WO2018134852A1

WO2018134852A1 - Improved vesicular formulation of thymoquinone for the treatment of dermal inflammatory disorders and method thereof

Info

Publication number: WO2018134852A1
Application number: PCT/IN2018/050035
Authority: WO
Inventors: Dr. Poonam NEGI; Charul RATHORE; Sharma Ishita SHARMA
Original assignee: Shoolini University Of Biotechnology And Management Sciences
Priority date: 2017-01-23
Filing date: 2018-01-23
Publication date: 2018-07-26

Abstract

The inventor of the present invention discloses an improved formulation of thymoquinone for the treatment of dermal inflammatory disorders like psoriasis and eczema etc. Many characterization tests were performed to check the activity of the thymoquinone loaded ethosomal vesicles. In-vivo and in-vitro experiments showed that the bioavailability and effectiveness of the present invention is very high as compared to the existing formulations used for treatment of dermal inflammatory disorders.

Description

IMPROVED VESICULAR FORMULATION OF THYMOQUINONE FOR THE TREATMENT OF DERMAL INFLAMMATORY DISORDERS AND METHOD THEREOF

FIELD OF INVENTION The invention generally relates to the field of pharmaceutical formulations. More specifically it relates to an improved vesicular formulation of thymoquinone for the treatment of dermal inflammatory disorders and method thereof.

BACKGROUND OF THE INVENTION Thymoquinone (TQ) is an active ingredient isolated from seeds of the plant Nigella sativa and has structure as shown in Fig. 1. The therapeutic benefits of Thymoquinone have been extensively reviewed (Khader, M., & Eckl, P. M. (2014). Thymoquinone: an emerging natural drug with a wide range of medical applications. Iranian Journal of Basic Medical Sciences, 17(12), 950—957). It has been investigated for its anti-oxidant, anti-inflammatory, and anticancer activities in both in vitro and in vivo models since its first extraction in 1960s. Its anti-oxidant/anti-inflammatory effect has been reported in various disease models, including encephalomyelitis, diabetes, asthma and carcinogenesis.

Despite very good therapeutic potential, Thymoquinone has not been used in formulations because it is a highly hydrophobic drug having poor aqueous solubility. This problem has been solved in an innovative manner in the present invention by use of vesicular formulation of Thymoquinone resulting in very good solubility, bioavailability and enhanced therapeutic benefits.

Inflammatory diseases and disorders associated with inflammation affect a significant portion of the world population. Skin disorders such Psoriasis, Eczema osteoarthritis, and rheumatoid arthritis etc. vary greatly in symptoms and severity. They can be temporary or permanent and may be painless or painful. Some have situational causes while others may be genetic. Some skin conditions are minor, and others can be life-threatening. Skin Inflammation is a vital part of the body's immune response. It is the body's attempt to heal itself after an injury, defend itself against foreign invaders, such as viruses and bacteria; and repair damaged tissue.

Common terms used in the context of the present invention are defined below:

Psoriasis: It is a common inflammatory skin-disease and is also long-lasting. It is characterized by patches of abnormal skin which are typically red, itchy and scaly.

Eczema: Eczema is a term for a group of medical conditions that cause the skin to become inflamed or irritated. Affected skin is always itchy. The rash most commonly appears on the face, back of the knees, wrists, hands, or feet. It may also affect other areas as well. Affected areas usually appear very dry, thickened, or scaly.

(http://www.webmd.eom/skin-problems-and-treatments/guide/atopic-dermatitis-eczema#2).

Vesicular system: It refers to a system used to improve the therapeutic index of both existing and new drug molecules by encapsulating the active medicament inside the vesicular structure of the system due to which existence of the drug in the systemic circulation is prolonged and toxicity is reduced. The vesicular structures commonly known are liposomes, niosomes, sphingosomes, ethosomes, transferosomes and pharmacosomes.

(Reference: www.japsonline.com/ admin/php/uploads/367 _pdf.pdf). Ethosomes: Ethosomes are phospholipid-based elastic nanovesicles containing a high content of ethanol (20^-5%). Ethanol is known as an efficient permeation enhancer and has been added in the vesicular systems to prepare elastic nanovesicles.

(https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3255417/)

Problems in the existing dermal treatment processes A number of ointments or dermal antibiotics are present for the treatment of skin inflammatory disorders. However, they suffer from the limitations of lower bioavailability, poor efficacy and poor physiochemical properties like hydrophobicity, aqueous solubility and stability. Hence there is a need to develop an ointment/ topical formulation for skin inflammatory disorders which can overcome these problems. Introduction to the present invention

The present invention has addressed this problem in an innovative manner. It discloses an improved ethosomal vesicle (EV) formulation of thymoquinone for the treatment of dermal inflammatory disorders and its method of manufacture. The improved formulation is highly effective, has high bioavailability, easy method of manufacture and overcomes the problems of existing formulations relating to poor bioavailability.

