Pharmaceutical wound healing composition

Abstract

The present invention discloses a biodegradable and biocompatible pharmaceutical composition comprising silk Sericin, sophorolipid, a gelling or thickening agent and one or more pharmaceutically acceptable carriers or excipients for faster wound healing and limit scarring.

Claims

1. A pharmaceutical composition for wound healing, comprising: a) 8% w/w of silk sericin; b) 0.1% of sophorolipid; c) 2-5% of a gelling or thickening agent; and d) one or more pharmaceutically acceptable ingredients.

2. The pharmaceutical composition as claimed in claim 1, wherein the composition is prepared as a topical formulation; wherein the formulation is selected from the group consisting of an aqueous solution, suspension, dispersion, salve, ointment, gel, cream, lotion, spray or paste.

3. The pharmaceutical composition as claimed in claim 1, wherein the gelling or thickening agent is selected from the group consisting of carboxy methylcellulose sodium, sodium alginate, hydroxypropyl methylcellulose (HPMC).

4. A process for the preparation of a pharmaceutical composition according to claim 1, comprising the steps of: i) providing cocoons of Bombyx mori; ii) heat extracting the cocoons as provided in step (i) to forma sericin gel; iii) providing a culture of Candida bombicola in 10% glucose solution and 1% vegetable oil to obtain a cell broth; iv) incubating the cell broth as obtained in step (iii) at nearly 28 C., up to 180 rpm for 6-7 days followed by extracting with ethyl acetate to obtain sophorolipid; v) adding sodium alginate to the sericin gel obtained in step (ii) with continuous stirring; vi) heating the mixture obtained in step (v) in a water bath at 40-70 C.; vii) adding sophorolipid obtained in step (iv) to the mixture of step (vi) to form a uniform gel; and viii) sterilizing the uniform gel as obtained in step (vii) under UV light for nearly 20-25 minutes to obtain the wound healing composition.

5. A method of treating wounds comprising applying a pharmaceutical composition according to claim 1 to a wound area.

6. A method of healing wounds or removing scars comprising applying a pharmaceutical composition according to claim 1 to a wound or scar.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIGS. 1 to 5 depicts the visible observation of comparative wound healing of the Wister rats by the application of control vis--vis group B. The group B (presented on the right side) treated with the gel according to the invention completely diminishes the scar of the wound.

(2) Table 1 shows body weights of the animals in grams.

(3) Table 2 depicts different test formulations.

(4) Table 3 represents wound size measurement in rats post application of test formulations at day 2, 4, 6, 8 and 10.

DETAILED DESCRIPTION OF THE INVENTION

(5) The invention will now be described in detail in connection with certain preferred and optional embodiments, so that various aspects thereof may be more fully understood and appreciated.

(6) The present invention provides biodegradable and biocompatible pharmaceutical composition comprising silk Sericin, sophorolipid, a gelling or thickening agent and one or more pharmaceutically acceptable carriers or excipients for faster wound healing and limit scarring.

(7) Sericin as used in the present composition is a protein created by Bombyx mori (silkworms) in the production of silk. Silk cocoons were purchased from Sai Techni silk Industry located in Jejuri near Pune. Use of 8 to 10% sericin is permitted by USFDA. Sericin gel is obtained by heat extraction of the cocoons of Bombyx mori. It is reported that Sericin containing food relives constipation, suppresses development of bowel cancer and accelerates the absorption of minerals. Sericin is also used in the products such as Skin, hair and nail cosmetics.

(8) The second active, sophorolipid as used in the invention is produced using Candida bombicola (ATCC 22214) in 10% glucose solution and 1% vegetable oil by incubating the cell broth at 28 C. followed by extracting with ethyl acetate to obtain a brown colored viscous product (sophorolipid) that was stored at 4 C. One preferred vegetable oil is oleic acid. Sophorolipid produced using any other vegetable oil is also suitable for the purpose of the present invention including Linoleic, linolenic, stearic, arachidonic, arachidic and such like.

(9) The active components viz., sericin and sophorolipids as used in the invention can be produced from renewable sources and are completely biocompatible and biodegradable. Since, the production of sophorolipid is very cost effective and the required amount in the present wound healing composition is very negligible, the invention disclosed herein is also cost-effective.

(10) The sericin is by-product of silk industry and can be obtained easily as it is considered as waste. sericin and sophorolipid, both the components as used in the instant compositions are biocompatible and completely absorbed by the skin without any side effects. The composition according to the invention causes faster wound healing as compared to commercially available wound healing formulations. Also, the wound healing compositions leaves minimal scar in the wound area after the healing process.

