention and does not limit the scope of the invention. While the present disclosure has been described in terms of its speicfic embodiments, certain modifications and equivalents will be apparent to those skilled in the art and are intended within the scope of the present invention.

EXAMPLES
Example 1: Synthesis of hydroquinone liposomal composition
The phospholipids essential fatty acids (EFA) were dissolved in an amount of 1 wt.% in water 5 wt.% followed by addition of ethoxydiglycol 8 wt.%, propylene glycol 65 wt. %, and Hydroquinone 2 wt. % in a beaker. The mixture was stirred at 60°C for 25-30 min, at 600 rpm (until a fine paste is obtained) to create a smooth lipid paste. Citric acid was added and sodium citrate buffering solution in 18-20 drops (buffer ratio of Citric Acid and Sodium Citrate is 1:2) in the paste. The cooling was followed by that resulted in the formation of suspension of lipids. The suspended lipids were left for 1 hr to hydrate at room temperature resulting into the formation of multilamellar vesicle (MLVs). The hydroquinone in liposomes forms were prepared by sonicating liposomes for 3 min in sonication bath. The obtained hydroquinone in liposomes forms are stored for up to 2 days in the fridge (4°C) and aerated with nitrogen gas of 2.0 barg.

Example 2: Synthesis of hydroquinone liposomal composition
The phospholipids essential fatty acids (EFA) were dissolved in an amount of 3 wt.% in water 6 wt.% followed by addition of ethoxydiglycol 10 wt.%, propylene glycol 80 wt. %, and Hydroquinone 4 wt. % in a beaker. The mixture was stirred the at 60 °C for 25-30 min, at 800 rpm (until a fine paste is obtained) to create a smooth lipid paste. Citric acid was added and sodium citrate buffering solution in 18-20 drops (buffer ratio of Citric Acid and Sodium Citrate is 1:2) in the paste. The cooling was followed by that resulted in the formation of suspension of lipids. The suspended lipids were left for 1 hr to hydrate at room temperature resulting into the formation of multilamellar vesicle (MLVs). The hydroquinone in liposomes forms were prepared by sonicating liposomes for 3 min in sonication bath. The obtained hydroquinone in liposomes forms are stored for up to 2 days in the fridge (4°C) and aerated with nitrogen gas of 2.0 barg.

Example 3: Comparative assessment of the toxicity and Tyrosinase Inhibitory Activity of the hydroquinone liposomal composition as compared to conventional hydroquinone (HQ) composition
Cell culture: The Human Keratinocytes (HaCaT) and Melanocytes (WM 266-4) cells were cultured in Dulbecco’s modified eagle medium (DMEM) containing 2 mM L-glutamine, antibiotic-antimycotic solution (1000 mg/ml streptomycin sulphate, 1,000 units/ml, penicillin G sodium, and 2.5 mg/ml of amphotericin B) and 10% Fetal Bovine Serum (FBS) until they reached the confluency of 70-80%. Cells were maintained inside incubator at 37? with 5% CO2 conditions.

Preparation of hydroquinone liposomal composition
Hydroquine liposomal composition (HLC) and hydroquinone (HQ) was dissolved in sterile distilled water at stock concentration of 50% v/v.

Treatment of Cells
The 2x103 HaCaT were seeded on each well of 96 well plate and WM 266-4 cells (2×105) were seeded on 60 mm cell culture plates in 10 % FBS containing DMEM media and incubated inside incubator at 37? with 5% CO2 conditions. Cells were further treated with the 0.4 % working concentration of hydroquinone liposomal composition or hydroquinone (HQ) only. Cells were further incubated for different time intervals as per study requirements. HaCaT Cells were processed for MTT assay as per standard procedure. WM 266-4 cells were trypsinized and cell suspension was pelleted through centrifugation at 10,000 rpm for 10 mins. Finally, cell pellet was resuspended in 500 µl of RIPA (radioimmunoprecipitation assay) buffer. Cells were further sonicated for 10 mins and lysate was prepared. The protein concentration of cell lysate was estimated using the Bradford method.

TYROSINASE ACTIVITY ESTIMATION AND CALCULATIONS
Assay procedure was followed as per the standard method. Briefly 20 µL samples (cell lysate), tyrosinase aqueous solution (10 µL, 50 units/mL), and phosphate buffer (pH 6.8, 80 µL) were mixed and pre-incubated at 37°C for 5 min. Then L-DOPA (90 µL, 2 mg/mL) was added. The mixture was then incubated for 20 min at 37 °C. The amount of dopachrome was measured at the wavelength of 475 nm for the samples and blank, using a spectrophotometer. Following that absorbance values were put into the equation to determine the percent inhibition of the tyrosinase enzyme by the sample compared to the positive control.