Prior Art

S.No. Patent Application No. Prior Art Patent Present Invention

1. WO 2013030669 A2 The present invention The present invention discloses treatment of discloses an ethosomal inflammatory diseases and vesicle system of disorders using thymoquinone alone which thymoquinone (TQ) alone or is easy to manufacture at in combination with other industrial scale, shows compounds, including improved physicochemical eicosapentaenoic acid properties and good (EPA). There is no efficacy for treatment of disclosure about vesicles. dermal inflammation.

2. WO 2016061117 Al The present invention The present invention does discloses the compositions not disclose an oil based of black cumin (Nigella formulation. Rather it sativa seed) oil, olive oil, discloses an ethosomal and tea tree (Melaleuca vesicle system of alternifolia) oil, Cocoa thymoquinone only for use butter and vitamin A or a as dermal formulation for derivative thereof. There is inflammatory diseases. no disclosure about vesicles. 3. US 20160213727 Al The present invention The present invention does discloses a therapeutic oil not disclose an oil based comprising essential oil. 1% formulation. Rather it monarda, 40% discloses an ethosomal thymoquinone and vesicle system of thymohydroquinone thymoquinone only for use combined having anti- as dermal formulation for inflammatory effects. inflammatory diseases.

The improved formulation of the thymoquinone of the present invention has not been disclosed in the prior art.

OBJECTS OF THE PRESENT INVENTION

The object of the present invention is to disclose an improved ethosomal vesicle (EV) formulation of thymoquinone for the treatment of dermal inflammatory disorders.

Another object of the present invention is to disclose highly effective improved formulation of thymoquinone with higher bioavailability.

Yet another object is to disclose its method of manufacture.

SUMMARY OF THE INVENTION

The present invention discloses an improved ethosomal vesicle (EV) formulation of thymoquinone for the treatment of dermal inflammatory disorders like psoriasis and eczema etc. Many characterization tests were performed to check the activity of the thymoquinone loaded ethosomal vesicles. In-vivo and in-vitro experiments showed that the bioavailability and effectiveness of the present invention is very high as compared to the existing formulations used for treatment of dermal inflammatory disorders.

DETAILED DESCRIPTION OF DRAWINGS

Fig. 1 Structure of Thymoquinone (TQ)

Fig 2. Standard curves of TQ in:

(a) Distilled water (b) Ethanol

(c) Phosphate buffer (pH 7.4)

(d) 0.1 N (HC1)

Fig 3. Solubility studies of TQ in different buffer Fig 4. TEM photographs (21000 X) of the prepared ethosomes of TQ Fig 5. In-vitro drug release of ethosomal formulation of TQ Fig 6. In vivo drug activity of different formulations of TQ

Fig 7. Histology of the orthokeratosis mouse tail skin action: parakeratotic skin section of the mouse tail

(a) Control

(b) Nigella Sativa

(c) TQ loaded ethosome

(d) Tazarotene (Reference Drug)

Note: Tazarotene is a third- generation prescription topical retinoid sold as a cream, gel, or foam. Tazarotene is a member of the acetylenic class of retinoids. This medication is approved for treatment of psoriasis, acne, and sun damaged skin

( Source: https://en. wikipedia. org/wiki/Tazarotene ).

DETAILED DESCRIPTION OF THE INVENTION

The present invention discloses an improved ethosomal vesicle (EV) formulation of thymoquinone for the treatment of dermal inflammatory disorders like psoriasis and eczema etc. The formulation is in gel form and consists of hydrogel in which the ethosomal vesicles loaded with thymoquinone are incorporated. The invention is now described in detail with regard to its method of manufacture, quality analysis methods and assessment of its efficacy.

1. Preparation of thymoquinone loaded ethosomal vesicles (EVs)

Thymoquinone - loaded ethosomal vesicles (EVs) were prepared by cold method. The steps for the synthesis of thymoquinone loaded ethosomal vesicles are given below: i. Solubilizing thymoquinone in presence of phospholipids- 3-4% Phospholipid and 2% thymoquinone are dissolved in 10-30% ethanol -phosphate buffer (pH. 7.4) solution (1:4, v/v) to yield a clear solution.

ii. Formation of ethosomal vesicles: To the clear solution of drug and phospholipids, appropriate quantity of water is added (q.s. to 100 %) and the mixture is continuously stirred till the formation of milky ethosomal vesicles (EVs) dispersion. The suspension is kept at room temperature for 2 hours for complete hydration.

iii. Incorporation of the ethosomal vesicles in hydrogel: Thereafter, the suspension is gelled by addition of 0.05% carbopol, to form a hydrogel for easy application on the skin (topical application).