(11) The commercially available formulations cause side effects such as erythema, erosion, edematous swelling, allergy etc. Contrary to the commercial formulations, the instant formulation does not show any side effects as described earlier. The rationale behind the instant formulation encompasses the synergistic activity of the combination of both the active ingredients. The antibacterial and cell proliferation activity of the sericin and sophorolipid enhances cell proliferation and eventually vascularization. Therefore, the instant formulation shows better wound healing capacity.

(12) In an embodiment, the said biodegradable and biocompatible pharmaceutical composition comprises sericin in the range of 8-10% w/w; sophorolipid in the range of 0.01 to 10%; a gelling or thickening agent in the range of 2 to 5%, together with one or more pharmaceutically acceptable carriers/excipients.

(13) In a preferred embodiment, the said gelling or thickening agents may be selected from the group consisting of various gums, Carboxy methylcellulose sodium, sodium alginate, Hydroxypropyl Methylcellulose (HPMC).

(14) In another preferred embodiment, sodium alginate is used as a gelling agent.

(15) In yet another preferred embodiment, the said topical composition may be formulated into variety of formulations selected from the group consisting of cream, ointment, gel, spray or solution using appropriate pharmaceutical carriers/excipients.

(16) In another preferred embodiment, the said composition is formulated as topical gel.

(17) In yet another preferred embodiment, pH of said formulations is maintained at 5.5-6.1.

(18) In yet another embodiment, the invention provides a process for preparation of topical gel comprising the steps: i) providing cocoons of Bombyx mori; ii) heat extracting the cocoons as provided in step (i) to form sericin gel; iii) providing culture of Candida bombicola in 10% glucose solution and 1% vegetable oil to obtain cell broth; iv) incubating the cell broth as obtained in step (iii) at nearly 28 C., upto 180 rpm for 6-7 days followed by extracting with ethyl acetate to obtain sophorolipid; v) adding sodium alginate to the sericin gel obtained in step (ii) with continuous stirring; vi) heating the mixture obtained in step (v) in a water bath at 40-70 C.; vii) adding sophorolipid obtained in step (iv) to the mixture of step (vi) to form a uniform gel; viii) sterilizing the uniform gel as obtained in step (vii) under UV light for nearly 20-25 minutes to obtain the wound healing composition.

(19) The formulation thus prepared is further evaluated for its extrudability, swelling index, in-vitro diffusion study, and release kinetics and ex-vivo bio-adhesive strength.

(20) In still yet another embodiment, the present invention provides methods of treating wounds comprises applying to said wound a wound-healing amount of said pharmaceutical composition to the affected area. The period of application will depend on the size and severity of the wound.

(21) In still yet another embodiment, the present invention provides methods of administering said pharmaceutical compositions.

(22) In another preferred embodiment, the said composition is useful for diabetic scar healing and in burns.

(23) In yet another embodiment, the invention provides wound healing testing protocol of the compositions in vitro and in vivo and its efficacy based on the parameters viz., visible observations; wound size measurement; physical examination; drug content determination; viscosity measurement (Rheology); In vitro drug release studies; Drug release kinetic studies; Skin irritation test and Histopathological studies.

(24) In Incision model, the results confirm the faster wound-healing activity of Sericin plus sophorolipids gel. The antioxidant activity is assessed by DPPH scavenging method wherein, the sericin plus sophorolipids gel is found to be most potent antioxidant than the standard gels. Thus the gel composition comprising Sericin plus sophorolipids possesses not only antimicrobial activity but also antioxidant activity and hence provides faster wound healing than the standard gels.

EXAMPLES

(25) The following examples are given by way of illustrations and should not be construed to limit the scope of the present invention.

Example 1

(26) A. Sophorolipid Production

(27) Seed culture was developed by transferring loopful of Candida bombicola ATCC 22214 cells from slant, in 10 mL Malt Extract-Glucose-Yeast extract-Peptone (MGYP) medium, followed by incubation at 28 C., 180 rpm for 24 h. This seed culture was transferred to 90 mL of fresh media and incubated for 48 h at 180 rpm, 28 C. After 48 h of growth the culture media was centrifuged at 5000 rpm, for 20 minutes at 10 C. The cell pellet was collected and further subjected to resting cell method.