The percent inhibition of the tyrosinase enzyme was calculated using the following equation Percent Inhibition = [1- {(Asample- Ablank) / (Acontrol - Ablank)}] × 100%

Where:
Asample is the absorbance of the sample (mixture of tyrosinase, L-DOPA, and phosphate buffer).
Ablank is the absorbance of the blank (phosphate buffer).
Acontrol is the absorbance of control.

Table 1: % Cell Viability measured through MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay in HaCaT cells after 96 hrs treatment of HLC or HQ.

S.No. Sample % Cell Viability
1 Control 100
2 0.4% HQ 81.37
3 0.4% HLC (present work) 95.82

Table 2: Inhibition percentage of Tyrosinase activity in Melanocytes (WM 266-4) cells.

S.No. Concentration (v/v) Inhibition
HQ Control 37.94
HLC (present work) 0.4% HQ 61.73

Concentrations of 0.4% (v/v) of test sample in the 100 µL enzymatic reaction did not interfere in the assay. Therefore, this concentration was selected for the study procedure. The Inhibition of Tyrosinase with HLC was significantly increased as compared to 0.4 % HQ alone, which confirms significant improvement in its whitening activity.

Results: Study indicates, HLC was not toxic (% cell viability>70%) even after 96 hrs of continuous exposure to human keratinocytes cells. (See Fig.1) Moreover % cell viability was found to be significantly higher as compared to HQ treatment for the same time duration which shows less toxicity (or improved safety) of HLC as compared to HQ over long term use. The HLC is found to be very safe with 95.82% cell viability as compared to hydroquinone which shows cell viability 81.37% when tested at the same concentration.

Further, the Inhibition of Tyrosinase due to HLC was 61.73 % which was significantly increased as compared to 0.4 % HQ alone (37.94 %), which confirms significant improvement in its whitening activity.

Example 4: To assess the effect of HLC on Proliferation of the Human Melanocyte cells using WM 266-4 cells Cell Line.

This study includes proliferation of potential cells and expression analysis of cell cycle marker cyclin D1 through RT PCR.

Cell culture: The Human Melanocytes (WM 266-4) cells were cultured in DMEM (Dulbecco's Modified Eagle Medium) containing 2 mM L-glutamine, antibiotic-antimycotic solution (1000 mg/ml streptomycin sulphate, 1,000 units/ml, penicillin G sodium, and 2.5 mg/ml of amphotericin B) and 10% FBS until they reached the confluency of 70-80%. Cells were maintained inside incubator at 37? with 5% CO2 conditions.

Preparation of HLC
HLC and hydroquinone (HQ) was dissolved in sterile distilled water at stock concentration of 50% v/v.

Treatment of Cells
WM 266-4 cells (1×105) were seeded on 60 mm cell culture plates in 10 % FBS containing DMEM media and incubated inside incubator at 37? with 5% CO2 conditions. Cells were further treated with the 0.4 % working concentration of HLC or hydroquinone (HQ) only. Cells were further incubated for different time intervals (3, 5 and 7 days) as per the study requirements and cells were counted using trypan blue dye following standard procedure.

RT PCR analysis of cyclin D1 expression
Expression analysis of the WM 266-4 cells was done using the cell culture and the RT PCR based method. Briefly WM 266-4 cells were sub-cultured and incubated inside incubator at 37? with 5% CO2 for 24 hours. After reaching the confluence of 60– 70%, the cells were treated with the HLC or hydroquinone (HQ). After treatment, the cells were further incubated for 24 hours, and RNA extraction was carried out. Post RNA quantification and cDNA conversion, expression analysis was done using the RT PCR with specific primers for the cyclin D1.

Cell proliferation assay
The Human Melanocytes (WM 266-4) cells count was substantially decreased at day 5 and 7 after incubation with the indicated concentration of HLC as compared to hydroquinone (HQ). (See FIG. 2)

Table 3: Data for cell proliferation rate after incubating for different number of days post treatment.
Number of cells (×105)
Day 0 Day 3 Day 5 Day 7
Control 1 1.83 2.94 3.58
0.4% HQ 1 1.58 2.4 2.96
0.4% HLC 1 1.29 1.45 1.83

RT PCR analysis of cyclin D1 expression
mRNA expression of cyclin D1 was decreased in Human Melanocytes (WM 266-4) cells after 24 hrs of incubation with the indicated concentration of HLC as compared to hydroquinone (HQ). See FIG.3

Table 4: Data for Real time PCR analysis of cyclin D1 in Human Melanocytes (WM 266-4) cells after 24 hrs treatment with HLC or hydroquinone (HQ).