The composition of the ethosomal vesicles is given below in Table 1.

Table 1. Composition of various ethosomal vesicle (EVs)

Out of the six formulations, EV 4 was found to be best in performance viz., optimum particle size (477.6), Percent drug entrapment value (79.52), and cumulative Drug Release value (71.11%). The steps for the synthesis of EV 4 are now illustrated in Example 1.

Example 1

i. Solubilizing thymoquinone in presence of phospholipids- 4 g Phospholipid and 2 g thymoquinone were dissolved in 30 ml of Ethanol- Phosphate solution to yield a clear solution. (The ethanol-phosphate solution was prepared by mixing 10 ml of ethanol and 40 ml of phosphate buffer, pH 7.4) ii. Formation of ethosomal vesicles: To the clear solution of drug and phospholipids, appropriate quantity of water is added (q.s. to 100 %) and the mixture is continuously stirred till the formation of milky ethosomal vesicles (EVs) dispersion. The suspension is kept at room temperature for 2 hours for complete hydration.

iii. Incorporation of the ethosomal vesicles in hydrogel: Thereafter, the suspension is gelled by addition of 50 mg carbopol, to form a hydrogel for easy application on the skin (topical application).

Analysis of Thymoquinone solubility in different solvents

TQ is a highly hydrophobic drug having poor aqueous solubility. The different solvents viz., ethanol, Phosphate buffer (pH 7.4), Phosphate buffer (pH 5), 0.1 N (HQ) and distilled water were analysed to check the solubility of the thymoquinone. The spectrophotometric analysis and Equilibrium solubility method was used to find out the solvent having maximum solubility of thymoquinone.

Spectrophotometric analysis: UV spectrophotometer was chosen as the method of analysis in order to quantify the absorbance values obtained during in vitro and in vivo studies. A stock solution of TQ was prepared in various solvents and scanning was carried out in the range starting from 200 nm to 400 nm. λ max was selected from this range and absorbance values of suitably prepared dilutions of the stock were taken at the chosen wavelength. A standard curve of TQ was prepared in different buffer solutions and analyzed in UV spectrophotometer at _max 253-254 nm. The UV scan and calibration curve for TQ was obtained and depicted in Table 2 and Figure 2.

Table 2. Standard curve values of TQ in various Solvents

Sr. No. Concentration Absorbance

^g/mL)

Ethanol Phosphate Phosphate 0.1 N (HC1) Distilled buffer buffer water (pH 7.4) (pH 5)

1 0 0 0 0 0 2 5 0.200 0.210 0.230 0.190 0.245

3 10 0.390 0.350 0.400 0.350 0.390

4 15 0.555 0.536 0.557 0.490 0.597

5 20 0.701 0.669 0.724 0.623 0.770

6 25 0.874 0.811 0.890 0.785 0.940

7 30 1.020 0.974 1.040 0.898 1.170

Solubility studies: Equilibrium solubility method was used to determine the solubility of TQ in different solvents, i.e., phosphate buffer pH 7.4, phosphate buffer pH 5, 0.1 N HCl, pH 1.2 and distilled water for 72 hours at 37 °C employing an orbital shaker. The equilibrium solubility data on the TQ in various aqueous and hydro alcoholic solvent system at 37 °C is depicted in Table 3 and Figure 3.

Table 3. Solubility Studies of TQ in different buffers

Result: It is clear from the Table 3 that the solution of ethanol and phosphate buffer had maximum solubilty of thymoquinone. So this solution was used as a solvent for the thymoquinone and also employed as a diffusion media in the in vitro drug release and ex vivo drug permeation studies. Characterization of ethosomal vesicles (EV)

Transmission electron microscopy: The final ethosomal vesicles formulation was observed for its morphology under the transmission electron microscope (TEM). The vesicles were invariably round. TEM photograph is shown in Figure 4.

Percent drug entrapment (% PDE): The PDE of TQ-loaded EVs was measured by the ultracentrifugation method. Vesicular preparations were centrifuged at 40,000 rpm for 2 hours, at 4°C. Clear supernatant as well as the vesicular supernatant was assayed for the drug (TQ) content. PDE of TQ was calculated using Equation: PDE =— X100

T

TQ is expected to entrap in the non-polar lipid bilayers because it is a hydrophobic drug. Whereas, the effect of ethanol on the PDE was different. At 3% phospholipid level there was an increase in concentration of ethanol up to 20%, which got decreased slightly afterwards, i.e., at 30% ethanol concentration. At 4% phospholipid level there was an increase in concentration up to 10% ethanolic concentration, i.e., EV1 revealed the maximum PDE values followed by decrease at high ethanol concentration (20% and 30%). This could possibly be related to the drug leakage from phospholipid membranes at higher ethanol concentration.