(28) B. Resting Cell Method

(29) The cell pellet was re-suspended in 10% of glucose solution. 1% oil (Oleic acid) was added to the above solution. For the production of sophorolipids, the cell broth was incubated at 28 C., 180 rpm for 6-7 days with continuous monitoring. This procedure was repeated three times and sophorolipids was extracted for further yield estimation and characterization.

(30) C. Extraction of Sophorolipids

(31) Extraction of sophorolipid was ensued after 7 days when the oil film was visibly vanished from the culture medium. Further the medium was centrifuged at 5000 rpm for 20 minutes at 10 C. to pellet down the cells. Supernatant was collected and extracted with equal volume of ethyl acetate as described in prior art. Anhydrous Sodium sulphate was added to remove any traces of water left. The ethyl acetate was filtered and reduced by rotary evaporation under vacuum to yield a brown colored viscous product that was stored at 4 C.

Example 2: Formulation Details and Process of Preparation

(32) In order to optimize the concentration of gelling agent to achieve proper consistency of the gel, formulations were prepared with different gelling or thickening agents such as various gums, Carboxy methylcellulose sodium, sodium alginate, Hydroxypropyl Methylcellulose (HPMC) with different concentrations of 1 to 8% were tried. The formulations that showed good spreadability and consistency was selected for further studies.

(33) A. Composition of One Preferred Formulation

(34) TABLE-US-00001 Ingredients Quantity Sericin 8-10% SL(sophorolipid) 1 mg/ml Sodium alginate 2-3%
B. Process for Preparation:

(35) Gelling agent sodium alginate was slowly added to the sericin gel with continuous stirring and heating on water bath (Temp: 40-70 C.). SL (sophorolipid) was added with continuous stirring till a uniform gel was formed. Formed gel was placed under UV light for sterilization upto 20-25 min and stored in plastic container at room temperature.

(36) The prepared gel was inspected visually for their colour and homogeneity. The spreadability (n=3) of the gel formulation was determined by measuring the spreading diameter of 1 g of gel between two horizontal plates (20 cm20 cm) after one min. The standardized weight tied on the upper plate was 125 g.

(37) The pH was measured at room temperature, in each gel sample using digital pH meter which was calibrated before each use with standard buffer solutions. The pH of the gel formulations was performed at 1, 10, 45 and 60 days after preparation to detect any pH changes with time and the observations on the formulation prepared areas below:

(38) a) Colour: Brown

(39) b) pH determination: 5.50.06 to 6.10.33

(40) c) Spreadability: 47 to 600.50 mm

(41) d) Homogeneity: Homogeneous

Example 3: Wound Healing: Testing Protocol and Results: In Vitro and In Vivo

(42) A. Testing Protocol:

(43) Wister rats (Male) obtained from National Institutional of Bioscience, Pune, Maharashtra weighing 25020 gm were used and all the studies performed as per CPCSEA guidelines (CPCSEA Reg No. SSBS/AH/04-2015). The animals were housed in a standard individual metal cages and room was maintained at 221 C. with an alternating 12 h light-dark cycle. Food and water were provided ad libitum. All the experiments on animals were conducted after obtaining permission from Institutional Animal Ethical Committee of the Institute.

(44) B. Incision Wound Model:

(45) Animals were divided into two groups (six animals each). Body weights of the animals in grams are shown in following table:

(46) TABLE-US-00002 TABLE NO. 1 GROUPS (wt in grams) ANIMAL A B 1 270 259 2 250 266 3 256 269 4 245 268 5 270 240 6 244 267

(47) All animals of two groups were anesthetized with an aesthetic ether, and a paravertebral long incision of 4.4 cm length were made through the skin and cutaneous muscle at a distance about 1.5 cm from the middle on right side of the depilated back.

(48) All groups (A and B) of animals received sufficient amount of formulation applied externally (as depicted in table No. 2). All the test formulations were applied once a day for 10 days starting from the day of incision. Wound-healing property was evaluated by wound length and wound closure time.