Sample Fold Change in Relative Expression of Cyclin D1
Vehicle Control 1
0.4% HQ 0.73
0.4% HLC 0.42

Results: Study suggests Cell Proliferation of the Human melanocytes Cells with the HLC is decreased as compared to the hydroquinone (HQ). Study also indicates that the test article HLC downregulates the synthesis of DNA during the S phase of the cell cycle as cyclin D1 expression has been found to be decreased as compared to hydroquinone (HQ) treatment in melanocyte cells analyzed through RT PCR method.

Particularly, it is noted that the Human Melanocytes (WM 266-4) cells count was substantially decreased at day 5 and day 7 after incubation with the indicated concentration of the novel molecule miniHQ-2 as compared to hydroquinone (HQ), which shows that the HLC is quite potent than HQ.

Example 5: To assess the effect of test article on melanin content modulation using in vitro model of melanocyte cell line (WM 266-4).

Cell culture
The WM 266-4 cells were cultured in DMEM containing 2 mM L-glutamine, antibiotic-antimycotic solution (1000 mg/ml streptomycin sulphate, 1,000 units/ml, penicillin G sodium, and 2.5 mg/ml of amphotericin B) and 10% FBS until they reached the confluency of 70-80%. Cells were maintained inside incubator at 37? with 5% CO2 conditions.

Preparation of HLC
HLC and hydroquinone (HQ) was dissolved in sterile distilled water at stock concentration of 50% v/v.

Treatment of Cells
The WM 266-4 cells (2×105) were seeded on 60 mm cell culture plates in 10 % FBS (Fetal Bovine Serum) containing DMEM (Dulbecco's Modified Eagle Medium) media and incubated inside incubator at 37? with 5% CO2 conditions. Cells were further treated with the 0.4 % working concentration of HLC or hydroquinone (HQ) only. Cells were further incubated for 48 hrs. Cells were trypsinized and cell suspension was pelleted through centrifugation at 10,000 rpm for 10 mins. Finally, cell pellet was resuspended in 500 µl of RIPA buffer (Radioimmunoprecipitation assay buffer). Cells were further sonicated for 10 mins and lysate was prepared. Protein concentration of cell lysate was estimated using Bradford method.

Preparation of Melanin Standard Reaction
1. Prepared 5 mg/mL stock solution of melanin in DMSO.
2. Used Melanin stock solution and Assay Buffer to generate 500 µg/mL concentration of Melanin standard solution (M1).
3. Performed 1:2 serial dilutions to produce the remaining serially diluted Melanin standards (M2-M7).

Assay Reaction
1. Prepared the standards and test samples (250 microgram cell lysate/per well) in assay buffer and made up the volume to 50 µL of each sample in separate well in a microplate.
2. Added 50 µL Signal Enhancer to all the wells.
3. Incubated the reaction mixture at room temperature for 30 to 60 minutes.

Measurement
Fluorescence was measured using plate reader at ?Ex/?Em = 470/550 nm with cutoff at 515 nm.

Calculations were done using the formula as follows:
% change in melanin content = [(Actr – Atreated) /Actr] x 100
Observed values of Actr (Absorbance of vehicle treated or control sample & Atreated (Absorbance of treated sample)

Table 5: Values of absorbance and % Change
Sample Absorbance % Change
Control 36.28 NA
Treated with 0.4% HQ 24.92 31% decrease
Treated with 0.4% HLC 14.53 59.95% decrease

Results: Study suggests HLC treatment substantially decreases melanin content in melanocyte (WM 266-4) cells. Association of melanocytes (melanin containing cells) with keratinocytes cells is responsible for blackening of the skin. Study results indicate significant anti-melanin potential of test article as compared to hydroquinone (HQ) treatment only. Therefore, in the assessment of efficacy test, the novel molecule (miniHQ-2) substantially decreases melanin content in melanocyte (WM 266-4) cells by 59.95% as compared to the conventional molecule (Hydroquinone) which was found to reduce the melanin concentration by 31%, when tested on the same concentrations.