Table 4: Composition of various evaluation parameters of ethosomal formulation

Formulation Lipid Particle % PDE % Transmittance Sedimentation

(mg) size(nm) volume

TQ EV 1 300 253.7 45±0.75 64.0 0.4

TQ EV 2 300 308.0 62±0.53 71.5 1

TQ EV 3 300 395.4 59.88±0.64 60.0 0.8

TQ EV 4 400 477.6 79.52±0.24 73.5 0.1 TQ EV 5 400 507.7 70.96±0.19 65.0 0.3

TQ EV 6 400 531.3 62.28±0.53 57.0 0.5

In vitro drug release studies: In vitro release studies employing dialysis membrane was carried out on the various formulations as shown in Table 4 and Fig 5. Phosphate buffer pH 7.4 and ethanol in ratio i.e. 4: 1 was used as the releasing medium.

Table 5. In vitro drug release of ethosomal formulation

7¾ £V4 revealed the optimum particle size (477.6), high PDE (Particle drug entrapment) value (79.52), and % CDR value i.e. Cumulative Drug Release value (71.11%). Characteristics of the optimized vesicular formulation in gel form

The optimized formulation is in gel form, in which vesicles are present and the drug is entrapped in the vesicles which are referred to as 'ethosomes' owing to presence of alcohol. The vesicles are having optimal phospholipid content 4%, and thymoquinone content 2%. The optimal characteristics of the ethosomes are as below: i. Particle size - 477.6 nm

ii. Percent Drug Entrapment (PDE value) - 79.52%

iii. Percent Cumulative Drug Release (CDR) value - 71.11%.

Efficacy Evaluation of the formulation

Healthy male rats, aged between four to six weeks and weight between 20 to 25 grams were used to perform in vivo studies. The animals were maintained at a room temperature of 25±2°C and allowed to acclimatize to the environment for 7 days prior to the experiment. Then the different formulations were applied on the mouse. The in vivo drug activity of different formulations obtained is shown in Fig. 6. i. Anti-psoriatic activity in mouse tail: Treatment with various combinations of the formulation for two weeks showed marked histological changes in the mouse tail skin. The results clearly demonstrated that all formulations possessed anti-psoriatic activity as reflecting from their respective DA (drug activity) values, and % OK (orthokeratotic) values in Fig 7(a), 7(b), 7(c), 7(d) and 7(e). Induction of orthokeratosis in those sections of the adult mouse tail having normal parakeratotic differentiation was evaluated by measuring the length of granular layer (A) and the length of the scale (B).

The percent orthokeratosis was calculated using equation:

A

% orthokeratosis =— Ί00

B

Drug activity (DA) was calculated using equation:

Mean OK of treated group— Mean OK of control group

100 — mean OK of control group

The percent orthokeratotic values are reported in the Table 6. Results:

It was observed that the TQ formulation of the invention has efficacy (in terms of % orthokeratosis or OK) comparable to the standard reference drug tazarotene (55.2% vs 51.3%) whereas for TQ alone it was 52.2%.

The OK value for normal saline treated skin was 44%, 52.2% for TQ, 50.52% for Nigella sativa extract, 55.2% for TQ loaded EV gel, and 51.3% for marketed formulation, i.e., tazarotene.

The percent Drug Activity (DA) values have been reported in the Table 7 showing 17.1% for TQ, 13% Nigella sativa, 25% for TQ-loaded EV gel and 19.2% for marketed formulation. % OK and % DA values of TQ and TQ-loaded ethosomal vesicles gel revealed much better anti-psoriatic activity. Ethosomal vesicles were able to enhance the anti-psoriatic efficacy of TQ and its percent orthokeratosis induction was comparable to that of the marketed product. This showed favorable effect of the vesicular carriers by virtue of their high affinity for skin lipids and flexibility, which undergo a preferential uptake and enable a better skin penetration through epithelium. Nano size range of ethosomal vesicles owing to their higher surface area maintain a closer and more intimate contact with the epithelium to be able to sustain the appreciable amount of drug during the entire period of treatment.

Table 6. Effect of different formulations on percent orthokeratosis

Group Formulation % Ortokeratosis

No.