(49) TABLE-US-00003 TABLE NO. 2 GROUP FORMULATION A Positive control (povidone iodine ointment, commercially available) B Test group (formulation of sophorolipid with sericin) C Sericin alone D Negative control (untreated) E Sophorolipid alone
C. Results: Visible Observations

(50) The area of wound was measurement on the days 2, 4, 6, 8, 10 days of post-surgery in all the groups (as shown in FIGS. 1 to 5 respectively). The group, treated with Sericin plus sophorolipids gel, showed fast contraction and healing when compared with control and standard compounds. There was very rapid closure of the wound treaded with Sericin plus sophorolipids ointment when compared with control and standard compounds. FIGS. 1 to 5 depicts the visible observation of comparative wound healing of the Wister rats (control vis--vis group B) by the application of the formulations. The group B treated with the formulation according to the invention not only reduces wound size (in diameter) but also completely diminishes the scar of the wound.

(51) When a wound occurs and is exposed to external environment, it is more prone to attack by microbes, which invade through the skin and delay the natural wound-healing process. Reactive oxygen species (ROS, includes oxygen-derived radicals known as well as non-radical oxidants), often loosely termed oxidants, are vital part of healing and serve as cellular messengers that drive numerous aspects of molecular and cell biology. ROS can trigger the various beneficial pathways of wound healing, for example, at micro molar concentrations of hydrogen peroxide can promote vascular endothelial growth factor (VEGF) expression in keratinocytes (Khanna et al., 2001). Results obtained in this study confirm the faster wound-healing activity of Sericin plus sophorolipids gel. The antioxidant activity was assessed by DPPH scavenging method wherein, Sericin plus sophorolipids gel was found to be most potent antioxidant than the standard gel. This will confirm that the Sericin+sophorolipids gel not only possesses antimicrobial activity but also possesses antioxidant activity.

Example 4: In Vitro Testing Protocol

(52) 1. Wound Size Measurement:

(53) Every alternative days wound size contraction was measured. By placing transparent blotting paper carefully on the wounded part of rats marking were done by permanent marker and size of reduction in wound noted.

(54) TABLE-US-00004 TABLE NO. 3 SR NO A B C D E DAY 2 1 34 17 24 39 26 2 27 27 25 40 28 3 26 27 30 44 25 4 24 26 30 43 24 5 35 23 26 43 27 6 20 24 26 36 26 AVG: 27.66667 24 26.83333 40.83333 26.000 DAY-4 1 33 9 21 36 25 2 27 20 23 35 26 3 25 13 27 38 22 4 24 18 28 38 21 5 34 15 24 37 24 6 20 14 23 31 24 AVG: 27.16667 14.83333 24.33333 35.83333 23.666 DAY-6 1 31 5 20 34 23 2 24 10 22 33 24 3 23 8 25 35 18 4 22 9 27 34 19 5 33 9 24 33 21 6 19 8 22 30 18 AVG: 25.33333 8.166667 23.33333 33.16667 20.5 DAY-8 1 27 2 18 29 18 2 22 4 16 28 18 3 19 6 17 28 15 4 20 8 16 30 16 5 29 3 16 26 17 6 18 4 15 25 15 AVG: 22.5 4.5 16.33333 27.66667 16.5 DAY 10 1 19 1 9 20 10 2 15 0 6 22 11 3 15 0 7 23 7 4 15 0 6 24 7 5 16 1 6 19 8 6 9 2 5 18 6 AVG: 14.83333 0.666667 6.5 21 8.11

(55) From the above, it is evident that the group B treated with the formulation comprising sericin and sophorolipid provides faster and complete wound healing at day 10, from the date of incision, when compared to control/standard/other test formulations.

(56) 2. Photographic Comparison

(57) From the above, it is evident that the group B treated with the formulation comprising sericin and sophorolipid provides faster and complete wound healing at day 10 (FIG. 5), from the date of incision, when compared to control and other test formulations.

Example 5: Skin Irritation Test

(58) The skin irritation test was carried out on male Wistar Rats 25020 gm. The animals were kept under standard laboratory conditions, with temperature of 22 C.1 C. and relative humidity of 55%5%. The animals were housed in standard individual metal cages with free access to a standard laboratory diet.

(59) Hair was shaved from back and area of 4 cm.sup.2 was marked on both the sides, one side served as control while the other side was test. Gel was applied (500 mg/animal) twice a day for 7 days and the site was observed for any sensitivity and the reaction if any, was graded as 0, 1, 2, 3 for no reaction, slight patchy erythema, slight but confluent or moderate but patchy erythema and severe erythema with or without edema, respectively.

(60) The above test confirms that the formulation according to the invention is safe as it is not provoked any allergic response when applied on the skin.

ADVANTAGES OF INVENTION

(61) Wound healing compositions with naturally derived materials Free of skin irritation Economically viable compositions No side effects