SPECIFIC EMBOIDIMENTS OF THE PRESENT DISCLOSURE
[0055] The present disclosure relates to a process of preparing hydroquinone liposomal composition comprising the steps of:
a) dissolving phospholipids in water followed by the addition of ethoxydiglycol, propylene glycol, and hydroquinone to form a mixture;
b) stirring the mixture of step a) to form a homogenous lipid paste;
c) adding citric acid and sodium citrate buffering solution to the paste of step b) followed by cooling to form a suspension of lipids;
d) hydrating the suspension at a room temperature to form multilamellar vesicles;
e) preparing hydroquinone liposomes by sonicating liposomes in sonication bath; and
f) storing the prepared hydroquinone liposomes at a temperature between 2-6 ? followed by aerating with nitrogen gas to form the hydroquinone liposomal composition.

[0056] Such process is disclosed, wherein an amount of the phospholipids is in the range of 1 wt. % to 3 wt.% and an amount of ethoxydiglycol is in the range of 8 wt.% to 10 wt.%

[0057] Such process is disclosed, wherein an amount of the water is in the range of 5wt. % to 6 wt.%, an amount of propylene glycol is in the range of 65 wt.% to 80 wt.%, and an amount of hydroquinone is in the range of 1.90 wt.% to 4 wt.%.

[0058] Such process is disclosed, wherein the stirring is carried out at a temperature of 50oC to 70oC.

[0059] Such process is disclosed, wherein sodium and citrate in the sodium citrate buffering solution is in the ratio of 1:1 to 1:2.
[0060] Such process is disclosed, wherein the hydration is carried out for a time period in the range 46 hours to 48 hours.

[0061] Such process is disclosed, wherein the sonication is carried out for a time period in the range of 2 minutes to 4 minutes.

[0062] Such process is disclosed, wherein aerating with nitrogen in an amount of 1.0 barg to 3.0 barg.

[0063] The present disclosure also relates to a hyoquinone liposomal composition comprising
phospholipids in an amount 1 wt.% to 3 wt.%;
water in an amount 5 wt.% to 6 wt.%;
ethoxydiglycol in an amount 8 wt.% to 10 wt.%;
propylene glycol in an amount 65 wt.% to 80 wt.%; and
hydroquinone in an amount 1.90 wt.% to 4.0 wt.%, and
wherein the hydroquinone liposomal composition is aerated with nitrogen gas.

[0064] Such composition is disclosed, wherein the nitrogen is supplied in an amount of 1.0 barg to 3 barg. , Claims:WE CLAIM:

1. A process of preparing hydroquinone liposomal composition comprising the steps of:
a) dissolving phospholipids in water followed by addition of ethoxydiglycol, propylene glycol, and hydroquinone to form a mixture;
b) stirring the mixture of step a) to form a homogenous lipid paste;
c) adding citric acid and sodium citrate buffering solution to the paste of step b) followed by cooling to form a suspension of lipids;
d) hydrating the suspension at a room temperature to form multilamellar vesicles;
e) preparing hydroquinone liposomes by preparing by sonicating liposomes in sonication bath; and
f) storing the prepared hydroquinone liposomes at a temperature between 2-6 ? followed by aerating with nitrogen gas to form the hydroquinone liposomal composition.

2. The process as claimed in claim 1, wherein an amount of the phospholipids is in the range of 1 wt.% to 3 wt.% and an amount of ethoxydiglycol is in the range of 8 wt.% to 12 wt.%.

3. The process as claimed in claim 1, wherein an amount of the water is in the range of 5 wt. % to 6 wt.%, an amount of propylene glycol is in the range of 65 wt.% to 80wt.%, and an amount of hydroquinone is in the range of 1.90 wt.% to 4.0 wt.%.

4. The process as claimed in claim 1, wherein the stirring is carried out at a temperature of 50°C to 70°C for a time period in a range of 2 minutes to 4 minutes.
5. The process as claimed in claim 1, wherein sodium and citrate in the sodium citrate buffering solution is in the ratio 1:1 to 1:2.

6. The process as claimed in claim 1, wherein the hydration is carried out for a time period in the range 46 hours to 48 hours.

7. The process as claimed in claim 1, wherein the sonication is carried out for a time period in the range of 2 minutes to 4 minutes.

8. The process as claimed in claim 1, wherein aerating with nitrogen in an amount of 1.0 barg to 3.0 barg.

9. A hydroquinone liposomal composition comprising
phospholipids in an amount 1 wt. % to 3 wt.%;
water in an amount 5 wt.% to 6 wt.%;
ethoxydiglycol in an amount 8 wt.% to 10 wt.%;
propylene glycol in an amount 65 wt.% to 80 wt.%; and
hydroquinone in an amount 1.90 wt.% to 4.0 wt.%, and
wherein the hydroquinone liposomal composition is aerated with nitrogen gas.

10. The composition as claimed in claim 9, wherein the nitrogen is supplied in an amount of 1.0 barg to 3.0 barg.