1 Normal psoriatic skin 44%

2 TQ 52.2%

3 Nigella sativa extract 50.52%

4 TQ loaded ethosomal gel 55.2%

5 Tazarotene 51.3% Table 7. Effect of different formulations on percent drug Activity

Efficacy against edema: The efficacy of the formulation was checked by inducing local inflammation by intra-plantar inj ection of 0.1 ml homogenous suspension of 1 % carrageenan in sterile normal saline into the right hind paw of rats. Immediately after the administration of carrageenan, the paw volume was measured (i.e., time zero). The formulations as mentioned in Table 8 were applied to rat's paws.

Table 8: Effect of different formulations on Percent increase in swelling

Results: In the saline control group (Group-I) and TQ groups (Group-II and Group-Ill), a rapid and continuous increase in paw volume (i.e. edema) was observed and the inflammation was sustained during the entire period of 8 hours of the study. In the groups receiving active formulations (Group-Ill), the percentage increase in paw volume was lower vis-a-vis control and groups, indicating that TQ EV gel formulation are anti-inflammatory. iii. Drug permeation and retention evaluation: The animals were sacrificed by cervical dislocation to obtain skin tissue. The hair on the dorsal side of animal skin were removed with the help of 0.1 mm animal hair clipper in the direction of tail to head. Subcutaneous fat were removed from the full thickness abdominal rat skin. Dermis part of the skin was wiped, 3 to 4 times with a cotton swab soaked in iso-propanolol to remove any adhering fat material.

Skin tissue was mounted, with the stratum corneum surface facing upwards, and the donor compartment was kept open to atmosphere. The skin tissue was allowed to equilibrate with the sink medium. All the formulation viz-, TQ suspension in normal saline and TQ-loaded EV were applied uniformly, on the dorsal side of skin, in donor compartment. Aliquots of 1 mL were withdrawn periodically for a period of 5 h, and replaced with equal volume of the receptor medium, to maintain the receptor phase volume at a constant level. At the end of this study (5 h), the donor compartment and the skin surface(s), were washed thrice with receptor medium. Samples obtained were suitably diluted, and quantified for drug using UV spectrophotometric analysis.

Results: The skin permeation value for thymoquinone ethosomal formulation was found to be 36.6 and 11.6 for thymoquinone suspension. The amount of thymoquinone ethosomal formulation retained on skin was 88.714μg/cm² which was higher from rest of the formulations.

It is clear from the above results that the thymoquinone ethosomal formulation has better retention and permeation properties thereby enhancing bioavailability of the drug.

Novelty, Inventive Step and Industrial Application

Novelty- The present invention discloses an improved vesicular formulation of thymoquinone for the treatment of dermal inflammatory disorders e.g. psoriasis, eczema etc. etc. Inventive Step- The technical advancement of knowledge lies in disclosing a method for preparing an improved formulation of thymoquinone for the treatment of dermal inflammatory disorders and overcoming the technical barriers relating to poor bioavailability of the drug in an innovative and low-cost manner, by use of ethanolic vesicles which are easy to prepare and do not require any specialized machinery or equipment. The improved formulation was more effective and showed higher bioavailability than existing standard formulation Tazarotene.

Industrial Application- The improved formulation of the thymoquinone of present invention has widespread application in pharmaceutical industries, for treatment of dermal inflammatory disorders.

Claims

I claim:

1. An improved ethosomal vesicle (EV) formulation of thymoquinone for the treatment of dermal inflammatory disorders WHEREIN the said formulation consists of ethosomal vesicles having phospholipid content 4% and thymoquinone content 2% and wherein the optimal characteristics of the ethosomes are as below:

- Particle size - 477.6 nm;

- Percent Drug Entrapment (PDE value) - 79.52%;

- Percent Cumulative Drug Release (CDR) value - 71.11%.

2. The improved formulation of thymoquinone as claimed in Claim 1 WHEREIN the method of preparation of the same consists of the following steps:

Solubilizing thymoquinone in presence of phospholipids- 3-4% Phospholipid and 2% thymoquinone are dissolved in 10-30% ethanol -phosphate buffer (pH 7.4) solution (1:4, v/v) to yield a clear solution.

Formation of ethosomal vesicles: To the clear solution of drug and phospholipids, appropriate quantity of water is added (q.s. to 100 %) and the mixture is continuously stirred till the formation of milky ethosomal vesicles (EVs) dispersion. The suspension is kept at room temperature for 2 hours for complete hydration.

Incorporation of the ethosomal vesicles in hydrogel: Thereafter, the suspension is gelled by addition of 0.05% carbopol, to form a hydrogel for easy application on the skin (topical application).